Clinical Chemistry

Clinical chemistry (also known as chemical pathology, clinical

biochemistry or medical biochemistry)
- is the area of chemistry that is generally concerned
with analysis of body fluids for diagnostic and
therapeutic purposes.

Types of sample that are used in testing:

Body Fluids: whole blood
serum
plasma
urine
CSF
 Three General Procedures / Methods for obtaining Blood
1. Skin Puncture / Capillary puncture / Prick Method
- usually performed on the middle or ring finger of an adult

2. Arterial Puncture
- process by which blood is obtained from patient’s artery.

Sites of collection:
radial artery scalp artery
brachial artery umbilical artery
femoral artery
3. Venipuncture
- process by which blood is obtained from patient’s vein.

Sites of Collection:
a) antecubital fossa region:
median cubital vein
cephalic vein
basilic vein
B) veins in the dorsal part of hand or wrist

c) veins on the ankle

Two General Methods of Doing Venipuncture

1. Syringe Method
– used for single blood collection only

2. Vacutainers Method
– used for single and multiple blood collect
General Precautions in Collecting Blood Samples
1. Always read the request first to see whether the examination is
complete, to determined the amount of blood needed.
2. If fasting specimen is required, confirm that the fasting order has
been followed.
3. Never extract blood from patients while they are receiving
intravenous medications.
4. Blood specimens obtained must be placed in appropriate
containers for each specific test . Ensure prompt and adequate
mixture of blood and anticoagulant to prevent formation of
“unwanted clot”.
5. Avoid prolonged application of tourniquet to avoid
hemoconcentration.
6. Never draw out the syringe without removing first the tourniquet to
avoid hematoma.
Proper Steps in Doing Venipuncture

1. Patient’s Identification
- ask patient’s full name
- verify diet restrictions

2. Preparation of materials
- syringe w/ needle
- needle/tube/holder
- tourniquet, cotton with alcohol

3. Position the Patient

- Extend the patient’s arm so that the shoulder to wrist forms a straight line
- make them comfortable
4. Application of tourniquet
- used to increase intravascular pressure
- helps with the palpitation of vein
- suggested time - 1 minute
- 3 – 4 inches above the collection site or 4 finger widths
5. Selection of the vein to be puncture
- locate a vein that is visible, straight, clear and with a good size
- most frequently used veins are the arm veins.

Important Note:
In choosing a collection site avoid areas with …..
- hematoma
- artevenous (AV) shunt or fistula
- partial or radical mastectomy
- edema
- thrombosed veins
- burned or scarred areas
6. Sterilization of the Selected vein
- clean the site using cotton w/ 70% alcohol .Start from the center and then moving
downward or outwards.
- allow it to air dry

7. Insertion of the needle

- Hold arm below the venipuncture site
- Make sure the bevel of the needle is pointing up
- Insert the needle quickly following the direction of the vein

8. Withdrawal of the blood

- for syringe pull back the plunger as the blood begin to flow into the syringe
- for vacutainer tube , push each tube into holder
- for multidraw , carefully remove each tube from the holder with gentle twist and full motion
then follow the order of draw

9. Releasing of the Tourniquet

10. Withdrawal of the needle

11. Application of dry cotton / gauze pad

- instruct patient to apply pressure (2-3 mins)

12. Label the tube with patient’s name, date, time and initial of
person collecting sample.

13. Dispose the needle in safety disposal unit.

Important Note:
Venipuncture should never be done on an artery and should not be performed on the feet unless specified.
 Blood Collection Tubes:
 Two Major types of Collecting Tubes:
1. SST - Serum Separating Tubes
2. PST – Plasma Separating Tubes

Serum Separating Tubes (SST)

Top Tubes Additives Principle Uses
Red -------- Serology
Sometimes it has gel or Enhancing the formation of -Antibodies
silicon at the bottom of blood clot -Hormones
tube to reduce hemolysis -Drugs
-Virology
-Blood Cross matching before
blood transfusion
-Chemistry
Gold ---------
It has gel at the bottom of Serum separating from the -Serology
the tube to separate serum blood through the gel in -Chemistry
from the blood the tube
 Plasma Separating Tubes
Top Color Additives Principle Uses
-the stongest anti-coagulant -Hematology
Lavander EDTA -Ca+² chelating agent -Blood Bank (ABO)
-To preserve blood cells components -HBA1C (glycosylated Hb)
PT: Protrombin Time
Light Blue Sodium Citrate Ca+² chelating agent PTT: Partial Thromboplastin Time (increase of
unexplained bleeding and liver disease)
Heparin binds to Thrombin and inhibits Enzymes
Green Sodium Heparin the second step in coagulation Hormones
or cascade Electrolytes (Na+, K+,Mg+, Cl-)
Lithium Heparin (Prothrombin Thrombin
Heparin
Fibrinogen Fibrin
ESR (Erythrocyte Sedimentation Rate)
Black Sodium Citrate Ca+² chelating agent to test how much inflamation in the patient,
unexplained fever, arthritis, autoimmune disorder
Gray Sodium Flouride Glycolysis inhibitor Glucose Test
Potassium Oxalate Anti-coagulant
Royal Heparin Anti-coagulant tube should not be Toxicology
Blue Na-EDTA contaminated with metals Trace element and metals
DNA studies, Paternity test
Yellow ACD (Acid Citrate Anti-coagulant HLA Tissue Typing (Human Leukocyte antigen)
Dextrose) The body used this protein to differentiate the
Recommended Order of Drawing/Filling Tubes as recommended by CLSI

Procedure Order
Drawing from Tubes/syringe Sterile, blue, red, gold, green,
Lavander, gray
Filling microcantainers from Lavander, green, red
fingers/heel-stick
 Preparation of Blood Sample
 One of Three Specimens may be used:
- whole blood
- serum
- plasma
These must be separated from cells and analyzed within limited time (why?)
* over time, cells will lyse which will change the conc. some analytes like potassium ,
phosphate and lactate dehydrogenase
- to prevent hemolysis
- to prevent glycolysis
- to prevent lipolysis
- certain substances are very unstable
Procedure of Serum Preparation
 1. Draw blood from patient. Put blood to vacutainer tube without anticoagulant .
2. Allow to stand for 20-30min for clot formation
3. Centrifuge the sample for about 10 min. at 2000 -2500 rpm.
4. Separate the supernatant (serum) from the clot by transferring to another
clean test tube.

Procedure of Plasma Preparation

1. Draw blood from patient. Put blood to vacutainer tube with an appropriate
anticoagulant .
2. Mix well with anticoagulant
3. Allow to stand for 10 min.
4. Centrifuge the sample for about 10 min. at 2000 -2500 rpm.
5. Separate the supernatant (plasma ) from the red cells by transferring to
another clean test tube.
 Difference Between Serum and Plasma
SERUM PLASMA
Liquid portion of clotted blood Unclotted blood

Does not contain fibrinogen Contain fibrinogen

Lipemia clearing factor is absent Present
Optically clear Less clear
SERUM - best specimen in most Clinical Chemistry test (Why?)
- has less protein
- No anticoagulant
- Most reagent are more compatible with serum
- Can be more stable than plasma with certain substances
Specimen Rejection Criteria
- specimen improperly labelled or unlabelled
- inappropriate volume of blood
- using wrong collection tube
- improper storage
- hemolysed sample

*Hemolysis – liberation of hemoglobin due to rupture of red blood cell

Causes of Hemolysis:
A. Collection of blood
- through prolonged application of tourniquet
- cleansing the venipuncture site with alcohol and not allowing
the site to dry
- the use of small bore needle
- moisture or contamination in needle, syringes or blood
containers
- excessive traction on syringe plunger.
B. Transfer of Blood
- expel blood from syringe through a needle
- allow blood to fall into the container through the air
- vigorously shaking / mixing with anticoagulant

C. Separation of Blood
- prolonged contact of serum or plasma with rbc
- exposure to excessive hot or colds can cause rupture of rbc
- the use of applicator stick to dislodge the fibrin may also cause
rupture of rbc
 PIPETTING
Pipette - is a cylindrical glass tube used in measuring fluids.
- It is calibrated to deliver or transfer a specified volume from
one vessel to another.
Classification
I. Types of pipetes:
1. Transfer or Volumetric Pipet
- consist of a cylindrical bulb joined at both ends to narrower
glass tubing
- used for accurate measurements of aliquots of nonviscous
samples, filtrates, controls, and standard solutions.

Ex. Ostwald-Folin Pipet (TD, Blownout )

Pasteur pipet
2. Measuring or Graduated
- plain narrow tube drawn out to a tip
- graduated uniformly along its length

Ex.
Serological Pipette - the graduation marks continue to the tip

Mohr pipette – the graduation marks on these always ends to

before the tip.
Mechanical / Automatic macro- or micropipette

- this pipette operate by piston-driven air displacement.

- it can be set to transfer any volume within the individual

pipette’s volume range

- it is a continuously adjustable or fixed digital pipette

II. Calibration Marks / Design
a) To Deliver (TD) – delivers the exact amount it holds into a
container.
- let drain along the side of the receiving
vessel

b) To Contain (TC)– holds the particular volume but does not

dispense the exact volume
- needs to be blown out
III. Drainage Characteristics:
a) Blowout – has a continuous etched rings on top of the
pipet

b) Self-draining – absence of etched ring, liquid is allowed to

drain by gravity.
Types of pipettes Micropipette
Pipetting Techniques using Manual Pipettes

1. Check the pipette to ascertain its correct size,

and to inspect if there is broken delivery or
suction tips.
2. Wearing protective gloves, hold lightly between the thumb and the last 3
fingers, leaving the index finger.
3. Place the tip of the pipette well below the surface of the liquid to be pipetted
4. Using aspirator bulb ,carefully draw the liquid up into the pipette until the
level of liquid is well above the calibration mark.
5. Quickly cover the suction opening at the top of the pipette with the index
finger
6. Wipe the side of the pipette, dry with a piece of gauze or tissue
paper to remove excess fluid.
7. Hold the pipette in a vertical position with the delivery tip against
the side of the original vessel. Carefully allow the liquid in the
pipette to drain by gravity until the bottom of the meniscus is exactly
at the calibration mark.
8. While still holding the pipette in vertical position, touch the index
finger from the top of the pipette to permit free drainage.
9. To be certain that the drainage is as complete as possible touch the
delivery tip of the pipette to another are on the side wall of the
receiving vessel.
10. Remove the pipette from the receiving vessel and place it in the
pipette washer.
Micropipettes (uL)
Pipette operation:
*When operating the plunger
two points o resistance are met:

- The 1st stop is the point

when the piston reaches the
calibrated volume on the dial

- The 2nd stop is used to

totally expel the sample
volume into a receiving
vessel
 Steps of Liquid transfer using an Automatic micropipette

1. Set the volume

2. Attach a disposable tips
3. Depress the plunger to the first stop
4. Immerse the tip in the sample and draw up sample
5. Pause (wait for few seconds to ensure that the full volume of sample is
drawn into the plastic tip)
6. Withdraw the pipette from the sample
7. Dispense the sample into the receiving vessel (2nd stop)
8. Withdraw the pipette from the receiving vessel
9. Release the plunger
10. Discard the tip ( depressing the tip ejector)
1 ml (milliliter) contains 1000 ul( microliter)
Important terms in CC
Blank - used to set the spectrophotometer to zero absorbance and 100%Transmittance
water blank (dist. Water) - seen mostly in UV procedures
reagent blank - seen in colorimetric procedures
- contains all the reagents used in the `tests’.
Standard - used to calibrate an assay method
standard solutions (also called Calibrator)- a solution that contains known amount of analyte
Control - are used to verify the accuracy and acceptability of a run
- it should be tested in the same manner as the patient samples .
control Soln- can be a liquid or freeze dried (lyophilized material) and
composed of 1 or more constituents (analytes) of known concentration.

Types of Control solution/reagent : 1. Commercially prepared 2. Non-commercially prepared

normal control – contains normal level for the analyte being tested
abnormal Control – contains analyte at a concentration above or below the NR for the analyte being tested

*Good Laboratory practice requires testing normal and abnormal controls for each test daily *
unknown (patients sample)
Fasting Blood (FBS)
Method : Glucose Oxidase
Principle:
D- Glucose is oxidized by glucose oxidase to produce D-Gluconic acid and hydrogen is then
oxidatively coupled with-aminothypyrine and phenol substitute, p-HBS in the presence of peroxidase
to yield to a red quinonimine dye. The amount of colored couple formed is proportional to glucose
concentration and can be photometrically measured
Wavelength : 500 – 520 nm
Temperature: 37 0C
Procedure:
1. Label 5 test tubes : Blank , Standard, Control (N), Control (Ab) Unknown
2. Put the following:
Blank - 10 ul dist. water
Standard - 10 ul std. Soln.
Control (N) - 10 ul control rgt
Control (Ab) - 10 ul control rgt
Unknown - 10 ul pt. serum
3. Pipette 1.5 ml of glucose reagent to all tubes.
4.Mix and stand for exactly 10 minutes .
5.Read all tubes in TC 84+ at 500 nm against reagent blank.
Normal Value: 3.9 – 5.8 mmol/l
Cholesterol:
Method : CHOD-PAP ,Enzymatic photometric test

Principle :
Cholesterol and its esters are released from lipoproteins by detergents. Cholesterol esterase hydrolyzes the
esters. In the subsequent enzymatic oxidation by cholesterol oxidase , H 2 O is formed. This is converted into a
colouredquinonimine in a reaction with 4-aminoantipyrine and phenol catalyzed by peroxidase.
Wavelength : 500 – 520 nm
Temperature: 37 0C

Procedure
1. Label 5 test tubes: Blank, Standard, Control (N), Control (Ab) Unknown
2. Put the following:
Blank - 10 ul dist. water
Standard - 10 ul std. Soln.
Control (N) - 10 ul control rgt
Control (Ab) - 10 ul control rgt
Unknown - 10 ul pt. Serum
3. Pipette 1.0 ml of cholesterol reagent to all tubes.
4. Mix well and incubate for 5 minutes at 37 ˚C.
5. Read all tubes in TC 84 Plus machine at 500 nm against reagent blank.

Normal Value : 0 – 5.2 mmol/l

Blood Uric Acid (BUA)
Method : Enzymatic , Colorimetric
Principle :
Enzymatic determination of Uric Acid according to the following reactions:
Uricase
Uric Acid + 2H2O + O2------------------------Allantoine + CO2 + H2O2
Peroxidase
2 H2O2 + 4 –AAP + DHBS ---------------------- Quinoneimine + H2O + HCL
DHBS = 3,5 – Dichloro-2Hydroxy-Bensenesulfonic Acid
4-AAP = Amino-4antipyrine
Wavelength : 500 nm
Temperature : 37 0C
Procedure:
atic determination of Uric Acid according to the following reactions: DHBS = 3,5 – Dichloro-2Hydroxy-Bensenesulfonic Acid 4-AA = Amino-4antipyrine

En
Blank Standard Control (N) Control (Ab) Unknown
Reagent 1000 ul 1000 ul 1000 ul 1000 ul 1000 ul
Standard -- 25 ul -- -- --
Control (N) -- -- 25 ul -- --
Control (Ab) -- -- -- 25 ul --
Unknown -- -- -- -- 25 ul
Mix and incubate for 5 mins in TC84 Plus at 500 nm against reagent blank.

Normal Value : 155 – 357 umol/l (Female)

208 – 428 umol/l (Male)
 Semi- Automatic Clinical Chemistry Analyzer
MAP LAB PLUS / TC 84 Plus

Spectrophotometer (instrument)- which measure an amount of light that sample absorbs

Spectrophotometry – is a method to measure how much a chemical substance absorbs light

by measuring the intensity of light beam of light passes through sample solution.
The basic principle is that each compound absorbs or transmits light over a certain range of
wavelength.
 Fully Automatic Clinical Chemistry Analyzer
BT 1500

Automated Analyzer ( BT 1500) based upon the spectrophothometry principles

Principles of operation:
After the tray is loaded with sample, a pipette aspirates a precisely measured aliquot of sample and
discharges it into the reaction vessel ;A measured volume of diluent rinses the pipette. Reagent are
dispensed into the reaction vessel. After the solution is mixed (and incubated, if necessary) , it is either
passed through a colorimeter, which measures its absorbance while it is still in its reaction vessel, or
aspirated into a flow cell, where its absorbance is measured by a flow-through colorimeter. The analyzer
then calculates the analyte’s chemical concentration.
 Quality Assurance (QA) in Laboratory Testing

 Introduction:

- Clinical Labs are important in diseases diagnosis, determination its severity and
patient response to specific treatment. Diagnosis of any disease is first done by
the physical examination by doctor and confirmed by laboratory diagnostic test.

- Laboratory values are very important in determination of disease

severity and in follow-up.

- The pathologist and Laboratory people are very much involve in:
correct diagnosis, effective treatment and follow-up of the patient

- For correct diagnosis QA and QC are very important

 Quality Control (QC) – it refers to the measures that must be included during each
 assay run to verify that the test is working properly

 Quality Assurance (QA) – is defined as the overall program that ensures that the final
results reported by the laboratory are correct.

*The aim of QC is simply to ensure that the results generated by the

test are correct. However QA is concerned with much more: that the
- right test is carried out on the
- right specimen and that the
- right result and
- right interpretation is delivered to the
- right person at the
- right time
Quality Control can be implemented in 2 ways

I. Internal QC (Intralaboratory )
- performed by individual labs at their own levels. It forms the day to day basis working QA
- involves the analysis of control sample together with patient sample
- It is important for daily monitoring of accuracy and precision of analytical methods

II. External QC (Interlaboratory)

- performed by many labs at the same time, monitored by one (DOH)
- It involves proficiency testing programs (NEQAS) that periodically provide samples of
unknown concentration of analytes to participating laboratories
- It is a way to compare the performance of a laboratory with reference to other laboratories
.
*For Clinical Chemistry QC Accuracy and precision of test is important.

Accuracy – is defined as closeness of the test result and accepted true value

Precision – reproducibility of a results, whether accurate or inaccurate within

a define frame time.

*Can be controlled by replicate test, check test on previously measured specimens and statistical
evaluation of results.

Daily QC Chart preparation is mandatory (Levy-Jennings Chart)

Control value within ±2SD is good sign and patient result obtained are reliable and
can be reported
Control value beyond ±2SD the result must not be reported
  Laboratory work Flow Cycle

 Three Phases : Pre-analytical, Analytical , Post –analytical

 (Component of Quality Assurance)

I. Pre-analytical - everything that precedes actual test performance

example:
Process Potential error

Test ordering wrong patient ID

inappropriate test
partly erased or illegible label
disparity between request form and specimen
specimen collected in container without proper
anticoagulant
II. Analytical - everything related to actual testing process
Example :
Analytical measurement : - Instrument not calibrated properly
- If the principle and procedure of the test is
not strictly followed; reagents, standards,
- QC materials are not prepared, mixed and
processed properly
III. Post-analytical - everything that comes after the test analysis
Example:
Test Reporting : - may occur if reporting , checking and verification are not
done properly (transcription error)
- abnormally/unexpected result is not taken seriously not
reviewed and performed again.(report not legible)
- report delayed
Clinical Chemistry Requirements :

Bring Patient- (must be an OUTSIDER and at least 8 hours fasting)

5 ml syringe with 21 gauge needle (bring extra) or vacutainer

tube (Red Top)
Tourniquet
Alcohol
cotton (wet and dry)
gum label / masking tape
Dropper
aspirator bulb
laboratory gown / head cap

Note:
The actual extraction will be done at the Clinical Laboratory
Thank you for your
attention
Clinical Microscopy
CLP
 Urine or Urine Analysis
• One of the oldest laboratory procedure
• Is the physical, chemical, and microscopic examination of
urine involving a number of tests to detect and measure
various compounds that passes through the urine.

• Reasons for urinalysis:

• General evaluation of health
• It screens asymptomatic populations for undetected disorders.
• It helps in monitoring the progress of disease and the
effectiveness of therapy.
• Screening for Drug abuse
URINE COMPOSITION

 95-97% Water
 3-5% Solutes

 Urea- major organic

component
 Chloride – major inorganic
component
TYPES OF URINE SPECIMEN:
1. Random/Occasional/Single
- for routine and qualitative urinalysis
2. First Morning
 - is the most concentrated, most
acidic
- Ideal specimen for routine UA and
pregnancy test.
3. Second Morning/ Fasting
- second voided urine after a period of
fasting; for glucose determination
 4. 2-hr postprandial
 - for diabetic monitoring
 5. Glucose Tolerance test

 - optional with blood samples in

Glucose tolerance test
 6. Midstream clean-catch Ffor URINE CULTURE

 7. Catheterized

 8. Suprapubic aspiration

 - for anaerobic urine culture and urine

cytology
9. Three-glass technique
- for prostatic infection
1. First portion of voided urine
2. Middle portion of voided urine
3. Urine after prostatic massage
• Examine the 1st and 3rd specimen
microscopically, then compare the no. of WBC
and bacteria.
• Prostatic Infection- If the no. of WBC and
bacteria in the 3rd specimen is 10x greater
than that of the 1st (3rd > 1st)
• 2nd specimen- control for kidney & bladder
infection. If (+), the result for the 3rd specimen
is invalid.
10. Pediatric specimen
- uses a soft, clear plastic bag w/
adhesive.
11. Timed specimen
• 24hr (Ex:8am to 8am)- requires
preservative
• 12hr (ex: 8am to 8pm) – for Addis count
• 4hr - for Nitrite determination
• Afternoon(2pm to 4pm)- for Urobilinogen
determination
 Specimen Evaluation
Before one can proceed with any examination,
the urine specimen must be evaluated in
terms of its acceptability. Considerations
include:
 Proper labelling
 Proper specimen for the requested test.
 Proper preservatives (if necessary)
Visible signs of contaminations
Transportation delay
 SPECIMEN INTEGRITY
 In general, randomly collected urine specimen
are satisfactory for microscopic evaluation,
however, it is recommended that the
examination takes place when the sample is
fresh, particularly if no preservative has been
added. Cells and cast begin to lyse within 2
hours of collection.
 Changes in
unpreserved/deteriorated urine
• 1. increase in bacterial content
• 2. increase in turbidity
• 3. increase pH (Alkaline)
• 4. increase nitrite
• 5. decrease glucose
• 6. decrease ketones
• 7. decrease bilirubin
• 8. decrease urobilinogen
• 9. disintegration of RBCs, WBCs and cast,
particularly in dilute alkaline urine
• 10. changes in color due to oxidation and
reduction of metabolites
 URINE PRESERVATION
PHYSICAL CHEMICAL

 REFRIGERATION  Formalin
 (Addis count)
 FREEZING
 Boric acid
 (Urine culture)

 NaF/benzoic acid
 (Glucose)

 Saccomano’s fixative
 (Urine cytology)
PHYSICAL EXAMINATION

I. Volume
II. Color
III. Turbidity/Clarity/Transparency
IV. Odor
COLOR
TURBIDITY
ODOR
Odor Cause
Aromatic Normal
Foul, ammoniacal UTI
Fruity, sweet Ketones
Caramelized sugar, curry, maple MSUD
syrup PKU
Mousy Tyrosinemia
Rancid butter Isovaleric acidemia
Sweaty feet MM
Cabbage Contamination
Bleach Cystine disorder
Sulfur Trimethylaminuria
Rotting fish Ingestion of onions, garlic &
Pungent asparagus
 Chemical Examination
•Reagent Strip Reading time
•Leukocyte esterase 120s
•Nitrite 60s
•Urobilinogen 60s
•Protein 60s
•pH 60s
•Blood 60s
•Specific Gravity 45s
•Ketones 45s
•Bilirubin 30s
•Glucose 30s
CLINICAL SIGNIFICANCE
Macroscopic
Color urine concentration, presence of pigments
Odor diet, bacteria, amino acid disorders
Turbidity crystals, cells, contamination

Reagent strip
pH Useful in interpretation of microscopic crystals

Hemoglobin RBC trauma or enzyme deficiencies, unstable hgb, immune

mediated
Specific gravity Useful in microscopic results
Protein Glomerular diseases
Glucose Diabetes mellitus
Ketones Ketoacidosis
Nitrite Bacterial presence
Leukocyte esterase Pyuria
MICROSCOPIC
EXAMINATION

CLASSIFICATION OF
URINARY SEDIMENTS
SEDIMENT CONSTITUENTS
II. Casts
The major constituent is Tamm-Horsfall
protein, a glycoprotein excreted by the RTE
cells of the distal convoluted tubules and
upper collecting ducts.
Other proteins present in the urinary
filtrate such as albumin and
immunoglobulins are also incorporated into
the cast matrix.
 Cylindroids – casts with tapered ends
produced at the junction of ascending
loop of Henle & DCT
 Cylindriduria or Cylinduria - presence of
cast in the urine

Hyaline cast
Cellular cast
Granular cast : coarsely-finely
Waxy cast (final degenerative form)
Types of Casts:

a. Hyaline casts
-colorless, homogenous and has same refractive
index as urine
-made up of entirely Tamm-Horsfall Protein
-Indicates “mild renal damage”
-0-2/lpf is considered normal
Clinical Significance:
1. non-pathogenic: strenuous exercise, dehydration,
heat exposure, emotional stress
2. pathogenic: acute glomerulonephritis,
pyelonephritis, chronic renal disease, congestive
heart failure
b. RBC casts

 Indicates hemorrhage in the renal tubules,

“active acute nephritis” and “glomerulonephritis”
 Found in healthy individual following participation in
“contact sports” or strenous exercises.
c.WBC casts
 Presence signifies “infection and
inflammation within the nephron”
 Most frequently seen in pyelonephritis
 Presence indicates the need to perform
bacterial cultures

d. Epithelial cell casts

 Observed in conjunction with RBC and
WBC cast in glomerulonephritis and
pyelonephritis
e. Granular casts
Usuallyseen accompanying hyaline cast following periods of stress
and strenuous exercise

f.Waxy cast
Indicates “extreme stasis of urine flow”
Refractile with rigid texture, which causes them to be fragmented or
jagged as they pass through the tubules
g. Fatty cast
Seen in conjunction with oval fat bodies in disorders
causing “lipiduria” such as nephrotic syndrome

h. Broad cast
Presence in urine presents grave prognosis, referred to as
“renal failure cast”
III. CRYSTALS
A. Crystals in Acidic Urine
1. Amorphous urates
 Macroscopic pink upon refrigeration
 Microscopic brick red or yellow brown in color
 2. Uric acid
• Product of purine metabolism
• Rhombic, wedge, hexagonal, whetstone, lemon-
shaped
• Mistaken as cystine crystals
• Present in pxs with Lesch-Nyhan syndrome
3. Calcium oxalate
May also be seen in neutral/sl. Alkaline urine
Mostly appear as envelope or pyramidal (dihydrate)
Dumbbell and ovoid rectangle seen in cases of ethylene
glycol poisoning (monohydrate)
Derived from various food notably spinach, rhubarb,
berries and tomatoes
B. Crystals in Alkaline Urine
1. Amorphous phosphate
Granular, similar to amorphous urates, macroscopic white
turbidity
2. Calcium carbonate
Also seen in neutral urine
Colorless granules larger than amorphous phosphates
Often appearing as “dumbbell forms or spherical forms”
3. Ammonium biurate
Yellow-brown/golden
“thorny apples” / spicule-covered spheres
Only urate found in alkaline urine
Converts to uric acid crystal when glacial acetic acid is
added
4. Calcium phosphates
Colorless, flat rectangular plates on thin prisms often in
rosette formation
Constituent of renal calculi

5. Triple phosphate
Referred to as coffin lid crystals
Also seen in neutral/ sl. Acidic urine
Seen in highly alkaline urine associated with presence of urea-
splitting bacteria
 C. Abnormal Acidic Urine
1. Cystine
Hexagonal plates, colorless, highly refractile and thick/thin
Often mistaken with uric acid but dissolves in diluted HCl
(uric acid does not)
* A noticeable “sulfur odor” may be present in the urine in
disorders of cystine metabolism
2. Cholesterol
Large flat plates with one or more corners cut-off; notched
plates
Seen in nephrotic conditions and lipid necrosis
Soluble in chloroform
Associated with: disorders producing lipiduria such as
nephrotic syndrome
3. Leucine
Yellow or brown spheres resembling fat globules with
delicate radiating and concentric striations
Less seen than tyrosine, soluble in hot alkali/alcohol
“scallop-lily looking crystal”
4. Tyrosine
 Colorless fine needles grouped in clusters (may appear
black in the center). Rosettes or sheaves crossing at
various angles
 Seen in conjuction with leucine crystal, (+) bilirubin test
 Soluble in alkali and heat
 Has “sour or rancid” odor
5. Bilirubin
Yellow-rhombic/ruby red crystals/clumped needles or granules
with characteristic yellow
Soluble in HAc, HCl, NaOH, ether and chloroform

6. Sulfonamides
Cause of formation: inadequate patient hydration
Green color, soluble in acetone
Less encountered: needles, rhombic, whetsones, sheaves of wheat,
rosettes with colors ranging from colorless to yellow brown
Clinical significance: tubular damage
 7. Radiographic dye
• Similar to cholesterol crystals
• To differentiate with cholesterol:
• 1. patient’s history
• 2. correlation with other UA results
• 3. Radio. Dye = SG > 1.040 (refractometer)
CLINICAL RELEVANCE
Manner of reporting

RBCs Ave. number per 10 hpfs

WBCs Ave. number per 10 hpfs
Squamous cells Rare, few, mod.,many / lpf
Bacteria Few, moderate, or many/hpf;
presence of WBCs must be
required
Mucus threads Rare, few, mod., many / lpf
Casts Ave. number/lpf
FECALYSIS
Importance:
1.Detection of gastrointestinal bleeding
2.Detection of biliary and liver disorders
3.Detection of malabsorption and maldigestion
syndrome
4.Identification of bacteria and parasites.
Components of Normal Stool:
1.Bacteria
2.Undigested food stuff
3.Bile pigments
4.Water and electrolytes
5.Enzymes
Macroscopic Screening
Normal color: Brown
Normal consistency: Formed
Color/Appearance Possible Cause
Black Upper GI bleeding; Iron
therapy; Charcoal; Bismuth
Red Lower GI bleeding; Beets and
food coloring; Rifampin
Pale yellow, white, gray Bile-duct obstruc.; Barium
SO4
Green Biliverdin/ oral antibiotics;
green vegetables
Bulky/frothy Bile-duct obstruc.; Pancreatic
disorder
Ribbon-like Intestinal constriction
Mucus/ blood-streaked mucus Colitis; Dysentery; Malignancy;
Constipation
 Microscopic Screening
Fats
Muscle fibers
Fecal leukocytes
 Parasites
Chemical examination
Fecal occult blood test
APT test
Test for fecal enzymes
Test for fecal carbohydrates
 •Ascaris •Hookworm •Trichuris

•E. Histolytica cyst

•E. Histolytica •G. Lamblia troph
Troph

•G. Lamblia cyst