Nursing, Healthcare and Medical Certifications Guide

NURSING*RADTECH*DENTISTRY*CRIMINOLOGY*MIDWIFERY*MEDTECH
LET*PSYCHOMET*RESPIRATORY THERAPY*CIVIL SERVICE*NAPOLCOM

NCLEX*DHA*HAAD* PROMETRIC* UK-CBT
NOTES IN CLINICAL MICROSCOPY: By Gerard Andrew Ramos, RMT,MSMT, ACPi (CM),2nd Placer
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
FOUR MAJOR RENAL FUNCTIONS:

1. Renal Blood Flow
 Based on an average body size of 1.73 m2 of surface
 total renal blood flow =
 total renal plasma flow =
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
 Pressure is regulated by R-A-A system
↓ Blood Pressure
↓ Glomerular Pressure
Angiotensinogen Angiotensin I Angiotensin II
3. Tubular Reabsorption
 Essential substances and water are reabsorbed back to circulation (PCT→ Blood capillaries)
 Mechanisms:
 Active transport
o substance to be reabsorbed must combine with a carrier protein
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.
4. Tubular Secretion
 Elimination of unfiltered waste products
 Regulation of acid base balance
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
 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
3. Creatinine
 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
 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
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
 EXOGENOUS procedure (125I-iothalamate)
 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
 NOT useful indicator of Early Renal Disease
Calculation of plasma cleared / min (C) (Adjusting Body Size)

C = Urine creatinine (Urine volume) C = UV x 1.73
Plasma creatinine P Px BSA
Reference Range:
Serum Creatinine: 0.5 – 1.5 mg / dL Urine
Creatinine Clearance: 120 mL / min
o Male: 107 – 139 mL / min
o Female: 87 – 107 mL / min
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 Male:
Female: multiply the answer by 0.85

Ccr = 140 – age serum
x Px
creatinine
w
2. eGFR using Modification of Diet in Renal Disease (MDRD) Formula:

 most widely-used equation to estimate GFR in adults
 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
If Female: multiply the answer by 0.742
If African-American: multiply the answer by 1.202
TUBULAR REABSORPTION TESTS
Concentration Tests - tests to determine the ability of the tubules to reabsorb essential salts & H2O
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
Specific Gravity = affected by number of particles present and its densities
Osmolality = affected only by number of particles present
Solute dissolved in Solvent: (changes in colligative properties)

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

 measuring FREEZING POINT DEPRESSION was the first principle utilized by clinical osmometers
 clinical osmometers uses solutions of known NaCl conc. as their reference standard
Reference ranges:
Serum osmolality : 275 – 300 mOsm Urine
osmolality : 50 – 1400 mOsm
Ratio of Urine to Serum osmolality

 should be at least 1:1
 after controlled fluid intake/restriction, it should reach 3:1
INTRODUCTION TO URINALYSIS
Urine Composition
95% Water
5% Solutes
 MAJOR Organic Component:
 MAJOR Inorganic Component:
Urine Volume
 Normal Range (24 hrs): 600 – 2000 mL
 Average (24 hrs): 1200 mL
> 2000 mL / 24 hrs Increased fluid intake

Diuretics
Diabetes mellitus
Diabetes insipidus
< 400 mL / 24 hrs Dehydration / burns

Renal calculi / tumor
< 100 mL / 24 hrs Complete obstruction (stones, carcinomas)

Toxic agents (MERCURY POISONING)
Hemolytic Transfusion Reaction
>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

2. Clarity – decreased bacterial growth; precipitation of urates
3. Odor – increased breakdown of urea to ammonia
4. pH – increased breakdown of urea to ammonia
5. Glucose – decreased glycolysis and bacterial use
6. Ketones – decreased volatilization
7. Bilirubin – decreased light sensitive
8. Urobilinogen – decreased oxidation to urobilin
9. Nitrite – increased multiplication of nitrate-reducing bacteria
10. RBC,WBC & casts – decreased disintegration in dilute, alkaline urine
11. Bacteria – increased multiplication
URINE PRESERVATION
1. - most routinely used method (2-8C) up to 24 hrs; precipitates amorphous urates, phosphates
2. – keeps pH at 6.0; Bacteriostatic; used in transport for urine culture
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. - used for CYTOLOGY; made up of 50% ethanol & 2% carbowax
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
TYPES OF URINE SPECIMEN
Routine screening for obvious abnormality, may produce erroneous results
IDEAL specimen; useful for Pregnancy test and Orthostatic proteinuria
Recommended for glucose monitoring (for DM)
24-Hour specimen = used for Quantitative Chemical tests

12-Hour specimen = ADDIS COUNT using hemocytometer
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 through the
urethra into the bladder; can be used for bacterial culture
safer, less traumatic method for obtaining urine for bacterial culture and routine urinalysis;
less contaminated by epithelial cells and bacteria
Specimen is collected by external introduction of a needle through the abdomen into the
bladder; useful for Anaerobic Culture
Useful to determine PROSTATIC INFECTION

Prostatic infection: 3rd bottle - WBC is 10x more than that of the 1st bottle 2nd bottle
(midstream urine) = serves as “control” for bladder infection
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
 Required volume: 30 – 45 mL
 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:
 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 _
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
 imparts an orange-brown color to urine that is not fresh
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)
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

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 Phenylketonuria
6. Rancid butter Tyrosinemia
7. Sweaty feet Isovaleric acidemia
8. Cabbage/Hops Methionine malabsorption
9. Rotting fish Trimethylaminuria
10. Sulfur Cystine disorder
Odorless urine
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
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)
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
4. 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
5. Reagent Strip method

 based on the change in pKa (dissociation constant) of a polyelectrolyte
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
30 sec Double sequential

Glucose oxidase & peroxidase 75-125 Brown / Purple
enzyme reaction
mg/dL
30 sec Diazo reaction 2, 4 – dichloroaniline 0.4-0.8 Pink / violet

diazonium salt mg/dL
Sodium
40 sec Na nitroprusside, glycine 5-10 mg/dL Purple
nitroprusside
reaction
pKa change of a Bromthymol blue 1.000-1.030 Blue (1.000)

45 sec polyelectrolyte Yellow (1.030)
Protein error of tetrabromphenol blue /

1 min 15-30 mg/dL Green / Blue
indicators tetrachlorophenol
tetrabromosulfophthalein
pH
Double indicator Yellow (4-6)
1 min Methyl red / bromthymol blue pH 5-9
system
Blue (6-9)
5-20
Pseudoperoxidase
1 min Tetramethylbenzidine RBCs/mL Green/ Blue
activity of Hgb
1 min Ehrlich's Aldehyde p-dimethylaminobenzaldehyde, 4- 0.2 mg/dL Dark Pink

reaction
methoxybenzene-diazonium-
tetrafluoroborate
1 min Greiss reaction p-arsanilic acid or 0.06-0.1 Pink

sulfanilamide mg/dL
2 mins Leukocyte esterase Indoxylcarbonic/pyrrole acid ester 5-15 Purple

WBCs/hpf
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:
o (-) Blue
o (+) Orange-red
 Pass-through phenomenon
o Occurs when there is high urine glucose levels
o Blue to Orange-red to Green-Brown color
o Minimized by using two-drop urine method instead of five-drops
Sulfosalicylic acid test

 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.
Reporting SSA Turbidity (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)
 Micral-Test reagent strips contain a gold-labeled antihuman albumin antibody-enzyme conjugate
 The amount of color produced represents the amount of albumin present in the urine.
 Early methods require 24-hour urine collection:
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 .
Acetest
 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
OTHER INFORMATION:
1. pH
 pH of normal random samples: 4.5 to 8.0
 pH of 9 = unpreserved urine
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
2. Protein
 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
Proteinuria Renal Proteinuria Proteinuria
(Glomerular Disorders) (Tubular Disorders)
intravascular hemolysis (Hb) immune complex disorder Fanconi’s syndrome lower UTI
muscle injury (myoglobin) microalbuminuria toxic agents / prostatic fluid

heavy metals
vaginal secretion
severe inflammation (APRs) strenuous exercise
spermatozoa
multiple myeloma (bence orthostatic proteinuria*

jones protein)*
*Bence Jones protein - monoclonal Ig light chains associated with multiple myeloma
*Orthostatic proteinuria - occurs following periods spent in a vertical posture and disappears when a horizontal
position is assumed.
3. Glucose
 most frequently performed chemical analysis on urine
 Renal threshold: 160 – 180 mg/dL
 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.
Glucose Oxidase Clinitest Interpretation

1+ Negative Only small amount of glucose is present
4+ Negative Possible oxidizing agent interference on rgt strip
Negative Positive Non-glucose reducing substance present;
Possible interfering substance for rgt strip
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
 Clinical significance of ketonuria:
o Diabetic acidosis; insulin dosage monitoring
o Starvation, vomiting
o Strenuous exercise (overuse of CHO)
o Inborn errors of amino acid metabolism
5. Blood
Hematuria Hemoglobinuria Myoglobinuria
renal calculi transfusion reactions muscular trauma / crush syndromes

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

Microscopic exam
Plasma examination
Blondheim’s test
6. Bilirubin
 degradation product of Hemoglobin
 Unconjugated bilirubin – B1 – Water Insoluble bilirubin
o bound to albumin
o cannot be excreted by the kidneys
 Conjugated bilirubin – B2 – Water soluble bilirubin
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
Bile duct obstruction (-) but reported

+++
as normal
Liver damage + or – ++
Hemolytic disease (-) +++
7. Urobilinogen
 bile pigment that result from Hb degradation
 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.
 WATSON-SCHWARTZ DIFFERENTIATION TEST

o done after using Ehrlich’s reagent & Na acetate (test for urobilinogen)
o differentiates urobilinogen & porphobilinogen
Urobilinogen Porphobilinogen Other Ehrlich-Reactive subs

CHLOROFORM
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
Soluble to Chloroform:
Soluble to Butanol:
 HOESCH TEST ( Inverse Ehrlich Test )

o used for RAPID SCREENING for urine porphobilinogen ( > 2 mg/dL )
o Reagent: Ehrlich’s reagent dissolved in 6 M HCl
o Positive result: Red Color
8. Nitrite
 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
 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
FALSE RESULTS IN REAGENT STRIPS
FALSE POSITIVE FALSE NEGATIVE
Protein highly alkaline urine

quaternary ammonium compounds proteins other than albumin
detergents
Glucose Oxidizing agents Ascorbic acid
Phthalein dyes (PSP, bromsulphtalein) Highly

Ketones pigmented red urine Levodopa Improperly preserved urine
Blood Oxidizing agents high ascorbic acid / nitrite

Formalin, captopril, crenated
RBCs
Bilirubin Highly Pigmented urines 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
Nitrite -large quantities of bacteria further
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
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 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
II. Descriptive
None Rare Few Moderate Many
Epithelial cells / LPF 0 0-5 5-20 20-100 >100
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
 and
 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 and
 addition of 2% Acetic acid
 Also enhances NUCLEAR detail
 Lyses RBCs
 distinguishes RBCs from WBCs, yeasts, oil droplets, & crystals
3. Lipid stains: Oil Red O & Sudan III

 Lipiduria (triglycerides, neutral fats, cholesterol)
o Lipid stains
 triglycerides & neutral fats = orange-red
 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
 stains EOSINOPHILS in the urine in cases of
6. Prussian Blue stain

 stains structures containing iron
 stains the hemosiderin granules in urine sediment blue color
CytoDiagnostic Urine Testing

 stain w/ Papanicolaou’s
 performed independently of urinalysis for detection of MALIGNANCIES of the lower urinary tract
 Specimen required is
 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)
SEDIMENT CONSTITUENTS
Normal: Strenuous Exercise:
0 – 2 or 0 – 3 RBC / hpf - RBCs or RBC cast
0 – 5 or 0 – 8 WBC / hpf 0 – 2 - Hyaline casts
hyaline cast / lpf
- Granular casts
RBCs
 non-nucleated biconcave disks (7 um)
 presence of RBCs in urine is associated with:
o glomerular membrane damage
o vascular injury within the genitourinary tract
in hypertonic urine (cells shrink due to loss of water)

in hypotonic urine (cells swell, absorb water, & lyse rapidly)
primarily associated w/ glomerular bleeding
 frequently confused with: yeast cells, oil droplets, air bubbles
WBCs
 larger than RBCs (12 um)
 increased WBCs in urine is termed as:
o Bacterial infections - Pyelonephritis, cystitis, prostatitis, urethritis
o Non-bacterial d/o – Glomerulonephritis, SLE, tumors, interstitial nephritis
Neutrophil
 predominant WBC (granulated, multi-lobed nuclei)
 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
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 _ 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
 not normally present in urine
 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
MUCUS
 protein material produced by the glands & ECs of the lower genitourinary tract

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 ducts
 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

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
 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/
 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
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
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 whetstones
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:
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
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
 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)
 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
For Liver Disorders :

4. Tyrosine crystals
 fine colorless to yellow needles frequently in clumps or rosettes
 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:
 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 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, whereas casts, other than fatty casts, do NOT
6. Fecal contamination
URINE SCREENING FOR METABOLIC DISORDERS
 The appearance of abnormal metabolic substances in the urine can be caused by a variety of disorders that can generally
be grouped into two categories:
o Overflow type - result from disruption of a normal metabolic pathway that causes increased plasma
concentrations of the nonmetabolized substances
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 Følling in 1934
 most well-known of the aminoacidurias; autosomal recessive inheritance
 if undetected: causes severe mental retardation
 Urine Findings: ↑ Keto acids, including phenylpyruvic acid
 MOUSY ODOR of urine
 lacks the enzyme:
 Screening test:
o Guthrie’s Bacterial Inhibition test
 blood-impregnated disks are placed on culture media streaked w/ B. subtilis
 if there is ↑ 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
o Urine Screening test
 Ferric chloride tube test: PERMANENT BLUE-GREEN
2. Tyrosyluria
 there is excess tyrosine in the plasma producing urinary overflow
 Causes:
o inherited form (enzymes required in metabolic pathway are not produced)
o metabolic form (caused by acquired severe liver disease)
 Types:
o Type I - deficiency of fumaryl acetoacetate hydrolase (FAH)
o Type II - deficiency of tryrosine aminotransferase
o Type III - deficiency of p-hydroxyphenylpyruvic acid dioxygenase
 Urine findings:
o ↑ tyrosine, or metabolites:
 p-hydroxyphenylpyruvic acid
 p-hydrozyphenyllactic acid
 Urine Screening test
o 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.
 Causes brown-stained or black-stained cloth diapers
 lacks the enzyme:
 in later life, brown pigment deposits in tissues, cartilage (arthritis) & ears
 URINE SCREENING TEST
o Ferric chloride tube test: TRANSIENT BLUE
o Benedict’s / Clinitest: YELLOW PRECIPITATE
o Alkalinization of urine: DARKENING OF URINE
 add alkali to freshly voided urine;
 or add silver nitrate + NH4OH
4. Melanuria
 increased urinary melanin
o DARKENING OF URINE (after exposure to air)
o overproliferation of melanocytes (melanin-producing cells), producing a malignant melanoma
o these tumors secrete 5,6-dihydroxyindole (precursor of melanin), which oxidizes to melanogen, and then to
melanin
o Ferric chloride tube test: GRAY/BLACK PRECIPITATE
o Sodium nitroprusside: RED COLOR
o Ehrlich’s reagent: RED COLOR
Branched-Chain Amino Acid Disorders

1. Maple syrup urine disease (MSUD)
 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:
o 2,4 – dinitrophenylhydrazine (DNPH) test: YELLOW PRECIPITATE
2. Organic Acidemias
 Three most common disorders:
o Isovaleric acidemia
 “sweaty feet odor” of urine
 deficiency of enzyme: isovaleryl CoA in the leucine pathway
o 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
o indican
o 5-hydroxyindoleacetic acid (5-HIAA)
1. Indicanuria
 Intestinal defects that cause increased amounts of tryptophan converted to INDOLE
o intestinal obstruction
o presence of abnormal bacteria or malabsorption syndromes
o 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”)
o 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)
o Nitroso-naphthol test: PURPLE (with nitric acid)
o Ferric chloride tube test: BLUE-GREEN
 Patients must be given explicit dietary instructions before collecting any sample to be tested for 5-HIAA:
o avoid foods such as bananas, pineapples, and tomatoes.
o medications, including phenothiazines and acetanilids, also interfere with results
CYSTINE DISORDERS
 Two disorders:
o cystinuria – defect in renal tubular transport of COLA amino acids
o cystinosis – inborn error of metabolism
Cystinuria
 caused by the INABILITY of the renal tubules to reabsorb cystine filtered by glomerulus
 amino acids involved:
 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
o Cyanide-nitroprusside test: RED-PURPLE
 False (+): ketones and homocystine
Cystinosis
 “Genuine inborn error of metabolism”
 Categories:
o 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
 if 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 to a deficiency of
 clinical signs:
o cataracts
o thrombosis
o mental retardation
o Cyanide-nitroprusside test: RED-PURPLE
o Silver-nitroprusside test: RED-PURPLE
Porphyrin disorders
 Disorders of porphyrin metabolism are collectively termed porphyrias.
 Forms:
o Inherited: absence of a particular enzyme involved in heme synthesis
o Acquired: lead poisoning, excessive alcohol intake, iron deficiency, chronic liver disease
 Causes PORT WINE COLOR of urine
 Tested in Urine: ALA, Porphobilinogen. Uroporphyrin , Coproporphyrin
 Tested in Feces or Bile: Coproporphyrin, Protoporphyrin
 Tested in Blood: Protoporphyrin
o Ehrlich’s reaction: RED COLOR = detects ALA & Porphobilinogen
o Fluorescent test: VIOLET / PINK / RED fluorescence
Mucopolysaccharide disorders
 mucopolysaccharides or glycosaminoglycans
o large compounds located in connective tissues
o 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:
o Hurler syndrome

mucopolysaccharides deposits in the Cornea

abnormal skeletal structure

mental retardation
o Hunter syndrome
 abnormal skeletal structure
 mental retardation
o Sanfilippo synbdrome
 mental retardation ONLY

o Cetyltrimethylammonium bromide (CTAB) Test = WHITE TURBIDITY after 5 mins.
o Acid-albumin test = WHITE TURBIDITY after 30 mins.
o 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
o disorder of purine metabolism
o sex-linked recessive
o lacks the enzyme:
o causes massive excretion of urinary uric acid crystals
 Clinical signs:
o severe motor defects and mental retardation
o tendency toward self-destruction
o gout
o renal calculi
 first sign is often uric acid crystals resembling “orange sand in diapers”
RENAL DISEASES
I. GLOMERULAR DISORDERS
 immunologic
o 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
o blood, protein, and casts are seen in the urine
1. Acute Post-streptococcal Glomerulonephritis

 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
 clinical symptoms: fever, edema, fatigue, HPN, oliguria & hematuria
 Urine findings: RBC casts, dysmorphic RBC, granular casts, WBCs
 there is elevated serum ASO
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
3. Goodpasture Syndrome
 due to attachment of Antiglomerular basement membrane antibody to the basement membranes following viral
respiratory infections
 Clinical findings: hemoptysis, dyspnea, hematuria
4. Vasculitis
 disorders affecting systemic vascular system can result in glomerular involvement
 Wegener’s granulomatosis
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
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)

 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
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
 Lab findings: DECREASED SERUM COMPLEMENT LEVELS
8. Chronic Glomerulonephritis
 marked decrease in renal function resulting from glomerular damage precipitated by other renal disorders
 Urine findings: hematuria, proteinuria, glucosuria, BROAD CASTS
 Lab findings: decreased GFR, increased BUN & crea
9. Nephrotic Syndrome
 disruption of electrical charges that produce the tightly fitting podocyte barrier resulting in massive loss of
proteins & lipids
 Urine findings:
o MASSIVE PROTEINURIA ( > 3.5 g/dL )
o RTEs & oval fat bodies
o fatty & waxy casts
o hematuria
 Lab findings: Low serum Albumin, High serum Lipids
 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 children
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
II. TUBULAR DISORDERS

1. Acute Tubular Necrosis
 Two causes:
o Ischemic
 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. Fanconi Syndrome
 most frequently assoc. w/ tubular disorder
 generalized FAILURE OF TUBULAR REABSORPTION in the PCT
 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”
 Lower UTI – involves urethra & bladder
 Upper UTI – involves the renal pelvis, tubules & interstitium
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
4. Acute Pyelonephritis
 Upper UTI involving both the tubules & interstitium
 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
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

 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
III. VASCULAR DISORDERS

1. Renal Failure
 2 forms:
o Acute Renal Failure
 exhibits sudden loss of renal function
 frequently REVERSIBLE
 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
o Chronic Renal Failure (END-STAGE RENAL DISEASE)

 gradual progression from the original disorder
 Lab findings:
 marked decreased GFR
 electrolyte imbalance
 Azotemia: ↑ BUN & Crea
 lack of renal conc. ability
 proteinuria & glycosuria
 abundance of granular, waxy, broad casts =
2. Renal Lithiasis
 may form in the calyces & pelvis of the kidneys, ureters, & bladder
 small calculi maybe passed in the urine, large stones cannot
 Lithotripsy – uses high-energy shock waves to break stones in the upper urinary tract
 conditions favoring formation of renal calculi: pH, chemical conc. & urinary stasis
 Analysis of renal calculi:
o chemical test
o X-ray Crystallography – for comprehensive analysis
 Renal calculi:
o 80% -
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 PHOTOMETRY
o a spectrophotometric measurement of light reflection
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,
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 .
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
o Arachnoid Mater
Subarachnoid space (it is where CSF flows)
o Pia Mater
 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: mL
o Neonates mL
 Mode of collection: (between 3rd, 4th or 5th vertebrae)

 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 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
o Malignancy
 Designated CSF sterile tubes:
Tube # Lab Test (STAT) Storage requirement
1 Freezing temp
2 Room temp
3 Refrigerator temp
 Appearance of CSF:
o Normal CSF: 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
Intracranial hemorrhage Traumatic Tap

Distribution of Blood Even distribution Uneven distribution
Bottle 1 = 2 = 3 Bottle 1 > 2 > 3
Clot Formation – +
Color of Supernatant Xantochromic Clear
D-dimer Test + –
Erythrophagocytosis + –
Cell Count
 should be done immediately because WBCs & RBCs will begin to lyse in 1 hour
 40% of leukocytes will disintegrate within 2 hrs.
 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%)
 The presence of increased numbers of normal cells is termed as & indicate abnormality.
 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
 Ref Value: 15 – 45 mg dL
 Major protein is Albumin, followed by prealbumin
 Major beta globulin present is Transferrin
 Unique to CSF is the 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:
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
Evaluation of integrity of blood-brain barrier:
CSF/serum albumin index = CSF albumin (mg/dL) * An index value of < 9 represents an an intact
blood brain barrier.
Serum albumin (g/dL)
Indication of IgG production within CNS
CSF IgG index = CSF IgG (mg/dL) / serum IgG (g/dL) * An index value of > 0.7 indicate IgG
synthesis within CNS
CSF albumin (mg/dL) / serum albumin (g/dL)
CSF Electrophoresis
 primary purpose is to detect 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)
 Guillain-Barré syndrome
 Encephalitis, Cryptococcal meningitis
 Neurosyphilis, Neuroborreliosis, Trypanosomiasis
 Neoplastic disorders
o (+) CSF and (+) serum
 HIV infection
Myelin 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: 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
 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 that is present in CNS
 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)
 Reye’s syndrome (75% of cases)
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 –
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: “ ”
 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
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 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
SEMEN ANALYSIS
Fractions of Semen
60-70% provides Fructose – for motility of sperm cells provides

Flavin – gray appearance of semen
20-30% contains ACP, citric acid, zinc, proteolytic enzymes responsible

for coagulation and liquefaction of semen
5% Testes – for production of spermatozoa (seminiferous tubules)

Epididymis – for maturation and storage of sperm cells
5% forms a thick, alkaline mucus that helps to neutralize acidity from the
prostate secretions & vaginal acidity
SPECIMEN COLLECTION
 Sexual abstinence of DAYS
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
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
 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
Sperm Concentration
 Normal: million / mL (10-20 million is the borderline)
 2 chambers:
o Neubauer Counting Chamber (counted in the same manner as CSF)
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
 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
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
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
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 for flagellar motion
 Acrosomal Cap: contains for ovum penetration (1/2 of the Head)
 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
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:
 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
 Zinc = > 2.4 μmol/ejaculate
 Citric acid = > 52 μmol/ejaculate
 Acid phosphatase = > 200 Units/ejaculate
Antisperm antibodies
 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
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 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

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
 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:
 Deeper yellow – Inflammation
 Greenish – Bacterial infection
 Blood – hemorrhagic arthritis VS traumatic tap (uneven distribution of blood)
 Turbidity – leukocytosis; cell debris and fibrin
 MILKY –
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
 ROPES OR MUCIN CLOT TEST

o measurement of the degree of polymerization of the HA
o Reagent:
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
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
Neutrophil with ingested round body Lupus erythematosus
Vacuolated macrophage w/ ingested neutrophils Reactive arthritis / Reiter’s syn
Neutrophil with dark cytoplasmic granules Rheumatoid arthritis

containing immune complexes
Macroscopic: polished rice appearance Microscopic: Tuberculous arthritis /

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

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
Condition associated Shape Polarized light Compensated

Polarized Light
Gout needles strongly Negative
(↑ serum uric acid or ↓ renal birefringent Birefringence
excretion) (yellow color)
Pseudogout rhomboid weakly Positive

(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
OTHER TESTS:
1. Microbiologic Tests
 Gram stain & culture
 include a chocolate agar – for fastidious Haemophilus & Neisseria gonorrhoeae
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%
Crystals present
III Septic condition Cloudy, YELLOW-GREEN fluid

- microbial infection Variable viscosity
WBCs 50,000 - 100,000 uL
Neutrophils >75%
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
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
 = Accumulation of fluid in the body cavity
Fluids in closed body cavities

Collection: needle aspiration
Pleural fluid Lungs
Pericardial fluid Heart
Peritoneal / Ascitic fluid Abdominal
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 the
balance in fluid filtration/reabsorption membranes of a particular cavity
Process Mechanical Inflammatory
Pathologic causes Increased Hydrostatic pressure; Decreased Increased Capillary permeability;

Oncotic pressure Deceased Lymphatic resorption
Examples Congestive heart failure Infections

Malignancy
Hypoproteinemia Nephrotic
Membrane inflammations
syndrome
Lab Tests for differentiation

TRANSUDATE EXUDATE
Appearance Clear Cloudy
Fluid:Serum protein ratio < 0.5 > 0.5
Fluid:Serum LDH ratio < 0.6 > 0.6
WBC count < 1000 / uL > 1000 / uL
Spontaneous clotting No Possible
Pleural fluid cholesterol < 45-60 mg / dL > 45- 60 mg / dL
Pleural fluid:serum cholesterol ratio < 0.3 > 0.3
Pleural fluid:serum bilirubin ratio < 0.6 > 0.6
Serum-ascites albumin gradient > 1.1 < 1.1
PLEURAL FLUID
Appearance of fluid
Associated condition
Normal
Turbid, white Microbial infection (e.g. tuberculosis)
Bloody Hemothorax or Hemorrhagic effusion
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
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
↑ levels in Tuberculosis (> 40 U/L)
↑ 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
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
PERITONEAL FLUID
Appearance of fluid
Normal
Turbid Microbial Infection
Blood-streaked Trauma, infection, malignancy
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
Malignancy of gastrointestinal origin
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
Detection of intra-abdominal bleeding in Blunt Trauma; RBC ct:
>100,000 / uL
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 Neural Increased fetal swallowing of urine
Tube defect Urinary Tract deformities
Fetal Distress Membrane Leakage
Specimen Collection
 AMNIOCENTESIS
o Transabdominal amniocentesis – most frequently performed
o Vaginal amniocentesis
 Usually performed AFTER WEEK OF GESTATION
 Chromosome Analysis – Fluid is collected at 16th Week Gestation
 Volume collected
o Maximum of mL
o First 2 or 3 mL = Contaminated by maternal blood = Discarded
Specimen Handling
 Fetal Lung Maturity (FLM) Tests
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
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.
Amniotic Fluid Creatinine

 Has been used to determine fetal age
 Creatinine value of mg/dL in amniotic fluid signifies fetal age of > 36 WEEKS.
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 + -
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
2. Neural Tube Defects

 Examples of defects are Anencephaly and Spina bifida
 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)
o A value two times the median value (greater than 2 MoM) is considered abnormal
 ↑ Amniotic fluid AFP levels are followed by measuring Amniotic
o more specific test but specimen should NOT be performed on a bloody specimen. (false +)
Tests for Fetal Maturity

 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
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
 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
*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
 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
FECES
 Normally contains bacteria, cellulose, undigested foodstuff, GI secretions, bile, electrolytes & water
 Approximately 100 to 200 g of feces is excreted in a 24-hour period.
 Normal stool pH = 7 - 8
Diarrhea
 defined as an increase in daily stool weight above 200 g, increased liquidity of stools, and frequency of more than three
times per day
 Mechanisms:
o Secretory Diarrhea
 due to bacterial, protozoan infection causing increase secretion of water & electrolytes,
which override the resorptive capability of the large intestine
o Osmotic Diarrhea
 caused by poor absorption that exerts osmotic pressure across the intestinal mucosa
 Incomplete breakdown of food presents increased fecal material to the large intestine
 Results to excessive watery stool.
 Maldigestion (impaired food digestion) and malabsorption (impaired nutrient absorption by
the intestine) contribute to osmotic diarrhea
o Altered motility
 describes conditions of enhanced motility (hypermotility) or slow motility (constipation).
Both can be seen in irritable bowel syndrome (IBS)
Steatorrhea
 defined as an increase in 6 g per day)
 associated with the ff conditions:
o pancreatic insufficiency (e.g. cystic fibrosis)
o small-bowel disorders that cause malabsorption
o chronic pancreatitis and carcinoma
COLOR OF FECES
Brown from intestinal oxidation of urobilinogen to urobilin

Black Upper GI bleeding, Iron therapy, Charcoal, Bismuth (antacids)
Red Lower GI bleeding, Beets & food coloring, Rifampin
Pale yellow / White / Gray Bile duct obstruction, Barium Sulfate (diagnostic procedure)
Green Biliverdin / oral antibiotics, Green vegetables
APPEARANCE
Bulky / Frothy Bile duct obstruction, Pancreatic disorder

Ribbon-Like Intestinal constriction
Mucus / Blood-streaked Colitis, Dysentery, Malignancy
Salmonella infection
Vibrio infection
Fecal Leukocytes
 primary WBC observed are NEUTROPHILS
 Microscopic screening is performed as a preliminary test to determine whether diarrhea is being caused by
invasive bacterial pathogens
o / HPF = indicates invasive condition
o Preparations:
 WET PREPARATION – uses Methylene blue
 DRY PREPARATION – uses Wright’s or Gram stain
 Lactoferrin Latex Agglutination Test
o detects fecal leukocytes
o Lactoferrin is present in secondary granules of granulocytes
Muscle Fibers
 microscopic exam for presence of undigested striated muscle fiber
 helpful in diagnosing and monitoring pancreatic insufficiency such as
 Procedure:
o Specimen is emulsified w/ 10% Alcoholic Eosin
o slide is examined for exactly 5 minutes, and the number of red-stained fibers with
well-preserved striations is counted
 undigested fiber – have horizontal & vertical striations
 partially digested fiber – striations in one direction only
 digested fibers – no visible striations
o Only UNDIGESTED FIBERS are counted (> 10 / HPF = reported as INCREASED)
Qualitative Fecal Fats

 Microscopic screening for presence of excess fecal fat (steatorrhea)
 Lipids included: Neutral fats (TGL), Fatty acid salts, Fatty acids, Cholesterol
 Stains: (routinely used), Sudan IV, Oil Red O
Neutral Fat Stain

 Reagents:
o 95% alcohol (emulsifier of stool sample)
o Sudan III (stain)
 Results – examination under HPF
o Count the orange -red droplets
o > 60 Droplets / HPF = STEATORRHEA
Split Fat Stain

 Provide a better indication of total fat content in stool
 Reagents:
o 36% acetic acid (emulsifier of stool sample)
o Sudan III (stain) + gentle heating of slide
 Results – examination under HPF
o Normal: 100 small droplets (< 4 um)
o INCREASED = 100 Droplets (6-75 um)
Quantitative Fecal Fats

 confirmatory test for steatorrhea
 requires a 3-Day collection of stool sample
 patient must maintain a regulated intake of fat 100 g/day before & during collection
 Methods:
o
 method routinely used for fecal fat measurement and is also the gold standard
 fecal lipids are converted to fatty acids & titrated to a neutral end-point w/ NaOH
 Reference value: 1 – 6 g/day
o Acid Steatocrit
 rapid test to estimate the amount of fat excretion
 more convenient since it required spot collection of feces
 similar to the microhematocrit test
 Results:
 <31% is considered normal
 >31% indicates steatorrhea in adults
Fecal Occult Blood

 Bleeding in excess of 2.5 mL / 150 g of stool is pathologically significant
 Testing is necessary if there is no visible signs of bleeding on suspected patients
 Used also as a mass screening procedure for early detection of colorectal cancer
 Guiac-based test for Occult Blood
o most frequently used and preferred screening test for fecal blood
o Principle is based on detection of Pseudoperoxidase activity of Hb
o Hb + pseudoperoxidase = H2O2 (oxidizes) Guiac = Oxidized Guiac (BLUE color) + H2O
 To prevent False-Positive, obtain sample from the center of the Stool
Interferences
Result Remarks
Vitamin C (>250 mg/dL) Must be avoided 3 days before collection
Aspirin & anti-inflammatory medications Must be avoided 7 days before collection
Red meat, horseradish, melons, raw broccoli, Must be avoided 3 days before collection
cauliflower, radish, turnips
APT Test
 Distinguishes between the presence of fetal blood or maternal blood in an infant’s stool or vomitus
 Fecal Material or Vomitus is emulsified, centrifuged, 1% NaOH is added to the pink supernatant
o Fetal Hb = ALKALI-RESISTANT – solution remains color
o Maternal Hb = will be DENATURED – solution becomes color after 2 mins
 Stool specimens should be tested when fresh
 The presence of maternal thalassemia major would produce erroneous results owing to the high
concentration of HbF
Other Fecal Tests
Method/Principle Interpretation
Clinitest Addition of Clinitest tablet on emulsified stool detects Reaction of 0.5 g/dL reducing substances suggests
presence of reducing substances carbohydrate intolerance
Trypsin Emulsified specimen placed on x-ray paper Inability to digest gelatin indicates lack of
determines ability to digest gelatin trypsin
Elastase I Immunoassay using an ELISA test Sensitive indicator of exocrine pancreatic

insufficiency

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NURSING*RADTECH*DENTISTRY*CRIMINOLOGY*MIDWIFERY*MEDTECH

LET*PSYCHOMET*RESPIRATORY THERAPY*CIVIL SERVICE*NAPOLCOM

FOUR MAJOR RENAL FUNCTIONS:

Angiotensinogen Angiotensin I Angiotensin II

GLOMERULAR FILTRATION TESTS

Glomerular Filtration Rate (GFR)

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

Female: multiply the answer by 0.85

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

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

If Female: multiply the answer by 0.742

If African-American: multiply the answer by 1.202

TUBULAR REABSORPTION TESTS

Solute dissolved in Solvent: (changes in colligative properties)

Freezing Point Osmometers

Ratio of Urine to Serum osmolality

> 2000 mL / 24 hrs Increased fluid intake

< 400 mL / 24 hrs Dehydration / burns

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

URINE SPECIMEN COLLECTION

CHANGES IN UNPRESERVED URINE

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

TYPES OF URINE SPECIMEN

Routine screening for obvious abnormality, may produce erroneous results

IDEAL specimen; useful for Pregnancy test and Orthostatic proteinuria

Recommended for glucose monitoring (for DM)

24-Hour specimen = used for Quantitative Chemical tests

Useful to determine PROSTATIC INFECTION

DRUG SPECIMEN COLLECTION

PHYSICAL EXAMINATION OF URINE

URINE COLORS AND ASSOCIATED CONDITIONS

Colorless Very dilute urine

URINE COLOR AND COMMONLY USED DRUGS

Yellow Mepacrine (antimalarial)

Brown Phenol poisoning

URINE CLARITY / TURBIDITY

Hazy few particulates, print easily seen through urine

Turbid print cannot be seen through urine

Lab Correlations in Urine Turbidity

2. Alkaline urine Amorphous phosphates, carbonates

3. Soluble with Heat Amorphous urates, Uric acid crystals

4. Soluble in Dilute Acetic acid RBCs

5. Insoluble in Dilute Acetic acid WBCs

6. Soluble in Ether Lipids, Lymphatic fluid, chyle

METHODS OF DETERMINATION OF SPECIFIC GRAVITY

2. Refractometry (TS meter)

4. Harmonic Oscillation Densitometry

5. Reagent Strip method

CHEMICAL EXAMINATION OF URINE

Time Test Principle Reagent Sensitivity (+) Result

30 sec Double sequential

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

pKa change of a Bromthymol blue 1.000-1.030 Blue (1.000)

Protein error of tetrabromphenol blue /

1 min Ehrlich's Aldehyde p-dimethylaminobenzaldehyde, 4- 0.2 mg/dL Dark Pink

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

2 mins Leukocyte esterase Indoxylcarbonic/pyrrole acid ester 5-15 Purple

Sulfosalicylic acid test

Reporting SSA Turbidity (mg/dL)

ACID URINE ALKALINE URINE

muscle injury (myoglobin) microalbuminuria toxic agents / prostatic fluid

multiple myeloma (bence orthostatic proteinuria*

NURSINGRADTECHDENTISTRYCRIMINOLOGYMIDWIFERY*MEDTECH

LETPSYCHOMETRESPIRATORY THERAPYCIVIL SERVICENAPOLCOM