lOMoARcPSD|19521100

NOTES IN CLINICAL MICROSCOPY: By Gerard Andrew Ramos, RMT

RENAL FUNCTION
Kidneys
• The kidneys selectively clear waste products from the blood & simultaneously maintain the body’s
essential water & electrolyte balances.
• Each kidney contains 1 to 1.5 million functional units called NEPHRONS

2. UNFILTERAD BLOOD
ACTUAL
2.
FILTRATON
6. RENAL VEIN
BACK TO THE PERIPHERAL
ENTRY BLOOD CIRCULATION
RENAI I
Brop H.
PERITBUM
ARIES

LEXCHANGE
FoN

OFSUBSTMEN BULES
M

FILTRAG (S6: 1,010)

-GWMERULAR

Lo ACTUAL FILRATION OF THE BLOR

FOUR MAJOR RENAL FUNCTIONS:

1. Renal Blood Flow
• Based on an average body size of 1.73 m2 of surface
1200 mL/min. (25% OF CARDIAC OUTPUT
▪ total renal blood flow = _______________ *

▪ total renal plasma flow = _______________

300 700 mL/min. -

2. Glomerular Filtration
• Glomerulus is consists of a coil of approximately eight capillary lobes, the walls of which
are referred to as the glomerular filtration barrier. It is located within Bowman’s capsule.
• Responsible for non-selective filtration of substances in plasma
▪ Filtered substances – MW of less than 70,000 da > 70,00: I 2 3
· NOT FILTERAD CO2 NO BIG

▪ Pressure is regulated by R-A-A system (RENIN-ANGIOTENSIN-ALDOSTERONE)

L IMPORTANT NA CONSISTENT
PRESSURE NEEDED
NOTE: MAINTAINED

MONITORSHIEPRESSURE
VOUm TR

↑ra
↓ Blood Pressure AFFECTS THE GLOMERULAR PRESSURE
PHYSIO LOGIC EFFECTS:
EX: MASSIVE BLEEDING PROMOTING A BLOOD FLOW
SOURCE: JUX TAGLOMERULAR
↓ Glomerular Pressure I. VASODICATION OF AFFERENT ARTERIOLES
OF HO & SODIUM
IN THE PROXIMAL CONSULATED

1APPARAN(EN2ymE) STIMULATES REABSORPTION HLO RETENTON

RIMMOREENY
on
2.
ACE IANGIOTENSIN_CONVERTING ENZYME) TUBULE_. CORRECTS BLOOD VOLUME
SOURCE: LUNGS
3. STIMULATES RELEASE OF 2 HORMoWEs
& polEnion minim

Angiotensinogen SUBSTRATE
Angiotensin I Angiotensin II RESULT: CORRECTS
THE PROBLEM INSIDE

BLOOD-BORNE

3. Tubular Reabsorption
• Essential substances and water are reabsorbed back to circulation (PCT→ Blood capillaries)
• Mechanisms:
▪ Active transport
O
o substance to be reabsorbed must combine with a carrier protein
2

o the electrochemical energy created transfers the substance across membranes

▪ Passive transport
o movement of molecules across a membrane as a result of differences in their
concentration or gradient on opposite sides of the membrane.

MAJORIM OF ESSENTIAL SUBSTANCES ARE

T
E AB SORBED
R

L
PARTNER

WALLS ARE
IMPERMEABLE O- NaCl
7 T
WATER
*

&

I NFLUENCED
↓ BY ADH

PCT FAII N SECRETE HYDROGEN

ION:

HERINEELINE
IN
> HCOG WILL BE ELIMINARD
URINE

4. Tubular Secretion(BLOOCAPILLARIES TUBULES)

=RENAL TUBULAR
CIDOSIS
A

Lo LEAD TO METABOLK

ACIOspH, HCOR
• Elimination of unfiltered waste products;
LEVEL
*

EX: MEDICATIONS
9 901 CHOULD BE
E AB SOR BED

• Regulation of acid base balance

R CARBONIC ACID

BUFIEE
->
SECRETION OF HYDROGEN IONS BUT
& NOT ELIMINATED

REABSORBES
> ESSENTAL

REAB SOR BED

TO

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GLOMERULAR FILTRATION TESTS

Clearance Test
• standard test for the filtering capacity of glomerulus
• measures the rate by which the kidneys are able to remove a filterable substance from the blood
• Substance must be one that is NEITHER reabsorbed nor secreted by the tubules
• Improperly-Timed Specimens = GREATEST SOURCE OF ERROR in any clearance procedure

1. Urea
USED IN

AE PAST
• earliest glomerular filtration test
• 40% of filtered urea is reabsorbed
• REPLACED by newer methods

2. Inulin
• Original REFERENCE METHOD
• polymer of fructose; extremely stable; used in the past
• EXOGENOUS procedure
24-AR URINE

3. Creatinine SAMPLE: X PLASMAISERUM

• waste product of muscle metabolism, produced enzymatically by creatine phosphokinase from creatine
• increased intake of meat can raise the urine and plasma levels of creatinine during the 24-hr collection
• not a reliable indicator in patients suffering from muscle-wasting diseases

4. Cystatin C ↑= ↓GFR
• MW: 13,359 – produced at a constant rate by all nucleated cells
• readily filtered by the glomerulus and reabsorbed and broken down by the renal tubular cells.
• No cystatin C is secreted by the tubules, and serum concentration can be directly related to the GFR
• Advantage: Independent of Muscle Mass ↑SERUM LEVEL CAN DIRECTLY REFLECT THE OFR

5. Beta2 microglobulin
• MW: 11,800 – dissociates from HLA at a constant rate and is rapidly filtered by glomerulus
• Advantage: more sensitive indicator of decreased GFR than creatinine clearance
• Disadvantage: not reliable in patients w/ history of immunologic disorders / malignancy

6. Radionucleotides
• enables visualization of the filtration in one or both kidneys FIRST FUNCTION
TUBULAR
AFFECTED IN RENAL DISEASE:
REABSORPTON
• EXOGENOUS procedure (125I-iothalamate)
1.

• valuable to measure the viability of a transplanted kidney

Glomerular Filtration Rate (GFR)

• volume of plasma from w/c the clearance substance is completely removed per minute
• reported in mL / min 15mL/min:
A <COML/min:
<
KIDNEY DISEASE

KIDNEY FAILURE

• NOT useful indicator of Early Renal Disease

Calculation of plasma cleared / min (C) (Adjusting Body Size)

C = Urine creatinine (Urine volume) C = UV x 1.73Me -
CONSTANT

Plasma creatinine P Px BSA (BODY AREAT SURFACE

mL/min

URINE CREATINE: 120 mg/dL c:N.1.055 1.29)

Reference Range: PLASMA CREATNINE: 1.2 mgldL
mL/min. CFR =
105 mL/minT
Serum Creatinine: 0.5 – 1.5 mg / dL URINE VOLUME FOR 24-HORS; 1520 mL

mL/min.
Urine Creatinine Clearance: 120 mL / min omd.comor:
# 1.055

o Male: 107 – 139 mL / min

o Female: 87 – 107 mL / min

24-HOUR URINE COLLECTION

DAY 1 TAM - FIRST MORNING DISCARDED

10 AM -
INCLUDED

b
WHOLE DAY-INCLUDED

FAM. FIRST MORNING CLAST URINE COLLECTED

DA+ 2
-
COLLECT BLOOD FOR PLASMA/SERUM

MEASURES
* THE TOTAL VOLUME
ALIQUOT OF 24-HR URINE: REPRESENTATIVE SAMPLE
*

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Other Formula:
1. COCKCROFT & GAULT Formula
• Predicts creatinine clearance
• Historical and not used clinically nowadays
• will likely result to 10-20% higher than current methods
2 ERROR

DOES NOT REQUIRE URINE

Male: Ccr = 140 – age x Px weight

serum creatinine 72

Female: multiply the answer by 0.85

2. eGFR using Modification of Diet in Renal Disease (MDRD) Formula:

• most widely-used equation to estimate GFR in adults ISOTOPE-DILUTION MASS SPECTROMETRY

• correspond more closely to the IDMS reference method

• most accurate when results are lower than 60 mL/min
• Reporting of results:
o Lower values (< 60 mL/min) = numerical reporting
o Higher values = reported as > 60 mL/min

GFR = 175 × serum creatinine–1.154 × age–0.203

NO NEED OF URINE

If Female: multiply the answer by 0.742

If African-American: multiply the answer by 1.202

TUBULAR REABSORPTION TESTS

RENAL Concentration Tests - tests to determine the ability of the tubules to reabsorb essential salts & H2O
↑INCONVENIENT
1. FISHBERG METHOD = patients were deprived of fluids for 24 hrs. prior to measuring sp. gr.
2. MOSENTHAL METHOD = compares the volume & sp. gr. of day and night urine samples
FIRST MORNING: 4URINE
SO

NURMAL
b Specific Gravity = affected by number of particles present and its densities USEFUL AS SCREENING PROCEDURE FOR URINE CONCENTRATON

REN AL CONCENTRAT.
BlUtY
A Osmolality = affected only by number of particles present
REABSORB
c ABILITY TO &PROVIDES MORE ACCURATE EVALUARON OF DENAL CONCENTRATING ABILIT

HW & ESSENTIAL SALT LoREQUESTED BY THE DOCTOR

Solute dissolved in Solvent: (changes in colligative properties)

↓ Freezing pt. ↑ Boiling pt.
↓ Vapor Pressure ↑ Osmotic Pressure

Freezing Point Osmometers USED FIRST & MOST

• measuring FREEZING POINT DEPRESSION was the first principle utilized by clinical osmometers
• clinical osmometers uses solutions of known NaCl conc. as their reference standard
T

Reference ranges: EVALUATION OF: CLOMERULAR FILTRATION-CLEARANCE

TEST

Serum osmolality : 275 – 300 mOsm REABSORPTON OF H2O C NaCI-CONCENTRATION

EST
BULAR
M

Urine osmolality : 50 – 1400 mOsm RENAL BLOOD FLOW

AMBULAR SECREMON
-P-AMINOHIPPURIC
SPAH TESTS
ACID EST

>URINE & SERU

Ratio of Urine to Serum osmolality

• should be at least 1:1 NORMAL

• after controlled fluid intake/restriction, it should reach 3:1 INDICATES NORMAL RCA CRENAL CONCENTRATING ABILITY

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INTRODUCTION TO URINALYSIS
Urine Composition
95% Water
5% Solutes
• MAJOR Organic Component: ________
UREA

• MAJOR Inorganic Component: _______

CHLORIDE INGT K

Urine Volume ↑
AFFECTED BY BODY HYDRATION STANS

• Normal Range (24 hrs): 600 – 2000 mL

• Average (24 hrs): 1200 mL
&IABETS INSIPIDUS
DIABETS MELLINUS
POLYURIA
> 2000 mL / 24 hrs Increased fluid intake VOLUME POLYURIA
↑ALE YELLO
POLYURIA COLOR ALE YELLOW
↑

Diuretics DEFECTIVE HORMONE ↓ INSULIN

ADH
↓

EXCRI)
CLARGE AMOUNT OF HIO
↑(GLYCOSURIA)
Diabetes mellitus
↓
SC

Diabetes insipidus

OLIJURIA < 400 mL / 24 hrs Dehydration / burns

Renal calculi / tumor

ANURIA < 100 mL / 24 hrs Complete obstruction (stones, carcinomas)

Toxic agents (MERCURY POISONING)
Hemolytic Transfusion Reaction
NOCTURIA >500 mL at night Diuretics
Sp. Gr. < 1.018 Low nocturnal bladder capacity

URINE SPECIMEN COLLECTION

• Specimens must be collected in clean, dry, leak-proof containers.
• The recommended capacity of the container is 50 mL
• Volume required is 12 mL of urine
• Specimens should be tested WITHIN 2 HOURS after collection

CHANGES IN UNPRESERVED URINE

1. Color – modified/darkened oxidation/reduction of metabolites BACTERIA

2. Clarity – decreased bacterial growth; precipitation of urates NITITE

PI (UREA AMMONIA)
3. Odor – increased breakdown of urea to ammonia
>

ODOR

4. pH – increased breakdown of urea to ammonia

5. Glucose – decreased glycolysis and bacterial use
CLUCOSE
BILIRUBIN
UROBILINOGEN
6. Ketones – decreased volatilization ICETONES
RBC, WBC, CAST (DILUTED, ALKALINE URINE
7. Bilirubin – decreased light sensitive ↓RINE CLARITY

8. Urobilinogen – decreased oxidation to urobilin WHAT PARAMETER IS LEAST AFFECTED?

9. Nitrite – increased multiplication of nitrate-reducing bacteria - URINARY PROTEIN

10. RBC,WBC & casts – decreased disintegration in dilute, alkaline urine

11. Bacteria – increased multiplication

URINE PRESERVATION
- CAUSES MORE CRYSTALS
REFRIGERATION - most routinely used method (2-8C) up to 24 hrs; precipitates amorphous urates, phosphates
1. __________ INHIBITS BACTERIAL MULTIPULATION

2. __________ – keeps pH at 6.0; Bacteriostatic; used in transport for urine culture

BORIC ACID

3. Formalin – excellent sediment preservative (Addis count); but acts as reducing agent
4. Sodium fluoride – good preservative for drug analysis; inhibits rgt strips for glucose, blood, leukocytes
5. Phenol – does not interfere w/ chemical tests; causes an odor change
6. _____________
SACUMANNO'S - used for CYTOLOGY; made up of 50% ethanol & 2% carbowax
FIXATIVE

7. Light Gray and Gray C&S tube – sample is stable at room temp for 48 hrs; has boric acid
8. Yellow UA Plus tube – used in automated instruments; must refrigerate within 2 hrs
9. Cherry Red/Yellow Preservative Plus tube – stable for 72 hrs at room temp; instrument-compatible

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TYPES OF URINE SPECIMEN

RANDOM URINE COLLECTION

Routine screening for obvious abnormality, may produce erroneous results

FIRST MORNING URINE IDEAL specimen; useful for Pregnancy test and Orthostatic proteinuria
> MOST CONCENTRATED

OR
FASTING URINE SAMPLE
Recommended for glucose monitoring (for DM)
2 HOUR
- ↑USTPRANDIAL URINE

24-Hour specimen = used for Quantitative Chemical tests FORMED SPECIMENS

12-Hour specimen = ADDIS COUNT using hemocytometer
BACTERIURIA/ ID ETECTS POSSIBLE BLADDER INFECTION
TIMED-URINE SPECIMEN 4-Hour specimen = Nitrite Determination – for presence of bacteria
2pm – 4pm specimen = Urobilinogen Determination

Specimen is collected under sterile conditions by passing a hollow tube

CATHETERIZED URINE
through the urethra into the bladder; can be used for bacterial culture

safer, less traumatic method for obtaining urine for bacterial culture and
COLLECTION
MIDSTREAM
routine urinalysis; less contaminated by epithelial cells and bacteria

Specimen is collected by external introduction of a needle through the

SUPRAPUBIC ASPIRATION abdomen into the bladder; useful for Anaerobic Culture

↑HREE-CLASS COLLECTION
PORTION OF URINE IS COLLECTED
TECHNIQUE
Useful to determine PROSTATIC INFECTION
FIRST: FIRST

SECOND: MIDDLE PORTION OF URINE

Prostatic infection: 3rd bottle - WBC is 10x more than that of the 1st bottle
....-

THIRD:
PROSTATIC MASSAGE--..........
REMAINING PORTION OF URINE 2nd bottle (midstream urine) = serves as “control” for bladder infection
~ WIC COUNT
CES

DRUG SPECIMEN COLLECTION

• Ensure that no tampering of the specimen occurred, such as substitution, adulteration, or dilution.
• Chain of Custody refers to procedures to account for each specimen by tracking its handling and
storage from point of collection to final disposal
CHROMANGRAPHY) FOR SCREENING (THIN LATER

• Required volume: 30 – 45 mL
-

(OC/MS -CONFIRMATORY

• Temperature required after 4 minutes: 32.5 to 37.7°C

PHYSICAL EXAMINATION OF URINE

COLOR
• roughly indicates the degree of hydration & should CORRELATE WITH URINE SP. GR.
• Normal Urine Color: _______________________
IDIFFERENT SHADES TELLOW OF

• Urine Pigments:
o UROCHROME
▪ MAJOR urine pigment; yellow color; amount is dependent on metabolic state
▪ named by Thudichum in 1864; product of endogenous metabolism
▪ increased production in ___________________________
StARVAMONIFASTING, FEVER, THROTOXICOSIS (PTHY ROID HURMONES

o
o Uroerythrin
▪ pink color; most evident in refrigerated specimen (ppt of urates)
▪ attaches to the urates, producing pink color to the sediment
o
o Urobilin
▪ oxidation product of normal urinary constituent, urinobilinogen OMM

▪ imparts an orange-brown color to urine that is not fresh

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URINE COLORS AND ASSOCIATED CONDITIONS

Colorless Very dilute urine

Cloudy Phosphates, carbonates, urates, uric acid, WBCs, RBCs (“smoky”)
Bacteria, yeasts, spermatozoa, prostatic fluid, mucin, mucous threads,
Calculi (“gravel”) phosphates, oxalates, radiographic dye
Milky Many neutrophils (pyuria), Fat (Lipiduria), Chyluria
Pale yellow Dilute urine, Diabetes insipidus; Diabetes mellitus
Yellow-orange Concentrated urine, dehydration, fever
Yellow-brown / Beer-brown Bilirubin-biliverdin
Dark yellow / Amber Concentrated urine (white foam)
Bilirubin (yellow foam)
Red Hemoglobinuria, Myoglobinuria, Hematuria
Beets, fuscin, menstrual contamination
Red-purple Porphyrins (port-wine)
Red-brown RBCs, Hemoglobin on standing, Methemoglobin,
Myoglobin, Muscle injury, Bilifuscin (dipyrrole) BILIRUBIN;
- DERIVED FROM GALISTONES

Brown-black Methemoglobin, Homogentisic acid, Melanin

Blue-green Indicans, Pseudomonas infection, chlorophyll

URINE COLOR AND COMMONLY USED DRUGS

Yellow Mepacrine (antimalarial)

Fluorescein sodium (examination of retina)
Bright Yellow Riboflavin (multivitamins)
Orange Acriflavine (antiseptic)
Pyridium (urinary analgesic)
Phenindione (anticoagulant)
Orange-yellow Sulfasalazine (ulcerative colitis)
Red Chlorzoxazone (muscle relaxant)
Deferoxamine (chelates iron)
Orange-red Rifampin (antituberculosis)
Red-brown Levodopa (parkinsonism)
Methyldopa (antihypertensive)
Metronidazole (Amebiasis, trichomonas infection)
Brown Phenol poisoning

URINE CLARITY / TURBIDITY

Clear no visible particulates, transparent
Hazy few particulates, print easily seen through urine
Cloudy many particulates, print blurred through urine
Turbid print cannot be seen through urine
Milky may precipitate or clot

Lab Correlations in Urine Turbidity

1. Acidic urine Amorphous urates
Radiographic contrast media
2. Alkaline urine Amorphous phosphates, carbonates
3. Soluble with Heat Amorphous urates, Uric acid crystals

4. Soluble in Dilute Acetic acid RBCs

Amorphous phosphates, carbonates
5. Insoluble in Dilute Acetic acid WBCs
Bacteria, yeast, Spermatozoa

6. Soluble in Ether Lipids, Lymphatic fluid, chyle

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URINE ODOR

1. Aromatic Normal
2. Ammoniacal Bacterial decomposition; UTI
3. Fruity/sweet Ketones (DM, starvation, vomiting)
4. Maple syrup/burnt sugar MSUD
5. Mousy A Phenylketonuria
6. Rancid butter Tyrosinemia
7. Sweaty feet Isovaleric acidemia
8. Cabbage/Hops Methionine malabsorption
9. Rotting fish A Trimethylaminuria
10. Sulfur Cystine disorder

Odorless urine ________________________

ASSOC. W/ ACUTE TUBULAR NECROSIS

Specific Gravity
• density of a solution compared w/ the density of a similar volume of distilled water at similar temp.
• influenced by number & density of particles in solution
• Normal random specimen: 1.002 – 1.035
• 1.010 = isosthenuric; < 1.010 = hyposthenuric; > 1.010 = hypersthenuric
• Dilutions = just multiply the decimal portion of the urine specific gravity by the dilution factor

METHODS OF DETERMINATION OF SPECIFIC GRAVITY

1. Urinometry
• less accurate; not recommended by NCCLS; requires large volume: 10 – 15 mL
• Calibration Temperature (20°C)
• TEMP. CORRECTION
o -0.001 for every 3°C that the specimen temp. is BELOW the urinometer calibration temp.
o +0.001 for every 3°C that the specimen temp. is ABOVE the urinometer calibration temp.
• GLUCOSE & PROTEIN CORRECTION
o 1 g/dL Glucose – 0.004 (subtract)
o 1 g/dL Protein – 0.003 (subtract)
• CALIBRATION
o K+ sulfate – (20.29 g K+ sulfate to 1 L H2O) = sp. gr. 1.015
TOTAL SOUD

2. Refractometry (TS meter)

• Refractive index is a comparison of the velocity of light in air with the velocity of light in a solution.
• Uses only a small volume of specimen (one or two drops).
• DOES NOT NEED TEMPERATURE CORRECTION
• Temperature is compensated between 15°C and 38°C
• GLUCOSE & PROTEIN CORRECTION
o 1 g/dL Glucose – 0.004 (subtract) - IF NOT EXACT THEN ADNST THE

INSTRUMENT

o 1 g/dL Protein – 0.003 (subtract

• CALIBRATION
o Distilled water ____
o 5% NaCl ____ + 0.001
o 9% Sucrose ____ + 0.001
• Abnormally high results (>1.040) are seen in patients who have recently undergone an IV pyelogram

3. Harmonic Oscillation Densitometry

• based on the principle that the frequency of sound wave entering a solution will change
in proportion to the density of the solution. Yellow IRIS automated urinalysis uses this method

4. Reagent Strip method

• based on the change in pKa (dissociation constant) of a polyelectrolyte

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CHEMICAL EXAMINATION OF URINE

Reagent Strips
• chemical-impregnated absorbent pads attached to a plastic strip. A color-producing chemical reaction
takes place when the absorbent pad comes in contact with the urine.
• Usually composed of 10 pads; if there is 11th rgt pad – intended for detecting ascorbic acid
• To ensure against run-over, blotting the edge of the strip on absorbent paper and holding the strip
horizontally while comparing it with the color chart is recommended.
• Storage:
o Reagent strips are packaged in opaque containers w/ desiccant to protect from light & moisture
o Stored at room temperature below 30°C but never refrigerated
• Reagent strips must be checked with both (+) and (-) controls a minimum of once every 24 hours, when
a new bottle is opened, or questionable results are obtained.
• Manufacturers:
o MULTISTIX – Siemens Healthcare Diagnostics
o CHEMSTRIP – Roche Diagnostics

Time Test Principle Reagent Sensitivity (+) Result

Double sequential
↓ LULUSE Glucose oxidase & peroxidase 75-125 Brown / Purple
30 sec enzyme reaction
mg/dL

BILIRUBIN Diazo reaction 2, 4 – dichloroaniline 0.4-0.8 Pink / violet

30 sec
diazonium salt mg/dL
Sodium
KITONE
nitroprusside Na nitroprusside, glycine 5-10 mg/dL Purple
40 sec
reaction

56
pKa change of a Bromthymol blue 1.000-1.030 Blue (1.000)
45 sec polyelectrolyte Yellow (1.030)

A tetrabromphenol blue /
Protein error of
PROTEIN tetrachlorophenol 15-30 mg/dL Green / Blue
1 min indicators
tetrabromosulfophthalein
pH
Double indicator
1 min pH Methyl red / bromthymol blue pH 5-9 Yellow (4-6)
system
Blue (6-9)
5-20
Pseudoperoxidase
1 min BLOOD Tetramethylbenzidine RBCs/mL Green/ Blue
activity of Hgb

1 min URUBILINOGEN Ehrlich's p-dimethylaminobenzaldehyde, 0.2 mg/dL Dark Pink

Aldehyde reaction 4-methoxybenzene-diazonium-
tetrafluoroborate

1 min NITR ITE Greiss reaction p-arsanilic acid or 0.06-0.1 Pink

sulfanilamide mg/dL UNIFORMED PINK

LEUIU CYTE Leukocyte esterase Indoxylcarbonic/pyrrole acid 5-15 Purple

2 mins
ester WBCs/hpf

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Other Tests:

Clinitest
• Purpose: Non-specific test for reducing sugars:
o (+) Glucose
o (+) Galactose, lactose, fructose, maltose, pentoses
o Interference: (+) ascorbic acid, drug metabolites, and antibiotics ex. (Cephalosporins)
• Principle: Copper reduction (copper sulfate to cuprous oxide)
• Color results: CLINITEST TABLETS
USES

o (-) Blue
o (+) Orange-red
• Pass-through phenomenon
o Occurs when there is high urine glucose levels 2g(d( -

o Blue to Orange-red to Green-Brown color

o Minimized by using two-drop urine method instead of five-drops
- - - -

Sulfosalicylic acid test SSA ALBUMIN/GLOBULIN

• cold precipitation test that reacts equally with all forms of protein
o Add 3 mL of 3% SSA reagent to 3 mL of centrifuged urine.
o Mix by inversion and observe for cloudiness.
CONC.
QUIVALENT
E

Reporting SSA Turbidity & ESCRIPTON (mg/dL)

Negative No increase in turbidity <6
Trace Noticeable turbidity 6 – 30
1+ Distinct turbidity w/ no granulation 30 – 100
2+ Turbidity w/ granulation w/ no flocculation 100 – 200
3+ Turbidity w/ granulation and flocculation 200 – 400
4+ Clumps of protein > 400

Microalbuminuria
• Useful in predicting early renal complications brought by DM
• Uses an immunochemical assay for detection of low albumin levels in urine (0-10 mg/dL)
• D Micral-Test reagent strips contain a gold-labeled antihuman albumin antibody-enzyme conjugate (ELISA)
• The amount of color produced represents the amount of albumin present in the urine.
• Early methods require 24-hour urine collection: REPORTING UNIT OF

o results were reported in mg of albumin/24 hours or as the albumin excretion (AER) in μg/min.
o Significant result: 30 to 300 mg of albumin is excreted in 24 hours or AER is ____________.
20-200 ng/min.

Acetest CONFIRMATORY
• Tablet test used to confirm questionable results in urine ketone rgt strip test
- -

• Can be used also in serum and body fluids

• Content: sodium nitroprusside, glycine, disodium phosphate, lactose
• (+) result: Purple

Ictotest
• Tablet test used to confirm results in urine bilirubin rgt strip test
• Content: p-nitrobenzenediazonium, SSA, sodium carbonate, boric acid
• (+) result: Blue / Purple

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OTHER INFORMATION:
1. pH
• pH of normal random samples: 4.5 to 8.0
• pH of 9 = unpreserved urine
ENHANCES THE EXCRERON OF UROBILINOGN
↑
ACID URINE ALKALINE URINE
Emphysema Hyperventilation
DM Vomiting
Starvation, dehydration, diarrhea Renal tubular acidosis
Acid-producing bacteria (E. coli) Vegetarian diet
High protein diet Old specimens
Cranberry juice (remedy for minor UTI) Alkaline tide 2PM -
4PM

L RECOMMENDED FOR UROBILINOGEN DETERMINATION

2. Protein RTAGENT STRIP IS ONLY SENSINUE TO ALBUMIN

• ALBUMIN – major serum protein found in urine

• Normal protein excretion: less than 10 mg/dL or 100 mg per 24 hours is excreted
• Clinical proteinuria is indicated at 30 mg/dL or greater

Pre-renal Post-renal
Renal Proteinuria
Proteinuria Proteinuria
(Glomerular Disorders) (Tubular Disorders)
intravascular hemolysis (Hb) immune complex disorder Fanconi’s syndrome lower UTI
RE-ECLAMPSIA, DEFY DRATON
↑

muscle injury (myoglobin) microalbuminuria toxic agents / prostatic fluid

↓YPERTENSION, AMY LOIDOSIS, heavy metals
AGENTS, DABENC NEPHOPANH
XIL
↑
SEVERE VIRAL INFECTIONS vaginal secretion
severe inflammation (APRs) strenuous exercise
spermatozoa
multiple myeloma
orthostatic proteinuria*
(bence jones protein)* rIDENTIFIED THROUGH
FIXATION 8 40-60' IMMUNO PRECIPITATES

*Bence Jones protein - monoclonal Ig light chains associated with multiple myeloma : (-) REAGENT STRIP; N 8 DISSONES 100°

*Orthostatic proteinuria - occurs following periods spent in a vertical posture and disappears
when a horizontal position is assumed.
3. Glucose
PLASMA CONCENTRATON
BY WHICH
SUBSTANCE
• most frequently performed chemical analysis on urine
OF A
• Renal threshold: 160 – 180 mg/dL
REABSORPION MBULAR
OF
TRANSPORT EFECTIVE RENAL 1
ACTIVE
SLOPS p
REABSORPRON
• Glycosuria in the absence of hyperglycemia is frequently referred to as “renal glycosuria” and is seen in
end-stage renal disease, cystinosis, and Fanconi syndrome.
SPECIFIC ONLY FOR GUCUSE

REAGENT STRIP
Glucose Oxidase Clinitest Interpretation
1+ Negative Only small amount of glucose is present
4+ Negative Possible oxidizing agent interference on rgt strip
Negative Positive FOR NON-
Non-glucose reducing substance present;
-> NON-SPECIFIC
REDUCING SUGAR
Possible interfering substance for rgt strip
t f EXPECTED RESULT IN SLUCOSE IN URINE) GLYCOSURIA)

4. Ketones
• results from increased fat metabolism due to compromised carbohydrate utilization
• 3 ketone compounds:
o 78% beta-hydroxybutyric acid
o 20% acetoacetic acid
o 2% acetone
• Reagent strip primarily detects _______________________________
ACETOACEMC ACID (DIACERC ACID), ACETONE
*

• Clinical significance
YPE DM
of ketonuria:
↑

o Diabetic acidosis; insulin dosage monitoring

o Starvation, vomiting
o Strenuous exercise (overuse of CHO)
o Inborn errors of amino acid metabolism

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5. Blood -

NO INTACT RBCS
I I

Hematuria Hemoglobinuria Myoglobinuria

- PRESENCE OF INTACT RBCS IN URINE

renal calculi transfusion reactions muscular trauma / crush syndromes

glomerulonephritis haemolytic anemia rhabdomyolysis
tumors / trauma severe burns muscle wasting diseases
exposure to toxic chemicals infections / Malaria prolonged coma, convulsions
strenuous exercise strenuous exercise / RBC trauma drug abuse / extensive exertion

Tests for differentiation:

SMOdy/ CLOUDY RED CLEAR DED CLEAR MD

Hematuria Hemoglobinuria Myoglobinuria

Microscopic exam t -
-

Plasma examination RED PLA SMA CLEAR PLASMA

Blondheim’s test SUPERNATANT

REAGENT STRIP
IS NEGATIVE FOR BLOOD
SUPERNATANT IS POSINVE

> AMMONIUM SULFATE

Lo PRECIPITAES IF PRESENT Fe2+

PROTOPHRINOXYGENASES BRIVERDINGMEOXINDIRECT
HOB

HEME B HEME UNCONJUSATD

6. Bilirubin t
H2O INSOLUBLE

• degradation product of Hemoglobin

OBIN-2 6) PAIRS OF GLOBIN CHAINS
NON-POLAR
-
HEMOBILIRUBIN PRINCIPAL PIGMENT
CONNGATION

• Unconjugated bilirubin – B1 – Water Insoluble bilirubin B1-ALBUMINATO-P* EULSIFICATION ABSORDTON OF

&OF
FATS

o bound to albumin GLUCONYL >STORED IN GALIBLADDER

DIRECT

SALLBLAPDiCEIZIA oorCto
TRANSFERASE
-

EBOY'Ruc
- CONJUGATAD

o cannot be excreted by the kidneys -

-
POST-HEPATC
H2O

POLAR
SOLUBLE

• Conjugated bilirubin – B2 – Water soluble bilirubin

REABSORBA
-

50% IS

- CHOVEBILIRUBIN

o a bilirubin diglucuronide, by the action of glucuronyl transferase

o normally doesn’t appear in the urine
o liver bile duct intestine (reduction to urobilinogen) feces
• CS: Hepatitis, Cirrhosis, Biliary obstruction (gallstones, carcinoma)

URINE BILIRUBIN & UROBILINOGEN IN JAUNDICE

Urine Bilirubin Urine Urobilinogen

(-) but reported

Bile duct obstruction +++
as normal

Liver damage + or – ++

Hemolytic disease (-) +++

NORMAL: <IngIdL or ERLICH UNIT

↑ IN DISEASE
LEVELS LIVER DISEASE & HEMOHRC
URINE
7. Urobilinogen UROBILINOGAN LEVELS/ ABSENT:
↓
BILE DUCT OBSTRUCTION CERAYACHOUC STAL colon)

• bile pigment that result from Hb degradation DEFECTED BY REAGENT STRIP

* ABSENCE OF UROBILINOGAN CANNOT BE

• 50% is excreted as urobilin (feces)

• 50% is reabsorbed:
o intestine blood back to liver
o as it circulates into the blood, some urobilinogen is filtered by the glomerulus, appearing in the
urine ( Normal is < 1 mg/dL or Ehrlich unit)

• Increased urine urobilinogen (greater than 1 mg/dL) is seen in liver disease and hemolytic disorders.
• Although it cannot be determined by reagent strip, the absence of urobilinogen in the urine and feces is
also diagnostically significant and represents an obstruction of the bile ducts.

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• WATSON-SCHWARTZ DIFFERENTIATION TEST

o done after using Ehrlich’s reagent & Na acetate (test for urobilinogen) -

o differentiates urobilinogen & porphobilinogen

CAN INDICATE PORPHYRIAS DDEFECT IN HEME SYNTHESIS

COMPONENTS: Urobilinogen Porphobilinogen Other Ehrlich-Reactive subs

CHLOROFORM Lo INSOLUBLE TO BOR CHLOROFORM
AND BUT NOL

EXTRACTION

Urine (Top Layer) Colorless Red Red

Chloroform (Bottom Layer) Red Colorless Colorless

BUTANOL
EXTRACTION

Butanol (Top Layer) Red Colorless Red

Urine (Bottom Layer) Colorless Red Colorless

URINE BECOMES COLORIESS: SOLUBLE

URINE STATS RED: INSOLUBLE

UROBILINOGEN ONLY
Soluble to Chloroform: ___________________
Soluble to Butanol: ______________________
UROBILINOGEN & OTHER ERLICH SUBSTANCES
REACTIVE

• HOESCH TEST ( Inverse Ehrlich Test )

o used for RAPID SCREENING
PDAB
for urine porphobilinogen ( > 2 mg/dL )
o Reagent: Ehrlich’s reagent dissolved in 6 M HCl
o Positive result: Red Color

8. Nitrite DUNIFORM PINK MUM)

• provides rapid screening for presence of UTI
• detects BACTERIURIA (E. coli, Klebsiella, Enterobacter, Proteus, Staph)
• Enterococcus is UNABLE to reduce nitrate to nitrite
• valuable for detecting initial bladder infection (cystitis) - px are asymptomatic

9. Leukocyte
• advantage of chemical test for this parameter is that it will detect the presence of leukocytes that have
been lysed, particularly in dilute, alkaline urine LYMPIOCYT- NEGATI

• detects presence of esterase in granulocytic WBCs (neutro, eo, baso, mono)

• Esterases are also present in Trichomonas and histiocytes

10. Specific Gravity

• Reagent strip:
o pKa (dissociation constant) change of a Polyelectrolyte
o the polyelectrolyte ionizes releasing hydrogen ions in proportion
to the NUMBER OF IONS in the solution
o the higher the conc. of urine, the more H+ are released, thereby lowering the pH
o Bromthymol blue on the rgt pad measures the change in pH
▪ Blue (1.000 – alkaline)
- -

▪ Yellow (1.030 – acid) . .

▪ manufacturers recommend adding 0.005 to sp. gr. reading when

pH is 6.5 or higher due to interference w/ bromthymol blue indicator

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FALSE (+) IN OXIDIZING AGENTS; FALSE Ct) IN HIGHLY PICMENTED:

> PROTEIN
- GLUCOSE
- KETONES
- BLOOD
> BILIRUBIN
- LEUILOCYTE
> NITRITE
BILIRUBIN 5. LEUKOCHE
> LEUKOCYE 1.
GLUCOSE 3.
4)NITRITE
BLOOD
FALSE RESULTS IN REAGENT STRIPS 2.

DUE TO EXCESSIVE LEVELS OF ASCORBIC ACID

FALSE POSITIVE FALSE NEGATIVE

highly alkaline urine

Protein
quaternary ammonium compounds proteins other than albumin
detergents

Glucose Oxidizing agents

Ascorbic acid

Phthalein dyes (PSP, bromsulphtalein)

Ketones Highly pigmented red urine
Improperly preserved urine
Levodopa

high ascorbic acid / nitrite

Blood Oxidizing agents
Formalin, captopril,
crenated RBCs

Highly Pigmented urines

Bilirubin exposure to light
Indican
high ascorbic acid / nitrite
Metabolites of Lodine

Other Ehrlich Reactive Compounds

porphobilinogen
indican
p-aminosalicylic acid
-Old specimens (photo-oxidation)
Urobilinogen sulfonamides
-Formalin & increased nitrite
methyldopa
procaine
chlorpromazine

-nonreductase-containing bacteria
improperly preserved specimen -insufficient contact time between bacteria
(multiplication of bacteria) & urinary nitrate
-large quantities of bacteria further
Nitrite
highly pigmented urines converting nitrite to nitrogen
-lack of urinary nitrate
-presence of antibiotics
-high ascorbic acid
-high sp. gr.

Oxidizing agents
Formalin -High conc. of protein, glucose,
Leukocyte Highly pigmented urines oxalic acid, ascorbic acid
Nitrofurantoin -Gentamicin, cephalosporins, tetracyclines

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MICROSCOPIC EXAMINATION OF URINE

• 12 mL = frequently used because reagent strips are easily immersed in this volume
• Centrifugation: 5 minutes, 400 RCF
• RCF = 1.118 x 10-5 x radius in centimeters x RPM2
• Recommended volume for glass-slide method of microscopic analysis: 20 uL
• Recommended cover slip: 22 x 22 mm glass cover slip (5040 hpfs)
• Examination of sediment: MINIMUM OF ________________under
1 FIELDS both low & high power
• In glass-slide method of analysis, CASTS have tendency to locate near the EDGES of the coverslip.
• Formed elements – primarily RBCs, WBCs, & Hyaline Casts DISINTEGRATE rapidly, particularly
in a DILUTE, ALKALINE URINE
• Addis Count – first procedure to standardize the quantitation of formed elements in urine microscopy
o named after a British scientist Thomas Addis
o uses a hemocytometer to count the number of RBCs, WBCs, casts, and epithelial cells present
in a 12-hour specimen
o Normal values have a wide range and are approximately:
▪ 0 to 500,000 RBCs
▪ 0 to 1,800,000 WBCs and epithelial cells
▪ 0 to 5000 hyaline casts

Reporting of Microscopic analysis

I. Numerical Ranges (sample reporting: 0-2, 2-5, 5-10, 10-25, 25-50, 50-100, > 100)
a. Casts / LPF
b. RBCs, WBCs / HPF
c. RTE cells / HPF MOST CLINICALLY SIGNIFICANT E.C
·

II. Descriptive

SQUAMOUS
None Rare Few Moderate Many
Epithelial cells / LPF 0 0-5 5-20 20-100 >100
NORMAL
Crystals / HPF 0 0-2 2-5 5-20 >20
Bacteria / HPF 0 0-10 10-50 50-200 >200
Mucus threads LPF 0-1 1-3 3-10 >10

SEDIMENT STAINS
1. Sternheimer-Malbin
SAFRANIN
• ____________
CRYSTAL
and ______________
VIOLET

• most frequently used stain

• The stain is available commercially under a variety of names, including:
o Sedi-Stain (Becton Dickinson)
o KOVA stain (Hycor Biomedical, Inc)
• delineates structure & contrasting color of nucleus & cytoplasm
• identifies WBCs, ECs, and casts
o WBC – purple
o Glitter cells – light blue
o SECs – orange-purple
o RTE cells – blue-purple
o Hyaline casts – pale-pink
o RBCs – pink (acid); purple (alk)
o Bacteria – no stain (motile); purple (nonmotile)

2. Toluidine blue
• used as 0.5% solution; a metachromatic stain
• enhances NUCLEAR detail
• differentiates ____________
WBC (3-5COBULES) and ______________
RTECREWAL TUBULAREC)

• addition of 2% Acetic acid

• Also enhances NUCLEAR detail
• Lyses RBCs
• distinguishes RBCs from WBCs, yeasts, oil droplets, & crystals

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&

PROTEINURIA

3. Lipid stains: Oil Red O & Sudan III EX: NEPHRORC XLIPIDURIA SYNDROME

• Lipiduria (triglycerides, neutral fats, cholesterol)

o Lipid stains
▪ triglycerides & neutral fats = orange-red "MALTESE.CROSS "FORMATON -

▪ cholesterol = does not stain (but capable of polarization)

4. Gram stain
• limited to identification of BACTERIAL CASTS
• differentiates gram-positive & gram-negative bacteria

5. Hansel stain
• ____________ and ______________ ALLERGIC REACTION MEDICATIONS
EOSIN Y TO
METHLENEBLUE ↑

• stains EOSINOPHILS in the urine in cases of _______________

ACUTE INTERSTMAL NEPHRITIS DRUS-INDUCED INERSTITIAL NEPHRIDS

6. Prussian Blue stain

• stains structures containing iron
• stains the hemosiderin granules in urine sediment blue color
to IRON CRANULES CASSOC. EXCESSIVE INTRAVASCULAR HEMOLYSIS

HARRIS HEMATOXYLIN-NUCLEAR STAIN

CytoDiagnostic Urine Testing ORANGE-GREEN 6-KERATINIZED CELLS

• stain w/ Papanicolaou’s EA-50-NON-HERARNIZED

-
CELLS, MATURE SUPERFICIAL CALS

• performed independently of urinalysis for detection of MALIGNANCIES of the lower urinary tract
• Specimen required is ____________________
FIRST MORNING URINE

• also provides information in transplant rejection; viral, fungal, parasitic infections; cellular inclusions;
pathologic casts; inflammatory conditions

MICROSCOPIC TECHNIQUES
1. Bright-Field Microscopy
• objects appear dark against a light background
• used for routine urinalysis; sediments w/ a low refractive index may be overlooked
• sediments must be examined using decreased light controlled by adjusting the rheostat on the light source

2. Phase-Contrast Microscopy
• Light passes to the specimen through the clear circle in the phase ring in the condenser, forming a halo of
light around the specimen.
• enhances visualization of elements w/ low refractive indices, such as hyaline casts, mixed cellular casts,
mucous threads, Trichomonas

3. Polarizing Microscopy
• aids in the identification of crystals and lipids
o crystals – characteristic colors
o lipids – MALTESE CROSS formation
• “Birefringent”
o a property indicating that the element can refract light in two dimensions at 90° to each other
o Positive Birefringence - rotates the plane of polarized light 90 degree in a clockwise direction
o Negative Birefringence - rotates the plane on a counterclockwise direction

4. Dark-field microscopy
• object appears light against the black background
• often used for unstained specimens, and in particular, to identify the spirochete Treponema pallidum

5. Fluorescence microscopy
• Used in visualization of structures stained by a fluorescent dye including labeled antigens and antibodies

6. Interference-Contrast Microscopy
• provides a three-dimensional image showing very fine structural detail by splitting the light ray so that
the beams pass through different areas of the specimen.
• The advantage is that an object appears bright against a dark background but without the diffraction halo
• Two types:
o modulation contrast (Hoffman)
o differential interference contrast (Nomarski)

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SEDIMENT CONSTITUENTS
Normal: Strenuous Exercise:
0 – 2 or 0 – 3 RBC / hpf - RBCs or RBC cast
0 – 5 or 0 – 8 WBC / hpf - Hyaline casts
0 – 2 hyaline cast / lpf - Granular casts

RBCs ENAL LITHIASIS R

OF URINARY TRACTS
INTURES BLOOD VESSES
• non-nucleated biconcave disks (7 um)
-

HEMATURIA ->

• presence of RBCs in urine is associated with:

o glomerular membrane damage
o vascular injury within the genitourinary tract

_______________
CRENATED RBCS in hypertonic urine (cells shrink due to loss of water)
_______________
6HOST CELLS in hypotonic urine (cells swell, absorb water, & lyse rapidly)
_______________ primarily associated w/ glomerular bleeding
DYSMORPHIC RBCS

• frequently confused with: yeast cells, oil droplets, air bubbles

WBCs
• larger than RBCs (12 um)
• increased WBCs in urine is termed as: _________________
PYURIA
PoCONERnt
o Bacterial infections - Pyelonephritis, cystitis, prostatitis, urethritis
o Non-bacterial d/o – Glomerulonephritis, SLE, tumors, interstitial nephritis
& INFECTION OF RENAL INTERSTUM (UPPER UAT

Neutrophil
• predominant WBC (granulated, multi-lobed nuclei)
• ________________
GLITTER CELLS in hypotonic urine (neutrophils absorb water & exhibit Brownian movement)

Eosinophil
• primarily associated with Acute/Drug-induced interstitial nephritis
• also seen in UTI & renal transplant rejection
• preferred stain is Hansel’s stain (Wright’s stain can also be used)
• > 1 % is considered significant

Mononuclear cells
• lymphocytes – seen in early stages of renal transplant rejection
• monocytes, macrophages, histiocytes – may appear vacuolated (inclusions)

Platelets in urine – HEMOLYTIC-UREMIC SYNDROME

EPITHELIAL CELLS
• derived from the linings of the genitourinary system
• represent normal sloughing of old cells

1. Squamous Epithelial Cells

• LARGEST cells found in the urine sediment
• Originate from the linings of the vagina & female urethra; & lower portion of male urethra
• Clue cells – appear as SECs covered w/ many Gardnerella coccobacillus
• reported in terms of rare, few, moderate or many per lpf

2. Transitional Epithelial (Urothelial) Cells

• smaller than SECs
• several forms: spherical, polyhedral, caudate
• all forms have distinct, CENTRALLY LOCATED NUCLEI
• originate from the lining of the renal pelvis, calyces, ureters, bladder, and upper portion of male urethra
• increased numbers present following invasive urologic procedures (catheterization)
. . . .
- -

• transitional epithelial cells in clumps = SYNCYTIA

• reported in terms of rare, few, moderate or many per hpf

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3. Renal Tubular Epithelial Cells

• most CLINICALLY SIGNIFICANT of the epithelial cells
• RTE cells can originate from:
o PCT – rectangular in shape; referred to as columnar or convoluted cells
o DCT – round or oval in shape; mistaken for WBCs and transitional EC
o Collecting ducts – cuboidal & has flattened edges; referred to as renal fragments when in groups
• Most forms have small, dense, ECCENTRIC NUCLUES
• presence of MORE THAN __________________
1 RTE/npf indicates TUBULAR INJURY
o exposure to heavy metals, drug-induced toxicity, hemoglobin and myoglobin toxicity,
o viral infections (hepatitis B), pyelonephritis, allergic reactions, malignant infiltrations,
o salicylate poisoning, acute allogenic transplant rejection
• increased amounts: Necrosis of renal tubules
• may sometimes contain substances for reabsorption:
o bilirubin (deep yellow color) – liver damage
o hemoglobin (yellow brown hemosiderin) - hemoglobinuria
• reported as average number of RTE cells per 10 hpf

OVAL FAT BODIES

• these are Lipid-containing RTE cells (lipids were absorbed from the glomerular filtrate)
• appear highly refractile and nucleus is difficult to observe
• usually seen in conjunction with free-floating fat droplets
• Identification:
o Lipid stains (Sudan III or Oil Red O)
▪ Triglycerides & neutral fats = orange-red droplets
o Polarized light
▪ Cholesterol = Maltese Cross formation
. . . . .

• reported as average number per hpf

• LIPIDURIA
o nephrotic syndrome, severe tubular necrosis
o DM, trauma (release of bone marrow fat from long bones)
• *Acute tubular necrosis: “Bubble cells”
o RTE cells containing large NONLIPID-filled vacuoles
o Represent injured cells in w/c the endoplasmic reticulum has dilated prior to death

BACTERIA ASCENDING UT

• not normally present in urine -> E. COL

• to be considered significant for UTI, must be accompanied by WBCs

• The bacteria most frequently associated with UTI are the Enterobacteriaceae
• reported as few, moderate, or many per hpf

YEASTS
• appear as small, refractile oval structures (may contain a bud)
• in severe infections, they may appear as branched, mycelial forms
• the acidic, glucose-containing urine of px w/ DM provides an ideal medium for their growth
• a true yeast infection should be accompanied by WBCs
• reported as few, moderate, or many per hpf

PARASITES
• most frequent parasite encountered in urine: T. vaginalis
• Trichomonas trophozoite
o pearl-shaped w/ undulating membrane; rapid darting motility
o when not moving, Trichomonas may resemble a WBC, transitional, or RTE cell
o Use of phase microscopy may enhance visualization of the flagella or undulating membrane
o reported as few, moderate, or many per hpf
• bladder parasite ova that can be found in urine: S. haematobium
• most common fecal ova contaminant: E. vermicularis

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MUCUS
• protein material produced by the glands & ECs of the lower genitourinary tract
• ____________________________________________________
UROMODULIN

o major constituent of mucus

o glycoprotein excreted by RTE cells of the DCT and upper collecting ducts.
• thread-like structures w/ a low refractive index
• reported as rare, few, moderate, or many per lpf

CASTS
• CYLINDRURIA – presence of urinary casts
• formed within the lumens of the DCT & collecting ductsA
• their shape is representative of the tubular lumen
• in conventional glass-slide analysis, perform along the edges of coverslip
• has LOW REFRACTIVE INDEX – use subdued light
• cast matrix dissolves quickly in DILUTE, ALKALINE URINE
• reported as the average number per 10 lpfs

• TAMM-HORSFALL PROTEIN/ UROMODULIN

________________________________
o major constituent of casts
o glycoprotein excreted by the RTE cells of the DCT & upper collecting ducts
o excreted at a constant rate in normal conditions
o increased excretion seen in stress & exercise
• the width of the cast depends on the size of the tubule in which it is formed:
o Broad casts = result from tubular distention or in extreme urine stasis
o Casts with tapered ends = formed at the junction of ascending LOH and DCT (cylindroids)
• Any elements present in the tubular filtrate, including cells, bacteria, granules, pigments and crystals,
may become “EMBEDDED” or attached to the cast matrix

1. Hyaline casts
• most frequently seen cast
• consists almost entirely of Tamm-Horsfall protein
• NV: 0 – 2 hyaline cast / lpf
• Pathologically, they are increased in:
o acute glomerulonephritis
o pyelonephritis
o chronic renal disease
o CHF
• appear colorless in unstained sediment
• have a refractive index similar that of urine
• stains light pink w/ Sternheimer-Malbin

2. RBC casts
• presence indicates ________________________
GLOMERULONEPHRITIS

• RBC casts associated w/ glomerular damage are usually accompanied by proteinuria & dysmorphic
RBCs
• detected by their orange-red color
• more fragile & may exist as fragments
• in massive hemoglobinuria = granular, dirty, red-brown casts

3. WBC casts
• mostly associated w/ ________________________
P-ELONEPHRITS

• may also be associated w/ acute interstitial nephritis (bacteria is absent)

• casts tightly packed w/ WBCs may have irregular borders
• structures should be carefully examined to determine the presence of cast matrix

4. Bacterial casts
• seen in pyelonephritis
• presence should be considered when many free WBCs & WBC casts are seen

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5. Epithelial Cell casts

• casts containing RTE cells = Advanced Tubular Destruction
• associated w/ metal/chemical/drug-induced toxicity, allograft rejection
• cells visible on the cast matrix: smaller, cuboidal or columnar-shaped
• Bilirubin-stained RTE cells are seen in hepatitis

6. Fatty casts
• seen in conjunction w/ oval fat bodies & free fat droplets
• frequently associated with:
o Nephrotic syndrome
o toxic tubular necrosis, DM, crush injuries
• fatty casts are HIGHLY REFRACTILE
• triglycerides & neutral fats = orange-red in fat stains
• cholesterol = Maltese cross formation under polarized light
• Fats do NOT stain w/ Sternheimer-Malbin

7. Mixed Cellular casts

• frequently encountered:
o RBC & WBC casts in glomerulonephritis
o WBC & bacterial casts in pyelonephritis

8. Granular casts
• non-pathologic cause:
o origin appears to be in lysosomes excreted by RTE cells during metabolism
o increased cellular metabolism: strenuous exercise
o accompanies the hyaline casts in strenuous exercise
• pathologic cause:
o represent disintegration of cellular casts or protein aggregation filtered by the glomerulus
o urinary stasis must be present in order for the granules to result from disintegration of casts
• when granular casts remain in the tubules for extended periods, the granules further disintegrate, & the
cast matrix develops a WAXY appearance

9. Waxy casts
• FINAL DEGRADATION FORM of all types of casts
• represents “Stasis of urine flow” (chronic renal failure)
• appears brittle, HIGHLY REFRACTIVE cast matrix
• often appear fragmented w/ jagged ends & notches in their sides

10. Broad casts

• represent EXTREME URINE STASIS
• indicates Destruction (widening) of the tubular walls
• often referred to as “RENAL FAILURE CASTS”
• most commonly seen broad casts: Granular & Waxy

URINARY CRYSTALS
• Reporting:
o Routinely reported as rare, few, moderate, or many per hpf
o Abnormal crystals may be averaged and reported per lpf
• formed by the precipitation of urine solutes (inorganic salts, organic compounds, medications)
• Solutes precipitate more readily at low temperatures (crystals are abundant in refrigerated specimens)
• first consideration when identifying crystals: URINE pH
• All abnormal crystals are found in ACID URINE
• additional aids for identification:
o Polarized microscopy
o Solubility characteristics
• Changes in temp & pH contribute to crystal formation, and REVERSAL of these changes can cause
crystals to DISSOLVE

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NORMAL CRYSTALS IN ACIDIC URINE

1. Amorphous urates
• yellow-brown granules
• frequently encountered in refrigerated specimen: PINK SEDIMENT (Uroerythrin)

2. Uric acid crystals

• shapes:
o rhombic
o four-sided plates – “lemon-shaped” or whetstonesA
o wedges
o rosettes
• maybe colorless or yellow-brown in color
• may also appear as hexagonals (like cystine)
• HIGHLY BIREFRINGENT (cystine crystals are not birefringent)
• increased amounts:
o leukemia patients receiving chemotherapy
o Lesch-Nyhan syndrome
o Gout

3. Calcium oxalate crystals

• 2 forms:
o Dihydrate (weddelite)
▪ most common form
▪ colorless, octahedral envelopes, pyramidal
o Monohydrate (whewellite)
▪ less frequently seen
▪ oval or dumbbell shaped
• Both forms are BIREFRINGENT under polarized light
• clumps of CaOx = related to formation of renal calculi
• increased CaOx: foods high in oxalic acid / ascorbic acid (tomatoes, asparagus)
• increased amounts of monohydrate form of CaOx:___________________________________ COMPONENT OF ANH-FREEZE / COOLANT
ETHYLENE GLYCOL POISONING (COMMON
USED IN CAR
RADIATORS)

NORMAL CRYSTALS IN ALKALINE URINE

1. Amorphous phosphates
• granular in appearance
• causes white precipitate on refrigeration and does NOT dissolve on warming
• differentiated from urates by the color of sediment & urine pH

2. Triple phosphate (Magnesium ammonium phosphate)

• “STRUVITE”
• colorless, prism shape
• resembles a “COFFIN-LID”
• BIREFRINGENT under polarized light
• seen in highly alkaline urine assoc. w/ presence of urea-splitting bacteria

3. Calcium phosphate
• colorless, flat rectangular plates or thin prisms in “rosette forms”
• rosette forms are confused w/ sulfonamide crystals when pH is neutral
o calcium phosphate will dissolve in dilute acetic acid
o sulfonamides will not dissolve in dilute acetic acid
• common constituent of renal calculi

4. Calcium carbonate
• small, colorless, dumbbell or spherical shapes
• resemble amorphous material
• distinguishing characteristic: formation of GAS (carbon dioxide) after the addition of acetic acid
• also BIREFRINGENT under polarized light w/c differentiates them from bacteria

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5. Ammonium biurate
• exhibit yellow-brown color
• described as “THORNY APPLES” (spicule covered spheres)
• like other urates, they dissolve in 60°C
• they will be convert to uric acid crystals when glacial acetic acid is added
• found in OLD SPECIMENS
• also assoc. w/ the presence of ammonia produced by urea-splitting bacteria

SUMMARY OF NORMAL URINARY CRYSTALS

Crystal pH Color Solubility

Uric acid acid Yellow-brown alkali-soluble

Amorphous urates acid brick dust (yellow brown) alkali & heat
Calcium oxalate acid/neutral colorless (envelopes) dilute HCl
(alkaline)
Amorphous phosphate alkaline/neutral white - colorless dilute acetic acid
Calcium phosphate alkaline/neutral colorless dilute acetic acid
Triple phosphate alkaline colorless (coffin-lids) dilute acetic acid
Ammonium biurate alkaline yellow-brown (thorny-apples) acetic acid with heat
Calcium carbonate alkaline colorless (dumbbells) Gas from acetic acid

ABNORMAL URINE CRYSTALS

1. Cystine crystals
• found in persons w/ cystinuria
o inherited metabolic disorder that prevents reabsorption of cystine
o tendency to from renal calculi, at an early age
• colorless, HEXAGONAL plates (maybe thick or thin)
• maybe difficult to differentiate from colorless uric acid crystals
o uric acid crystals are very birefringent under polarized light
o only thick cystine crystals have polarizing capability
• confirmatory test: CYANIDE-NITROPRUSSIDE TEST

2. Cholesterol crystals CONSIDER

·
PATIENT
HISTORY

• rarely seen unless specimens have been refrigerated

• rectangular plate w/ a NOTCH in one or more corners
• “STAIRCASE PATTERN”
• associated w/ disorders causing lipiduria (nephrotic syndrome) ACCOMPANIE BY: X
• HIGHLY BIREFRINGENT under polarized light

3. Radiographic Dye crystals

• very similar appearance to cholesterol crystals
• they are also highly BIREFRINGENT
o differentiation: patient history & other urinalysis results
o if cholesterol is present, it should be accompanied by other lipid elements
• specimen containing radiographic media has elevated sp.gr. in a refractometer

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For Liver Disorders :

BORT
PRECIPIA 4. Tyrosine crystals
WHEN ALcoI
E
• fine colorless to yellow needles frequently in clumps or rosettes
ADDERINE • seen in conjunction w/ leucine crystals in specimens w/ positive bilirubin
• may also be encountered in inherited disorders of amino-acid metabolism

5. Leucine crystals
• yellow-brown spheres w/ concentric circles & radial striations
• seen less frequently than tyrosine crystals
• if seen, it should be accompanied by tyrosine crystals

6. Bilirubin crystals
• present in hepatic disorders causing bilirubinuria
• characteristic yellow clumped needles or granules

7. Sulfonamide crystals
• primary cause: ______________________________________
INADEQUATE PATIENT HYDRATION

• if found in a fresh urine, it can suggests possible tubular damage

• needles, rhombics, whetstones, sheaves of wheat, rosettes
• colorless to yellow-brown in color
• if necessary, a diazo reaction can be performed for further confirmation

SUMMARY OF ABNORMAL URINARY CRYSTALS

Crystal pH Color Solubility

Cystine acid Colorless ammonia, dilute HCl

Cholesterol acid colorless (notched plates) chloroform
Leucine acid/neutral - yellow hot alkali or alcohol
Tyrosine acid/neutral colorless – yellow alkali or heat

Bilirubin acid d yellow acetic acid, HCl, NaOH,

ether, chloroform

Sulfonamides acid/neutral varied acetone

Radiographic dye acid colorless 10% NaOH

Ampicillin acid/neutral colorless refrigeration forms bundles

URINARY SEDIMENT ARTIFACTS

• they are often highly REFRACTILE
• reporting of artefacts is not necessary

1. Starch granules
• most common contaminant; occasionally confused w/ RBCs
• from corn starch in powdered gloves
• usually with a DIMPLED CENTER
• resemble fat droplets – also produce Maltese cross formation
• differentiation: chemical test for blood & presence of oval fat bodies
2. Oil droplets (may also resemble RBCs)
3. Air bubbles (from coverslip)
4. Pollen grains (seasonal contaminants)
5. Fibers
• from hair fibers, clothing, and diapers
• mistaken for casts
• but they are much longer & more refractile
• will often POLARIZE, whereasC casts, other than fatty casts, do NOT -

6. Fecal contamination

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I. GLOMERULAR DISORDERS
• immunologic
o B majority of the cases
o deposition of immune complexes
• non-immunologic
o exposure to chemicals
o disruption of electrical membrane charges (nephrotic syndrome)
o deposition of amyloid material
o thickening of basement membrane assoc. w/ diabetic nephropathy
• Glomerulonephritis
o sterile, inflammation that affects the glomerulus
RBC
o blood, protein, and casts are seen in the urine iRBC CAST THE URINE; DYSMORPHIC
-
IN
> INDICATES BLEEDING IN THE GLOMERULUS

1. Acute Post-streptococcal Glomerulonephritis 5. PYOGENES

• symptoms usually appear following resp. infections of Group A Strep.

• nephrogenic strains of streptococci form immune complexes w/ the Ab & become deposited on the
glomerular membrane <400mL

• clinical symptoms: fever, edema, fatigue, HPN, oliguria & hematuria

- . . . . . . . .

• Urine findings: RBC casts, dysmorphic RBC, granular casts, WBCs

L FROM CELLULAR CASTS

• there is elevated serum O

ASO INDICATIVE
STREP.
-

INFECTION
FOR
OYO GENES

2. Rapidly Progressive (Crescentic) Glomerulonephritis

• deposition of immune complexes from systemic immune disorders on the glomerular membranes
• possibility of acute renal failure
• Urine findings: Proteinuria & Very Low GFR <60 (KIDNEY DISEASE);
<15-KIDNEY FAILURE

3. Goodpasture Syndrome HAS RESPIRATORY

-
SYNDROME

• due to attachment of Antiglomerular basement membrane antibody to the basement membranes

following viral respiratory infections
• Clinical findings: hemoptysis, dyspnea, hematuria
-BLOOD IN SPUTUM

4. Vasculitis
• disorders affecting systemic vascular system can result in glomerular involvement
• Wegener’s granulomatosis -AFFECTS
KIDNEYS THE LUNGS AND

o Antineutrophilic cytoplasmic autoantibody (ANCA) binds to neutrophils in vascular walls

producing damage to vessels in lungs & glomerulus
o patients usually present first w/ pulmonary symptoms
o clinical findings: hematuria, RBC casts, elevated BUN & crea
• Henoch-Schonlein purpura BLOOD VESSELS, LUNGS, KIDNEYS

o occurs primarily in children following upper respiratory infections

o a decrease in platelets disrupts vascular integrity
o clinical findings: red patches on skin, blood on sputum & stools

5. IgA Nephropathy (Berger’s Disease) D

• most common cause of glomerulonephritis

• deposition of IgA on the glomerular membrane resulting from increased levels of IgA
• Urine findings: macroscopic hematuria

6. Membranous Glomerulonephritis
• pronounced thickening of the glomerular basement membrane resulting from deposition of IgG
immune complexes
• disorders associated: SLE, Sjogren’s syndrome, Secondary syphilis, hepatitis B. malignancy
• most common is frequent development to nephrotic syndrome in adults A

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7. Membranoproliferative Glomerulonephritis
• cellular proliferation affecting capillary walls or the glomerular basement membrane, possible
immune-mediated
• Type I
o displays increased cellualrity in the subendothelial cells of the mesangium, causing
thickening of capillary walls
o could progress to nephrotic syndrome
• Type II
o displays extremely dense deposits in the glomerular membrane
o experiences symptoms of chronic glomerulonephritis
• Urine findings: hematuria & proteinuria +(3
• Lab findings: DECREASED SERUM COMPLEMENT LEVELS

8. Chronic Glomerulonephritis
• marked decrease in renal function resulting from glomerular damage precipitated by other renal
disorders CHRONIC GLOMERULONEPHRINS

• Urine findings: hematuria, proteinuria, glucosuria, BROAD CASTS CHRONIC

CHRONIC
PYE LONEPHRIMS

RENAL FAILURE

• Lab findings: decreased GFR, increased BUN & crea (APPEAR DISEASES
>RENAL FAILURE CAST IN CHRONIC KIDNEY

9. Nephrotic Syndrome
• disruption of electrical charges that produce the tightly fitting podocyte barrier resulting in
massive loss of proteins & lipids HEAVY PROTEINURIA AND HEAVY LIPIDURIA
>FAT PROPLETS

• Urine findings: >OVAL FAT BODIES

~
FATTY CASTS

o MASSIVE PROTEINURIA ( > 3.5 g/dL ) ↓PROTEIN-LOW

EDEMAS PLASMAL
ONCORC PRESSURE

PRESSURE
LEAKAGE OF

LONCORC
-

o RTEs & oval fat bodies

o fatty & waxy casts
o hematuria
• Lab findings: Low serum Albumin, High serum Lipids B
• Clinical findings: PRONOUNCED EDEMA

10. Minimal Change Disease

• “Lipid Nephrosis”
• disruption of the podocytes occurring primarily on children following ALLERGIC REACTIONS
& IMMUNIZATIONS
• Urine findings: Heavy Proteinuria
• Lab findings: normal BUN & crea
• Clinical findings: EDEMA
• most common nephrotic syndrome in childrenD

11. Focal Segmental Glomerulosclerosis

• disruption of podocytes in certain areas of glomeruli associated with
HEROIN & ANALGESIC ABUSE, and also AIDS
• Urine findings: Heavy proteinuria, hematuria

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II. TUBULAR DISORDERS

1. Acute Tubular Necrosis

• Two causes: ↓Oz ↓ i NUTRIENS

o Ischemic LOSS BLOOD SUPPLY-

OF
DEATH OF IMBULAR
CELLS

▪ causes include shock, trauma & surgical procedures

▪ decreased blood flow throughout the body
o Direct Toxic
▪ caused by nephrotoxic agents like aminoglycosides, amphotericin, cyslosporine,
radiographic dye, ethylene glycol, heavy metals
• Urine findings: presence of RTE cells & casts, mild proteinuria, hematuria,
-> 2(upf

2. Fanconi Syndrome IDISORDER

• most frequently assoc. w/ tubular disorder MAJORITY OF THE ESSENTIAL
SUBSTANCES

• generalized FAILURE OF TUBULAR REABSORPTION in the PCT REABSORB ARE

HERE

• maybe inherited in association w/ cystinosis & Hartnup disease

• may also be acquired through exposure to toxic heavy metals & outdated tetracycline
• Urine findings: glucosuria & possible cystine crystals

INTERSTITIAL DISORDERS
• disorders affecting the interstitium also affect the tubules
• “Tubulointerstitial Disease” CYSTITIS
URETHRITIS

• Lower UTI – involves urethra & bladder

• Upper UTI – involves the renal pelvis, tubules & interstitium iPYECONEPHRITS

3. Cystitis
• bacterial infection of the bladder (Lower UTI)
• seen more often in women & children
• Symptoms: urinary frequency & burning
• Urine findings: WBCs, bacteria, mild proteinuria, increased pH
CONVERSION OF UREA < AMMONIA BY BACTERIA

4. Acute Pyelonephritis
• Upper UTI involving both the tubules & interstitium E.COLL
• most frequently occurs as a result of ascending movement of bacteria from a lower UTI (untreated
cystitis) into the renal tubules & interstitium
• enhancing conditions include renal calculi, pregnancy, and “reflux” of urine from the bladder back into
the ureters (vesicoureteral reflux)
• Symptoms: urinary frequency, burning & lower back pain
• a recommended test beside urine culture is blood culture (possible bacteremia)
• Urine findings: WBCs, WBC casts, bacteria, mild proteinuria
• WBC casts = primary diagnostic value * + BACTERIAL CAST

5. Chronic Pyelonephritis
• more serious disorder that can result in permanent damage to the renal tubules
• most frequently casue by Congenital urinary structural defects producing reflux nephropathy
• often diagnosed in children (congenital origin)
• Urine findings: similar to acute pyelonephritis + increased proteinuria,
hematuria, granular, WAXY CASTS, BROAD CASTS

6. Acute Interstitial Nephritis CDRVC INDUCED INTERSTMAL NEPARITS)

• marked by inflammation of the renal interstitium
• primarily assoc. w/ an ALLERGIC REACTION to medications that occurs within the renal
interstitium (binding of the medication to interstitial protein)
• medications assoc: penicillin, methicillin, cephalosporins, sulfonamides
• Urine findings: hematuria, proteinuria, WBCs - INCREASED EOSINOPHILS
WBC casts, ABSENT BACTERIA
RBC O WBC BECAUSE OF ALLERGIC REACTIONS
> PRESENT
BUE
GOSINOPHIL IN DIIN WE USE HANSEL STAIN-METHYLINE
TO VISUALIZE
S IN Y
*

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III. VASCULAR DISORDERS

1. Renal Failure
• 2 forms:
o Acute Renal Failure
▪ exhibits sudden loss of renal function
▪ frequently REVERSIBLE I
▪ Primary causes:
• Prerenal – ↓ blood flow to the kidney / hemorrhage
• Renal – acute glomerular & tubular disease
• Postrenal – renal calculi, tumor obstruction
▪ Urine findings: (varied)
• RTE cells & casts – suggests tubular origin
• RBCs – indicate glomerular injury
• WBC casts – interstitial infection / inflammation
• Abnormal urothelial cells – malignancy / postrenal obstruction
DIALYSIS
o Chronic Renal Failure (END-STAGE RENAL DISEASE) IRREVERSIBLE TRANSPLANT
▪ gradual progression from the original disorder
VRBC COUNT
▪ Lab findings: RENAL FAILURE-IEPO=↓HOB

• marked decreased GFR <15mL/min

• electrolyte imbalance
• Azotemia: ↑ BUN & Crea
FIRST MORNING URINE CDILURD)
• lack of renal conc. ability ↓URINE SO EVEN IN

• proteinuria & glycosuria

• abundance of granular, waxy, broad casts = ________________________
FLESCOPE URINE SEDIMENT

RENAL STONE ANALYSIS:

2. Renal Lithiasis A 1. URIC ACID -

FELLOW-BROWNISH RAS

COMMON FINDINGS: •
HARB

may form in the calyces & pelvis of the kidneys, ureters, & bladder
-
MODERATELY

2. PHOSPHATE STONE-PALE O FRIABLE

HEMATRIA
1.

URINECRYSTAIS
• small calculi maybe passed in the urine, large stones cannot 3. CALCIUM OXALAEE STONES
VERY HARD, DARK COLOR
2.
• Lithotripsy – uses high-energy shock waves to break stones in the upper urinary tract
-

-
ROUGH SURFACE

3.466 4. CYSTINE STONES

• conditions favoring formation of renal calculi: pH, chemical conc. & urinary stasis -
ELCON-BROWN
-SOMENHAT GREAST

• Analysis of renal calculi:

o chemical test
o X-ray Crystallography – for comprehensive analysis
• Renal calculi:
o 80% - _________________________
CALCIUM OXALATE

o 15 % - Triple phosphate (struvite) – caused by urea-splitting organisms (Proteus)

o 5 – 10% - Uric acid or Cystine

URINALYSIS AUTOMATION
Automated Reagent Strip Readers*
• uses REFLECTANCE PHOTOMETRYS
o a spectrophotometric measurement of light reflection ~ CHEMICAL

o uses the principle that light reflection from the test pads decreases in proportion to the intensity of
color produced by the conc. of the test substance
• also uses a photodetector and an analog/digital converter

Automated Microscopy
• Sysmex UF-1000i Urine Cell Analyzer
o uses laser-based flow cytometry along w/ impedance detection,A
light scatter, and fluorescence to identify stained urine sediment particles
o To perform an automated microscopy, 4 mL of uncentrifuged urine is aspirated into the instrument
o Particles are identified by measuring height and width of the fluorescent and light scatter signals
o The main particles enumerated are RBCs, WBCs, epithelial cells, hyaline casts, and bacteria
o Flagged particles include pathologic casts, crystals, small round cells (RTE or transitional ECs),
sperm, mucus, and yeast-like cells, and must be confirmed by ____________________.
MANUAL MICROSCOPY

INDICATES ABNORMALITY IN THE SAMPLE

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 URINE SCREENING FOR METABOLIC DISORDERS

The appearance of abnormal metabolic substances in the urine can be caused by variety of disorders that
can generally be grouped into two categories: I metabolli
reted in urine

Overflow type. result from disruption of normal metabolic pathway that causes increased
plasma concentrations of the nonmetabolized substances ; 4here is missing enuyme
O Renal type caused by malfunctions in the tubular reabsorption mechanism

Disruption of enzyme function can be caused by failure to inherit the gene to produce a particular
enzyme, referred to as an inborn error of metabolism (IEM).
Tandem
mass spectrophotometry (MS/MS) is capable of screening the infant blood sample for specific
substances associated with particular IEMs.
Amino Acid Disorders
1. Phenylketonuria
first identified in Norway by Ivan Folling in 1934
most well-known of the aminoacidurias; autosomal recessive inheritance
if undetected: causes severe mental retardation
Urine Findings: 1 Keto acids, including phenylpyruvic acid
MOUSY ODOR of urine
lacks theenzyme: PHEN LALANINE HOROXLASE
Screening test:
o Guthrie's Bacterial Inhibition test : SPECIMEN: Blood
blood-impregnated disks are placed on culture media streaked w/ B. subtilis
if there is 1 phenylalanine: there will be Bacterial growth (+)
phenylalanine counteracts the action of beta-2-thienylalanine,
which is an inhibitor of B. subtilis that is present in the media
• Urine Screening test
Ferric chloride tube test: PERMANENT BLUE-GREEN

2. Tyrosyluria
there is excess tyrosine in the plasma producing urinary overflow
Causes:
inherited form (enzymes required in metabolic pathway are not produced)
metabolic form (caused by acquired severe liver disease)
Types:
Type I - deficiency of fumaryl acetoacetate hydrolase (FAH)
Type II - deficiency of tryrosine aminotransferase
° Type III - deficiency of p-hydroxyphenylpyuvic acid dioxygenase
• Urine findings:
tyrosine, or metabolites:
p-hydroxyphenylpyruvic acid
p-hydrozyphenyllactic acid
• Urine Screening test
Nitroso-naphthol test: ORANGE-RED

3. Alkaptonuria
one of the six original inborn errors of metabolism described by Garrod in 1902

Urine Findings: DARKENED URINE after becoming alkaline in room temp. CO-OR OF JRINE:
Causes brown-stained or black-stained cloth diapers Brown and Black

lacks the enzyme: HOMOGENTISIC ACID ONIDASE

in later life, brown pigment deposits in tissues, cartilage (arthritis) & ears
URINE SCREENING TEST
• Ferric chloride tube test: TRANSIENT BLUE
Benedict's / Clinitest: YELLOW PRECIPITATE
Alkalinization of urine: DARKENING OF URINE
add alkali to freshly voided urine;
in or add silver nitrate + NH.OH
 4. Melanuria

increased urinary melanin respons bis for the increate

DARKENING OF URINE (after exposure to air) (Brown or Black)

overproliferation of melanocytes (melanin-producing cells), producing a malignant melanoma
these tumors secrete 5,6-dihydroxyindole (precursor of melanin), which oxidizes to melanogen,
and then to melanin
URINE SCREENING TEST
• Ferric chloride tube test:
GRAY/BLACK PRECIPITATE

111
Sodium nitroprusside: RED COLOR
Ehrlich's reagent: RED COLOR
Branched-Chain Amino Acid Disorders
1. Maple syrup urine disease (MSUD) EM

caused by inborn error of

metabolism- autosomal recessive trait
amino acids involved: Leucine, Isoleucine, & Valine
absent branched-chain ketoacid decarboxylase, blocks metabolism of leucine, isoleucine & valine
KETOACIDS accumulate in blood, urine, CSF
Maple syrup odor of urine
URINE SCREENING TEST:

2. Organic Acidemias
Three most common disorders: - TEM
o Isovaleric acidemia
"sweaty feet odor" of urine
deficiency of enzyme: isovaleryl CoA in the leucine pathway
Propionic and methylmalonic acidemias
result from errors in the metabolic pathway converting isoleucine, valine, threonine,
and methionine to succinyl coenzyme A.

Tryptophan disorders
major concern is urinary excretion of the metabolites
indican
O 5-hydroxyindoleacetic acid (5-HIAA)

1. Indicanuria -intestinal defects

Intestinal defects that cause increased amounts of tryptophan converted to INDOLE
intestinal obstruction
o presence of abnormal bacteria or malabsorption syndromes
HARTNUP DISEASE
excess indole is reabsorbed & processed in the liver for conversion to INDICAN
Urine Findings: INDIGO BLUE color when exposed to air ("Blue diaper syndrome")
URINE SCREENING TEST
Ferric chloride tube test: BLUE-VIOLET w/ CHLOROFORM

2. 5-HIAA (metabolite of serotonin)

serotonin is produced from tryptophan by ARGENTAFFIN CELLS in the intestine & is carried

through the body primarily by the platelets

elevated urinary levels is caused by a Carcinoid Tumor involving the
argentaffin or enterochromaffin cells (ARGENTAFFINOMA)
URINE SCREENING TEST concer of the

Nitroso-naphthol test: PURPLE (with nitric acid)

Ferric chloride tube test; BLUE-GREEN
Patients must be given explicit dietary instructions before collecting any sample to be tested for 5-HIAA:
avoid foods such as bananas, pineapples, and tomatoes. A i these food are rich in Serulonins

medications, including phenothiazines and acetanilids, also interfere with results

 CYSTINE DISORDERS
Two disorders:
cystinuria - defect in renal tubular transport of COLA amino acids
cystinosis - inborn error of metabolism

Cystinuria defective ralbsotphion of omino adds

caused by the INABILITY of the renal tubules to reabsorb cystine filtered by glomerulus
amino acids involved: Custine. Ornithing. Lysine, Arginine
cystine IS much less soluble than the other three amino acids
rules out inborn error of metabolism but the condition is inherited
patients have tendency to form RENAL CALCULI
URINE SCREENING TEST
Cyanide-nitroprusside test: RED-PURPLE
False (+): ketones and homocystine

Cystinosis
"Genuine inborn error of metabolism"
Categories:
Nephropathic
incomplete metabolism of cystine results in crystalline deposits of cystine in many areas
of the body, including the cornea, bone marrow, lymph nodes, and internal organs.
a major defect in renal tubular reabsorption mechanism (Fanconi syndrome) also occurs
i f untreated, will result in renal failure
O Non-nephropathic
relatively benign but may cause some ocular disorders.
Renal transplants & use of cystine-depleting medications are extending lives.

Homocystinuria
inherited disorder of the metabolism of amino acid methionine due o a deficiency of cystathionin -B-symthase
clinical signs:
cataracts
thrombosis
mental retardation
URINE SCREENING TEST
• Cyanide-nitroprusside test: RED-PURPLE
Silver-nitroprusside test: RED-PURPLE

Porphyrin disorders
Disorders of porphyrin metabolism are collectively termed porphyrias.
Forms: Problem in heme Synthesis
Inherited: absence of a particular enzyme involved in heme synthesis
Acquired: lead poisoning, excessive alcohol intake, iron deficiency, chronic liver disease
Causes PORT WINE COLOR of urine

Tested in Feces or Bile: Coproporphyrin, Protoporphyrin

Tested in Blood: Protoporphyrin
URINE SCREENING TEST
Ehrlich's reaction: RED COLOR = detects ALA & Porphobilinogen
0 Fluorescent test: VIOLET / PINK /RED fluorescence
 Hurler

Mucopolysaccharide disorders a k a MUCOPOLYSACCHARIDOSES

large compounds located in connective tissues

consists of a protein core w/ numerous polysaccharide branches
inherited disorders prevent the breakdown of the polysaccharide portions
results in accumulation in the lysosomes of connective tissue cells & their excretion in the urine
most frequently found in urine: dermatan sulfate, keratin sulfate, & heparin sulfate
types:
LEUKOGITE ABNORMALITIES:
Hurler syndrome
Alder - Rilcy Bodies
appearance ot
mucopolysaccharides deposits in the Cornea
abnormal skeletal structure
mental retardation
O Hunter syndrome
abnormal skeletal structure
mental retardation

Sanfilippo synbdrome
mental retardation ONLY

URINE SCREENING TEST

Cetyltrimethylammonium bromide (TAB) Test = WHITE TURBIDITY after 5 mins.
Acid-albumin test = WHITE TURBIDITY after 30 mins.
MPS paper test
Whatman no.1 filter dipped in 0.59% Azure A dye in 2% acetic acid
BLUE SPOT that cannot washed away by acidified methanol

PURINE DISORDERS
LESCH-NYHAN DISEASE
disorder of purine metabolism
sex-linked recessive
lacks the enzyme: Hspoxanthine Guanine Phosphorile 1 TransFerase
causes massive excretion of urinary uric acid crystals
Clinical signs:
severe motor defects and mental retardation
tendency toward self-destruction
gout
renal calculi

first sign is often uric acid crystals resembling "orange sand

in diapers"
 lOMoARcPSD|19521100

CSF
• Major fluid in the body which has the ff functions:
o supply nutrients to the nervous tissue
o removes metabolic wastes
o produce a mechanical barrier to cushion the brain & spinal cord
• Meninges are membranous coverings of the brain and spinal cord and has the ff layers:
o Dura Mater OUTER -

o Arachnoid Mater Subarachnoid space (it is where CSF flows)

o Pia Mater INNER -

• CSF is produced by Choroid plexuses at a rate of 20 mL / hr

o performs “selective” filtration of plasma under hydrostatic pressure
o has tightly fitting junctures that prevent passage of many molecules (BBB)
• Normal CSF Volume
o Adults: ______ 150
mL 90 -

o Neonates _____ mL
10-60

•
• LUMBARI SPINAL TAP (between 3rd, 4th or 5th vertebrae)
Mode of collection: _______________
• Considerations during CSF collection:
o Opening pressure: (using manometer)
▪ Lateral position – 90-180 mmH2O (adults)
▪ Sitting up/Obese px – slightly higher than normal
▪ Infants and Children – 10-100 mmH2O
o Amount of CSF removed
▪ up to ____
20 mL of CSF can be normally removed (w/ stable pressure)
▪ > 200 mmH2O (relaxed px) Do NOT aspirate more than 2 ml CSF
• Indications of CSF analysis:
o Infectious meningitis
o Subarachnoid hemorrhage
o Demyelinating disease MULDPLE SCLEROSIS -

PROCESS
o Malignancy 1.
MICRO

• Designated CSF sterile tubes: STAT IME (SOMINS. -I HOURS 2. HEMA

CHEMISACO
Tube # Lab Test (STAT) Storage requirement 3.

1 CHEMISTR/ SEROLOGY Freezing temp

2 MICROBIOLOGY Room temp
3 HEMATOW6Y
Refrigerator temp
2) A TUBE MAY ALSO BE DRANN FOR MICROBIOLOGY FOR BETTER EXCLUSION OF SKIN CONTAMINATON

• Appearance of CSF:
o Normal CSF: _______________
CRYSTAL CLEAR
with viscosity similar to water
o Cloudy – presence of WBCs or ↑ Protein
o Oily – Radiographic contrast media
o Bloody – Hemorrhage or Traumatic tap
o Clot formation – traumatic tap, spinal block, suppurative meningitis
o Pellicle/web-like formation in CSF – Tubercular meningitis
o Xantochromic CSF
▪ pink to yellow color of CSF supernatant usually due to RBC degradation products
• Pink – slight amount of oxyhemoglobin
• Yellow – oxyhemoglobin converted to unconjugated bilirubin
• Orange – heavy hemolysis or carotenoids
NO PANOLOGIC SIGNIFICANCE

Intracranial hemorrhage Traumatic Tap

Distribution of Blood Even distribution Uneven distribution
Bottle 1 = 2 = 3 Bottle 1 > 2 > 3
Clot Formation – +
INTRODUCTION OF FIBRINOGEN TO CSF SAMPLE

Color of Supernatant Xantochromic Clear

D-dimer Test + –
Erythrophagocytosis + –
-> MACROPHAGES W/ INGESTED RBC

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Cell Count
• should be done immediately because WBCs & RBCs will begin to lyse in 1 hour
• 40% of leukocytes will disintegrate within 2 hrs.A
• The standard Neubauer calculation formula for blood cell counts is also applied to CSF cell counts
• Clear specimens may be counted undiluted, but for turbid specimens, dilution is made with NSS.
• For WBC count, diluent is 3% Acetic acid and Methylene blue for better differentiation of WBCs.
• For Differential cell count, recommended is the Cytocentrifuge method
• RBC count is only done in case of traumatic tap

Normal Values Cell count Differential

Adult 0-5 cells / uL Lymphocytes & Monocytes (70:30)
Newborns / Children 0-30 cells / uL Monocytes (80%)
PLEOMORPHIC- ASSUMES DIFFERENT SHAPES, ORIENTATION (MORPHOLOGY CORYNEBACTERIUM

• The presence of increased numbers of normal cells is termed as _________ & indicate abnormality. PLEOCTOSIS

• Cells in CSF and their significance:

o Lymphocytes – Normal; Viral, Tubercular, Fungal meningitis; Multiple sclerosis
o Monocytes - Normal; Viral, Tubercular, Fungal meningitis; Multiple sclerosis
o Neutrophils – Bacterial meningitis; EARLY cases of viral, tubercular, fungal meningitis
o Macrophages with ingested RBCs – RBCs in spinal fluid
o Eosinophils – parasitic or fungal infections (C. immitis)
o Ependymal, choroidal cells, spindle-shaped cells – Diagnostic procedures
o Blast forms – acute leukemia
o Malignant cells – metastatic CA; primary CNS carcinoma (clusters & fusing of borders)

CSF Protein SERUM PROTEIN

• Ref Value: 15 – 45 mg dL mg/d 6 8.3

-

• Major protein is Albumin, followed by prealbumin

• Major beta globulin present is Transferrin
• Unique to CSF is the _____
TA U
protein which is a “carbohydrate-deficient Transferrin fraction
o beta-1 transferrin – seen in all body fluids
o beta-2 transferrin – seen only in nervous system
• CSF Ig include only IgG & IgA
• Not found in normal CSF: __________________________
IgM, FIBRINOGEN, BETA LIPOPROTEIN

Elevated CSF Protein Low CSF Protein

Meningitis CSF leakage / trauma
Hemorrhage Rapid CSF production
Multiple sclerosis Water intoxication
Primary CNS tumors Recent puncture
Guillain-Barré syndrome Hyperthyroidism
Neurosyphilis
Polyneuritis
Myxedema

• CSF Protein Methods:

o Turbidimetric Methods
▪ Trichloroacetic acid – reagent of choice; ppt. both albumin & globulin
▪ SSA – unless combined with sodium sulfate, it will ppt albumin more
o Dye Binding Methods
▪ Coomassie Brilliant Blue – rapid and highly sensitive
▪ Ponceau S

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Evaluation of integrity of blood-brain barrier:

CSF/serum albumin index = CSF albumin (mg/dL) * An index value of < 9 represents an
Serum albumin (g/dL) an intact blood brain barrier.

Indication of IgG production within CNS

CSF IgG index = CSF IgG (mg/dL) / serum IgG (g/dL) * An index value of > 0.7 indicate
CSF albumin (mg/dL) / serum albumin (g/dL) IgG synthesis within CNS

CSF Electrophoresis
• primary purpose is to detect _______________
OLIGOCLONAL BANDS in the gamma region
• To ensure that the oligoclonal bands are present as the result of neurologic inflammation, a
simultaneous serum electrophoresis must be performed
o (+) CSF and (-) serum
▪ Multiple sclerosis (2 or more bands)
FPRESENT
R

INFLAMMATON
▪ Guillain-Barré syndrome
IN THE ▪ Encephalitis, Cryptococcal meningitis
CNS
▪ Neurosyphilis, Neuroborreliosis, Trypanosomiasis
▪ Neoplastic disorders
o (+) CSF and (+) serum
▪ HIV infection

Myelin Basic Protein & AUN ANTIBODIES ANTI-ANTIMYELIN BASIC PROTEIN)

-

• Presence in CSF indicates recent destruction of the myelin sheath (demyelination)

• Measurement of MBP in CSF can be used to monitor the course of multiple sclerosis

CSF Glucose
• Ref value: _________
60-70% of Plasma Glucose
• for comparison, blood glucose should be drawn 2 hours PRIOR to the spinal tap
o Elevated CSF glucose levels = due to plasma glucose elevations
o Decreased CSF glucose levels
▪ With numerous neutrophils – Bacterial meningitis
▪ With numerous lymphocytes – Tubercular meningitis (pellicle form)
o Normal glucose levels with numerous lymphocytes – Viral meningitis

CSF Lactate
• Ref value: 10 – 24 mg/dL
• destruction of tissue w/in the CNS due to oxygen deprivation causes ↑ Lactate levels
• it is also frequently measured to monitor severe head injuries
• Findings:
o Elevated CSF lactate levels (> 25 mg/dL) = cases of bacterial, tubercular, fungal meningitis
o Lower or Normal levels (< 25 mg/dL) = cases of viral meningitis
• Falsely elevated = xantochromic or hemolyzed fluid (RBCs have high conc.)

CSF Glutamine LESS TOXIC SUBSTANCE

-

• Ref value: 8 – 18 mg/dL

• produced in the CNS by the brain cells from the toxic ammonia & a-ketoglutarate
• Increased synthesis is caused by __________________
EXCESS AMMONIAC NEUROTOXIC) that is present in CNS ⑦

• it is preferred over direct measurement of ammonia because it is more stable

• Findings:
o Elevated CSF Glutamine levels
▪ Coma / disturbance of consciousness (> 35 mg/dL)
MULTIPLE SCLEROSIS ▪ Reye’s syndrome (75% of cases)
↑CSF PROTEIN

PCSF Ig6 INDEX

CSF IMPHOCYE/ MONOCYTE

↑

OLIGOCIONAL BANDS: (t) CSF. (-) SERUM

It) MBP IN CSF

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CSF Microbiologic Analysis

• All specimens should be concentrated by centrifugation (1500 x g for 15 minutes) before
performing Gram stain and culture
• Most common cause of bacterial meningitis:
o Neonates – _________________
STREPTOCOCLUS ASALACMAE (CAMP EST POSITIVE)

o 3 months and older – Neisseria meningitidis, Streptococcus pneumoniae

o Newborn to 1 month – Escherichia coli and other gram-negative bacilli
o 3 months to 18 years – Haemophilus influenzae
o Neonates, elderly, alcoholics, immunosuppressed – Listeria monocytogenes
• Viral meningitis:
o Enteroviruses (echoviruses, coxsackieviruses, polioviruses) and arboviruses
o RT-PCR is significantly more sensitive than cell culture
• Fungal meningitis
o Cryptococcus neoformans – most frequently isolated
▪ Gram stain: “_______________”
STAR BURST APPEARANCE

▪ India ink: thickly encapsulated / halos

▪ Latex agglutination – has higher sensitivity
• Tuberculous meningitis
o Available tests done on CSF:
▪ Acid fast staining
▪ Dot ELISA – for detection of MTB antigens
▪ ADA (Adenosine deaminase) activity A

Limulus amebocyte lysate

• An aqueous extract of blood cells (amoebocytes) from the horseshoe crab Limulus polyphemus
• Principle:
o ENDOTOXIN found in the cell wall of gram-negative bacteria coagulates the
amebocyte lysate within 1 hr. if incubated at 37°C
• The test is sensitive even in minute amounts of endotoxin present

Serologic Tests
• Done mainly to detect presence of NEUROSYPHILIS
• CDC recommends _________
UDRL to detect neurosyphilis
o standard nontreponemal test: VDRL
o treponemal test: FTA-ABS

Bacterial antigen tests (BAT)

• Latex agglutination on CSF
• Used to detect H. influenzae, N. meningitidis, S. agalctiae, S. pneumoniae

Primary Amebic Meningoencephalitis

• caused by free-living amoeba (MOT: Intranasal)
o Naegleria fowleri, Acanthamoeba species, and Balamuthia mandrillaris
• Intact and degenerating organisms may be identified on Wright’s- or Giemsa-stained cytospins
• Acridine Orange
o amoeba – brick red fluorescence
o WBC – bright green fluorescence

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SEMEN ANALYSIS

Fractions of Semen

60-70% provides Fructose – for motility of sperm cells

SEMENAL VESICLE provides Flavin – gray appearance of semen

20-30% contains ACP, citric acid, zinc, proteolytic enzymes

PROSTATE GLAND
responsible for coagulation and liquefaction of semen

5% Testes – for production of spermatozoa (seminiferous tubules)

FOIDIDYMIS
↑ESTES
Epididymis – for maturation and storage of sperm cells

BULBOUREMRAL GLAND'S 5% forms a thick, alkaline mucus that helps to neutralize acidity
OR
from the prostate secretions & vaginal acidity
COWPER'S 6)AND

SPECIMEN COLLECTION
• Sexual abstinence of ________ DAYS
2 -
7

o Prolong abstinence: ↑ Volume and ↓ Motility

• Fertility Testing: 2-3 samples tested at 1-3week intervals
o 2 abnormal results are significant
• Warm sterile glass or plastic containers, kept at room temp, delivered within 1 hour of collection
• Specimens awaiting analysis should be kept at 37°C
• Liquefaction Time: 30 – 60 minutes after collection
IF DELAYED LIQUEFACTION
-
PROBLEM IN PROSTAE SLAND

SEMEN ANALYSIS

Appearance
• Normal: Gray-white color and translucent
• ↑ White Turbidity: Presence of WBC and Infection
o > 1 million WBCs / mL = INFECTION
o Screening: Leukocyte esterase reagent strip
o WBCs must be differentiated from immature sperm (spermatids)
• Yellow Coloration:
o Urine contamination
o Prolonged abstinence
o Medications

Volume
• 2 – 5 mL
• ↓ volume: Infertility

Viscosity
• Normal: Pours in droplets
• Ratings: 0 (watery) to 4 (gel-like)
• Viscosity can also be reported as low, normal, or high.
• ↑ Viscosity can impede testing for sperm motility, sperm concentration

pH
• Normal: 7.2 – 8.0 RESPONSIBLE:
- BULBOURATHRAL GLAND

• should be measured within 1 hour of ejaculation; pH pad of a urinalysis rgt strip can be used
o ↓ pH: associated with increased prostatic fluid
o ↑ pH: indicative of infection within reproductive tract

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Sperm Concentration
• Normal: ____________
20-10 million / mL (10-20 million is the borderline)
• 2 chambers:
o Neubauer Counting Chamber (counted in the same manner as CSF) >NEEDS DILUTON
o Makler counting chamber – undiluted seminal fluid; immobilized by heating
• Diluting Fluid (immobilizes the sperm): NaHCO3, formalin, saline, distilled water
• 1:20 is commonly used dilution (Positive Displacement Pipette)
• Only fully developed sperm should be counted RECOMMENDED FOR VISCOUS BODY FLUID

wo...lo
Y
• Both sides of the chamber is counted & must agree within 10%

• CC x DF = # Sperm / uL x 1,000 to get # Sperm / mL

SC x VS EX:ox2o

Sperm Count
• Normal: ≥ 40 million / ejaculate
• Formula: Sperm Conc. (sperm/mL) x Volume of specimen (mL)

Sperm Motility
• Should be examined in liquefied semen within 1 hour of collection
• 10 μL of semen is placed under a 22 × 22 mm cover slip using a calibrated positive-displacement
pipette, and allow it to settle for 1 minute. Evaluate 20 HPFs

Motility Grading
Grade WHO Criteria Sperm Motility Action
4.0 a Rapid, straight-line motility
3.0 b Slower speed, some lateral movement
2.0 b Slow forward progression, noticeable lateral movement
1.0 c No forward progression MORILE ↑

0 d No movement
*Normal result: Minimum motility of 50% with a rating of 2.0 after 1 hour

Alternative Motility Grading

Progressive motility (PM) Sperm moving linearly or in a large circle
Nonprogressive motility (NP) Sperm moving with an absence of progression
Immotility (IM) No movement
& FFERENCE LAR,
CASA – Computer-assisted Semen Analysis
• provides objective determination of both sperm velocity and trajectory (direction of motion).
• Sperm concentration and morphology are also included in the analysis

Sperm Morphology
• Head: 5 um long x 3 um wide = affects ovum penetration
• Tail: 45 um long = affects motility
• Middle Piece: 7 um long and thickest part of the tail; houses the ___________
MITOCHONDRIA for flagellar motion

• Acrosomal Cap: contains __________ for ovum penetration (1/2 of the Head)
ENIYMES

• Examination:
o Thinly Smeared (10 uL) and stained slide observed using OIO
o Stained using Wright’s, Giemsa, or Papanicolau (stain of choice)
o At least 200 sperm cells should be evaluated
• Kruger’s Strict Criteria
o uses morphometry or stage micrometer for measurement & size
o not routinely performed but recommended by WHO
• Head abnormalities: giant, double, amorphous, tapered, pinhead, constricted head
• Tail abnormalities: double, coiled, bent
• Neck (middlepiece) abnormalities: sperm head bends backward
• Normal Values:
o > 30 % Normal Forms – using Routine Criteria
o > 14 % Normal Forms – using Strict Criteria

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Sperm Vitality Test

• Indicated when a specimen has a normal sperm conc. with markedly decreased motility.
• Sperm vitality should be assessed within 1 hour of ejaculation.
• Stain used: ______________
EOSIN-NIGROSIN

• Evaluate 100 sperm cells:

o Dead cells = stained red against purple background
o Living cells = not stained, remained bluish white
• Normal: 50% living cells

Seminal Fluid Fructose

• RESORCINOL TESTS (Seliwanoff’s)
• Positive: Orange-Red color
• Lack of support medium may cause LOW SPERM COUNT
• Should be tested within 2 hours to prevent fructolysis
• Normal: ≥ 13 umol / ejaculate
• Low fructose levels are caused by:
o abnormalities of the seminal vesicles, congenital absence of the vas deferens,
o obstruction of the ejaculatory duct, partial retrograde ejaculation
o androgen deficiency

Other Substances found in Semen

• Neutral α-glucosidase = > 20 mU/ejaculate :SOURCE: EPIDIDYMIS
• Zinc = > 2.4 μmol/ejaculate
SOURCE: PROSTATE GLAND
• Citric acid = > 52 μmol/ejaculate
• Acid phosphatase = > 200 Units/ejaculate

Antisperm antibodies IREQUESTED IF THERE IS CLUMPING OF SPERM CEUS

• MAR Test (Mixed Agglutination Reaction)
o screening test to detect IgG antibodies
o semen w/ motlile sperm is incubated w/ AHG + suspension of coated-latex
o Normal: < 10 % attachment of the motile sperm to the particles
• Immunobead Test
o more Specific; Detects IgG, IgM, & IgA
o detects the presence of IgG, IgM, and IgA antibodies and will demonstrate what area of the sperm
(head, neck, tail) the autoantibodies are affecting.
o sperm are mixed with polyacrylamide beads known to be coated with either anti-IgG,
anti-IgM, or anti-IgA.
o Normal: < 50 % attachment of beads to the sperm

POSTVASECTOMY ANALYSIS
• routinely tested at monthly intervals
• done 2 months post vasectomy, continuing until 2 consecutive monthly specimens show no sperm
• Recommended testing includes examining a wet preparation using phase microscopy for the
presence of motile and nonmotile sperm

SPERM FUNCTION TESTS

1. Hamster Egg Penetration – sperm are incubated w/ hamster eggs & observed for penetration
2. Cervical Mucus Penetration – observe sperm penetration on partner’s cervical mucus
3. Hypo-Osmotic Swelling – sperm are exposed to low-Na conc. for membrane integrity
4. In Vitro Acrosome Reaction – evaluation of acrosome to produce enzymes needed

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SYNOVIAL FLUID

• Also known as “Joint Fluid”

• A viscous fluid found in the cavities of movable joints or diarthroses
• Lubricates joints and supplies nutrition to chondrocytes of the cartilage
• Lessens the shock of joint compression (ex. walking)
• Normal viscous synovial fluid resembles egg white.
o The word “synovial” comes from the Latin word for egg / ovum.
o The fluid’s noticeable viscosity is due to _____________
HALURONIC ACID
secreted by synoviocytes
• Fluid Volume:
o Normal: < 3.5 mL
o > 3.5 mL = inflammation/infection
• An ultrafiltrate of plasma
o Filtration is non-selective except for the exclusion of High M.W. proteins
o most of the chemical constituents have concentrations similar to plasma values

Specimen Collection
• _________________
ARTHROCENTESIS
o Needle aspiration
o Can be done on an inflamed knee/joint
o Site of aspiration: Area of greatest distention
o Volume of fluid collected should be recorded
o Syringe is moistened with ______________
HEPARIN

• Fluid
o Normally does NOT clot
o Diseased joint – contain fibrinogen and will clot
o Collected in different tubes

Lab Test Ac-preservative

Microbiology Sterile heparinized tube or
Sodium polyanethol sulfonate

Hematology / Cell count Liquid EDTA

Glucose Analysis Sodium Fluoride

Other tests Plain tube (no Ac)

*Considerations for Crystal analysis

• Do not refrigerate specimen (will cause production of more crystals)
• Do not use powdered anticoagulants (may produce artifacts that will interfere with crystal analysis)

Appearance
• Normal: __________________
CLEAR, PALE ELIOW

• Deeper yellow – Inflammation

• Greenish – Bacterial infection
• Blood – hemorrhagic arthritis VS traumatic tap (uneven distribution of blood)
• Turbidity – leukocytosis; cell debris and fibrin
• MILKY – ________________
CRYSTAIS

Viscosity
• Polymerization of the Hyaluronic Acid (proper joint lubrication)
• Measure of viscosity
o String that measures 4-6 cm = Normal
o < 3 cm = ↓ viscosity (Depolymerization of Hyaluronic Acid)
▪ Ex. Arthritis
▪ affects both production of HA and its ability to polymerize

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• ROPES OR MUCIN CLOT TEST

o measurement of the degree of polymerization of the HA
o Reagent: _________________
2-5% ACETIC ACID

o Normal result: SOLID CLOT surrounded by CLEAR fluid

o Reported as
▪ Good (solid clot),
▪ Fair (soft clot)
▪ Poor (friable clot)
▪ Very poor (no clot)
o not routinely performed
o can be used to identify a QUESTIONABLE fluid as synovial fluid

Cell Count
• Normal WBC count = < 200 wbc/uL
• RBC count – unless evidence of traumatic tap exists
• performed < 1hr or sample should be refrigerated
• If fluid is very viscous, it is pretreated by adding 0.05% Hyaluronidase in PO4 buffer and then
incubating at 37°C for 5 mins
• Clear fluids can usually be counted undiluted, but dilutions are necessary when fluids are turbid
• Neubauer counting chamber – manual count
• WBC count OK FOR CSF COUNT
o Acetic acid as diluting fluid CANNOT be used
o use NORMAL SALINE, may add methylene blue to separate WBCs and RBCs
o use Hypotonic saline (0.3%) with Saponin to lyse RBCs
• Automated cell counts
o Fluid is highly viscous and may block the apertures
o debris and tissue cell = falsely elevated counts

Differential Count
• should be performed on cytocentrifuged preparations or on thinly smeared slides
• 65% - mononuclear cells
• < 25% - neutrophils (↑ septic condition)
• < 15% - lymphocytes (↑ non-septic inflammation)

Other Cell abnormalities

LE CELL Neutrophil with ingested round body ANOTHER

Lupus erythematosus
> NEUROPHIL THAT HAS ENGULFED Ab.COATD NUCLEUS OF
NEUTROPHIL

EITER
R CELLS Vacuolated macrophage w/ ingested neutrophils Reactive arthritis / Reiter’s syn

Neutrophil with dark cytoplasmic granules Rheumatoid arthritis

RACELL/ RAGOCYTE containing immune complexes
- AUTOIMMUNE
TOINTS
DISEASE IMMUNE COMPLEX DEPOSIT

ON
RHEUMATOID FACTOR + FC PORTION OF 196 -> ACTIVATS COMPLEMENT

Macroscopic: polished rice appearance Tuberculous arthritis /

RICE BODIES
Microscopic: presence of collagen and fibrin Septic or rheumatoid arthritis

Fat Droplets refractile intracellular & extracellular globules Traumatic injury

Hemosiderin inclusions within clusters of synovial cells Pigmented villonodular synovitis

Cartilage Cells large, multinucleated cells Osteoarthritis

- DEGENERATION OF ARLCULAR CARMUAGE
> OCCURS IN OLD AGE

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Crystal formation
• frequently results in an acute, painful inflammation
• Causes:
o Metabolic disorder
o Decreased renal excretion
o Degeneration of cartilage and bones
o Injection of medication
• Crystal examination should be performed soon after fluid collection
o examined as an unstained wet preparation
o reported as being located extracellularly and intracellularly (within neutrophils)
• Primary Crystals
& IFFERENDATES THE TO

Condition associated Shape Polarized light Compensated

Polarized Light
MONOSODIUM URATE (MSU) Gout needles strongly Negative
TURIC ACID] (↑ serum uric acid or ↓ birefringent Birefringence
renal excretion) (yellow color)

CALCIUM PTROPHOSPHATE Pseudogout rhomboid weakly Positive

DIHYDRATE (CPPD) (Degenerative arthritis or square, rods birefringent Birefringence
↑ Ca levels) (blue color)

• Compensated polarized light

o uses red compensator which is placed in the microscope between the crystal & analyzer.
o the compensator separates the light ray into slow-moving and fast-moving vibrations
▪ Color produced by each crystal when it is aligned with the slow vibration:
• MSU crystals – produces yellow color
• CPPD crystals – produces blue color

• Additional crystals
Condition associated Shape CPL
Cholesterol RA / joint inflammations notched, rhomboid plates Negative birefringence

Corticosteroid following injection flat, variable-shaped Positive & Negative

Birefringence

Calcium oxalate Renal dialysis envelopes Negative birefringence

Hydroxyapatite / Osteoarthritis small particles NO birefringence

Calcium Phosphate

Glucose determination
• ↓ values – inflammatory (group 2) or septic disorders (group 3)
• 8 hours fasting
• NV: 0-10 mg/dl lower than plasma glucose

Lactate
• Differentiation between inflammatory and septic arthritis
• NV: <250 mg/dl
• INCREASE: septic arthritis

Protein
• NV: Less than 3 g/dL
• INCREASE: inflammatory and hemorrhagic disorders

Uric acid
• For Gout; used to confirm Dx in the absence of crystals in fluid
• NV: Equal to blood value

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OTHER TESTS:
1. Microbiologic Tests
• Gram stain & culture
• include a chocolate agar – for fastidious Haemophilus & Neisseria gonorrhoeae
CAUSES GONOCLOCAL ARARINS

2. Serologic Tests
• majority of the tests are performed on serum
• but autoantibodies of SLE & RA can also be demonstrated in synovial fluid
• Lyme Disease (B. burgdorferi) : frequent complication is Arthritis

Classification and Pathologic Significance of Joint Disorders

Group Classification Lab Findings

I Non-Inflammatory CLEAR, yellow fluid

-degenerative joint GOOD viscosity
disorder (osteoarthritis) WBCs <1,000 uL
Neutrophils <30%
NORMAL glucose level

II Inflammatory Cloudy, yellow fluid

(Immunologic) Poor viscosity
- RA, SLE WBCs 2,000 - 75,000 uL
Neutrophils >50%
DECREASED glucose level
Possible autoantibodies present

(Crystal-induced) Cloudy or MILKY fluid

- gout / pseudogout Low viscosity
WBCs up to 100,000 uL
Neutrophils <70%
DECREASED glucose level
Crystals present

III Septic condition Cloudy, YELLOW-GREEN fluid

- microbial infection Variable viscosity
WBCs 50,000 - 100,000 uL
Neutrophils >75%
DECREASED glucose level
Positive culture and GS

IV Hemorrhagic Cloudy, RED fluid

- trauma, hemophilia, Low viscosity
coagulation deficiency WBCs <5,000 uL
Neutrophils <50%
NORMAL glucose levek
RBCs present

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SEROUS FLUIDS
• the fluid between Parietal membrane & Visceral membrane
• provides lubrication as the surface moves against each other
• normally, only a small amount is present and is formed as an ULTRAFILTRATE of plasma
• Fluid production and reabsorption are subject to hydrostatic pressure and
colloidal pressure (oncotic pressure) from the capillaries
• __________________
SEROUS EFFUSION = Accumulation of fluid in the body cavity

Fluids in closed body cavities

Collection: needle aspiration
Pleural fluid Lungs MORACENTESIS

Pericardial fluid Heart ERICARDIOCENTESIS

P

Peritoneal / Ascitic fluid Abdominal PARACENTESIS

Specimen collection and Handling

Ac-preservative Lab Tests
EDTA Cell counts and Differential
Sterile tube / SPS Microbiology, Cytology
Heparin or Plain tube Chemistry

*Specimens for pH – must be maintained anaerobically on ICE

*For better recovery of microorganisms & abnormal cells, centrifuge at least 100 mL of fluid

Types of Effusion
TRANSUDATE EXUDATE
Mechanism due to systemic disorder; disruption of due to conditions that directly involved
balance in fluid filtration/reabsorption the membranes of a particular cavity

Process Mechanical Inflammatory

Pathologic causes Increased Hydrostatic pressure; Increased Capillary permeability;

Decreased Oncotic pressure Deceased Lymphatic resorption

Examples Congestive heart failure Infections

Hypoproteinemia Malignancy
Nephrotic syndrome Membrane inflammations

Lab Tests for differentiation

TRANSUDATE EXUDATE
Appearance Clear Cloudy
Fluid:Serum protein ratio < 0.5 > 0.5 PROTEIN
↑

↑
Fluid:Serum LDH ratio < 0.6 > 0.6 LDH
WBC count < 1000 / uL > 1000 / uL WBC COUNT
↑

Spontaneous clotting No Possible LEURAL FLUID

↑

Pleural fluid cholesterol < 45-60 mg / dL > 45- 60 mg / dL ↑CHOVESTROL

Pleural fluid:serum cholesterol ratio < 0.3 > 0.3 4 BILIRUBIN

Pleural fluid:serum bilirubin ratio < 0.6 > 0.6

Serum-ascites albumin gradient > 1.1 4 SAAG < 1.1 SAA6
↓

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PLEURAL FLUID

Appearance of fluid INJURY TO TORACI CAVIT

Associated condition
CLEAR, PALE YELLOW Normal
Turbid, white Microbial infection (e.g. tuberculosis)
Bloody -
Hemothorax or Hemorrhagic effusion CANCER, TB
->

Milky Chylous or Pseudochylous effusion

Brown Rupture of amebic liver abscess
Black Aspergillus
Viscous Malignant mesothelioma

Milky Pleural Fluid

Chylous Effusion Pseudochylous Effusion
Cause leakage from thoracic duct chronic inflammation
Appearance Milky/white Milk/green tinge
Lipid content ↑ Triglycerides ↑ Cholesterol
Triglyceride content > 110 mg/dL < 50 mg/dL
Cholesterol crystals Absent Present
Sudan III staining t -

Bloody Pleural Fluid

Hemothorax Hemorrhagic Effusion
Cause traumatic injury chronic membrane disease
Blood Distribution uneven even
Fluid Hematocrit > 50% of whole blood Hct lower Hct than whole blood

Differential Cell Count

↑ Neutrophils Bacterial pneumonia, pancreatitis, pulmonary infarction
↑ Lymphocytes Viral infections, tuberculosis, malignancy
↑ Plasma cells Tuberculosis
↑ Eosinophils Parasitic infection; Pneumothorax / Hemothorax
Malignant cells Primary adenocarcinoma; metastatic carcinoma

Chemistry Tests
Glucose ↓ levels in purulent infection & rheumatoid inflammation
A
DENOSINE DEAMINASE CADAS ↑ levels in Tuberculosis (> 40 U/L)
MYLASE
A

↑ levels in Pancreatitis, esophageal rupture, malignancy

pH < 7.2 = need for chest-tube drainage in cases of pneumonia
< 6.0 = esophageal rupture

PERICARDIAL FLUID

Appearance of fluid
Associated condition
CLEAR, PAIF YELLOW Normal
Turbid Infection or Malignancy
Blood-streaked Malignancy
Grossly bloody Cardiac puncture, anticoagulant medications
Milky Chylous or Pseudochylous effusion

Important Lab Tests

↑ Neutrophils Bacterial endocarditis
Malignant cells Metastatic carcinoma
CEA Metastatic carcinoma
Acid Fast stain Tubercular effusion
Adenosine deaminase (ADA) Tubercular effusion
↑
CSF ADA-TUBERCULAR MENINGINS
EFFUSIO
N SAROUS FWID ADA-MBERCULAR

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PERITONEAL FLUID

Appearance of fluid
Associated condition
CLEAR, PALE YELLOW
Normal
Turbid Microbial Infection
Blood-streaked Trauma, infection, malignancy
GREEN Presence of bile; gallbladder or pancreatic disorder
Milky Chylous or Pseudochylous material

Ascites (Peritoneal fluid effusion)

• Ascitic Transudate: due to Hepatic disorders / cirrhosis
• Ascitic Exudate: due to Bacterial infections or peritonitis, malignancy
• SAAG (Serum-Ascites Albumin Gradient)
o Recommended to detect transudates of hepatic origin
o Difference of 1.1 or greater suggests a transudate effusion

Important Lab Tests

↑ Neutrophils Bacterial peritonitis
WBC count > 500 cells /uL Bacterial peritonitis; cirrhosis
CEA Malignancy of gastrointestinal origin
CA 125 Malignancy of ovarian origin
↓ Glucose Tubercular peritonitis; malignancy
Psammoma bodies w/ striations Benign conditions & ovarian/thyroid malignancies
Acid Fast stain Tubercular peritonitis
Adenosine deaminase (ADA) Tubercular peritonitis
↑ Amylase Pancreatitis; GI perforation
↑ Alkaline phosphatase Intestinal perforation / hollow visceral injury
DIAGNOSTC PERITONEAL LAVAGE (DPL) Detection of intra-abdominal bleeding in Blunt Trauma;
RBC ct: >100,000 / uL

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AMNIOTIC FLUID

• Present in the AMNION, a membranous sac that surrounds the fetus

o Provides a protective cushion for the fetus
o Allows exchange of water & chemicals

Fluid Volume
• Increases in quantity throughout pregnancy; reaches a peak of approximately 800 to 1200 mL
during the third trimester, and then gradually decreases prior to delivery.

Polyhydramnios Oligohydramnios
Fluid Volume Increased (> 1,200 mL) Decreased (< 800 mL)
Causes Decreased fetal swallowing of urine Increased fetal swallowing of urine
Neural Tube defect Urinary Tract deformities
Fetal Distress Membrane Leakage

Specimen Collection
• AMNIOCENTESIS
o Transabdominal amniocentesis – most frequently performed
o Vaginal amniocentesis
• Usually performed AFTER ______
14 TH WEEK OF GESTATION
• Chromosome Analysis – Fluid is collected at 16th Week Gestation
• Volume collected
o Maximum of ____38 mL
o First 2 or 3 mL = Contaminated by maternal blood = Discarded

Specimen Handling LECITHIN, SPHINGOMYELIN

x:
E

• Fetal Lung Maturity (FLM) Tests (MEASUREMENT OF LUNG SURFACTANTS)

o Must be placed on ICE for delivery to Lab & refrigerated prior to testing
o Low-speed Centrifugation is required (500 – 1000) for less than 5 mins.
• Bilirubin testing – specimen must be protected from light
• Cytogenetic studies - Maintained at Room Temp. or Body Temp. to prolong the life of the cells
• Chemistry – fluid must be separated from cellular elements & debris

COLOR SIGNIFICANCE

___________
COLORLESS Normal

Blood – Streaked Traumatic Tap

Abdominal Trauma
Intra-amniotic Hemorrhage

Yellow Bilirubin (HDN)

Dark – Green Meconium

Dark Red-Brown Fetal Death

Meconium
• dark green, mucus-like material and is the newborn’s first bowel movement
• formed in the intestine from fetal intestinal secretions and swallowed amniotic fluid.
• It may be present in the amniotic fluid as a result of fetal distress.

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Amniotic Fluid Creatinine

• Has been used to determine fetal age
• Creatinine value of ______
72. mg/dL in amniotic fluid signifies fetal age of > 36 WEEKS.
0

Differentiation between Amniotic Fluid & Maternal Urine

• Determines possible premature membrane rupture or possible accidental puncture of maternal
bladder during specimen collection

AMNIOTIC MATERNAL
ANALYTE FLUID URINE

Creatinine < 3.5 mg/dL > 10 mg/dL

Urea < 30 mg/dL > 300 mg/dL

Glucose + -

Protein + -

Fern test + -
"PALM-LEAF" APPEARANCE

TESTS FOR FETAL DISTRESS

1. Bilirubin Scan for HDN

• Performed by Spectrophotometric Analysis
• ↑ Optical Density (OD) at 450 nm – Bilirubin is present in the fluid
o Wavelength of Maximum Bilirubin absorption
o “Absorbance Difference at 450 nm” = ∆A450
o Result is plotted on a Liley graph

Result Remarks
Zone I ∆ OD: 0.025 – 0.09 Nonaffected or mildly affected fetus
Zone II ∆ OD: 0.1 – 0.2 Moderately affected fetus requiring close monitoring
Zone III ∆ OD:: 0.2 – 1.0 Severely affected fetus requiring intervention

*∆A450 > 0.025 = usually associated with HDN

• Specimens must be protected from Light

• Markedly decreased values are observed to as low as 30 mins. exposure to Light
• Avoid contamination of Hemoglobin, cells, & debris that might interfere w/ analysis
o Maximum Absorbance of Oxyhemoglobin occurs at 410 nm
o can interfere w/ the bilirubin absorption peak
o Interference can be removed by extraction with chloroform if necessary
I NCOMPLET MATURATON
NTD 2. Neural Tube Defects CNTD) OF BRAIN TISSUE OSPINAL &
COLUMN

MATERNAL
↑
*
/ SERUN
AFPL PAMNIORC
• Examples of defects are Anencephaly and Spina bifida
#

UUID
• Increased Levels of alpha-1 fetoprotein (AFP) are found in both maternal serum & Amniotic fluid
o Both serum & amniotic fluid AFP levels are reported as multiples of the median (MoM)
AChE
<MORE SPECIFIC o A value two times the median value (greater than 2 MoM) is considered abnormal
•
OF A. F
<PRESENCE
INDICATED NTD
↑ Amniotic fluid AFP levels are followed by measuring Amniotic __________________
ACETYL CHOLINESTERASE CAChE)

o more specific test but specimen should NOT be performed on a bloody specimen. (false +)
LUNG

Tests for Fetal Maturity

A

• Respiratory Distress
o most frequent complication of early delivery
o also called “Hyaline Membrane Disease of the Newborn”
o caused by an insufficiency of lung surfactant production & structural immaturity fetal lungs

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1. Lecithin-Sphingomyelin Ratio (L/S Ratio)

• Lecithin
o primary component of surfactants that make up the alveolar lining for alveolar stability.
o It is produced at constant rate until 35th week of gestation, then its production increases
• Sphingomyelin
o a lipid that is produced in a constant rate after about 26 weeks’ gestation.
o It can serve as a control on which to base the rise in Lecithin.
• Results:
o < 35 weeks’ gestation = L/S ratio is < 1.6
o > 35 weeks’ gestation = L/S ratio is > 2.0 FETAL LUNG MATURIY ->

• False elevated results are found in specimen contaminated with Blood or Meconium

2. Phosphatidyl Glycerol
• another lung surface lipid is also essential for FLM
• Its production parallels that of Lecithin, but DELAYED in Diabetic Mothers.
• Amniostat-FLM uses antisera specific for phosphatidyl glycerol
o The size of the agglutinates is read macroscopically and the results are reported as
▪ Negative - indicating pulmonary immaturity
▪ Low positive or high positive - indicating pulmonary maturity
o NOT AFFECTED by specimen contamination w/ Blood and Meconium

3. Foam stability Index

• a mechanical screening test called the “foam” or “shake” test to measure individual lung-surface
lipid concentrations.
• Procedure:
shake - 15 sec stand - 15 mins
o AF + 95% ethanol = + BUBBLES
PRIMARY REAGENT
NORMAL:
*presence of continuous line of bubbles indicates
sufficient amount of phospholipids is present

• A modified Foam test uses 0.5 mL of amniotic fluid added to increasing amounts of 95% ethanol
o provide a semiquantitative measure of the amount of surfactant present.
o A value of 47 or higher indicates FLM.

4. Microviscosity
• The presence of phospholipids decreases the microviscosity of the AF
PRINCIPLE
• This change in microviscosity can be measured by Fluorescence Polarization:
• Albumin is used as an internal standard (remains at a constant level throughout gestation).
• Fluid should be filtered rather than centrifuged prior to examination
• A Ratio of 70 or greater predicts FLM & lower values maybe considered

5. Lamellar Bodies and Optical Density

• Type II Pneumocytes
o produce & secrete surfactants responsible for FLM
o they are in the form of structures called “Lamellar bodies”
▪ correlates with the amount of phospholipid present in the fetal lungs
▪ presence of lamellar bodies increases the OD of the amniotic fluid.
▪ Specimens are centrifuged at 2000 g for 10 mins
▪ measured using a wavelength of 650 nm
▪ O.D. of 0.150 has been shown to correlate well w/ an L/S ratio of 2.0
• Lamellar bodies can be counted using “Resistance-pulse Counting”
o Lamellar bodies can be counted using the platelet channel
o Lamellar bodies range from 1.7 to 7.3 fL
o Sample must be free of particle contamination such as meconium or blood
o A count of 32,000/uL or more represents adequate FLM

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 A

ETAL FAT
#

NORMAL
I STERCOBILIN

CANTI-TB DRUG

CCLAY-COLORE/ ACHOLI SO

PEA-SOUP

RICE WATER
 3
=> NEUTROPHIKS

CYSTC FIBROSIS

SUDANI

VAN DE KAMER TTRATON

*

ADVANTAGE:

REAGENT

FALSE-NEGATIVE

ALSE-POSITIVE
#

FALSE-POSITIVE

REACENT

PINK
ECLOW-BROWN
↓

SUPERNATANT