RENAL FUNCTIONS

1. Renal blood flow 3. Tubular reabsorption

2. Glomerular filtration 4. Tubular secretion
Flow of Urine formation
Blood→Renal arteries (Afferent arteriole)→Glomerulus (Efferent arteriole)→Proximal convoluted tubule (PCT)→Descending
LOH→Ascending LOH→Distal convoluted tubule (DCT)→Collecting duct→Renal pelvis→Ureter→Bladder→Urethra
Basic structural and functional unit of the kidney Nephron
Tuft of 8 capillaries within the Bowman’s capsule Glomerulus (sieve, nonselective filter of plasma substances <70000 Da MW
Major site of reabsorption of plasma substances PCT (65%, salts, water, AA, Glucose, urea)
No solutes reabsorbed, water permeable DLOH
Reabsorbs solutes, impermeable to water ALOH
Regulates water reabsorption in the DCT and CD ADH/Vasopressin
Regulates sodium reabsorption Aldosterone
Inabilty to produce an acid urine Renal tubular acidosis (H ions are not excreted in the urine)
Renal threshold for glucose 160-180 mg/dL
Renal blood flow 1200 mL/min
Renal plasma flow 600-700 mL/min
Major organic substance in urine Urea
Major inorganic substance in urine Cl >Na>K
Principal salt in urine NaCl
RENAL FUNCTION TESTS
1. Glomerular Filtration Creatinine Clearance
CC-Creatinine Clearance
Clearance Test
U-Urine creatinine (mg/dL)
 Urea (old) CC = UxV x 1.73m2
V-Urine volume (mL/min)
 Creatinine (most common) P A
P-Plasma creatinine
 Inulin (gold standard) A-Body surface area
 B2-microglobulin
 Cystatin C
 Radioisotopes *The greatest source of error in Clearance test -Improperly timed urine specimens
2. Tubular Reabsorption
Concentration Test
 Specific gravity (influenced by the number and density of particles)
 Osmolality (influenced by the number of particles)
 Mosenthal test (compare the SG and volume of day and night urine)
 Fishberg test (24 hours fluid deprivation, SG measured ≥1.026) *12 hours ≥1.022
3. Tubular Secretion and Renal Blood Flow
 p-aminohippuric acid (PAH) test
 Phenolsulfonphthalein (PSP) test (obsolete, hard to interpret)
TYPES OF URINE SPECIMEN
Single, Occasional, Random Most commonly used, for routine urine analysis
24-hour Timed: Creatinine clearance
12-hour Timed: Addis count
PM (2pm-4pm) Timed: Urobilinogen determination
4-hour Timed: Nitrite testing
First morning/8-hour specimen Ideal for screening, Routine urinalysis and ACP determination
(most concentrated, acidic) Pregnancy, HCG and Orthostatic proteinuria
Second morning/Fasting Collected after a period of fasting, collected in parallel with glucose specimen test
Midstream clean-catch Routine screening and aerobic bacterial culture
Catheterized Aerobic bacterial culture
Suprapubic aspiration Bladder urine for urine cytology and aerobic and anaerobic bacterial culture
Pediatric specimen Use of soft, clear and clean plastic bag with adhesive attached
3 Glass technique Prostatic infection Normal state: 1>3 bacterial and WBC count (10x)
1. (first portion of voided urine) Infection state: 3>1 bacterial and WBC count (10x)
2. (middle portion/Midstream) If #2 is (+) what must be the proper response?
3. (after prostatic massage) (control for kidney and bladder infections, result
from 3rd specimen is invalid)
Requires a Chain of Custody Required volume: 30-45 mL
(COC) –a process that provides container: 60 mL
correct documentation and Temperature: 32.5-37.7°C within 4 mins
DRUG TESTING
proper sample identification. Other preliminaries: blue dye, non-watered urinal
Commonly tested chemicals:
Methamphetamine,
tetrahydrocannabinol (THC)
CHANGES OCCURING IN UNPRESERVED URINE
*Urine specimens should be tested within 2 HOURS
Increased 1. pH (Urea-(urease)-Ammonia)

↑
2. Bacteria (Multiplication)
3. Odor (Urea-(urease)-Ammonia)
4. Nitrite (Due to bacterial multiplication)
Decreased 1. RBC/WBC/Cast (Disintegrate in alkaline urine)

↓
2. Bilirubin (CB/B2-Light exposure)
3. Clarity (Bacterial multiplication, precipitation of amorphous material)
4. Ketones (Volatilization)
5. Glucose (Glycolysis)
6. Urobilinogen (Oxidation to urobilin)
7. Trichomonas (become immobile/die, misidentification as WBCs)
Changed (Darkened) Color (Oxidation/reduction of metabolites)
Least affected Proteins
TYPES OF URINE PRESERVATION
Physical Chemical
1. Refrigeration *with special concerns
2. Glucose 1. Formalin- for Addis count
2. Benzoic acid/Sodium fluoride- for glucose
3. Boric acid- best for culture
4. Saccomano’s fixative- for cytology
 PHYSICAL EXAMINATION OF URINE
URINE VOLUME
24-hour urine volume: 600-2000 mL Day:night ratio: 2:1 to 3:1
Average volume: 1200-1500 mL Volume required for RUA: 10-15 mL (12 mL)
INCREASED URINE VOLUME DECREASED URINE VOLUME COMPLETE CESSATION OF BLOOD FLOW
Polyuria (>2000mL or >2.5L/day) Oliguria (<500mL or <400mL/day) Anuria (<100mL/24 hours)
↑IN-DDDD ↓CIRDD -Toxic agents (mercury)
Increased fluid intake Calculus/tumors of the kidney/UT -Complete UT obstruction (stones and carcinomas)
Nervousness Increased ADH production -Decreased renal blood flow
Diuretic medications Renal stone/tumors INCREASED URINE OUTPUT AT NIGHT
Diuretic drinks (coffee,tea,alc) Dehydration (Nocturia)
DM (↑Vol,↑SG,↓Insulin) Decreased fluid intake >500 ml w/ SG <1.018
DI (↑Vol,↓SG,↓ADH) -Pregnancy, Early DM
URINE COLOR
Urochrome Yellow pigment, Major pigment (production is proportional to metabolic rate)
Pigments Uroerythrin Pink pigment (deposits found in Amorphous urates and uric acid crystals
Urobilin Dark yellow/orange pigment (product of RBC degradation)
Color Cause
Colorless Recent fluid consumption
Pale yellow Polyuria, dilute random urine
Dark yellow Concentrated specimen
Amber Dehydration (fever, burns)
Orange Acriflavin, Nitrofurantoin, Phenindione, Pyridium, Bilirubin
Yellow-green, Yellow-brown Bilirubin oxidized to Biliverdin
Green Pseudomonas infection
Blue-green Indican, Methylene blue, Methocarbamol, Phenol, Chlorophyll, Clorets, Amitriptyline
Pink, Red RBCs, Hemoglobin, Myoglobin, Rifampin, Beets, Menstrual contamination
Portwine, Burgundy/Purplish red Porphyrins
Brown, Black Homogentisic acid, Methemoglobin, Melanin, Phenol, Methyldopa, Argyrol, Metronidazole
What is the most common abnormal urine color? -Red
Clear red urine due to intravascular hemolysis -Hemoglobinuria
Clear red or reddish brown urine due to muscle damage (rhabdomyolysis) -Myoglobinuria
Cloudy or smoky red urine due to intact RBCs -Hematuria
URINE CLARITY/TURBIDITY
PATHOLOGIC CAUSES OF TURBIDITY NONPATHOLOGIC CAUSES OF TURBIDITY
BRWN CLLAY SMARTS FV
Bacteria Squamous Epithelial cells
RBC Mucus
WBC Amorphous (urates,phosphates & carbonates)
Nonsquamous Epithelial Cells Radiographic Contrast Media
Chyle Talcum powder
Lymph fluid Semen/Spermatozoa
Lipids Fecal contamination
Abnormal crystals Vaginal cream
Yeast
TRANSPARENCY TERMS
Clear No visible particulates
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 be clotted
LABORATORY CORRELATIONS IN URINE TURBIDITY
Acidic urine Amorphous urates, RCM
Soluble with heat Amorphous urates, uric acid crystals
Alkaline urine Amorphous phosphates and carbonates
Soluble in dilute HAc Amorphous phosphates and carbonates, RBCs
Insoluble in dilute HAc WBCs, bacteria, yeast, spermatozoa
Soluble in ether Chyle, lymphatic fluid, lipids
URINE ODOR
Slightly aromatic to odorless Normal
Fruity or sweet Ketones (DM, starvation, vomiting)
Rancid butter Tyrosinemia (Tyrosiluria)
Sweaty feet Isovaleric academia
Rotting fish Trimethylaminuria
Sulfur Cystinuria
Ammoniacal Bacterial decomposition, UTI (Proteus vulgaris)
Mousy Phenylketonuria
Cabbage Methionine malabsorption
Maple syrup MSUD
Bleach Contamination
Odorless RTA
URINE SPECIFIC GRAVITY
Terms related to SG: Isosthenuria (1.010) Hyposthenuria (<1.010) Hypersthenuria (>1.010)
Normal SG: 1.003/1.002-1.035 SG <1.003: NOT urine (DI,SG 1.001) SG>1.040: RCM (refractometer)
Urinometry (Hydrometer) Refractometry (TS Meter) Harmonic Oscillation Densitometry
Required urine volume: 10-15 mL Indirect method based on RI - The frequency of the sound wave that
Requires temperature correction (20°C) Already compensated to 15-38°C enters a solution will change in proportion
3°C↑--(+) 0.001 in SG reading Requires correction for glucose & protein to the density of the solution.
3°C↓--(-) 0.001 in SG reading For 1 g/dL glucose—(-) 0.004
Requires correction for glucose & protein For 1g/dL protein—(-) 0.003 Example: Yellow IRIS (automated)
For 1 g/dL glucose—(-) 0.004 Calibration (International Remote Imaging System)
For 1g/dL protein—(-) 0.003 Distilled water (SG=1.000)
Calibration 5% NaCl (Saline) (SG=1.022+0.001)
Potassium sulfate solution (SG=1.015) 9% Sucrose (SG= 1.034+0.001)
 CHEMICAL EXAMINATION OF URINE
REAGENT STRIP TEST
Urine Parameter Reading Principle Positive color Correlation
Time
Glucose 30 secs Double sequential enzyme reaction M: blue to green to brown Ketones
C: yellow to green
Bilirubin 30 secs Diazo reaction Tan or pink to violet Urobilinogen
Ketones 40 secs Sodium nitroprusside reaction (Legal’s test) Purple Glucose
Specific Gravity 45 secs pKa change in polyelectrolyte pad Blue (SG:1.000) to
yellow (SG:1.030)
Protein 60 secs Protein/Sorensen’s error of indicators Blue-green *MNL-B
pH 60 secs Double indicator system Orange (pH:5) to *MNL
blue (pH:9)
Blood 60 secs Pseudoperoxidase activity of hemoglobin Uniform green/blue (Hb/Mb), *PM
Speckled/spotted (intact RBCs)
Urobilinogen 60 secs Ehrlich reaction Red Bilirubin
Nitrite 60 secs Greiss reaction Uniform pink *PLM
Leukocytes 120 secs Leukocyte esterase reaction Purple *MPN
*M-microscopic,N-nitrite,L-leukocytes,B-Blood,P-protein,
REAGENT STRIP REAGENTS AND SENSITIVITY
Urine Reagents Sensitivity
Parameter
Glucose M: Glucose oxidase, Peroxidase, Potassium iodide 75-125 mg/dL
C: Glucose oxidase, Peroxidase, Tetramethylbenzidine 40 mg/dL
Bilirubin M: 2,4-dichloroaniline diazonium salt 0.4-0.8 mg/dL
C: 2,6-dichlorobenzene diazonium salt 0.5 mg/dL
Ketones M: Sodium nitoprusside 5-10 mg/dL AAA
C: Sodium nitoprusside, Glycine 9 mg/dL AAA, 70 mg/dL Acetone
Specific Gravity M: Poly (methyl vinyl ether/maleic anhydride) bromthymol blue SG: 1.000 to 1.030
C: Ethyleneglycoldiaminoethylethertetraacetic acid bromthymol blue SG: 1.000 to 1.030
Protein M: Tetrabromphenol blue 15-30 mg/dL
C: Tetrachlorophenol, tetrabromosulfonphthalein 6 mg/dL
pH Methyl red, Bromthymol blue pH: 5.0-9.0
Blood M:Diisopropylbenzene dehydroperoxide tetramethylbenzidine 5-10 RBCs/uL or 0.015-0.062 mg/dL of Hb
C: Dimethyldihydroperoxyhexane tetramethylbenzidine 5 RBCs/uL, with Hb corresponding to 10 RBCs/uL
Urobilinogen M: p-dimethylaminobenzaldehyde/ Ehrlich’s reagent 0.2 mg/dL urobilinogen
C: 4-methoxybenzene-diazonium-tetrafluoroborate 0.4 mg/dL urobilinogen
Nitrite M: p-arsinilic acid, Tetrahydrobenzo(h)-quinolin-3-0l 0.06-0.1 mg/dL nitrite ion
C: Sulfanilamide, 3-hydroxy-1234-tetrahydro-78-benzoquinoline 0.05 mg/dL nitrite ion
Leukocytes M: Diazonium salt, Derivatized pyrrole amino acid ester 5-25 WBC/hpf
C: Diazonium salt, Indoxylcarbonic acid ester 10-25 WBC/hpf
Important in the identification of crystals and identification of unsatisfactory specimens- pH
Normal random urine: (4.5-8.0) 1st morning urine: (5-6) Unpreserved urine: (9)
URINE pH
ACIDIC URINE ALKALINE URINE
CHEMPS DDD HPROVV
Cranberry juice HypERventilation
HypOventilation Presence of alkaline-producing bacteria
Emphysema Renal Tubular Acidosis
Medications Old specimen
-Methenamine mandelate (mandelamine) Vomiting
-Fosfomycin tromethamine (monurol) Vegetarian diet
Presence of acid-producing bacteria
Starvation
DM, Diarrhea, Dehydration
Produces white foam in urine when shaken: protein RST indicator is sensitive to: (albumin)
Analyte most indicative of renal disease: protein Normal urine concentration: <150 mg/day; <10mg/dL or 100mg/day
A cold precipitation test that reacts equally with all forms of protein: SSA Precipitation test
Procedure: GRADING
3 mL of 3% SSA + Grade Turbidity Protein (mg/dL)
3 mL centrifuged urine Negative No increase in turbidity <6
→(+) Cloudiness Trace Noticeable turbidity 6-30
1+ Distinct turbidity with no granulation 30-100
2+ Turbidity with granulation with no flocculation 100-200
3+ Turbidity with granulation and flocculation 200-400
4+ Clumps of protein >400
Most frequently detected substance in urine: Glucose (RT: 160-180 mg/dL)
Nonspecific test for reducing sugars: Copper reduction test (Clinitest/Benedict’s test)
GLUCOSE OXIDASE AND CLINITEST REACTIONS
Glucose oxidase Clinitest Interpretation
(Oxidation reaction, spc. Glucose) (Reduction reaction, all reducing sugars)
Negative Positive Non-glucose reducing sugars present/
interference in glucose oxidase test (ascorbic acid)
1+ Positive Negative No enough glucose to reduce CuSO4
4+ Positive Negative Possible oxidizing agents (detergents)
By-products of increased fat metabolism due a defect in carbohydrate metabolism: Ketones (Acetest)
78% Major ketone (not detected in RST) B-hydroxybutyric acid
20% Parent ketone Acetoacetic acid
2% Minor ketone (detected w/ glycine) Acetone
COMPARISON BETWEEN HEMOGLOBINURIA AND MYOGLOBINURIA
Plasma examination Blondheim’s test
Reagent: Ammonium sulfate
Hemoglobin Red plasma (↓haptoglobin) Precipitated
Myoglobin Pale yellow, clear plasma (↑aldolase,CK) Not precipitated
Early indication of liver disease, significant in the screening for diseases such as Hepatitis, Cirrhosis and Biliary duct obstruction:
Bilirubin-B2/conjugated (amber urine w/ yellow foam when shaken)
A more sensitive and less subjected to interference determination of biirubin: Ictotest
A bile pigment resulting from hemoglobin degradation: Urobilinogen (the only analyte that is not reported as negative)
Specimen: PM urine (2-4 pm peak) usual amount: <1mg/dL/Ehrlich unit
UROBILINOGEN AND PORPHOBIINOGEN DIFFERENTIATION: (WATSON SCHWARTZ TEST)
Urobilinogen Porphobilinogen Other Ehrlich reactive substances
Chloroform extraction
Urine Colorless Red Red
Chloroform Red Colorless Colorless
Butanol extraction
Butanol Red Colorless Red
Urine Colorless Red Colorless
Soluble in chloroform and Insoluble in chloroform Soluble in butanol but not in
butanol and butanol chloroform
*Other Ehrlich reactive substances: Indican, Methyldopa, Procaine, Sulfonamide
Rapid screening test for Porphobilinogen: Hoesch test (Inverse Ehrlich reaction)
Rapid screening test for UTI/bacteriuria: Nitrite (specimen 1 st morning/4-hr urine)
Good for determination of UTI and inflammation, screening of urine culture specimen: Leukocytes
11th reagent pad: Ascorbic acid Causes false negative reactions on: BBLNG (blood, bilirubin, leukocyte, nitrite, glucose)
MICROSCOPIC EXAMINATION OF URINE
CELLULAR CONSTITUENTS
Quantitative 12-hour urine sediment count: Addis count Preservative: Formalin
Normal values→ Hyaline casts: 0-5000 RBCs: 0-500 000 WBCs: 0-1 200 000
Sediment constituents Term Normal urine findings (Henry & Strasinger)
RBCs Hematuria 0-2/0-3 RBCs/HPF
WBCs Pyuria/Leukocyturia 0-5/0-8 WBCs/HPF
Casts Cylinduria 0-2 Hyaline cast/LPF
RBCs in dilute alkaline hypotonic urine: Ghost cells (swell/hemolyzed) (hypertonic-crenate/shrink)
WBCs in dilute alkaline hypotonic urine: Glitter cells (Brownian movement-sparkling appearance)
Associated with drug-induced interstitial nephritis: Eosinophils (>1%)
Largest cell with abundant, irregular cytoplasm and prominent nucleus: Squamous Epithelial Cells
Squamous epithelial cells covered with G. vaginalis: Clue cells
Increased numbers are seen following catheterization: Transitional EC/Urothelial (renal pelvis,bladder,upper urethra-male)
Most clinically significant epithelial cell: Renal Tubular EC ( nephron, >2 RTE/hpf indicates tubular injury)
RTE cells w/ non-lipid filled vacuoles: Bubble cells
RTE cells w/ lipid filled vacuoles: Oval fat bodies
Most common cause of UTI: Enterobacteriaceae-E.coli (UTI- Bacteria and WBCs)
Seen in DM and vaginal moniliasis: Yeast- Candida albicans
Most frequent parasite encountered in the urine: T. vaginalis (pear-shaped, ping-pong disease)
Specimen of choice for S. haematobium ova determination: 24-hour unpreserved urine
Most common fecal contaminant: E. vermicularis
Major constituent of mucus threads: Tamm Horsfall Protein/ Uromodulin
Seen in urine after sexual intercourse: Spermatozoa
CASTS
Formed in the: DCT and CD (unique to the kidney)
Major constituent, it is a glycoprotein produced by the RTE cells of DCT and CD: THP/Uromodulin
Performed along the edges the coverslip with subdued light
With tapered end cast: Cylindroid
Disintegration of Casts: Hyaline→Cellular→Coarsely Granular→Finely Granular→Waxy
Casts Composition/Information Associated Conditions
Hyaline cast Beginning of all types of casts Stress and exercise
(NV: 0-2/LPF) Made up of THP/Uromodulin Glomerulonephritis, Pyelonephritis, CRD,
CHF
Epithelial cast A hyaline cast with trapped RTE cells Glomerulonephritis, Pyelonephritis, Renal
tubular damage
RBC cast A hyaline cast with RBCs and free RBCs Glomerulonephritis, Strenuous exercise
WBC cast White blood cells (generally neutrophils) are present Pyelonephritis (Kidney infection)
within or upon casts
Bacterial cast casts composed of bacteria in a protein (hyaline) matrix Pyelonephritis (Kidney infection)
Coarsely/Finely Granular cast Result either from the degeneration of cellular casts, or Stress and exercise
direct aggregation of plasma proteins or Glomerulonephritis, Pyelonephritis
immunoglobulin light chains
Fatty cast Formed by the breakdown of lipid-rich epithelial cells. Nephrotic syndrome, Toxic tubular
A hyaline cast with OFB. Visualized through Polarizing necrosis, DM, Crush injuries
microscope and staining with ORO/Sudan III
Waxy cast The final stage of degeneration of cellular casts Stasis of urine flow, Renal failure
Chronic Renal disease (CRF)
Broad cast A very large cast, AKA ―Renal Failure Cast‖ Extreme stasis of urine, RF, ESRD
CRYSTALS (Crystalluria)
Factors that contribute to crystal formation: pH, Temperature, Solute concentration (↑CHONs,salts) N°-RFMM/hpf, AbN°-ave/lpf
NORMAL URINARY CRYSTALS
ALKALINE
Crystal Ph Color Characteristic Solubility
C Calcium carbonate ALK Colorless Dumbbell/spherical-shaped Gas from Hac
(Calute/Aragonite)
C Calcium phosphate ALK/NEU Colorless Flat plates, thin prism in rosette form. Dilute Hac
(Apatite) Arranged like sheaves of wheat
(resembling sulfonamide crystals)
A Amorphous phosphates ALK/NEU White-colorless Granular white precipitates Dilute Hac
A Ammonium biurate ALK Yellow-brown Thorny apples Hac w/ heat
T Triple phosphate ALK Colorless Coffin-lid, prism-shaped, feathery, Dilute Hac
(Magnesium ammonium fern leaf
phosphate/Struvite)
 NORMAL URINARY CRYSTALS
ACIDIC
Crystal Ph Color Characteristic Solubility
C Calcium oxalate ACD/NEU/ Colorless Monohyadrate (Whewellite)-oval/dumbbell Dilute HCl
rarely ALK *Associated with Ethylene glycol poisoning
Dihydrate (Wheddelite)-envelope/pyramidal
C Calcium sulfate ACD Colorless Long, thin needles or prisms (identical Hac
to calcium phosphate). Resembles
cigarette butt
H Hippuric acid ACD Colorless/Yello Elongated prism or plates. They may Water, Ether
w-brown be so thin as to resemble needles and
they often cluster together
U Uric acid ACD Yellow-brown Rhombic, four sided plates Alkali
(whetstones),wedges, rosettes, lemon-
shaped
A Amorphous urates ACD Brick Yellow-red granules Alkali and heat
dust/Yellow Pink sediment
ABNORMAL URINARY CRYSTALS
ACIDIC
Crystal Ph Color Characteristic Solubility
B Bilirubin ACD Yellow Clumped needles or granules HAc, HCl, NaOH,
Ether, Chloroform
L Leucine ACD/NEU Yellow/Yellow- Oily spheres/concentric circles with Hot alkali/
brown radial striations Alcohol
T Tyrosine ACD/NEU Colorless- Fine needles in clumps or rosettes Alkali/heat
yellow
C Cholesterol ACD Colorless Rectangular plates with a notch in one Chloroform
or more corners (staircase). Resembles
RCM crystals
R Radiographic dye (RCM) ACD Colorless Flat/slightly notch plates (similar to 10% NaOH
cholesterol)
A Ampicillin ACD/NEU Colorless Needles, Bundles upon refrigeration Acid/Neutral
C Cystine ACD Colorless Hexagonal plates, mistaken as Ammonia, Dilute
uric acid HCl
S Sulfonamide ACD/NEU Colorless to Sheaves of wheat with central binding Acetone
yellow-brown rosettes, arrowheads, petals
(varied)
Test for sulfonamide crystals which uses newspaper: Lignin test (Urine+25% HCl→(+) yellow)
Increased in cases of Lesch-Nyhan syndrome, Chemotherapy and Gout: Uric acid
Seen in urine after taking massive doses of penicillin: Ampicillin crystals
Crystals seen in liver disease: Bilirubin, Leucine, Tyrosine
CYSTINE AND URIC ACID DIFFERENTIATION
Cystine Uric acid
Color Coloress Yellow-brown
Solubility in ammonia Soluble Soluble
Solubility in dilute HCL Soluble Insoluble
Birefringence Not birefringent Birefringent
Cyanide-nitroprusside reaction Positive Negative
URINARY SEDIMENT ARTIFACTS
Fecal contamination Starch granules Air bubbles
Hairs and fibers Oil droplets Pollen grains
Spheres with dimpled center, Maltese cross formation on polarizing microscope: Starch granules
Most common urinary artifacts: Starch granules

OTHER BODY FLUIDS

METHODS OF COLLECTION AND NORMAL VOLUME OF BODY FLUIDS
Body Fluid Method of Collection Normal Volume
Amniotic fluid Amniocentesis 800-1200 mL (3rd trimester)
(up to 30 mL is collected in sterile syringe)
Synovial fluid (joint fluid) Arthrocentesis <3.5 mL (inf.>25mL)
Pleural fluid Thoracentesis <30 mL
Pericardial fluid Pericardiocentesis <50 mL
Peritoneal fluid Paracentesis <100 mL
Gastric fluid Aspiration 20-50 mL fasting specimen
Passed through the nose: Levine
Passed through the mouth: Rehfuss
Seminal fluid -Masturbation (best) 2-5 mL
-Coitus interruptus (withdrawal)
-Condom method (nonlubricant)
CSF Lumbar puncture (Between 3rd,4th or 5th Adults: 90-150 mL (140-170mL)
lumbar vertebrae) up to 20 mL can be collected Neonates: 10-60 mL
Sputum First morning-Routine (most preferred) *Tracheobronchial secretions
24-hour-for volume measurement
Throat swab-for pediatric patient
Sputum induction-for non-cooperative patients
Tracheal aspiration-for debilitated patients
Sweat Gibson and Cooke Pilocarpine Iontophoresis *Sweat Na+ and Cl- values: <40
Diagnosis of Cystic fibrosis/Mucovisidosis Pilocarine + mild current (induce sweat mEq/L
production) 40 mEq/L- Borderline gor CF
>70 mEq/L- Diagnostic for CF
Bronchoalveolar secretions Bronchoalveolar Lavage * Diagnostic test for: Pneumocystis
Cellular milieu of the alveoli + Saline carinii/jiroveci
Predominant cells: Macrophage
 AMNIOTIC FLUID
Amniotic fluid volume is from: fetal urine and lung fluid
Major contributor to the volume after the 1st trimester of pregnancy: fetal urine
Increased amniotic fluid volume caused by decreased fetal swallowing of urine and NTD: Polyhydramnios
Decreased amniotic volume caused by increased fetal swallowing of urine, membrane leakage and urinary tract deformities: Oligohydramnios
Assessment of genetic defects such as Down syndrome: 2nd trimester amniocentesis
Assessment of FLM and HDN: 3rd trimester amniocentesis
Test used to detect ruptured amniotic membranes and to diagnose early pregnancy: Fern test (vaginal fluid→slide, airdry +fern-like crystal)
Amniotic fluid color: Normal →Colorless
Amniotic fluid color: Traumatic tap/abdominal trauma →Blood-streaked
Amniotic fluid color: HDN →Yellow
Amniotic fluid color: Meconium →Dark-green
Amniotic fluid color: Fetal death →Dark red-brown
Amniotic fluid test for HDN: O.D. 450 Results are plotted on a: Liley graph
NTD: there is incomplete closing of the backbone and membranes around the spinal cord →Spina bifida
NTD: absence of major portion of the brain, skull and scalp during embryonic development →Anencephaly
Screening test for NTD: Alpha fetoprotein (AFP) (Increased in NTD, Decreased in Down syndrome)
Confirmatory test for NTD: Acetylcholinesterase
FLM test reference method: Lecithin/Sphingomyelin ratio (mature>2.0 ) (L-alveolar stability, S- serves as control-constant production)
Immunologic test for Phosphatidyl glycerol: Amniostat-FLM (not affected by blood or meconium, delayed prod’n in diabetic mothers)
Test that uses ethanol, production of foam or bubbles indicates positive result: Foam stability/foam/shake test *shake-15s, stand-15mins
Test measured by fluorescence polarization: Microviscosity (presence of phospholipids decreases microviscosity)
Lamellar bodies produced by type II pneumocytes are counted: Lamellar body count (>32 000/uL adequate FLM)
FLM absorbance test: O.D. 650 nm (OD ≥0.150 equivalent to L/S ration ≥2.0)
SYNOVIAL FLUID
Synovial fluid viscosity is due to: Hyaluronic acid (produced by synoviocytes) (normal level:0.3-0.4 g/dL, able to form string 4-6cm long)
Colorless to pale yellow: Normal
Red: Traumatic top, hemorrhagic arthritis
Deeper yellow: Inflammation
Milky: Presence of crystals
Greenish tinge: Bacterial infection
Synovial fluid viscosity test: Ropes/Mucin clot test (Hyaluronate polymerization test) Reagent: 2-5% acetic acid
Grading: good→solid clot; fair→soft clot; low→friable clot; poor→No clot
Characteristic of Synovial Fluid Crystals
Crystal Shape Compensated Polarized Light Significance
Monosodium urate Needles Negative birefringence Gout
Calcium pyrophosphate Rhombic square, rods Positive birefringence Pseudogout
Cholesterol Notched, rhombic plates Negative birefringence Extracellular
Corticosteroid Flat, variable-shaped plates Positive and Negative birefringence Injections
Calcium oxalate Envelopes Negative birefringence Renal dialysis
Apatite (Ca phosphate) Small particles No birefringence Osteoarthritis
Laboratory Findings in Joint Fluid Disorders
Group I IIa IIb III IV
Non-Inflammatory Inflammatory Inflammatory Septic Hemorrhagic
(Immunologic) (Crystal-Induced)
Significance Degenerative joint Immunologic Gout, Pseudogout Microbial infection Traumatic injury
disorder disorders Coagulation
(Osteoarthritis) deficiencies
Color and Clear, yellow Cloudy, yellow Cloudy/milky Cloudy, yellow- Cloudy, red
Clarity green
Viscosity Good Poor Low Variable Low
WBC count <1000/uL 2000-75 000/uL Up to 100 000/uL 50 000-100 000/uL Equal to blood
Neutrophils < 30% > 50% < 70% >75 % Equal to blood
Glucose Similar to blood (N°) Decreased Decreased Decreased Normal
Others + Autoantibodies + Crystals + Culture and GS + RBCs
SEROUS FLUID
Fluid between parietal and visceral membrane: Serous fluid
Accumulation of fluid between the membranes: Effusion (classified as exudate or transudate)
Are usually bilateral owing to systemic conditions leading to ↑capillary hydrostatic pressure or ↓plasma oncotic pressure: Transudate
Are more unilateral associated with localized disorders that ↑vascular permeability or interfere with lymphatic resorption: E xudate
A test that differentiates exudates from transudates: Rivalta’s test (serosamucin clot test) (HAc+water+unk.fluid→Heavy pption=Exudate)
TRANSUDATE VS EXUDATE
Transudate Exudate
Causes CHF, Hypoproteinemia, Hepatic cirrhosis, NS Infection, Malignancy, Inflammation
Appearance Clear Cloudy
Fluid:serum protein ratio < 0.5 > 0.5
Fluid:serum LD ratio < 0.6 > 0.6
WBC count < 1,000/uL > 1,000/uL
Pleural fluid:bilirubin ratio < 0.6 > 0.6
Pleural fluid cholesterol (mg/dL) < 45-60 > 45-60
Pleural fluid:serum cholesterol ratio < 0.3 > 0.3
Serum-ascites albumin gradient (SAAG) > 1.1 < 1.1
Spontaneous clotting No Possible
Glucose Decreased Increased
Rivalta’s test Negative Positive
CHARACTERISTIC FEATURES OF CHYLOUS AND PEUDOCHYLOUS EFFUSIONS
Chylous Pseudochylous
Cause Thoracic duct leakage Chronic inflammation
Onset Sudden Gradual
Appearance Milky white, pale yellow, bloody Milky, greenish, gold paint
Microscopic examination Lymphocytes Mixed cells, cholesterol crystals
Triglyceride >110 mg/dL <50 mg/dL
Sudan III staining (+++) (-)/weakly (+)
Lipoprotein electrophoresis (+) (-)
 BLOODY PLEURAL FUID
Hemothorax (traumatic tap) Hemorrhagic effusion
Blood distribution Uneven Even
Hematocrit Pleural fluid hct is ≥1/2 of the WB hct Pleural fluid hct is <1/2 of the WB hct
GASTRIC FLUID
Normal: pale gray with mucus Abnormal volume (fasting state): >50 mL
With large amount of bile: yellow-green Test meal using oatmeal: Boa’s
With small amount of fresh blood: red Test meal using bread, tea or water: Ewald’s
With large amount of blood: coffee ground Test meal using beef steak and mashed potato: Riegel’s
Normal volume (fasting state): 20-50 mL Most preferred chemical stimulant: Pentagastrin
Total gastric secretion during unstimulated, fasting state: BAO (basal acid output)
Total gastric secretion after gastric stimulation: MAO (maximum acid output)
BAO (mEq/hr) MAO (mEq/hr) BAO/MAO
Normal 2.5 25.0 10%
Pernicious anemia 0 0 0
Duodenal ulcer 5.0 30.0 17%
Zollinger-Ellison syndrome 18.0 25.0 72%
SEMINAL FLUID
Site of spermatogenesis: Seminiferous tubules (testes)
Serve as nurse cells for developing sperm cells: Sertoli cells
Site of sperm maturation: Epididymis
Provide nutrients for sperm, rich in fructose: Seminal fluid (seminal vesicle)
Contains ACP, zinc, citric acid and other enzymes for coagulation and liquefaction: Prostate fluid
Secretes thick alkaline mucus that neutralizes the acidity from the prostate secretions and vagina: Bulbourethral gland
Normal Results/Values
Color: Gray-white, translucent Quality: >2.0 0r a,b,c
Volume: 2-5 mL Morphology: >30% normal forms (routine criteria)
>14% normal forms (Kruger’s strict criteria)
pH: 7.2-8.0 Round cells: <1 million/mL
Viscosity: Pour in droplets (0 watery;4 gel-like) Sperm concentration: 20-160 million/mL
Motility: >50% within 1 hr Sperm count: ≥40 million/ejaculate
Sperm Motility Grading
Grade
4.0 a Rapid, straight 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
Uses heat to immobilize sperm cells, used for undiluted specimen: Makler counting chamber
*Improve Neubauer counting chamber- Dilution-1:20, Diluents-formalin, sodium bicarbonate,saline, distilled water, tap water (cold)
Stains used for sperm morphology assessment: Papanicolau’s stain, Wright, Giemsa
Test for sperm viability: Modified Bloom’s test (Rgts: Eosin and Nigrosin, unstained/bluish white-Living, Red-dead sperms)
Test for antisperm antibodies: Mixed agglutination reaction (IgG <10% Motile sperm-attch to particles), Immunobead test (IgG, IgM, IgA)
Test for seminal fluid fructose: Resorcinol/Seliwanoff (tested w/in 2 hours/frozen to avoid fructolysis)
Test for detection of semen: Florence test (choline, iodine crystals, KI, dark brown rhombic crys), Barbiero’s (spermin, SPA, TCA, yellow leaf-like crys)
Head: 5umx3um (LxW)
Acrosomal cap: 1/2 of head, 2/3 of nucleus
Tail: 45 um
Midpiece: contains mitochondria

Most common cause of male infertility: Varicocele (hardening of veins that drain the testes, tapered head sperm)
Complete lack of semen with ejaculation: aspermia Presence of immotile/dead sperm cells: Necrospermia
Lack of sperm cells in the semen: azoospermia Low sperm concentration or sperm count: Oligospermia
CSF
Specific part of the brain that produces CSF: Choroid plexus Rate of CSF production: 20mL/hr
Protects the brain from chemicals and other substances circulating in the blood: Blood Brain Barrier
Order of draw (3 CSF tubes): 1-Chemistry (frozen), 2-Microbiology(RT), 3-Hematology (Ref), *4-Micro/Sero
If 1 CSF tube only: Micro-Hema-Chem/Sero
TRAUMATIC TAP VS INTRACRANEAL HEMORRHAGE CSF DILUTION (DF: 3%HAc w/MB)
Traumatic tap Intracraneal hemorrhage Appearance Dilution
Blood distribution on 3 tubes Uneven (1>2>3) Even (1=2=3) Clear Undiluted
Clot formation (+) (-) Slightly hazy 1:10
Supernatant Clear Xanthochromic Hazy 1:20
Erythrophages (-) (+) Slightly cloudy 1:100
D-dimer (-) (+) Cloudy/Slightly bloody 1:200
Bloody/Turbid 1:10 000
Predominant cells in CSF: Lymphocyte and Monocyte (Adults:70%L:30%M, Neonates inverse ratio up to 80%M)
Increased number of normal cells in CSF: Pleocytosis
Major CSF protein: albumin
2nd most prevalent protein: Prealbumin
Carbohydrate deficient transferring found only in CSF: Tau transferrin (B2)
The presence of 2 or more oligoclonal bands in CSF but not in serum is valuable diagnosis for: Multiple sclerosis (demyelinat ing disorder)
Used to monitor the course of MS: Myelin Basic Protein (MBP) lipid protein complex that insulate nerves, destruction of myeli n sheath
Preferred method for CSF protein determination: TCA (3% ) (ppts both albumin and globulin)
Precipitates albumin only, to precipitate globulin add sodium sulfate: SSA (3%)
Indirect test for the presence of excess ammonia in CSF: Glutamine Increased seen in: Reye’s syndrome (disturbance in consciousness)
NORMAL CSF RESULTS
Appearance: crystal clear Protein Adults: 15-45 mg/dL Glucose: 60-70% of blood glucose (50-80mg/dL)
WBC count Adults: 0-5 WBCs/uL Infants: 150 mg/dL Lactate: 10-22 mg/dL
Neonates: 0-30 WBCs/uL Immature: 500 mg/dL Glutamine: 8-18 mg/dL
CSF/Serum albumin index: <9 IgG index: <0.77 CSF LDH: LD1>2>3>4>5
 DIFFERENTIAL DIAGNOSIS OF MENINGITIS
Bacterial Viral Tubercular Fungal
Predominant WBC ↑Neutrophils ↑Lymphocyte ↑Lymphocytes and ↑ Lymphocytes and
Monocytes Monocytes
Protein ↑ ↑ ↑ ↑
Glucose ↓ N° ↓ ↓
Lactate ↑ N° ↑ ↑
Others (+) Culture Agents: Enteroviruses Agent: MTB Agent: C. neoformans
(+) GS Coxsackievirus (+) AFB (+) GS (Classic starburst
(+) Limulus Lysate test Echovirus (+) pellicle/weblike clot pattern)
Poliovirus formation after (+) India ink
refrigeration (+) Immunologic test
SPUTUM
Odorless: normal Fecal: liver abscess, enteric G (-) infection
Foul/putrid: cavitary TB, lung abscess, lung gangrene Cheesy: necrosis, tumors, empyema
Colorless/translucent: normal Olive green/grass green: lung carcinoma
Greenish: P. aeroginosa infection Currant, jelly-like: K. pneumonia infection
Anchovy sauce/rusty brown: Lobar pneumonia Rusty w/ pus: Lobar pneumonia
Prune juice: pneumonia, chronic lung cancer Rusty w/o pus: Congestive heart failure
Macroscopic structures Microscopic structures
*Dittrich’s plugs Yellow/gray material, pinhead size *Charcot-Leyden crystals From disintegration of eosinophils
Lungstones Hard concretions in a bronchus *Curschmann spiral Coiled mucus strands
Bronchial cast Branching tree-like cast of the brochi *Creola bodies Cluster of columnar cells
Foreign bodies Bronchial calculi, asbestos, silica particles Pigmented cells Heart failure cells (hemosiderin-laden MAC)
Myelin globules Colorless globules mistaken as Blastomyces
*seen in Bronchial asthma
FECALYSIS
Quantity of stool passed per day: 100-200g
Macroscopic Stool Characteristics
Brown Normal
Black (Melena) Upper GI bleeding, iron, charcoal, bismuth
Red (Hematochezia) Lower GI bleeding, beets, rifampin, food color.
Pale yellow, white, gray Bile duct obs, barium sulfate
Green Biliverdin, oral antibiotics, green vegetables
Blue Prussian blue, grape soda
Violet/purple Porphyria
Bulky/frothy Bile duct obs, pancreatic disorders, steatorrhea
Butter-like Cystic fibrosis
Mucus, blood-streaked mucus Colitis, dysentery, malignancy, constipation
Ribbon-like Intestinal constriction
Rice watery Cholera
Pea-soup Typhoid
Scybalous (Goat droppings) Constipation
Increased fats in stool: Steatorrhea (>6g/day)
Gold standard for fecal fat determination: Van de Kamer titration (NaOH) (sample 3-day/72-hr stool, N° 1-6g/day, S>6g/day)
Abnormal excretion of muscle fibers in feces: Creatorrhea
Neutrophil count that indicates invasive condition: ≥3 neutrophils/hpf
Screening test for colorectal cancer: Fecal Occult Blood Test (occult-hidden, Significant ≥2.5 mL blood/150g stool)
Principle of FOBT: Pseudoperoxidase activity of hemoglobin
Test that differentiates fetal blood and maternal blood: Apt test (Apt-Downey test) specimen-fetal stool/vomitus. Reagent: 1% NaOH,
Positive: Pink color (fetal blood-HbF), Negative: yellow brown supernatant (maternal blood-HbA)

METABOLIC DISORDERS
Phenylalanine-Tyrosine Disorders
Disorder Tests Positive results
Phenylketonuria FeCl3 tube test Blue-green
Lacks gene that codes for Phenylalanine hydroxylase Phenistix strip Gray to gray-green
Mousy odor urine
Guthrie bacterial inhibition test B. subtilis growth
Severe mental retardation
Confirmatory: Ion exchange HPLC
Tyrosiluria/Tyrosinemia FeCl3 tube test Transient green
Rancid butter odor urine Nitroso-naphthol Orange-red
Confirmatory: Chromatography, serum assay
Alkaptonuria FeCl3 tube test Transient blue
Lacks gene that codes for Homogentisic acid oxidase Clinitest Yellow precipitate
Urine darkens after alkalinization from standing at RT Confirmatory: Paper/Thin layer chromatography,
capillary electrophoresis
Melanuria FeCl3 tube test Gray/black precipitate
Overproliferation of melanocytes Sodium nitroprusside test Red
Urine darkens upon air exposure Ehrlich’s test Red
Branched-Chain Amino Acid Disorders
Maple Syrup Urine Disease 2,4-dinitrophenylhydrazine (DNPH) test Yellow
↑Leucine, Isoleucine and Valine in blood & urine turbidity/precipitate
Caramelized sugar/Maple syrup urine odor
Organic acidemias: Isovaleric academia
Propionic academia
Methylmalonic acidemia p-nitroaniline test Emerald green
Tryptophan Disorders
Indicanuria Obermeyer’s test Violet
Indigo color urine after air exposure
Seen in intestinal disorders and Hartnup disease
Argentaffinoma FeCl3 tube test Blue-green
Carcinoid tumor of argentaffin/enterochromaffin cells Nitroso-naphthol w/ nitrous acid Violet
Production of 5-HIAA (hydroxyindoleacetic acid)
 Cystine Disorders
Cystinuria (defective tubular reabsorption of Cystine, Brand’s Modification of Legal’s nitroprusside Red-purple
Ornithin, Lysine and Arginine)
Cystinosis (Lacks gene that codes for an enzyme Brand’s Modification of Legal’s nitroprusside Red-purple
responsible for cystine metabolism)
Homocystinuria Silver-nitroprusside test Red
(Lacks gene that codes for cystathione B-synthase)
Mucopolysaccharide Disorders (Mucopolysaccharidosis)
Hurler syndrome (skeletal abn & mental retardation, Acid albumin test White turbidity
MPSs accumulate in cornea) Cetyltrimethylammonium bromide (CTAB) test White turbidity
Hunter syndrome (skeletal abn & mental retardation) Mucopolysaccharide (MPS) paper test Blue
Sanfilippo syndrome (mental retardation)
Purine Disorders
Lesch-Nyhan disease →Lacks gene that codes for Hypoxanthine-guanine phosphoribosyltransferase (HPRT)
Orange sand in diapers, ↑uric acid in blood and urine

Porphyrin Disorders (Porphyrias)

Disorder
ALA hydratase deficiency porphyria
Acute intermittent porphyria
Congenital erythropoietic porphyria
Porphyria cutanea tarda
Hereditary coproporphyria
Variegate porphyria
Lacks gene that codes for
Aminolevulinic (ALA) synthetase
Uroporphyrinogen synthase
Uroporphyrinogen cosynthase
Uroporphyrinogen decarboxylase
Coproporphyrinogen oxidase
Protoporphyrinogen oxidase
Ehrlich’s reaction
Detects D-ALA, porphobilinogen
Fluorescence at 550-600 nm Violet/Pink/Red
Test for uroporphyrin, coproporphyrin
and protoporphyrin
Free Erythrocyte Protoporphyrin
*recommended by CDC for lead
poisoning

“For I know the plans I have for you,” declares the LORD, “plans to prosper you
and not to harm you, plans to give you hope and a future.”
Jeremiah 29:11