ANALYSIS OF

URINE AND BODY FLUIDS

UNOFFICIAL COMPILATION NOTES

SY 2020-2021 BY AIRAH MANZA

 ANALYSIS OF URINE & BODY FLUIDS So, if you look at your
TOPIC 01 URINARY SYSTEM REVIEW ANATOMY AND PHYSIOLOGY back, since your kidney
is a retroperitoneal
Course Outline:
organ, it can be easily
▪ Functional Anatomy of the Urinary System
accessed at the back.
▪ Functions of the Urinary System You have this angle
▪ Parts of the Nephron here in between your
▪ Mechanisms of Urine Formation 12th rib and vertebrae
▪ Characteristics of Urine which is called costal-
▪ General Principles of Urine Formation vertebral angle where
you can locate your
KIDNEY kidney.
FUNCTIONS
filter blood, allowing wastes and excess ions to leave the body in Here is a CT scan of your
1 urine while returning needed substances to the blood in just the abdomen showing your
right proportions kidneys here. Basically,
as one of the excretory organs of the body, it eliminates nitrogenous retroperitoneal: it is at the
2 (nitrogen-containing) wastes, toxins, and drugs from the body (most back. They are slightly anterior
of the drugs are either eliminated on the kidney or the liver) to the psoas muscles (P). The
regulate the blood’s volume and chemical makeup to maintain the right kidney is found behind
3 proper balance between water and salts and between acids and the liver (L).
bases (kidneys will determine your acid-base balance)
4 by producing the enzyme renin, they help regulate blood pressure
▪ size of the kidney:
the hormone, erythropoietin, stimulates the RBC production in
o 12 cm (5 inches) long
bone marrow (that’s why it is very important for patients who have
5 o 6 cm (2.5 inches) wide
a damaged kidney, especially in chronic kidney diseases, to have
o 3 cm (1 inch) thick
erythropoietin injections to prevent anemia)
o about the size of a large bar of soap
6 kidney cells convert Vitamin D into its active form
▪ renal hilum: a very important structure
HOMEOSTATIC FUNCTIONS ▪ enclosed in a fibrous capsule
1 excretion of metabolic waste products and foreign chemicals ▪ surrounded by perirenal fats BLOOD SUPPLY OF KIDNEYS
2 regulation of water and electrolyte balance ▪ renal cortex (cortex=bark): coat-side portion of the kidney receives about 22% (in some books, ¼ or 25%) of the cardiac output, or
3 regulation of body fluid osmolality and electrolyte concentrations ▪ renal medulla (middle) 1100 ml/min
▪ pyramids (in the nnedulla)
4 regulation of arterial pressure
▪ renal pelvis
5 regulation of acid-base balance
▪ calyces (collect into and becomes the renal pelvis)
6 regulation of erythrocyte production
7 secretion, metabolism, and excretion of hormones
8 gluconeogenesis
LOCATION AND STRUCTURE
▪ the kidney is a retroperitoneal organ
▪ located in the superior lumbar region
▪ marker: T12 to the L3 vertebra

Added info: Your kidney has two capillary beds: your glomerulus (first
capillary bed) and your peritubular capillaries (second).

structural and functional unit

nephrons
800,000 to 1,000,000 of nephrons per kidney

AIRAH M.
 has two types: Note: Read the function of the juxtaglomerular apparatus on blood pressure.
▪ cortical nephrons: found in the cortex Here is another microscopic view of the nephron. You can see that the distal
▪ juxtamedullary nephrons convoluted tubules are slightly pale. The collecting ducts are lined with
cuboidal cells.

Here is an electron microscopic view of the

renal capsule. Here are the foot-like
processes (marked with yellow circles).
Then, these are your capillaries (marked
with red circles). In between is your
has two main structures: basement membrane. The substances
▪ renal corpuscle passes through the capillary, then to the
basement membrane, towards the
▪ renal tubule filtration slits in your podocytes.
contains the:
▪ glomerulus
▪ Bowman’s capsule (encloses the glomerulus)
▪ podocytes: called so because it contains foot-like
processes; “pod” means foot

renal
corpuscle URETER
bilateral slender tubes
25-30 cm length
lumen is about 6mm diameter which is lined by the
ureters
transitional epithelium
function: act as passageways that connect the kidney to
the bladder

divided into four segments: RENAL TUBULE: After the glomerulus, the proximal convoluted tubule is
▪ proximal convoluted tubule immediately found. Then, it forms a loop called the nephron loop (loop of
renal Henle), which has thick and thin segments, going to the distal convoluted
▪ nephron loop or loop of Henle
tubule
▪ distal convoluted tubule tubule, then it drains into the collecting ducts.
▪ collecting duct

URINARY BLADDER
smooth, collapsible, muscular sac that stores urine
urinary
temporarily
bladder
function: storage of urine

AIRAH M.
 located retroperitoneally in the pelvic cavity =, just in males, it is quite longer because of its This is the reason why there are two capillary beds: the glomerular capillaries
posterior to the symphysis pubis surrounding organs and the peritubular capillaries.
3 openings: 2 urethral orifices and internal urethral ▪ prostatic urethra
orifice ▪ membranous urethra non-selective, passive process in which fluid passes
trigone: formed together by the orifices ▪ penile (spongy) urethra from the blood into the glomerular capsule part of the
renal tubule
capillaries of the glomerulus are under high pressure
glomerular
compared to the glomerular capsule: as a result, fluids
filtration
move down the pressure gradient, from the blood into
the glomerular capsule
▪ oliguria: 100-400 ml/day of urine
▪ anuria: <100ml/day of urine
begins as soon as the filtrate enters the proximal
convoluted tubule
tubule cells are “transporters”, taking up needed
substances from the filtrate and then passing them out
in females, it’s shorter, just a very short area in tubular their posterior aspect into the extracellular space, from
which it drains into the vagina reabsorption which they are absorbed into peritubular capillary
has three layers of detrusor muscle
blood
epithelium: transitional epithelium (has an
most reabsorption occurs in the proximal convoluted
umbrella-shaped or dome-shaped structure in which
tubules, but the distal convoluted tubule and the
this epithelium is
specifically designed collecting duct are also active
for exposure to reverse of tubular reabsorption
urine) (has three this process seems to be important for getting rid of
tubular
layers: basal, substances not already in the filtrate, such as certain
secretion
intermediate, outer) drugs, excess potassium, or as an additional means for
controlling blood pH)

RENAL HANDLING OF FOUR HYPOTHETICAL SUBSTANCES

freely filtered but not reabsorbed nor secreted
excretion rate = filtration rate
filtration only
e.g. creatinine (is filtered, not reabsorbed nor secreted,
goes directly into the urine)
filtration, urinary excretion < rate of filtration
partial excretion rate = filtration rate – reabsorption rate
URINE FORMATION
reabsorption e.g. electrolytes such as Na+, Cl-
Rates at which substances are excreted in the urine are due to: all the filtered substance is reabsorbed
▪ glomerular filtration filtration, occurs for some of the nutritional substances in the
▪ reabsorption of substances back into the blood complete blood (amino acids, and glucose), allowing them to be
URETHRA ▪ secretion of substances from the blood into the renal tubules reabsorption conserved in the body fluids (you notice that your urine
*urine formation is due to these processes* doesn’t have glucose unless your kidneys are damaged)
thin-walled tube that carries urine by peristalsis substance is freely filtered and additional quantities are
from the bladder to the outside of the body urinary excretion rate = filtration rate – reabsorption rate + secretion rate
secreted from the peritubular capillaries into the
different structures: ① glomerular filtration: water and tubules
▪ internal urethral sphincter filtration, often occurs for organic acids and bases, permitting
solutes smaller than proteins are forced secretion
▪ external urethral sphincter them to be rapidly cleared from the blood and excreted
through the capillary walls and pores of
in large quantities
the glomerular capsule into the renal
tubule excretion rate = filtration rate + tubular secretion rate
urethra ② tubular reabsorption: water, glucose,
amino acids, and needed ions are
transported out of the filtrate into the
tubule cells and then enter the capillary
blood
③ tubular secretion: H+, K+, creatinine,
and drugs are removed from the
peritubular blood and secreted by the tubule cells into the filtrate

AIRAH M.
 Summary of the Renal Handling of Substances As your bladder accumulates more urine, you will start to have your The table above is a sample urinalysis result. The left table is for the physical
micturition contractions until it increases. examination of urine, while the right table is for the microscopic
⬇ examination.
When the bladder is only partially filled, these micturition contractions
you normally have a yellow urine.
usually relax spontaneously after a fraction of a minute, the detrusor Sometimes, it can get in a range of
muscles stop contracting, and pressure falls back to the baseline. red to orange
⬇ color
can even get to other colors (e.g.
As the bladder continues to fill, the micturition reflexes become more black) which indicates abnormal
frequent and cause greater contractions of the detrusor muscle. results
⬇ transparency clear, turbid, or cloudy
Initial contraction of the bladder activates the stretch receptors to cause a is the comparison of the urine (has
greater increase in sensory impulses from the bladder and posterior urethra, suspended solutes) to distilled
which causes a further increase in reflex contraction of the bladder. (Cycle is specific gravity water (has zero solutes)
repeated again & again until the bladder has reached a strong degree of the higher the specific gravity, the
contraction.) higher your solutes
⬇ the parameters from the right table, including the others not mentioned
here, can be tested with the dipstick method or with the use of
After a few seconds to more than a minute, the self-regenerative reflex
automation (eliminates reader bias)
begins to fatigue and the regenerative cycle of the micturition reflex ceases,
RBCs WBCs
permitting the bladder to relax (urination).
e.g. Your water is filtered, and is sometimes absorbed (in the proximal However, you can control your flow of micturition. This is done by the PONS
tubule, loop of Henle, and near the collecting ducts). (Review the diagram.) and cerebral cortex which can override the micturition reflex. Higher centers
MICTURITION REFLEX (how do we urinate?) (PONS and cerebral cortex) normally exert final control of micturition as
Your urinary bladder is controlled by the autonomic nervous system: the follows:
sympathetic and parasympathetic. The higher centers keep the micturition reflex partially inhibited,
1
except when micturition is desired. casts: protein-based structures crystals
The higher centers can prevent micturition, even if the micturition
2 reflex occurs, by tonic contraction of the external bladder sphincter
until a convenient time presents itself.
When it is time to urinate, the cortical centers can facilitate the
sacral micturition centers to help initiate a micturition reflex and at
3
the same time inhibit the external urinary sphincter so that urination
can occur.
mucus threads EC (epithelial cells)
VOLUNTARY URINATION
Person voluntarily contracts his or her abdominal muscles, which
increases the pressure in the bladder and allows extra urine to enter
4
the bladder neck and posterior urethra under pressure, thus
stretching the walls.
sympathetic allows relaxation of the detrusor muscles that’s why This stimulates the stretch receptors, which excites the micturition
5
ANS the urine will be collected in the bladder reflex and simultaneously inhibits the external urethral sphincter.
parasympathetic allows relaxation of the sphincter (when we pee) ALL the URINE will be emptied, with rarely more than 5 to 10
6 NOTE for CRYSTALS: when reporting crystals, you have to know the pH
ANS excretion rate = filtration rate – reabsorption rate milliliters left in the bladder.
of the urine because some crystals form only in acidic urine, others in
FILLING OF THE BLADDER AND URINE: OVERVIEW OF URINALYSIS basic urine. Each crystal has a different name when ssyou are in an acidic
BLADDER WALL TONE or a basic pH.
Color Yellow RBC 2-4/hpf NOTE for EC: In the EC parameter, you can sometimes see squamous EC,
▪ beyond 300 to 400 ml, Transparency Clear WBC 5-10/hpf
collection of more urine tubular cells, or transitional EC. That is very important to report.
Specific Gravity 1.005 Casts None
in the bladder causes the pH 6.5 Crystals None
pressure to rise rapidly Glucose Negative Mucus threads None
▪ micturition waves: Protein Negative EC 1-2/hpf
periodic acute increases Ketone Negative
in pressure that last from Nitrites Negative
a few seconds to more Leukocytes Trace
than a minute Blood Trace

AIRAH M.
Youtube Video Title: Anatomy and Physiology of the kidneys, Urinary renal papilla—or nipples, pointing towards the in each kidney, there are about 1 million nephrons
Bladder, Ureters, Urethra, and Nephron center of the kidney nephron each nephron is made up of a renal corpuscle and a
Link: https://www.youtube.com/watch?v=805VoHIIQCs
renal papilla: project into minor calyces which join renal tubule
workhorses of the urinary system (like a water together to form major calyces which funnel into the where blood filtration starts
purification plant that helps clean the drinking water renal pelvis includes the:
for a city) renal pelvis: where urine is collected which then renal corpuscle ▪ glomerulus (tiny bed of capillaries)
main function: filter all of the body’s blood about 30 heads out of the kidney through the ureter ▪ Bowman’s capsule (made of renal cells that
times a day, and to produce urine the outside rim which can be divided into an outer surround the glomerulus)
twin, bean-shaped organs in your body that clear cortical zone and an inner juxtamedullary zone
harmful substances by filtering your blood it also has sections called renal columns which As blood flows into the glomerulus, water and some solutes in the blood like
produces hormones and also regulate: renal cortex extend down into the medulla separating the renal sodium are able to pass through the endothelial lining of the capillary, move
▪ blood pH pyramids from one another across the basement membrane, through the epithelial lining of the
▪ volume above each renal pyramid and renal cortex is the nephron and finally into the Bowman’s space of the nephron itself—at
kidneys ▪ pressure renal lobe which point it is called filtrate.
▪ osmolality
specialized cells that made up the epithelium of the
located between the T12 and L3 vertebrae, and are
podocytes nephron which wrap around the basement
partially protected by ribs 11 and 12 (floating ribs)
membrane like the tentacles of an octopus
roughly the size of a fist and are retroperitoneal,
tiny gaps found between the tentacle-like
meaning they sit behind the peritoneal membrane
projections
alongside the vertebral column
filtration slits act like a sieve allowing only small particles (e.g.
right kidney is pushed down by the liver, so it sits
water, glucose and ionic salts) to pass through while
slightly lower than the left kidney
blocking large proteins and RBCs
in its middle, there is an indentation that forms the
renal hilum
the entry and exit point for the:
▪ ureter So an adult’s kidneys filter about 150 liters of blood every day. If we assume
renal hilum ▪ renal artery and renal vein that there are 5 liters of blood in the body, that means that the entire blood
▪ lymphatics volume gets filtered about 30 times a day, which is more than once every
▪ nerves going into and come out of the kidney hour. Because of this the kidneys get about ¼ of the cardiac output which is
blood getting pumped out of the left ventricle.
BLOOD FLOW IN KIDNEYS
aorta → left and right renal arteries → (further divide into) segmental
arteries → interlobar arteries (pass through the renal columns) → arcuate
arteries (go over the bases of the renal pyramids) → cortical radiate
arteries (supply the cortex) → (continue to divide forming) afferent
arterioles → (split into a tiny bundle of capillaries) glomerulus (blood
filtration site) → (blood does not enter into venules, instead into) efferent
where the filtrate from the Bowman’s capsule flows
arterioles → (divide into capillaries, 2nd time) peritubular capillaries → into
cortical radiate veins → arcuate veins interlobar veins → left and right surrounded by the peritubular capillaries
THREE LAYERS OF TISSUE IN THE KIDNEY
located on the outside which is a thin layer of dense renal veins → inferior vena cava can be divided into:
▪ proximal convoluted tubule
renal fascia connective tissue that anchors the kidney to its The flow of the veins is similar to the arteries but in reverse, the only ▪ nephron loop or loop of Henle
surroundings difference is that there’s a segmental artery but no segmental vein. o descending limb
the middle layer which is a fatty layer that protects o ascending limb
adipose capsule
the kidney from trauma renal tubule ▪ distal convoluted tubule
the deepest layer which is a smooth transparent ▪ collecting duct (ultimately sends urine to the
renal capsule sheet of dense connective tissue that gives the minor calyces)
kidney its distinctive shape here, the filtrate becomes fine-tuned based on what
TWO MAIN PARTS OF THE KIDNEY (cross-section) the body wants to keep versus what it wants to
the inner portion which is made up of 10 to 18 renal discard, with water and solutes getting passed back
renal medulla pyramids with the base of the pyramids facing the and forth between the filtrate in the lumen of the
renal cortex and the tips of the pyramids, called renal tubule and the blood in the peritubular
capillaries

AIRAH M.
 a really unique region each nephron has The ureters insert into the bladder at the ureterovesical junction at a in men and women, around the internal urethral orifice,
involved in the regulation of blood pressure and the sideways angle so that. It is basically. the detrusor muscle thickens to form the internal
glomerular filtration rate—or the amount of blood sphincter
that passes through the glomeruli each minute has a muscular lining which helps to push urine along internal sphincter: an involuntary sphincter is controlled
juxtaglomerular located in between the distal convoluted tubule and insert into the bladder at the ureterovesical junction at a by the autonomic nervous system and keeps the urethra
complex the afferent arteriole sideways angle closed when the bladder isn’t full
has three types of cells: ▪ when the bladder becomes full, it compresses the external sphincter: found at the level of the urogenital
▪ macula densa cells ureter diaphragm in the floor of the pelvis which is under
openings to the ureters preventing the backflow of
▪ juxtagmolerular cells urine voluntary control
▪ extraglomerular mesangial cells ▪ a one-way valve that prevents urine from refluxing ▪ by contracting the skeletal muscles around it,
backwards from the bladder into the ureters urination can be stopped voluntarily (kegel exercise:
looks like a balloon done to strengthen the pelvic floor)
its muscular wall has many folds called rugae that can
contract when the bladder is emptied of urine and can
expand when it is filled with urine
in women, it is in front of the vagina, uterus, and rectum
in men, it is just in front of the rectum
can hold around 750 millilitres of urine
▪ slightly less in women because of crowding from the
uterus (especially true during pregnancy)
bladder
the floor of the bladder has a smooth triangular region
called the trigone region with two corners at the
ureterovesical junctions and the third corner being the
internal urethral orifice, where the bladder meets the
located in the distal convoluted tubule urethra
they can sense when levels of sodium & chloride trigone region:
are low very sensitive to expansion and once it stretches to a The act of urination involves close coordination between the nervous
macula densa in the case of hypovolemia and hypotension, certain point, the bladder sends a signal to the brain - that system and muscles of the bladder. Once the volume of bladder is greater
cells these sense the low sodium and chloride levels it’s time to pee than about 300-400 millilitres, basically when it’s half full, pressure on the
and send a signal over to the juxtaglomerular cells
bladder walls increase and send signals to the urination or micturition centre
which are located in the wall of the afferent LAYERS OF THE BLADDER
arteriole in the spinal cord, located at S2 and S3.
a mucosal layer that is stretchy allowing for bladder
transitional
extraglomerular help with the signaling between macula densa distention while maintaining a barrier between urine and
epithelium This sets off a reflex arc called the micturition reflex which causes
mesangial cells cells and juxtaglomerular cells the body
receive the signal and also independently sense detrusor a thick muscular layer called that helps with bladder contraction of the bladder and relaxation of the internal sphincter and
the low pressure in the blood vessels and secrete muscle contraction during urination external sphincter.
an enzyme called renin fibrous
juxtaglomerular
▪ increases sodium reabsorption which helps adventitia -
cells
raise the blood volume outer layer
▪ also causes constriction of blood vessels which
helps raise blood pressure a thin muscular tube that drains urine from the bladder,
starting from the internal urethral orifice to the external
Once millions of nephrons have each made urine, it flows into the minor opening
calyces, then major calyces, and finally into the renal pelvis. From there, it in males, urethra passes in this order:
goes down the ureter. ▪ through the prostate (prostatic urethra)
▪ through deep muscles of peritoneum (intermediate
urethra)
urethra ▪ through the penis (spongy urethra)
*male urethra is also used during ejaculation, except
there - semen enters into the urethra via the ejaculatory
ducts* Now, the pontine storage center and pontine micturition center are two
in females, the urethra runs through the perineal floor of areas in the pons part of the brain that help control urination. When you
the pelvis and exits between the two labia minora, above can’t find a toilet and want to hold urine in, you activate the pontine storage
vaginal opening and below the clitoris in an area called center and that stops the micturition reflex. When you finally do find that
the vulval vestibule toilet, and you’re ready to urinate, the pontine micturition center is active
and it allows the the micturition reflex to happen - and you can finally pee.

AIRAH M.
 AUBF: URINALYSIS 1 (INTRODUCTION TO URINALYSIS & PHYSICAL ▪ urine examinations can be used to detect other diseases (e.g. sample at the beginning of the collection period, then collect the
EXAMINATION OF URINE) liver disorders can be diagnosed of the presence of a certain succeeding samples. The last sample will be collected at 7am on the
crystal in the urine sample; pregnancy testing for the presence of following day.
Learning Objectives:
the hormone hCG) After the collection, the specimen is brought to the lab for examination.
▪ State the reasons for performing urinalysis
monitoring the progress of disease & effectiveness of therapy The MT checks the label and measures the volume of the urine. Hence,
▪ Differentiate the types of urine specimens, the correct procedure of ▪ UTI then the physician is giving him antibiotics, there is an initial the first examination done is the determination of urine volume. After
collection & the diagnostic use of each. 3 urine analysis prior to the medication and after treatment, there that, aliquots are taken. You get several small samples of the 24-hr
▪ List the different urine specimen containers & their usage. is a follow up urinalysis to check whether the drug is effective or collection and store it in the freezer if it cannot be examined right away.
▪ Briefly discuss methods of preserving urine specimens of the patient is improving You can’t keep the whole container in the freezer for a long period (takes
▪ List the changes that happen when urine is allowed to stand at room up lots of space and can be mistaken as juice bottles since we use
temperature for more than 2 hours. urine sample must be collected in a clean, dry container
recycled bottles for their volume capacity).
& should be examined when freshly voided, within 2
▪ Describe methods for identifying an unknown fluid as urine. urine
hours of voiding
▪ State possible reasons for rejecting a urine sample. collection NOTE: During the 24-hour collection, the sample should be refrigerated.
If analysis is to be delayed, urine should be refrigerated
▪ List the components of Routine Urinalysis Each time a patient voids, the sample is added to the container.
or preserved
▪ Discuss the relationship of urochrome to normal urine color. COLLECTION ERROR PROBLEMS
▪ Discuss the abnormal urine colors & their significance. A. ROUTINE URINALYSIS loss of voided specimen: whenever px forget that they are doing
▪ List pathologic & non-pathologic causes of urine turbidity. ▪ most common examination the 24hr collection and fails to collect the urination.
1
▪ State possible cause of abnormal urine odor. ▪ method of collection: midstream collection (midstream spx) ▪ The MT should not accept specimen. Discontinue the
▪ Describe the principle of pH determination & significance. procedure and start over again.
advantage: can be collected any time of the day
▪ Describe the principle of Specific Gravity determination & the failure to discard the first specimen: at the start of the collection
and is susceptible for routine examination 2
significance of the test. period, always remind the px to discard the first sample
disadvantage: the concentration of the formed
poor preservation: aside from refrigerating the sample, you also
▪ Recognize normal & abnormal daily urine volume. elements (e.g. urinary casts, WBCs, RBCs) will be
random need to add in preservative
affected by the degree of hydration of the 3
BRIEF HISTORY AND IMPORTANCE specimen ▪ e.g. addition of acid to prevent the growth of bacteria
patient (if the px takes in a lot of fluid during that
Urine analysis was actually the beginning of laboratory medicine. It began day, the urine is dilute; if less water, the patient because we will be storing it for 24hrs
6000 years ago with the analysis of human urine which was called Uroscopy will have concentrated urine sample: it varies as 4 inadequate refrigeration: should not stay at room temp.
until the 17th century and today is termed as URINALYSIS. it depends on the degree of hydration) C. BACTERIOLOGIC or MICROBIOLOGIC EXAMINATION
the specimen of choice because the urine sample spx of choice: clean-catch spx (collected clean catch and midstream)
Examination of urine back then was based only on the physical examination
is more concentrated and stable A clean-catch midstream specimen is desirable but sometimes
of the urine sample: the color, turbidity, odor, volume, and viscosity. This
▪ Why stable? Because the urine spx has an catheterization or suprapubic aspiration of the bladder is necessary. If
was based on the drawings they saw from Egyptian hieroglyphics and the acidic pH in early mornings in which the the px is unable to void, another alternative is catheterized sample or
drawings of cavemen. They saw physicians examining a bloody-shaped glass formed elements are better preserved. suprapubic aspiration of the bladder is done by the physician (not by
of urine. first morning MT).
also recommended for pregnancy testing to
specimen
Later on, it progressed to ant testing. If the urine attracts ants, it means the prevent a false negative pregnancy test: the One important consideration: The spx container should be sterile unlike
urine is sweet (it contains a lot of glucose). Then, it progressed to taste hormone hCG is more concentrated in the first in routine urinalysis (can use a clean, dry, chemical-free container).
testing, they taste if the urine is sweet or not (lolol idk I really can’t hear). morning sample However, in bacteriologic examinations, a sterile container is needed.
After that, it progressed to chemical testing to the examination of protein: this is also used for the evaluation of orthostatic
proteinuria Bacterial culture should be done immediately but if not possible, urine
the determination of albuminuria wherein if urine is boiled, the proteins will
should be refrigerated at 4°C until cultured.
coagulate. If it coagulates, there is an increased amount of protein in the B. QUANTITATIVE ANALYSIS
sample. spx of choice: timed specimen Strong bacterial agents (e.g. hexachlorophene or povidone iodine)
▪ 2 to 12-hour timed collection should not be used. Mild antiseptic towelletes are recommended.
After that, the microscope was invented in the 17th century which led to the
▪ 24-hour specimen SPECIAL COLLECTION TECHNIQUE
examination of the urine sediments wherein they developed methods to
usually used for urobilinogen determination because 1 Urethral catheterization
quantitate these sediments. Now, we have what we called Addis Count
2hr it is the time of peak excretion of urobilinogen Introduce a catheter into the urethra & bladder. Necessary for
wherein a 12hr urine sample is collected, and the cells are counted. patients who are unable to void.
take between 2-4 pm (afternoon specimen)
referred to as a liquid tissue biopsy of the urinary tract – 2 Suprapubic aspiration
used for quantitative cell count, one example is
painlessly obtained 12hr Addis counting (the cells, WBCs and RBCs, are Urine is aspirated with a syringe & needle above the symphysis pubis
urine it yields a great deal of information quickly & counted) through the abdominal wall into the full bladder. This is used for:
specimen economically ▪ anaerobic cultures
a clearance testing (for glucose, protein, or
but like any other laboratory procedure, it should be 24hr ▪ problem cultures (contamination cannot be ruled out)
creatinine)
carefully performed & properly controlled ▪ infants (difficulty to collect sample, and is contaminated)
For timed specimens, collection instructions are very important. For
3 Glass Collection
REASONS FOR PERFORMING URINALYSIS example, a big brown plastic container (3000ml urine sample) is needed.
This is used to determine prostatic infection. Instead of discarding
1 diagnosis & management of renal or urinary tract diseases Depending on the type of examination needed, a urine preservative is
the 1st urine passed, it is collected in a sterile container & the
detection of metabolic or systemic disease not directly related to also added.
2 midstream portion in another container (2nd container). Prior to the
the kidney Example situation: A patient for 24hr testing started at 7am. You have to 3rd collection, the prostate is then massaged so that prostatic fluid
instruct the patient that the urine is voided but discard the first urine

AIRAH M.
will be passed with the remaining urine into the 3rd bottle. (These 3 usually for pregnant women to check for 3 chemical preservatives
containers should be sterile.) diabetes, they prefer 2hr glucose tolerance commonly used are toluene, formalin, thymol
After collection of samples, quantitative cultures are made on each state wherein a 75g glucose load is given for preservation of acetone, diacetic acid,
E. DRUG SPECIMEN COLLECTION toluene
specimen. reducing substances & protein
Urine collection is the most vulnerable part of a drug testing program. for preservation of formed elements
1st container
examined microscopically Correct collection procedure & documentation are necessary to ensure formalin (40%) an excess will cause pption of urea
2nd container that drug testing results are those of the specific individual submitting
used as control for bladder or kidney infection. will cause a false (+) Clinitest & Fehling’s test
the specimen.
in prostatic infection, this will have a WBC/hpf & good preservative for most chemical tests
rd
3 container is the process that provides this documentation of
bacterial count 10x that of the 1st specimen will cause a false (+) reaction for protein w/ heat
thymol
4 Ureteral catheterization proper sample ID from time of collection to the and acetic acid test
This is used to differentiate bladder infection from kidney infection. receipt of lab results does not affect the dipstick method
Ureteral catheters are inserted via a cytoscope into each ureter. is a standardized form that must document and delay the decomposition of chemical as well as
chain of
Bladder urine is first collected, then a bladder washout specimen. accompany every step of drug testing, from formed elements but does not stop the growth
custody (COC) boric acid
Ureteral urine specimens are obtained separately from each kidney collector, to courier, to lab, to medical review of yeast
pelvis & carefully labeled right ureter & left ureter. officer, to employer (every step is documented so it will precipitate uric acid
each person who has taken part during the test preserve glucose in 24 hr. collection to inhibit
should affix their signature) glycolysis by cells & bacteria
sodium fluoride
Urine specimen collections may be “witnessed” or “unwitnessed”. The it will inhibit the reagent strip for glucose
laboratory should also make sure that the person to be tested should not it is a good preservative for drug analyses
have any access to water in the toilet to avoid dilution or adulteration of cytologic
specimen. 50% alcohol – for evaluation of tumor cells
preservative
1. Collect sample about 30 to 45 ml. urine. SACCOMANNOS preserve cellular elements
2. Urine temperature must be taken within 4 min. of collection to fixative used for cytology studies
confirm that the specimen has not been adulterated. Temperature urine C & S for U/A and C&S on the same specimen,
should read between 32.5 °C to 37.7 °C. transport kit decreases pH – preservative is boric A
3. Urine color is inspected to identify any sign of contaminants. Toilet used for transportation of urine for routine
lid + faucet handles are taped to eliminate source of H2O. Toilet water screening U/A, preserve glucose & other
reservoir is added with bluing agent (dye) to prevent adulterated constituents by releasing formaldehyde (will not
specimen. react with the copper reduction test: remember
SPECIMEN CONTAINERS the formalin we use in the lab will affect the
D. DIABETIC MONITORING
▪ disposable plastic containers, 100 to 200 ml. with Clinitest which is a test for sugar)
TYPES OF SPECIMEN
lids for routine screening also contain benzoate & mercury & have an acid
2nd morning specimen
▪ 12 ml. capped plastic disposable tubes are also reaction
2nd voided specimen after a period of fasting
available if you know that glucose is to be tested and the
fasting specimen this specimen will not contain any metabolite
▪ rigid brown plastic containers, 3000 ml. with wide urine contains a preservative table, you need to
from food taken or ingested prior to the preservative
use another method of determination: 95 mg.
beginning of the fasting period mouths & screw caps for 24 hr. collection tablet
tablet is used with 20 ml. urine (in this
patient is instructed to void shortly before ▪ pediatric urine collectors of clear pliable polyethylene are available for
concentration, formaldehyde will not react with
consuming a routine meal and to collect a male & female infants
the copper reduction tests (Clinitest) & the
2hr postprandial specimen 2 hrs. after eating ▪ sterile containers are used for cultures: make sure that the px is properly preservative properly used does not interfere
specimen specimen is tested for glucose and used informed regarding the collection (e.g. prior to collection of urine, for with common reagent strips)
primarily for monitoring insulin therapy in female patients, the genitals have to cleansed with mild antiseptic; for this formaldehyde tablet will slightly increase the
person w/ diabetes mellitus uncircumcised male, the foreskin should be retracted) specific gravity of the sample (0.002/tablet):
specimens are collected to correspond with the that’s why if you know that the spx contains a
HANDLING AND PRESERVING SPECIMENS
blood samples drawn during a GTT preservative tablet, you subtract 0.002 from the
1 refrigeration
tested for glucose & ketones specific gravity
▪ if sample is not tested within 2 hours, refrigeration is needed
most tests include fasting, ½ hr., 1hr., 2hrs., and ▪ (2-8 °C) most routinely used – decrease bacterial growth a very low pH (< 3) will prevent bacterial growth
3hrs ▪ in general, many substances for qualitative or quantitative chemical pH adjustment & stabilize substances such as catecholamines,
Glucose
e.g. The patient is asked to fast. A fasting urine determinations (cells & casts) preserve best when refrigerated at an VMA or 5 HIAA
Tolerance Test
(GTT) specimens spx is collected together with the blood acid pH (about 6) w/out preservation
sample. After the blood is drawn, the px is 2 freezing
asked to drink a glucose load (usually 100g). 30 ▪ useful for aliquots of urine to be used for quantitative chem. tests
minutes after, blood sample is drawn and ▪ light sensitive pigments are preserved in dark-colored containers
another urine sample is also collected. (1 hr, o will help retard loss of labile subs. such as urobilinogen, bilirubin,
2hrs, and 3 hrs after) & porphobilinogen but not completely

AIRAH M.
 CHANGES IN URINE WITH DELAYED TESTING The presence of considerable amounts of urea nitrogen and creatinine is an acid urine containing hemoglobin will
What will happen when urine is allowed to stand at room temperature? highly suggestive of urine, as most other body fluids contain only small darken on standing (because hemoglobin is
RESULT REASON amounts of these substances. dark brown or
already converted to methemoglobin)
breakdown or alteration of chromogen or other black urine
TESTS TO IDENTIFY URINE can also be caused by homogentisic acid and
urine constituents (e.g. hemoglobin, melanin, melanin
test for urea test for creatinine
homogentisic acid, porphyrins)
change in color in urine: 600 mg/100 ml in urine: 50 mg/100 ml color of urine can be due to certain food and
e.g. hemoglobin converted into methemoglobin: candy dyes as well as drugs (e.g. red urine:
(will darken) in blood: 20mg% in blood: 1-2mg%
brown color ingestion of beets)
other factors
e.g. homogentisic acid: urine turns black upon COMPONENTS OF ROUTINE URINALYSIS note: red is the most common abnormal color
standing A SPECIMEN EVALUATION (presence of blood)
▪ bacterial growth Before doing any testing, the MT should evaluate its acceptability.
changes in odor
▪ decomposition A red color of urine can be caused by the two mentioned below, but
1 proper labelling; name, date, time of collection, birthday of px
▪ increased bacteria (multiplication) what are their differences?
increased proper specimen for the requested test (e.g. urine culture for
▪ crystal formation may appear cloudy, smoky, pink, red or
turbidity 2 C&S test, make sure that it was properly collected through clean
▪ precipitation of amorphous material brown
catch, or if urine container is sterile)
glucose converted to acids and alcohols by bacteria smoky: most common description
3 proper preservative (e.g. for timed spx) hematuria
producing ammonia = CO2 lost
visible signs of contamination (e.g. very turbid with foul smelling commonly caused by infections
commonly, the pH of the sample increases (the 4
odor or ammoniacal: the urine has been stood for some time) (glomerulonephritis), tumors, trauma (stones
falsely low pH usual phenomenology of the sample)
transportation delays (that’s the reason that there is also the & injury), poisoning
however, in patients who have increased glucose in
time of collection labelled: if it’s more than two hours from the clear red, clear red brown, or dark brown
the urine, the glucose is converted to acids and 5
time of delivery to the lab, reject it and ask the px to provide clear: most common description
alcohols (urine becomes acidic instead)
another sample) caused by severe burns, incompatible blood
▪ volatilization of acetone
false negative in specimens submitted for multiple testing, bacteriologic exam transfusion, fever, snake venom
▪ breakdown of acetoacetate by bacteria hemoglobinuria
ketone should be done first (e.g. tests for both routine urinalysis and
yields false negative result congenital erythropoietic porphyria
6 C&S, you should first give it to the microbiology section first for
▪ destroyed by light drugs (ex. Rifampin: used in treating TB) and
false negative the C&S, prior to performing the routine urinalysis, to make sure
▪ oxidation to biliverdin dyes in diagnostic tests (ex.
bilirubin it won’t be contaminated)
yields false negative result Phenolsulfonphthalein)
B PHYSICAL TESTS
destroyed by light 2 CHARACTER (CLARITY)
false negative 1 COLOR
to avoid, samples should be placed in brown- ▪ presence of particulate
urobilinogen ▪ normally yellow, however, variations in color may be caused by
colored bottles then wrap the container with foil matter in unspun urine
diet, medication, and disease
it will be false positive because when the urine is needs to be explained
▪ sometimes provide a clue for the diagnosis of certain diseases
allowed to stand after voided, the bacteria will microscopically
false positive ▪ yellow color is due to the pigment urochrome and to small
multiply, then nitrite, being produced by bacteria, ▪ cloudy urine may not be
nitrite amounts of urobilin and uroerythrin
will turn the reagent strip result positive pathologic
▪ urochrome excretion is increased during fever, thyrotoxicosis,
side note: nitrite can be tested with a reagent strip and starvation ▪ turbidity might be due to:
nitrite converts to nitrogen and evaporates ▪ color indicates the degree of hydration and urine concentration a precipitation of crystals or non-pathologic salts
when the patient really has a bacterial infection seen in a normal person with high fluid intake ▪ amorphous phosphates (occasionally carbonates) in alkaline
(when urine is freshly voided and is allowed to pale urine urine but redissolve when acetic acid is added
false negative pale color: can be an indication of low sp.gr.
stand, instead of the bacteria producing nitrite, the ▪ uric acid & urates in acid urine and redissolve on warming to
nitrite maybe seen when fluids are withheld
nitrite will convert into nitrogen so by the time you 60°C (do not boil)
will examine it, it is no longer there, it has darker urine can be due to the presence of other ▪ under the microscope, uric and phosphate looks the same,
evaporated) substances (e.g. bilirubin) hence, you have to check the pH (if alkaline, it is phosphate)
increased bacteria multiply in specimen before analysis pale urine of is found in patients with diabetes mellitus and then confirm (addition of acetic acid: if it redissolves, it is
bacteriuria high sp.gr. after the use of radiographic media phosphate)
“uria”: present in the urine in an increased amount
often associated with bile pigments, chiefly b leukocytes
unstable environment, especially in alkaline urine,
bilirubin ▪ especially in patients with UTI, it forms a white cloud similar
disintegration of hypotonic urine or both
yellow brown side note: an easy test for checking bilirubin: to phosphates but cloud remains after the addition of dilute
cells/casts e.g. if the urine is alkaline or not hypotonic, the
or greenish the shake test (you simply put urine on a test acetic acid
formed elements will disintegrate
brown urine tube and shake it, if a yellow foam is ▪ confirmed microscopically
Sometimes, the doctor will send an unknown fluid into the lab. He wants it produced, bilirubin might be present) (white c bacterial growth
to be tested to verify if the sample is urine or not. What will the MT do to foam indicates withheld urine only) ▪ causes a uniform opalescence that is not removed by
identify if the fluid is urine? The best way is to check for the presence of urea dark green severe obstructive jaundice acidification or by filtering
and creatinine, the waste products of metabolism. They are also present in orange red or ▪ e.g. E. coli, Proteus, Enterococcus, yeast, Staph. (skin
large amounts of urobilin
the blood but can be also found on urine in large amounts. orange brown contaminant)

AIRAH M.
 d increased number of epithelial cells ▪ normal odor: slightly aromatic odor o acid indicator: methyl red
▪ usually, this will affect female patients ▪ chiefly important in the recognition of contaminated specimens & o alkaline indicator: bromthymol blue
▪ doctors will try to confirm and ask the patient to submit on standing are ammoniacal, fetid so unsuitable for laboratory o read 60 seconds after dipping (don’t dip for a long period
another sample examination because the reagents also will be washed out)
▪ sometimes, not a midstream urine sample ▪ urine odors associated with amino acid disorders: o pH ranges from 5.0 to 8.5 in half units
e RBCs (hematuria) sweaty feet isovaleric acidemia & glutaric acidemia ▪ in healthy individuals: first morning specimen (pH 5-6), after
does not clear on warming and needs to be confirmed maple syrup maple syrup urine disease meals (a more alkaline pH: alkaline tide)
microscopically cabbage methionine malabsorption ▪ normal random samples: pH 4.5-8.0
f spermatozoa and prostatic fluid mousy phenylketonuria CLINICAL SIGNIFICANCE OF URINE pH
g mucus from the urinary passages rotting fish trimethyl aminuria 1 respiratory or metabolic acidosis/ketosis
increased in inflammatory states of the lower urinary tract rancid tyrosinemia 2 respiratory or metabolic alkalosis
h fecal material in urine (especially during LBM) 4 URINE VOLUME defects in renal tubular secretion & reabsorption of acids &
3
contamination with powders or antiseptics that become ▪ is being measured when you perform the ____ testing (can’t hear) bases – renal tubular acidosis
i
opaque with water (e.g. phenol) ▪ if you have timed specimens, you need to measure the volume 4 renal calculi formation
in its concentrated form, phenol is clear, but if you will dilute it ▪ not measured during routine urinalysis 5 treatment of urinary tract infection
after, it will become turbid ▪ average daily volume in normal adult: 1200-1500 ml 6 precipitation/ identification of crystal
j chyluria normal range 600-2000ml 7 determination of unsatisfactory specimens
▪ maybe normal, opalescent, milky (same with lipiduria) normal cause nocturia (at night) and the excretion of
▪ urine contains lymph associated with obstruction to lymph pregnancy a dilute urine sample POSSIBLE CAUSES OF:
flow & rupture of lymphatic vessels into the renal pelvis, excretion by an adult of more than 500 ml. ACID URINE ALKALINE URINE
ureter, bladder, or urethra nocturia urine with a specific gravity of less than 1.018 emphysema hyperventilation
k lipiduria at night diabetes mellitus vomiting
▪ fat globules appear in the urine increase in urine volume of more than 2000 starvation renal tubular acidosis
▪ caused by nephrotic syndrome and crush injury (due to major polyuria
ml. in 24 hours presence of urease-producing
skeletal trauma with one or more fractures to major long oliguria decrease in urine output dehydration
bacteria
bones or pelvis)
anuria patient can no longer void diarrhea vegetarian diet
▪ one way of checking this: mix it with ether (both chyluria and
lipiduria are soluble in ether, resulting to a clear solution) high protein diet old specimens
cranberry juice
Since in the physical examination, you need to describe the color and
the transparency, here is a guide on describing clarity. You need to medications for UTI
check the appearance of the urine sample by placing a print at the (ex. Mandelamine)
back of the sample. presence of acid-producing
HOW TO DESCRIBE URINE CLARITY bacteria (E. coli)
clear no visible particulates, transparent 6 SPECIFIC GRAVITY AND OSMOLALITY
▪ considered as a part of the physical examination, however, it can
hazy few particulates, print easily seen through urine
5 HYDROGEN ION CONCENTRATION OF URINE (pH) still be a part of the chemical examination because of another
cloudy many particulates, print blurred through screen method (several methods of determining sp.gr.)
o the pH of the urine reflects the ability of the kidney to maintain
turbid print cannot be seen through urine ▪ a measure of the concentrating and diluting power of the kidney
normal H+ concentration in the plasma and ECF
milky may precipitate or be clotted ▪ inability to concentrate & dilute urine is an indication of renal
TWO BASIC METHODS disease or hormonal deficiency
potentiometric unsuitable for routine urinary pH ▪ measurement of both tests should give an indication of the urinary
determination measurement but should be used for quality total solute concentration
(pH meter) control
depends on the number of solutes in a unit of
rapid, inexpensive but still gain useful
solution
information osmolality
e.g. Multistix & Chemstrip brands: make use a more exact measurement of urine
concentration than sp.gr.
indicator of a double-indicator system of Methyl red
paper strips and Bromthymol blue depends on the number of particles present in
Methyl Red + H+ ---- Bromthymol Blue -- H+ a solution & the density of these particles
specific
red -- yellow yellow -- blue is influenced by the size of the molecules such
gravity
(pH 4-6) (pH 6-9) as urea, glucose, & protein that are not
3 URINE ODOR significant in renal conc.
▪ not usually included in the physical examination but if you notice
hyposthenuric patients having constant urines of low Sp.Gr.
something different in the odor, you need to check it also because
urine (less than 1.007)
this can be caused by a certain disease(s)

AIRAH M.
 isosthenuric This is the calibration that you will see on the stem. The manufacturer and multiply your result for the reading of the diluted sample
patients having constant urine of fixed Sp.Gr. will put the temperature of calibration on the stem. The urinometer
urine by the dilution.)
Sp.Gr. of the float is also manufactured and calibrated. urinary preservative: preservatives increase the urinary
free glomerular filtrate is 1.010
Protein specific gravity (One good example is the preservative tablet
Freezing Point for urine. The use of this tablet can increase the specific
commonly employed method for osmolality 5 gravity by .002. That’s why if this tablet has been added to
Depression
determination the urine sample being examined, you need to subtract .002
Method
to your specific gravity reading. Let’s say if the reading is
METHODS OF SPECIFIC GRAVITY MEASUREMENT 1.015, subtract .002 so that what you will report is 1.013.)
In the laboratory, we have several methods to measure specific
gravity. We have the refractometer method, urinometer method, Example: determine urine’s true sp.gr. in the urinometer method:
reagent strip, and the automated method. Name of Patient – Maria Cruz
Temp. of urine – 36.0 degrees Centigrade
A. REFRACTOMETER (TS meter): measures the refractive index of a
solution and advantages include: Temp. of calibration- 21.0 degrees Centigrade
▪ temperature compensated between 60°F-100°F Urinometer reading – 1.015
▪ requires only 1 drop of urine Difference between the 2 temperatures =15÷3=5
Calibration: 5 is multiplied by .001 & will then be added to the urinometer reading
1. distilled water - 1.000 So, the True Sp.Gr. is 1.020.
2. 5% NaCl - 1.022 ± .001
If the patient Maria Cruz has proteinuria, her urine contains 2
3. 9% Sucrose - 1.034 ± .001
gm.protein/100ml.What is the final Sp.Gr.to be reported?
The disadvantage of this method if compared to that of the 1.020 – (.003x2) = 1.014
refractometer is that you will need a larger volume of urine because
you need to fill the urinometer cylinder about ¾ths full, while in the For urine samples with very high Sp.Gr., which can’t be read on both
refractometer, you need only a drop. Also, the refractometer is not Urinometer & Refractometer, simply dilute the urine with distilled
affected by temperature changes because it gives an accurate reading water and multiply the Sp.Gr. Reading of the diluted sample with the
between 60-100°F whereas in urinometer, it is affected by dilution.
temperature. If it is calibrated at 20°C, it means that if the urine Ex. 5 ml.urine was diluted with 10 ml.dist.water.
temperature is 20°C, the reading you see on the stem is fairly Sp.Gr.reading= 1.020 x 3(dilution) = 1.060
accurate. However, if the urine temperature is higher or lower, then Explanation: The dilution of the 5ml urine added with 15 ml water
you need to compute for this temperature change. (nani? In the example, it is 10 idk why she said 15) is 1:3. So, you
SOURCES OF ERROR multiply the last three 3 digits by 3. Do not include the 1 because
This is the appearance of the refractometer. The one on the left will
sp.gr. always starts with 1. There is no sp.gr. which begins with 2 or
have a light source, an electric bulb inside. The right one is handheld. temperature differences: Most urinometers are calibrated
3, it only starts with 1. What you will multiply with 3 is only the .020,
You face the light source so that you can read the calibration inside. at 200°C. A difference of 30°C between urine temperature &
hence, you will have 1.060.
calibration temperature requires a correction of 0.001 to be
B. URINOMETER
added if the temperature of the urine is above the C. REAGENT STRIP METHOD
▪ the image on the right shows a
urinometer: the urine container is
temperature of calibration & subtracted if below the proper ▪ based on the change in pKa (dissociation constant) of a
temperature. (For example, calibrated at 20°C, and the polyelectrolyte in an alkaline medium
called the urinometer cylinder
temperature of the urine is 30°C, hence, it is above. So, the ▪ the polyelectrolyte ionizes releasing hydrogen ions in proportion
while the instrument inside is called
1 difference between the two is 10°C which will then be divided to the number of ions in the solution
the urinometer float
by 3 since it is for every 3-degree difference. The quotient will ▪ the higher the concentration of urine, the more hydrogen ions are
▪ a hydrometer adapted to measure
then be multiplied by .001. So, that is about .003 to be added released, thereby lowering the pH
sp.gr. at room temperature: this is
to the specific gravity reading.) (However, if the temperature ▪ Indicator Bromthymol Blue measures the change in pH
calibrated at a specific temperature
of urine is 17°C so that it is below the temperature of
unlike your refractometer wherein
calibration. In this case, the difference is 3 divided by 3 x .001, D. HARMONIC OSCILLATION DENSITOMETRY
it gives accurate result if the urine
will give .001 which will then be subtracted to the specific
temperature is between 60-100°F Principle:
gravity reading.)
▪ here, the urinometer is affected by temperature difference The frequency of a sound wave entering a solution will change in
▪ consists of a weighted float with a calibrated stem 2 proteinuria: subtract 0.003 for every 1g/100ml protein proportion to the density of the solution.
▪ the float, the one inside, consists of a weighted float having a 3 glycosuria: subtract 0.004 for every 1g/100ml glucose
▪ used by the Yellow IRIS (International Remote Imaging Systems)
calibrated stem where you will read the calibrations x-ray contrast media: sp.gr. may exceed 1.050 (There is a
▪ A portion of urine enters a U-shaped tube. A sound wave of
▪ the float displaces a volume of liquid equal to its weight & has contrast media in the urine which is used in the examination
specific frequency is generated at one end of the tube, and as the
been designed to sink to a level of 1.000 in distilled water of patients to follow the course of a dye. A certain dye is being
sound wave passes thru the urine, its frequency is altered by the
▪ it will displace an equal amount of liquid depending on the injected and the course of the dye is being followed through
4 density of the solution.
amount of solute in the urine sample: the additional mass x-ray. So that, this dye is excreted later on through the urine.
provided by the dissolved substance causes the float to displace a The urine passed out now will have a very high specific gravity
volume of urine smaller than that of distilled water that sometimes, you cannot reqd it on the calibration. What
▪ requires a larger volume of urine, unlike refractometer you need to do is to simply dilute the urine with distilled water

AIRAH M.
E. FALLING DROP METHOD
▪ a direct method for measuring sp.gr.
▪ this is a more historical method
▪ more accurate than the refractometer and
more precise than the urinometer

Falling Drop Method by M.Winstead

Utilizes a specially designed column filled with
water–immiscible oil. A measured drop of urine
is introduced into the column, and as the drop
falls, it encounters two beams of light; breaking
the first beam starts a timer, while breaking the second turns it off.
The falling time is measured electronically & expressed as a specific
gravity.
C CHEMICAL EXAMINATION
D SEDIMENT EXAMINATION

AIRAH M.
 ANALYSIS OF URINE AND BODY FLUIDS o hemoglobin RENAL PROTEINURIA CAUSED BY IMPAIRED FILTRATION
TOPIC 03: CHEMICAL ANALYSIS OF URINE (PROTEIN AND GLUCOSE) o myoglobin A GLOMERULAR PROTEINURIA
▪ acute phase reactants (APR)
PATHOPHYSIOLOGY OF PROTEIN IN THE URINE ▪ when the glomerular membrane is damaged, selective filtration is
▪ Bence Jones Protein (MM): found in patients with
multiple myeloma1 impaired
▪ an increase amounts of serum, protein, and even WBCs and RBCs, pass
1
a proliferative disorder of the immunoglobulin- through the membrane and are excreted in the urine
producing plasma cells wherein the serum contains ▪ major causes of proteinuria due to glomerular proteinuria are:
markedly elevated levels of monoclonal immunoglobulin
light chains ▪ amyloid material
renal - ▪ toxic substances
protein can be added to a urine specimen as it passes ▪ immune complexes
exposure to
through the lower urinary tract and these proteins o lupus erythematosus (L.E.)2
abnormal
include: substances o streptococcal glomerulonephritis3
▪ exudates: from bacterial and fungal infections and
post-renal 2
an autoimmune disease
inflammations 3
a complication of streptococcal infection
▪ blood: result of injury and menstrual contamination
▪ prostatic fluid increased pressure from the blood entering the
▪ spermatozoa glomerulus that may override the selective
notation of the glomerulus
Of all the proteins found in the blood, only few molecular weight proteins are BENCE JONES PROTEIN (MM) this condition is reversible and does not
increased
filtered out which includes our star protein, the albumin. However, most of 1 Bring to a boil. necessarily mean glomerular damage
pressure
these low molecular weight proteins that are filtered out are readily 2 Filter. caused by:
reabsorbed by the kidneys leaving only a small percentage to be found in the 3 Cool down to 60°C or lower down to 40°C. Observe for turbidity. ▪ strenuous exercise
urine. Although albumin is the major protein in urine, serum microglobulin RESULT: ▪ dehydration
and proteins produced in the GUT are also found. turbid: positive reaction clear: negative reaction ▪ hypertension
proteinuria that occurs during the latter months
PROTEINS PRODUCED IN THE GUT RENAL PROTENURIA of pregnancy may indicate a pre-eclamptic state
tubular microglobulins prostatic secretions Proteinuria associated with true renal disease may be the result of either pregnancy and should be considered by the physician in
Tamm-Horsfall Protein ← RTE* seminal/vaginal secretions glomerular or tubular damage. Although the term glomerular is used, you will conjunction with other clinical symptoms to
* also known as uromodulin, produced by the renal tubular epithelial see later on that some of these conditions do not necessarily damage the determine if this condition really exists
cells glomerulus but simply override filtration. Values go as high as 4 grams a day note: benign proteinuria is transient which means
other causes of
for glomerular damage. In disorders affecting tubular reabsorption, the it diminishes over time
benign
normally filtered albumin and other low-molecular-weight proteins can no ▪ high fever
proteinuria
longer be reabsorbed. However, in this case, markedly elevated protein levels ▪ exposure to colds
are seldom seen. B MICROALBUMINURIA
▪ in both type I and type II diabetes mellitus, a common occurrence is a
development of diabetic nephropathy which causes a reduced
glomerular filtration which can be detected by microalbuminuria
▪ the detection of microalbuminuria in patients with type I and type II
When urine protein is tested, a positive result requires additional testing to DM can predict onset of renal complications (e.g. renal failure)
determine whether the protein represents a normal or pathologic condition. ▪ the progression of renal disease can be prevented by stabilizing blood
glucose and control of hypertension because prevention is always
is indicated at ≥ 30mg/dL better than cure
its causes can be grouped into three major ▪ microalbuminuria is also associated with increased risk of
categories based on the origin of the protein: cardiovascular disease
clinical proteinuria ▪ pre-renal*
▪ renal Before reagent strips method were available, detection of
▪ post-renal microalbuminuria can be done by these steps:
*not indicative of renal disease Using a 24-hour urine specimen, albumin is measured quantitatively. The
values of 30-300mg albumin per 24 hours or an albumin excretion rate of
THREE MAJOR CAUSES OF CLINICAL PROTEINURIA 20-200ug per minute is considered significant.
caused by conditions prior to reaching the kidneys
▪ specimen: 24-hr urine
this condition is frequently transient and happens when
▪ testing: quantitative albumin measurement
filtration of proteins exceeds the normal reabsorptive
pre-renal results mg albumin/24hrs AER in ug/min
capacity of the renal tubules resulting in an overflow of
proteins into the urine which includes: 30-300mg
significant values 20-200ug/min
▪ low-molecular-weight CHONS albumin/24 hours

AIRAH M.
C ORTHOSTATIC PROTEINURIA turbidity, granulation, no
REAGENT STRIPS BRANDS 2+ 100-200
▪ also called postural proteinuria flocculation
REAGENT SENSITIVITY
▪ refers to long periods spent in vertical position which is believed to turbidity, granulation,
MULTISTIX Tetrabromophenol blue 15-30 mg/dL albumin 3+ 200-400
cause pressure in renal vein and overwrite filtration causing benign flocculation
3’,3’’,5’,5’’-
proteinuria 4+ clumps of protein greater than 400
tetrachlorophenol,
▪ this condition is common in young adults CHEMSTRIP 6 mg/dL albumin C MICRAL TEST
3,4,5,6-
▪ benign proteinuria is transient and, in this case, would disappear when ▪ a new method for microalbuminuria testing
tetrabomosulfophthalein
a horizontal position is assumed ▪ this test is specific for albumin
Reaction Interference:
Patients expected of orthostatic proteinuria are requested to ▪ several semi-quantitative reagent strip methods have been developed
1 ▪ Since the reagent strip method for proteins highly depends on the pH
empty their bladder before going to bed. so that patient at risk for renal disease can be monitored using
indicators, the main reaction interference would come from any
Upon arising in the morning, a urine specimen is collected random or first morning specimens and this includes the new method
2 condition that would change the pH of the urine specimen.
called MICRAL TEST manufactured by Roache Diagnostics
immediately. ▪ The major source of error with reagent strips occurs with highly
▪ note: the word “monitoring” is different from “screening” and
3 A second specimen is collected several hours in vertical posture. buffered alkaline urine since our pH buffer is set at an acidic pH (3.0).
“diagnosing”
Both specimens are tested for protein which would indicate the ▪ Prolonged contact with the reagent pad and urine causes bleaching
4 Manufactured by: Roache Diagnostics
presence of orthostatic proteinuria. out of reagent, or the removal of the reagent in the reagent pad.
Prolonged application is different from prolonged reading. Principle: Enzyme Immunoassay (EIA)5
first morning second specimen Sensitivity: 0-10 mg/dL (or in mg/L: just adjust the value)
FALSE POSITIVE FALSE NEGATIVE
orthostatic proteinuria NEGATIVE POSITIVE Specimen: first morning
highly buffered alkaline urine CHON other than albumin
Detects: albumin
RENAL PROTEINURIA CAUSED BY TUBULAR DAMAGE prolonged contact with urine microalbuminuria Application Time: 5 secs
In tubular proteinuria, other low-molecular-weight proteins that are contamination with quaternary Reading Time: 1 min
usually reabsorbed are also present. ammonium compounds (QAC), Color range: white to red
detergents, & antiseptics End color: RED
CAUSES
spx with high sp.gr.
▪ exposure to toxic substances & heavy metals 5
if there is the word “immune”, it always means there is antigen-antibody
highly pigmented urine4
▪ severe viral infections binding (ask yourself: What is the antibody and what is the antigen?)
Side notes:
▪ Fanconi syndrome 4
does not interfere with the pH buffer system but it interferes with
METHODOLOGY (PROTEIN IN URINE: PROTEIN DETERMINATION) the color reaction REAGENT STRIP DIAGRAM
B SULFOSALICYLIC ACID (SSA) PRECIPITATION TEST
A REAGENT STRIP METHOD
Principle: cold precipitation
Principle: protein error of indicators
▪ pH indicators have a characteristic color at certain pH levels: it Specimen: centrifuged urine – to remove any extraneous contamination
changes color according to the pH of the solution and it detects all protein, not just albumin
▪ however, CHONS (1°ly albumin) accept H+ from the indicator causing Detects: all forms of protein
a change of color even in a medium with constant pH
Add 3 ml of 3% sulfosalicylic acid reagent to 3ml centrifuged
▪ more sensitive to albumin (albumin has more amino group > other 1
urine.
CHONS)
2 Mix by inversion and observe for cloudiness.
Mode of Measurement: colorimetric Rate the degree of turbidity using the table found in the
3 COMPOSITIONS OF THE REAGENT STRIP
Reaction Principle: reference book. reagent gold-labeled antihuman albumin-
pH 3.0
enzyme (𝞫-galactosidase) conjugate
indicator + protein -----------> protein + H+
side note: when there is a word “anti”, it means
(yellow) indicator - H+ Zone 1:
there is an antibody, hence, the antibody
(blue to green) Conjugate Matrix
present is directed against human albumin
At a constant acid pH, protein in the urine causes a changed color in the Fleece
enzyme: 𝞫-galactosidase
indicator resulting to an end color of blue to green depending on the
overall, it is an antibody-enzyme conjugate with
concentration of protein in the urine.
a gold label
Reading Time: 60s Zone 2: Capture embedded/fixed in the strip of Zone 2 is the
Reading and Interpretation: Matrix Fleece human serum albumin
where the last reagent, substrate chlorophenol
detection pad
REPORTING OF SSA TURBIDITY red galactoside, can be found
PROTEIN RANGE STEPS
GRADE TURBIDITY
(mg/dL)
Strips are dip into the urine up to a level mark on the strip and
Negative no increase in turbidity less than 6 1
held for 5 seconds.
Trace noticeable turbidity 6-30 Place the strip on a neon absorbent surface or across the top of
distinct turbidity, no 2 the collection cup to allow excess urine’s drain and wait for one
1+ 30-100
granulation minute.

AIRAH M.
 Compare the color of the reaction pad on the strip with the color Watch the video for this tho: Sensitivity: 8-15 mg/dL (80-150 mg/L)
3
scale of the test strip vial. Both the bound and unbound Reaction Interference: (causes change of color in the reagent pad)
CROSSOVER TO IMMUNOLOGY-SEROLOGY particles migrate up the strip. First, a. visibly bloody urine
Note: This is just a sample illustration, meaning, the strips reflected are a blue band is formed by the b. abnormally colored urine
just depictions to help you understand how micral test works. unbound particles. Since the Other Compositions
migration is controlled by the size of a. polymethyl vinyl: increase specificity of reagent pad to albumin by
Principle:
the particles, the bound particles decreasing nonspecific binding of polyamino acids to the albumin
We have the anti-human albumin (A) (no need to mention antibody
continue to migrate up and form a pad
because it is understood when you say anti-human albumin, it is an
second blue band further up the strip. The top band represents the bond b. treatment with bis-(heptapropylene glycol) carbonate prevents
antibody, but you can also say anti-human albumin antibody) which is an
particles and the bottom band represents the unbound particles. reaction interference by highly buffered alkaline urine (as you
antibody directed against human albumin. Next, we have our gold label
noticed, highly buffered alkaline urine is not mentioned in the
(B). We have our enzymes (C). We also have our albumin (D), which serves STEPS
interference)
as the antigen. Together, they form gold labelled anti-human albumin Place container in urine specimen for three minutes. Control the
1 Reagent Strip Reaction: Creatinine
antibody enzyme conjugate (E). amount of urine and press the container through a vent hole.
2 Wait for 3 minutes. Principle: pseudoperoxidase activity of copper-creatine complexes
3 Read the results. Reagents:
a. CuSO4
b. 3,3’,5,5’-tetramethyl-benzidine (TMB)
c. diisopropyl benzene dihydroxyperoxide (DBDH)
Reaction Principle: (memorize since methodology revolves around it)
side note: that is orange to green to blue (on the product side)
Watch the video for this tho:
The albumin of the urine bounce to the antibody. They will bind together.
Both the bound and unbound conjugates move up the strip by wicking
action. The unbound conjugate is captured or removed in a captive zone
by combining with the albumin embedded in the strip. Leaving only the
albumin-bound conjugates to continue up the strip and reach an area
containing an enzyme substrate which is called the detection pad. This The purpose of the creatinine measurement is to correlate the albumin
contains the substrate chlorphenolred-galactoside. So, the albumin concentration to the urine concentration, producing a semi-quantitative
bound conjugate moves up the detection pad. The enzyme 𝞫- albumin:creatinine ratio.
Reading and Interpretation: (Textbook vs. Manufacturer)
galactosidase will catalyze the reaction, and break down our substrate, The color intensity of the bands is compared against the manufacturer’s Reaction Interference:
chlorphenolred-galactoside, to produce a color which is red. Most color chart. However, these values are different from the manufacturer’s false high
methods use only the colloidal gold and no more enzyme conjugates package insert. For our long exams, refer to the reference values in the a. visibly bloody urine
because the colloidal gold is enough to produce a red color. But some textbook. But, when performing actual laboratory tests and reporting b. abnormally colored urine
methods enhance color reaction by enzymatic reaction, producing outpatient results, always refer to the manufacturer’s instructions. c. gastric acid-reducing medication cimetidine (Tagamet)
stronger red colors. Normal Value: 10-300 mg/dL
darker bottom band NEGATIVE less than 1.2 mg/dL
equal band colors BORDERLINE 1.2 – 1.8 mg/dL Albumin/Protein : Creatinine Ratio
darker top band POSITIVE 2.0 – 8.0 mg/dL Clinitek Multistik
manufacturer-supplied
MANUAL chart (higher of the
X
READING protein-low or protein
high result is used)
INSTRUMENT Clinitek Urine Clinitek Urine Chemistry
D IMMUNODIP READING Chemistry Analyzer Analyzer
creatinine, protein-high,
ANALYTES albumin and
Manufactured by: Sekisui Diagnostics E ALBUMIN : CREATININE RATIO protein-low (albumin)
MEASURED creatinine only
Principle: immunochromographic and many more*
▪ estimation of 24-hour microalbumin excretion
Specimen: first morning protein-high: protein
▪ done by comparing albumin excretion to the consistent excretion rate
Detects: albumin PRINCIPLE dye-binding error of indicator
of creatinine
Reagents: blue latex particles coated protein low: dye-binding
▪ albumin reading can be corrected for over-hydration and
with antihuman albumin Ab TLTR. Clinitek microalbumin reagent strips are designed for instrument
dehydration in a random sample
▪ brands avliable: Clinitek Microalbumin reagent strips and Multistix use only whereas Multistix can be manually read. However, using the
Pro reagent strips (Siemens Healthcare Diagnostics) manufacturer-supplied chart, the higher the protein-low or protein high
result is used in calculating the A:C ratio. The Clinitek reagent strips are
Reagent Strip Reaction: Albumin read on Clinitek Urine Chemistry Analyzer in which the Siemens Multistix
Principle: dye-binding reaction Pro 10 reagent strips can also be read on the same analyzer. Clinitek
Indicator: bis(3’3’’-diiodo-4’4’’-dihydroxy-5’5’’-dinitrophenyl)-3,4,5,6- measures albumin and creatinine only, whereas Multistix measures
tetrabromosulphonphthalein (DIDNTB)

AIRAH M.
 creatinine, protein-high, protein-low (which is the albumin), and many This is the pathway of glucose in different situations. In the body’s normal METHODOLOGY (GLUCOSE IN URINE: GLUCOSE DETERMINATION)
more (see reference textbook for more details). The Clinitek uses the state, the body produces insulin in response to increased blood glucose,
1 REAGENT STRIP (GLUCOSE OXIDASE REACTION)
principle dye-binding, whereas for Multisticks, protein-high utilizes the converting glucose to glycogen. However, in disorders affecting hormonal
▪ uses the same principle with glucose oxidase method utilized in CC1
protein error of indicator principle, while for protein-low, we use dye- functions (e.g. pancreatitis, acromegaly, Gaucher’s syndrome, for determination and measurement of glucose in blood
binding. hyperthyroidism, pheochromocytoma, thyrotoxicosis), an increase of ▪ considered as an indirect method because it does not directly
CLINITEK MULTISTIX hormones that work in opposition to insulin happens and these include the measure glucose
displays albumin, glucagon, epinephrine, cortisol, tyrosine, and growth hormone. These so-
protein: creatinine ratio Principle: double sequential enzymatic reaction (utilizes two enzymes)
creatinine, & A:C ratio called “opposing hormones” break down glycogen to glucose by a process Detects: glucose
results (using protein-low in the
in both SI and called glycogenolysis and there will be excess glucose that will be excreted in Reagents:
calculation)
conventional units the urine. ▪ glucose oxidase
reported as normal or SEVERE STRESS ▪ peroxidase
values of 30-300mg/g
abnormal Another situation is when the body is subjected to severe stress. In this case, ▪ chromogen – KI (Multistix) or tetramethylbenzidine (Chemstrip)
or 3.4033.9 mg/mmol
interpretation “normal dilute”: spx the body will release epinephrine and, apart from being an “opposing ▪ buffer
are considered
should be recollected, Reaction Principle:
significant hormone” to insulin, epinephrine also blocks secretion of insulin and when
pref. first morning
there is no insulin, there will be excess glucose that will be excreted in the
albumin: albumin:
urine.
10-150 mg/L 10-150 mg/L
sensitivity creatinine: creatinine: PREGNANCY
10-300mg/dL, 10-300mg/dL, Hyperglycemia, that occurs during pregnancy and disappears after delivery,
0.9 to 26.5 mmol/L 0.9 to 26.5 mmol/L is called gestational diabetes. The onset of hyperglycemia and glycosuria is In the first step, glucose oxidase catalyzes a reaction between glucose
normally around the 6th month of pregnancy. Although glycosuria may occur, and room air to produce gluconic acid and peroxide. In the second step,
PATHOPHYSIOLOGY OF GLUCOSE IN THE URINE
the hormones placed in the placenta blocked the action of insulin, therefore, or what we call indicator reaction, since we cannot visualize your
insulin could not convert glucose to glycogen and there will be excess glucose peroxide, we utilize chromogen. The peroxidase enzyme catalyzes the
excreted in the urine. Some of the glucose would enter the placenta and, reaction between the peroxide and the chromogen to form an oxidized
sadly, insulin could not pass through the placenta. The body will be exposed colored compound that is in direct proportion to the concentration of
to high levels of glucose so that they will produce more insulin. The excess glucose. The end product is the oxidized colored chomogen and the end
color depends on the chromogen utilized.
glucose presented to the baby is stored as fat, resulting in a large baby called
macrosomia. This baby is at risk for obesity and, later time, to diabetes. Also, END PRODUCT COLOR
women who have gestational diabetes are prone to developing type II POSITIVE NEGATIVE
diabetes mellitus in later years. Multistix green brown
Chemstrip yellow green
RENAL GLYCOSURIA
▪ happens when reabsorption of glucose by the renal tubules is Reading and Interpretation:
compromised Urine glucose may be reported in terms of:
▪ caused by diseases (e.g. end stage renal disease, cystinosis, Fanconi ▪ negative ▪ 2+
syndrome) ▪ trace ▪ 3+
Glucose in the blood is readily filtered by the glomerulus and just like albumin,
▪ there is another condition wherein tubular reabsorption is not ▪ 1+ ▪ 4+
most of it is reabsorbed. However, for glucose, reabsorption is done by active impaired but rather, it is a temporary lowering of renal threshold such However, the color chart also provide qbuantitative measurements
transport in the proximal convoluted tubule with a renal threshold of 160-180 that of pregnancy and this is not associated with gestational diabetes ranging from 100 mg/dL to 2g/dL or .1% to 2%.
ml/dL. This value represents the blood level of glucose at which tubular
reabsorption stops. Since most of the glucose is reabsorbed, urine contains
only minute amounts of glucose.

The American Diabetes Association recommends quantitative reporting

of urine glucose. However, please take note that reagent strips only
provide semi-quantitative measurements since they only estimate the
amount of glucose present in the urine.
Reaction interference:
Since glucose oxidase method is highly specific for glucose, false positive
reactions are not obtained from other urinary constituents, including
reducing sugars, that may be present. False positive reactions may occur,
however, if containers become contaminated with peroxide or strong
oxidizing detergents, they work by oxidizing the chromogen leading to
NORMAL STATE false positive or false high results.

AIRAH M.
 contamination with peroxide and strong rakc and not held in hand because the reaction heat could cause
false positive (+)
oxidizing agents burns.)
strong reducing agent (e.g. ascorbic acid) 4 Wait 15 seconds after conclusion of effervescence.
increased ketone, S.G. 5 Shake tube gently.
false negative (-)
decreased temperature Compare results to the color chart and report results in mg/dL or
6
bacterial degradation of glucose percent.
Strong reducing agents (e.g. ascorbic acid) prevents oxidation of the Reading and Interpretation:
chromogen and may produce false (-) results. To minimize interference
from ascorbic acid, reagent strips incorporated additional chemicals into
the test pad such as iodate that oxidizes ascorbic acid so that it cannot
interfere with the oxidation of the chromogen. The colors would range from the negative blue to a positive orange or
High levels of ketone, specific gravity, and low temperatures also cause red color.
false (+) results, however, because high levels of ketones are usually Care must be taken to observe the reaction closely as it is taking place,
accompanied by marked glycosuria, this seldom presents a problem. High because at high glucose levels, a phenomenon known as “pass through”
specific gravity and low temperature may decrease the sensitivity of the may occur. When this happens, the color produced passes through the
test. orange or red stage quickly and returns to green brown color and, if not
By far, the greatest source of post-negative error is the technical error of observed, a high glucose level may be reported as negative.
allowing specimen to remain unpreserved at room temperature for
extended periods, subjecting glucose to bacterial degradation. Remedy: using two drops instead of fibrous urine
Therefore, urine must be processed immediately after collection. ▪ minimizes the occurrence of “pass through”
2 CU REDUCTION TEST (CLINITEST) ▪ a separate color chart, which provides value up to 5 grams instead of
▪ also called Benedict’s test the usual 2g/dL, maybe used to interpret the reaction
Principle: CuSO4 is reduced to Cu2O by reducing substances in the Sensitivity: 200mg/dL (hence, Clinitest cannot be used as a confirmatory
presence of alkali and heat test for glucose)
Reagents: Benedict’s solution Reaction Interference:
▪ copper sulfate ▪ reducing sugars (galactose, lactose, fructose, maltose, pentoses)
▪ sodium carbonate ▪ ascorbic acid
▪ sodium citrate: buffer ▪ certain drug metabolites
▪ sodium hydroxide (added ingredient in the Clinitest Tablet) ▪ antibiotics (cephalosporins)
Reaction principle: Storage and Handling: the tablets should be stored in their tightly closed
packages (since these tablets are very hygroscopic)
▪ a strong, blue color in unused tablets signifies deterioration due to
moisture and vigorous tablet fizzing
Clinical Significance:
CuSO4 is reduced by the reducing substance which includes glucose and ▪ usually performed on pedia px up to 2 years old
other reducing substances. ▪ detection of galactose in NB represents “inborn error of metabolism”
Benedict’s Test (in addition to glucose, the reducing sugar galactose is also detected
and is most significant especially for NB)
▪ lack of enzyme galactose-1-phosphate uridyl transferase
▪ prevents breakdown of galactose → complications → death (hence,
screening for galactosemia is required in newborn reading programs
because early detection followed by dietary restriction can control the
condition)
PROCEDURE The appearance of other reducing sugars is usually of medical
1 Add 5 drops of urine into a thick glass test tube placed in a rack. significance and lactose is frequently found in the urine of nursing
2 Add 10 drops of distilled H2O. mothers.
Drop 1 Clinitest tablet and observe reaction until cessation of
boiling. (In Clinitest, you no longer subject the test tube into
flame. The Clinitest tablet itself reproduce the heat. This happens
upon the addition of tablet to water and urine. Heat is produced
3
by the hydrolysis of sodium hydroxide and its reaction with
sodium citrate. Carbon dioxide is released on the sodium
carbonate to prevent room air from interfering the reduction’s
reaction. Thick walled tubes should be placed in a heat-resistant

AIRAH M.
 ANALYSIS OF URINE AND BODY FLUIDS Observe for the appearance of a reddish-purple ring at the PROCEDURE
TOPIC 04: UA 2 & 3 - SPECIAL TESTS IN URINE 4
interphase of the 2 layers within 1 min & 30 secs. 1 Take 3 ml of urine in a test tube.
Objectives: 2 Add 1-2 drops of concentrated nitric acid.
▪ Enumerate the different analyzes present in urine sample & their clinical NOTE: 3 Boil and then cool the test tube.
significance. ▪ brown ring: NEGATIVE 4 Then add 1 ml of 10% ferric chloride.
▪ Discuss the different principles involved in the examination of the ▪ if red/purple color fades during
chemicals in target. first 5 minutes: NEGATIVE RESULT: FeCl3 + acetoacetate/ethyacetoacetate → purple color,
▪ Differentiate the manual method from the reagent strip testing. ▪ width of the ring: roughly diacetate/acetoacetic acid
proportional to the concentration
Topic Outline:
of ketone bodies BOIL URINE: acetoacetate into acetone → test negative
I. Ketone 1.0ml of 10% FECl3 solution → reddish color is formed after the mixture
II. Blood 4 HART’S TEST
III. Bile Pigments sensitive to beta-hydroxybutyric acid
IV. Nitrite READING AND INTERPRETATION
HART’S REAGENT: acetic acid and hydrogen peroxide
V. Leucocyte negative no ring/brown ring is present
VI. Ascorbic Acid RESULT: formation of red ring
(+) faint pinkish purple ring appearing slowly
VII. Calcium 5 URINE KETONES CONFIRMATORY TEST
(++) narrow dark purple ring
VIII. Urine Reagent Strip Correlation ▪ determination of ketone bodies is all more targeted as ketone bodies
(+++) wide dark purple ring appearing very rapidly ▪ acetone plus beta-hydroxybutyric acid are produced from
KETONES (KETONE BODIES) 2 REAGENT STRIP METHOD acetoacetic acid and/or acetic acid
▪ the test does not measure beta-hydroxybutyric acid and is only
▪ intermediate products of fat metabolism
PRINCIPLE: slightly sensitive to acetone when glycine is present
acetone (2%) acetoacetic acid (20%) β- hydroxybutyrate (78%)
Sodium Nitroprusside Decomposition ▪ as much as these compounds are derived from acetoacetic acid, their
shows a deficiency in insulin indicating the need to presence can be assumed and is
regulate dosage and in patients with diabetes who COMPOSITION: not necessary to perform
experience medical problems in addition to diabetes ▪ 7.1% (w/w) sodium nitroprusside individual tests
▪ 92.9% (w/w) buffer ▪ large amounts of Levodopa and
increased accumulation of ketones in the blood leads to:
▪ electrolyte imbalance other medicines containing
▪ dehydration PROCEDURE sulfhydryl groups may produce
ketonuria atypical color reactions
▪ acidosis & eventual diabetic coma (if not corrected) Immerse all test areas of reagent strip in fresh urine specimen
weight loss and eating disorder clinics can make use of a 1 ▪ false negative results:
& remove strip immediately.
practical application of ketonuria produced by the volatilization of acetone and
Run edge of strip against the rim of the container to remove breakdown of acetoacetic acid by
avoidance of carbohydrates to monitor patients 2
excess urine. bacteria
frequent strenuous exercise can cause overuse of
Hold strip and compare the ketone test area closely to the color
available carbohydrates and produce ketonuria 3 ACETEST TABLET: (specific for diacetic acid and acetone)
chart label at exactly 40 secs later.
▪ sodium nitroprusside
CLINICAL SIGNIFICANCE: KETONURIA ▪ glycine
READING AND INTERPRETATION
1 management/monitoring of Diabetes mellitus (Type 1) ▪ disodium phosphate
negative test area remains tan-colored after 40 secs
2 loss of carbohydrate from vomiting ▪ lactose (color enhancer)
inadequate intake of carbohydrate associated with starvation & trace to (+++) test area turns pink to purple within 40 secs
3 SENSITIVITY DETECT KETONE BODIES IN FRESH SERUM AND URINE
malabsorption/pancreatic disorders
Multistix 5-10 mg/dl or 0.25-0.5 mmol/L of acetoacetic acid A. Nitroprusside test
TESTS FOR KETONE BODIES
Chemstrip 9 mg/dl acetoacetic acid & 70 mg/dl acetone
1 ROTHERA’S TEST
SOURCE OF ERROR
PRINCIPLE: Sodium Nitroprusside Decomposition ▪ acetone or acetoacetate reacts with nitroprusside to form violet color
Nitropusside in alkaline medium reacts with a ketone group to form a ▪ highly pigmented red urine
▪ if the reagent contains glycerin, acetone can be detected by Acetest
purple ring. It is given by acetone and acetoacetate, but not by beta ▪ phthalein dyes
false positive tablets or ketostix
hydroxy butyric acid. ▪ Levodopa (medication for Parkinson’s disease)
▪ medication containing free sulfhydryl groups B. Enzymatic
REAGENT: Rothera’s reagent (overlay with 1ml conc. NH4 hydroxide)
false negative improperly preserved specimen
▪ 7.5 g sodium nitroprusside
▪ 200 g ammonium sulfate 3 GERHARDT’S FERRIC CHLORIDE TEST
PRINCIPLE: A purplish color is given by acetoacetate. On boiling,
PROCEDURE acetoacetate is converted to acetone and does not give this test positive.
1 Prepare Rothera’s Reagent. This test is only given by acetoacetate and not by beta hydroxy butyric
2 Add 1 gram of Rothera’s reagent to 5ml urine. Mix well. acid directly. This test is also sensitive to acetoacetic/diacetic acid.
3 Overlay w/ 1ml conc. ammonium hydroxide. REAGENT: Concentrated Nitric Acid

thankful to have PATRICK YRAY again with me, and he said he’ll
still be here for the upcoming ones 🥺 such a constant tysm ♡
AIRAH M.
 BLOOD 5ml of centrifuged urine + 2.8g ammonium sulfate. Dissolve by Make a smear(s) of the sediment and allow to air dry. (NOTE: All
3
hematuria hemoglobinuria myoglobinuria mixing. Filter. glassware, slides, coverslips, etc., should be iron free. Water should
be demineralized.)
CLINICAL SIGNIFICANCE RESULT TEST FOR BLOOD IN URINE USING REAGENT STRIP
presence of RBC in urine pigment is precipitated
(+) hemoglobin PRINCIPLE: pseudoperoxidase activity of hemoglobin
▪ bleeding due to trauma or damage of renal or supernatant shows normal color ▪ detects the presence of hemoglobin (Hb) in urine
genitourinary tract
pigment is precipitated ▪ hemoglobin functions as a peroxidase that catalyzes reduction of
▪ renal calculi or stones presumptive of
supernatant is red/red-brown peroxides in the presence of a hydrogen donor
▪ glomerulonephritis hemoglobin
hematuria proceed for protein electrophoresis ▪ with intact RBCs, spotting occurs since the RBCs for aggregates
▪ pyelonephritis (clumps) and the Hb remains inside the RBCs
▪ tumors Prior to the development of sensitive serum immunoassay test for
▪ qualitative interpretation is by color and spotting after 60 seconds
▪ exposure to toxic chemicals myoglobin, a procedure was used to differentiate hemoglobin from
▪ anticoagulants myoglobin in the urine.
▪ strenuous exercise A precipitation test was used to screen the presence of myoglobin.
presence of free HEMOGLOBIN in urine
▪ lysis of RBC in the urinary tract 1 2.8 grams of ammonium sulfate plus 5 ml of centrifuged urine.
▪ transfusion reaction After mixing and allowing the specimen to sit for 5 minutes, the
2
▪ hemolytic anemia urine is filtered and centrifuged.
hemoglobinuria
▪ severe burns 3 The supernatant is then tested for blood with reagent strip.
▪ infection/malaria
▪ strenuous exercise PRINCIPLE: Larger hemoglobin molecules are precipitated by ammonium
▪ brown recluse spider bites sulfate and myoglobin remains in the supernatant. MULTISTIX CHEMSTRIP
presence of MYOGLOBIN in urine: produce clear READING AND INTERPRETATION ▪ diisopropylbenzene ▪ dimethyldihydroperoxy-
red-brown urine reagents dihydroperoxide hexane
supernatant retains the red color
▪ muscular trauma ▪ tetramethylbenzidine ▪ tetramethylbenzidine
(+) myoglobin supernatant gives a positive reagent strip test
▪ crush syndrome 5 to 20 RBC’s/ml 5 RBCs/ml
▪ prolonged coma for blood
hemoglobin is found in the red precipitate sensitivity hgb corresponding to 10
myoglobinuria ▪ convulsion 0.015 – 0.062 mg/dL hgb
(+) hemoglobin RBC’s /mL
▪ muscle-wasting diseases supernatant tests negative for blood
▪ alcoholism/overdose FOR HEMOSIDERIN ▪ strong oxidizing agent/bleach
▪ drug abuse Free granules of hemosiderin and inclusions in a cast false positive ▪ bacterial peroxidases
▪ extensive exertion ▪ patient had hemolytic event (e.g. ▪ menstrual contamination
▪ cholesterol lowering statin medications incompatible transfusion, DIC, ▪ high specific gravity
Muscle destruction, as in rhabdomyolysis, could be an effect for patients acute hemolytic anemia such as ▪ presence of crenated cells
sickle-cell anemia, etc.) ▪ formalin
taking cholesterol lowering statin medications. The heme portion of
▪ picture shows free granules of false negative ▪ captopril
myoglobin is toxic to the renal tubules and high concentrations can cause
hemosiderin (400x) ▪ high concentrations of nitrite
acute renal failure. ▪ unstained and Prussian blue stain ▪ ascorbic acid greater than 25mg/dl
hematuria hemoglobinuria myoglobinuria REAGENTS: Prussian Blue Reagent (made fresh) ▪ unmixed specimen
occult blood + + +
Prussian blue stain: Add concentrated hydrochloric acid (HCl) to an
spun serum normal pink normal
aliquot of the potassium ferrocyanide solution (20% in demineralized
spun urine clear red pink orange
water) until a white precipitate forms that remains stable on shaking.
microscopic
RBCs negative negative Filter through Whatman No. 5 filter paper.
exam
CPK, LDH Working counterstain: Dilute 1 mL safranin O stain (0.5 g in 100 mL
haptoglobin aldorase distilled water) to 50 mL with phosphate buffer (pH: 6.4–4.7).
others
U-hemosiderin creatinine PROCEDURE
bone scan
The Prussian blue reaction is used to demonstrate iron in
SCREENING TEST FOR HEMOGLOBINURIA AND MYOGLOBINURIA hemosiderin using a dry smear or wet preparation.
1 AMMONIUM SULFATE SOLUBILITY TEST OR BLONDHEIM’S TEST 1 DRY PROCEDURE
SCREENING FOR PROTEIN When stained with Prussian blue reagent, hemosiderin appears as
1 1 ml urine + 3% sulfosalicylic acid. Filter. blue granules, 1–3 µm, singly or in groups, in renal tubular epithelial BLUE PIGMENTS
Check filtrate. cells, as amorphous sediment, or as blue granules in casts.
2 WET PROCEDURE bilirubin urobilinogen
2 NEGATIVE: PROCEED TEST:
colored filtrate precipitated protein Urine is collected in an iron-free glass container overnight. Let stand Bilirubin is a degradation product of hemoglobin. After a lifespan of 120
for 2 hours. Decant three fourths of it and centrifuge the remainder. days, the RBCs are destroyed in the spleen and liver by the phagocytic cells
thankful to have PATRICK YRAY again with me, and he said he’ll
still be here for the upcoming ones 🥺 such a constant tysm ♡
AIRAH M.
of reticuloendothelial cells. The liberated hemoglobin is broken down into FOUCHET’S TEST/HARRISON’S SPOT TEST REAGENTS:
iron, protein, and protoporphyrin: FOUCHET’S REAGENT: ▪ p-nitrobenzene-diazonium-p-toluenesulfonate
▪ the protein and iron are reused by the body ▪ trichloroacetic acid (25 gms) ▪ SSA (sulfosalicylic acid)
▪ the cells of the reticuloendothelial system convert protoporphyrin to ▪ 10% ferric chloride solution (10ml) ▪ sodium carbonate
bilirubin and is being released in the circulation where it combines with ▪ distilled water (100ml) ▪ boric acid
albumin and transported to the liver SENSITIVITY: 0.05-0.1 mg/dl
PRINCIPLE: Barium chloride added to urine combines with sulfate
The kidneys cannot excrete the circulating bilirubin because, not only is it radicals in urine to form precipitate of barium phosphate. If bile pigments no false positive: interfering substances are
false positive
bound to albumin, but it is also water-soluble (meaning, it is not a are present in urine, they will adhere to these large molecules. Ferric corrected
conjugated bilirubin). In the liver, bilirubin is conjugated with glucuronic acid chloride present in Fouchet reagent then oxidizes yellow bilirubin in ▪ specimen exposure to light
by the action of glucuronyl transferase to form water-soluble bilirubin presence of trichloroacetic acid to green biliverdin. false negative ▪ ascorbic acid greater than 25 mg/dl
diglucuronide (conjugated bilirubin). ▪ high concentration of nitrite
Usually, the conjugated bilirubin does not appear in the urine because it is
being passed from the liver into the bile duct and onto the intestine. In the
intestine, intestinal bacteria reduce bilirubin to urobilinogen which is then
oxidized and excreted in the feces in the form of stercobilinogen & urobilin.
▪ possible to have a negative reagent strip test and positive icotest
CLINICAL SIGNIFICANCE
o difference in sensitivity levels
▪ conjugated bilirubin appears in urine 2 REAGENT STRIP METHOD ▪ always perform Ictotest when
▪ bile duct obstruction (gallstone/carcinoma) PRINCIPLE: Diazotization or azo-coupling of bilirubin with diazonium salt
bilirubin o urine bilirubin test specifically ordered
▪ liver disease or damage such as hepatitis & in an acid medium to form azo dye o urine appearance is amber: even if bilirubin reagent strip test is
cirrhosis
negative
URINE BILIRUBIN AND UROBILINOGEN IN JAUNDICE o positive reagent strip test
URINE BILIRUBIN UROBILINOGEN 4 GMELIN’S TEST
bile duct obstruction +++ normal PRINCIPLE: Nitric acid oxidizes Bilirubin to Biliverdin giving different
liver damage + or - ++ colors from green to violet. In other words, it is the oxidation of bilirubin
hemolytic disease negative +++ by nitric acid.

MULTISTIX CHEMSTRIP REAGENT: concentrated nitric acid

TESTS FOR BILE PIGMENTS (BILIRUBIN)
1 FOUCHET’S TEST (not a sensitive test) 2,4-dichloroaniline 2,6-dichlorobenzene- PROCEDURE
reagents
PRINCIPLE: diazonium salt diazonium salt To about 5ml of concentrated HNO3 in at least tube, add an
▪ oxidation of bilirubin to biliverdin by the ferric chloride in Fouchet’s sensitivity 0.4 - 0.8 mg/dl 0.5 mg/dl 1 equal volume of urine carefully so that the two liquids do not
reagent mix.
▪ BaCl reacts with sulfate in urine to form barium sulfate. If bilirubin is ▪ highly pigmented urines At the junction of two liquids, various colored rings (green, blue,
present in urine, it adheres to precipitate and is detected by ▪ phenazopyridine 2
false positive red, violet etc.) will be formed
oxidation to form biliverdin (green) with FeCl3 in the presence of ▪ indican (intestinal disorders)
trichloroacetic acid. Nitric acid oxidizes bilirubin to biliverdin giving ▪ metabolites of Iodine
different colors from green to violet. ▪ specimen exposure to light
FOUCHET’S REAGENT: false negative ▪ ascorbic acid greater than 25 mg/dl
▪ trichloroacetic acid ▪ high concentration of nitrite
▪ ferric chloride solution 3 ICTOTEST
▪ barium chloride solution • Tes means tablet
• confirmatory for bilirubin
PROCEDURE
o tablets containing p-nitrobenzene-diazonium-p-toluenesulfonate.
1 Take 5 ml of 10% BaCl2 to 10 ml of urine and filter. SSA, sodium carbonate and boric acid
Dry the filter paper and add a few drops of Fouchet’s reagent • use specified mat for test
2
(prepared by adding 10mg of 10% FeCl3 to 100 ml of 25% TCA). o the mat keeps bilirubin on surface for
A green color is obtained due to oxidation of bilirubin to reaction meaning it doesn’t settle down
3
biliverdin. • positive reaction: blue to purple colour
CLINICAL SIGNIFICANCE
• interfering substances are washed into
RESULTS AND INTERPRETATION the mat and only bilirubin remains on the ▪ less than 1mg/dl or Ehrlich unit normally found in
negative no green color on the filter paper surface urine which means the presence of urobilinogen in
positive green color appear on the filter paper the urine will never reach zero
urobilinogen
▪ early detection of liver disorders, hepatitis, cirrhosis
PRINCIPLE: diazotization or azo-coupling of bilirubin with diazonium salt & carcinoma
in an acid medium to form azo dye ▪ hemolytic of liver disease disorders

thankful to have PATRICK YRAY again with me, and he said he’ll
still be here for the upcoming ones 🥺 such a constant tysm ♡
AIRAH M.
 TESTS FOR BILE PIGMENTS (UROBILINOGEN) 4-methoxybenzene- 2 Read results below.
1 ERLICH’S TEST p-dimethyl-
reagents diazonium- 3 Pink layer is then decanted and shaken with butanol.
PRINCIPLE: aminobenaaldehyde
tetrafluoroborate
▪ urobilinogen reacts with dimethylamino-benzaldehyde in choloform RESULTS AND INTERPRETATION (1st SET)
to form a pink coloured aldehyde complex sensitivity 0.4 - 0.8 mg/dl 0.5 mg/dl (+) urobilinogen pink color in the chloroform layer
▪ based on Ehrlich aldehyde reaction (+) porphobilinogen pink coloration of aqueous portion
MULTISTIX
REAGENTS: ▪ porphobilinogen
▪ Ehrlich’s reagent ▪ indican
o P-dimetyhlaminodenzaldehyde false positive ▪ p-aminosalicylic acid
o conc. hydrochloric acid ▪ sulfonamides
▪ highly pigmented urine
o distilled water
▪ old specimens
▪ saturate sodium acetate false negative
▪ preservation in formalin
▪ chloroform
CHEMSTRIP
PREFERRED SPECIMEN: fasting urine
false positive ▪ high pigmented urine
PROCEDURE ▪ old specimen
1 5ml urine + 5ml Ehrlich’s reagent false negative ▪ preservation in formalin
▪ high concentration of urine
2 mix and allow to stand for 10 minutes
3 + 5ml saturated sodium acetate and mix ▪ The red color is more of red purple
▪ Increased urobilinogen greater than 1 mg/dl is seen in liver disease PROCEDURE (2nd SET?)
4 shake vigorously and allow layers to separate
and haemolytic disorders. But if it is routinely performed on 1% of
1 Label 2 tubes #1 & #2.
non-hospitalized population and 9% of hospital population exhibit
RESULTS AND INTERPRETATION to EACH tube, add:
elevated results because of constipation
appearance of pink color in the chloroform layer ▪ 2ml urine
(+) urobilinogen colour is easily detected when view from the top 2 ▪ 2ml chloroform (in TUBE 1 only)
of the test tube ▪ 2ml butanol (in TUBE 2 only)
▪ 4ml sodium acetate
3 Vigorously shake both bottles.
4 Place in rack for layers to settle.
5 Observe both tubes for red color in the layers.

RESULTS AND INTERPRETATION (2nd SET)

cherry red complex is extractable with n-
(+) urobilinogen
butanol
cherry red complex is not extractable with
(+) porphobilinogen
n-butanol & chloroform
2 FOAM TEST no cherry red complex is formed with
negative
screening for urobilinogen is useful in the diagnosis of liver function 4 WATSON-SCHWARTZ TEST Ehrlich’s aldehyde reagent
disorders ▪ the Ehrlich’s aldehyde reaction and Watson-Swartz tests are based on
PRINCIPLE: The urine is a yellowish-brown or greenish-yellow color and solubility differences between urobilinogen and porphobilinogen
bilirubin is suspected, shake the urine. If a yellow or greenish-yellow ▪ urobilinogen can be extracted by chloroform and or butanol whereas
foam develops, then bilirubin is most likely present. Bilirubin alters the porphobilinogen will remain in an aqueous phase
surface tension of urine and foam will develop after shaking. PRINCIPLE: solubility difference of porphobilinogen and urobilinogen In
3 REAGENT TEST STRIPS Ehrlich’s aldehyde reagent
screening tests for urobilinogen are based on the Erlich Aldehyde
REAGENTS:
reaction:
▪ P-dimetyhlaminodenzaldehyde
p-dimethylaminobenzaldehyde + urobilinogen = red-colored azo dye
▪ conc. hydrochloric acid
MULTISTIX CHEMSTRIP ▪ distilled water
azo-coupling (diazo) reaction ▪ additional: saturated sodium acetate and N butanol
using 4-methoxybenzene- PROCEDURE (1st SET)
Ehrlich’s aldehyde
principle tetrafluoroborate with Add 1-2 ml of chloroform, shake for 2 minutes and allow to
reaction 1
urobilinogen forming red stand.
azodye
thankful to have PATRICK YRAY again with me, and he said he’ll
still be here for the upcoming ones 🥺 such a constant tysm ♡
AIRAH M.
5 HOESCH’S TEST ▪ high conc. protein, glucose, oxalic acid,
▪ based on the inverse Ehrlich’s reaction (e.g. of maintaining an acid ▪ improperly preserved specimen
false positive false negative ascorbic acid
solution by adding a small urine volume to a relatively large reagent ▪ highly pigmented urine
▪ inaccurate timing
volume), eliminating the problem of urobilinogen reactivity ▪ non-reductase containing bacteria
▪ sensitivity is similar to that of the Watson-Schwartz test, but the ▪ insufficient contact time between bacteria &
MISCELLANEOUS CHEMICAL SCREENING TESTS
reaction is for porphobilinogen urinary nitrate
false negative
▪ detects about 20 – 100 mg/L of porphobilinogen, and urobilinogen in ▪ presence of antibiotics individuals taking therapeutic doses of vitamin C or
amounts up to 200 mg/L does not give a positive result (red color) ▪ high concentration of ascorbic acid other preparations containing abundant ascorbic acid
▪ a yellow color may be caused by urea ▪ high specific gravity ascorbic acid has reducing properties
PRINCIPLE: inverse Ehrlich’s reaction inhibit several reagent strip reactions causing False
LEUCOCYTE
(+)/(-) results
▪ urorosein: urinary pigment related to detection of leucocyte esterase from intact and lysed WBC
false positive indoleacetic acid REAGENT STRIPS FOR ASCORBIC ACID IN URINE
CLINICAL SIGNIFICANCE
▪ dark pigmented urine 1 C-STIX REAGENT STRIPS
▪ bacterial & nonbacterial UTI
NOTE: False-positive problems may be excluded by testing the pyuria ▪ inflammation of the urinary tract ▪ phosphomolybdates buffered in an acid medium
specimen with concentrated HCl (6 mol/L) separately in conjunction ▪ screening of urine culture specimen ▪ the phosphomolybdates are reduced by ascorbic acid to molybdenum
with the Hoesch’s test. blue
TESTS FOR LEUCOCYTE SENSTIVITY: 5 mg/dL of ascorbic acid in urine after 10 seconds.
NITRATE 1 URINE LEUKOCYTE ESTERASE
conversion of nitrate to nitrite in bacteria INTERFERING SUBSTANCE:
▪ leukocyte esterase is present only in neutrophils
Gentisic acid and l-dopa may cause false (+) results
CLINICAL SIGNIFICANCE ▪ few neutrophils can be seen in normal urine
2 STIX REAGENT STRIPS
▪ rapid screening test for UTI ▪ increased numbers of neutrophils usually indicate the presence of a
25 mg/dL of ascorbic acid at 60 seconds
▪ cytitis urinary tract infection
REAGENTS:
▪ pyelonephritis ▪ screening for UTI also includes pH, protein, and nitrite
Methylene green, which is reduced to its colorless form with ascorbic
bacteriuria ▪ evaluation of antibiotic therapy ▪ mix specimen well and test at room temperature
acid. Neutral red provides a background color, and the overall color
▪ monitoring of patients at high risk for urinary PRINCIPLE: changes from blue to purple at levels of 150 mg/dL.
tract infection action of leucocyte esterase to catalyze the hydrolysis of an acid ester to 3 MULTISTIC MULTIPLE REAGENT STRIPS
▪ screening of urine culture specimen
an aromatic compound and acid Large amounts of bilirubin and pH greater than 7.5 interfere with the
TESTS FOR NITRATE color. False-positive results are not seen with urates, salicylates, gentisic
1 URINE NITRATE TESTING acid, or creatinine.
▪ aromatic amine in reagent strip reacts with nitrite producing
▪ increased dietary intake
diazonium salt
▪ hyperthyroidism
▪ the diazonium salt reacts with sulfanilic acid and acetic acid to
calcium ▪ osteolytic bone diseases
produce a pink azo dye ▪ steatorrhea
▪ Vit. D deficiency
REAGENT STRIPS FOR CALCIUM IN URINE
1 SULKOWITCH TEST
PRINCIPLE: ▪ test for serum calcium
Greiss Reaction: nitrite at acidic pH reacts with aromatic amine to form ▪ Sulkowitch test: equal quantity of reagent is added to urine
a diazonium compound ▪ test urine 5ml + 5 drops of 1% acetic acid + 5ml potassium oxalate
SULKOWITCH REAGENT:
▪ 2.5g oxalic acid
▪ 2.5g ammonium oxalate
▪ glacial acetic acid
RESULT: degree of precipitates
MULTISTIX CHEMSTRIP
if no ppt hypocalcium: 7.5 ml/dl
▪ derivatized pyrrole indoxylcarbonic acid
reagent amino acid ester fine cloud ppt 9 – 11 mg/dl
ester and diazonium salt heavy white ppt hypercalcemia
▪ diazonium salt
sensitivity 5 – 15 wbc/hpf 10 – 25 wbc/hpf SENSITIVITY: 5-20 mg/dl
INTERFERENCE: Calcium is precipitate as calcium oxalate
MULTISTIX CHEMSTRIP ▪ strong oxidizing agents
p-arsanilic acid Sulfanilamide, ▪ formalin or 5-hydroxyindoleacetic acid
false positive serotonin HPLC
reagent Tetrahydobenzo(h)- Hydroxytetrahydro ▪ highly pigmented urine
quinoline-3-ol benzoquinoline ▪ nitrofurantoin SCREENING TEST

thankful to have PATRICK YRAY again with me, and he said he’ll
still be here for the upcoming ones 🥺 such a constant tysm ♡
AIRAH M.
 ▪ based on the development of a purple color specific
for 5-hydroxyindoles with nitrous acid and 1-nitroso-
2-naphthol
▪ ethylene dichloride is used to remove interfering
chromogens
neoplastic cells (e.g., nevus, melanoma)
SCREENING TEST
melanin are based on nonspecific color reactions produced with
ferric chloride, Ehrlich’s aldehyde reagent, and
nitroferricyanide

DIPSTICK CORRELATIONS OF CHEMICAL ANALYSIS OF URINE:

POSITIVE TEST CORRELATE OTHER TESTS
pH nitrite, leucocyte esterase, microscopic
protein blood, nitrite, leucocyte esterase, microscopic
glucose ketone
blood protein, microscopic
bilirubin urobilinogen
urobilinogen
protein, leucocyte esterase, microscopic
nitrite
specific gravity turbidity, microscopic

SIDE COMMENTS:
She never understood why Ketone only detect acetone and acetoacetic acid.
To think they only consist of small percentages while 78% beta
hydroxybutyric acid is not being tested for anything. Why is that?

REFERENCES:
▪ S.K. Strasinger, Urinalysis and Body Fluids, 6th Ed
▪ N. Brunzel, Fundamentals of Urine and Body Fluid Analysis, 4th Ed
▪ Henry’s Clinical Diagnosis and Management by Laboratory Methods,
22nd Ed

thankful to have PATRICK YRAY again with me, and he said he’ll
still be here for the upcoming ones 🥺 such a constant tysm ♡
AIRAH M.
 AUBF 1: 3 POLARIZED MICROSCOPY
standardization of size: 12-15 and with a use of centrifuge tube which
MICROSCOPIC EXAMINATION OF URINE confirms presence of fat, specifically cholesterol
contains almost this amount
Learning Objectives: ▪ average of 12ml 4 INTERFERENCE CONTRAST
▪ ratio of sediments in: produces a 3-dimensional image & layer by layer imaging of specimen
▪ Explain the clinical significance of performing the microscopic
o manual method - 12:1 5 CYTODIAGNOSITC UA
examination of urine sediments.
o automated is 12:1 or 30:1 employing cytocentrifuge & Papaniculau stain
▪ Discuss the procedure in specimen preparation for microscopic exam.
VOLUME 6 FLOURESCENCE MICROSCOPY
▪ Explain the effects of stains used to enhance visualization of each type
1 10-15 ml (12ml average) visualization of natural fluorescence microorganisms or stained with
of urinary structures
2 a representative of the elements in the urine fluorescence dye
▪ Identify the different normal & pathologic microscopic structures in the
insufficient volume should be noted so correction be made prior to 7 DARKFIELD MICROSCOPY
urine sediments and the manner of reporting such findings 3
reporting for identification of T. pallidum
RATIONALE Side notes: 8 COMMERCIAL SYSTEMS
▪ to detect if abnormal physical & chemical tests results is due to renal ▪ There are times that you cannot expect to have the right volume ▪ KOVA ▪ Quick-Prep UA System
dss or lower UTI ▪ If the volume didn’t reach 10, just get 6 of it, spin it and examine it ▪ Urisystem ▪ R/S Workstations
▪ to provide information to the type of renal disease directly and them multiply the result by 2 (considering that you use ▪ Censlide 2000 ▪ Count 10
half) Side notes:
MICROSCOPIC EXAMINATION ▪ If less than 6, you can mix the sediment and examine it directly, put a ▪ there is a technique: when you charge (do not overcharge because
▪ is a viral part of the routine urinalysis note that: (a) quantity is insufficient, (b) urine was examined directly sediments will overflow on the side; just enough)
▪ a diagnostic tool for the detection and renal urinary tract disorders as and centrifuged ▪ just use a small drop (Pasteur pipette with a smaller bore)
well as other systemic disorders Centrifugation ▪ usually we use the bright field microscope, polarizing microscope, the
▪ verify the positive chemical tests 5 min at 400 (RCF) relative centrifugal force or 1500-2000 rpm, for interference, and other microscopes that can aid in the proper
▪ the value is dependent on two main factors: 1 optimum amount of sediment & least chance of damaging the visualization of the sediments
o the examination of a suitable specimen sediments ▪ soon as you have your sample on the slide, there are two things that
o the knowledge of the person performing the examination 2 0.5 to 1.0 ml sediment volume then gently agitate you have to do in fresh preparation examination:
3 20 ul (.02 ml) sediments used in conventional slide method o focus at LPO where you look at the sides for the presence of casts
GENERAL CONSIDERATIONS
Side notes: since they are reported in the LPO: as you examine, lower down
1 method of specimen preparation ▪ CENTRIFUGATION is standardized to be done at a specific time that your light (unlike in staining preparation wherein you use a
2 volume of specimen actually examined will completely settle the sediments without disrupting the structures: brighter light)
3 methods & Equipment for visualization 1,500 to 2000 rpm, 5 minutes spinning o keep on moving up and down the fine adjustment knob in order
4 manner of reporting results ▪ centrifuge tube: a test tube with conical bottom (not rounded because to enable the viewer to see the depth of the object as well as
it is made in such a way to facilitate the settling of sediments upon other structures that may be in other focal planes
SPECIMEN PREPARATION
centrifugation) MANNER OF REPORTING RESULTS
1 freshly voided or adequately preserved urine
2 midstream clean catch ▪ cover slip also standardized: 22 by 22 mm and also, its thickness is RBC, WBC average no./HPF reporting results
3 mix properly before centrifugation standardized casts average no./LPF
▪ exact volume used: more or less 20 mm sediments used epithelial cells, uses semiquantitative-rare, few,
4 RBCs, WBCs, & casts disintegrate rapidly in dilute alkaline urine
✓ you put the volume which is 12-15 ml (standardized volume) crystals, bacteria moderate & plenty or plus (+) values
refrigeration ppts amorphous urates & phosphates and non-
5 ✓ after spinning, do not apply the break: it will disrupt the
pathologic crystals
structures, let it stop naturally TERM PLUS VALUE DESCRIPTION
6 warming at 37°C dissolves some of the crystals
✓ upon complete stop, you decant the supernatant and what is left rare 1+ present but hard to fine
Side notes: at the bottom is more or less .5 to 1 ml for the sediments
The best spx for routine urinalysis is 1st morning specimen 1 or more present in almost every
✓ try to tap it on your palm or flick it slowly to resuspend the few 1+
FOV
▪ casts and WBC tend to dissolve or lyse in spx with low specific
sediment but not in a way that you will disrupt the structures
gravity or alkaline pH easy to find, number present in
(resuspend slowly and carefully) moderate 2+
▪ usually provides the concentrated and acidic environment needed ✓ then add one drop to the slide and cover with a cover slip, be sure
FOV varies
for these structures that there is no overflowing because you will be losing the casts many 3+ prominent, large number present
The sediments should be examined as soon as possible after collection that will mostly settle at the edges of the coverslip TNTC 4+ packed, crowded, or overwhelming
but may be refrigerated for a few hours if the examination cannot be METHOD AND EQUIPMENT FOR VISUALIZATION
performed immediately (however, refrigeration also gives way in Side notes:
1 BRIGHTFIELD MICROSCOPY
formation of crystals). ▪ LPO for casts then turn to HPO to visualize what kind of casts
A using unstained sediment ▪ RBC, WBC: reported in the average of 10 views per high power field
ADVANTAGES MADE IN AN EFFORT TO AID MT in MICROSCOPIC exam B stained sediment by supravital stain ▪ For MVC: sometimes you have to report TNTC when they are close
▪ staining (supravital stains)
examples: to each other (congested field)
▪ development of phase and interference contrast microscopy
▪ Sternheimer Malbin stain ▪ KOVA stain ▪ for epithelial cells and bacteria: you can report +1, +2, or rare, few,
▪ automation
▪ SEDI stain ▪ Sudan III Prescott Brodie moderate, many
You now have the urine sample. You try to visualize (QC of urine
sample) if there are any faecal contamination (reject if present). 2 PHASE CONTRAST MICROSCOPY Take note of the laboratory’s protocol!
examine for old urine: when urine is freshly collected, it is somewhat permits more detailed visualization of elements with low refractive
warm index (e.g. hyaline casts & crystals)

AIRAH M.
A CONVERSION OF AVERAGE NOS TO NOS PER ML enhances WBCs, yeast, oil CONDITIONS ASSOCIATED WITH HEMATURIA
1 Calculate the area of a lpf or hpf for the microscope used nuclei of WBCs droplets & crystals renal disease/tumor hemorrhagic disease
area of a cirlce = 𝜋𝑟 2 Lipid stains: Oil stains triglycerides & identifies free fat pyelonephritis violent exercise
Example: Red O, Sudan neutral fats—orange droplets & lipid- trauma to kidneys Tb of the gut
diameter of hpf: 0.35 mm III red containing cells & casts traumatic catheterization prostatitis
3.14 𝑥 0.1752 = 𝟎. 𝟎𝟗𝟔 𝐦𝐦𝟐 Gram stain
differentiates Gm (+)
identifies bacterial casts
passage of stones/calculi cystitis
& Gm (-) bacteria contamination with menstrual blood
2 Calculation of maximum no. of lpf or hpf in viewing area:
methylene blue & B dysmorphic RBCS
identifies urinary
area (22 x 22 mm)cover slip 484 mm2 Hansel stain eosin Y stains
eosinophiles variation of size, cellular protrusions & sometimes fragmented, is
eosinophilic granules associated with glomerular bleeding
484
= 𝟓𝟎𝟒𝟎 𝐡𝐩𝐟𝐬 identifies yellow
0.096 Prussia blue stains structures
granules of hemosiderin
stain (ROUS) containing iron
Calculate the no. of hpfs per ml urine tested using the in cells & casts
3
concentration factor & volume of sediment examined.
SEDIMENT CONSTITUENTS
5040 5040
= = 𝟐𝟏, 𝟎𝟎𝟎 𝐡𝐩𝐟/𝐦𝐥 You have no difficulty for those negative in the chemical tests, you just
0.02 𝑚𝑙 𝑥 12 0.24 have to run through the field wherein you can see little to none. But if
Calculate the number of formed elements per ml urine by positive with bacteria, then you have to scan the field properly.
4 multiplying the no. of hpfs/ml by the average no. of formed
1 WHITE BLOOD CELLS (PUS CELLS)
elements per field.
increase number of pus cells in urine Side notes:
Example: ▪ there is bleeding and must be differentiated if it is in the lower or
▪ acute glomerulonephritis
4 wbc/hpf x 21,000 = 𝟖𝟒, 𝟎𝟎𝟎 𝐰𝐛𝐜/𝐦𝐥 pyuria upper part of the urinary system (e.g. hematuria)
▪ urinary tract infection
Note: The number of hpf or lpf/ml of urine remains the same in the ▪ leukocyte esterase ▪ supplement the RBC with other structures
same microscope and volume of the sediments used. (constant) ▪ disregard contaminant due to menstruation (recollect at another
Brownian movement of the granules w/n the
FACTORS THAT INFLUENCE APPEARANCE OF URINARY day)
B glitter cells neutrophil w/c absorbed water when exposed to
SEDIMENTS ▪ sometimes, RBC appearance is affected by concentration (hypotonic,
hypotonic urine
1 cells & casts in various stages of development or degradation hypertonic): appearance may vary (sometimes bloated, sometimes
normal value: 0-5 wbc/hpf
crenated)
distortion of cells & crystals by the chemical content of the
2 ▪ we have also these morphic RBCs where you can correlate later on
specimen
with glomerular bleeding
3 presence of inclusions in cells & casts 3 EPITHELIAL CELLS
4 contaminants by arfifacts
A SQUAMOUS EPITHELIAL CELLS
C CONVENTIONAL SLIDE METHOD ▪ derives from the lower urinary tract, not present in the kidney,
▪ 22 x 22 mm cover slip lines the distal 1/3 of both male & female urethra &entire vagina.
▪ overflowing may result in the loss of heavier elements Large flat cell with small central spherical nucleus
▪ casts have a tendency to locate near the edges of the cover slip Side notes:
▪ Clue Cells: Gardnerella vaginalis infection, coccobacilli covered
▪ many sediments have a reflective index same as as urine and it is ▪ there is infection wherein bacteria can also be seen
most of the cell surface
essential that examination must be done under reduced light ▪ you can also differentiate of WBC or infection if it is upper or lower
urinary tract infection Side notes:
Thomas Addis in 1926 introduced a standardize method of urine ▪ correlate with other structures ▪ normally found at the lower part of urinary tract (vagina,
sediments identification called the ADDIS count. It utilized a ▪ you usually see bacteria which is reported as few, moderate, plenty urethra) and sometimes cannot be considered abnormal because
hemacytometer to count RBC, WBC, casts & epithelia in 12-hr urine ▪ usually positive in routine and chemical tests so, you have to check it is sometimes due to the sloughing off of the epithelial linings
sample. with the microscopic exams ▪ when found in significant amounts, it is also with clinical
Identification is sometimes difficult even for experience Lab personnel 2 RED BLOOD CELLS significance
so refractile, biconcave discs ▪ big or flat cells, with rounded centrally located nucleus
reading of sediments is enhanced by the use of sediment stains: ▪ sometimes the nucleus will be seen as WBC or RBC especially
unfresh urine colorless, ̏shadow cells ̋ when the epithelial cells are folded and there are plenty, then
STAIN ACTION FUNCTION concentrated urine you cannot identify it properly
hypersthenuric urine
Sternheimer delineates structures small & crenated B TRANSITIONAL (URETHELIAL) CELLS
identifies WBCs,
–Malbin & contrasting colors dilute urine ▪ originate from the lining of renal pelvis, calyces, ureters, bladder &
epithelial cells, and hyposthenuric urine
(crystal violet of the nucleus & large swollen “Ghost” from the upper portion of the male urethra
casts
& safranin O) cytoplasm ▪ smaller than squamous appear in several forms with centrally
differentiates WBCs & A HEMATURIA located nucleus
enhance nuclear
Toluidine Blue
details
RTE (renal tubular ▪ presence of intact RBC in urine
Side notes:
epithelial) cells ▪ Note: It is important to take note of the appearance of urine if
▪ the cells that can be seen from the bladder, urether going up to
2% acetic acid lyses RBCs & distinguishes RBCs from RBCs is suspected
the calyx of kidney

AIRAH M.
 ▪ significant, not normally found in urine lipid-containing RTE cells, highly refractile, usually seen in conjunction
▪ there is sloughing off in the cells of specific portions in urinary with free-floating fat droplets
tract
▪ usually seen with bleeding in cases of stone that goes down to LIPIDURIA
▪ is most frequently associated with damage of
the bladder into the urine which will irritate the ureter wherein
the glomerulus caused by nephrotic syndrome
you will find RBC and transitional epithelial cells B RBC CASTS
▪ also seen in:
▪ smaller than squamous, rounded, centrally located nucleus
o severe tubular o trauma that
C RENAL TUBULAR EPITHELIAL CELLS ▪ associated with glomerular damage with
necrosis releases bone
columnar/convoluted large rectangular from the proximal o diabetes mellitus o marrow fat from proteinuria & dysmorphic erythrocytes
cells convoluted tubule long bones ▪ orange red, positive (+) reagent strip for
round or oval, smaller than those from blood
DCT (distal Side notes:
the PCT C WBC CASTS
convoluted tubule) OVA: BODIES
nucleus is round and eccentric ▪ are epithelial cells that contains fats
cuboidal never round, eccentric and ▪ you can check that one with staining or polarizing microscope
presence of at least one straight edge o if it is cholesterol, it will portray a maltase cross appearance signifies inflammation within the
collecting duct RTE
differentiating it from spherical or 5 BACTERIA nephron, distinguishes pyelonephritis
polyhedral transitional cells from lower UTI
▪ bacteriuria: increase
bacteria = UTI
▪ renal fragments: cells from the collecting duct that appears in
▪ reported as few, D BACTERIAL CASTS
groups of 3 or more
moderate or many/
▪ presence of more than 2 RTE cells/hpf indicates tubular injury
Hpf
▪ presence of increased amounts is indicative of necrosis of the
▪ findings to suspect UTI:
renal tubules, with the possibility of affecting overall renal
o increase pus cells (pyuria) containing bacilli both w/n & bound
function
o nitrate & leukocyte esterase (+) to the protein matrix seen in
▪ if u see RBC, WBC with your RTE, then the cause of the bleeding
6 CASTS PYELONEPHRITIS
or infection is in the kidney
▪ formed within the lumens of the distal convoluted tubules &
CONDITIONS PRODUCING TUBULAR NECROSIS collecting ducts
exposure to heavy metals pyelonephritis ▪ major component: Tamm-Horsfall Protein, a glycoprotein excreted by E EPITHELIAL CASTS
drug-induced toxicity allergic reactions the RTE of the DCT & Upper collecting ducts (another name is
uromodulin)
hemoglobin & myoglobin toxicity salicylate poisoning
▪ mucus: major constituent cast containing RTE cells represent the
viral infections (hep.B) malignant infiltrations ▪ cylindruria: presence of urinary casts presence of advance tubular destruction
acute allogenic transplant rejection Side notes:
▪ we have the basic structure which is hyaline casts
▪ empty, usually elongated rounded ends F GRANULAR CASTS
▪ when there are inclusions in the hyaline casts, it is no longer called in disease granules may
that (now depends on the inclusion) represent disintegration of
o If RBC: RBC casts cellular casts & tubule cells
o If WBC: wbc casts or protein aggregates
o If RTE: RTE casts (usually inside the casts) filtered by the glomerulus
▪ you can never say as squamous casts since it is not found in the
kidney where casts are found G WAXY CASTS
▪ try to compare the clumps of each: or the casts, you can see the
matrix (inside)
A HYALINE CASTS: 0-2/lpf are representative of extreme urine stasis,
▪ increased in the ff: indicating renal failure
o strenuous exercise o heat exposure
o dehydration o emotional stress
4 OVAL FAT BODIES
▪ pathologic increase: H BROAD CASTS
o acute glomerulonephritis o pyelonephritis
o chronic renal disease o congestive heart failure
indicates destruction of the tubular
wall

7 CRYSTALS FOUND IN URINE

AIRAH M.
 ▪ formed by precipitation of urinary solutes B AMPICILLIN CRYSTALS
▪ subject to changes in temp, solute conc & pH
▪ presence in freshly voided urine is associated with concentrated
colorless, dumbbell shape
specimens (high sp. gravity) long colorless, thin prism or needles tend to form bundles
Side notes: 8 OTHER STRUCTURES SEEN IN URINE
▪ are somewhat insignificant but if seen with a freshly voided urine (e.g.
D AMMONIUM BIURATE A YEAST CELLS
calcium oxalate crystals, uric acid crystals), it is significant
▪ may indicate renal stone formation thorn apple crystal
▪ these crystals with their solubility can aid in the identification of the
crystals since there are also crystals that are abnormal especially smaller than RBCS, budding
when found with hepatic disorders
NORMAL CRYSTALS IN ACID URINE
A AMORPHOUS URATES ABNORMAL URINE CRYSTALS
found in acid urine or rarely in neutral urine B SPERMATOZOA
brick red granules/pink sediment
B URIC ACID A CYSTINE
▪ yellow brown, rhombic, colorless hexagonal plates for males: early ejaculation
four-sided flat plates
(whetstones), wedges and for females: postcoital
rosettes
▪ increased levels of purines
C PARASITES
& nucleic acids B CHOLESTEROL
▪ seen in leukemia under broken windowpanes or stairstep appearance
chemotherapy, gout, & in
Lesch-Nylan syndrome ▪ Trichomonas vaginalis- trophozoite
▪ Schistosoma haematobium- ovum
C SODIUM URATE
▪ NOTE: presence of parasites suggest
crystals are needle-shaped fecal contamination reject specimen
CRYSTALS ASSOCIATED WITH LIVER DISORDERS
A TYROSINE CRYSTALS
D STARCH GRANUELES
fine colorless to yellow needles that with a dimpled center
D CALCIUM OXALATE frequently form clumps or rosettes (can be seen on infants whose parents
octahedral envelope, may be related to formation of renal calculi are fond of putting powder in the
inguinal area)
B LEUCINE CRYSTALS
E OIL DROPLETS AND AIR BUBBLES

yellow brown spheroids with

NORMAL CRYSTALS FOUND IN ALKALINE URINE striations
A AMORPHOUS PHOSPHATES
granular as urates, does not dissolve in warming C BILIRUBIN
F POLLEN GRAINS

reddish brown cubes

B TRIPLE PHOSPHATE
prism shape, coffin- lid DRUG RELATED CRYSTALS
G HAIR AND FIBERS
A SULFONAMIDE CRYSTALS

▪ appear yellow to brown as bundles of

C CALCIUM CARBONATE needles or sheaves of wheat
▪ inadequate patient hydration

CRYSTALS pH COLOR SOLUBILITY

AIRAH M.
 cystine acid colorless ammonia, dilute HCl ▪ specimen evaluation
cholesterol acid colorless chloroform ▪ physical examination
leucine acid/N yellow hot alkali or alcohol o sp.gravity (refractometer)
tyrosine acid/N colorless/yellow alkali and heat o w/ distilled water = 1.000
acid HAc, HCl, NaOH, o 3% NaCl =1.015
bilirubin yellow
ether, chloroform o 9% sucrose = 1.034
sulfonamides acid/N varied acetone o 5% NaCl=1.022
radiographic acid ▪ chemical examination - - reagent strip
colorless 10% NaOH
dye ▪ confirmatory test controls
acid/N refrigeration forms ▪ proficiency testing
ampicillin colorless
bundles

TYPE APPEARANCE CLINICAL SIGNIFICANCE AUTOMATION OF URINALYSIS

▪ colorless ADVANTAGES EXAMPLES
▪ strenuous exercise
▪ homogenous protein improves productivity Clinitek and Atlas
▪ stress
hyaline cast ▪ matrix w/ rounded minimize variability Chemstrip Urine Analyser
▪ inflammation of the
ends, various sizes & improve on TAT Rapimat II
urogenital tract
shapes
▪ protein matrix filled w/ ▪ glomerulonephritis ASPECTS
RBC cast RBCs ▪ acute interstitial nephritis A CHEMICAL TESTS
▪ often many free RBCs ▪ strenuous exercise use of test strip readers like Clinitek (Ames)
▪ pyelonephritis B COMPLETE URINALYSIS (chemistry/sediment,sp. gr.)
▪ kidney infection (w/ ▪ YELLOW IRIS (International Remote Imaging System)
protein matrix filled w/
WBC cast bacteria, protein, RBC) ▪ chemistry: test strip reader using reflectance photometry
WBCs
▪ glomerulonephritis w/ ▪ sp.gravity: sound wave frequency
RBC casts ▪ sediment examination: uses planar hydrodynamic positioning
matrix filled w/renal
epithelial cast renal tubular damage KOVA SYSTEM SEDIMENT EXAM
epithelial.cells
matrix filled w/ provides a standardized urine sediment examination using a standard
▪ urine flow stasis volume of 12ml and aspiration of a fixed amount of sediment placed in a
degenerating cells,
fine/coarsely ▪ UTI chamber of a constant volume for microscopic evaluation
amorphous crystals or
granular ▪ strenuous exercise
bacteria, colorless-
▪ stress Side notes:
yellowish
Pre-analytical QC: observation of physical appearance
▪ tubular obstruction/dss
▪ homogenous w/ waxy, Analytical QC:
w/extreme urine flow
thick appearance ▪ expiration date of strips (analytical)
waxy/broad stasis
▪ often w/blunt uneven, ▪ close the container tightly
▪ severe nephron damage
brittle-looking edges ▪ never remove desiccant since it will disturb the moisture
▪ nephrotic syndrome
▪ nephrotic syndrome ▪ keep stability of reagent strip
▪ RTE cell death
for specific gravity: use different chemicals to calibrate your urinometer if
protein matrix w/oval ▪ severe crush injury w/
fatty it gives you the right SG
fat bodies disruption of body fat
accompanied w/ Automated:
proteinuria ▪ read the readings of the strip semi-quantitively
▪ incorporated bilirubin ▪ sediments are examined (sometimes may plug down hence you have
hyaline matrix w/
pigmented (golden brown) to do the manual reading)
coloration due to
casts ▪ Hgb or myoglobin (yellow
pigment incorporation ✓ IDENTIFICATION depends on the training of the MT doing the exam
to red brown)
▪ 2 or more cell types 2 cell types in filtrate during ✓ REMEMBER TO CORRELATE THE RESULTS!
enmeshed in a protein cast formation initial cast REFERENCES
matrix formation in one tubule,
mixed ▪ Strasinger/Di Lorenzo, 6th Ed. Urinalysis & Body Fluids p.86-140 Chap6
▪ part granular & part followed by stasis in another
▪ Mundt/Shanahan 2nd Ed.55-170 Ch5
waxy, any combination wider lumen & further cast
possible formation ▪ Graff’s Textbook of Routine Urinalysis & Body Fluids
▪ Brunzel, Urine & Body Fluid Analysis2013 p.157- 210 Ch 8
QUALITY CONTROL IN ANALYSIS
GUIDELINES:

AIRAH M.
 URINE SEDIMENTS
SEDIMENTS DEFINITION IMAGE SEDIMENTS DEFINITION IMAGE
WHITE BLOOD CELLS
(PUS CELLS)
lipid-containing RTE cells, highly refractile,
▪ can be pyuria or glitter cells
OVAL FAT BODIES usually seen in conjunction with free-
▪ normal value: 0-5 wbc/hpf
floating fat droplets

▪ bacteriuria: increase bacteria = UTI

▪ reported as few, moderate or many/ Hpf
▪ refractile, biconcave discs
RED BLOOD CELLS BACTERIA ▪ findings to suspect UTI:
▪ caused by hematuria and dysmorphic RBCs
o increase pus cells (pyuria)
nitrate & leukocyte esterase (+)
▪ formed within the lumens of the distal
convoluted tubules & collecting ducts
▪ major component: Tamm-Horsfall
can be squamous, transitional, or renal tubular Protein, a glycoprotein excreted by the
EPITHELIAL CELLS CASTS elaborated below
epithelial cells RTE of the DCT & Upper collecting ducts
(another name is uromodulin)
▪ mucus: major constituent
▪ cylindruria: presence of urinary casts
▪ formed by precipitation of urinary solutes
▪ subject to changes in temp, solute conc & pH
CRYSTALS FOUND IN OTHER STRUCTURES SEEN
▪ presence in freshly voided urine is associated elaborated below elaborated below
URINE IN URINE
with concentrated specimens (high sp.
gravity)
CASTS
TYPES DEFINITION APPEARANCE IMAGE TYPES DEFINITION APPEARANCE IMAGE
▪ increased in the ff:
o strenuous exercise
o dehydration
o heat exposure ▪ colorless
containing bacilli both w/n &
HYALINE o emotional stress ▪ homogenous protein BACTERIAL
bound to the protein matrix
CASTS ▪ pathologic increase: ▪ matrix w/ rounded ends, CASTS
seen in PYELONEPHRITIS
o acute glomerulonephritis various sizes & shapes
o chronic renal disease
o pyelonephritis
o congestive heart failure
▪ associated with glomerular
damage with proteinuria & cast containing RTE cells
protein matrix filled w/ RBCs EPITHELIAL matrix filled w/renal
RBC CASTS dysmorphic erythrocytes represent the presence of
often many free RBCs CASTS epithelial.cells
▪ orange red, positive (+) advance tubular destruction
reagent strip for blood

in disease granules may

fine/coarsely granular:
signifies inflammation within represent disintegration of
GRANULAR matrix filled w/ degenerating
WBC CASTS the nephron, distinguishes protein matrix filled w/ WBCs cellular casts & tubule cells or
CASTS cells, amorphous crystals or
pyelonephritis from lower UTI protein aggregates filtered by
bacteria, colorless- yellowish
the glomerulus

▪ homogenous w/ waxy, thick ▪ homogenous w/ waxy, thick

are representative of extreme
WAXY appearance indicates destruction of the appearance
urine stasis, indicating renal BROAD CASTS
CASTS ▪ often w/blunt uneven, brittle- tubular wall ▪ often w/blunt uneven,
failure
looking edges brittle-looking edges

AIRAH M.
 CRYSTALS FOUND IN URINE
NORMAL CRYSTALS FOUND IN ACID URINE
TYPES DEFINITION IMAGE TYPES DEFINITION IMAGES

AMORPHOUS URATES brick red granules/pink sediment SODIUM URATE crystals are needle-shaped

▪ yellow brown, rhombic, four-sided flat plates

(whetstones), wedges and rosettes
octahedral envelope, may be related to
URIC ACID ▪ increased levels of purines & nucleic acids CALCIUM OXALATE
formation of renal calculi
▪ seen in leukemia under chemotherapy, gout, &
in Lesch-Nylan syndrome

NORMAL CRYSTALS FOUN IN ALKALINE URINE

AMORPHOUS
granular as urates, does not dissolve in warming CALCIUM CARBONATE colorless, dumbbell shape
PHOSPHATES

TRIPLE PHOSPHATE prism shape, coffin- lid AMMONIUM BIURATE thorn apple crystal

ABNORMAL URINE CRYSTALS

broken windowpanes or stairstep

CYSTINE colorless hexagonal plates CHOLESTEROL
appearance

CRYSTALS ASSOCIATED WITH LIVER DISORDERS

fine colorless to yellow needles that frequently

TYROSINE CRYSTALS LEUCINE CRYSTALS yellow brown spheroids with striations
form clumps or rosettes

BILIRUBIN reddish brown cubes

DRUG RELATED CRYSTALS

▪ appear yellow to brown as bundles of needles

SULFONAMIDE long colorless, thin prism or needles tend to
or sheaves of wheat AMPICILLIN CRYSTALS
CRYSTALS form bundles
▪ inadequate patient hydration

AIRAH M.
 OTHER STRUCTURES SEEN IN URINE

for males: early ejaculation

YEAST CELLS smaller than RBCS, budding SPERMATOZOA
for females: postcoital

▪ Trichomonas vaginalis- trophozoite

with a dimpled center
▪ Schistosoma haematobium- ovum
PARASITES STARCH GRANULES (can be seen on infants whose parents are
▪ NOTE: presence of parasites suggest fecal
fond of putting powder in the inguinal area)
contamination reject specimen

OIL DROPLETS AND

POLLEN GRAINS
AIR BUBBLES

HAIR AND FIBERS

AIRAH M.
 ANALYSIS OF URINE AND BODY FLUIDS ▪ filtration and complete reabsorption in young adults, this clearance has a mean value of:
TOPIC 06: RENAL FUNCTION TESTS ▪ filtration and secretion
MEN WOMEN
Objectives: Remember this, because this are the processes that we are going to discuss 127mL per minute per 118mL per minute per
▪ Identify the laboratory procedures used to evaluate glomerular in the renal function test. 1.73m2 1.73m2
▪ Describe the creatinine clearance test RENAL HANDING OF SUBSTANCES These values decline with age and the method
▪ Calculate the creatinine clearance and determine whether the result is requires intravenous infusion of inulin and timed
normal urine collections over many hours which makes the
▪ Discuss the clinical significance of the glomerular filtration rate tests procedure clinically impractical.
▪ Describe and contrast MDRD, cystatin C, and beta2-macroglobulin
This is the general formula of clearance in which clearance is equal to the
▪ Define osmolarity and discuss its relationship to urine concentration
urine concentration of the substance times the volume of urine per unit time
RENAL PHYSIOLOGY divided by the concentration of the substance in the plasma. Always
Back to the basics! remember this. This is the basis of almost all clearance formulas.

Ux V
CLEARANCE TESTS 𝐜𝐥𝐞𝐚𝐫𝐚𝐧𝐜𝐞 =
Px
▪ standards tests used to measure the filtering capacity of the glomeruli
▪ measures the rate at which the kidneys are able to remove or ‘clear’ a UX and Px = concentration of substances in urine and plasma
filterable substance from the blood V = volume of urine per unit time
▪ substance must be one that is neither absorbed nor secreted by the
CREATININE CLEARANCE
tubules, to ensure accuracy
o Substance must be one that is neither absorbed nor secreted by the ▪ the first glomerular filtration test
tubules to ensure accuracy. In other words, that substance must be ▪ waste product of muscle metabolism that is produced enzymatically by
filtered only by the kidney. creatine phosphokinase from creatine
▪ normally found at a relatively constant level in the blood, it provides the
laboratory with an endogenous procedure for evaluating glomerular
function

So, we have these substances:

▪ creatinine, beta2-macroglobulin, cystatin C, and radioisotopes
THREE BASIC RENAL PROCESSES ▪ exogenous procedure vs endogenous procedure
this is how water and solutes (smaller than proteins) are o These exogenous procedures uses infused substances not produced DISADVANTAGES
forced through the capillary walls and pores of the by the body for testing and this is seldom used because we have some creatinine is secreted by the tubules and secretion increases
glomerular 1
glomerular capsule enter the renal tubules endogenous substances that is already present in the body that is as the blood levels also rise
filtration
in the glomerular filtration, always remember that there normally cleared by the glomeruli. Chromogens present in human plasma react in the chemical
is the pressure gradient ▪ urea clearance* 2 analysis. Their presence, however, also may help counter act to
starts at the proximal tubules ▪ inulin clearance* falsely elevated rates caused by tubular secretion.
tubular this is where water, glucose, amino acids, and needed * So basically, we have this urea clearance, inulin clearance. These are used Some medications inhibit tubular secretion of creatinine, thus
reabsorption ions are transported out of the filtrate into the tubule historically as CLEARANCE TEST. causing falsely low serum levels. Your medications such as
cells and then enter the capillary blood 3 gentamicin, cephalosporins (like cephalexin,
was used because it was present in all urine
the opposite of tubular reabsorption cefuroxime,ceftriaxone,and cimetidine) will also inhibit tubular
specimen and the existence of routinely used
tubular this is how the hydrogen ions, potassium ions, secretion of creatinine causing falsely low serum creatinine levels.
methods of chemical analyses
secretion creatinine, and drugs are removed from the peritubular Bacteria will break down urinary creatinine if specimens are kept at
approximately 40% of routinely filtered urea is 4
blood and secreted by the tubule cells into the filtrate UREA room temperature for extended periods
reabsorbed, values suggested to reflect the
(urea clearance) Heavy meat consumption during collection of a 24-hour urine
So, the net effect of those 3 processes is excretion equal to filtration minus absorption
▪ therefore, it is not an ideal clearance test specimen will influence the results. If the plasma specimen is drawn
the reabsorption rate plus the secretion rate.
its value as a measure of GFR is not very good for 5 before the collection period because the increase intake of meat can
EXCRETION = FILTRATION – REABSORPTION + SECRETION raise the urine and plasma levels of creatinine during the24hr
several reasons
So, remember the substances, hypothetical substances, handled by your collection period.
is also a complex polysaccharide produced by
INULIN Measurements of creatinine clearance is not a reliable indicator in
kidneys. We have through certain plants
(inulin 6 patients suffering from muscle-wasting diseases or persons
▪ filtration only has been widely regarded as the gold standard for
clearance) involved in heavy exercise or athletes supplementing with creatine.
▪ filtration and partial reabsorption measuring GFR
always amazed by how kind this girl is, GENEVIEVE RUSTE, thank
you so much for helping me on this one, I truly appreciate it, SML ♡
AIRAH M.
 Accurate results depend on the accurate completeness of a 24-hour MDRD-IDMS CYSTATIN C
7
collection. The previous equation was derived before routine creatinine methods were ▪ was used in the modified Schwartz formula
Must be corrected for body surface area unless normal is assumed commonly made traceable to the IDMS standard. For methods traceable to ▪ a small protein (molecular weight 12,259) produced at a constant rate by
8 and must always be corrected for children. So that’s why we have the IDMS standard, creatinine results tend to be lower and the equation all nucleated cells
different formula in using creatinine clearance in children. should be used with the smaller initial constant. So, this is a derivation of ▪ readily filtered by the glomerulus and reabsorbed and broken down by
the MDRD using the IDMS traceable standard which is the renal tubular cells
ESTIMATED GLOMERULAR FILTRATION RATES
the two most widely used formulas for estimated glomerular filtration rates ▪ no cystatin C is secreted by the tubules, and the serum concentration can
▪ Cockroft-Gault formula be directly related to the GFR
▪ Modification of Diet in Renal Disease (MDRD( ▪ monitoring levels of cystatin C is recommended for pediatric patients,
o also known as levi formula persons with diabetes, the elderly, and critically ill patients because this
o there is a modification: MDRD-IDMS traceable formula SCHWARTZ FORMULA substance is not independent (but contradicting to what’s in the ppt
Both Cockroft-Gault formula and the MDRD formula estimate GFR in adults which said that it is independent of muscle mass) in muscle mass because
for children:
and are not applicable to measurement of GFR in children. The following most of your patients, your elderly and critically ill patients have
▪ Schwartz Formula
formulas are used most often for estimating creatinine clearance in decreased muscle mass.
▪ Counahan-Barrett Formula (pronounced as hunahan?)
children. It is much simpler using only these 2 variables: ▪ an advantage of cystatin C is that it is independent of muscle mass
COCKROFT-GAULT FORMULA
o height o serum-creatinine ▪ not widely used clinically (measurements are difficult and expensive)
is a widely used formula usually used by medical residents or by nephrologist BETA2-MICROGLOBULIN
in getting the GFR but you have to take note of the ideal body weight ▪ molecular weight: 11,800
▪ dissociates from human leukocyte antigens at a constant rate and is
rapidly removed from the plasma by glomerular filtration
If your serum-creatinine is in micromoles per liter, you can use the other ▪ more sensitive indicator of a decrease in GFR than creatinine clearance,
formula (the second one). however, the test is not reliable in patients who have a history of
male IBW 50 kg + 2.3 for each inch over 5 feet immunologic disorders of malignancy
MODIFIED SCHWARTZ FORMULA
female IBW 45.5 kng + 2.3 kg for each inch over 5 feet NOTES OF GFR
In 2009, Schwartz et.al, further modified their formula to estimate GFR more
Side notes: Classical formula for getting the GFR, we have the Cockroft-Gault accurately in children. The new formula is very similar to the MDRD equation GFR is determined not only by the number of functioning nephrons
1
Formula in which: in that it uses multiple exponential function. Use of the formula requires but also by the functional capacity of these nephrons.
140 minus age times the ideal body weight (IBW) divided by 72 times the knowing the values of: 2 its value does not lie in the detection of early renal disease
serum creatinine and multiply with 0.5 if the patient is female (different on o serum-creatinine o BUN usage:
the slide tho). o height o serum cystatin C (new substance) ▪ to determine the extent of nephron damage in known cases of
renal disease
MDRD FORMULA ▪ to monitor the effectiveness of treatment designed to prevent
3
further nephron damage
The MDRD formula is quite complicated. In the MDRD study, Levi and co- ▪ to determine the feasibility of administering medications, which
workers measure GFR by iodothalamine to derive a formula for estimating can build up to dangerous blood levels if the GFR is markedly
GFR using 6 variables: reduced
o age o serum-creatinine TUBULAR REABSORPTION TESTS
o sex o race This is quite complicated. We have several variables. You can arrive at a more
determine the ability of tubule to reabsorb
o serum-urea nitrogen o serum-albumin concentration accurate GFR using this modified Schwartz formula in children.
CONCENTRATION TESTS
COUNAHAN-BARRETT FORMULA ▪ tests to determine the ability of the tubules to reabsorb the essential salts
and water that have been non-selectively filtered by the glomerulus
▪ Fishberg and Mosenthal concentration tests
patients were deprived of fluids for 24 hours before
Fishberg test
measuring specific gravity
compared the volume and specific gravity of day
SIMPLIFIED MDRD FORMULA
Mosenthal test and night urine samples to evaluate concentrating
In the same time in 2000, the same investigators, Levi et-al, produced a
ability
simplified MDRD formula based on the 4 variables
o age o serum-creatinine OSMOLALITY
o sex o race ▪ osmolality measures only the number of particles in a solution, whereas
specific gravity is influence by the number and density (molecular weight)
of the particles
▪ osmolality is measured by these two machines:
o Freezing Point Osmometers o Vapor Pressure Osmometers
always amazed by how kind this girl is, GENEVIEVE RUSTE, thank
you so much for helping me on this one, I truly appreciate it, SML ♡
AIRAH M.
 ADH CHALLENGE REFERENCES
ADH Challenge is applicable in tubular reabsorption test. It used to
differentiate neurogenic and nephrogenic diabetes insipidus. So, this is how
your ADH challenge is performed.

CLINICAL SIGNIFICANCE
1 evaluating renal concentrating ability
2 monitoring the course of renal disease
3 monitoring fluid and electrolyte therapy
establishing the differential diagnosis of hypernatremia and
4
hyponatremia
5 evaluating the secretion of and renal response to ADH
Reference serum osmolality values: 275 – 300 mOsm

TUBULAR SECRETION AND RENAL BLOOD FLOW TESTS

measure tubular secretion of nonfiltered substances and renal blood flow
P-AMINOHIPPURIC ACID (PAH) TEST
▪ this nontoxic substance is loosely bound to plasma proteins, which
permits its complete removal as the blood passes through the peritubular
capillaries
▪ except for a small amount of PAH contained in plasma that does not come
in contact with functional renal tissue, all the plasma PAH is secreted by
the proximal convoluted tubule
▪ therefore, the volume of plasma flowing through the kidneys
determines the amount of PAH excreted in the urine

always amazed by how kind this girl is, GENEVIEVE RUSTE, thank
you so much for helping me on this one, I truly appreciate it, SML ♡
AIRAH M.
 ANALYSIS OF URINE AND BODY FLUIDS HISTOLOGY: STOMACH
TOPIC 07: GASTRIC AND DUODENAL SECRETIONS
Objectives:
▪ Discuss briefly the anatomy and histology of the stomach and duodenum
▪ Discuss the different gastric and duodenal secretions with their functions
▪ To briefly discuss few gastric and pancreatic disorders
STOMACH AND ITS CONTENTS
STOMACH AND DUODENUM ANATOMY

This is a picture of an oxyntic or a stomach gland and a pyloric gland. The upper
cardiac zone includes the fundus and contains surface epithelial cells and For the inhibitors, we have:
endocrine secreting? cells. The body of the stomach contains mucus secreting INHIBITORS OF GASTRIC SECRETION
cells and the parietal oxyntic cells. These are your surface mucous cells, parietal high gastric acidity
cells, and mucous neck cells. Also found on your oxyntic glands are your: 1
▪ decreases release of gastrin
enterochromaffin-like cells produces histamine GIP: gastric inhibitory polypeptide
D cells produces somatostatin 2 ▪ K cells, duodenum, and jejunum
chief cells produces pepsinogen ▪ secondary to fat, glucose, and amino acid
enterochromaffin cells produces ANP VIP: vasoactive intestinal polypeptide
Schematic drawing of the stomach with major zones.
3 ▪ H cells
For your pyloric gland, which is seen in the pyloric zone, are divided into
The stomach is located at the left side of the upper abdomen and the stomach ▪ inhibits gastric secretion, gastric release, and motility
antrum and pyloric canal sphincter. These pyloric glands secrete mucus,
receives food from the esophagus. As food reaches the esophagus, it enters First is the high gastric acidity. In the presence of high acid environment or low
pepsinogen, serotonin, and gastrin, except HCl. Please remember that cells in
the stomach through a muscular valve called the lower esophageal sphincter. pH environment, it decreases the release of gastrin. Another is the presence of
all three zones of the stomach produces pepsinogen. They also produce HCl
The stomach secretes acids and enzymes that digest the food. The stomach is GIP which is produced by the duodenum and jejunum. This is produced
except in the glands found on the pyloric zone.
divided into three major zones: secondary to the presence of fat, glucose, and acid. Lastly, the presence of VIP
PHYSIOLOGY OF GASTRIC SECRETION
cardiac zone directly adjacent to the esophagus which is produced by the H cells. This inhibits gastric secretion, gastric release,
The stimuli for gastric secretion can be due to:
body of the stomach the major component and stomach’s motility.
pyloric zone immediately adjacent to the small intestine STIMULI FOR GASTRIC SECRETION
neurogenic impulses from the brain via vagal nerves (e.g. sight, smell, GASTRIC SECRETIONS
1
or anticipation of food) The mucus membrane of the stomach has tubular gastric glands that secrete:
2 distention of the stomach with food or fluid ▪ from the parietal cells of the cardiac zone and the
contact or protein breakdown products: secretagogues with gastric hydrochloric body of the stomach
3
mucosa acid ▪ converts pepsinogen to pepsin
4 gastrin: most potent stimulus to gastric secretion ▪ hydrolyze polypeptides and disaccharides directly
1. CEPHALIC PHASE: For the stimulation of gastric secretion, it can be due to ▪ from the goblet cells and the mucus glands
the neurogenic impulses from the brain (cephalic phase) which is via the vagus ▪ main function: protective to stomach wall
mucus
nerve and it is usually associated with the sight, smell, or anticipation of food. ▪ complex mixture of mucoproteins and
2. GASTRIC PHASE: Next is the gastric phase wherein the gastric secretion is mucopolysaccharides
stimulated by the distention of the stomach, secondary to the filling up. As we ▪ from the parietal cells
intrinsic factor
▪ is required for Vit. B12 absorption
eat, food goes down to the stomach (so there is distention), and there is
gastric secretion contains all the electrolytes found in
contact of protein breakdown products (e.g. secretagogues with gastric
electrolytes other body fluids in a combined osmolar concentration
Efficient digestion of food and absorption of nutrients are the result of the mucosa). ??? as well as gastrin and histamine stimulation is a ?? stimuli for the
equal to or slightly greater than plasma
correlative function that occurs in the GIT. Ordination and regulation of these production of gastric secretions. Also, gastrin is the most potent stimulus for composed of: (elaborated on Table A)
functions depends on the four bones that inhibit or stimulate gastric and gastric secretion. digestive ▪ pepsinogen
duodenal secretions. They usually contain HCl, bile acids, bicarbonate, and 3. INTESTINAL PHASE enzymes ▪ pepsin
other digestive enzymes. ▪ gastric lipase
dearest ANJELA SOLIS never leaving my side,
grateful to you have you b, thank you ♡
AIRAH M.
 ▪ LDH ▪ time interval is determined from titratable acidity and INSULIN HYPOGLYCEMIA TEST (HOLLANDER’S TEST) ④
non-digestive ▪ aspartate amino transferase total acid volume ▪ hypoglycemina: administration of insulin
enzymes ▪ isocitric dehydrogenase output ▪ specimen is obtained as 15-min collection for 1 hour o principle: INSULIN is a potent stimulus to gastric secretion
▪ alkaline phosphatase ▪ N: 0.6 mEq of acid; 10-100 ml o in the presence of insulin, there is an increase of the production of the
▪ usually secondary to gastric response to IV secretin gastric contents
Table A. DIGESTIVE ENZYMES post
stimulation o this stimulus is transmitted by the vagus nerve and abolished by
the most important enzyme found in the gastric secretion stimulation
▪ after 1hr B: 1-40 mEq of acid; 40-350 mL (less in women vagotomy
is the pepsinogen gastric
and older persons)
▪ biologically inactive proenzyme of PEPSIN acidity ▪ blood glucose <50 mg/dL (after insulin administration)
▪ the hourly acid output: maximum acid output (MAO)
▪ produced by chief cells ▪ assess completeness of vagotomy
▪ remains stable in the blood GASTRIC FLUID o COMPLETE if acid output in 2hr post-insulin is less than the greater of
pepsinogen TYPICAL REFERENCE INTERVALS
▪ converted into active form by gastric acid (< pH 5) 2-hour basal output
▪ usually destroyed by alkaline pH COMPONENT CONVENTIONAL RECOMMENDED o INCOMPLETE if acid output in 2hr post-insulin exceeds than the greater
FACTOR
▪ 2 distinct forms with ratio (4:1) UNITS SI UNITS of 2hr basal period by 0.5 mEq
o Pepsinogen I (PG I) fasting residual
20-100 ml 0.001 0.02-0.10 L ⑤ TESTING FOR H. PYLORI
o Pepsinogen II/Gastricsin (Pepsinogen C) volume
▪ active form of pepsinogen pH <2 1 <2 ▪ H. pylori
▪ the major component of gastric fluid basal acid o urease producing, gram (-) bacilli
0-6 mEq/hr 1 0-6 mmol/hr
▪ responsible for the hydrolysis of proteins to output (BAO)* ▪ most important cause of PUD (peptic ulcer disease) and is also associated
pepsin polypeptides maximum acid with other GI pathology
▪ primary function: digestion of food output (MAO) ▪ most common tests include:
▪ active form is easily inactivated in blood (in contrast with (after 5-40 mEq/hr 1 5-40 mmol/hr o biopsy
pepsinogen which is more stable in blood) histamine o breath tests
▪ hydrolyze triglycerides (long-chain fatty acid esters of stimulation) o fecal antigen detection
gastric
glycerol BAO/MAO
lipase <0.4 1 <0.4 A RAPID UREASE TESTING
▪ helps in the digestion of food ratio
* varies between male and female and ages ▪ sample: antral biopsy (used in basic procedure because we use ??
TESTS FOR GASTRIC SECRETION tissue from the antrum)
③ GASTRIN ▪ agar gel or reaction strip with urea (pH-sensitive color changing
① GASTRIC ANALYSIS
▪ produced by the antral G cells at pH 5 to 7 indicator)
▪ nowadays, this is usually not done
▪ regulates acid secretion and growth of gastric mucosa ▪ POSITIVE: suggests urease activity (presence of H. pylori)
▪ is used clinically mainly to:
o detect hypersecretion characteristic of the Zöllinger-Ellison syndrome ▪ neutral environment because it helps protect the stomach from over-
o evaluate pernicious anemia acidification
o aid in the determination of the type of surgical procedure required for ▪ main function: gastric secretion in aiding digestion of food
ulcer treatment ▪ N: 50-150 pg/mL
▪ the collection of gastric secretion is usually done by intubating the patient ▪ usually seen in abnormal stats such as:
and suctioning of the gastric content o ZE (Zollinger–Ellison) syndrome
o PUD
A VOLUME AND APPEARANCE o pernicious anemia
▪ the gastric content, when empty, is translucent, pale gray, slightly ▪ ZE syndrome
viscous B UREA BREATH TEST
o produces abnormally high levels of gastrin: >1000-400,000 pg/mL
▪ faintly acrid odor ▪ PRINCIPLE: presence of urease in H. pylori
o usually secondary to a gastrin-producing tumor
▪ after suctioning of the content, there are usually residual or basal ▪ labeled urea and CO2 is ingested, detected, and quantified in expired
▪ PUD: normal, pernicious anemia breath
specimens that remains in the stomach
o there is considerable <75 ml LABORATORY DETERMINATION
o may contain flacks of blood or are green, brown, or yellow A RIA (radioimmonassay) or EIA (enzyme-linked immunosorbent assay)
o sometimes bloody because intubation of the patient is usually an confirmation of gastrin secreting tumors
invasive procedure (blood will mix with the gastric content) ▪ fasting for 12 hours
o REMEMBER: food particles present in a well-fasted residual gastric ▪ processed immediately or frozen – gastrin hormone is unstable in
specimen are abnormal! serum
MEASURING GASTRIC ACID ② B SECRETIN STIMULATION TEST
▪ ability of the stomach to secrete against a hydrogen ion gradient (ability of confirmation of equivocal gastrin results in diagnosis of gastrin-secreting
the stomach to produce acid and acid environment) tumor
▪ PRINCIPLE: this ability is determined by measuring the pH ▪ N: drop in gastrin after secretin infusion
▪ volume, pH, and tritratable acidity and calculated acid output are also ▪ in patients with gastrin secreting tumor: this is usually not seen; you
measured when measuring gastric acid will still see high levels of gastrin after injection or administration of
secretin (which inhibits gastrin production)
dearest ANJELA SOLIS never leaving my side,
grateful to you have you b, thank you ♡
AIRAH M.
 C ANTIBODIES vs H. pylori Endocrine cell hyperplasia (stimulate parietal cell growth and gastric (usually seen on the shaded blue on the image)
8
▪ enzyme immunoassay acid secretion) also called Major Duodenal Papilla
▪ used to screen for H. pylori Zollinger-Ellison syndrome (PUD of stomach, duodenum, and one of the major secretions of the duodenum which
▪ while UREA BREATH used to confirm eradication after treatment 9 jejunum): remember the triad of peptic ulceration, hyperchloridia, Ampulla Vater comes from the pancreas
and gastrin producing tumor usually drains pancreatic contents into the duodenum
DISORDERS
10 Viral infections (Cytomegalovirus (CMV) and Herpes simplex virus) it is part of the jejunum
① ZOLLINGER-ELLISION SYNDROME
The distinction between duodenum and jejenum are arbitrary. Histologically
▪ TRIAD: peptic ulceration, hyperchlorhydria, and non-B islet cell tumor
speaking, it consists of the typical three layers common to hollow organs of
(gastrinoma)
the GIT, but the Brunner’s glands is the main characteristic of the duodenum.
▪ 25% of ZE occur in MEN I
Remember the three components of the duodenal contents which are:
gastric secretion (sucrose and glucose, nani?? not sure), pancreatic exocrine
DIAGNOSIS OF GASTRINOMA
secretion, and bile.
▪ gastrinoma: most common cause is a gastrin secreting tumor that
causes hyperchlorhydria and eventual peptic ulceration HISTOLOGY: SMALL INTESTINE
▪ basis: presence of gastrin levels (most common cause) On the top are the normal gastric epithelial cells/gastric glands. And the one
▪ secretin is an inhibitor of plasmid secretion below is the ulceration (which is the defect) (ulceration filled with
o if you have administration of secretin; normal response of the inflammatory infiltrates).
body is to decrease the gastric levels, since an abnormal source
of gastrin is seen in this patient, usually in tumor, even with DUODENUM AND ITS CONTENTS
secretin administration there is no decrease in gastrin production composed by:
1 ZE is HIGHLY SUGGESTIVE if: ▪ Exocrine Pancreatic Secretions
▪ high gastrin levels >150 ng/L ▪ Bile
▪ gastric pH <3 ▪ Intestinal Secretion (Succus entericus) mixed with gastric secretion
2 FOR EQUIVOCAL The pancreatic secretion contains multiple enzymes for digesting all of the
▪ does not confirm or lie between the criteria, administration is usually three major types of food: proteins, carbohydrates, and fats. It also contains
done large quantities of bicarbonate irons which play an important role in
▪ secretin (2 U/kg in 10 ml 0.9% NaCl) given IV – serial gastrin neutralizing the acidity of the chyme emptied by the stomach in the
measurement duodenum. This provides a large quantity of alkali in the pancreatic tubes
▪ positive test: gastrin >200 ng/L after 15 minutes which helps to neutralize the HCl emptied into the duodenum from the
3 other modalities used: LOCALIZATION OF TUMOR This is the histology of the small intestine. It is similar throughout the small
stomach.
these methods are used before imaging study to localize the tumor: intestine. There is a villus projection (blue lines) of the epithelial cells that are
▪ octreotide Ulcerations from the stomach (PUD) can be seen as duodenal ulcer disease. lined with columnar epithelial cells. The mucosa, muscularized mucosae, and
▪ somatostatin (STT) receptor: current modality of choice This is usually secondary to an increased acid production that is decreased by submucosa can be seen. What is unique to the duodenum is the presence of
bicarbonate production. the Brunner’s glands found in the submucosa. You will know that you are in
PEPTIC ULCER DISEASE ②
Peptic ulcer disease (PUD) refers to chronic mucosal ulceration Bicarbonate neutralizes the acidity of the stomach contents that is passed the duodenum because of the presence of Brunner’s glands.
▪ affects the duodenum and stomach down as you digest food content. small finger-like projections formed by the internal
▪ pathophysiology: increased gastric acid secretion and decreased duodenal surface
bicarbonate secretion ANATOMY: STOMACH AND DUODENUM aids to increase the surface area of the small intestine
▪ remember: what helps in the neutralization of the stomach is the villus projection of the small intestine helps increase
formation of bicarbonate secretion; it helps the stomach’s acidity (pH the absorptive capacity/absorptive surface of the
villi
environment) to be in check small intestine
▪ gross and microscopic: side note: if these are laid out in a flat surface, it
o mucosal defect in the gastric mucosa usually covers the absorptive surface of the small
o ulcerations demonstrate predominantly neutrophilic inflammatory intestine will be about 250 m2 similar to that area of a
infiltrate tennis court

RISK FACTORS FOR PUD SMALL INTESTINE SECRETIONS

1 Helicobacter pylori infection a C-shaped organ 1 MUCUS-SECRETING GOBLET CELLS/BRUNNER’S GLAND
2 Cigarette Use (synergizes with H. pylori for gastric PUD) is the first of the 3 parts of the small intestine that ▪ thick, alkaline mucus respond
duodenum 2 ENZYMES IN THE MICROVILLI
3 Chronic Obstructive Pulmonary Disease receives the partially digested food from the stomach
4 Use of Illicit Drugs that decrease mucousal blood flow (e.g. cocaine) and begins with absorption of nutrients peptidase break down peptides into amino acid
5 Use of NSAIDs (potentiated by corticosteroids) directly attached to the superior region of the sucrase, maltase, break down disaccharides to
6 Alcoholic Cirrhosis (primarily duodenal PUD) pyloric zone of duodenum lactase monosaccharides
7 Physiological Stress (can increase gastric acid secretion) the stomach is divided/guarded by the pyloric sphincter lipase breakdown fats into fatty acids and glycerol
also closely related to the head of the pancreas enterokinase converts trypsinogen to trypsin
dearest ANJELA SOLIS never leaving my side,
grateful to you have you b, thank you ♡
AIRAH M.
 somatostatin inhibits acid secretion by stomach 2 BILE ▪ stimulation with secretin & pancreozymin
inhibits gastric glands, stimulates pancreas ▪ approximately 500-1000 ml. Enters the duodenum daily ▪ includes measurement of the volume & quantitative analysis of
CCK/cholecystokinin to release enzymes that acids in the release ▪ alkaline (pH 7.0-8.5) pancreatic juice for HCO3 & enzyme content specifically Amylase
of bile by the gall bladder ▪ yellow to brown/ green activity
▪ contains bile salts (chiefly sodium glycholate & taurocholate) also ▪ remember: secretin & pancreozymin are responsible for stimulate
stimulates the pancreas to release
secretin includes bilirubin, pigments, cholesterol, phospholipids, various bile exocrine secretion in the duodenum
bicarbonate iron
inorganic salts) D AUGMENTED SECRETIN TEST
DUODENAL CONTENTS ▪ alkaline phosphatase ▪ of particular value since augmented stimulation enhance secretory
1 PANCREATIC EXOCRINE SECRETION o only enzyme present deficiencies as in inflammation & CA/malignancy
▪ major contributor to duodenal contents o no function in digestion
E MISCELLANEOUS TESTS
▪ exceeds 1,500 ml per day in normal adult 3 SUCCUS ENTERICUS
▪ ▪ leukocytosis & WBC count sometimes reaching 30,000/ cu mm. In
▪ colorless, clean, non-viscid, alkaline solution (pH ≈ 8) unknown daily volume
▪ acute pancreatitis
▪ main function is to neutralize the acid chyme/acid fluid that is passed contains a variety of digestive enzymes- break down ingested food
▪ decreasing serum calcium: points to a more serious form of
down in the stomach TESTS FOR DUODENAL SECRETION pancreatitis
enzymes or their precursors 1 EXAMINATION OF STIMULATED BILE ▪ hyperbilirubinemia & other liver function tests: with abnormal
▪ trypsinogen ▪ elastase ▪ duodenal intubation is performed results
1 to 2%
▪ chymotrypsinogen ▪ collagenase ▪ following aspiration of the residual content ▪ test for malabsorption syndrome (caused by inadequacy of
Organic
▪ amylase ▪ leucine o 50 to 100 ml of sterile 25% saturated MgSO4 is introduced through pancreatic secretion resulting from chronic pancreatitis or
Material
▪ lipase aminopeptidase the duodenal tube pancreatic CA)
▪ lecithinase ▪ various esterases ▪ after a minute, the MgSO4 & duodenal contents are aspirated o Serum carotenoid level
▪ Sodium: major cation o the collections are pooled and discarded until a yellow bile first o Glucose tolerance test
1% ▪ Bicarbonate: major anion appears (requiring about 2-10 ml) o C-labeled triglyceride breath test
Inorganic ▪ Na+ & K+ are present in the same concentration in ▪ three fractions of the bile are subsequently collected in separate o Starch tolerance test
Material serum while Ca++ & Mg+ are present in lower containers o 3-day fecal fat determination
concentration the first to appear which is light yellow & watery (5 to o D-xylose test (useful to distinguish malabsorption syndrome
A Bile cause by pancreatic disorders from that of intestinal disorders)
20 ml)
STIMULI TO PANCREATIC EXOCRINE SECRETION ▪ tests for cystic fibrosis (mucoviscidosis) of the pancreas
appears 1 to 3 minutes after aspiration, viscid deep
for the regulation of pancreatic secretion B Bile
yellow brown bile (30-75 ml) DISORDERS: CYSTIC FIBROSIS
A VAGAL STIMULUS C Bile pale yellow and watery Characterized by abnormal secretion by the various exocrine glands of the
releases in enzymes in the acini and eventually the duodenum
ABSENCE OF “B” BILE: body including the pancreas, salivary glands, peritracheal & peribronchial
B HORMONAL STIMULI (2 subst. Secreted by duodenal mucosa) glands. The laboratory diagnosis depends largely of the increase of Na and Cl
▪ advanced Cholecystitis [inflammation/infection of the gallbladder]
▪ Secretin: HCl stimulates its release and results in a copious flow of ▪ Choleithtiasis with obstruction the cystic duct (secondary to in the sweat.
pancreatic secretion that is low in enzyme content & high in HCO 3 obstruction by a stone or tumor)
▪ Pancreozymin (Cholecystokinnin): stimulate the pancreas to SWEAT TESTS: (primary test)
▪ absence of gallbladder secondary to Cholecystectomy (there is no bile
secrete enzymes which aids in the digestion of food PRINCIPLE: Pilocarpine is iontophoresed into the skin to stimulate locally-
coming from the gall bladder either by inflammation, obstruction of the
C GASTRIN increased sweat gland secretion. The resulting sweat is absorbed by filter
duct system, or absence of gall bladder)
weak stimulus but may cause pancreatic exocrine secretion paper or gauze pad, weighed, diluted with water and analyzed for Na & Cl
content.
SANDY BILE If there is an increase in the Na and Cl content in the sweat it means that
▪ deep red-brown/black bile
there is a possibility for cystic fibrosis [since this is not the only test that will
▪ cholesterol crystals or calcium
diagnose cystic fibrosis}
bilirubinate
▪ highly suggestive of cholelithiasis or REFERENCES
calculus in the biliary tract

2 TEST FOR PANCREATIC SECRETION

A SERUM AMYLASE
▪ for the diagnosis of acute & chronic pancreatitis (increased levels of
pancreatic enzymes in the blood and urine)
▪ remember serum amylase rises 2-3 hours after the onset of
pancreatitis and peaks at 48 hours and returns to normal in 3-5 days
B immunoreactive TRYPSIN det. in plasma by RIA
C SECRETIN TEST
▪ exocrine secretory function
dearest ANJELA SOLIS never leaving my side,
grateful to you have you b, thank you ♡
AIRAH M.
 Table A. MODE OF ACTION
MODE OF READING
TESTS PRINCIPLE SENSITIVITY SPECIMEN REAGENTS DETECTS INTERPRETATION FALSE HIGH FALSE LOW
MEASUREMENT TIME
PROTEINS
mg AER in
results
24-hour albumin/24hrs ug/min
quantitative albumin
for microalbuminuria urine 30-300mg
measurement significant 20-
specimen albumin/24
values 200ug/min
hours
for orthostatic first morning second specimen
proteinuria NEGATIVE POSITIVE
highly buffered alkaline
urine
MULTISTIX: prolonged contact with
MULTISTIX: 15-30 mg/dL
Tetrabromophenol blue urine
albumin CHON other than
contamination with
reagent strip method protein error of indicators colorimetric 60s albumin albumin
CHEMSTRIP: quaternary ammonium
CHEMSTRIP: microalbuminuria
3’,3’’,5’,5’’-tetrachlorophenol, 3,4,5,6- compounds (QAC),
6 mg/dL albumin
tetrabomosulfophthalein detergents, & antiseptics
spx with high sp.gr.
highly pigmented urine4
REPORTING OF SSA TURBIDITY
PROTEIN
GRADE TURBIDITY RANGE
(mg/dL)
no increase in
Negative less than 6
turbidity
noticeable
Trace 6-30
turbidity
sulfosalicylic acid centrifuged all forms distinct turbidity,
cold precipitation 1+ 30-100
precipitation test urine of protein no granulation
turbidity,
2+ granulation, no 100-200
flocculation
turbidity,
3+ granulation, 200-400
flocculation
greater
4+ clumps of protein
than 400
gold-labeled antihuman albumin-enzyme
first (𝞫-galactosidase) conjugate
micral test Enzyme Immunoassay : 0-10 mg/dL 1 min albumin
morning human serum albumin
chlorophenol red galactoside,
darker NEGATIVE
less than
bottom
1.2 mg/dL
band
first blue latex particles coated with
immunodip immunochromographic albumin equal band BORDERLINE 1.2 – 1.8
morning antihuman albumin Ab
colors mg/dL
darker top POSITIVE 2.0 – 8.0
band mg/dL
bis(3’3’’-diiodo-4’4’’-dihydroxy-5’5’’-
a. visibly bloody urine
8-15 mg/dL (80-150 dinitrophenyl)-3,4,5,6-
albumin ratio dye-binding reaction b. abnormally colored urine
mg/L) tetrabromosulphonphthalein (DIDNTB)

a. visibly bloody
urine
a. CuSO4 b. abnormally
b. 3,3’,5,5’-tetramethyl-benzidine colored urine
pseudoperoxidase activity of normal value: 10-300 (TMB) c. gastric acid-
creatinine ratio
copper-creatine complexes mg/dL c. diisopropyl benzene reducing
dihydroxyperoxide (DBDH) medication
cimetidine
(Tagamet)

GLUCOSE
strong reducing
agent (e.g.
▪ glucose oxidase ascorbic acid)
▪ peroxidase increased ketone,
REAGENT STRIP POSITIVE NEGATIVE contamination with
double sequential enzymatic ▪ chromogen – KI (Multistix) or S.G.
(GLUCOSE OXIDASE glucose Multistix green brown peroxide and strong
reaction tetramethylbenzidine (Chemstrip) decreased
REACTION) Chemstrip yellow green oxidizing agents
▪ buffer temperature
bacterial
degradation of
glucose

AIRAH M.
 Benedict’s solution
▪ reducing sugars (galactose, lactose, fructose,
▪ copper sulfate
CuSO4 is reduced to Cu2O by maltose, pentoses)
▪ sodium carbonate
CU REDUCTION TEST reducing substances in the ▪ ascorbic acid
200mg/dL ▪ sodium citrate: buffer
(CLINITEST) presence of alkali and heat ▪ certain drug metabolites
▪ sodium hydroxide (added
▪ antibiotics (cephalosporins)
ingredient in the Clinitest Tablet)

KETONES
Sodium Nitroprusside
Decomposition negative no ring/brown ring is present
Rothera’s reagent (overlay with 1ml
Nitropusside in alkaline faint pinkish purple ring
conc. NH4 hydroxide) (+)
medium reacts with a ketone appearing slowly
ROTHERA’S TEST ▪ 7.5 g sodium nitroprusside
group to form a purple ring. It (++) narrow dark purple ring
▪ 200 g ammonium sulfate
is given by acetone and wide dark purple ring
acetoacetate, but not by beta (+++)
appearing very rapidly
hydroxy butyric acid.
▪ highly pigmented red
Multistix:
urine
5-10 mg/dl or 0.25-0.5
▪ phthalein dyes
Sodium Nitroprusside mmol/L of acetoacetic test area remains tan-colored
negative ▪ Levodopa
REAGENT STRIP Decomposition acid ▪ 7.1% (w/w) sodium nitroprusside after 40 secs improperly preserved
(medication for
METHOD ▪ 92.9% (w/w) buffer trace to test area turns pink to purple specimen
Parkinson’s disease)
Chemstrip (+++) within 40 secs ▪ medication
9 mg/dl acetoacetic acid
containing free
& 70 mg/dl acetone
sulfhydryl groups
A purplish color is given by
acetoacetate. On boiling,
acetoacetate is converted to
acetone and does not give this FeCl3 + acetoacetate/ethyacetoacetate → purple color,
test positive. This test is only diacetate/acetoacetic acid
GERHARDT’S FERRIC
given by acetoacetate and not Concentrated Nitric Acid
CHLORIDE TEST
by beta hydroxy butyric acid 1.0ml of 10% FECl3 solution → reddish color is
directly. This test is also formed after the mixture
sensitive to
acetoacetic/diacetic acid.

sensitive to beta-
acetic acid and hydrogen peroxide
HART’S TEST hydroxybutyric acid formation of red ring

▪ large amounts of
▪ the test does not
Levodopa and other ▪ volatilization of
measure beta-
medicines acetone and
URINE KETONES hydroxybutyric acid
containing breakdown of
CONFIRMATORY and is only slightly
sulfhydryl groups acetoacetic acid
TEST sensitive to
may produce by bacteria
acetone when
atypical color
glycine is present
reactions
▪ sodium nitroprusside
ACETEST TABLE diacetic
▪ glycine
(UNDER THE ONE acid and
▪ disodium phosphate
ABOVE) acetone
▪ lactose (color enhancer)
▪ acetone or acetoacetate
Nitroprusside test
reacts with nitroprusside to form violet color
(STILL UNDER)
form violet color
BLOOD
pigment is precipitated
(+) hemoglobin supernatant shows normal
AMMONIUM color
SULFATE SOLUBILITY pigment is precipitated
TEST OR supernatant is red/red-
BLONDHEIM’S TEST presumptive of
brown
hemoglobin
proceed for protein
electrophoresis
supernatant retains the red
Larger hemoglobin molecules color
(+) myoglobin
PRECIPITATION TEST are precipitated by ammonium supernatant gives a positive
(UNDER ABOVE) sulfate and myoglobin remains reagent strip test for blood
in the supernatant. hemoglobin is found in the
(+) hemoglobin
red precipitate

AIRAH M.
 supernatant tests negative
for blood
for hemosiderin Prussian Blue Reagent (made fresh)
▪ high specific gravity
MULTISTIX
▪ presence of
5 to 20
crenated cells
MULTISTIX RBC’s/ml
▪ formalin
diisopropylbenzene dihydroperoxide 0.015 – 0.062 ▪ strong oxidizing
▪ captopril
tetramethylbenzidine mg/dL hgb agent/bleach
pseudoperoxidase activity of ▪ high
REAGENT STRIP ▪ bacterial peroxidases
hemoglobin concentrations of
CHEMSTRIP CHEMSTRIP menstrual
nitrite
dimethyldihydroperoxy-hexane 5 RBCs/ml contamination
▪ ascorbic acid
tetramethylbenzidine hgb
greater than
corresponding
25mg/dl
to 10 RBC’s /mL
unmixed specimen
BILIRUBIN
▪ oxidation of bilirubin to
biliverdin by the ferric
chloride in Fouchet’s
reagent
▪ BaCl reacts with sulfate in
urine to form barium
sulfate. If bilirubin is
present in urine, it
adheres to precipitate and
is detected by oxidation to
FOUCHET’S REAGENT:
form biliverdin (green)
▪ trichloroacetic acid
with FeCl3 in the presence
▪ ferric chloride solution no green color on the filter
of trichloroacetic acid. negative
FOUCHET’S TEST (not ▪ barium chloride solution paper
Nitric acid oxidizes
a sensitive test) green color appear on the filter
bilirubin to biliverdin positive
or HARISSON’S SPOT FOUCHET’S REAGENT: paper
giving different colors
TEST ▪ trichloroacetic acid (25 gms)
from green to violet.
▪ 10% ferric chloride solution (10ml)
▪ distilled water (100ml)
Barium chloride added to urine
combines with sulfate radicals
in urine to form precipitate of
barium phosphate. If bile
pigments are present in urine,
they will adhere to these large
molecules. Ferric chloride
present in Fouchet reagent
then oxidizes yellow bilirubin
in presence of trichloroacetic
acid to green biliverdin.
MULTISTIX ▪ specimen exposure
▪ highly pigmented
2,4-dichloroaniline diazonium salt to light
Diazotization or azo-coupling of urines
M: 0.4 - 0.8 mg/dl ▪ ascorbic acid
REAGENT STRIP bilirubin with diazonium salt in ▪ phenazopyridine
greater than 25
METHOD an acid medium to form azo CHEMSTRIP ▪ indican (intestinal
C: 0.5 mg/dl mg/dl
dye disorders)
2,6-dichlorobenzene-diazonium high concentration of
salt metabolites of Iodine
nitrite
REAGENTS: ▪ specimen exposure
▪ p-nitrobenzene-diazonium-p- to light
diazotization or azo-coupling of
toluenesulfonate no false positive: ▪ ascorbic acid
bilirubin with diazonium salt in
ICTOTEST 0.05-0.1 mg/dl ▪ SSA (sulfosalicylic acid) interfering substances greater than 25
an acid medium to form azo dye
▪ sodium carbonate are corrected mg/dl
▪ boric acid high concentration of
nitrite
Nitric acid oxidizes Bilirubin to
Biliverdin giving different colors
concentrated nitric acid
from green to violet. In other the junction of two liquids, various colored rings
GMELIN’S TEST
words, it is the oxidation of (green, blue, red, violet etc.) will be formed
bilirubin by nitric acid.

UROBILINOGEN
▪ urobilinogen reacts with
dimethylamino-
▪ Ehrlich’s reagent
benzaldehyde in appearance of pink color in
o P-dimetyhlaminodenzaldehyde
choloform to form a pink the chloroform layer
o conc. hydrochloric acid (+)
ERLICH’S TEST coloured aldehyde colour is easily detected
o distilled water urobilinogen
complex when view from the top of
▪ saturate sodium acetate
▪ based on Ehrlich the test tube
▪ chloroform
aldehyde reaction

AIRAH M.
 The urine is a yellowish-brown
or greenish-yellow color and
bilirubin is suspected, shake
the urine. If a yellow or
greenish-yellow foam
FOAM TEST
develops, then bilirubin is most
likely present. Bilirubin alters
the surface tension of urine and
foam will develop after
shaking.
MULTISTIX
Ehrlich’s aldehyde ▪ M: old specimens
reaction preservation in
▪ M: porphobilinogen
formalin
▪ indican
M: p-dimethyl-aminobenaaldehyde
CHEMSTRIP M: 0.4 - 0.8 mg/dl ▪ p-aminosalicylic acid
C:
REAGENT TEST STRIPS azo-coupling (diazo) ▪ sulfonamides
C: 4-methoxybenzene- diazonium- ▪ old specimen
reaction using 4- C: 0.5 mg/dl highly pigmented urine
tetrafluoroborate ▪ preservation in
methoxybenzene- formalin
C: high pigmented urine
tetrafluoroborate with high concentration of
urobilinogen forming red urine
azodye

pink color in the

(+) urobilinogen
chloroform layer
(+) pink coloration of
porphobilinogen aqueous portion

▪ P-dimetyhlaminodenzaldehyde
solubility difference of 2nd set
▪ conc. hydrochloric acid
porphobilinogen and cherry red complex is
WATSON-SCHWARTZ ▪ distilled water (+) urobilinogen extractable with n-
urobilinogen In Ehrlich’s
TEST ▪ additional: saturated sodium acetate butanol
aldehyde reagent
and N butanol cherry red complex is
(+)
not extractable with n-
porphobilinogen
butanol & chloroform
no cherry red complex is
negative formed with Ehrlich’s
aldehyde reagent
▪ detects about 20 – 100 mg/L of porphobilinogen, ▪ urorosein: urinary
and urobilinogen in amounts up to 200 mg/L does pigment related to
HOESCH’S TEST inverse Ehrlich’s reaction
not give a positive result (red color) indoleacetic acid
a yellow color may be caused by urea dark pigmented urine
NITRATE
▪ non-reductase
MULTISTIX containing bacteria
p-arsanilic acid ▪ insufficient contact
Greiss Reaction: nitrite at Tetrahydobenzo(h)- time between
acidic pH reacts with aromatic quinoline-3-ol ▪ the diazonium salt reacts with sulfanilic acid and ▪ improperly preserved bacteria & urinary
URINE NITRATE
acetic acid to produce a pink azo dye specimen nitrate
TESTING amine to form a diazonium
highly pigmented urine ▪ presence of
compound CHEMSTRIP antibiotics
Sulfanilamide, ▪ high concentration
Hydroxytetrahydro benzoquinoline of ascorbic acid
high specific gravity
LEUCOCYTE
▪ strong oxidizing
action of leucocyte esterase to MULTISTIX CHEMSTRIP
agents ▪ high conc. protein,
catalyze the hydrolysis of an ▪ derivatized pyrrole amino
URINE LEUKOCYTE indoxylcarbonic acid ester and ▪ formalin glucose, oxalic acid,
acid ester to an aromatic reagent acid ester
ESTERASE diazonium salt ▪ highly pigmented ascorbic acid
compound and acid ▪ diazonium salt
urine inaccurate timing
sensitivity 5 – 15 wbc/hpf 10 – 25 wbc/hpf
nitrofurantoin
ASCORBIC ACID
▪ phosphomolybdates
buffered in an acid medium
: 5 mg/dL of ascorbic Gentisic acid and l-dopa
C-STIX REAGENT ▪ the phosphomolybdates are
acid in urine after 10 may cause false (+)
STRIPS reduced by ascorbic acid to
seconds. results
molybdenum blue

Methylene green, which is reduced to its

25 mg/dL of ascorbic acid colorless form with ascorbic acid.
STIX REAGENT STRIPS at 60 seconds Neutral red provides a background color,
and the overall color changes from blue
to purple at levels of 150 mg/dL.

AIRAH M.
 False-positive results are
Large amounts of bilirubin and
MULTISTIC MULTIPLE not seen with urates,
pH greater than 7.5 interfere
REAGENT STRIPS salicylates, gentisic acid,
with the color.
or creatinine.
CALCIUM
RESULT: degree of precipitates
SULKOWITCH REAGENT: if no ppt hypocalcium: 7.5 ml/dl
▪ 2.5g oxalic acid
fine cloud 9 – 11 mg/dl
SULKOWITCH TEST ▪ test for serum calcium 5-20 mg/dl ▪ 2.5g ammonium oxalate Calcium is precipitate as calcium oxalate
▪ glacial acetic acid ppt
heavy hypercalcemia
white ppt
or 5-hydroxyindoleacetic acid
HPLC
serotonin SCREENING TEST
▪ based on the development of a purple color specific for 5-
hydroxyindoles with nitrous acid and 1-nitroso-2-naphthol
▪ ethylene dichloride is used to remove interfering chromogens
neoplastic cells (e.g., nevus, melanoma)
melanin SCREENING TEST
are based on nonspecific color reactions produced with ferric chloride,
Ehrlich’s aldehyde reagent, and nitroferricyanide

AIRAH M.
 Table A. MODE OF ACTION
TESTS PRINCIPLE REAGENTS PROCEDURE INTERPRETATION
KETONE BODIES
Ketone bodies are the products of incomplete fat metabolism. The three ketones present in the urine are acetone, acetoacetic acid and β-hydroxybutyric acid. The presence of increased amounts of ketone bodies in urine indicates
excessive fat catabolism, such as can occur in patients with diabetes mellitus and during starvation.
A positive test is the appearance of a reddish
purple ring at the interphase of
two layers within 1 minute and 30 seconds.
A brown ring is NOT a positive reaction. If
the red to purple color fades
during the first 5 minutes, the test is
Sodium nitroprusside is decomposed to
1. To 5 ml of urine in a test tube (16x150 considered negative.
Na4Fe(CN)6, NaNO2 and Fe(OH)3 in 1. Pulverize and mix 7.5 grams Sodium
mm), add approximately 1 gram of The width of the ring is roughly proportional
a buffered ammonium sulfate solution. In the Nitroprusside and 200 grams
ROTHERA’S TEST Rothera’s reagent (premeasured). to the concentration of the
presence of acetone and acetoacetic Ammonium Sulfate.
2. Overlay with about 1 ml of concentrated ketone bodies.
acid, these compounds yield a complex 2. Concentrated Ammonium Hydroxide.
ammonium hydroxide.
having a rose or purple color.
NEGATIVE : No ring or a brown ring
Trace to + : a faint pinkish purple ring
appearing slowly
++ : narrow dark purple ring
+++ : wide dark purple ring appearing very
rapidly
1. Immerse ;all test areas of reagent strip in
NEGATIVE : Test area remains tan-colored
fresh urine specimen and remove
after 40
strip immediately.
seconds
Follows the Sodium Nitroprusside 7.1% w/w sodium nitroprusside; 92.9% w/w 2. Run edge of strip against the rim of the
REAGENT STRIP METHOD POSITIVE :
Decomposition reaction in Rothera’s test. buffer container to remove excess urine.
(Trace to Large)
3. Hold strip and compare the ketone test
Test area turns pink to purple within 40
area closely with the color chart on
seconds
bottle label exactly 40 seconds later.
URINARY BILE PIGMENTS
The appearance of bilirubin in the urine can provide an early indication of liver disease and is often detected long before the development of jaundice. Bilirubin provides early detection of hepatitis, cirrhosis, gallbladder disease, and cancer
and ideally should be included in every routine urinalysis.

There are two major groups of methods for the detection of bilirubin in urine:
1. Oxidation Methods involving the conversion of bilirubin to colored derivatives.
2. Diazo Methods involving coupling of bilirubin with a diazonium salt in appropriate buffer.
1. Fouchet’s reagent
Dissolve 25 gm trichloroacetic acid, rgt
grade, in 50 ml distilled
water in a 100 ml volumetric flask. Dilute to
volume.
Place the 100 ml TCA solution in a glass-
The phosphate ion present in urine reacts stoppered bottle. Add 10 Procedure:
with the barium in the filter paper ml Ferric chloride solution. 1. Mix 5 ml of urine with an equal amount of
FOUCHET’S TEST (Oxidation Method) to form barium phosphate. Bilirubin is 2. Ferric chloride solution 10% Barium chloride solution. NEGATIVE : No green color on filter paper
adsorbed on the surface of the barium Dissolve 10 gm of ferric chloride, A.C.S., in 2. Filter. The precipitated bile will remain on POSITIVE : Green color appears on filter
Materials: coarse, thick filter paper phosphate, then oxidized to colored 50 ml distilled water in the filter paper paper.
derivatives (largely biliverdin) by the ferric another 100 ml volumetric flask. Dilute to 3. Air-dry the filter paper with precipitates.
chloride in Fouchet’s reagent. volume with distilled water. 4. Add 2 drops of Fouchet’s reagent.
3. Barium chloride solution, 10% w/v
Weigh 10 gm of barium chloride, A.C.S.
Place in a 100 ml
volumetric flask and dissolve in 50 ml
distilled water. Dilute to volume
with distilled water.
NEGATIVE : Test area remains pale lemon
Perform simultaneously with reagent strip
Bilirubin combines with 2,4 - dichloroaniline 0.4 % w/w 2,4 - dichloroaniline diazonium in color after
REAGENT STRIP METHOD (Diazo for ketones. Same as “Dipstick
diazonium salt in an acid salt; 37.3% w/w buffer; 62.3% 30 seconds
Method) Method” of Reagent Strip for ketones except
medium to form azobilirubin. w/w nonreactive ingredients. POSITIVE :
that the reading of color on the
(small to large)

AIRAH M.
 bilirubin test area should be done 30 seconds Test area turns cream to mocca brown in
later after dipping strip in urine. 30 seconds
Hemoglobin in Urine
The presence of abnormal number of red blood cells in urine is Hematuria, whereas the term Hemoglobinuria indicates the presence of hemoglobin in solution in urine.
NEGATIVE : Test pads remains yellow after
1. Immerse all test areas of the reagent strip
60 seconds
in urine specimen and remove strip
POSITIVE :
The oxygen liberated by the catalytic 6.8% w/w diisopropylbenzene immediately.
(Hemolyzed Trace to Large)
decomposition of hydrogen peroxide by the dihydroperoxide 2. Run edge of strip against the rim of the
Test pad turns green after 60 seconds with
REAGENTS STRIP METHOD peroxidase activity of hemoglobin oxidizes 4% w/w 3,3’ , 5,5’ - tetramethylbenzidine container to remove excess urine.
free
Tetramethlybenzidine to produce a green 48% w/w buffer 3. Hold strip against the reagent strip bottle
hemoglobin
color. 41.2 w/w nonreactive ingredients and compare test area for blood
Nonhemolyzed Trace : Green spots appear
closely with the color chart at 60 seconds
on yellow test pad in the
after dipping strip into urine.
presence of intact red blood cells.
MYOGLOBIN IN URINE
When there is rapid destruction of skeletal muscles, myoglobin appears in the urine. The urine appears red or brown. This is similar to the color seen with
hemoglobin and other substances like porphyrins, homogentisic acid and dyes (pyridium). Myoglobin also reacts with the different chemical tests for hemoglobin in the urine and therefore must be distinguished from the latter when
suspected.
1. Mix 1 ml of urine and 3 ml of 3%
sulfosalicylic acid.
2. Filter.
If the pigment remains in the filtrate, the test
If the pigment is precipitated and the
is negative.
supernatant shows a normal color, the
If the pigment is precipitated, it is a protein.
pigment is hemoglobin. If the supernatant is
Blondheim’s Test Proceed to step 3.
Hemoglobin is precpitated from urine that is colored red or red-brown, this is
DIFFERENTIATION BETWEEN Granular Ammonium Sulfate, 2.8g If the filtrate is normal in color, no
80% saturated with ammonium presumptive of myoglobin.
MYOGLOBINURIA AND 3% SSA nonprotein-pigment is present.
sulfate but myoglobin is not.
HEMOGLOBINURIA 3. To exactly 5 ml of urine in a test tube, add
Protein electrophoresis and other methods
exactly 2.8g of ammonium sulfate.
must confirm the presumptive test for
Dissolve by mixing. The urine is now 80%
myoglobin.
saturated with ammonium sulfate. This is
optimum for the precipitation of
hemoglobin.
4. Filter.
CALCIUM IN THE URINE
The urinary output of calcium depends upon dietary intake of calcium, skeletal weight and endocrine factors.
High levels of clacium may be seen in the urine in increased dietary intake of calcium, primary hyperparathyroidism and in osteolytic bone disease especially after immobilization.
Urinary calcium levels are low in hypoparathyroidism, reduced calcium absorption, steatorrhea and vitamin D deficiency.
NEGATIVE : No precipitate Less than 5
1. The pH of the sample should be adjusted mg/dl
to pH 5 before testing by adding
Sulkowitch Reagent drops of acetic acid. Check pH with (+) : Slight clouding 5-10 mg/dl
Calcium is precipitated as calcium oxalate at Weigh out 2.5g oxalic acid and 2.6g indicator provided. If urine is turbid,
a pH where phosphates are soluble. ammonium oxalate into 1 liter volumetric centrifuge (++) : Definite clouding 10-15 mg/dl
SULKOWITCH TEST The degree of precipitation is roughly flask. Add 200 ml distilled water and or filter.
proportional to the amount of calcium in the dissolve the solids. Then add 5 ml glacial 2. Place 3 ml of urine in a test tube and add 3 (+++) : Clouding throughout but
specimen and is noted visually. acetic ml of sulkowitch reagent. not opaque
acid. Make up 1 liter with distilled water. 3. Mix and allow to stand for a few minutes. 15-20 mg/dl
4. Note the density of the precipitates as
early as two minutes after mixing. (++++) : Opaque precipitate More than 20
mg/dl
ALBUMIN IN THE URINE
The bulk of albumin precipitated by various reagents can be used as an approximation of the quantity of albumin present in a urine sample.
The method of Esbach or the later one of Pfeifer may be used. These two methods use the Albuminometer.
precipitation of protein (albumin) in urine 1. Fill the albuminometer with urine to mark
The level of the precipitates indicates
by a picric acid solution “U”
directly on the scale the parts of albumin
(Esbach's Reagent), this procedure Picric acid 10g. 2. Add Esbach’s reagent to the mark “R”
per 1000 parts urine.
ESBACH’S METHOD being carried out in a special but simple Citric acid 20g. 3. Stopper the tube
If the reaction of the urine is not acid, make
piece of glass apparatus called an Distilled water 1000 ml 4. Mix the contents well without shaking
it so before precipitation by adding a
albuminometer tube. 5. Let stand for 24 hours at room
few drops of acetic acid.
temperature

AIRAH M.
 albumin + picric acid = albumin (The set-up shall be submitted to the If the specific gravity of the urine is over
piariate (get deposited) Technician’s counter for safe keeping. 1.006 - 1.008, it should be diluted
Results should be read the next day). and the results corrected in proportion.
UROBILINOGEN IN URINE
Urobilinogen is formed in the gut by the action of bacteria on bilirubin. It is reabsorbed in the small bowel and excreted partially by the liver. Up to 4 mg is
excreted in the urine daily by a healthy adult.
Urinary urobilinogen is increased when there is increased destruction of red blood cells as in hemolytic anemia. It is also increased when there is parenchymal liver disease where the hepatic cells may not reabsorb or excrete the circulating
urobilinogen.
In complete obstructive jaundice, as in carcinoma of the head of the pancreas, urobilinogen may be entirely absent from the urine since bilirubin cannot reach the gut to be changed to urobilinogen.
1. Immerse all test areas of the reagent strip NORMAL
in urine specimen and remove strip (3.2 - 16 umol/L)
Urobilinogen reacts with p- immediately. : Test pad appears very light orange in color.
diethylaminobenzaldehyde to produce a 2. Run edge of strip against the rim of the 32 umol/L : Test pad appears light orange in
0.2% w/w p-diethylaminobenzaldehyde
REAGENT STRIP METHOD colored container to remove excess urine. color.
99.8% w/w nonreactive ingredients
urobilinogen-aldehyde complex on the test 3. Hold strip against the reagent strip bottle 64 umol/L : Test pad turns light pink in
pad. and compare test area for color.
urobilinogen closely with the color chart at 128 umol/L : Test pad turns dark pink in
60 seconds after dipping strip into urine. color.
PORPHOBILINOGEN
This is the monopyrrole precursor of all porphyrins. Porphyrinuria occurs usually in heavy metal poisoning however porphobilinogen is found in the urine in acute intermittent porphyria but not in lead poisoning.
Porphobilinogen also reacts in the ehrlich-aldehyde reaction and must be distinguished from urobilinogen.
1. Into a test tube, place 2.5 ml of urine and
add 2.5 ml of Ehrlich’s aldehyde
reagent.
Porphobilinogen and urobilinogen form a Ehrlich’s aldehyde reagent 2. Mix and observe: NEGATIVE : No cherry-red complex is
“cherry-red” pigment with Ehrlich’s Weigh 0.7g of p- 3. Add 5.0 ml of saturated sodium acetate formed with
aldehyde reagent. The urobilinogen- dimethylaminobenzaldehyde and mix with and shake. Ehrlich’s aldehyde reagent.
aldehyde complex is soluble in n-butanol 150 ml conc. HCL 4. If a pink or red color develops, add 5.0 ml POSITIVE for UROBILINOGEN : Cherry-
Watson-Schwartz Test while and 100 distilled water of n-butanol. red complex is extractable with
the porphobilinogen-aldehyde complex is 5. Shake vigorously. n-butanol
not. Color due to other ehrlich reactive Saturated Sodium Acetate 6. If n-nutanol becomes pink or red and color POSITIVE for PORPHOBILINOGEN :
substance is also soluble in n-butanol so that remains in the aqueous phase, Cherry - red complex is not extractable
the test is specific for porphobilinogen. N-butanol remove the colored n-butanol and discard. with n-butanol
Re-extract with fresh n-butanol until no
more color is extracted into the n-butanol
from the aqueous layer.

AIRAH M.
 SUMMARY OF THE CHEMICAL TESTS
MODE OF READING
TESTS PRINCIPLE SENSITIVITY SPECIMEN REAGENTS DETECTS INTERPRETATION FALSE HIGH FALSE LOW
MEASUREMENT TIME
PROTEINS
mg AER in
results
24-hour albumin/24hrs ug/min
quantitative albumin
for microalbuminuria urine 30-300mg
measurement significant 20-
specimen albumin/24
values 200ug/min
hours
for orthostatic first morning second specimen
proteinuria NEGATIVE POSITIVE
highly buffered alkaline
urine
MULTISTIX: prolonged contact with
MULTISTIX: 15-30 mg/dL
Tetrabromophenol blue urine
albumin CHON other than
contamination with
reagent strip method protein error of indicators colorimetric 60s albumin albumin
CHEMSTRIP: quaternary ammonium
CHEMSTRIP: microalbuminuria
3’,3’’,5’,5’’-tetrachlorophenol, 3,4,5,6- compounds (QAC),
6 mg/dL albumin
tetrabomosulfophthalein detergents, & antiseptics
spx with high sp.gr.
highly pigmented urine4
REPORTING OF SSA TURBIDITY
PROTEIN
GRADE TURBIDITY RANGE
(mg/dL)
no increase in
Negative less than 6
turbidity
noticeable
Trace 6-30
turbidity
sulfosalicylic acid centrifuged all forms distinct turbidity,
cold precipitation 1+ 30-100
precipitation test urine of protein no granulation
turbidity,
2+ granulation, no 100-200
flocculation
turbidity,
3+ granulation, 200-400
flocculation
greater
4+ clumps of protein
than 400
gold-labeled antihuman albumin-enzyme
first (𝞫-galactosidase) conjugate
micral test Enzyme Immunoassay : 0-10 mg/dL 1 min albumin
morning human serum albumin
chlorophenol red galactoside,
darker NEGATIVE
less than
bottom
1.2 mg/dL
band
first blue latex particles coated with
immunodip immunochromographic albumin equal band BORDERLINE 1.2 – 1.8
morning antihuman albumin Ab
colors mg/dL
darker top POSITIVE 2.0 – 8.0
band mg/dL
bis(3’3’’-diiodo-4’4’’-dihydroxy-5’5’’- a. visibly bloody urine
8-15 mg/dL (80-150
albumin ratio dye-binding reaction dinitrophenyl)-3,4,5,6- b. abnormally colored urine
mg/L)
tetrabromosulphonphthalein (DIDNTB)
a. visibly bloody
urine
a. CuSO4 b. abnormally
b. 3,3’,5,5’-tetramethyl-benzidine colored urine
pseudoperoxidase activity of normal value: 10-300 (TMB) c. gastric acid-
creatinine ratio
copper-creatine complexes mg/dL c. diisopropyl benzene reducing
dihydroxyperoxide (DBDH) medication
cimetidine
(Tagamet)

GLUCOSE
strong reducing
agent (e.g.
▪ glucose oxidase ascorbic acid)
▪ peroxidase increased ketone,
REAGENT STRIP POSITIVE NEGATIVE contamination with
double sequential enzymatic ▪ chromogen – KI (Multistix) or S.G.
(GLUCOSE OXIDASE glucose Multistix green brown peroxide and strong
reaction tetramethylbenzidine (Chemstrip) decreased
REACTION) Chemstrip yellow green oxidizing agents
▪ buffer temperature
bacterial
degradation of
glucose

AIRAH M.
 Benedict’s solution
▪ reducing sugars (galactose, lactose, fructose,
▪ copper sulfate
CuSO4 is reduced to Cu2O by maltose, pentoses)
▪ sodium carbonate
CU REDUCTION TEST reducing substances in the ▪ ascorbic acid
200mg/dL ▪ sodium citrate: buffer
(CLINITEST) presence of alkali and heat ▪ certain drug metabolites
▪ sodium hydroxide (added
▪ antibiotics (cephalosporins)
ingredient in the Clinitest Tablet)

KETONES
Sodium Nitroprusside
Decomposition negative no ring/brown ring is present
Rothera’s reagent (overlay with 1ml
Nitropusside in alkaline faint pinkish purple ring
conc. NH4 hydroxide) (+)
medium reacts with a ketone appearing slowly
ROTHERA’S TEST ▪ 7.5 g sodium nitroprusside
group to form a purple ring. It (++) narrow dark purple ring
▪ 200 g ammonium sulfate
is given by acetone and wide dark purple ring
acetoacetate, but not by beta (+++)
appearing very rapidly
hydroxy butyric acid.
▪ highly pigmented red
Multistix:
urine
5-10 mg/dl or 0.25-0.5
▪ phthalein dyes
Sodium Nitroprusside mmol/L of acetoacetic test area remains tan-colored
negative ▪ Levodopa
REAGENT STRIP Decomposition acid ▪ 7.1% (w/w) sodium nitroprusside after 40 secs improperly preserved
(medication for
METHOD ▪ 92.9% (w/w) buffer trace to test area turns pink to purple specimen
Parkinson’s disease)
Chemstrip (+++) within 40 secs ▪ medication
9 mg/dl acetoacetic acid
containing free
& 70 mg/dl acetone
sulfhydryl groups
A purplish color is given by
acetoacetate. On boiling,
acetoacetate is converted to
acetone and does not give this FeCl3 + acetoacetate/ethyacetoacetate → purple color,
test positive. This test is only diacetate/acetoacetic acid
GERHARDT’S FERRIC
given by acetoacetate and not Concentrated Nitric Acid
CHLORIDE TEST
by beta hydroxy butyric acid 1.0ml of 10% FECl3 solution → reddish color is
directly. This test is also formed after the mixture
sensitive to
acetoacetic/diacetic acid.

sensitive to beta-
acetic acid and hydrogen peroxide
HART’S TEST hydroxybutyric acid formation of red ring

▪ large amounts of
▪ the test does not
Levodopa and other ▪ volatilization of
measure beta-
medicines acetone and
URINE KETONES hydroxybutyric acid
containing breakdown of
CONFIRMATORY and is only slightly
sulfhydryl groups acetoacetic acid
TEST sensitive to
may produce by bacteria
acetone when
atypical color
glycine is present
reactions
▪ sodium nitroprusside
ACETEST TABLE diacetic
▪ glycine
(UNDER THE ONE acid and
▪ disodium phosphate
ABOVE) acetone
▪ lactose (color enhancer)
▪ acetone or acetoacetate
Nitroprusside test
reacts with nitroprusside to form violet color
(STILL UNDER)
form violet color
BLOOD
pigment is precipitated
(+) hemoglobin
AMMONIUM supernatant shows normal color
SULFATE SOLUBILITY pigment is precipitated
TEST OR presumptive of supernatant is red/red-brown
BLONDHEIM’S TEST hemoglobin proceed for protein
electrophoresis
supernatant retains the red color
(+) myoglobin supernatant gives a positive
Larger hemoglobin molecules reagent strip test for blood
PRECIPITATION TEST are precipitated by ammonium hemoglobin is found in the red
(UNDER ABOVE) sulfate and myoglobin remains precipitate
in the supernatant. (+) hemoglobin
supernatant tests negative for
blood
a

AIRAH M.
 for hemosiderin Prussian Blue Reagent (made fresh)
▪ high specific gravity
MULTISTIX
▪ presence of
5 to 20
crenated cells
MULTISTIX RBC’s/ml
▪ formalin
diisopropylbenzene dihydroperoxide 0.015 – 0.062 ▪ strong oxidizing
▪ captopril
tetramethylbenzidine mg/dL hgb agent/bleach
pseudoperoxidase activity of ▪ high
REAGENT STRIP ▪ bacterial peroxidases
hemoglobin concentrations of
CHEMSTRIP CHEMSTRIP menstrual
nitrite
dimethyldihydroperoxy-hexane 5 RBCs/ml contamination
▪ ascorbic acid
tetramethylbenzidine hgb
greater than
corresponding
25mg/dl
to 10 RBC’s /mL
unmixed specimen
BILIRUBIN
▪ oxidation of bilirubin to
biliverdin by the ferric
chloride in Fouchet’s
reagent
▪ BaCl reacts with sulfate in
urine to form barium
sulfate. If bilirubin is
present in urine, it
adheres to precipitate and
is detected by oxidation to
FOUCHET’S REAGENT:
form biliverdin (green)
▪ trichloroacetic acid
with FeCl3 in the presence
▪ ferric chloride solution no green color on the filter
of trichloroacetic acid. negative
FOUCHET’S TEST (not ▪ barium chloride solution paper
Nitric acid oxidizes
a sensitive test) green color appear on the filter
bilirubin to biliverdin positive
or HARISSON’S SPOT FOUCHET’S REAGENT: paper
giving different colors
TEST ▪ trichloroacetic acid (25 gms)
from green to violet.
▪ 10% ferric chloride solution (10ml)
▪ distilled water (100ml)
Barium chloride added to urine
combines with sulfate radicals
in urine to form precipitate of
barium phosphate. If bile
pigments are present in urine,
they will adhere to these large
molecules. Ferric chloride
present in Fouchet reagent
then oxidizes yellow bilirubin
in presence of trichloroacetic
acid to green biliverdin.
MULTISTIX ▪ specimen exposure
▪ highly pigmented
2,4-dichloroaniline diazonium salt to light
Diazotization or azo-coupling of urines
M: 0.4 - 0.8 mg/dl ▪ ascorbic acid
REAGENT STRIP bilirubin with diazonium salt in ▪ phenazopyridine
greater than 25
METHOD an acid medium to form azo CHEMSTRIP ▪ indican (intestinal
C: 0.5 mg/dl mg/dl
dye disorders)
2,6-dichlorobenzene-diazonium high concentration of
salt metabolites of Iodine
nitrite
REAGENTS: ▪ specimen exposure
▪ p-nitrobenzene-diazonium-p- to light
diazotization or azo-coupling of
toluenesulfonate no false positive: ▪ ascorbic acid
bilirubin with diazonium salt in
ICTOTEST 0.05-0.1 mg/dl ▪ SSA (sulfosalicylic acid) interfering substances greater than 25
an acid medium to form azo dye
▪ sodium carbonate are corrected mg/dl
▪ boric acid high concentration of
nitrite
Nitric acid oxidizes Bilirubin to
Biliverdin giving different colors
concentrated nitric acid
from green to violet. In other the junction of two liquids, various colored rings
GMELIN’S TEST
words, it is the oxidation of (green, blue, red, violet etc.) will be formed
bilirubin by nitric acid.

UROBILINOGEN
▪ urobilinogen reacts with
dimethylamino- ▪ Ehrlich’s reagent
appearance of pink color in
benzaldehyde in o P-dimetyhlaminodenzaldehyde
the chloroform layer
choloform to form a pink o conc. hydrochloric acid (+)
ERLICH’S TEST colour is easily detected
coloured aldehyde o distilled water urobilinogen
when view from the top of
complex ▪ saturate sodium acetate
the test tube
▪ based on Ehrlich ▪ chloroform
aldehyde reaction
The urine is a yellowish-brown
FOAM TEST or greenish-yellow color and
bilirubin is suspected, shake

AIRAH M.
 the urine. If a yellow or
greenish-yellow foam
develops, then bilirubin is most
likely present. Bilirubin alters
the surface tension of urine and
foam will develop after
shaking.
MULTISTIX
Ehrlich’s aldehyde ▪ M: old specimens
reaction preservation in
▪ M: porphobilinogen
formalin
▪ indican
M: p-dimethyl-aminobenaaldehyde
CHEMSTRIP M: 0.4 - 0.8 mg/dl ▪ p-aminosalicylic acid
REAGENT TEST C:
azo-coupling (diazo) ▪ sulfonamides
STRIPS C: 4-methoxybenzene- diazonium- ▪ old specimen
reaction using 4- C: 0.5 mg/dl highly pigmented urine
tetrafluoroborate ▪ preservation in
methoxybenzene- formalin
C: high pigmented urine
tetrafluoroborate with high concentration of
urobilinogen forming red urine
azodye
pink color in the
(+) urobilinogen
chloroform layer
(+) pink coloration of
porphobilinogen aqueous portion

▪ P-dimetyhlaminodenzaldehyde
2nd set
solubility difference of ▪ conc. hydrochloric acid
porphobilinogen and cherry red complex is
WATSON-SCHWARTZ ▪ distilled water (+) urobilinogen extractable with n-
urobilinogen In Ehrlich’s
TEST ▪ additional: saturated sodium acetate butanol
aldehyde reagent
and N butanol cherry red complex is
(+)
not extractable with n-
porphobilinogen
butanol & chloroform
no cherry red complex is
negative formed with Ehrlich’s
aldehyde reagent
▪ detects about 20 – 100 mg/L of porphobilinogen, ▪ urorosein: urinary
and urobilinogen in amounts up to 200 mg/L does pigment related to
HOESCH’S TEST inverse Ehrlich’s reaction
not give a positive result (red color) indoleacetic acid
a yellow color may be caused by urea dark pigmented urine
NITRATE
▪ non-reductase
MULTISTIX containing bacteria
p-arsanilic acid ▪ insufficient contact
Greiss Reaction: nitrite at Tetrahydobenzo(h)- time between
acidic pH reacts with aromatic quinoline-3-ol ▪ the diazonium salt reacts with sulfanilic acid and ▪ improperly preserved bacteria & urinary
URINE NITRATE
acetic acid to produce a pink azo dye specimen nitrate
TESTING amine to form a diazonium
highly pigmented urine ▪ presence of
compound CHEMSTRIP antibiotics
Sulfanilamide, ▪ high concentration
Hydroxytetrahydro benzoquinoline of ascorbic acid
high specific gravity
LEUCOCYTE
▪ strong oxidizing
action of leucocyte esterase to MULTISTIX CHEMSTRIP
agents ▪ high conc. protein,
catalyze the hydrolysis of an ▪ derivatized pyrrole amino
URINE LEUKOCYTE indoxylcarbonic acid ester and ▪ formalin glucose, oxalic acid,
acid ester to an aromatic reagent acid ester
ESTERASE diazonium salt ▪ highly pigmented ascorbic acid
compound and acid ▪ diazonium salt
urine inaccurate timing
sensitivity 5 – 15 wbc/hpf 10 – 25 wbc/hpf
nitrofurantoin
ASCORBIC ACID
▪ phosphomolybdates
buffered in an acid medium
: 5 mg/dL of ascorbic Gentisic acid and l-dopa
C-STIX REAGENT ▪ the phosphomolybdates are
acid in urine after 10 may cause false (+)
STRIPS reduced by ascorbic acid to
seconds. results
molybdenum blue

Methylene green, which is reduced to its

25 mg/dL of ascorbic acid colorless form with ascorbic acid.
STIX REAGENT STRIPS at 60 seconds Neutral red provides a background color,
and the overall color changes from blue
to purple at levels of 150 mg/dL.
Large amounts of bilirubin and
MULTISTIC MULTIPLE False-positive results are not seen with urates,
pH greater than 7.5 interfere
REAGENT STRIPS salicylates, gentisic acid, or creatinine.
with the color.

AIRAH M.
 CALCIUM
RESULT: degree of precipitates
SULKOWITCH REAGENT: if no ppt hypocalcium: 7.5 ml/dl
▪ 2.5g oxalic acid
fine cloud 9 – 11 mg/dl
SULKOWITCH TEST ▪ test for serum calcium 5-20 mg/dl ▪ 2.5g ammonium oxalate Calcium is precipitate as calcium oxalate
▪ glacial acetic acid ppt
heavy hypercalcemia
white ppt
or 5-hydroxyindoleacetic acid
HPLC
serotonin SCREENING TEST
▪ based on the development of a purple color specific for 5-
hydroxyindoles with nitrous acid and 1-nitroso-2-naphthol
▪ ethylene dichloride is used to remove interfering chromogens
neoplastic cells (e.g., nevus, melanoma)
melanin SCREENING TEST
are based on nonspecific color reactions produced with ferric chloride,
Ehrlich’s aldehyde reagent, and nitroferricyanide

AIRAH M.
 SUMMARY OF THE TESTS FOR EXERICISES 2,3,4,5
TESTS PRINCIPLE REAGENTS PROCEDURE INTERPRETATION
KETONE BODIES
Ketone bodies are the products of incomplete fat metabolism. The three ketones present in the urine are acetone, acetoacetic acid and β-hydroxybutyric acid. The presence of increased amounts of ketone bodies in urine indicates
excessive fat catabolism, such as can occur in patients with diabetes mellitus and during starvation.
A positive test is the appearance of a reddish purple
ring at the interphase of
two layers within 1 minute and 30 seconds.

A brown ring is NOT a positive reaction. If the red

to purple color fades during the first 5 minutes, the
Sodium nitroprusside is decomposed to
1. To 5 ml of urine in a test tube (16x150 mm), add test is considered negative.
Na4Fe(CN)6, NaNO2 and Fe(OH)3 in a
1. Pulverize and mix 7.5 grams Sodium approximately 1 gram of Rothera’s reagent
buffered ammonium sulfate solution. In the
ROTHERA’S TEST Nitroprusside and 200 grams Ammonium Sulfate. (premeasured). The width of the ring is roughly proportional to the
presence of acetone and acetoacetic acid,
2. Concentrated Ammonium Hydroxide. 2. Overlay with about 1 ml of concentrated concentration of the
these compounds yield a complex having a
ammonium hydroxide. ketone bodies.
rose or purple color.
NEGATIVE : No ring or a brown ring
Trace to +: a faint pinkish purple ring appearing
slowly
++ : narrow dark purple ring
+++ : wide dark purple ring appearing very rapidly
1. Immerse ;all test areas of reagent strip in fresh
urine specimen and remove strip immediately. NEGATIVE : Test area remains tan-colored after
2. Run edge of strip against the rim of the container 40 seconds
REAGENT STRIP Follows the Sodium Nitroprusside 7.1% w/w sodium nitroprusside; 92.9% w/w
to remove excess urine.
METHOD Decomposition reaction in Rothera’s test. buffer
3. Hold strip and compare the ketone test area POSITIVE : (Trace to Large)
closely with the color chart on bottle label exactly Test area turns pink to purple within 40secs
40 seconds later.
URINARY BILE PIGMENTS
The appearance of bilirubin in the urine can provide an early indication of liver disease and is often detected long before the development of jaundice. Bilirubin provides early detection of hepatitis, cirrhosis, gallbladder disease, and cancer
and ideally should be included in every routine urinalysis.

There are two major groups of methods for the detection of bilirubin in urine:
1. Oxidation Methods involving the conversion of bilirubin to colored derivatives.
2. Diazo Methods involving coupling of bilirubin with a diazonium salt in appropriate buffer.
1. Fouchet’s reagent
Dissolve 25 gm trichloroacetic acid, rgt grade, in
50 ml distilled water in a 100 ml volumetric flask.
Dilute to volume.
Place the 100 ml TCA solution in a glass-
The phosphate ion present in urine reacts with Procedure:
stoppered bottle. Add 10 ml Ferric chloride
FOUCHET’S TEST the barium in the filter paper to form barium 1. Mix 5 ml of urine with an equal amount of 10%
solution.
(Oxidation Method) phosphate. Bilirubin is adsorbed on the Barium chloride solution. NEGATIVE : No green color on filter paper
2. Ferric chloride solution
surface of the barium phosphate, then 2. Filter. The precipitated bile will remain on the
Dissolve 10 gm of ferric chloride, A.C.S., in 50 ml
Materials: coarse, thick oxidized to colored derivatives (largely filter paper POSITIVE : Green color appears on filter paper.
distilled water in another 100 ml volumetric flask.
filter paper biliverdin) by the ferric chloride in Fouchet’s 3. Air-dry the filter paper with precipitates.
Dilute to volume with distilled water.
reagent. 4. Add 2 drops of Fouchet’s reagent.
3. Barium chloride solution, 10% w/v
Weigh 10 gm of barium chloride, A.C.S. Place in
a 100 ml volumetric flask and dissolve in 50 ml
distilled water. Dilute to volume with distilled
water.
Perform simultaneously with reagent strip for NEGATIVE : Test area remains pale lemon in color
REAGENT STRIP Bilirubin combines with 2,4 - dichloroaniline 0.4 % w/w 2,4 - dichloroaniline diazonium salt; ketones. Same as “Dipstick Method” of Reagent after 30 seconds
METHOD (Diazo diazonium salt in an acid medium to form 37.3% w/w buffer; Strip for ketones except that the reading of color on
Method) azobilirubin. 62.3% w/w nonreactive ingredients. the bilirubin test area should be done 30 seconds POSITIVE : (small to large)
later after dipping strip in urine. Test area turns cream to mocca brown in 30 secs
Hemoglobin in Urine
The presence of abnormal number of red blood cells in urine is Hematuria, whereas the term Hemoglobinuria indicates the presence of hemoglobin in solution in urine.

AIRAH M.
 NEGATIVE : Test pads remains yellow after 60s
1. Immerse all test areas of the reagent strip in urine
POSITIVE :
The oxygen liberated by the catalytic specimen and remove strip immediately.
6.8% w/w diisopropylbenzene dihydroperoxide (Hemolyzed Trace to Large)
decomposition of hydrogen peroxide by the 2. Run edge of strip against the rim of the ontainer
REAGENTS STRIP 4% w/w 3,3’ , 5,5’ - tetramethylbenzidine Test pad turns green after 60 seconds with free
peroxidase activity of hemoglobin oxidizes to remove excess urine.
METHOD 48% w/w buffer hemoglobin
Tetramethlybenzidine to produce a green 3. Hold strip against the reagent strip bottle and
41.2 w/w nonreactive ingredients
color. compare test area for blood closely with the color
Nonhemolyzed Trace :
chart at 60 seconds after dipping strip into urine.
Green spots appear on yellow test pad in the
presence of intact red blood cells.
MYOGLOBIN IN URINE
When there is rapid destruction of skeletal muscles, myoglobin appears in the urine. The urine appears red or brown. This is similar to the color seen with hemoglobin and other substances like porphyrins, homogentisic acid and dyes
(pyridium). Myoglobin also reacts with the different chemical tests for hemoglobin in the urine and therefore must be distinguished from the latter when suspected.
1. Mix 1 ml of urine and 3 ml of 3% sulfosalicylic
acid.
2. Filter. If the pigment remains in the filtrate, the
If the pigment is precipitated and the supernatant
test is negative. If the pigment is precipitated, it is a
BLONDHEIM’S TEST shows a normal color, the pigment is hemoglobin. If
protein. Proceed to step 3. If the filtrate is normal in
DIFFERENTIATION Hemoglobin is precpitated from urine that is the supernatant is colored red or red-brown, this is
Granular Ammonium Sulfate, 2.8g color, no nonprotein-pigment is present.
BETWEEN 80% saturated with ammonium sulfate but presumptive of myoglobin.
3% SSA 3. To exactly 5 ml of urine in a test tube, add exactly
MYOGLOBINURIA AND myoglobin is not.
2.8g of ammonium sulfate. Dissolve by mixing. The
HEMOGLOBINURIA Protein electrophoresis and other methods must
urine is now 80% saturated with ammonium sulfate.
confirm the presumptive test for myoglobin.
This is optimum for the precipitation of
hemoglobin.
4. Filter.
CALCIUM IN THE URINE
The urinary output of calcium depends upon dietary intake of calcium, skeletal weight and endocrine factors.
High levels of clacium may be seen in the urine in increased dietary intake of calcium, primary hyperparathyroidism and in osteolytic bone disease especially after immobilization.
Urinary calcium levels are low in hypoparathyroidism, reduced calcium absorption, steatorrhea and vitamin D deficiency.
1. The pH of the sample should be adjusted to pH 5 NEGATIVE : No precipitate Less than 5 mg/dl
before testing by adding
Sulkowitch Reagent drops of acetic acid. Check pH with indicator (+) : Slight clouding 5-10 mg/dl
Calcium is precipitated as calcium oxalate at a
Weigh out 2.5g oxalic acid and 2.6g ammonium provided. If urine is turbid, centrifuge
pH where phosphates are soluble. The degree
oxalate into 1 liter volumetric flask. Add 200 ml or filter. (++) : Definite clouding 10-15 mg/dl
SULKOWITCH TEST of precipitation is roughly proportional to the
distilled water and dissolve the solids. Then add 5 2. Place 3 ml of urine in a test tube and add 3 ml of
amount of calcium in the specimen and is
ml glacial acetic acid. Make up 1 liter with sulkowitch reagent. (+++) : Clouding throughout but not opaque
noted visually.
distilled water. 3. Mix and allow to stand for a few minutes. 15-20 mg/dl
4. Note the density of the precipitates as early as two
minutes after mixing. (++++) : Opaque precipitate More than 20 mg/dl
ALBUMIN IN THE URINE
The bulk of albumin precipitated by various reagents can be used as an approximation of the quantity of albumin present in a urine sample.
The method of Esbach or the later one of Pfeifer may be used. These two methods use the Albuminometer.
The level of the precipitates indicates directly on the
precipitation of protein (albumin) in urine by 1. Fill the albuminometer with urine to mark “U” scale the parts of albumin per 1000 parts urine.
a picric acid solution (Esbach's Reagent), this 2. Add Esbach’s reagent to the mark “R”
procedure being carried out in a special but 3. Stopper the tube If the reaction of the urine is not acid, make it so
Picric acid 10g.
simple piece of glass apparatus called an 4. Mix the contents well without shaking before precipitation by adding a few drops of acetic
ESBACH’S METHOD Citric acid 20g.
albuminometer tube. 5. Let stand for 24 hours at room temperature acid.
Distilled water 1000 ml
(The set-up shall be submitted to the Technician’s
albumin + picric acid = albumin piariate counter for safe keeping. Results should be read the If the specific gravity of the urine is over 1.006 -
(get deposited) next day). 1.008, it should be diluted and the results corrected
in proportion.
UROBILINOGEN IN URINE
Urobilinogen is formed in the gut by the action of bacteria on bilirubin. It is reabsorbed in the small bowel and excreted partially by the liver. Up to 4 mg is
excreted in the urine daily by a healthy adult.
Urinary urobilinogen is increased when there is increased destruction of red blood cells as in hemolytic anemia. It is also increased when there is parenchymal liver disease where the hepatic cells may not reabsorb or excrete the circulating
urobilinogen.
In complete obstructive jaundice, as in carcinoma of the head of the pancreas, urobilinogen may be entirely absent from the urine since bilirubin cannot reach the gut to be changed to urobilinogen.
NORMAL
REAGENT STRIP Urobilinogen reacts with p- 0.2% w/w p-diethylaminobenzaldehyde 1. Immerse all test areas of the reagent strip in urine
(3.2 - 16 umol/L) : Test pad appears very light
METHOD diethylaminobenzaldehyde to produce a 99.8% w/w nonreactive ingredients specimen and remove strip immediately.
orange in color.

AIRAH M.
 colored urobilinogen-aldehyde complex on 2. Run edge of strip against the rim of the ontainer 32 umol/L : Test pad appears light orange in color.
the test pad. to remove excess urine. 64 umol/L : Test pad turns light pink in color.
3. Hold strip against the reagent strip bottle and 128 umol/L : Test pad turns dark pink in color.
compare test area for urobilinogen closely with the
color chart at 60 seconds after dipping strip into
urine.
PORPHOBILINOGEN
This is the monopyrrole precursor of all porphyrins. Porphyrinuria occurs usually in heavy metal poisoning however porphobilinogen is found in the urine in acute intermittent porphyria but not in lead poisoning.
Porphobilinogen also reacts in the ehrlich-aldehyde reaction and must be distinguished from urobilinogen.
1. Into a test tube, place 2.5 ml of urine and add 2.5
ml of Ehrlich’s aldehyde reagent.
2. Mix and observe:
Porphobilinogen and urobilinogen form a Ehrlich’s aldehyde reagent NEGATIVE : No cherry-red complex is formed
3. Add 5.0 ml of saturated sodium acetate and
“cherry-red” pigment with Ehrlich’s aldehyde Weigh 0.7g of p-dimethylaminobenzaldehyde with Ehrlich’s aldehyde reagent.
shake.
reagent. The urobilinogen-aldehyde complex and mix with 150 ml conc. HCL
4. If a pink or red color develops, add 5.0 ml of n-
WATSON-SCHWARTZ is soluble in n-butanol while the and 100 distilled water POSITIVE for UROBILINOGEN : Cherry-red
butanol.
TEST porphobilinogen-aldehyde complex is not. complex is extractable with n-butanol
5. Shake vigorously.
Color due to other ehrlich reactive substance Saturated Sodium Acetate
6. If n-nutanol becomes pink or red and color
is also soluble in n-butanol so that the test is POSITIVE for PORPHOBILINOGEN : Cherry -
remains in the aqueous phase, remove the colored
specific for porphobilinogen. N-butanol red complex is not extractable with n-butanol
n-butanol and discard. Re-extract with fresh n-
butanol until no more color is extracted into the n-
butanol from the aqueous layer.

AIRAH M.
 3920_Ch14_255-268 23/01/14 10:38 AM Page 256

@asherum_ | BSMT-3E 2020 : ANALYSIS OF URINE AND BODY FLUIDS 8.1

Basic methods and specimen collection in hematology

256 Part Three | Other Body Fluids

LESSON 8 In the minds of most laboratory personnel, fecal specimen analysis

metabolized by bac
fits into the category of a “necessary evil.” However, as an end prod-
amounts of flatus. E
Date of Lecture : 26-30 Octiber 2020. Ms. Michelle Angelica D. Fulgencio, RMT, (ASCPi)CM, AMT uct of body metabolism, feces do provide valuable diagnostic intolerant people w
References : Strasinger, S. K., & Di Lorenzo, M. S. (2014). Fecal Analysis. In Urinalysis and Body Fluids (6th ed., pp. 255-265).
information. Philadelphia,
Routine fecal PA: F.A.
examination includes Davis.
macroscopic, lactose from consum
microscopic, and chemical analyses for the early detection of gas- Although diges
trointestinal (GI) bleeding, liver and biliary duct disorders, fats takes place thr
• In the minds of most laboratory personnel, fecal specimen analysis fits into the category of a “necessary evil.”
maldigestion/malabsorption syndromes, pancreatic diseases, testine is the prima
inflammation, and causes of diarrhea and steatorrhea. Of equal tion of these comp
• However, as an end product of body metabolism, feces do provide valuable diagnostic information. Routine fecal examination includes macroscopic,
diagnostic value is the detection and identification of pathogenic small intestine by t
microscopic, and chemical analyses for the early detection of various disorders. bacteria, viruses, and parasites; however, these procedures are best amino peptidase, a
covered in a microbiology textbook and are not discussed here. in the digestion of
creates an inability
OUTLINE Physiology foods. Excess undig
1. Physiology 2.2. Steatorrhea 5. Microscopic Screening 6.1. Occult (Hidden) Blood in the feces, and th
The normal fecal specimen contains bacteria, cellulose, undigested and malabsorption
2. Diarrhea and Steatorrhea 3. Specimen Collection 5.1. Fecal Leukocytes foodstuffs, GI secretions, 6.2. Quantitative
bile pigments, cells from Fecal Fat 9000 mL of ingeste
the intestinal
2.1. Diarrhea 4. Macroscopic Screening 5.2. Muscle Fibers Testing 100 to 200 g of feces intestinal secretions
walls, electrolytes, and water. Approximately
2.1.1. Secretory Diarrhea 4.1. Color 5.3. Qualitative Fecal Fats
is excreted in a 24-hour6.3. APTspecies
period. Many Testof(HbF)bacteria make up mal conditions, on
the normal flora of the intestines. Bacterial metabolism produces reaches the large in
2.1.2. Osmotic Diarrhea 4.2. Appearance 6. Chemical Testing 6.4. Fecal Enzymes
the strong odor associated with feces and intestinal gas (flatus). the feces. Water and
2.1.3. Altered Motility Carbohydrates, especially6.5.oligosaccharides,
Carbohydratesthat are resistant to small and large inte
digestion pass through the upper intestine unchanged but are that is similar to tha

1
physiology
• Proteins, carbohydrates, and fats
o Digestion – throughout the alimentary tract
o small intestine is the primary site for the final breakdown and reabsorption
§ Digestive enzymes are secreted into the small intestine by the pancreas (trypsin,
chymotrypsin, amino peptidase, and lipase)
§ Bile salts provided by the liver aid in the digestion of fats.
* A deficiency in any of these substances creates an inability to digest and,
therefore, to reabsorb certain foods.
* Excess undigested or unreabsorbed materials then appear in the feces, and the
patient exhibits symptoms of maldigestion and malabsorption.
• Among the large volume of fluid entering the digestive tract each day, under normal conditions,
only between 500 to 1500 mL of this fluid reaches the large intestine.
o only about 150 mL is excreted in the feces.
• The large intestine is capable of absorbing approximately 3000 mL of water.
o Excess – excreted with the solid fecal material, producing diarrhea.
o Constipation provides time for additional water to be reabsorbed from the fecal material,
producing small, hard stools.
• Normal fecal specimen constituents:
o Bacteria o Cellulose o Undigested foodstuffs
Figure 14
o Bile pigments o Electrolytes o Water tract.
o Cells from intestinal walls o GI secretions
o ≈ 100-200g of feces is excreted in a 24-hour period
o Many species of bacteria make up the normal flora of the intestines.
o Bacterial metabolism produces the strong odor associated with feces and intestinal gas (flatus).

2
diarrhea and steatorrhea
2.1
DIARRHEA
• “① an ↑ in daily stool weight above 200g, ② increased liquidity of stools, and ③ frequency of > 3x/day.”
• Diarrhea classification can be based on:
1. Illness duration 2. Mechanism 3. Severity 4. Stool characteristics
o acute diarrhea < 4 weeks < chronic diarrhea

MAJOR MECHANISMS Secretory Osmotic Intestinal Hypermotility

DIFFERENTIAL LAB TESTS Fecal electrolytes (Fecal Na and fecal K) Fecal osmolality Stool pH
• The normal total fecal osmolarity is close to the serum osmolality (290 mOsm/kg), normal fecal sodium is 30 mmol/L, and fecal potassium is 75
mmol/L.
o The fecal sodium and fecal potassium results are used to calculate the fecal osmotic gap.
Osmotic gap = 290 – [2 (fecal sodium + fecal potassium)]

2.1.1
Secretory Diarrhea
• < 50 mOsm/kg; ↑electrolytes
• caused by increased secretion of water
• ① Bacterial, ② viral, and ③ protozoan infections produce increased secretion of water and electrolytes
o override the reabsorptive ability of the large intestine à secretory diarrhea.
• Various enterotoxin-producing organisms (determined by stool culture) can stimulate these water and electrolyte secretions:
@asherum_ | BSMT-3E 2020 : ANALYSIS OF URINE AND BODY FLUIDS 8.2

o Campylobacter o Cryptosporidium o Protozoa o Shigella o Vibrio cholerae

o Clostridium o Escherichia coli o Salmonella o Staphylococcus
• Other causes of secretory diarrhea:
o collagen vascular disease o endocrine disorders o inflammatory bowel disease o stimulant laxatives
o Drugs o hormones o neoplasms

2.1.2
Osmotic Diarrhea
• caused by poor absorption that exerts osmotic pressure across the intestinal mucosa.
• Incomplete breakdown or reabsorption of food presents increased fecal material to the large intestine
o resulting in water and electrolyte retention in the large intestine (osmotic diarrhea) à excessive watery stool
• Maldigestion (impaired food digestion) and malabsorption (impaired nutrient absorption by the intestine) contribute to osmotic diarrhea.
• The presence of unabsorbable solute à ↑ stool osmolality and ↓ concentration of electrolytes [negligible] à increased osmotic gap (>50 Osm/kg)
• fecal fluid pH < 5.6 indicates malabsorption of sugars à osmotic diarrhea
• Causes of osmotic diarrhea:
o amebiasis o laxatives o poorly absorbed sugars
o antibiotic administration o malabsorption (celiac sprue) (lactose, sorbitol, mannitol)
o disaccharidase deficiency (lactose intolerance) o Mg-containing antacids

OSMOTIC DIARRHEA SECRETORY DIARRHEA

• Clinitest • Muscle fiber detection
• D-xylose tolerance test • Muscle fiber detection
• Fecal leukocytes
• Fecal electrolytes • Qualitative fecal fats
• Ova and parasite examinations
COMMON FECAL TESTS • Fecal osmolality • Quantitative fecal fats
• Rotavirus immunoassay
• Lactose tolerance test • Stool pH
• Stool cultures
• Microscopic fecal fats • Trypsin screening
• Microscopic fecal fats
Osmotic gap >50 Osm/kg <50 Osm/kg
LABORATORY TEST

Stool Na <60 mmol/L >90 mmol/L

Stool output in 24 hours <200 g >200 g
pH <5.3 >5.6
Reducing substances Positive* Negative
• The presence of reducing sugars in patients with disaccharidase deficiency (lactose intolerance) yields a positive result in the test for reducing
substances for osmotic diarrhea.

2.1.C
Altered Motility
• Altered motility describes conditions of enhanced motility (hypermotility) or slow
motility (constipation)
o Both can be seen in irritable bowel syndrome (IBS)
§ a functional disorder in which the nerves and muscles of the bowel are
extra sensitive
• bloating • cramping • flatus
SYMPTOMS
• constipation • diarrhea
• chemicals • exercise
TRIGGERS
• emotional stress • food
• Intestinal hypermotility is the excessive movement of intestinal contents through the GI tract that can cause diarrhea because normal absorption of
intestinal contents and nutrients cannot occur.
o It can be caused by ① enteritis, the ② use of parasympathetic drugs, or ③ with complications of malabsorption.
• Rapid gastric emptying (RGE) dumping syndrome describes hypermotility of the stomach and the shortened gastric emptying half-time
o causes the small intestine to fill too quickly with undigested food from the stomach
o hallmark of early dumping syndrome (EDS)
o Healthy people have a gastric emptying half-time range of 35-100 minutes, which varies with age and gender.
§ A gastric emptying time of < 35 minutes is considered RGE.

2.2
STEATORRHEA
• Detection of steatorrhea (fecal fat) is useful in diagnosing pancreatic insufficiency and small-bowel disorders that cause mal- absorption.
• Absence of bile salts that assist pancreatic lipase in the breakdown and subsequent reabsorption of dietary fat (primarily triglycerides) produces an
increase in stool fat (steatorrhea) > 6g/day.
• Likewise, pancreatic disorders (cystic fibrosis, chronic pancreatitis, and carcinoma) that ↓ production of pancreatic enzymes, are also associated
with steatorrhea.
• Steatorrhea may be present in both maldigestion and malabsorption conditions and can be distin- guished by the D-xylose test.
o D-Xylose is a sugar that does not need to be digested but does need to be absorbed to be present in the urine.
URINE D-XYLOSE Low High
CONDITION Malabsorption Pancreatitis
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3
Specimen Collection
• Collection of a fecal specimen (stool specimen) is not an easy task for patients.
o Detailed instructions and appropriate containers depending on the type of test and amount of feces required should be provided.
o For certain tests, dietary restrictions are required before fecal specimen collection.
• Patients should be instructed to collect the specimen in a clean container (bedpan or disposable container) and transfer the specimen to the
laboratory container.
o Patients should understand that the specimen must not be contaminated with urine or toilet water
§ may contain chemical disinfectants or deodorizers – can interfere with chemical testing.
o Containers that contain preservatives for ova and parasites must not be used to collect specimens for other tests.
• Random specimens suitable for qualitative testing for blood and microscopic examination for ① leukocytes, ② muscle fibers, and ③ fecal fats are
usually collected in plastic or glass containers with screw-tops similar to those used for urine specimens.
o Material collected on a physician’s glove and samples applied to filter paper in occult blood testing kits are also received.
• For quantitative testing (fecal fats), timed specimens are required.
o Because of the variability of bowel habits and the transit time required for food to pass through the digestive tract, the most representative sample
is a 3-day collection.
§ Spx are frequently collected in large containers to ① accommodate the specimen quantity and ② facilitate emulsification before testing.
§ Care must be taken when opening any fecal specimen to slowly release gas that has accumulated within the container.
§ Patients must be cautioned not to contaminate the outside of the container.

4
Macroscopic Screening
4.1
COLOR
• The first indication of GI disturbances can often be changes in the brown color and formed consistency of the normal stool.
• Of course, the appearance of abnormal fecal color may also be caused by ingestion of highly pigmented foods and medications, so a differentiation
must be made between this and a possible pathologic cause.
• results from intestinal oxidation of stercobilinogen à urobilin.
• As was discussed, conjugated bilirubin formed in the degradation of hemoglobin passes through the bile duct to the small intestine,
Brown stools where intestinal bacteria convert it to urobilinogen and stercobilinogen.
o Therefore, stools that appear pale (acholic stools) may signify a blockage of the bile duct.
§ also associated with diagnostic procedures that use BaSO4
• may be observed in patients taking oral antibiotics, because of the oxidation of fecal bilirubin to biliverdin.
Green stools
• Ingestion of increased amounts of green vegetables or food coloring also produces green stools.

• A primary concern is the presence of blood in a stool specimen

o Depending on the area of the intestinal tract from which bleeding occurs, the color can range from bright red to dark red to black.
• Blood that originates from the esophagus, stomach, or duodenum takes approximately 3 days to appear in the stool
• Likewise, blood from the lower GI tract requires less time to appear and retains its original red color.
Black, tarry stool • degradation of Hb • Upper GIT bleeding • Iron, charcoal, bismuth ingestion
Red stool • Lower GIT bleeding • Beets and other foods • rifampin
Pale yellow, white, gray • Bile-duct obstruction • Barium sulfate (BaSO4)

4.2
APPEARANCE
watery consistency diarrhea
small, hard stools constipation
slender, ribbon-like stools Intestinal obstruction (obstruction of the normal passage of material through the intestine)
bulky and frothy (pale stools) with foul odor biliary obstruction and steatorrhea; stools may appear greasy and may float.
• damage to the intestinal walls, possibly caused by bacterial or amebic dysentery or malignancy
Mucus- or blood-streaked mucus
• colitis; constipation
• The presence of mucus-coated stools indicates intestinal inflammation or irritation.
• Mucus-coated stools may be caused by pathologic colitis, Crohn disease, colon tumors, or excessive straining during elimination. suggests.
o The presence of mucus should be reported

5
Microscopic Screening
• Microscopic screening of fecal smears is performed to detect the presence of leukocytes associated with microbial diarrhea and undigested muscle
fibers and fats associated with steatorrhea.

5.1
FECAL LEUKOCYTES
• Leukocytes, primarily neutrophils, are seen in the feces in conditions that affect the intestinal mucosa, such as ulcerative colitis and bacterial dysentery.
• The presence or absence of fecal neutrophils can help the physician presumptively determine the cause of the patient’s diarrhea.
@asherum_ | BSMT-3E 2020 : ANALYSIS OF URINE AND BODY FLUIDS 8.4

Neutrophils present Neutrophils absent

• Campylobacter • Salmonella • Yersinia • Staphylococcus aureus • viruses
• Enteroinvasive E. coli. • Shigella • Vibrio spp. • parasites
• Diarrhea caused by toxin production; usually do not cause the
• Diarrhea caused by invasive bacterial pathogens
appearance of fecal leukocytes

Wet Preparations Dry Preparations

Methylene blue Wright’s or Gram’s Stain
• Faster but may be more difficult to interpret • Provide permanent slides for preparation
• Distinguishes G(+) and G(-) bacteria – aids in initial t(x)
• All slide preparations must be performed on fresh specimens.
o In an examination of preparations under HPO, as few as 3 neutrophils/hpf can be indicative of an invasive condition.
o Using OIO, the finding of any neutrophils has ~70% sensitivity for the presence of invasive bacteria.
• A lactoferrin latex agglutination test is available for detecting fecal leukocytes and remains sensitive in refrigerated and frozen specimens.
o The presence of lactoferrin, a component of granulocyte secondary granules, indicates an invasive bacterial pathogen.

5.2
MUSCLE FIBERS
• Microscopic examination of the feces for undigested striated muscle fibers can be helpful in diagnosing and monitoring patients with pancreatic
insufficiency (i.e. cases of cystic fibrosis).
o It is frequently ordered in conjunction with microscopic examinations for fecal fats.
o ↑ amounts of striated fibers may also be seen in biliary obstruction and gastrocolic fistulas.
• Slides for muscle fiber detection are prepared by emulsifying a small amount of stool in 10% alcoholic eosin,
o which enhances the muscle fiber striations.
o The entire slide is exam- ined for exactly 5 minutes, and the number of red-stained fibers with well-preserved striations is counted.
§ Care must be taken to correctly classify the fibers observed.
TYPE STRIATIONS
• visible striations, vertically and horizontally
Undigested fibers
• only ones counted – presence of > 10 à reported as increased
Partially digested fibers • only one direction
Digested fibers • no visible striations
o To produce a representative sample, patients should be instructed to include red meat in their diet before collecting the specimen.
§ Specimens should be examined within 24 hours of collection.

5.3
QUALITATIVE FECAL FATS
• Specimens from suspected cases of steatorrhea can be screened microscopically for the presence of excess fecal fat (steatorrhea).
o The procedure can also be used to monitor patients undergoing treatment for malabsorption disorders
o In general, correlation between the qualitative and quantitative fecal fat procedures is good
§ However, additional unstained phospholipids and cholesterol esters are measured by the quantitative procedure.
• Lipids included in the microscopic examination of feces are neutral fats (triglycerides), fatty acid salts (soaps), fatty acids, and cholesterol.
o Their presence can be observed microscopically by staining with the dyes ① Sudan III (most routinely used), ② Sudan IV, or ③ oil red O; Sudan III
is the most routinely used.
§ The staining procedure consists of two parts: the neutral fat stain and the split fat stain.
§ Cholesterol is stained by Sudan III after heating à as the specimen cools à forms crystals that can be identified microscopically.

6
Chemical Testing of Feces
6.1
OCCULT (Hidden) BLOOD
• The most frequently performed fecal analysis
• Upper GI bleeding – produce a black, tarry stool; lower GI bleeding – overtly bloody stool.
o However, because any bleeding in excess of 2.5 mL/150g of stool is considered pathologically significant, and no visible signs of bleeding may be
present with this amount of blood, fecal occult blood testing (FOBT) is necessary.
§ Annual testing for occult blood has a high positive predictive value for detecting colorectal cancer in the early stages and is recommended
by the American Cancer Society, particularly for people older than age 50.
§ Methods for detecting fecal occult blood include the ① guaiac-based, ② immunochemical, and ③ fluorometric porphyrin quantification
tests – ② and ③ are more sensitive and specific than ①.

• most frequently used screening test for fecal blood

• Principle: pseudoperoxidase activity of hemoglobin
Guaiac-based

o This is the same principle as the reagent strip test for urinary blood, but uses a different indicator chromogen.
(gFOBT)

o The reaction uses the pseudoperoxidase activity of Hb reacting with H2O 2 to oxidize a colorless compound to a colored compound:
1 ,-./01,.213405-.
Hb + H% O% + guaiac 6⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯8 oxidized guaiac + H%O
o Several different indicator chromogens have been used to detect occult blood. All react in the same chemical manner but vary in
their sensitivity.
• Contrary to the norm, guaiac is the least sensitive yet preferred for routine testing.
@asherum_ | BSMT-3E 2020 : ANALYSIS OF URINE AND BODY FLUIDS 8.5

o Considering that a normal stool can

contain up to 2.5 mL of blood, a less
sensitive chemical reactant is
understandably more desirable.
o Pseudoperoxidase activity is
present from hemoglobin and
myoglobin in ① ingested meat and
fish, ② certain vegetables and
fruits, and ③ some intestinal
bacteria.
§ ∴ to prevent false(+) reactions,
test sensitivity must be decreased – accomplished by varying the amount and purity of the guaiac reagent used in the test
• The patient is asked to collect samples from stool specimens collected on 3 consecutive days.
o Two samples from three different stools should be tested before a (-) result is confirmed.
o To prevent the presence of dietary pseudoperoxidases in the stool, patients should be instructed to avoid eating the following for 3
days before specimen collection:
§ cauliflower § radishes § turnips
§ horseradish § raw broccoli § vitamin C; iron supplements
§ melons § red meats w/ vit. C1
• Screening for colorectal cancer.
1
Ascorbic acid is a strong reducing agent that interferes with the peroxidase reaction, causing a false(-) result.
• Many commercial testing kits are available for occult blood testing with guaiac reagent.
• The kits contain guaiac-impregnated filter paper enclosed in a cardboard slide, to which the fecal specimen and H2O2 are added.
o Two or three filter paper areas are provided for application of material taken from different areas of the stool, and (+) and (-) controls
are also included.
o Obtaining samples from the center of the stool avoids false-(+) reactions from external contamination.
o The sample is placed slide is closed.
o Adding H2O2 to the back of the filter paper slide that contains stool produces a blue color with guaiac reagent when
pseudoperoxidase activity is present (positive for occult blood).
• specific for the globin portion of human hemoglobin
• uses polyclonal anti-human hemoglobin antibodies
Immunochemical

• Because this method is specific for human blood in feces, it does not require dietary or drug restrictions.
(iFOBT)

• It is more sensitive to lower GI bleeding that could be an indicator of colon cancer or other GI disease
2
o can be used for patients who are taking aspirin and other anti-inflammatory medications.
• Hemoglobin from upper GI bleeding is degraded by bacterial and digestive enzymes before reaching the large intestine and is
immunochemically nonreactive.
• In contrast, there is little hemoglobin degradation in lower GI bleeding, so the blood is immunochemically active.

• HemoQuant offers a porphyrin-based FOBT fluorometric test for Hb based on the conversion of heme to fluorescent porphyrins.
Porphyrin-based FOBT

o measures both ① intact hemoglobin and the ② hemoglobin that has been converted to porphyrins.
o As hemoglobin progresses through the intestinal tract, bacterial actions degrade it to porphyrin that the gFOBT cannot detect
§ thereby making the HemoQuant test more sensitive to upper GI bleeding.
3
• False-negative results from upper GI bleeding can be seen with the gFOBT.
o the porphyrin-based test is not affected by the presence of reducing or oxidizing substances or the H2O content of the fecal spx
• False-positive results can occur with the porphyrin-based test when non-human sources of blood (red meat) are present
o ∴ patients should be instructed to avoid red meat for 3 days before the test

6.2
QUANTITATIVE FECAL FAT TESTING
• used as a confirmatory test for steatorrhea.
• requires the collection of at least a 3-day specimen.
o The patient must maintain a regulated intake of fat (100 g/d) before and during the collection period.
o The specimen is collected in a large, pre- weighed container.
§ Before analysis, the specimen is weighed and homogenized. Refrigerating the specimen prevents any bacterial degradation.
• method routinely used/gold standard for fecal fat measurement
o ① gravimetric, ② near-infrared reflectance spectroscopy, and ③ nuclear magnetic resonance spectroscopy methods are also
Van de
available
1 Kamer
• Fecal lipids are converted to fatty acids and titrated to a neutral endpoint with NaOH.
Titration
• Approximately 80% of the total fat content is measured by titration, whereas the gravimetric method measures all fecal fat.
o A drawback of the titration/gravimetric method is that it is ① time consuming and ② uses corrosive and flammable solvents.
Hydrogen Nuclear Magnetic Resonance Spectroscopy
• rapid (5 minutes) and safe procedure for analyzing quantitative fecal fat
• homogenized specimen is microwaved-dried and analyzed
H NMR
2 o The results correlate well with the gravimetric method.
method
• The fat content is reported as “grams of fat” or the “coefficient of fat retention per 24 hours”
o Reference values based on a 100 g/d intake are ① 1 to 6 g/d or a ② coefficient of fat retention of at least 95%.
o The coefficient of fat retention is calculated as follows:
@asherum_ | BSMT-3E 2020 : ANALYSIS OF URINE AND BODY FLUIDS 8.6

(dietary fat − fecal fat)

x 100
dietary fat
• rapid test to estimate the amount of fat excretion
Acid • It is similar to the microhematocrit test and is more convenient than a 72-hour stool collection
3
Steatocrit • a reliable tool to monitor a patient’s response to therapy
• screening test for steatorrhea in pediatric populations

6.3
APT TEST (Fetal Hemoglobin)
• Grossly bloody stools and vomitus are sometimes seen in neonates as the result of
swallowing maternal blood during delivery.
o Should it be necessary to distinguish between the presence of fetal blood
or maternal blood in an infant’s stool or vomitus, the APT test may be
requested.
1. The material to be tested is emulsified in water to release hemoglobin (Hb).
2. Centrifuge.
3. Divide pink Hb-containing supernatant into two tubes.
4. Add 1% sodium hydroxide (NaOH) to one tube; other is “control”. Wait 2 minutes.
5. Compare color with that in the control tube:
Pink solution Positive for presence of alkali-resistant fetal hemoglobin (HbF)
Yellow-brown supernatant Negative for HbF; denaturation of the maternal hemoglobin (HbA)
6. Prepare controls using ① cord blood and ② adult blood.
• The APT test can also aid in the presumptive dx of maternal thalassemia major.

6.4
Fecal Enzymes
• Enzymes supplied to the GI tract by the pancreas are essential for digesting dietary proteins, carbohydrates, and fats.
o ↓ production of these enzymes (pancreatic insufficiency) – associated with ① chronic pancreatitis and ② cystic fibrosis.
o Steatorrhea occurs, and undigested food appears in the feces.
• Analysis of the feces focuses primarily on the proteolytic enzymes ① trypsin, ② chymotrypsin, and ③ elastase I.
CHYMOTRYPSIN ELASTASE I
• an isoenzyme of the enzyme elastase
• the enzyme form produced by the pancreas
• capable of gelatin hydrolysis but is most frequently
• present in high concentrations in pancreatic secretions
measured by spectrophotometric methods
• strongly resistant to degradation
• accounts for about 6% of all secreted pancreatic enzymes
• strongly resistant to degradation
• pancreas specific
• more resistant to intestinal degradation • conc. is about 5x higher than in pancreatic juice
• more sensitive indicator of less severe cases of • not affected by motility disorders or mucosal defects
Fecal

pancreatic insufficiency • can be measured by immunoassay using the ELISA kit

• remains stable in fecal spx for up to 10 days @ RT o provides a very sensitive indicator of exocrine pancreatic insufficiency
o specific in differentiating pancreatic from nonpancreatic causes in
patients with steatorrhea

Carbohydrates
• ↑ carbohydrates in the stool produces osmotic diarrhea from the osmotic pressure of the unabsorbed sugar in the intestine drawing in fluid and
electrolytes.
• Carbohydrates in the feces may be present as a result of intestinal inability to reabsorb carbohydrates
o as seen in celiac disease or lack of digestive enzymes (e.g. lactase à lactose intolerance)
• Carbohydrate malabsorption or intolerance (maldigestion) is primarily analyzed by serum and urine tests
o However, an ↑ concentration of carbohydrate à detected by performing a Cu reduction test on the fecal specimen.
§ Testing for fecal reducing substances detects congenital disaccharidase deficiencies and enzyme deficiencies due to nonspecific
mucosal injury.
• Fecal carbohydrate testing is most valuable in assessing cases of infant diarrhea and may be accompanied by a pH determination.
o Normal stool pH = 7-8
§ ↑ use of carbohydrates by intestinal bacterial fermentation à ↑ lactic acid level + ↓ the pH < 5.5 in cases of carbohydrate disorders.
• Copper reduction test
o performed using a Clinitest tablet
§ can distinguish between diarrhea caused by abnormal excretion of reducing sugars and those caused by various viruses and parasites
§ does not detect sucrose (non-reducing sugar)
o one part stool : two parts water.
o A result of 0.5 g/dL is considered indicative of carbohydrate intolerance.
o This is a general test for the presence of reducing substances
o (+) result would be followed by more specific serum carbohydrate tolerance tests
§ D-xylose test for malabsorption (most common)
§ lactose tolerance test for maldigestion.
@asherum_ | BSMT-3E 2020 : ANALYSIS OF URINE AND BODY FLUIDS 8.7

§ Stool chromatography to identify the malabsorbed carbohydrate is available but rarely necessary to diagnose sugar intolerance.
§ Small-bowel biopsy specimens for histologic examination and the assay of disaccharidase enzyme activity differentiate primary from
secondary disaccharidase intolerance.

FECAL SCREENING TESTS

TEST METHODOLOGY/PRINCIPLE INTERPRETATION
Examination for Microscopic count of neutrophils in smear stained with methylene
3/hpf indicates condition affecting intestinal wall
neutrophils blue, Gram stain, or Wright's stain
Qualitative Microscopic examination of direct smear stained with Sudan III 60 large orange-red droplets indicate malabsorption
fecal fats Microscopic examination of smear heated with HAc and Sudan III 100 orange-red droplets measuring 6-75 μm = malabsorption
Pseudoperoxidase activity of hemoglobin liberates oxy- gen from
gFOBT Blue color indicates gastrointestinal bleeding
hydrogen peroxide to oxidize guaiac reagent
Uses polyclonal anti-human antibodies specific for the globin portion
iFOBT Positive test and control lines indicate GI bleeding
of human hemoglobin
Addition of sodium hydroxide to hemoglobin-containing emulsion
APT test Pink color indicates presence of fetal blood
determines presence of maternal or fetal blood
Emulsified specimen placed on x-ray paper determines ability to
Trypsin Inability to digest gelatin indicates lack of trypsin
digest gelatin
Elastase 1 Immunoassay using an ELISA test Sensitive indicator of exocrine pancreatic insufficiency
Addition of Clinitest tablet to emulsified stool detects presence of Reaction of 0.5 g/dL reducing substances suggests
Clinitest
reducing substances carbohydrate intolerance
 ANALYSIS OF URINE AND BODY FLUIDS normal blood brain barrier (BBB) ▪ endocrine medium for the brain: the CSF serves to transport
TOPIC 09: CEREBROSPINAL FLUID ANALYSIS the tight-fitting strucutre of the endothelial cells in the hormones to other areas of the brain
choroid plexuses ▪ hormones released into the CSF can be carried to remote sites
Objectives: is important for the normal chemistry results of CSF of the brain where they may act
▪ Describe CSF in terms of origin, functions and collection procedure maintaining its integrity is essential to protect the brain
CONCENTRATION OF VARIOUS SUBSTANCES IN HUMAN CSF & PLASMA
▪ Define terms related to CSF and laboratory tests & their significance from chemicals and other substances circulating in the
BBB SUBSTANCES CSF PLASMA
▪ Discuss the different diagnostic tests for CSF in terms of its procedure, blood that could harm the brain tissue
Na+ (meq/kg H2O) 147.0 150.0
principles and clinical significance also prevent the passage of helpful substances
K+ (meq/kg H2O) 2.9 4.6
including antibodies and medications
Topic Outline: Ca2+ (meq/kg H2O) 2.3 4.7
disruption of the BBB by diseases (e.g. meningitis,
I. CSF Formation and Function Cl- (meq/kg H2O) 113.0 99.0
multiple sclerosis) allows leukocytes, proteins, and
II. Specimen Collection & Handling HCO3- (meq/L) 25.1 24.8
additional chemicals to enter the CSF
III. Diagnostic Tests for CSF PCO2 (mmHg) 50.2 39.5
IV. Clinical Significance pH 7.33 7.40
osmolality
CSF FORMATION AND FUNCTION 289.0 289.0
(mosm/kg H2O)
a major fluid of the body protein (mg/dL) 20.0 6000.0
provides a physiologic system to supply nutrients to the glucose (mg/dL) 64.0 100.0
nervous tissue, removes metabolic wastes, and cholesterol (mg/dL) 0.2 175.0
produce mechanical barrier to cushion the brain and
the spinal cord against trauma SPECIMEN COLLECTION AND HANDLING
the liquid that surrounds the brain and spinal cord ▪ lumbar tap ▪ cisternal ▪ ventricular
the brain and spinal cord are surrounded by the
meninges that consist of three layers: LUMBAR TAP COLLECTION
▪ dura matter (hard mother) ▪ local anesthesia is employed for lumbar puncture
▪ arachnoid (spindle web-like) ▪ the patient is placed in the lateral recumbent position with the hips, knees,
▪ pia matter (gentle mother) and chin flexed toward the chest so as to open the inter-laminar spaces or
interspinous distance or he chould be in a sitting position
CSF flows between the arachnoid and pia matter in an
area referred to as the subarachnoid space Arachnoid granulation: where the circulating fluid is reabsorbed back at the ▪ a pillow may be used to support the head
cerebrospinal is formed mainly in the choroid plexuses of the lateral rate equal to its production ▪ inter-laminar splace
fluid (CSF) (two lumbar ventricles), third, and fourth ventricles ▪ inter-spinous diameter
RATIO OF FORMATION
some origniate from the ependymal cells lining the 0.2 – 0.7 ml per minute 20 ml per hour ≈ 600 - 700 ml per day
ventricles and from the brain substance 90 – 150 ml in adults 10 – 60 ml neonates
20 ml of CSF is produced every hour
to maintain a volume of 90-150 ml for adults and 10-60 MECHANISM OF EXCRETION (ABSORPTION)
ml in neonates, the circulating fluid is reabsorbed back excretion volume = production volume → constant CSF volume
into the blood capillaries in the arachnoid granulations absorption occurs at the arachnoid villi, protruding to the Dura to the
or villae at a rate equal to its production venous sinuses of the brain → bloodstream
▪ the cells in the arachnoid granulations act as one-way
FUNCTIONS OF CSF
valves that repsond to the presure within the CNS
protection: protects, lubricates the brain
and prevent reflux of the fluid
▪ the CSF protects the brain from damage by "buffering" the brain In the image below, the needle is being piereced through the L3 and L4
mechanism of formation: 1
▪ the CSF acts to cushion a blow to the head and lessen the (lumbar vertebral 3 and 4).
▪ selective ultrafiltration of plasma
impact
▪ active secretion by epithelial membranes
provides nutrients, removes waste
capillary networks that form the CSP from plasma by
▪ 90-150 ml adult
mechanisms of selective filtration under hydrostatic
▪ 10-60 ml newborn
pressure and active transport secretion 2
▪ excretion of waste products: the one-way flow from the CSF to
▪ therefore, CSF’s chemical composition does not
the blood takes potentially harmful metabolites, drugs and
resemble an ultrafiltrate of plasma
other substances away from the brain.
choroid ▪ capillary walls throughout the body are lined with
bouyancy: modulates pressure changes
plexuses endothelial cells that are loosely connected to allow
▪ because the brain is immersed in fluid, the net weight of the
passage of soluble nutrients and wastes between the 3
brain is reduced from about 1,400 gm to about 50 gm
plasma and tissues
▪ therefore, pressure at the base of the brain is reduced
in here, the endothelial cells have very tight-fitting
4 serves as a chemical buffer to maintain constant ionic environment
junctures, called BBB, that prevent the passages of may
serves as a transport medium for nutrients and metabolites,
molecules 5
endocrine substances, and even neurotransmitters

AIRAH M.
 THE CISTERNAL TAP hemorrhage1 2 protein (total and specific)
bloody red cells
trauma/traumatic tap 3 lactate
▪ old hemmorhage 4 lactate dehydrogenase
Hb
▪ lyzed cells 5 glutamine and acid-base parameters
▪ RBC breakdown
bilirubin CEREBROSPINAL PROTEIN
xantochromic2 ▪ increased serum bilirubin
TOTAL PROTEIN is the most common test
carotene increased serum levels
▪ normal 15 to 45 mg/dL (mg, not grams)
CHON RBC breakdown
o method dependent
melanin melanosarcoma o increased in infants and persons >40 years old
grayish or ▪ albumin is predominant, prealbumin is second
WBCs TB meningitis
greenish ▪ presence of alpha globulins, haptoglobin, and ceruloplasmin
1 uneven distribution of blood = traumatic, even distribution = hemorrhage
2
▪ transferrin is major beta globulin
term used to describe CSF supernatant that is pink, orange, or yellow
▪ TAU, carbohydrate-deficient transferrin seen in CSF, not in blood
▪ depending on the amount of blood and the length of time it has
o used to identify the CSF → differentiate water and CSF
Refer to image above. SUBOCCIPITAL PUNCTURE or CISTERNAL PUNCTURE is been present, the color will vary from:
▪ IgG: major gamma globulin
done by inserting a needle through the skin below the external occipital pink orange yellow
▪ fibrinogen and beta lipoprotein: absent in CSF
protuberance into the cisterna magna. This is done behind the neck. conersion of
very slight amount heavy PROTEINS (15-45 mg/dL)
oxyhemoglobin to
VENTRICULAR TAP of oxyhemoglobin hemolysis
unconjugated bilirubin INCREASED IN DECREASED IN
▪ RBCs usually remain in the CSF for appx. 2 hours before noticeable ▪ meningitis
hemoylsis begins, therefore, a xantochromic supernatant would be ▪ hemorrhage
the result of blood that has been present longer than that ▪ multiple sclerosis
introduced by the traumatic tap ▪ Guillan-Barre syndrome ▪ CSF leakage/trauma
▪ a very recent hemorrhage would produce a clear supernatant, and ▪ neurosyphilis ▪ recent puncture
introduction of serum protein from a traumatic tap could also ▪ polyneuritis ▪ rapid CSF production
cause the fluid to appear xantochromic ▪ myxedema ▪ water intoxication
▪ to examine a bloody fluid for the presence of xantochromia, the ▪ Cushing disease
fluid should be centrifuged in a microhematocrit tube and the ▪ diabetes
supernatant examined against a white background ▪ uremia
▪ additional testing for differentiation includes: microscopic
[

examination and the D-dimer test QUALITATIVE TESTS

▪ microscopic finding of macrophages containing ingested RBCs 1 ROSS-JONES TEST (excess of globulin in CSF)
(erythrophagocytosis) or hemosiderin granules indicates ▪ an obsolete test for an excess of globulin in the cerebrospinal fluid
This is usually done for babies wherein the fontenelles are still not closed and intracranial hemorrhage ▪ 1 mL of cerebrospinal fluid is carefully floated over 2 mL of a
are still not hardened. The puncture is made near the anterior fontenelle. ▪ detection of the fibrin degradation product D-dimer latext concentrated ammonium sulfate solution
agglutination immunoassay indicates fibrin formation at a ▪ if globulin is present in excess, a fine white ring appears at the line of
CSF PLACED IN 3 TUBES hemorrhage site junction in about 3 min.
1st chemistry and serology which are frozen 2 NONNE APELT
2nd microbiology remains at RT DIFFERENCES BETWEEN TRAUMA AND CEREBRAL HEMORRHAGE ▪ reagent: ammonium sulfate
3rd hematology which are refrigerated TRAUMATIC TAP HEMORRHAGE ▪ presence of white ring of ppt gives positive test (both 1 and 2)
4th may be collected for additional test ▪ more blood in test tube 1 than ▪ even distribution of blood in 3 3 PANDY’S TEST
▪ If only one tube can be collected, it must be tested first by tubes 2 & 3 tubes ▪ reagent: phenol
microbiology. ▪ form clots ▪ does not form clot ▪ presence of bluish white cloud gives positive test
▪ It is not unusual for cell counts requested to be performed on both ▪ no xantochromasia ▪ xantochromic supernatant ▪ done on the CSF to detect the elevated levels of proteins, mostly
Tubes 1 and 4 to check for cellular contamination by the puncture. pH specific gravity pressure globulins
MACROSCOPIC EXAMINATION 50-200 mm Hg ▪ named after Hungarian neorologist, Pandy Kalman who developed this
▪ volume:120-150 ml 7.3-7.45 (7.31) 1.006-1.008 (120 as average) test in 1910
* Quekenstedt test 1 ▪ principle: proteins, globulins, and albumin are precipitated by a
▪ coagulation: no clot 1
a test for spinal blockage of the subarachnoid space in which manual saturated solution of phenol in water
o TB meningitis: Cobweb’s or pine tree like clot (pellicle formation)
pressure is applied to the jugular vein to elevate venous pressure, which ▪ the reagents used are: phenol or carbolic acids (crystals dissolved with
▪ appearance: clear and colorless
indicates the absence of a block when there is a simultaneous increase in water) or pyrogallic acid (crystals usually terms as Pandy’s reagent or
VARIATION IN COLOR cerebrospinal fluid pressure, and which indicates the presence of a block solution)
COLOR CAUSES CONDITIONS when cerebrospinal fluid pressure remains the same or almost the same ▪ the procedure is 1 drop of CSF to 1ml of Pandy’s reagent
WBCS meningitis ▪ the turbid appearance signifies the presence of elevated levels of
CHEMICAL EXAMINATION globulin protein in the CSF and is regarded as POSITIVE
hazy/cloudy RBCs hemorrhage, trauma
milky/turbid disorders which affect blood ROUTINELY PERFORMED CSF BIOCHEMICAL TESTS ▪ no turbidity means NEGATIVE
CHONs 1 glucose
brain barrier

AIRAH M.
▪ proteins in the CSF, normally albumin and globulin, are present in the ▪ banding representing both systemic and neurologic involvement is SIGNIFICANCE:
ratio of 8:1 seen in the serum and CSF with HIV infection ▪ bacterial meningitis
▪ increases of protein levels are of diagnostic value in neurological ▪ presence of 2 or more bands in the CSF that are not present in the ▪ high CSF LS 76 hrs. ff. resuscitation predicts poor prognosis of hypoxic
diseases serum is a tool in the diagnosis of diseases with an increased IgG index brain injury
▪ a POSITIVE test shows a bluish white ring of precipitated proteins o multiple sclerosis o Guillain-Barre syndrome ▪ CK BB
▪ the degree of turbidity depends on the amount of protein in the CSF o encephalitis o neoplastic disorders o normal value: 17 mgs/ml or < 5 U/L
▪ it can vary from plain (?) turbidity, mild to moderate elevation in CSF o neurosyphilis o elevated levels: seizures, strokes, tumors, head injury
proteins so dense milky white where you can find high protein content ▪ oligoclonal bands (OCBs) are bands of immunoglobulins that are seen SIGNIFICANCE OF CHEMICAL EXAM
in CSF (??? I literally can’t decipher the words well on this bullet, i-) when a patient's blood serum, or cerebrospinal fluid (CSF) is analyzed REF. CSF SIGNIFICANCE SIGNIFICANCE
CHEMICAL
▪ POSITIVE results can be seen in diabetes mellitus, brain tumors, brain o are used in the diagnosis of various neurological and blood VALUE IF ⇧ IF ⇩
abscesses, neurosyphilis, and meningitis diseases, especially in multiple sclerosis ▪ meningitis
4 NOGUCHI’S TEST ▪ better resolution can be obtained using CSF immunofixation protein 15-45 mg/dl ▪ hemorrhage CSF leakage
▪ reagent: 10% of butyric acid electrophoresis (IEF) and ion electric focusing (IEF) followed by silver ▪ multiple sclerosis
▪ presence of ppt is positive test staining ▪ bacterial
o these techniques are also the method of choice when determining 60-70% of
glucose none ▪ tubercular
QUANTITATIVE TESTS whether a fluid is actually CSF plasma conc.
▪ fungal meningitis
o turbidimetric o dye binding technique >35 mg/dl:
1 GLUCOSE lactate 10-24 mg/dl none
o nephelometric o biuret bacterial meningitis
▪ 50-80 mgs./dlm or 2.75-4.4 mmol/L (60% - 70% of the plasma glucose)
▪ if the methods used in testing for proteins are turbidimetery and dye- ▪ note: for accuracy, must run with plasma glucose taken 2 hrs after the >35 mg/dl:
binding methods, the routine CSF protein procedures are desired to spinal tap glutamine 8-18 mg/dl some disturbance of none
measure TP concentration however, diagnosis of neurologic disorders ▪ glucose enters the CSF by selective transport across the BBB consciousness
associated with abnormal CSF protein often requires measurement of ▪ diagnostic significance of CSF glucose is confined to finding values that
individual protein fractions MICROSCOPIC EXAMINATION
are decreased relative to plasma
▪ diseases including multiple sclerosis that stimulate immunocompetent ▪ decreased CSF glucose values are caused primarily by alterations in the CELL COUNT* DIFFERENTIAL COUNT
cells in the CNS show a higher proportion of IgG mechanisms of glucose transport across the BBB and by increased use adult: 0-5/uL children: 0-30/uL newborn: 30 mononuclears/uL
▪ to accurately determine whether IgG is increased because it is of glucose by the brain cells * usually in very normal CSF, there is no cells present that is why we have
produced within the CNS or is elevated as a result of a defect in the a normal value of 0-5/uL
BBB, comparisons between serum and CSF levels of albumin and IgG QUALITATIVE QUANTITATIVE
must be made copper reduction tests ortho-toluidine 1 CELL COUNT
▪ the determination of CSF over serum albumin index is to evaluate the ▪ Fuch’s Rosenthal counting chamber: cells/cu.mm.= no. of cells/3
integrity of the BBB INCREASED IN DECREASED IN ▪ Neubauer Counting chamber:
▪ CSF IgG index is to measure IgG synthesis within the CNS ▪ decreased hypoglycemia o cells/cu.mm = cells counted x Area x Dilution x Depth
1 TURBIDIMETRIC ▪ pyogenic meningitis ▪ CSF Diluting Fluid:
▪ diabetes mellitus
principle: in the presence of ▪ fungal meningitis o crystal violet 0.200 gm
▪ infectious
sulphosalicylic acid and sodium ▪ subarachnoid hemorrhage o glacial acetic acid 10.000 ml
encephalitis
sulphate, proteins a uniform turbidity ▪ toxoplasmosis tumors in the brain o distilled water 90.000 ml
which absorbs maxium at 520 nm or disorders which affect blood brain barrier o final pH (at 25°C) 2.1 ± 0.05
green filter and is directly ▪ these specimens can be counted and diluted provided no overlapping
proportional to the protein Bacterial M. Tubercular Viral M of cells is seen during the microscopic examination
concentration glucose decreased decreased normal ▪ if needed to be diluted, use automatic pipettes with NSS as a dilute
cells PMNs lymphocyte lymphocyte ▪ count in 4 corners in the center square on both sides of the
composition:
2 LACTATE hemacytometer
▪ CSF protein reagent
2 DIFFERENTIAL COUNT
o sulphosalicylic acid: 30 gms/lt. normal value: 10-22 mgs/dl
o sodium sulphate: 70 gms/lt. ▪ lysis of RBC must be done on either diluted or undiluted specimens
if >25 mgs/dl if remain <25 increased levels ▪ use 3% glacial acetic acid to lyse the RBCs
▪ Standard: albumin fraction (100 ▪ bacterial ▪ standard dilution of blood for WBC is 1:20 (dilution factor is 20)
mgs/lt) ▪ tubercular viral meningitis hypoxia ▪ volume of diluted blood used is based on the area and depth of the
2 NEPHELOMETRIC ▪ fungal meningitis counting area
3 DYE BINDING TECHNIQUE
3 GLUTAMINE ▪ e.g area counted is 4 mm, depth is .1 mm = .4 cu/mm (volume factor)
dye: Coomassie Brilliant Blue G 250
▪ produced from ammonia & α-ketoglutaraldehyde ▪ utilizes stained smear of the sediments
4 BIURET ▪ can be from:
▪ for removal of toxic waste product of ammonia from CNS
ELECTROPHORESIS & IMMUNOPHORETIC TECHNIQUES ▪ normal value: 8-18 mgs./dl ▪ sedimentation ▪ centrifugation
▪ primary purpose: detection of oligoclonal bands o increased levels: excess ammonia in liver disorders ▪ filtration ▪ cytocentrifugation
(represent inflammation in the CNS) within the o elevated levels found in Reye’s syndrome ▪ 100 cells counted, classified & reported in %
CNS 4 LD ISOENZYMES ▪ normal lymphocytes:monocytes in adults: 70:30
▪ bands are located in the gamma region of the ▪ to observe pleocytosis (increased number of WBC in CSF)
NORMAL VALUES
protein electrophoresis, indicating immunoglobin
production 40 U/L (adults) 70 U/L (neonates)

AIRAH M.
 STAINS FOR CSF leukemia; are also found of nonhematologic origin (e.g. lung, breast, pH 7.4
Wright-Giemsa Stain (hema) India Ink Preparation (micro) renal, GIT malignancies) in which cells from primary CNS tumors daily secretion 450-500 ml
Gram Stain (micro) Modified Ziehl-Neelsen Stain (micro) include astrocytomas, retinoblastomas, and medulloblastomas specific gravity 1.006-1.007
images found on the last page DIFFERENTIAL DIAGNOSIS OF MENINGITIS NORMAL MICROSCOPIC EXAMINATION
BACTERIAL VIRAL TUBERCULAR FUNGAL lymphocytes 1-5/HPF
SIGNIFICANCE OF MICROSCOPIC EXAM
elevated WBC elevated WBC elevated WBC elevated WBC NORMAL CHEMICAL EXAMINATION
CELL TYPE SIGNIFICANCE FINDINGS
count count count count protein 15-45 mg/dl
▪ normal
▪ viral neutrophils lymphocytes lymphocytes lymphocytes glucose 50-80 mg/dl
all stages of development chloride 115-130 mmol/L
lymphocytes ▪ tubercular present present and monocytes and monocytes
may be found calcium 1.0-1.40 mmol/L
▪ fungal meningitis marked CHON moderate CHON mod to marked mod to marked
▪ multiple sclerosis elevation elevation elevation elevation phosphorus 0.4-0.7 mmol/L
▪ bacterial meningitis markedly normal to magnesium 1.2-1.5 mmol/L
normal glucose decreased potassium 2.6-3.0 mmol/L
▪ early cases of viral, ▪ granules may be less decreased decreased
level glucose
neutrophils tubercular, fungal prominent than in blood glucose level glucose NORMAL MICROBIOLOGICAL EXAMINATION
meningitis ▪ cells disintegrate rapidly lactate: normal lactate >25 mg/dl with no pathogenic microorganisms
>25 mg/dl
▪ cerebral hemorrhage >35 mg/dl level pellicle formation
Additional: During our internship soon ( ♥), when we will be assigned on
▪ normal (+) C. neoformans
gram stain (+) (+) immunologic histopath, please be very careful in handling the specimens. It’s not only
▪ viral found mixed in
monocytes BAT3 (+) test for C. because that some of them are very virulent and infectious, but it is more
▪ tubercular lymphocytes
neoformans important that we ask the supervisors and MTs as to whether we will discard
▪ fungal meningitis 3
bacterial antigen test the CSF. This is because some of them will look just like water and their
may contain phagocytized
OTHER LAB TESTS FOR CSF volume is very small in which sometimes you cannot see them. Other
RBC’s appearing as empty
macrophages RBCs in spinal fluid vacuoles or ghost cells, 1 MICROBIOLOGICAL TESTS: GRAM STAIN, C/S specimens to be very careful about are the needle biopsies wherein you can
hemosiderin granules & ▪ S. pneumoniae ▪ N. meningitidis barely see the tissue. So, we have to ask the MT as to what will be discarded
hematoidin crystals ▪ H. influenzae ▪ S. agalactiae or not. If there is a violation of negligence of duty, there is an extension of 7-
▪ lymphoblast ▪ E. coli ▪ L. monocytogenes 30 days. Hence, you have to be very careful in handling histopath specimens.
blast forms acute leukemia ▪ myelobast 2 BACTERIAL ANTIGEN TESTS (BAT) REFERENCES
▪ monoblast ▪ latex agglutination ▪ Urinalysis & Body Fluids by Strasinger, 6th ed
disseminated resemble lymphocytes ▪ use: community acquired meningitis that isnegative for Gram’s stain
lymphoma cells ▪ Fundamentals of Urine & Body Fluid Analysis by Brunzel, 4 th ed.
lymphomas with cleft nuclei 3 VDRL
▪ traditional & classic ▪ Clinical & Laboratory Management by Henry’s, 24th ed
▪ multiple sclerosis ▪ Venereal Disease Research Laboratories
plasma cells forms seen ▪ for diagnosis of neurosyphilis
▪ lymphocyte reaction
▪ reactive lymphocytes ▪ alternative test: Fluorescent Treponemal Antibody-Absorption Test
ependymal, (FTA-ABS)
seen in clusters with
choroidal and 4 LYMULUS LYSATE ASSAY
diagnostic procedures distinct nuclei and distinct
spindle-shaped ▪ test for the presence of endotoxin
walls
cells ▪ identifies all gram negative bacterial meningitis
▪ metastatic ▪ based on a gelation reaction between lysates of Limulus (horseshoe
seen in clusters with
carcinoma crab) amebocytes and bacterial endotoxin
malignant cells fusing of cell boarders and
▪ primary CNS
nuclei
carcinoma
Additional info:
▪ increase in eosinophils are seen in asosciation with parasitic
infections, ufngal infections (primarily Coccidioides immitis), and
introduction of foreign material, including medications and shunts,
into the CNS
▪ macrophages: purpose in the CSF is to remove cellular debris and
foreign objects such as RBCs
o are commonly seen following repeated taps
o increased macrophages indiciate previous hemorrhage
o further degradation of the phagocytized RBCs results in the
appearance of dark blue or black iron-containing hemosiderin
granules
NORMAL CSF ANALYSIS
o yellow hematoidin crystals represent further degeneration
▪ malignant cells can be found of hematologic origin (e.g. lymphoblasts, NORMAL PHYSICAL EXAMINATION
myeloblasts, monoblasts) in the CSF as serious complications of acute appearance and color clear, colorless

AIRAH M.
AIRAH M.
 ANALYSIS OF URINE AND BODY FLUIDS PRIMARY CAUSES OF EFFUSION ▪ pH determination specimens must be maintained anaerobically in ice
TOPIC 10: SEROUS FLUIDS 1 ⇧ hydrostatic pressure (congestive heart failure) ▪ for better recovery of microorganisms & abnormal cells, at least 100
found in closed cavities of the body, mainly the pleural, ⇩ oncotic pressure (hypoproteinemia, nephrotic syndrome, hepatic ml of the fluid is concentrated by centrifugation (preferable
2
peritoneal, and pericardial cavity, which are lined by two cirrhosis, malnutrition) cytocentrifugation) & used for analysis
membranes referred as “serous membranes” 3 ⇧ capillary permeability (inflammation, infection, malignancy)
▪ parietal membrane: lines the cavity wall
LABORATORY INVESTIGATION
4 lymphatic obstruction (tumors, lymphoma, thoracic duct injury)
▪ visceral membrane: covers the organs within the cavity 1 PHYSICAL EXAMINATION
the fluid is called “serous” because they resemble the serum TECHNIQUES OF SPECIMEN COLLECTION ▪ amount: measure volume in a graduated cylinder
the fluid between the membranes which provide lubrication as refers to the percutaneous puncture of a body
the surfaces move against each other paracentesis ▪ color & transparency:
cavity for the aspiration and removal of fluid o note blood (fresh or changed), chyle, bile
normally only a small amount of serous fluid is present because
thoracentesis collection of pleural fluid o fluids maybe described as:
production & reabsorption takes place at a constant rate
are formed as ultrafiltrates of plasma with no additional material collection of peritoneal fluid ✓ serous: when it looks like a serum
contributed by the membrane cells (unlike in synovial fluid peritoneocentesis most physicians this technique as abdominal ✓ fibrinous
wherein the memberane cells will produce hyaluronic acid which parasynthesis ✓ purulent: have an increased number of pus cells
serous fluid is responsible for its viscous nature) pericardiocentesis collection of pericardial fluid ✓ milky (chylous)
the small amount of filtered protein is removed by the lymphatic
✓ putrid: which has a foul order
system because there is a slight difference in the positive
pressure which will cause an excess of fluid in the cavity in which ✓ sanguinous: bloody
these are removed by the lymphatics ✓ or combination of these (e.g. serosanguinous)
production and reabsorption are subject to hydrostatic pressure ▪ specific gravity: use refractometer
& colloidal (oncotic) pressures from the capillaries serving the ▪ pH: reagent paper strips
cavities 2 BIOCHEMICAL EXAMINATION
under normal conditions, colloidal pressure from serum proteins routinely incude:
is the same in the capillaries on both sides of the membrane
▪ protein ▪ glucose ▪ cholesterol ▪ LDH
▪ therefore, the greater hydrostatic pressure in the systemic
capillaries on the parietal side favors fluid production through chemical tests performed on serous fluids are frequently compared with
the parietal membrane and reabsorption through the visceral plasma chemical concentrations because the fluids are essentially
membrane plasma ultrafiltrates
the accumulation of fluid between the membranes which is
▪ therefore, blood specimens should be obtained at the time of collection
caused by the disruption of the mechanisms of serous fluid
effusion ▪ the blood and the serous fluids are run together in the lab
formation & reabsorption
when this is observed, a serous fluid examination is done 3 BACTERIOLOGICAL EXAMNATION
Here is an illustration on the aspiration of pleural fluid (thoracentesis) using a
hydrothorax accumulation of fluid in the thoracic cavity depending on the suspected condition:
needle and a syringe. The needle is inserted into the pleural space where the ▪ Gram stain: for the other organisms
hydro
accumulation of fluid in the pericardial cavity fluid is found. The fluid is aspirated, careful not to damage or injure the organ.
pericardio ▪ Acid Fast stain: e.g. TB
also called as hypoperitoneum ▪ Culture ( Routine & Acid fast): e.g. TB
ascites accumulation of fluid in the peritoneum cavity in which the fluid ▪ Cytologic Exam: Cell Count & Differential Count (e.g. hema section: uses
is now called ascitic fluid hemacytometer)
▪ Papanicolaou Smear for malignant cells in which the techniques are:
o after centrifugation of the fluid, get a sedminent and make a smear,
do not allow it to dry (unlike it onter smears), and directly immerse
it in a fixative, then stained afterwards with Wright’s stain
CLOT FORMATION
▪ record presence or absence & state whether clot formation is slight,
weblike, or en masse (a hole, one big clot)
▪ reflects the fibrinogen content
o normally, fibrinogen cannot pass through the walls of the
capillaries since they are macromolecules
o fibrinogen is a large molecular protein & does not escape unless
The image above shows abdominal parasynthesis. The needle is inserted so there is considerable damage to the mesothelium
that fluid is aspirated in between the parietal and visceral membrane.
BIOCHEMICAL EXAMINATION
SPECIMEN COLLECTION 1 PROTEINS
Unlike CSF, an abundant fluid (greater than 100 ml) is usually collected, SCREENING TESTS
therefore suitable specimens are available for each section of the lab ▪ Refractometer: Total Solids (TS) meter
In the image above, we can see that there is a small space between the
EDTA tube heparinized tube Use one drop of the sample to read the total protein content in the
parietal membrane and the visceral memberane. In this space is where the refractometer.
for chemical, serologic,
serous fluid is found for lubrication as the organs move. This production of ▪ Rivalta Test:
used for cell counts & differential microbiologic & cytologic
serous fluid is governed by two important pressures called oncotic pressure Add 1 drop of glacial acetic acid to 100 ml.water in a graduated cylinder.
analysis
(plasma osmotic pressure) and the hydrostatic pressure. Allow 1 drop of fluid, free from cells, to fall into this solution.

AIRAH M.
 RESULTS AND INTERPRETATION (RIVALTA TEST) ② EXAMPLE 2 ROUTINE FLUID EXAM (including classification as transudate or exudates,
normal body fluids transudates inflammatory exudates ▪ specimen diluted: 1/20 appearance, cell ct. & diff., chem., micro procedure) is performed in the same
heavy precipitates often depends on the MT and how turbid the sample is (1/10, 1/20, 1/100, manner on all serous fluids. However, the significance of the test results and
no cloud only slight clouds
falling to the bottom 1/200 if it is very cloudy) in which you should know how to obtain the the need for specialized tests vary among fluids, that is why we test the fluids
QUANTITATIVE TESTING factor separately.
same as in blood determination ▪ WBC counted: 96
PLEURAL FLUID
2 GLUCOSE 10 1 1 10 𝟏 systemic disorders that result in production of:
▪ SCREENING TEST: reagent srip F= x x = =
1 10 20 200 𝟐𝟎
▪ QUANTITATIVE TEST: same procedure as blood transudates exudates
▪ INTERPRETATION: 96 x 20 = 𝟏, 𝟗𝟐𝟎 𝐖𝐁𝐂/𝐜𝐮. 𝐦𝐦. congestive heart failure pneumonia
o concentration = plasma glucose, the level of which should be hypoalbuminemia carcinoma (lung cancer)
TRANSUDATES AND EXUDATES
determined at the same time the level of serous fluid is measured
Many pathologic conditions can cause a build up (effusion) of serous fluid. ▪ a useful test in the differentiation:
o bacterial (including tuberculous) infections, RA & malignancies:
However, a general classification of the cause of the effusion can be o PF cholesterol o PF:serum cholesterol ratio o PF : bilirubin ratio
level is depressed or ⇩
accomplished by separating the fluid into the category of transudate or
o in SLE, the glucose level is normal (while rest of the parameters will APPEARANCE
exudate.
show exudation): a phenomenon that may help distinguish SLE clear, pale yellow normal and transudate pleural fluids
effusions that form because of a systemic disorder that
from RA since both conditions show exudation, however for RA, turbid presence of WBCs & indicates bacterial infection,
disrupts the balance in the regulation of fluid filtration
glucose is ⇩ (exudates) TB, or an immunologic disorder (e.g. RA)
and reabsorption such as:
3 LDH can signify hemothorax (traumatic injury:
▪ changes in hydrostatic pressure created by
▪ serum & serous fluid levels must be determined simultaneously inpuring? of blood into the thoracic cavity),
congestive heart failure, or bloody3
▪ primary & metastatic tumors involving the mesothelium produce transudates membrane damage (in malignancy), or a
▪ the hypoproteinemia associated with nephrotic
elevated LDH levels in serous fluid traumatic aspiration
syndrome (⇧ hydrostatic pressure or ⇩ plasma
▪ traditionally, SpGr & protein were considered to be the most valuable presence of chylous material from thoracic duct
oncotic pressure)
criteria for classification, however, fluid/blood ratios for protein & LDH leakage or to pseudochylous material produced
resulting from a mechanical process (either hydrostatic
have provided greater reliability milky in chronic inflammatory conditions
or plasma oncotic pressure is affected)
on grossly bloody specimens with high protein produced by conditions that directly involve the ▪ needs to be differentiated also (elaborated on
levels, the RBC should be lysed using hypotonic membranes of a particular cavity, including infections & table below)
3
cell count saline or saline with saponin prior to dilution exudates malignancies (⇧ capillary permeability or ⇩ lymphatic bloody:
▪ will prevent any unusual clumping or clotting of resorption such as tumor) ▪ in a traumatic tap: blood appears streaked and uneven, lesser
the fluid from an inflammatory process aspirated blood… (I can’t understand what maam’s saying i-)
Wright stained smear after cytocentrifugation ▪ to differentiate hemothorax from hemorrhagic exudates: run a
any suspicious cells seen on the diff. ct should be TRANSUDATE EXUDATE HEMATOCRIT on the bloody pleural fluid (run both fluids and compare
referred to the cytology lab or the pathologist appearance clear cloudy the result)
differential count specific gravity < 1.015 > 1.015 ▪ hemothorax: fluid hematocrit is more than 50% of the whole blood
if ever there is a cell you cannot identify, never
total protein1 < 3.0 g/dl > 3.0 g/dl hematocrit, because the effusion is actually occurring from the
consider it as a skip to side?? (skipping the cell
fluid : serum protein ratio < 0.5 > 0.5 inpouring of blood from the injury
without counting)
LDH < 200 IU > 200 IU ▪ chronic membrane disease: effusion contains both blood & increased
In the lab, we can use either a diluted or undiluted specimen. We charge the fluid : serum LD ratio < 0.6 > 0.6 pleural fluid, resulting in a much lower hematocrit
counting chamber directly, and count 10 big squares (1 square mmh) so that cell count2 < 1000 /uL > 1000 / uL EXAMPLES
in 1 chamber, you have a total area of 9mm2 wherein you can add the central spontaneous clotting no possible bloody pleural fluid chronic membrane
square of the second chamber to make it 10. You can also count 5 squares in hemathorax
pleural fluid cholesterol < 60 mg/dl > 60 mg/dl is due to: disease
1 chamber, the four corner squares and one central square, plus the 5 pleural fluid : serum chole ratio < 0.3 > 0.3 WB Hct 50 vol% 50 vol%
squares on the other chamber. pleural fluid : bilirubin ratio < 0.6 > 0.6 pleural fluid Hct 35 vol% 20 vol%
serum-ascites albumin gradient2 > 1.1 < 1.1
① EXAMPLE 1: CELL COUNT 1
total protein and cell count: DIFFERENTIATION BETWEEN CHYLOUS AND
▪ undiluted specimen (clear with slight cloudiness) ▪ the cut of value of 3.0 g/dl and 1000/uL, respectively, is used for PSEUDOCHYLOUS PLEURAL EFFUSIONS
▪ 125 WBCs counted in 10 big squares of the Neubauer counting chamber pleural and pericardial fluid CHYLOUS EFFUSION
PSEUDOCHYLOUS
▪ a lower value of 2.5 g/dl and 500/uL, respecitvely, is used for EFFUSION
F = Area x Depth X Dilution peritoneal fluid cause thoracic duct leakage chronic inflammation
2 appearance milky/white milky/green tinge
10 1 1 serum-ascites albumin gradient:
F= x x =1 predominantly
1 10 1 ▪ usually used in peritoneal fluid because it is harder to classify them, leukocytes mixed cells
lymphocytes
hence, serum albumin and ascitic fluid is determined
125 x 1 = 𝟏𝟐𝟓 𝐖𝐁𝐂/𝐜𝐮. 𝐦𝐦. cholesterol
absent present
crystals
triglycerides >110 mg/dl <50 mg/dl
Sudan III staining strongly positive negative/weakly positive

AIRAH M.
 HEMATOLOGY TESTS ⇧ amylase ⇧ adenosine deaminase (ADA) LABORATORY TESTS
▪ levels over 40 U/L are highly ▪ are primarily directed at determining if the fluid is a transudate or an
differential is the most diagnostically significant hematology test
indicative of TB exudate
cell count performed on serous fluids
▪ pancreatitis ▪ recommended for TB diagnosis ▪ fluid : serum protein ratio
⇧ eosinophils (>10%) ⇧ lymphocytes ▪ often elevated first in the pleural since you can have a faster result, ▪ LD ratio
▪ TB fluid compared to a TB (AFB) culture in ▪ WBC count: >1000WBCs/uL with a high percentage of neutrophils can be
▪ trauma resulting in the presence of ▪ PF amylase, including salivary which results can be given after
▪ viral infections indicative of bacterial endocarditis
air or blood (pneumothorax & amylase may also be elevated in three weeks to one month
▪ malignancy ▪ cytologic examination of pericardial exudates for the presence of
hemothorax) in the pleural cavity esophageal rupture & ▪ this test is considered 98% sensitive
▪ autoimmune disorders (e.g. RA, malignant cells: an important part of the fluid analysis
▪ also in allergic reactions & parasitic malignancy and 96% specific
SLE) ▪ pericardial fluid tumor marker levels correlate well with cytologic studies
infections ▪ also frequently elevated with
▪ L.E. cells may be seen
malignancy ▪ bacterial cultures and Gram stains are performed on concentrated fluids
⇧ PF neutrophils ⇧ PF plasma cells
triglyceride levels ⇧ PF lactate when endocarditis is suspected
▪ bacterial infection (pneumonia)
to confirm the presence of a ▪ infections are frequently caused by previous respiratory infections
▪ effusions resulting from bacterial infection
tuberculosis chylous effusion including:
pancreatitis and pulmonary
infarction o Haemophilus o Adenovirus
MICROBIOLOGIC AND SEROLOGIC TESTS o Streptococcus o Coxsackievirus
not an unusual finding since they line the serous cavities microorganisms primarily associated with pleural effusions: o Staphylococcus
are pleomorphic: they resemble lymphocytes, plasma ▪ Staphylococcus aureus ▪ Anaerobes
PERITONEAL FLUID
cells, & malignant cells, frequently making identification ▪ Enterobacteriaceae ▪ Mycobacterium tuberculosis
difficult accumulation of fluid between the peritoneal membranes
mesothelial LABORATORY EXAMINATIONS
⇧ mesothelial cells: not diagnostically significant but may performed when clinically indicated ascites fluid is commonly referred to as ascitic fluid rather than
cells
be increased in pneumonia & malignancy peritoneal fluid
1 gram stain, cultures (both aerobic and anaerobic)
▪ of more significance is the noticeable LACK of 2 acid- fast stains and mycobacteria cultures FREQUENT CAUSES OF:
mesothelial cells: associated with TB, which results
serologic testing of PF is used to differentiate effusions of ASCITIC TRANSUDATES EXUDATIVE FLUIDS
from exudates covering the pleural membranes 3
immunologic origin from noninflammatory processes ▪ bacterial infections (peritonitis):
normal mesothelial cells reactive mesothelial cells hepatic disorders often as a result of intestinal
TWO MOST FREQUENTLY PERFORMED SEROLOGICAL TESTS
appear in clusters; have varying (e.g. cirrhosis) perforation or a ruptured appendix
appear as single, small or large ANA (antinuclear antibody test): for SLE RF ( Rheumatoid Factor test)
amounts of cytoplasm, eccentric ▪ malignancy
round cells with abundant blue
nuclei & prominent nucleoli and be TUMOR MARKERS
cytoplasm & round nuclei with PERITONEAL LAVAGE ANALYSIS
multinucleated so they more provide valuable diagnostic information in effusions of malignant origin
uniform dark purple cytoplasm ▪ a sensitive test for the detection of intra-abdominal bleeding in blunt
closely resemble malignant cells CEA (Carcinoembryonic antigen) CA 125 (metastatic uterine cancer)
trauma cases
CA15.3 and CA 549 (breast cancer) CYFRA 21-1 (lung cancer)
DISTINGUISHING CHARACTERISTICS OF MALIGNANT CELLS ▪ lavage fluid: normal saline is sometimes introduced into the peritoneal
detection of malignant cells: primary concern in the exam of all serous PERICARDIAL FLUID cavity to act as a lavage for the detection of abdominal injuries that
effusions ▪ appearance is the same with the other two fluids however, this is the least have not yet resulted in the accumulation of fluid
1 nuclear and cytoplasmic irregularities performed among the three fluids because of the danger of accidental ▪ results of the RBC Count can be used along with radiographic
2 hyperchromatic nucleoli puncture procedures to aid in determining the need for surgery
3 cellular clumps with cytoplasmic molding (community borders) ▪ RBC counts: >100,000/uL are indicative of blunt trauma injuries
▪ normally, only a small amount (10 – 50 ml) of fluid is found between the
4 abnormal nuclear-to-cytoplasmic ratios pericardial serous membranes APPEARANCE
special staining techniques and flow cytometry may be used for positive ▪ pericardial effusions are primarily the result of changes in the permeability clear & pale yellow normal peritoneal fluid
identification of tumor cells of the membranes due to infection (pericarditis), malignancy, and trauma- turbid bacterial or fungal infections
CHEMISTRY TESTS producing exudates indicates presence of bile (can be confirmed
green or dark brown
most common tests performed ▪ the presence of an effusion is suspected when cardiac compression using standard chemical tests for bilirubin)
(aside from those used to differentiate transudates and exudates) (tamponade) is noted during the physician’s examination following trauma, & with tuberculosis,
blood streaked
intestinal disorders and malignancy
⇩ glucose pH PRIMARY CAUSES OF TRANSUDATES chylous or may be present with trauma or blockage of
▪ PF pH lower than 7.0 may indicate uremia hypothyroidism autoimmune disorders
the need for chest-tube drainage, in pseudochylous material lymphatic vessels
▪ TB addition to administration of APPEARANCE LABORATORY TESTS
▪ rheumatoid inflammation antibiotics in cases of pneumonia clear and pale yellow normal & transudate pericardial fluid
▪ purulent infections ▪ PF pH at least 0.30 degrees lower turbid infection and malignancy WBC COUNTS
▪ PF glucose parallel plasma levels than the blood pH is considered blood streaked malignant effusions ▪ normal WBC Count: less than 350 cells/uL
with values less than 60mg/dl significant associated with accidental puncture and misuse ▪ ⇧ WBC Counts: bacterial peritonitis and cirrhosis
considered decreased ▪ pH as low as 6.0 indicates an grossly bloody o to distinguish between the two, absolute neutrophil count is
of anticoagulant medications (e.g. Coumadin)
esophageal rupture that is allowing representing chylous and pseudochylous performed
the influx of gastric fluid milky
effusions

AIRAH M.
 o value greater than 250cells/uL or greater than 50% of the Total EXAMPLE 1 EXAMPLE 2
WBC Count is indicative of infection serum albumin 3.8mg/dl 3.8mg/dl
o e.g. absolute neutrophil count: 550, total WBC count: 900 = due to fluid albumin 1.2mg/dl 3.0mg/dl
bacterial infection gradient 2.6 0.8
o e.g. absolute neutrophil count: 200, total WBC count: 900 = result transudate effusion exudate effusion
cirrhosis
▪ lymphocytes: predominant cell in tuberculosis MICROBIOLOGIC TESTS

CELLULAR EXAMINATION gram stain and bacterial cultures for both aerobes and anaerobes:
1
when bacterial peritonitis is suspected
cytologic examination for malignant cells: important for the
1 inoculation of fluid into blood culture bottles at the bedside increases
detection of tumors of primary and metastatic origin 2
the recovery of anaerobic organisms
malignancies are most frequently of gastrointestinal, prostate or
2 3 acid-fast stains, adenosine deaminase, and cultures for tuberculosis
ovarian origin
cells present in ascitic fluid
▪ leukocytes
3 ▪ abundant mesothelial cells
▪ macrophages, including lipophages (macrophages containing fat
droplets)
microorganisms including bacteria, yeast, and Toxoplasma gondii
4
may be present
malignant cells of ovarian, prostatic, and colonic origin, often
5
containing mucin-filled vacuoles are frequently seen
psammoma bodies containing concentric striations of collagen-like
6 material can be seen in benign conditions & are also associated with
ovarian and thyroid malignancies
measurement of tumor markers CEA and CA125 is a valuable
procedure for identifying the primary source of tumors producing
7 ascitic exudates
▪ the presence of CA125 antigen with a negative CEA suggests the
source is from the ovaries, fallopian tubes, or endometrium
CHEMICAL TESTS
1 GLUCOSE
▪ decreased below serum levels in bacterial and tubercular peritonitis
and malignancy
2 AMYLASE
to ascertain cases of pancreatitis, however, it may be elevated in patients
with gastrointestinal perforations
▪ that’s why another examination is done to verify if it is pancreatitis
▪ do alkaline phosphatase (ALP) test
pancreatitis gastrointestinal perforations
amylase increased increased
ALP decreased increased
3 BUN AND CREATININE
▪ are renal function tests
▪ requested when a ruptured bladder or accidental puncture of the
bladder during the paracentesis
differentiation between ascitic fluid transudates and exudates is more
difficult in peritoneal than for pleural and pericardial effusions
▪ the Serum-Ascites Albumin Gradient (SAAG): is recommended over the
fluid: serum total protein and LD ratios for the detection of transudates of
hepatic origin
▪ a difference (gradient) of 1.1 or greater suggests a transudate effusion of
hepatic origin, and lower gradients are associated with exudative effusions
o gradient: the difference of the serum albumin and peritoneal fluid
albumin

AIRAH M.
 ANALYSIS OF URINE AND BODY FLUIDS The functions of these different organs include: ▪ performing the mixed antiglobulin reaciton (MAR) test (if
TOPIC 11: SEMEN ANALYSIS testis involved in spermatogenesis required)
Learning Objectives: epididymis where sperm maturation occurs ▪ assessing peroxidase-positive cells (if round cells are
▪ State the structures involved in sperm production and their function vas deferens propel sperm to ejaculatory ducts present)
▪ Describe the four components of semen with regard to source and seminal vesicles provide nutrients for sperm and fluid ▪ preparing spermatozoa for the immunobead test (if
function provide enzymes and proteins for coagulation required)
prostate gland ▪ centrifuging semen (if biochemical markers are to be
▪ Explain the procedures for collecting and handling semen specimens and liquefaction
Bulbourethal add alkaline mucus to neutralize prostatic acid assayed)
▪ Describe the normal appearance of semen and three abnormalities
glands and vaginal acidity within 3 hours
in appearance 3
▪ sending samples to the microbiology laboratory (if required)
▪ State two possible causes of low semen volume
after 4 hours
▪ Discuss the significance of semen liquefaction and viscosity 4
▪ fixing, staining, and assessing smears for sperm morphology
▪ Calculate a sperm concentration and count when provided with the later on the same day (or on a subsequent day if samples are
number of sperm counted, the dilution, the area of the counting frozen)
chamber used, and the ejaculate volume 5
▪ assaying accessory gland markers (if required)
▪ Define round cells, and explain their significance ▪ performing the indirect immunobead test (if required)
▪ Define round cells, and explain their significance
SAMPLE COLLECTION
▪ State the two parameters considered when evaluating sperm motility
▪ Describe the appearance of normal sperm, including structures and PREPARATION
their functions ▪ the sample should be collected in a private room near the
▪ Differentiate between routine and strict criteria for evaluating sperm laboratory or it can be done at home and should be immediately
morphology transported to the laboratory
▪ Give an abnormal result in a routine semen analysis, determine ▪ the sample should be collected after a minimum of 2 days and a
addiitonal tests that might be performed maximum of 7 days of sexual abstinence (instructed to the patient
SEMEN COMPOSITION: FOUR FRACTIONS before giving the sample to the laboratory)
▪ Describe the two methods routinely used to detect antisperm
spermatozoa 5% ▪ patient given clear written and spoken instructions
antibodies
60-70% ▪ the following information should be recorded on the report form:
▪ List two methods for identifying a questionable fluid as semen seminal fluid
(composes the bulk of the semen) o patient’s name
▪ State the WHO reference values for routine and follow-up semen o birth date and personal code number
prostatic fluid 20-30%
analysis o period of abstinence
Bulbourethral glands 5%
▪ Discuss the types and significance of sperm function tests o date and time of collection
▪ Describe methods of quality control appropriate for semen analysis STEPS IN SEMEN ANALYSIS o completeness of the sample
WHO Laboratory Manual for the Examination and Processing of Human Semen, 2010
▪ Know the fundamentals of setting up an andrology laboratory o any difficulties in producing the sample
PHYSIOLOGY: MALE GENITALIA A complete semen analysis would take less than about 4 hours or even o interval between collection and the start of the semen analysis
These are the structures composing the male genitalia. Semen is 1 day, depending on the tests required. The specimen has unique
SEMEN ANALYSIS PROPER
composed of the four fractions that are contributed by these following properties different from other body fluids that it requires time and
organs (the ones pointed by the arrow): adequate knowledge in performing the different procedures. 1 LIQUEFACTION
⇨ testis ⇨ prostate gland ▪ fresh semen specimen is clotted and should liquefy within 30-60
in the first 5 minutes after receiving the specimen:
⇨ epididymis ⇨ Bulbourethral gland minutes after collection
1 ▪ placing the specimen container on the bench or in an
⇨ seminal vesicles ▪ failure of liquefaction: caused by a deficiency in prostatic enzymes
incubator (37°C) for liquefaction ▪ analysis cannot begin until liquefaction has occurred
between 30 and 60 minutes ▪ this is the first thing you observe in the specimen
▪ assessing liquefaction and appearance of the semen ▪ if after 2 hours, the specimen has not liquified, you can do an equal
▪ measuring semen volume volume of physiologic dubicous??? phosphate buffer saline or
▪ measuring semen pH (if required) proteolytic enzymes (e.g. alpha chymotrypsin, bromelain) can be
▪ preparing a wet preparation for assessing microscopic added to induce liquefaction and to allow the rest of the analysis
appearance, sperm motility, and the dilution required for to be performed
2
assessing sperm number 2 VOLUME
▪ assessing sperm vitality (if the percentage of motile cells is
▪ normal semen volume: 2-5 ml
low)
▪ recommended period of abstininece (2-7 days): in order to report
▪ making semen smears for assessing sperm morphology
the correct semen volume
▪ making semen dilutions for assessing sperm concentration
▪ assessing sperm number

AIRAH M.
▪ increased volume may be seen following periods of extended 6 CALCULATING ROUND CELLS ▪ abnormalities in head morphology
abstinence ▪ differentiation and enumeration of round cells (immature sperm are associated with poor ovum
▪ decreased volume is more frequently assocaited with infertility and and leukocytes) can also be made penetration, wheras neckpiece,
may indicate improper functioning of one of the semen producing during the morphology midpiece, and tail abnormalities
organs, primarily the seminal vesicles (which contributes greatly to examination affect motility
the semen volume or seminal composition) ▪ peroxidase-positive granulocytes ▪ spermatozoa consist of a head, neck,
3 VISCOSITY are the predominant form of middle piece (midpiece), principal
▪ specimen viscosity refers to the consistency of the fluid and may lekocyte in semen and can be piece, and endpiece
be related to specimen liquefaction further differentiated from ▪ a normal sperm has:
▪ viscosity can be observed after liquefaction time has been recorded spermatogenic cells & lymphocytes o an oval-shaped head: around 5
▪ the normal semen specimen should be easily drawn into a pipette using a peroxidase stain um long and 3 um wide
and form small discrete droplets that do not appear clumped or ▪ by counting the number of o a long flagellar tail, approximately
stringy when falling by gravity from the pipette spermatids or leukocytes seen in 45 um long
▪ rating of 0 (watery) to 4 (gel-like) can be assigned to the viscosity conjunction with 100 mature o critical to ovum penetration is the enzyme containing acrosomal
report sperm, the amount per milliliter can cap (the one which penetrates): found in the head,
▪ viscosity can also be reported as low, normal, or high be calculated using the following encompassing half of its length, and cover approximately 2/3 of
▪ increased viscosity and incomplete liquefaction impede testing for: formula: the sperm’s nucleus
o sperm motility 𝐍𝐱𝐒 o the neckpiece attaches to the head to the tail and the midpiece
o sperm concentration 𝐜=
𝟏𝟎𝟎 o the midpiece is approximately 7 um long and is the thickest part
o anti-sperm antibody detection, and where: of the tail because it is surrounded by a mitochondrial sheet
o measurement of biochemical markers N: number of spermatids or which produces the energy required by the tail for motility
4 pH neutrophils counted per 100 o mitochondria: provides the sperm energy for its motility
▪ the pH should be measured within 1 hour of ejaculation due to the mature sperm ▪ for a spermatozoon to be considered normal, both its head and
loss of CO2 that occurs S: sperm concentration in millions tail must be normal
▪ normal pH of semen is alkaline with a range of 7.2 to 8.0 per milliliter ▪ all borderline forms should be considered abnormal
▪ spermatid is counted as one of the round cells
⇧ pH ⇩ pH 7 SPERM MOTILITY
▪ the presence of sperm capable of forward, progressive movement SPERM MORPHOLOGY (STRAS)
▪ increased prostatic fluid
infection within the ▪ ejaculatory duct obstruction is critical for fertility, because once presented to the cervix, the
reproductive tract ▪ poorly developed seminal sperm must propel themselves through the cervical mucosa to the
vesicles uterus, fallopian tubes, and ovum

SPERM MOTILITY GRADING

Grade WHO Criteria Sperm Motility Action
4.0 a rapid, straight-line motility
slower speed, some lateral
3.0 b
movement
slow forward progression,
5 SPERM CONCENTRATION AND SPERM COUNT 2.0 b
noticeable lateral movement
▪ reference values for sperm concentration: >20 to 250 million sperm 1.0 c no forward progression
per milliliter
0 d no movement
▪ concentrations between 10 and 20 million per milliliter are
considered borderline alternative SPERM MOTILITY GRADING CRITERIA
▪ the total sperm count for the ejaculate can be calculated by sperm moving linearly or in a
progressive motility (PM)
multiplying the sperm concentration by the specimen volume large circle
𝐭𝐨𝐭𝐚𝐥 𝐬𝐩𝐞𝐫𝐦 𝐜𝐨𝐮𝐧𝐭 = sperm conc. x specimen volume sperm moving with an In WHO classification, the defects are usually classified either into:
nonprogressive motility (NP)
absence of progression ▪ head defects
▪ total sperm counts >40 million per ejaculate are considered normal
immotility (IM) no movement ▪ neck and midpiece degects
(20 million per millilter x 2ml)
8 SPERM MORPHOLOGY ▪ tail defects
▪ in the clinical laboratory, sperm concentration is usually performed
▪ sperm morphology is evaluated with respect to the structure of the ▪ excess residual cytoplasm (sometimes)
using the Neubauer counting chamber
▪ only fully developed sperm should be counted head, neckpiece, midpiece, and tail consisting of a flagella It has more specific classifications for the sperm morphology. (shown
▪ immature sperm and WBCs, often referred to as “round cells”, must in the image below)
not be included

AIRAH M.
 SPERM MORPHOLOGY (WHO) 11 SEMINAL FLUID FRUCTOSE ⇥ finding of <10% of the motile sperm attached to the particles
▪ low sperm concentration may be caused by lack of the support is consired normal
medium produced in the seminal vesicles, which can be indicated o immunobead test
by a low to absent fructose level in the semen ⇥ is a more specific
▪ low fructose levels: procedure in that it can
o abnormalities of the seminal vesicles be used to detect the
o bilateral congenital absence of the vas deferens presence of either IgG,
o obstruction of the ejaculatory duct IgM, or IgA antibodies
o partial retrograde ejaculation, and ⇥ demonstrates what area
o androgen deficiency of the sperm (either head,
▪ resorcinol test that produces an orange color neckpiece, midpiece, or
▪ N°: ≥13 umol per ejaculate tail) the autoantibodies
are affecting
SEMINAL FRUCTOSE SCREENING TEST
⇥ head directed antibodies
Prepare reagent (50 mg resorcinol in 33 ml concentrated HCl can interfere with
1
diluted to 100ml with water). penetration into the
9 ADDITIONAL TESTS 2 Mix 1 ml of semen with 9 ml of reagent. cervical mucosa or ovum
3 Boil. ⇥ tail directed antibodies
possible
abnormal result test 4 Observe for orange-red color. affect movement through the cervical mucosa
abnormality
decreased motility 12 ANTISPERM ANTIBODIES ⇥ in this test, sperm are mixed with polyacrylamide beads
vitality eosin-nigrosin stain ▪ can be present in both men and women known to be coated with either anti-IgG, anti-IgM, or anti-
with normal count
lack of seminal ▪ may be detected in semen, cervical mucosa, or serum IgA
decreased count vesicle support fructose level ▪ are considered possible causes of infertility ⇥ the microscopic examination of the sperm shows beads
medium ▪ it is not unusual for both partners to demonstrate antibodies, attached to sperm at particular areas
mixed although male antisperm antibodies are more frequently ⇥ depending on the type of the beads used, the test could be
encountered reported as IgM tail antibodies, IgG head antibodies, etc.
agglutination
reaction and ▪ under normal conditions, the blood testis barrier separates sperm ⇥ the presence of beads <50% of the sperm is considered
decreased motility male antisperm from the male immune system: there is no connection or normal by WHO
immunobead tests
with clumping antibodies communication between the blood and the testis
sperm BIOCHEMICAL ASSAYS FOR ACCESSORY SEX ORGAN FUNCTION
▪ when this barrier is disrupted, usually following any surgery,
agglutination with 1 secretory capacity of the PROSTATE
vasectomy, trauma, or infection, the antigens on the sperm
male serum
produce an immune response which could damage the sperm ▪ zinc, citrate acid, or acid phosphatase
normal analysis sperm
female antisperm ▪ the damage may cause the production of antibodies in the female 2 secretory capacity of the SEMINAL VESICLES
with continued agglutination with
antibodies partner ▪ fructose
infertility female serum
▪ the presence of antibodies in the male subject can be suspected 3 secretory capacity of the EPIDIDYMIS
10 SPERM VITALITY
when clumps of sperm are observed during a routine semen ▪ L-Carnitine, glycerophosphocholine, neutral 𝝰-glucosidase
▪ should be ejaculated within 1 hour of ejaculation
analysis
▪ evaluated by mixing the specimen with an eosin-nigrosin stain, SPERM FUNCTION TESTS
▪ your sperm agglutinating antibodies cause sperm to stick to each
preparing a smear, and counting the number of dead cells in 100 TEST DESCRIPTION
other (either head to head, head to tail, or tail to tail pattern)
sperm using a brightfield or phase-contrast microscope sperm are incubated with species-
▪ the agglutination is either graded as few, moderate, or many in Hamster egg
▪ living cells are not infiltrated by the dye and remain bluish white, nonspecific hamster eggs and penetration is
microscopic examination penetration
whereas dead cells stain red against the purple background observed microscopically
▪ two frequently used tests to detect the presence of antibody
▪ normal vitality requires 50% or more living cells and should cervical mucus observation of sperm’s ability to penetrate
coated sperm:
correspond to the previously evaluated motility penetration partner’s midcycle cervical mucus
o mixed agglutination reaction (MAR) test:
⇥ a screening procedure used primarily to detect the presence sperm exposed to low-sodium
In this image, we hypo-osmotic
of IgG antibodes concentrations are evaluated for membrane
can see a greenish swelling
⇥ the semen sample containing motile sperm is incubated with integrity and sperm viability
tinge (living cells)
the IgG antihuman globulin and a suspension of latex in vitro acrosome evaluation of the acrosome to produce
but this was is
particles or treated RBCs coated with IgG reaction enzymes essential for ovum penetration
taken from a
⇥ the bivalent AHG binds simultaneously to both the antibody
computer-assisted REFERENCE VALUES
on the sperm and the antibody on the latex particles or RBCs
sperm analysis. volume 2 to 5 ml
forming the microscopically visible clumps of sperm and
particles of cells viscosity pours in droplets

AIRAH M.
 pH 7.2 to 8.0 ▪ storage
sperm concentration >20 million/ml o we use liquid nitrogen for cryopreservation
sperm count >40 million/ejaculate1 o has the cryopreservation materials and equipments
motility >50% within 1 hour
>2.0
quality
a, b, c in WHO grading system
> 14% normal forms (strict criteria)
morphology
> 30% normal forms (routine criteria)
round cells <1.0 million/ml
1
ejaculate: meaning we take note of the volume of the whole spx

SETTING UP AN ADROLOGY LABORATORY

Infertility is quite common nowadays. Male infertility comprises around
40% of couple’s infertility. That is why there is a growing need for REFERENCES
andrology labs especially in infertility clinics (in Cebu, there is one, where ▪ Urinalysis & Body Fluids by Strasinger, 6th ed
they also have cryopreservation). Your andrology laboratory is quite an ▪ WHO laboratory manual for the Examination and processing of
increasing demand in this field. human specimen (latest 5th edition)
1 COMMONLY PERFORMED TESTS ▪ Andrology Laboratory Manual by Rao, K.A., Agarwal, A., Srinivas, MS
Diagnostic Procedures Note: Doc keeps saying to read more on the WHO Lab Manual. Heads
▪ Complete Semen Analysis up lang :))
▪ Detection of Leukocytes in Semen (Endtz)
▪ Sperm Vitality and Membrane Vitality (Eosin-Nigrosin, Hypo
Osmotic Swelling Test: HOS)
▪ Biochemical Tests (e.g. fructose)
▪ Oxidative Stress (Reactive Oxygen SpeciesL: ROS)
▪ Azoospermia Screen Procedure
▪ Antibody Assessment
▪ Sperm DNA Damage Test (more advanced labs)
Therapeutic Procedures (advanced procedures)
▪ Sperm Preparation for Intrauterine Insemination (IUI) or In Vitro
Fertilizatiom (IVF)
▪ Preparation of Cryopreserved Semen for IUI or IVF
2 LABORATORY DESIGN

▪ private semen collection room

o must be quiet, away from noise
o there must be a sink or water supply
available
o waste disposal
o comfortable

▪ work stations
o quite small but is ideal (first image)
o in this station, sperm morphology can be assessed (2nd image)

AIRAH M.
 ANALYSIS OF URINE AND BODY FLUIDS ▪ test most frequently performed: LABORATORY FINDINGS IN JOINT DISORDERS
TOPIC 12: SYNOVIAL FLUID o WBC count o culture 1 NON-INFLAMMATORY
Learning Objectives: o differential count o crystal examination ▪ clear ▪ WBC <1000 uL
▪ Describe the formation and function of synovial fluid o gram stain ▪ yellow fluid ▪ neutrophils <30%
▪ To know the diagnostic tests most routinely performed on ▪ good viscosity ▪ similar to blood glucose
SPECIMEN COLLECTION AND HANDLING
synovial fluid 2 INFLAMMATORY
▪ To determine the appropriate speciemn collection and handling this is the procedure for the synovial fluid immunologic origin crystal induced origin
joint fluid aspiration ▪ cloudy ▪ cloudy
▪ To describe the appearance and cellular composition in a normal
uses sterile disposable needle with sterile ▪ yellow fluid ▪ milky fluid
and abnormal synovial fluid
disposable plastic syringe ▪ poor viscosity ▪ low viscosity
▪ To identify and describe crystals found in synovial fluid anthrocentesis
▪ WBC 2,000-75,000 uL ▪ WBC up to 100,000 uL
avoid contamination from exogenous bire-
▪ To know the clinical significance of glucose and alctose test on ▪ decreased glucose level ▪ neutrophils <70%
fringent materials (e.g. starch, glove
synovial fluid powders, powdered anticoagulants) which ▪ possible autoantibodies ▪ decreased glucose level
PHYSIOLOGY cause artifacts present ▪ crystal present
3 SEPTIC
also known as “joint fluid” REQUIRED TUBE TYPES FOR SYNOVIAL FLUID TESTS ▪ cloudy ▪ neutrophils >75%
found in the cavities of the movable joints or SYNOVIAL FLUID TEST REQUIRED TUBE TYPE ▪ yellow-green fluid ▪ decreased glucose level
synovial joints sterile heparinized or sodium ▪ variable viscosity ▪ positive culture and Gram
gram stain and culture
it is an ultrafiltrate of plasma across the synovial polyanethol sulfonate (SPS) ▪ WBC 50,000-100,000 uL stain
synovial fluid membrane cell counts heparin or liquid EDTA 4 HEMORRHAGIC
filtration is non-selective except for the exclusion glucose analysis sodium fluoride ▪ cloudy ▪ WBC is equal to blood
of HMW proteins all other tests non-anticoagulated ▪ red fluid ▪ neutrophils equal to blood
viscosity of synovial fluid is mainly caused by a ▪ low viscosity ▪ normal glucose level
large amount of hyaluronate molecules SYNOVIAL FLUID NORMAL VALUES
specialized cells found in the synovial membrane volume <3.5 mL GROSS EXAMINATION OF SYNOVIAL FLUID
synoviocytes secretes mucopolysaccharide containing color colorless to pale yellow I. COLOR AND CLARITY
hyaluronic acid and protein clarity clear ▪ normal synovial fluid appears colorless to pale yellow
viscosity able to form a string 4-6 cm long ▪ color becomes deeper yellow in the presence of non-
FUNCTIONS OF SYNOVIAL FLUID leukocyte count <200 cells/uL inflammatory and inflammatory effusions
1 reduces friction between the bones during joint movement neutrophils <25% of the differential
▪ presence of blood may be suggestive of a hemorrhagic arthritis,
2 provides nutrients to the articular cartilage crystals None
however it must be differentiated from a traumatic aspiration by
3 lessens the shock of joint compression glucose plasma <10 mg/dL lower than the blood
4 removal of waste material (metabolic waste and detritus) observing the uneven distribution of blood
difference glucose level
▪ turbidity is associated with the presence of WBC
total protein <3 g/dL
damage to the articular membrane that produces
pain and stiffness in the joints II. VISCOSITY
CLASSIFICATION OF JOINT DISORDERS
arthritis also associated with conditions like infection, ▪ reflection of hyaluronic acid content
inflammation, metabolic disorders, trauma, GROUP CLASSIFICATION PATHOLOGIC SIGNIFICANCE ▪ essential for the proper joint lubrication
physical stress and advanced age ▪ degenerative joint disorders ▪ the simplest test is to observe the fluid’s ability to form a string
non-inflammatory ▪ osteoarthritis from the tip of a syringe
▪ neurogenic joint disease
▪ immunologic problems (LE, RA) TESTS FOR VISCOSITY
inflammatory ▪ crystal induced gouts and 1 FALLING DROP TEST
pseudogout ▪ screening test
septic microbial infection ▪ fluid is aspirated into a pipet and is released to a tube
▪ traumatic injury ▪ if falling drop is drawn out into a 2 inch long or longer band or
hemorrhagic ▪ coagulation deficiencies string (4-6cm) it is considered normal
▪ hemophilia (factor VIII def.) ▪ if the drop falls like water or the string breaks before reaching
3 cm, the fluid has a poor or low viscosity
SYNOVIAL FLUID ANALYSIS
▪ indicate when there is effusion in the joint (hydroarthrosis)
▪ also used to determine the pathologic origin of arthritis
all my thanks to KEVIN CORDOVEZ for lending a
hand on this one ♡ P.S. I super love your work,
I’m such a fan! *starstruck* 🤩 AIRAH M.
 INTERPRETATION CELLS AND INCLUSIONS SEEN IN SYNOVIAL FLUID TYPES OF CRYSTALS (PRIMARY CRYSTALS)
markedly decreased viscosity is seen CELLS/ monosodium urate calcium pyrophosphate
DESCRIPTION SIGNIFICANCE
your joint fluid or fluid doesn't make INCLUSIONS (uric acid) (MSU) dihydrate (CPPD)
inflammatory effusion
or doesn't draw out a 2 inch or a 4- ▪ bacterial sepsis ▪ found in cases of gout ▪ found in cases of pseudogout
polymorphonuclear
6cm band/string neutrophil ▪ crystal induced ▪ increased serum uric acid ▪ associated with degenerative
leukocyte
2 MUCIN CLOT TEST/ROPES TEST inflammation (impaired metabolism of arthritis, cartilage
▪ by Marian Rivera (story of my layp~) jk it’s Marian Ropes mononuclear nonseptic purines, increase calcification and endocrine
lymphocyte
▪ not routinely performed leukocyte inflammation consumption of high purine disorders
▪ mucin is precipitated by 2% or 5% acetic acid large mononuclear content foods, ▪ appears rhomboid-shaped or
macrophage ▪ normal
▪ normal synovial fluid forms a solid clot leukocyte, may be chemotherapy, decreased square but may appear as
(monocyte) ▪ viral infection
vacuolated renal excretion of uric acid) short rods
INTERPRETATION ▪ needle shaped crystals ▪ positive birefringence (blue
similar to
solid clot surrounded by ▪ negative birefringence color)
good mucin macrophage, but may ▪ normal
clear fluid synovial (yellow color)
be multinucleated, ▪ disruption from
soft clot with shreds into lining cell
fair mucin resembling arthrocentesis
solution OTHER CRYSTALS FOUND IN SYNOVIAL FLUID
mesothelial cell
low mucin clot consists of shreds in a HYDROXYAPATITE
neutrophil containing CHOLESTEROL CRYSTALS
(Friable clot) turbid solution (basic calcium phosphate)
characteristic
poor mucin no clot LE cell
ingested “round
lupus erythematosus ▪ also known as apatite ▪ associated with chronic
body” crystals inflammation
III. MICROSCOPIC EXAMINATION ▪ associated with calcified ▪ appear as notched rhomboid
neutrophil with dark
▪ rheumatoid arthritis cartilage degeneration plates
1 CELL COUNT RA cell cytoplasmic granules
▪ immunologic (osteoarthritis) ▪ negative birefringence
▪ total leukocytes count: most frequently performed (ragocyte) containing immune
inflammation ▪ appear as a small particles
▪ counts should be performed as soon as possible complexes
▪ require electron microscopy
▪ very viscous fluid needs to be pretreated by adding 1 drop of cartilage large, multinucleated
osteoarthritis ▪ no birefringence
0.05% hyaluronidase in phosphate buffer per mL and cells cells
CORTICOSTEROIDS CALCIUM OXALATE
incubating at 37°C for 5 mins ▪ macroscopically
▪ clear fluids can be counted undiluted while turbid and bloody ▪ significance in corticosteroid ▪ associated with renal dialysis
resemble polished
fluids should be diluted with normal saline solution ▪ tuberculosis injections ▪ appear as envelopes
rice
RIce bodies ▪ septic and ▪ appear as flat, variable- ▪ negative birefringence
▪ to lyse RBCs, hypotonic saline solution (0.3%) or saline that ▪ microscopically
rheumatoid arthritis shaped plates
contains saponin is suitable show collage and
▪ NSS with methylene blue stains WBC nuclei ▪ positive and negative
fibrin
birefringence
2 DIFFERENTIAL COUNT ▪ refractile
▪ performed on “cytocentrifuged” for better preservation of cell intracellular and V. CHEMICAL ANALYSIS
morphology. extracellular ▪ traumatic injury
fat droplets GLUCOSE TOTAL PROTEIN
▪ primary cells seen in normal synovial fluids are the globules ▪ chronic inflammation
mononuclear cells (60%): ▪ stain with Sudan ▪ approximately same as ▪ not filtered through the
dyes serum values synovial membrane
o monocytes o synovial tissue cells ▪ markedly decreased glucose ▪ increased levels are found in
inclusions within pigmented
o macrophages indicate inflammatory inflammatory and
hemosiderin clusters of synovial villonodular synovitis
▪ lymphocytes (15%): increased lymphocytes suggests nonseptic cells (PVNS) (group II) or septic (group III) hemorrhagic disorders
inflammation ▪ NV: not more than 10mg/dL ▪ NV: less than 3g/dL
▪ neutrophils (25%): increased neutrophils indicate a septic IV. CRYSTAL IDENTIFICATION lower than blood value
condition ▪ identification should be performed soon after fluid collection LACTATE OR
▪ changes in temperature and pH in the synovial fluid affects the URIC ACID
ACID PHOSPHATASE
cell mononuclear lymphocytes neutrophils crystal’s solubility which leads to an erroneous result ▪ increased in cases of gout ▪ requested to monitor the
percentage 60% 15% 25% ▪ refrigeration may decrease the solubility of uric acid crystal, on the ▪ confirms the diagnosis of severity of rheumatoid
nonseptic septic other hand increases solubility of MSU (Monosodium urate) gout when crystals cannot arthritis
⇧ signifies
inflammation condition ▪ initial examination of crystals for crystals: wet preparation (1 drop be demonstrated ▪ also aid to the prognosis of
of freshly aspirated synovial fluid is placed on the slide and directly rheumatoid arthritis
examined under the microscope)
all my thanks to KEVIN CORDOVEZ for lending a
hand on this one ♡ P.S. I super love your work,
I’m such a fan! *starstruck* 🤩 AIRAH M.
 MICROBIOLOGIC TESTS ANALYSIS OF URINE AND BODY FLUIDS
(still part of the chemical analysis) TOPIC 13: PREGNANCY TESTS 21st day of gestation or
when hCG is first detectable
4-7 days after conception
▪ gram stain and culture most important microbiologic test for
determine the presence of human Chorionic 5th week of gestation rapid rise in urinary hCG begins
synovial fluid
Gonadotropin (hCG) that is produced by the 10th week peak level of 100,000-200,000
▪ infection may occur due to secondary complication of
pregnancy trophoblastic tissue (2 months and ½) mIU/mL
inflammation caused by trauma or dissemination of systemic
tests used to diagnose conditions other than 12th week tend to/gradually fall
infection
▪ uses enrichment medium such as chocolate agar pregnancy examples may be Hydatidiform mole falls to a plateau of 10,000-20,000
and testicular choriocarcinoma 15th – 16th week
▪ common organisms that infect synovial fluid: mIU/mL and persists up to delivery
o Staphylococcus o fastidious Haemophilus spp. CLINICAL APPLICATIONS SPECIMEN COLLECTION AND HANDLING
o Streptococcus o Neisseria gonorrhoeae of your pregnancy test kits may use urine, blood, amniotic fluid, colostrums and milk as a sample
VI. SEROLOGIC TESTS confirmation of a clinical diagnosis of early pregnancy in 1 st
1 PATIENT PREPARATION
▪ plays an important role in the diagnosis of joint disorders trimester
diagnosis of ectopic pregnancy in patient with lower 1 discontinue all medications 3-4 days prior to testing
▪ tests are performed in serum, synovial fluid analysis serves as a 2
abdominal pain dehydrate patient: no fluid intake starting 8 hours prior to
confirmatory measure 2
3 evaluation of threatened abortion during the 1st trimester urine collection
▪ autoimmune diseases rheumatoid arthritis and systemic lupus
diagnosis and treatment of trophoblastic tumors
erythematosus cause very serious joint inflammation 4 freshly voided in a disposable container or in a
(hydatidiform mole) clean and well rinsed bottle
▪ demonstrates presence of particular autoantibodies
evaluation of non-trophoblastic tumors (testicular
5 more concentrated, hCG deteriorate on
choriocarcinoma) 1st morning
prolonged standing at room temperature
urine
HUMAN CHORIONIC GONADOTROPIN (hCG) if it cannot be tested within 12 hours,
specimen
▪ produced from cytotrophoblastic cells of forming placenta shortly refrigeration is needed.
after nidation specific gravity must be >1.010
▪ a small molecule that is readily passed by the glomerulus into the cloudy specimen must be centrifuged
urine
TECHNIQUES FOR ASSAY OF hCG
▪ it is a glycoprotein consisting of a 2 polypeptide sub-unit: alpha
and beta ▪ bioassay ▪ radioreceptor assay
▪ immunoassays (HAI, LAI, & DAP) (RRA)
ALPHA BETA ▪ radioimmunoassay (RIA) ▪ immunometric assay
▪ has a molecular weight of 1 BIOASSAY
25,000-30,000 daltons
▪ has a molecular weight of ▪ crude method and difficult to standardize
▪ similar with LH but not with
15,000-20,000 daltons ▪ uses animals as test which makes the assay expensive
FSH and TSH
▪ nearly identical to the ▪ used as historic interest only
▪ also determines the biologic
hormones LH, FSH, and TSH
and immunologic specificity TEST DATE BASIS ANIMAL
of hCG corpus luteum
Aschheim prepubertal
1928 formation
& Zondek female mice
induced by hCG
mature
Friedman 1931 ovulation
female rabbit
mature
Xenopus female South
1934 ova release
laevis African clawed
toad
Rat ovarian ovarian prepubertal
1941
Hyperemia hyperemia female rat
Rana
1948 sperm release male frog
pipiens
Galli-
1948 sperm release male toad
Mainini
all my thanks to KEVIN CORDOVEZ for lending a
hand on this one ♡ P.S. I super love your work,
I’m such a fan! *starstruck* 🤩 AIRAH M.
2 IMMUNASSAY (discovered in the early 1960s) ▪ hematuria: grossly bloody urine PROCEDURE
relationship between the 3 ▪ conditions other than 1
Measure and record the total urine volume and withdraw
REPORTING
units expressed as: pregnancy: a 2 mL aliquot.
IU/mL urine or serum 1 IU/mL = 83.3 ng/mL o menopause or ovarian failure Using 0.9% NSS prepare a progressively doubled dilution
2
of aliquot (1:2, 1:4, 1:8, 1:16).
ng/mL urine or serum 1 ng/mL = 12 mIU/mL o H. Mole or chorioCA
3 Perform the qualitative test on each dilution.
mIU/mL urine or serum 1 mIU/mL = 0.08 ng/mL o ovarian cyst, Teratoma, CA
4 Endpoint: highest dilution with no agglutination
o testicular seminoma,
▪ has three types: (elaborated on Table A)
teratoma & embryonal CA Result:
o Hemagglutination inhibition (HAI) kyu? Aw jk ▪ amount of hCG/mL corresponds to the endpoint
o pituitary tumors and
o Latex Particle Agglutination-Inhibition (LAI) ▪ if total 24-hr hCG is desired, use the following formula:
bronchogenic CA
o Direct Latex Particle Agglutination (DAP)
o tubo-ovarian abscess HCG 24hrs = S x D x V
3 RADIOIMMUNOASSAY (RIA) where:
▪ uses antibody against B-subunit of hCG in serum o scrotal abscess
S = sensitivity of test (i.e. 0.8 IU/mL) (in the previous slide ni Ms.
▪ principle: competitive-binding assay in which both hCG in test it says 0.08 IU/mL. Idk which one we should follow. Just follow
serum and radiolabelled hCG compete for binding with anti- Table A. IMMUNOASSAY TYPES
1 Hemagglutination inhibition (HAI) your heart char)
hCG D = highest dilution of urine completely inhibiting the latex
▪ gamma Dab B-hCG: sensitivity 5 mIU/mL or 0.4 ng/mL ▪ test is done using test tube and is incubated for 1-2 hours at
agglutination
o not routinely done room temperature
V = volume of 24 hr specimen
o used to monitor hCG in Hydatidiform mole ▪ sensitivity: 150-4,000 mIU/mL
4 RADIORECEPTOR ASSAY (RRA) ▪ Wampole UCG: sensitivity of 1 IU/mL INTERPRETATION
▪ basis: specific binding of hCG on antigenic sites without ▪ advantage: not affected by proteinuria hCG in urine neutralizes anti-hCG reagent
competitive aspect positive no anti-hCG to react with latex particle coated
▪ sensitivity: 20-50 mIU/mL with hCG resulting to (-) agglutination
▪ employs Gonadotropin target issue: absence of hCG in urine renders anti-hCG
o rat or pig testes negative reagent free to react with latex particles coated
o ovaries from superovulating rats thus causing agglutination
o corpora lutea from ovaries of pregnant cows
▪ receptors on plasma membrane from these organs serve as ADVANTAGES DISADVANTAGES
binding sites for hCG (instead of anti-hCG reagent) increased incidence of false
rapid and simple
▪ less specific than RIA for B-subunit of hCG (+) with proteinuria
▪ RIA and RRA are more sensitive than HAI and LAI 3 Direct Latex Particle Agglutination (DAP)
▪ can be quantitative measurement of hCG INTERPRETATION ▪ test uses slide & is incubated for 2 minutes at room
5 IMMUNOMETRIC ASSAY hCG in urine neutralizes anti-hCG reagent temperature
▪ Solid Phase immunoassays no anti-hCG to react with SRBC ▪ e.g. Wampole DAP: sensitivity 2 IU/ml
positive
▪ labeled antibodies rather than labeled antigen resulting to (-) hemagglutination, SRBC settle to
the bottom forming a “doughnut pattern/ring” PROCEDURE
o Immunoradiometric Assay (IRMA)
absence of hCG in urine renders anti-hCG Urine is directly combined with latex particles coated with
o Immunoenzymatic Assay (IEMA) 1
negative reagent free to agglutinate SRBC forming a anti-hCG.
o Immunofiltration Assay
o Enzyme-linked immunosorbent assay (ELISA) diffuse mat of cells
INTERPRETATION
⇥ double-monoclonal antibody solid state ELISA using 2 Latex Particle Agglutination-Inhibition (LAI) hCG reacts with anti-hCG on latex particles
urine - as specific as RIA, sensitivity - 50 mIU/mL ▪ test uses slide and is incubated for 1-2 minutes at room positive
(+) agglutination on latex particles
temperature no hCG, no reaction with latex particles
FALSE POSITIVE FALSE NEGATIVE negative
▪ sensitivity: 500-3,500 mIU/mL (-) agglutination
▪ phenothiazine (bioassays & ▪ promethazine (ADP) ▪ Gravindex B hCG: sensitivity 0.08 IU/mL
immunoassays) ▪ deterioration of hCG
▪ promethazine: inhibit antisera Gravindex B hCG
▪ prozoning ▪ semi-quantitative method of excreted hormone
agglutination (HAI & LAI)
▪ proteinuria: >1g/24hrs (LAI & ▪ uses a 24-hour urine sample for higher accuracy
DAP) ▪ used to diagnose patient with hydatidiform mole or testicular
choriocarcinoma
all my thanks to KEVIN CORDOVEZ for lending a
hand on this one ♡ P.S. I super love your work,
I’m such a fan! *starstruck* 🤩 AIRAH M.
 COMPARISON OF BIOASSAY AND IMMUNOASSAY SYSTEMS IN
PREGNANCY TEST
Approx.
Sensitivity Time req. Test Type of
to hCG for Assay Specimen Test
(IU/mL)
BIOASSAY SYSTEM
Aschheim-
Zondek(mouse 1.5 - 6 96 hrs
follicle)
Friedman
(rabbit corpora 10 - 15 48 hrs
lutea)
Rat ovarian
1.2 - 20 6 - 24 hrs
(hyperemia)
X. laevis
75 18 hrs
(ovulation)
R. pipiens
22 - 45 4 hrs
(spermation)
B. americanus
12 - 45 4 hrs
(spermation)
B. marinas
35 - 50 5 hrs
(spermation)
IMMUNOASSAY SYSTEM
Pregnosticon 0.75 - 1.5 2 hrs Urine HAI
Wampole UCG
0.3 2 hrs
(titration)
Urine HAI
(undiluted
0.75 - 1.0 16 hrs
urine)
Perpuerin 0.75 - 1.5 2 mins Urine HAI
Immunoplate 6.0 2 mins Urine HAI
Pregnosis 1.5 - 2.5 2 mins Urine HAI
DAP test 2-3 2 mins Urine HAI
Placentex 1.0 1 ½ hrs Urine HAI
Sensi-Tex 0.25 1 ½ hrs Urine HAI
Gest Female
2-4 2 mins Urine HAI
State
Neocept 0.2 2.1 hrs Urine HAI
Posta - Tec 0.03 1 hr Serum RIA
HCG-B
0.015 1 hr Serum RIA
(Preg/STAT)
UCG Slide 2.0 3 mins Urine HAI
UCG Tube 1.0 2.2 hrs Urine HAI
Biocept - G 0.2 0.2 hrs Serum RRA
Tandem HCG Any 2.5 hrs Serum IMRA
▪ HAI: hemagglutination-inhibition ▪ IMRA: immunoradiometric
▪ RIA: radioimmunoassay (monoclonal Ab) assay
▪ RRA: radioreceptor assay

NOTE: For the procedures and principles of the test kits (card tests
and OBC), please refer to the laboratory PDF.

all my thanks to KEVIN CORDOVEZ for lending a

hand on this one ♡ P.S. I super love your work,
I’m such a fan! *starstruck* 🤩 AIRAH M.
 ANALYSIS OF URINE AND BODY FLUIDS 3 alllows movement POLYHYDRAMNIOS
TOPIC 14: AMNIOTIC FLUID stabilize temperature to protect the fetus from extreme ▪ >1200 ml
4
Table of Contents: temperature changes ▪ decrease fetal swallowing of fetal urine
I. General Characteristics 5 permit proper lung development ▪ failure of the fetus to begin swallowing results in excessive
accumulation of amniotic fluid
II. Specimen Collection and Handling CRUCIAL TESTS ON AMNIOTIC FLUID ▪ an indication of fetal distress, often associated with neural
III. Examination of Amniotic Fluid to assess the status of the fetus tube defects
AMNIOTIC FLUID 1 genetic and congenital disorders before birth OLIGOHYDRAMNIOS
▪ product of fetal metabolism fetal distress from hemolytic disease of the newborn (HDN) ▪ <800 ml
2
▪ the constituents that are present in the fluid provide information or infection ▪ possible causes of decreased amniotic fluid:
about the metabolic processes taking palce during, as well as the 3 fetal lung maturity o increased fetal swallowing
progress of, fetal maturation 4 assessment of the ability of the fetus to survive early delivery o urinary tract deformities
▪ when conditions that adversely affect the fetus arise, the danger o membrane leakage
to the fetus must be measured against the ability of the fetus to ▪ may be associated with congenital malformations, premature
survive an early delivery rupture of amniotic membranes, and umbilical cord
▪ the tests covered in this chapter are used to determine the extent compression, resulting to decelerated heart rate and fetal
death
of fetal distress and fetal maturity
▪ the fetus also secretes lung liquid and fetal pulmonary
clear, yellowish fluid that surrounds and protects substances into the amniotic fluid through fetal breathing
the fetus during pregnancy movements that circulate amniotic fluid
amniotic normal amniotic fluid is colorless to pale yellow ▪ exchange between amniotic fluid and the maternal plasma
fluid and slightly turbid due to fetal cells, vernix, and circulation equals the amount of amniotic fluid every 2-3 hours
hair
is present in the amnion A CHEMICAL COMPOSITION
a membranous sac that surrounds the fetus AMNIOTIC FLUID PRODUCTION ▪ the placenta is the ultimate source of amniotic fluid water and
is formed from the metabolism of fetal cells and 1 first and early second trimester solutes
fetal urine ▪ 35 ml is from maternal circulation (maternal plasma) ▪ amniotic fluid has a composition similar to that of the maternal
is metabolically active o in early pregnancy, the amniotic fluid is an ultrafiltrate of plasma and contains a small amount of sloughed fetal cells
amnion involved in the exchanges of water and maternal plasma from the skin, digestive system, and urinary tract
chemicals between the fluid, the fetus, and the ▪ after ealy pregnancy, the fluid arises from the transudation of o these cells provide the basis for cytogenetic analysis
maternal circulation fetal plasma thorugh the fetal skin and umbilical cord ▪ the fluid also contains biochemical substances that are
o up to 20 weeks gestation produced by the fetus which can be tested to determine the
produces peptides, growth factors, and
cytokines 2 from 20 weeks up to term health or maturity of the fetus
▪ during the latter part of pregnancy, fetal urine is the major ▪ a portion of the fluid arises from the fetal respiratory tract,
contributor to the amniotic fluid volume fetal urine, the amniotic membrane, and the umbilical cord
▪ at the time that fetal urine production occurs, fetal swallowing ▪ when fetal urine production begins, the chemical composition
of the amniotic fluid begins and regulates the increase in fluid of the amniotic fluid changes
from the fetal urine ▪ this change corresponds to the increased production of
▪ the fetus swallows amniotic fluid which is absorbed thorugh creatinine, at about 36 weeks of gestation
the gastrointestinal tract and re-excreted by the kidneys from ▪ measurement of amniotic fluid creatinine has been used to
the blood into fetal urine and back into amniotic fluid determine fetal age
▪ the volume is regulated by a balance between the production ▪ prior to 36 weeks gestation, the amniotic fluid creatinine level
of fetal urine and lung fluid and the absorption from fetal ranges between 1.5 and 2.0 mg/dl
swallowing and intramembraneous flow o it then rises above 2.0 mg/dl, thereby providing a means of
o intramembraneous flow is the absorption of amniotic fluid determining fetal age greater than 36 weeks
water and solutes into the fetal vascular system
increased decreased
amount of amniotic fluid increases in quantity throughout
FUNCTIONS OF AMNIOTIC FLUID 3 ▪ creatinine ▪ glucose
pregnancy
exchange of water and chemicals between the fluid, the ▪ urea ▪ protein
1 reaching a peak of approximately 800-1200 ml during third
fetus, and the maternal circulation ▪ uric acid
trimester
2 protective cushion for the fetus
4 gradually decreases prior to delivery

AIRAH M.
differentiation between amniotic fluid and maternal urine may be SPECIMEN HANDLING ▪ indicative of red blood cell destruction, resulting from
necessary to determine possible premature membrane rupture or ▪ the first 2 or 3 ml collected can be contaminated by maternal hemolytic disease of the newborn
accidental puncture of the maternal bladder during specimen blood, tissue fluid, and cells, in which they are discarded 4 DARK-GREEN
collection ▪ immediately after collection, the fluid is dispensed into sterile, meconium
plastic specimen containers ▪ a newborn’s first bowel movement, is formed in the intestine
amniotic fluid maternal urine
o glass containers are less desirable as cells have more of a from fetal intestinal secretions and swallowed amniotic fluid
▪ creatinine: <3.5 mg/dl ▪ creatinine: 10 mg/dl
tendency to adhere to the glass surface ▪ a dark-green mucus like material
▪ urea: <30 mg/dl ▪ urea: 300 mg/dl
▪ normal amniotic fluid is colorless to pale yellow, and slightly ▪ may be present in the amniotic fluid as a result of fetal distress
▪ glucose
turbid due to fetal cells, vernix, and hair ▪ fetal aspiration of meconium during fetal swallowing is a
▪ protein
▪ fluid for bilirubin analysis, in cases of HDN, must be protected concern when increased amounts are present in the fluid
▪ Positive Fern Test1
1 Positive Fern Test from light at all times 5 DARK RED-BROWN
associated with fetal death
▪ a microscopic test which is used to differentiate amniotic fluid o this can be accomplished by placing the specimens in amber-
▪ microscopic cytological examination of the amniotic fluid may
from maternal urine colored tubes, wrapping the collection tube in foil, or by use
yield information on the diagnosis of ruptured membranes or
▪ the amniotic fluid exhibits a positive fern test, wherein a of a black, plastic cover for the specimen container
chorioamnionitis
vaginal fluid specimen is spread on a glass slide and allowed to ▪ fetal lung maturity tests are placed on ice for delivery to the lab ▪ cytogenetic studies are also a common reason for performing
completely air dry at RT, then observed microscopically and refrigerated prior to testing amniocentesis
▪ the presence of a fern-like crystal due to the protein and NaCl o this involves phospholipid analysis
content is a positive screen? for amniotic fluid o the specimen here should undergo low speed centrifugation TESTS FOR FETAL DISTRESS
SPECIMEN COLLECTION (500 to 100xg) for no longer than 5 minutes only to prevent
1 HEMOLYTIC DISEASE OF THE NEWBORN
loss of phospholipids
AMNIOCENTESIS ▪ the oldest routinely performed lab test on amniotic fluid
▪ fluid for cytogenetic studies, which involves cell culture and
▪ amniotic fluid is obtained by needle aspiration into the evaluates the severity of the fetal anemia produced by the
chromosomal studies, must be processed aseptically and HDN
amniotic sac
maintained at RT or body temp. which is 37°C to prolong the life ▪ caused by an incompatibility of the Rh blood group between
▪ a thin hollow needle is inserted through the mother’s
abdomen into the mother’s uterus and into the amniotic sac of cells needed for analysis the mother and fetus
to aspirate the amniotic fluid ▪ amniotic fluid for chemical testing must be separated from ▪ initial exposure to foregin red cell antigens occurs during
▪ this procedure is usually trans abdominally with simultaneous cellular elements and debris ASAP to prevent distortion of gestation, delivery of the placenta, or a previous pregnancy
use of ultrasound chemical constituents by cellular metabolism or disintegration where fetal RBCs enter into the maternal cirulation and
▪ the addition of ultrasound has helped make this procedure o this can be performed using centrifugation or filtration stimulate the mother to produce antibodies to the antigen
safer, especially if performed after 14 weeks of gestation o the time and speed of centrifugation also depends on the test ▪ when these antibodies present in the maternal circulation
▪ the amount collected is usually 10-20 ml, with a maximum of and laboratory protocol cross the placenta into the fetal circulation and bind to the
30 ml, with collection into several different syringes to prevent o if blood is present, the specimen should be centrifuged to antigen on the fetal cells, the cells are destroyed
the contamination of all specimens with the blood from initial prevent hemolysis from altering the test results o this destruction results to the appearance of
puncture unconjugated bilirubin in the fluid
o all amniotic fluid samples for chemical analysis that must be
▪ amniocentesis is generally performed between 15 and 18 ▪ by measuring the amount of bilirubin in the fluid, the extent of
stored for any length of time must be centrifuged
weeks of gestation for genetic studies, although it may be used hemolysis taking place may be determined and the danger this
o samples for chemical analysis need to be stored more than 24 anemia present to the fetus may be assessed
later in the pregnancy in cases of fetal distress, fetal
hours must be frozen ▪ amniotic fluid bilirubin is measured by spectrophotometric
pulmonary maturity, or fetal hemolytic disease
▪ 14th week of gestation PHYSICAL EXAMINATION analysis using serial dilutions
▪ the optical density of the fluid is measured in intervals
done on the 2nd trimester done on the 3rd trimester 1 COLORLESS
between 365 nm and 550 nm
▪ genetic defects ▪ fetal pulmonary maturity normal amniotic fluid ▪ in normal fluid, the optical density (OD) is highest at 365 nm
▪ fetal hemolytic disease o might exhibit slight to moderate turbidity from cellular and decreases linearly, illustrated by a straight line
debris, particularly in later stages of fetal development) ▪ when bilirubin is present, a rise in OD is seen at 450 nm
2 BLOOD STREAKED because this is the wavelength of maximum bilirubin
▪ traumatic, tap, abdominal trauma, intra-amniotic hemorrhage absorption
▪ the source of the blood, if it’s either maternal or fetal, can be ▪ the difference between the OD of the theoretic baseline and
determined using the Kleihauer-Betke test for fetal the OD at 450 nm represents the amniotic fluid bilirubin
hemoglobin and is important for further case management concentration
3 YELLOW
▪ due to the presence of bilirubin

AIRAH M.
 o this difference in OD, referred to as the absorbance ▪ as mentioned, specimens must be protected from light at all o the test is more specific for neural tube disorders than AFP,
difference at 450 nm (D A450), is then plotted on a Liley times provided it is not performed on a bloody specimen,
graph to determine the severity of the hemolytic disease ▪ markedly decreased values will be obtained with as little as 30 because blood contains AChE
minutes of exposure to light
▪ contamination of the fluid by cells, hemoglobin, meconium, or TESTS FOR FETAL MATURITY
other debris will interfere with the spectrophotometric in the event of a fetal distress, the obstetrician to consider a
analysis preterm delivery wherein at this point, fetal maturity must be
▪ specimens should be immediately centrifuged to remove assessed
particulate interferences 1 FETAL LUNG MATURITY (FLM)
▪ specimens contaminated with meconium will cause falsely low respiratory distress syndrome (RDS)
results and are not acceptable for spectrophotometric analysis ▪ is the most frequent complication of early delivery and is the
▪ specimens that are contaminated with blood are generally 7th most common cause of morbidity and mortality in the
unacceptable because maximum absorbance of premature infant
oxyhemoglobin occurs at 410 nm and can interfere with the ▪ this disease is caused by an insufficiency of lung surfactant
bilirubin absorption peak production and structural immaturity of the fetal lungs
▪ this interference can be removed by extraction with ▪ surfactant normally appears in mature lungs and allows the
chloroform if necessary alveoli (air sacs of the lung) to remain open throughout the
▪ a control may be prepared by diluting commercial chemistry normal cycle of inhalation and exhalation
control sera 1 to 10 with normal saline and treating it in the ▪ surfactant keeps the alveoli from collapsing by decreasing
same manner as the patient specimen surface tension and allows them to inflate with air more easily
▪ bilirubin and protein levels approximate those in amniotic fluid ▪ if the surfactant concentrations are too low, the alveoli will
and can be varied by using low or high control sera collapse, causing RDS
2 NEURAL TUBE DEFECTS ▪ the incidence of RDS decreases with increasing gestational age
▪ one of the most common birth defects in the United States and lung maturity
▪ it can be detected by maternal serum alpha-fetoprotein ▪ therefore, laboratory tests must be performed to determine
(MSAFP) blood test, high-resolution ultrasound, and the maturity of the fetal lungs
amniocentesis ▪ the amount of surfactant in fetal lungs can be estimated by
▪ increased levels of alpha-fetoprotein (AFP) in both the measuring the amount of surfactants in amniotic fluid
maternal circulation and the amniotic fluid can be indicative of ▪ several laboratory tests are available to measure FLM
fetal neural tube defects, such as anencephaly and spina bifida 2 LECITHIN-SPHINGOMYELIN RATIO
▪ AFP is the major protein produced by the fetal liver during ▪ the reference method to which tests of FLM are compared is
early gestation (prior to 18 weeks) the lecithin-sphingomyelin (L/S) ratio
o it is found in the maternal serum due to the combined ▪ lecithin is the primary component of the surfactants
fetal-maternal circulations and in the amniotic fluid from (phospholipids, neutral lipids, and proteins) that make up the
diffusion and excretion of fetal urine alveolar lining and account for alveolar stability
▪ notice that the Liley graph plots the change in absorbance o measurement of amniotic fluid AFP levels is indicated ▪ lecithin is produced at a relatively low and constant rate until
against gestational age and is divided into three zones that when maternal serum levels are elevated or a family the 35th week of gestation, at which time a noticeable increase
represent the extent of hemolytic severity: history of previous neural tube defects exists in its production occurs, resulting in the stabilization of the
o normal values are based on the week of gestational age, as fetal lung alveoli
▪ non-affected/mildly affected the fetus produces maximal AFP between 12 and 15 weeks’
Zone 1 ▪ sphingomyelin is a lipid that is produced at a constant rate
▪ observe fetus for stress, repeat 2-4 weeks gestation, after which levels in amniotic fluid begin to after about 26 weeks’ gestation; therefore, it can serve as a
▪ moderately affected decline control on which to base the rise in lecithin
▪ requiring close monitoring and treatment, ▪ both serum and amniotic fluid AFP levels are reported in terms
Zone 2 ▪ both lecithin and sphingomyelin appear in the amniotic fluid
anticipating an early delivery, or exchanged of multiples of the median (MoM) in amounts proportional to their concentrations in the fetus
transfusion upon delivery o a value two times the median value is considered abnormal ▪ prior to 35 weeks’ gestation, the L/S ratio is usually less than
▪ severely affected (greater than 2 MoM) for both maternal serum and 1.6 because large amounts of lecithin are not being produced
▪ intervention through induction of labor or amniotic fluid at this time
Zone 3 intrauterine exchange transfusion must be ▪ elevated amniotic fluid AFP levels are followed by ▪ after 35 weeks’ gestation, the lecithin concentration increases
considered when it is in this zone measurement of amniotic acetylcholinesterase (AChE) while the sphingomyelin concentration remains constant
(deliver/treat) o AchE is a component of nerve tissues ▪ the L/S ratio will rise to 2.0 or higher as the lecithin production
increases to prevent alveolar collapse

AIRAH M.
▪ therefore, when the L/S ratio reaches 2.0, a preterm delivery ▪ the presence of bubbles indicates that a sufficient amount of o rapid TAT
is usually considered to be a relatively safe procedure phospholipid is available to reduce the surface tension of the o low reagent cost
▪ falsely elevated results are encountered in fluid contaminated fluid even in the presence of alcohol, an antifoaming agent o wide availability
▪ with blood or meconium because both these substances ▪ a modification of the foam test uses 0.5 mL of amniotic fluid o low degree of technical difficulty (easy)
contain lecithin and sphingomyelin added to increasing amounts of 95% ethanol, providing a o low volume of sample
▪ quantitative measurement of lecithin and sphingomyelin is gradient of ethanol/fluid ratios ranging from 0.42 mL to 0.55 o excellent clinical performance
performed using thin-layer chromatography (TLC) mL in 0.01-mL increments, which can be used to provide a ▪ some automated cell counters use both impedance and optical
▪ because the procedure is labor intensive and subject to high semiquantitative measure of the amount of surfactant present methods for counting
coefficients of variation, many laboratories have replaced the ▪ a value of 47 or higher indicates FLM ▪ amniotic fluid specimens containing whole blood, meconium,
L/S ratio with the quantitative phosphatidyl glycerol ▪ the Foam Stability Index has shown good correlation with the and mucus should not be used
immunoassays and lamellar body density procedures L/S ratio and tests for phosphatidyl glycerol o blood initially raises the LBC because of the presence of
3 PHOSPHATIDYL GLYCEROL ▪ the test cannot be used with contaminated amniotic fluid platelets and then can lower the LBC as lamellar bodies are
▪ the presence of another lung surface lipid, phosphatidyl because blood and meconium also reduce surface tension, trapped in the fibrin strands
glycerol (PG), is also essential for adequate lung maturity and yielding a falsely mature index result o meconium and mucus cause a false increase in the LBC and
can be detected after 35 weeks’ gestation 5 LAMELLAR BODIES should not be used for testing
▪ the production of PG normally parallels that of lecithin, but its ▪ surfactant is composed of approximately 90% phospholipid ▪ specimens may be stored at 2°C to 8°C but never frozen
production is delayed in cases of maternal diabetes and 10% protein and is packaged into layered storage granules ▪ results are reported in units of lamellar bodies per microliter
▪ in this circumstance (wherein the mother is diabetic), called lamellar bodies and should be accompanied by the laboratory’s established
respiratory distress occurs in the presence of an L/S ratio of values for maturity and the instrument that was used
lamellar bodies
2.0 because of the delay in PG production ▪ a consensus protocol for noncentrifuged samples considers
▪ are densely packed layers of phospholipids that represent a
▪ therefore, a thin-layer chromatography lung profile must LBCs:
storage form of pulmonary surfactant
include lecithin, sphingomyelin, and PG to provide an
▪ they are secreted by the type II pneumocytes of the fetal lung >50,000/uL <15,000/uL
accurate measurement of FLM
at about 24 weeks of gestation and are absorbed into the fetal lung maturity immature
▪ development of an immunologic agglutination test for PG has
alveolar spaces to provide surfactant results in between these two values are considered
provided a more rapid and easy to perform method for
▪ they enter the amniotic fluid at about 26 weeks of gestation indeterminate and further testing using alternate
assessment of fetal maturity that does not require a laboratory
and increase in concentration from 50,000 to 200,000 per methods is recommended
to be equipped to perform thin-layer chromatography
microliter by the end of the third trimester 6 MICROVISCOSITY
▪ the Aminostat-FLM (Irving Scientific, Santa Ana, CA) uses
▪ as the fetal lung matures, increased lamellar body production ▪ this assay provides a fluorescence polarization surfactant
antisera containing polyclonal anti-PG antibodies that are
is reflected by an increase in amniotic fluid phospholipids and albumin (S/A) ratio to measure FLM
specific for PG-containing lamellar bodies in the amniotic fluid
the L/S ratio ▪ principle: phospholipids decrease the microviscosity of the
o the size of the agglutinates is read macroscopically and the
▪ therefore, the number of lamellar bodies present in the amniotic fluid wherein this change in microviscosity was
results are reported as either:
amniotic fluid correlates with the amount of phospholipid measured using fluorescence polarization
positive (low/high) negative present in the fetal lungs ▪ the assay measured the polarization of a fluorescent dye that
indicating pulmonary indicating pulmonary ▪ the presence of lamellar bodies increases the OD of the combined with both surfactants and albumin in the amniotic
maturity immaturity amniotic fluid fluid
▪ specimens are centrifuged at 2000 g for 10 minutes and ▪ in this test, a fluorescent dye, that binds to both albumin andd
▪ the test is not affected by specimen contamination with blood
examined using a wavelength of 650 nm, which rules out surfactant, is added to the amniotic fluid sample
▪ and meconium
interference from hemoglobin but not other contaminants ▪ the addition of the fluorecent dye gives the sample a
▪ studies have shown good correlation with thin-layer
(e.g. meconium) measurable fluorescent polarization intesity value
chromatography but with a slightly higher incidence of false-
▪ an OD of 0.150 has been shown to correlate well with an L/S ▪ the degree of fluorescence polarization is inversely
negative results that may need to be followed up with further
ratio of greater than or equal to 2.0 and the presence of proportional to the quantity of pulmonary surfactant present
testing
phosphatidyl glycerol ▪ a value of >55 mg/g of phosphatidyl glycerol is indicative of
4 FOAM STABILITY INDEX
▪ used to determine the presence of lung-surface lipid Lamellar Body Count fetal lung maturity
concentrations ▪ lamellar body diameter is similar to that of small platelets ▪ the fluorescent dye bound to surfactant had a longer
▪ amniotic fluid is mixed with 95% ethanol, shaken for 15 ranging in size from 1.7 to 7.3 fL, or 1 to 5m fluorescence lifetime and a low polarization, whereas dye
seconds, and allowed to sit undisturbed for 15 minutes o therefore, lamellar body counts (LBCs) can be obtained bound to albumin had a decreased fluorescence lifetime and a
▪ at the end of this time, the surface of the fluid is observed for using the platelet channel of automated hematology high polarization
the presence of a continuous line of bubbles around the analyzers using either optical or impedance methods for ▪ the recorded changes in polarization produced a
outside edge counting surfactant/albumin ratio expressed in milligrams surfactant to
▪ advantages include: grams albumin that was compared with a fetal lung maturity II

AIRAH M.
 assay calibration standard curve using calibrators of known
surfactant/ albumin content
S/A ratio of:
55 mg/g 40 mg/g to 54 mg/g <39 mg/g
FLM indeterminate immature lungs
▪ the test required 1.0 mL of amniotic fluid
▪ specimens contaminated with blood, meconium, suspected
maternal urine, and visibly icteric sample could not be used

TESTS FOR FETAL AGE

1 CREATININE CONCENTRATION
▪ the creatinine levels rise as the baby nears term
▪ creatinine is measured by serum method (Jaffe’s method)
1.5 to 2.0 mg/dl >2.0 mg/dl
prior to 36th week of pregnancy is over
gestation 36 weeks

REFERENCES
▪ Strasinger, Urinalysis and Body Fluids 6 th Edition
▪ Graff’s, Textbook of Urinalysis and Body Fluids 2nd Edition

AIRAH M.
 ANALYSIS OF URINE AND BODY FLUIDS ▪ it is best collected in the morning soon after the patient wakes 2 CONSISTENCY
TOPIC 15: SPUTUM ANALYSIS up and before any mouthwash is used SEROUS OR
▪ early morning sputum samples should be obtained because MUCOID MUCOPURULENT
TABLE OF CONTENTS FROTHY
they contain pooled overnight secretions in which pathogenic asthma bronchiectasis
▪ Origin ▪ Variations lung edema
bacteria are more likely to be concentrated bronchitis TB
▪ Composition ▪ Other Features
▪ 24-hour collection should be discouraged because there is not 3 COLOR
▪ Collection ▪ Microscopic Examination
only a greater likelihood of contamination but also bacterial ▪ a deviation from the normal color of the sputum sample can
SPUTUM pathogens become diluted with the addition of subsequent and give us an idea or can aid us in identifying the underlying
can yield more watery specimens pathological condition of a patient
ORIGIN ▪ patient is requested to cough deeply and spit
▪ exudate: produced by conditions that directly involve the 2 sputum induction colorless and translucent which is made up
membranes of a particular cavity, including infections and of mucus only
▪ in the event that the patient is unable to cough a sputum
malignancies
induction can be done through the use of: ▪ this mucus-like secretion may become
normal
▪ secreted by: infected, blood stained, or contain
o 10% NaCl (aerosol spray)
o goblet cells of the bronchial lining o 10% propyleneglycol in saline solvent abnormal cells that may lead to a
o mucus-secreting granules glands of bronchial tree when ▪ what the patient do here is that they inhale a mist which can diagnosis
there is a respiratory infection cause a deep cough anchovy or lobar pneumonia, TB, pulmonary gangrene,
▪ a thick fluid produced in the lungs and in the adjacent airways rusty and hemorrhage from the lungs due to
3 bronchial lavage
COMPOSITION (old blood) pulmonary infarction
▪ bronchial lavage, together with induced sputum, are commonly
▪ a trachial-bronchial secretion that is a constant mixture of: used for diagnosing bacterial respiratory infections, usually in brown sputum congestive heart failure
plasma, electrolytes, mucin, and water immunocompromised HIV patients pseudomonas infection, caseous
▪ as these mixtures pass through the lower and upper respiratory bright green
▪ it is generally collected to detect any infectious agents present pneumonia, jaundice, and rupture of liver
trract, they become contaminated with cellular exfoliations, in the lower respiratory tract yellow green TB, bronchiectasis
nasal and salivary gland secretions, and normal bacterial flora olive
4 heated aerosol technique
of the oral cavity green/grass carcinoma of the lungs (chronic cancer)
5 throat swab (pediatric patients)
helps in the detection of: green
6 tracheal aspiration (debilitated patients)
▪ neoplastic diseases of the lungs, and other respiratory organs anthracosis, heavy smokers, dust, dirt,
▪ occurrence of infections [TB, upper respiratory tract infections ▪ remember that the sputum sample should black
carbon, or charcoal
(URTI)] be collected in: 4 ODOR
▪ respiratory disease (allergic reactions as in asthma, o dry, wide mouthed container normal, freshly
inflammation) o leak proof to prevent aerosols expectorated sputum odorless
SPUTUM COLLECTION o break resistant to prevent dessication sample
▪ a sputum culture can help in the identification of the causative ▪ the specimen should be sputum, NOT SALIVA cavitary TB, lung abscess, lung
organism in a suspected infections such as: gangrene, advanced necrotizing
▪ proper labeling of the specimen should be observed, such as: foul/putrid
o pneumonia o Pneumocystis carinii in HIV tumors due to anaerobic
o date
o TB o bronchiectasis bacteria
o name
o fungal infections fruity odor Pseudomonas aeruginosa
o time of collection
▪ a test ot detect and identify the fungi or bacteria that is infecting bronchiectasis, tuberculous
▪ sputum specimens should be examined in a biological safety sweetish
the lungs or breathing passages cavities
cabinet 5 OTHER FEATURES
▪ to be able to perform this test among others as well, an
appropriately collected specimen should be obtained SPUTUM VARIATIONS bronchial made of fibrin; branching lobar
▪ according to the Centers for Disease Control and Prevention, in multiple variations from the normal features of a sputum can be casts tree-like cast penumonia
order to get a good sputum sample, you shoud let the patient rinse suggestive of the nature of the condition of the patient pulmonary TB,
chessy fragments of necrotic
and spit with water to make sure there won’t be mouth bacteria pulmonary
1 AMOUNT masses pulmonary tissues
gangrene
in the sputum collected
SCANTY AMPLE ▪ yellowish or gray bronchectasis,
METHODS TO COLLECT SPUTUM ▪ bronchiectasis caseous matter, the bronchitis,
1 expectoration ▪ bronchial asthma ▪ lung abscess Dittrich’s size of a pinhead or chronic
▪ most common method ▪ acute bronchitis ▪ edema Plugs navy bean, which when bronchitis,
▪ a first morning is the most ideal or preferred ▪ early pneumonia ▪ gangrene crushed will give a foul bronchial
▪ advanced TB odor asthma,

AIRAH M.
 ▪ come from the bronchi healthy
or bronchioles persons
▪ formerly regarded as a
sign of TB
▪ grayish to yellowish
made up of cellular,
fatty acids, fat globules,
some bacteria
Lungstones/
formed from calcified
Pneumoliths/ histoplasmosis
pulmonary tissues
Broncholiths
bronchial
Curschmann’s mucoid threads that are
asthma, acute
Spirals twisted
bronchitis

MICROSCOPIC EXAMINATION

▪ wall of alveoli and bronchioles

elastic fiber
▪ TB, lung abscess, lung gangrene
Charcot-Leyden Crystals
▪ needle like and colorless
crystals ▪ form as a result of eosinophil degradation
▪ most significant
▪ characteristic of bronchial asthma
▪ large mononuclear cells with brown blood
pigment which is hemosiderin
o a characteristic of congestive heart
heart failure cells failure
o also occur in pulmonary infarction and
temporarily after pulmonary
hemmorhage
▪ seen in large numbers during antibiotic
yeasts
treatment with no significance
pathogenic fungi may also be seen, depending on the
bacteria, causative agent of the patient’s infection
parasites, viruses (if any)
neoplastic cells ▪ Papanicolau staining

REFERENCES

AIRAH M.
 ANALYSIS OF URINE AND BODY FLUIDS QUALITY ASSESSMENT ERRORS (VARIABLES) Viennese chemist Fritz Feigl (1892-1971) published
1920
TOPIC 16: AUTOMATION AND QUALITY ASSESSMENT Pre-Examination Examination Post-Examination his technique of “spot analysis”
Objectives: ▪ patient ▪ sample ▪ patient urine test strips in the sense used today were first
1950
▪ Define the different components of quality assessment in AUBF. misidentification misidentification misidentification made on industrial scale and offered commercially
▪ Distinguish between components of internal QC, external QC, ▪ wrong test order ▪ erroneous ▪ poor handwriting the company Boehringer Amnnheim (by Roche),
▪ incorrect urine instrument ▪ transcription 1964 launched its first Comburtest strips with new
electronic QC and Proficiency Testing
specimen type calibration error innovation
▪ Explain the principles involved in the automated analysis of urine.
collected ▪ reagent ▪ poor quality of Aution Analyzer UA-6 by Arkray, Japan launched the
▪ Differentiate the manual, semi-automated, and fully automated ▪ insufficient urine deterioration instrument
1972
world’s first automated urine analyser
methods in the urine analysis. volume ▪ poor testing printer
▪ Enumerate the advantages of automation in the analysis of urine. ▪ delayed technique ▪ failure to send THREE WAYS TO PERFORM TEST STRIP ANALYSIS
transport of ▪ instrument report manual the test is done by hand
Topic Outline:
urine to lab malfunction ▪ failure to call semi- the test strip is dipped in the urine manually and
I. Quality Assessment
▪ incorrect storage ▪ interfering critical values automated then analyzed by an instrument
II. Introduction to Automation
or preservation substances ▪ inability to fully- the test strip is analyzed completely by an
III. Principles Involved in Automation
of urine present identify automated instrument
IV. Implications/Advantages in Automation
▪ misinterpretation interfering
MANUAL PROCEDURE
I. QUALITY ASSESSMENT of QC data substances
Dip strip briefly, but completely into well-mixed, RT urine
1
quality guaranteeing quality patient care & regulated ACCREDITATION AGENCIES sample.
assessment (QA) total testing system 1 Joint Commission (JC) 2 Withdraw strip.
all laboratory's policies, procedures & ▪ few years back, Chong Hua is accredited by JC however, they 3 Blot briefly on its side.
resources in achieving quality testing were not able to live up to the standard for accreditation as Keep the strip flat, read results at the appropriate times by
laboratory's policies, processes, procedures mandated by JC 4 comparing the color to the appropriate color on the chart
& resources needed to achieve quality testing ▪ only Saint Lukes in Manila is accredited by JC provided.
includes quality control, preexamination, 2 College of American Pathologists (CAP)
examination, and post examination variables 3 Commission on Laboratory Assessment (COLA)
(these are new terms in which the old terms
quality system are pre-analytic, analytic, and post-analytic) II. INTRODUCTION TO AUTOMATION
(QS)
ANCIENT HISTORY
ISO: International 1st urologist, hypothesized that urine was a
Hippocrates
Organization for filtrate of the humors, which came from the Video: cobas u 411 urine analyzer | Roche Diagnostics USA
(460-355 BC)
Standardization blood and was filtered through the kidneys Youtube Video Link: https://www.youtube.com/watch?v=LeFSv6xIG-o
(in Cebu, one refined Hippocrates’ ideas, theorizing that
approved is Sotto) Galen urine represented, not a filtrate of the four
(AD 129-200) humors and overall condition, but rather, a
filtrate of the blood
VARIABLES Theophilus wrote De Urinis, manuscript from
Pre-Examination Examination Post-Examination Protospatharius Byzantiuum, first publication exclusively on
▪ specimen ▪ reagent status ▪ reporting of (610-641) the subject of urine
collection ▪ test performance results Johannes de wrote Fasiculus Medicinae, established a self-
▪ handling ▪ instrument ▪ interpretation Ketham of diasgnostic color wheel to self diagnose a
Germany (1491) disease using urine SPEED, SIMPLICITY, ACCURACY: those are the essentials for a small
▪ storage calibration ▪ turn around time
to mid-size lab that requires efficient user-friendly processing of
▪ maintenance ▪ documentation
Parisian chemist Jules Maumené (1818-1898) urine strips.
▪ personnel
requirements develop the first ”test strip” when he impregnated The semi-automated cobas u 411 urine analyzer delivers with
▪ technical 1850 a strip of merino wool (a first class wool taken from convenience that helps drive productivity. The cobas u 411 urine
competence the sheep) with “tinprotochloride” (stannous
analyzer is designed for speed. Two sensors detect the presence of a
chloride)
test strip automatically. This decreases handling and the risk of
English physiologist George Oliver (1841-1915)
1883 operator error. It also allows for faster operation, continuous strip
markets his “Urinary Test Papers”

AIRAH M.
loading, no warmup time between strip loading, allows for batch Maximize your laboratory performance with the AUCTION HYBRID FULLY-AUTOMATED
processing of a new strip every six seconds - that's up to 600 test AU-4050, a compact, fully automated, and integrated urine analyzer automated urine cell analyzers mix, aspirate, dilute and stain
1
strips an hour. The system also prints out results automatically, that consolidates two industry-proven technologies to perform both urine to classify urine sediment particles
about 60 seconds after the strip is detected and it stores up to 1,300 urine chemistry and sediment analysis in one small package. This automated urine systems performs complete urine analysis
results, a thousand samples, and 300 control tests. total walkaway system minimizes labor-intensive manual steps and 2 that includes the physical, chemical and microscopic parts of
frees operator time for other valuable activities. a routine urinalysis
Your staff will also appreciate the analyzers user-friendly features:
with a four-week calibration stability, a large LCD screen, a The easy-to-use analyzer features one-step loading of trusted aution
convenient interface that uses a touchscreen and stylus, and dry pad urine chemistry test strips with no calibration required.
intuitive operator software to access features such as urine controls, During analysis, the chemistry module aspirates the sample and
data management, and customizable reporting. precisely dispenses urine on each pad for measurement. The module
measures nine chemistries: glucose, protein, bilirubin, urobilinogen,
The unit also has a built-in barcode reader for easy input of patient pH, blood, ketones, nitrites, and leukocytes. For improved accuracy,
information with minimal handling. Plus the one-step cleaning the color compensation pad accounts for normal urine colors and
process helps keep maintenance to a minimum. The most important abnormal color detection notifies users of false positive reactions. It
feature is ACCURACY and the cobas u 411 delivers. Accurate efficient also performs specific gravity and color and turbidity measurements
urinalysis starts with Proven Chem Strip Technology and ends with
confidence in the results. Chem strip test strips are designed to help The sample rack then advances based on user defined criteria to the
avoid false negatives and eliminate vitamin C interference which can flow cytometry module. Here, a probe mixes the sample and
cause costly miss diagnosis. You can set up reporting for a complete aspirates into two separate chambers. The sediment chamber is
menu of testing parameters, including pH, protein, glucose, and heated to 35°C to remove any amorphous urates. The urine sample
ketones. And you can connect the instrument directly to your lis is mixed with diluent and fluorescent stain to accurately detect and
system. You can even set user-defined rules to flag certain results. enumerate formed elements in the sample. It can accurately detect
and enumerate RBCs, WBCs, epithelial cells, and casts. The analyzer
The cobus u 4:11 analyzer from Roche Diagnostics, comprehensive also flags six additional elements for review.
management of your urinalysis work area, designed for speed The latest is the IQ 200 which has more or less the same technology
simplicity and accuracy, to help drive productivity in your urinalysis The bacteria chamber mixes the urine sample with diluent and as the Arkray. The technology for both is said to be first
work area. nucleic acid stain and is heated to 42°C to suppress nonspecific discovered/from by Japan. Then Japan sold it to someone
staining. This, coupled with the anti-carryover rinse, offers (Arkray/Iris). The technology is where they get photographs of the
SEMI-AUTOMATED PROCEDURE different elements in the sediments. They will try to identify them
uncompromised bacteria discrimination. The combination of urine
1 Dip the reagent strip into a well mixture urine sample. and put them in the report.
chemistry and sediment analysis technologies generates
2 Blot the strip to remove excess urine.
standardized and accurate results in one consolidated report,
3 Place the strip onto the reagent strip platform. AUTOMATED ANALYZERS IN THE MARKET
providing operator confidence time after time. Instrument
4 Press the analyzer/enter button. ARKRAY URINE ANALYZER BY
parameters can be used to create effective reflux culture criteria to CLINITEK NOVUS BY SIEMENS
ARKRAY, INC.
minimize time and costs associated with reviewing contaminated or
negative urine cultures.

The high capacity system can meet the busiest Laboratories

workload demands by processing up to 200 chemistry samples an
hour and delivering rapid results in as little as 2 minutes and 15
seconds. The hybrid analyzer’s compact size makes it an ideal fit to
Video: Arkray Aution Hybrid AU-4050 Urinalysis Product Overview
meet space constraints of laboratories of all sizes and volumes.
Youtube Video Link: https://www.youtube.com/watch?v=GMVR5t6AzPs
COBAS U6500 BY ROCHE IRIS ANALYZER BY BECKMAN
Aution Hybrid AU-4050, proven technology you trust to achieve the
DIAGNOSTICS COUTLER
quality results you expect.

AIRAH M.
 III. PRINCIPLES IN AUTOMATION ▪ contains dissolved molecules and ion, that may not be turbid 6 LASER FLOW CYTOMETRY
▪ photometry ▪ refractometry but can be colored ▪ utilizes two stains with fluorescent dyes to stain cellular
▪ reflectance photometry ▪ laser flow cytometry ▪ principle of turbidimetry: Light is scattered by particles in the elements
▪ turbidity ▪ flow cytometry solution. A turbidimeter measures the intensity of transmitted ▪ separate bacteria channel for improved discrimination
▪ light transmission/light scatter light and a graph between to transmitted light intensity and ▪ forward scatter, hydrodynamic focusing, forward fluorescent
particle concentration can then be drawn. light, conductivity measurements, and adaptive cluster
1 PHOTOMETRY analysis to identify urine sediment particles
▪ measurement of light”
▪ substance is converted to a soluble, colored material, its
concentration is determined by the amount of color present in
the solution
▪ photometer & spectrophotometer are instruments used for
this type of measurement
4 LIGHT TRANSMISSION/LIGHT SCATTER
▪ amount of light scattered or reflected is directly proportional to
the product of the polymer molar mass & concentration
▪ angular variation of the scattered light is directly to the size of
the molecule (Mie Theory)
7 FLOW CYTOMETRY
2 REFLECTANCE PHOTOMETRY ▪ measures:
▪ uses the principle that light reflection from the test pads o Forward light scatter o Fluorescence Low Width 2
decreases in proportion to the intensity of color produced by o Side scatter (FLLW2)
the concentration of the test substance o Fluorescence staining o Fluorescence Low Width
▪ monochromatic light source is directed toward the reagent characteristic (FLLW)
pads o Adaptive Cluster analysis o Side Scatter (SSC)
▪ the light is reflected to a photodetector and an analog/digital o Forward Scatter (FSC) o Forward Scatter Width
converter o Fluorescence High (FLH) (FSCW)
▪ compare the amount of light reflection with that of known o Fluorescence Low (FLL)
concentration
▪ Ma’am worked first with Kodak (???), being a dry slide? ▪ identification of the stained urine sediment
technology, which turns out to be more expensive for the lab FACTORS INFLUENCING LIGHT SCATTER ▪ pictures for flow cytometry (with explanations) are at the last
than the liquid reagent technology 1 particle size page
2 concentration of particles MEASUREMENT TECHNOLOGY METHODS
Multi-layer Thin Film Dry Slide Technology 3 molecular weight of particles SPECIFIC
MANUFACTURER COLOR CLARITY
4 wavelength dependence GRAVITY
5 effect of polarization of incident light Refractive
Arkay, Inc. Photometry Light Scatter
index
6 distance of observation
Light Light
5 REFRACTOMETRY Transmission Transmission Refractive
Iris Diagnostics
▪ method of measuring or or Light Index
substances refractive Light Scatter Scatter
index Roche Reflectance
Turbidity Refractometry
Diagnostics photometry
▪ refractometer is the Light
instrument used to Siemens
Reflectance Transmission Refractive
Healthcare
measure refractive index photometry or Light Index
Diagnostics, Inc.
▪ measures the extent to Scatter
which light is bent when
it moves from air into a
3 TURBIDITY
sample
▪ measurement of decrease in light transmitter through a turbid
▪ refraction index: ratio of the speed of light in a vacuum to the
solution is measured
speed of light in another substance
▪ turbidity is a measure of suspended solids concentration

AIRAH M.
 SEMI-AUTOMATED URINE CHEMISTRY ANALYZERS IV. IMPLICATIONS IN AUTOMATION
list is not updated
MODEL MANUFACTURER FOR STANDARDIZATION OF:
Siemens Healthcare sample processing microscopy analysis
Clinitek Advantus biochemical test strip analysis reporting of results
Diagnostics,Inc.
Siemens Healthcare FEATURES OF AUTOMATED URINE ANALYZERS
Clinitek Status
Diagnostics,Inc. 1 on-line computer capability
Urisys 1800 system Roche Diagnostics 2 bar coding
COBAS u411 Roche Diagnostics 3 manual entry of color
DiaSreen 50 U.S Arkray 4 clarity
iChem 100 Iris Diagnostics 5 microscopic results
6 flagging of abnormal results
FULLY AUTOMATED URINE CHEMISTRY ANALYZERS
7 sorting of patients and control results
MODEL MANUFACTURER
8 minimal calibration
Siemens Healthcare
Clinitek Atlas 9 autocleaning
Diagnostics,Inc.
10 scheduled maintenance
Urisys 2400 System Roche Diagnostics
Aution Max AX-4300 U.S. Arkray DISADVANTAGES OF THE MANUAL METHOD
iChem Velocity Iris Diagnostics 1 subjective color reading
2 subjective element identification
FULLY AUTOMATED URINE MICROSCOPY ANALYZERS
3 poor reproducibility
MODEL MANUFACTURER
4 lack of standardization
UF-1000i Urine Cell Analyzer Sysmex Corporation
5 time consuming / labor intensive
iQ 200 Automated Urine
Iris Diagnostics
Microscopy REFERENCES
iQ 200 Sprint Iris Diagnostics ▪ S.K. Strasinger, Urinalysis and Body Fluids, 6th Ed
▪ N. Brunzel, Fundamentals of Urine and Body Fluids Analysis, 4th Ed
FULLY AUTOMATED URINE ANALYSER SYSTEMS
▪ Henry's Clinical Diagnosis and Management by Laboratory
MODEL MANUFACTURER
iRICELL Urinalysis System Iris Diagnostics Methods, 22nd Ed
CLINITEK AUWI System PHOTOS FOR FLOW CYTOMETRY
(Ma’am said they are being Siemens Healthcare (with the explanations)
manufactured by the two Diagnostics,Inc.
mentioned)
BODY FLUID ANALYSER SYSTEM
MODEL MANUFACTURER
iQ200 using Body Fluids
Iris Diagnostics
Software
ADVIA2120i with Body Fluids Siemens Healthcare
Software Diagnostics,Inc.
Sysmex XE-5000 using Body
Sysmex Corporation
Fluids mode

AIRAH M.
Explanation for image above. Using the flow cytometry, you have the
identification on how they stain urine sediments and are being
identified. Some of them are already being stained, so you can
identify enumerated parameters (RBC, WBC, epithelial cells, hyaline
casts, bacteria) and flagged parameters (pathological casts, crystals,
small round cells, yeast, mucus, sperm).

AIRAH M.