CLINICAL

MICROSCOPY
M EDICAL T ECHNOL OG Y ASSE S SM E NT PROGRAM (MTAP 1)
KRIST INA MARIA R. PETAL CO RI N, RMT, DTA, MSMT ( c)
 URINE FORMATION

PHYSICAL EXAMINATION OF URINE

TOPIC CHEMICAL EXAMINATION OF URINE

OUTLINE: MICROSCOPIC EXAMINATION OF URINE

RENAL DISEASES

OTHER BODY FLUIDS

RENAL FUNCTIONS:
Glomerular Filtration

Tubular Reabsorption

Tubular Secretion
RENAL BLOOD FLOW:
Renal artery supplies blood to the kidneys.

Receives 25% of blood

Blood enters the capillaries of the nephron though:

1. Afferent arterioles
2. Glomerulus
3. Efferent arterioles
GLOMERULAR FILTRATION:
The glomerulus function as a filter.

Nonselective filter of plasma substances.

Factors affecting the filtration process:

• Cellular structure of capillary walls and Bowman’s capsule.
• Hydrostatic and Oncotic pressure
• RAAS (Renin-Angiotensin-Aldosterone System)
Glomeruli receives blood through afferent
arterioles and an ultrafiltrate of plasma
passes through each glomerulus into
Bowman’s space.

The filtrate is passed through the tubules

and collecting ducts were reabsorption/
secretion of substances and urine occurs.
 TUBULAR REABSORPTION:
Plasma ultrafiltrate enters the Proximal Convoluted Tubule,
the nephrons will begin reabsorbing the essential
substances and water.

They are transported by these cellular transport:

ACTIVE TRANSPORT PASSIVE TRANSPORT
 CELLULAR TRANSPORT
MECHANISM:
1. ACTIVE TRANSPORT • Glucose • PCT
• Movement of a substance • AA
across a cell membranes • Salts
into the bloodstream by • Na • PCT and DCT
electrochemical energy. • Cl • A. Loop of Henle
2. PASSIVE TRANSPORT • Water • PCT, D. Loop of Henle, CD
• Movement of molecules • Urea • PCT, A. Loop of Henle
across a membrane by • Sodium • A. Loop of Henle
diffusion due to a physical
gradient.
TUBULAR SECRETION:
The passage of substances from blood in the peritubular
capillaries to the tubular filtrate.

Two major functions:

1.Eliminating waste products not filtered by the glomerulus.
2.Regulating the acid-base balance in the body through secretion
of H+ ions.
 URINE
RENAL FUNCTION TESTS
1. GLOMERULAR FILTRATION TEST

• Measure the rate at which the kidneys can remove a filterable

substance from the blood.
• Earliest GF test measured is Urea.
• Inulin was the original reference method.
• Clearance tests: Creatinine, 𝛽2- Microglobulin, Cystatin C and
radioisotopes
• Greatest source of error: Improperly timed urine spx.
CREATININE CLEARANCE:

• Urine Creatinine (U), Plasma Creatinine (P), Urine Volume (V).

• Formula:
1. UV/P
2. UV/ P x 1.73/ A
• Normal Creatinine Clearance = 120 mL/ min.
• Male = 107 to 139 mL/ min.
• Female = 87 to 107 mL/ min,
CYSTATIN C:

• Good procedure for screening and monitoring GFR.

• Readily filtered by the glomerulus and reabsorbed and broken
down by renal tubular cells.
• Recommended:
1. Patients with DM
2. Elderly
3. Critically ill patients
BETA-2-MICROGLOBULIN:

• Dissociates from HLAs and removed from plasma by GF.

• Rise in plasma levels shows a more sensitive indicator of
 in GFR than CC.
• Not reliable in patients with history of immunologic
disorders or malignancy.
2. TUBULAR REABSORPTION TEST

• The first function affected by renal disease.

• Specific Gravity Measurement
▪ Most useful as screening procedure.
▪ Quantitative measurement of renal conc. ability is best assessed
through Osmometry.
• Osmolality – Performed for a more accurate evaluation of renal
concentrating ability.
▪ Reported in milliosmoles (mOsm).
OSMOLALITY:

1. FREEZING POINT DEPRESSION

• One mole of a nonionizing substance dissolved in 1kg of water will
lower the freezing point 1.86℃.
• Volatile substances such as alcohol can interfere.
2. VAPOR PRESSURE DEPRESSION
• Actual measurement is the dew point (temperature at which vapor
condenses to a liquid) of the urine sample.
• No interference from volatile substances.
2. TUBULAR REABSORPTION TEST

• Obsolete Tests:
1. Fishberg Test – Patients were deprived of fluids for 24 hours
before measuring the specific gravity (SG 1.026 or higher).
2. Mosenthal Test – Compare the specific gravity and volume of
the day and night urine samples to evaluate concentrating ability.
3. TUBULAR SECRETION AND RENAL BLOOD FLOW

• Tests to measure tubular secretion of nonfiltered substances and

renal blood flow are closely related.
• Total renal blood flow through the nephron must be measured by
a substance that is secreted rather than filtered through the
glomerulus.
• Historically, PSP (Phenolsulfonphthalein) is used.
• Most common test: PAH (𝝆-aminohippuric acid).
URINE COMPOSITION
• 95% Water and 5% Solutes
COMPONENT: COMPOSITION:
Urea • Primary organic component.
• Product of CHON and AA metabolism.
Creatinine • Product of muscle metabolism.
Uric acid • Product of NA breakdown.
Chloride • Primary inorganic component.
• Found in combination of Sodium.
Sodium • Salt
Potassium • Combined with Chloride
Phosphate • Combines with Na to buffer blood.
Ammonium • Regulates blood and tissue fluid acidity.
Calcium • Combines with Cl, Sulfate and Phosphate.
 SPECIMEN
COLLECTION
SPECIMEN CONTAINERS:
Clean, dry, leak-proof and screw-top lids.
Disposable containers must be used.
Wide mouth and has a flat-bottom.
Clear material (color and clarity).
Capacity 50 mL
• 12 mL – Microscopic analysis, for
repeat analysis
 SPECIMEN LABEL:
Patient’s name and Identification number
Date and time of collection
Age and gender
Location
Healthcare provider’s name
Note: Labels must be attached to the body of the container not to the lid.
 SPECIMEN REJECTION:
Specimens in unlabeled containers.
Nonmatching labels and requisition forms.
Specimens contaminated with feces or toilet paper.
Containers with contaminated exteriors.
Specimens of insufficient quantity.
Specimens that have been improperly transported.
METHODS OF
COLLECTION
 TYPE OF SPECIMEN: PURPOSE:
1. Random Specimen • Routine screening
2. First Morning Specimen • Routine screening, Pregnancy test and
Orthostatic Protein
3. 24-hour/ Timed Specimen • Quantitative chemical tests
4. Catheterized Specimen • Bacterial culture
5. Midstream Clean catch • Routine screening, bacterial culture
6. Suprapubic Aspiration • Bladder urine for bacterial culture, Cytology
7. Three-glass Technique • Prostatic infection
8. Pediatric Specimen • Acquired by catheterization/ aspiration
9. Drug Specimen Collection • Drug testing
 URINE
PRESERVATIVES
 URINE PRESERVATION:
Urine specimens must be delivered and tested in the laboratory within 2 hrs.

PHYSICAL:
• Refrigeration
• Freezing/ ice
CHEMICAL:
1. Formalin 6. Phenol
2. Thymol 7. HCL
3. Boric acid 8. Sulfuric acid
4. Toluene 9. Saccamano’s fixative
5. Sodium Fluoride/ Benzoic acid
CHANGES IN UNPRESERVED URINE:

1. Color – modified/ darkened 7. Bilirubin - decreased

2. Clarity - decreased 8. Urobilinogen - decreased
3. Odor - increased 9. Nitrites - increased
4. pH - increased 10. RBC/ WBC - decreased
5. Glucose - decreased 11. Bacteria – increased
6. Ketones – decreased 12. Proteins – least affected
URINALYSIS
PHYSICAL EXAMINATION
(URINE VOLUME)
NORMAL URINE DAILY OUTPUT:
• 1200 to 1500 mL/ 600 to 2000 mL

OLIGURIA – Decrease in urine output

• Adult: <400 mL/day; Children: <0.5 mL/kg/hr; Infants: <1 mL/kg/hr

ANURIA – Cessation of urine flow

NOCTURIA – Increase in nocturnal secretion of urine

POLYURIA – Increase in daily urine volume

• Diabetes Mellitus – Glucose excretion
• Diabetes Insipidus – ADH dysfunction
COLOR
 URINE COLOR
• Roughly indicates the degree of hydration and correlate with urine
specific gravity.
• Normal urine color: Colorless to deep yellow
• Care should be taken to examine the specimen under good light
source, looking down through the container against a white
background.
PIGMENTS:
Urochrome Yellow
Uroerythrin Pink
Urobilin Dark yellow, orange
 ABNORMAL URINE COLOR:
Colorless • Recent fluid consumption
Pale yellow • Polyuria
• Diabetes Mellitus/ Diabetes Insipidus
Dark yellow • Concentrated specimen
Amber • Bilirubin (yellow foam when shaken)
Orange • Acriflavine, Pyridium, Nitrofurantoin, Phenindione
Yellow-green, yellow-brown • Bilirubin oxidized to Biliverdin
Green • Pseudomonas infection
Blue-green • Clorets, Indican, Methylene blue, Phenol
Pink • RBCs
Red • Hemoglobin, Myoglobin, Porphyrins, Beets, Rifampin and
menstrual contamination
Brown • RBCs oxidized to Methemoglobin
Black • Homogentisic acid, Melanin, Methyldopa, Flagyl
ABNORMAL URINE COLOR:
• A purple staining may occur in catheter bags and is caused by
indican in the urine or a bacterial infection, it is frequently caused by
Klebsiella/ Providencia spp.
• Port wine urine
1. Porphyrins
• Brown/ Black urine
1. Melanoma (malignancy)
2. Alkaptonuria – Homogentisic acid
3. Methemoglobin
 CLARITY AND
TRANSPARENCY
URINE CLARITY
• Refers to the transparency or turbidity of a urine sample.
• Specimen should be in a clear container.
• Clarity is determined by visually examining the mixed specimen while
holding it in front of a light source.
• Freshly voided urine is usually clear.
• A. phosphates and carbonates may cause white cloudiness.
• Faint cloud in urine after standing due to WBCs, epithelial cells and mucus
is called Nubecula.
CLARITY: DESCRIPTION:
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 be clotted.
 PATHOLOGIC CAUSES NONPATHOLOGIC CAUSES
OF URINE TURBIDITY: OF URINE TURBIDITY:
RBCs Squamous epithelial cells
WBCs Mucus
Bacteria Amorphous crystals, carbonates
Yeast Semen, spermatozoa
Nonsquamous epithelial cells Fecal contamination
Abnormal crystals Radiographic contrast media
Lymph fluid Talcum powder
Lipids Vaginal cream
 Acidic urine Amorphous urates, radiographic contrast media
Alkaline urine Amorphous phosphates, carbonates
Soluble with heat Amorphous urates, uric acid crystals
Soluble in dilute Acetic acid RBCs, Amorphous phosphates, carbonates
Insoluble in dilute Acetic acid WBCs, bacteria, yeast, spermatozoa
Soluble in Ether Lipids, Lymphatic fluid, Chyle

LABORATORY CORRELATIONS
IN URINE TURBIDITY:
SPECIFIC
GRAVITY
• Density of solution compared with density of similar volume of
distilled water at same temperature.
• Influence by number and size of particles in solution.
• Normal random specimens: SG = 1.002 to 1.035
• Specimens measuring lower than 1.002 are not urine.
• Isosthenuric – urine specific gravity is 1.010
• Hyposthenuric – below 1.010
• Hypersthenuric – above 1.010
• Specific gravity of plasma filtrate entering the glomerulus is 1.010.
• Determination of Specific Gravity:
1. Refractometry
2. Urinometry
3. Reagent strip
4. Harmonic Oscillation Densitometry
REFRACTOMETRY
• Principle: Refractive Index
• Temperature is compensated between 15°C and 38°C.
• Requires corrections for Glucose and Protein:
1. 1 g/dL Glucose: - 0.004
2. 1 g/dL Protein: - 0.003
• Calibration:
1. Distilled water: 1.000
2. 5% NaCl: 1.022 ± 0.001
3. 9% Sucrose: 1.034 ± 0.001
REAGENT STRIPS
PRINCIPLE: pKa change of a polyelectrolyte

RANDOM SPECIMENS:

• 1.003 to 1.030

SIGNIFICANCE:

• Indication of kidney’s concentrating ability and state of

hydration
•  in DM due to Glucose
•  in DI due to ADH dysfunction
HARMONIC OSCILLATION DENSITOMETRY
Frequency of sound wave entering a solution will changes
in proportion to the density of the solution.

Originally used in early automated urinalysis instruments.

 METHOD: PRINCIPLE:
Urinometry Density
Refractometry Refractive Index (RI)
Harmonic Oscillation Densitometry Density
Reagent strips pKa change of polyelectrolyte

SUMMARY OF SPECIFIC GRAVITY

MEASUREMENTS:
 URINE pH
NORMAL: pH 4.5 to 8.0 (random)
FIRST MORNING SPECIMEN: Slightly acidic (pH 5.0 to 6.0).
More alkaline pH is found following meals (Alkaline tide).
ACID URINE ALKALINE URINE
Emphysema, Diabetes Mellitus Hyperventilation, vomiting
Starvation, Dehydration, Diarrhea Renal tubular acidosis
Presence of acid-producing bacteria Presence of Urease-producing bacteria
High protein diet, cranberry juice Vegetarian diet
Medications Old specimens
 pH REAGENT
STRIP

pH REAGENT STRIP SUMMARY

Reagents: • Methyl Red, Bromthymol Blue
Sensitivity: • pH 5 – 9 • Principle: Double indicator
Sources of error/• No known interfering subs. system
Interference: • Runover from adjacent pads. 1. Methyl Red
• Old specimens (falsely alkaline) 2. Bromthymol blue
Correlation with • Nitrite, leukocytes
other tests: • Microscopic examination
ODOR
 AMMONIACAL Infection (Urea → Ammonia), UTI
FRUITY, SWEET Ketones (DM, starvation, vomiting)
ROTTING FISH Trimethylaminuria
RANCID Tyrosinemia, Tyrosiluria
URINE SWEATY FEET Isovaleric Acidemia

ODOR MOUSY Phenylketonuria

CABBAGE Methionine Malabsorption
Normal: MAPLE SYRUP Maple Syrup Urine Disease
• Aromatic BLEACH Contamination
ODORLESS Acute Tubular Necrosis
 URINALYSIS
CHEMICAL EXAMINATION
(REAGENT STRIPS)
PROTEIN
• Most indicative of renal disease.
• Normal: Negative or Trace (<10 mg/dL)
• Albumin – major serum protein found in urine.
• Other proteins include small amount of serum and tubular microglobulins:
1. Tamm-Horsfall (Uromodulin) – produced by renal epithelial cells,
produced in DCT.
2. Proteins from prostatic, seminal and vaginal secretions
PRE-RENAL PROTEINURIA:
• Not indicative of an actual renal disease.
• Conditions:
1. Intravascular hemolysis
2. Muscle injury
3. Severe infection and inflammation
4. Multiple Myeloma
▪ Bence Jones Protein – coagulates at temperature between 40 to
60℃ and dissolves when temperature reaches 100℃.
 RENAL PROTEINURIA:
GLOMERULAR DISORDERS
• CONDITIONS:
1. Immune Complex disorders, Amyloidosis
2. Toxic agents, strenuous exercise, Dehydration
3. Hypertension, Pre-eclampsia, Orthostatic Proteinuria
4. Diabetic Nephropathy – Microalbumin is considered as significant when 30
to 300mg of Albumin is excreted in 24 hours.
MICRAL TEST
Principle: Enzyme Immunoassay
Sensitivity: 0 to 10 mg/dL
Reagents: Gold-labeled Ab, 𝛽-Galactosidase, chlorophenol red galactoside
Interference: False negative: Dilute urine
 RENAL PROTEINURIA:
TUBULAR DISORDERS
• Conditions:
1. Exposure to toxic substances/ heavy materials
2. Severe viral infections
3. Fanconi’s Syndrome

POST-RENAL PROTEINURIA:
• Conditions:
1. Lower UTI/ inflammations
2. Injury/ trauma
3. Menstrual contamination, vaginal secretions
4. Prostatic fluid/ spermatozoa
 PROTEIN REAGENT STRIP SUMMARY
PRINCIPLE: • Protein error of indicator
SENSITIVITY: • Multistix: 15 to 30 mg/dL Albumin
• Chemstrip: 6 mg/dL Albumin

INTERFERENCE: FALSE POSITIVE FALSE NEGATIVE

• Highly buffered alkaline urine • Proteins other than Albumin
• Pigmented specimens
• Detergents
• Antiseptics, Chlorohexidine
• Loss of buffer from prolonged exposure to
reagent strip to the specimen.
• High specific gravity

CORRELATIONS: Blood, Nitrite, Leukocytes, Microscopic

 SULFOSALICYLIC ACID
PRECIPITATION TEST:
Cold precipitation test that reacts equally with all forms of proteins.

Procedures:
1. Add 3 mL of 3% SSA reagent to 3 mL of centrifuged urine.
2. Mix by inversion and observe for cloudiness.
3. Grade the degree of turbidity.
GRADE TURBIDITY PROTEIN RANGE
(mg/dL)
Negative No increase in turbidity <6
Trace Noticeable turbidity 6 – 30
1+ Distinct turbidity with no 30 – 100
granulation
2+ Turbidity, granulation 100 – 200
with no flocculation
3+ Turbidity, granulation, 200 – 400
flocculation
4+ Clumps of protein >400
GLUCOSE
Most frequently performed in urine analysis.
Renal threshold: 160 to 180 mg/dL
Other sugars in urine:
• Fructose
• Galactose
• Lactose
• Pentose
 CLINICAL SIGNIFICANCE:
HYPERGLYCEMIA- RENAL-ASSOCIATED:
ASSOCIATED:
Diabetes Mellitus, Pancreatic Fanconi Syndrome
cancer
Cushing’s Syndrome, Advanced renal disease
Pheochromocytoma
CNS damage, Pancreatitis, Osteomalacia
Acromegaly
Hyperthyroidism, Stress, GDM Pregnancy
REAGENT STRIP:
• Principle: Double sequential enzyme reaction
REAGENTS: CHROMOGENS:
1. Glucose oxidase 1. O-toluidine (pink – purple)
2. Peroxidase 2. Potassium Iodide (blue – brown)
3. Aminopropyl-Carbazol (yellow – orange brown)

• CHEMICAL REACTION:
 GLUCOSE REAGENT
STRIP SUMMARY:
Reagents: • Multistix: Glucose oxidase, Peroxidase Potassium iodide
• Chemstrip: Glucose oxidase, Peroxidase, Tetramethylbenzidine
Sensitivity: • Multistix: 75 to 125mg/dL
• Chemstrip: 40 mg/dL

Interference: FALSE POSITIVE: FALSE NEGATIVE:

• Contamination of oxidizing agents • High levels of ascorbic acid
• High levels of ketones
• High specific gravity
• Low temperatures
• Improperly preserved specimens
Correlations: • Ketones
 COPPER REDUCTION TEST:
Ability of Glucose and other substances to reduce Copper Sulfate to
Cuprous Oxide in the presence of alkali and heat.

A color change progressing from a negative blue (CuSO4) through green,

yellow and orange/ red (Cu2O) occurs when reaction takes place.

CHEMICAL REACTION:
 COPPER REDUCTION TEST:
• Tablets contain: CuSO4, Sodium Carbonates, Sodium Citrate and NaOH.
• GLUCOSE OXIDASE AND CLINITEST REACTIONS:

GLUCOSE OXIDASE: CLINITEST: INTERPRETATION:

(-) (+) • Non-glucose substance is present.

(+) (-) • Small amount of glucose present.

(-) (-) • No glucose is present.

(+) (+) • Glucose is present.

KETONES
Results from increased fat metabolism due to inability to metabolize
carbohydrates (DM).
Increased loss of carbohydrate from vomiting and inadequate intake of
carbohydrates associated with starvation and malabsorption.

Immediate products of fat metabolism:

• 78% BHA
• 20% Acetoacetic acid
• 2% Acetone
 CLINICAL SIGNIFICANCE:
Diabetes acidosis
Insulin Dosage monitoring
Starvation
Malabsorption/ pancreatic disorders
Strenuous exercise
Vomiting
Inborn errors of amino acid metabolism
REAGENT STRIP:
PRINCIPLE: Sodium Nitroprusside reaction
• Note: This cannot detect BHA and only sensitive to AAA. But if
Glycine is added, it is slightly sensitive to Acetone.
CHEMICAL REACTION:
REAGENTS: • Sodium Nitroprusside
• Chemstrip: Glycine
SENSITIVITY: • Multistix: 5 to 10 mg/dL Acetoacetic acid
• Chemstrip: 9 mg/dL Acetoacetic acid; 70 mg/dL Acetone
INTERFERENCE: FALSE POSITIVE: FALSE NEGATIVE:
• Phthalein dyes • Improperly preserved
• Highly pigmented red urine specimens
• Levodopa
• Medications containing free
sulfhydryl groups

CORRELATIONS: • Glucose

• Acetest tablets: Na Nitroprusside, Glycine, Disodium Phosphate and

Lactose (better color differentiation); hygroscopic.
 BLOOD
HEMATURIA • Cloudy red urine • Renal calculi,
• Microscopic: Intact RBCs Glomerulonephritis,
Pyelonephritis, Tumors,
Trauma...
HEMOGLOBINURIA • Clear red urine • Transfusion reactions,
Hemolytic anemias, severe
burns....
MYOGLOBINURIA • Clear red urine • Muscular trauma/ crush
syndromes, convulsions,
Rhabdomyolysis...
• Heme portion of Myoglobin
is toxic to the renal tubules
causing acute renal failure.
HEMOGLOBINURIA VS. MYOGLOBINURIA
• 1. PLASMA EXAMINATION:
HEMOGLOBIN Clear red urine Red/ pink plasma ↓ Haptoglobin Intravascular
Hemolysis
MYOGLOBIN Clear red urine Normal plasma ↑ CK, Aldolase Rhabdomyolysis

• 2. BLONDHEIM’S TEST (AMMONIUM SULFATE)

▪ 2.8g (NH₄)₂SO₄+ 5mL centri. urine → (NH₄)₂SO₄ ppts. Hgb.
HEMOGLOBIN Clear supernatant fluid SF (-) blood Red ppt.

MYOGLOBIN Red supernatant fluid SF (+) No ppt.

REAGENT STRIP:
• PRINCIPLE: Pseudoperoxidase activity of Hemoglobin
• Chromogen: Tetramethylbenzidine
• CHEMICAL REACTION:
 • Multistix: Diisopropylbenzene dihydroperoxide and 3,3’,5,5’- tetramethylbenzidine
REAGENTS: • Chemstrip: dimethyldihydroperoxyhexane and tetramethylbenzidine

• Multistix: 5 to 20 RBCs/mL, 0.015 to 0.062 mg/dL hemoglobin

SENSITIVITY: • Chemstrip: 5 RBCs/mL, hemoglobin corresponding to 10 RBCs/mL

INTERFERENCE: FALSE POSITIVE: FALSE NEGATIVE:

• Strong oxidizing agents • High specific
• Bacterial peroxidases gravity/crenated cells
• Formalin, Captopril, High
• Menstrual contamination
concentrations of nitrite
Ascorbic acid greater than
25 mg/dL
• Unmixed specimens
CORRELATIONS: • Protein
• Microscopic
BILIRUBIN
Early indication of Liver disease

Only Conjugated Bilirubin is excreted in the urine.

Significance:
• Hepatitis
• Cirrhosis
• Billiary obstruction (gallstones/ carcinoma)
• Other hepatic disorders
REAGENT STRIP:
PRINCIPLE: Diazo reaction
• 2,4-dichloroaniline diazonium salt
• 2,6-dichlorobenzene-diazonium-tetrafluoroborate
CHEMICAL REACTION:
REAGENTS: • Multistix: 2,4-dichloroaniline diazonium salt
• Chemstrip: 2,6-dichlorobenzene-diazonium salt
SENSITIVITY: • Multistix: 0.4 to 0.8 mg/dL bilirubin
• Chemstrip: 0.5 mg/dL bilirubin
INTERFERENCE: FALSE POSITIVE: FALSE NEGATIVE:
• Highly pigmented urines, • Specimen exposure to light
Phenazopyridine • Ascorbic acid greater than
• Indican (intestinal 25 mg/dL
disorders) • High concentrations of
• Metabolites of Lodine nitrite

CORRELATIONS: • Urobilinogen
ICTOTEST TABLET:

• Confirmatory test; more sensitive

• Contains:
1. 𝜌-nitrobenzene-diazonium-p-
toluenesulfonate
2. SSA
3. Sodium carbonate
4. Boric acid
• Positive result: Blue to purple
UROBILINOGEN
• Bile pigment that resulted from hemoglobin degradation.
• Small amount present in urine: 1mg/dL or 1 Ehrlich unit
• Clinical Significance:
1. Early detection of liver disease
2. Liver disorders
3. Hepatitis
4. Cirrhosis
5. Carcinoma
6. Hemolytic disorders
REAGENT STRIP:

• PRINCIPLE: Ehrlich reaction

• CHEMICAL REACTION:
REAGENTS: • Multistix: PDAB
• Chemstrip: 4-methoxybenzene-diazonium tetrafluoroborate

SENSITIVITY: • Multistix: 0.2 mg/dL urobilinogen

• Chemstrip: 0.4 mg/dL urobilinogen

INTERFERENCE: FALSE POSITIVE: FALSE NEGATIVE:

(Multistix) • Porphobilinogen, Indican,• Old specimens and
Sulfonamides, Methyldopa, formalin preservation
Procaine...
(Chemstrip) • Highly pigmented urine • Old specimens, formalin
preservation and high
conc. of Nitrates
CORRELATIONS: • Bilirubin
 WATSON-SCHWARTZ TEST:
• For differentiating Urobilinogen and Porphobilinogen.
OTHER EHRLICH-
UROBILINOGEN PORPHOBILINOGEN
REACTIVE SUBS.

1. CHLOROFORM EXTRACTION
• Urine (top layer) Colorless Red Red
• Chloroform (bottom) Red Colorless Colorless

2. BUTANOL EXTRACTION
• Butanol (top) Red Colorless Red
• Urine (bottom) Colorless Red Colorless
URINE BILIRUBIN AND UROBILINOGEN IN JAUNDICE
URINE BILIRUBIN URINE UROBILINOGEN

HEMOLYTIC DISEASE (-) (+++)

LIVER DAMAGE +/- ++

BILE DUCT OBSTRUCTION (+++) NORMAL

• HOESCH TEST:
▪ Rapid screening test for urine porphobilinogen (≥ 2mg/dL)
▪ Reagent: Ehrlich rgt dissolved in 6M HCl.
NITRITES
Detection of bacteriuria

REAGENT STRIP:
• Principle: Greiss reaction
• Positive Nitrite corresponds to 100, 000 organisms/mL
CHEMICAL REACTION:
REAGENTS: • Multistix: p-arsanilic acid, Tetrahydrobenzo(h)-quinolin-3-ol
• Chemstrip: Sulfanilamide, hydroxyte- trahydro benzoquinoline

SENSITIVITY: • Multistix: 0.06 to 0.1 mg/dL nitrite ion

• Chemstrip: 0.05 mg/dL nitrite ion

INTERFERENCE: FALSE POSITIVE: FALSE NEGATIVE:

• Improperly preserved spx. • Nonreductase-cont. bacteria

• Highly pigmented urine • Insufficient contact time between
bacteria and urinary nitrate
• Lack of urinary nitrate
• Large quantities of bacteria
converting nitrite to nitrogen
• Presence of antibiotics
• High concentrations of AA
• High specific gravity
CORRELATIONS: • Proteins, Leukocytes and Microscopic
LEUKOCYTE ESTERASE
SIGNIFICANCE:
• UTI/ Inflammation
• Screening of urine culture specimens
REAGENT STRIP:
PRINCIPLE: Leukocyte Esterase
• Detects presence of esterase in granulocytic WBCs (NEBM).
• Esterase is also present in Trichomonas and histiocytes.
• Lymphocytes, RBCs, bacteria and renal tissues (no esterase).
REAGENTS: • Multistix: Derivatized pyrrole amino acid ester Diazonium salt
• Chemstrip: Indoxylcarbonic acid ester Diazonium salt
SENSITIVITY: • Multistix: 5 to 15 WBC/hpf
• Chemstrip: 10 to 25 WBC/hpf
INTERFERENCE: FALSE POSITIVE: FALSE NEGATIVE:
• Strong oxidizing agents • High concentrations of protein,
• Formalin glucose, oxalic acid, ascorbic
• Highly pigmented urine, acid, gentamicin,
Nitrofurantoin cephalosporins, tetracyclines;
inaccurate timing

CORRELATIONS: • Proteins, Nitrites and Microscopic

 SUMMARY OF CHEMICAL TESTING BY
REAGENT STRIPS:
READING TEST: PRINCIPLE: REAGENTT STRIP
TIME: REACTION:
30 secs Glucose Double sequential • KI (Green – brown)
enzyme reaction • TMB (yellow – green)
30 secs Bilirubin Diazo reaction Azodye (tan/ pink to
violet)
40 secs Ketones Na Nitroprusside AAA in alk. Medium + Na
reaction Nitroprusside → purple

45 secs Specific Gravity pKa change of As SG ↑, indicator

polyelectrolyte changes from blue (Alk.)
to green, yellow (acid)
READING TEST: PRINCIPLE: REAGENTT STRIP REACTION:
TIME:
60 secs pH Double indicator • pH 5 (orange), yellow, green and
system pH 9 (deep blue).

60 secs Proteins Protein error of Protein conc. ↑ changes color from

indicator green to blue.

60 secs Blood Pseudoperoxidase • Free Hbg – yellow to green-blue.

activity of Hemoglobin • Lysed RBCs, Hgb produces
“speckled” pattern.

60 secs Urobilinogen Ehrlich’s reaction • Multistix: Aldehyde rxn (light to

dark pink)
• Chemstrip: Diazo rxn (white to
pink). More specific.
 READING TEST: PRINCIPLE: REAGENTT STRIP
TIME: REACTION:
60 secs Nitrite Greiss reaction Pink-colored azodye
120 secs Leukocytes Esterase reaction Aromatic compound +
diazonium salt → purple

DIAZO:
As a component of the reagent: Bilirubin, Leukocytes, Urobilinogen (Chemstrip)
As a product of the reaction: Nitrite

VITAMIN C/ ASCORBIC ACID

• Reducing agent
• Inference – Blood, Bilirubin, Glucose, Leukocyte Esterase and Nitrite).
REAGENT STRIPS:

1. Reagent strips are packed in opaque containers with

desiccant to protect them from light and moisture.
2. Strips are removed just prior to testing and the bottle
is tightly resealed immediately.
3. Bottles should not be opened in the presence of
volatile fumes.
4. Manufacturers recommend that reagent strips be
stored at room temperature below 30℃.
AUTOMATED REAGENT STRIP READERS:

1. Uses a spectrophotometric measurement of light reflection

termed Reflectance Photometry.
2. Reflectance Photometry uses the principle that light reflection
from the test pads decreases in proportion to the intensity of
color produced by the concentration of test substance.
MICROSCOPIC
EXAMINATION
MICROSCOPIC TECHNIQUES:

TECHNIQUES: FUNCTION:
Bright-field Used for routine urinalysis.
Phase-Contrast Visualization of elements with low refractive indices
(hyaline casts, mucous threads)
Polarizing Identification of OFB, fatty casts and crystals.
Dark-field Identification of Treponema pallidum.
Fluorescence Fluorescent microorganisms
Interference-Contrast 3D-microscopy image
 ROUTINE MICROSCOPIC EXAM:
• Centrifugation for 5 minutes at RCF of 400.
• Microscopic examination should be performed in a consistent manner.
• Observe a minimum of 10 fields under LPO and HPO.
• Reduced light when using Bright-field microscopy.
MUCUS CRYSTALS EPITH. CELLS BACTERIA
REPORTING:
LPF HPF LPF HPF
RARE 0-1 0-2 0-5 0-10
FEW 1-3 2-5 5-20 10-50
MODERATE 3-10 5-20 20-100 50-200
MANY >10 >20 >100 >200
 RBCs, WBCs Ave. number/ 10 HPF NORMAL FINDINGS:
Casts Ave. number/ LPF
Squamous EC Rare, few, moderate, many/ LPF
Transitional EC Rare, few, moderate, many/ HPF
0-2/0-3 RBCs/ hpf
RTE cells Ave. number/ 10 HPF
Oval fat bodies Ave. number/ HPF
Bacteria, yeast Rare, few, moderate, many/ HPF;
Presence of WBC is required. 0-5/ 0-8 WBCs/ hpf
Trichomonas Rare, few, moderate, many/ HPF
Spermatozoa Present, based on laboratory protocol
Mucus Rare, few, moderate, many/ LPF 0-2 Hyaline casts/ lpf
Normal crystals Rare, few, moderate, many/ HPF
Abnormal crystals Ave. and reported/ LPF
 SEDIMENT CONSTITUENTS:
CELLS
1. RED BLOOD CELLS • Non-nucleated biconcave disks.
• Crenated in conc. urine. (hypertonic).
• GHOST CELLS in hypotonic urine (dilute).
• Dysmorphic with glomerular membrane damage.
• Sources of errors:
1. Confused with yeast cells, oil droplets and air bubbles.
2. Add Acetic acid to a portion of sediment to lyse RBCs.

2. WHITE BLOOD CELLS • Larger than red blood cells, predominantly neutrophils.
• Granulated, multilobed neutrophils.
• GLITTER CELLS in hypotonic urine.
• “Pus cells”
• Pyuria – increase in urinary WBCs.
• Eosinophils >1% is considered significant.
• Mononuclear cells: Lymph, Mono, Macs and histiocytes
3. EPITHELIAL CELLS
SQUAMOUS EPITHELIAL • Largest cell in the sediment w/ abundant, irregular
cytoplasm and prominent nuclei.
• From linings of vagina and female urethra.
• Normal cellular sloughing; no clinical significance.
• Pathologic: CLUE CELLS
1. Vaginal infection (G. vaginalis).
2. Squamous EC w/ coccobacillus.

TRANSITIONAL EPITHELIAL • Spherical, polyhedral or caudate with centrally located

nucleus.
• From linings of renal pelvis, calyces, ureters and bladder.
• Present in small numbers.
• Normal cell sloughing.
3. EPITHELIAL CELLS
RTE • Most clinically significant.
• Indicative of necrosis of the renal tubules.
• Rectangular, polyhedral, cuboidal and columnar with eccentric nucleus.
• OVAL FAT BODIES – Lipid RTE cell; Nephrotic Syndrome
• BUBBLE CELLS – Nonlipid-filled; Acute tubular Necrosis
• Presence of >2 RTE/ hpf indicates tubular injury.

4. BACTERIA • Significant of UTI.

• Bacteria should be accompanied with WBCs.
• Bacteria motility – useful in differentiating Amorphous phosphates
and urates.

5. YEASTS • Small refractile oval structures that may/not contain bud.

• Severe infections:
✓ May appear as branched, mycelial forms.
6. PARASITES
Trichomonas • Most frequent parasite in urine.
vaginalis • Pear-shaped flagellate with an undulating membrane.
• When not moving, it resembles WBC, Transitional,
RTE cell.

Schistosoma • Associated with hematuria (cloudy, red urine).

haematobium • Bladder cancer

Enterobius • Most common fecal contaminant.

vermicularis
7. SPERMATOZOA • Vaginal contaminants – female (after
intercourse)
• Men: After recent sexual activity or
ejaculation.
 CASTS (CYLINDURIA)
• Only sediments found in the urinary sediment that are unique to the
kidneys.
• Formed at distal convoluted tubules and collecting ducts.
• Major constituent:
▪ TAMM-HORSFALL PROTEIN (UROMODULIN)
• Located near the edges of the cover slip.

Formation of casts: Hyaline casts → Cellular casts → Granular casts

(coarse/ fine) → waxy casts → broad casts
1. HYALINE • Most frequently seen cast. • Glomerulonephritis
• Consist of Uromodulin. • Pyelonephritis
• Presence of 0-2/ lpf is normal. • Chronic renal disease
• Pink in Sternheimer-Malbin stain. • Congestive heart failure
• Stress and exercise
2. RBC • Damage to the glomerulus • Glomerulonephritis
• Stress and exercise

3. WBC • Inflammation within the nephron. • Pyelonephritis

• Pyelonephritis • Acute Interstitial Nephritis
• Pyelonephritis (Upper UTI):
▪ WBC casts, WBCs, bacteria
• Cystitis (Lower UTI):
▪ WBCs, bacteria, no casts
• Acute Interstitial Nephritis
▪ WBC casts, no bacteria

4. BACTERIAL • Bacilli seen in Pyelonephritis • Pyelonephritis

5. EPITH. CELL • Containing RTE cell – advanced • Renal tubular damage
tubular destruction.
• Heavy metal and chemical/ drug-
induced toxicity, viral infections and
allograft rejection.
6. COARSE/ FINE • Maybe pathologic/ nonpatho. • Glomerulonephritis
• Stress and exercise
• Pyelonephritis
7. FATTY • Seen with OFB and free fat droplets • Nephrotic syndrome
causing – Lipiduria • Toxic tubular necrosis
• Confirmation by Polarized Microscopy • Diabetes Mellitus
and Sudan III/ Oil Red O fat stains • Crush injuries
8. WAXY • Urine stasis – Chronic renal failure • Stasis of urine flow
• Appear fragmented with jagged ends • Chronic renal failure
and have notches in the sides.

9. BROAD • Renal failure casts • Extreme urine stasis

• All cast may occur in broad form • Renal failure
URINARY CRYSTALS:
• Abnormal crystals may represent liver disorders,
inborn error of metabolism, or renal damage.
• Reported as rare, few, moderate, many per hpf.
• Abnormal crystals are reported as Averaged per lpf.
• Precipitation from solutes, including inorganic salts,
organic compounds, and medications.
 RENAL DISEASE
GLOMERULAR DISORDERS
 CLINICAL PRIMARY UA
DISORDER: ETIOLOGY: OTHER TESTS:
COURSE: RESULT:
• Deposition of • Rapid onset of • Hematuria • Antistreptolysin O titer
immune hematuria and • Proteinuria • Anti-Group A
complexes. edema. • RBC casts Streptococcal
ACUTE • GAS infection • Permanent renal • Granular casts enzymes
GLOMERULONEPHRITIS on glomerular damage seldom
membranes. occurs.

• Deposition of • Glomerular damage • Hematuria • BUN

immune • Progression to end- • Proteinuria • Creatinine
complexes from stage renal failure. • RBC casts • Creatine Clearance
systemic
RAPIDLY PROGRESSIVE immune
GLOMERULONEPHRITIS disorders on the
glomerular
membrane.
 CLINICAL PRIMARY UA
DISORDER: ETIOLOGY: OTHER TESTS:
COURSE: RESULT:
• Attachment of • Hemoptysis • Hematuria • Anti-glomerular
cytotoxic Ab • Dyspnea • Proteinuria basement
formed during • Hematuria • RBC casts membrane antibody
viral respiratory • Progression to
GOODPASTURE’S infx. To ESRD.
SYNDROME glomerular and
alveolar
basement
membranes.

• ANCA binds to • Hemoptysis • Hematuria • Antineutrophilic

neutrophils in • Renal involvement • Proteinuria Cytoplasmic
WEGENER’S vascular walls • Progression to • RBC casts Antibody
GRANULOMATOSIS damaging the ESRD.
vessels of lungs
and glomerulus.
 PRIMARY UA
DISORDER: ETIOLOGY: CLINICAL COURSE: OTHER TESTS:
RESULT:
• Primarily on • Appearance of purpura • Hematuria • Stool occult blood
children • Blood in sputum and • Proteinuria
• Viral respiratory stools • RBC casts
HENOCH- infections • Complete recovery is
SCHONLEIN • ↓ Platelets disrupts common.
PURPURA vascular integrity • Progress to ESRD

• Deposition of IgA • Hematuria • Early stages: • Serum IgA

on glomerular • Chronic Hematuria
IgA NEPHROPATHY membrane Glomerulonephritis • Late stages:
(BERGER’S resulting in ↑ Chronic
DISEASE) serum IgA. Glomerulo-
nephritis
 CLINICAL PRIMARY UA
DISORDER: ETIOLOGY: OTHER TESTS:
COURSE: RESULT:
• Thickening of the • Slow • Hematuria • ANA
glomerular progression to • Proteinuria • HBsAg
membrane ff. IgG Nephrotic • FTA-ABS
MEMBRANOUS immune complex syndrome.
GLOMERULONEPHRITIS deposition assoc.
w/ systemic
disorders.

• Cellular • Progression to • Hematuria • Serum Complement

proliferation Chronic • Proteinuria levels
affecting the Glomerulo-
capillary walls/ nephritis to
MEMBRANOPROLIFERATIVE
glomerular Nephrotic
GLOMERULONEPHRITIS
basement Syndrome
membrane.
• Immune mediated
 CLINICAL PRIMARY UA
DISORDER: ETIOLOGY: OTHER TESTS:
COURSE: RESULT:
• Marked ↓ in renal • Decrease in renal • Hematuria • BUN
function resulting function • Proteinuria • Serum Crea
to glomerular progressing to • Glucosuria • Crea Clearance
CHRONIC damage renal failure. • Cellular and • Electrolytes
GLOMERULONEPHRITIS precipitated by granular casts
other renal • Waxy and broad
disorders. casts

• Disruption of the • Acute onset • Heavy Proteinuria • Serum Albumin

electrical charges following systemic • Hematuria • Cholesterol
that produce the shock. • Renal tubular cells • Triglycerides
tightly fitting • Progression from • Oval fat bodies
NEPHROTIC podocyte barrier other glomerular • Fat droplets
SYNDROME resulting in disorders to renal • Fatty and waxy
massive loss of failure. casts
CHON and Lipids.
 CLINICAL PRIMARY UA
DISORDER: ETIOLOGY: OTHER TESTS:
COURSE: RESULT:
• Disruption of • Corticosteroid • Heavy • Serum Albumin
podocytes occurring treatment proteinuria • Cholesterol
MINIMAL CHANGE
primarily in children ff. • Transient • Triglycerides
DISEASE
allergic reactions and hematuria
immunization. • Fat droplets
• Disruption of • Resemble Nephrotic • Proteinuria • Drug of abuse
podocytes in some Syndrome of • Hematuria • HIV test
FOCAL SEGMENTAL
areas of glomeruli Minimal Change (Microscopic)
GLOMERULOSCLE
assoc. w/ heroin and Disease
-ROSIS
analgesic abuse and
AIDS
 RENAL DISEASE
TUBULOINTERSTITIAL DISORDERS
 CLINICAL PRIMARY UA
DISORDER: ETIOLOGY: OTHER TESTS:
COURSE: RESULT:
• Damage to the • Acute renal • Hematuria • Hgb
renal tubular cells dysfunction is • Proteinuria • Hct
caused by resolved when • RTE cells • Cardiac enzymes
Ischemia or toxic underlying cause • RTE cell casts
ACUTE TUBULAR
agents. is corrected. • Hyaline, waxy,
NECROSIS
broad, granular
casts

• Inherited in • Defect in renal • Glucosuria • Serum and urine

assoc. w/ tubular • Cystine crystals electrolytes
Cystinosis and reabsorption • Amino acid
FANCONI’S Hartnup disease. requiring chromatography
SYNDROME • Exposure to toxic supportive
agents therapy.
 CLINICAL PRIMARY UA
DISORDER: ETIOLOGY: OTHER TESTS:
COURSE: RESULT:
• Ascending bacterial • Acute onset of • Leukocyturia • Urine culture
infection of the urinary frequency • Bacteriuria
CYSITITIS bladder. and burning • Hematuria
(LOWER UTI) resolved w/ • Mild Proteinuria
antibiotics. • Increased pH

• Infection of renal • Acute onset of • Leukocyturia • Urine and blood

tubules and urinary frequency, • Bacteriuria cultures
interstitium related to burning and lower • WBC casts
interference of urine back pain resolved • Bacterial casts
ACUTE
flow to the bladder, with antibiotics. • Hematuria
PYELONEPHRITIS
reflux of urine from the • Proteinuria
(UPPER UTI)
bladder and untreated
Cystitis.
 CLINICAL PRIMARY UA
DISORDER: ETIOLOGY: OTHER TESTS:
COURSE: RESULT:
• Recurrent infection of • Diagnosed in • Leukocyturia • Urine culture
the renal tubules and children. • Bacteriuria • Blood culture
interstitium caused by • Progression to renal • Hematuria • BUN
structural failure. • Proteinuria • Creatinine
CHRONIC
abnormalities affecting • WBC casts • Crea Clearance
PYELONEPHRITIS
urine flow. • Bacterial casts
• Granular, waxy,
broad casts

• Allergic inflammation • Acute onset of renal • Leukocyturia • Urine Eosinophils

of the renal interstitium dysfunction • WBC • BUN
ACUTE INTERSTITIAL in response to certain accompanied by • Hematuria • Creatinine
NEPHRITIS medications. skin rash. • Proteinuria • Crea. Clearance
 RENAL DISEASE
(RENAL CALCULI/ STONES)
 RENAL CALCULI/ STONES:
Kidney stones may form in the calyces and pelvis of the kidney,
ureters and bladder.

Upper (renal) stones are common in industrialized countries; bladder

stones are uncommon.

80% Calcium Oxalate or mixture of Oxalate and Calcium Phosphate

3 to 10% Mixed Calcium Phosphate, Magnesium Ammonium Phosphate, Uric acid

1 to 2% Cystine stones
 CAUSES OF VARIOUS CALCULI COMPOSITIONS:
CALCIUM: CALCIUM OXALATE: CALCIUM PHOSPHATE:
• Idiopathic Hypercalciuria • Oxaluria • Same as Calcium
• Primary Hyperthyroidism • Incomplete catabolism of Oxalate
• Bone disease Carbohydrates • Alkaline infection
• Excessive milk, alkali or • Excessive glycogen • Persistently alkaline urine
Vitamin D intake breakdown
• Renal tubular Acidosis
• Sarcoidosis
• Berylliosis
 CAUSES OF VARIOUS CALCULI COMPOSITIONS:
MAGNESIUM AMMONIUM URIC ACID AND
CYSTINE
PHOSPHATE URATE
• Alkaline infection with Urea- • Gout • Transient acute phases of
splitting bacteria • Polycythemia chronic renal diseases
• Leukemia • Heavy metal nephrotoxicity
• Lymphoma • Aminoaciduria
• Liver disease • Renal tubular acidosis
• Theophylline and thiazide syndromes
therapy
• Conditions associated with
rapid Protein catabolism
 VALUES OF pH ASSOCIATED WITH CALCULI FORMATION
pH <5.5 Uric acid, Cystine, Xanthine calculi
pH 5 – 6 Calcium Oxalate and Apatite calculi
pH >7 Magnesium Ammonium Phosphate/ Calcium Phosphate

PHYSICAL CHARACTERISTICS OF CALCULI:

URIC ACID AND URATE Yellow to brownish red; moderately hard
PHOSPHATE Pale and friable
CALCIUM OXALATE Very hard, often dark in color; rough surface
CYSTINE Yellow-brown; greasy
RENAL DISEASE
(METABOLIC DISORDERS)
Phenylalanine – Tyrosine Disorders
Branched-Chain Amino acid Disorders
Tryptophan Disorders
Cystine Disorders
Mucopolysaccharide Disorders
Purine Disorders
Carbohydrates Disorders
Porphyrin Disorders
 PHENYLALANINE – TYROSINE DISORDERS
• PHENYLKETONURIA
1. Failure of inherit a gene to produce enzyme Phenylalanine
hydroxylase
2. Guthrie test - Bacterial inhibition
3. FeCl3 tube test: Blue-green

• TYROSYLURIA
1. FeCl3 tube test: Transient green
2. Nitroso-naphthol: Orange red
ALKAPTONURIA
• Failure to inherit the gene to produce the enzyme
Homogentisic acid oxidase.
• Benedict’s/ Clinitest: Yellow ppt.

MELANURIA
• Over-proliferation of melanocytes
• FeCl3 tube test: Gray/ black ppt.
• Sodium Nitroprusside: Red
• Ehrlich’s reagent: Red
BRANCHED-CHAIN AMINO ACID DISORDERS:

MAPLE SYRUP URINE DISEASE (MSUD)

• Accumulation of Leucine, Isoleucine and Valine in blood and urine.
• 2,4-Dinitrophenylhydrazine: Yellow turbidity/ ppt.

ORGANIC ACIDEMIAS
• Symptoms include early severe illness, often with vomiting accompanied by
metabolic acidosis, hypoglycemia, ketonuria and increased serum Ammonia.
• Isovaleric, Propionic and Methylmalonic Acidemias
 TRYPTOPHAN DISORDERS
• INDICANURIA
1. Intestinal disorders
2. Hartnup disease – Blue-diaper disorder (rare inherited)
3. FeCl3 – Violet with Chloroform

• ARGENTAFFINOMA
1. ↑ 5-HIAA (Hydroxyindoleacetic acid; metabolite of serotonin)
2. FeCl3 – Blue-green
3. Nitrosonaphthol – Violet with Nitric acid
 CYSTINE DISORDERS
CYSTINURIA
• Defect in tubular transport of Cystine, Ornithine, Lysine, Arginine.

CYSTINOSIS
• Inborn error of metabolism
• Cystine deposits in BM, cornea, lymph nodes and internal organs.
• Cyanide-Nitroprusside: Red - purple
HOMOCYSTINURIA
• Defects in metabolism of Methionine
• Cataracts, mental retardation
• Silver Nitroprusside: Red - purple
 MUCOPOLYSACCHARIDE DISORDERS
Glycosaminoglycans; inherited disorder
Resulting in accumulation of the incompletely metabolized polysaccharide portions in the
lysosomes of the connective tissue cells and their increased excretion in the urine.
The products most frequently found in the urine are dermatan sulfate, keratan sulfate, and
heparan sulfate.
HUNTER’S SYNDROME HURLER’S SYNDROME SANFILLIPO’S SYNDROME
• Skeletal structure is abnormal. • Skeletal structure is • Mental retardation
• Severe mental retardation abnormal.
• Sex-link recessive; females • Severe mental retardation
PORPHYRIAS
Disorders or Porphyrin metabolism
Port wine urine
Congenital Porphyria – red discoloration diapers
Can be inherited or acquired from erythrocytic and hepatic
malfunctions or exposure to toxic agents.
Screening tests for Porphyrinuria:
• Ehrlich reaction (ALA and Porphobilinogen)
• Fluorescence over UV light in 500 to 600nm range.
PURINE DISORDERS
• LESCH-NYHAN DISEASE
▪ Sex-linked recessive
▪ Results in massive excretion of urinary uric acid crystals.
▪ Failure to inherit the gene to produce hypoxanthine guanine
phosphoribosyltransferase is responsible for the accumulation
of uric acid throughout the body.
▪ Orange sand diapers
▪ Severe motor defect, gout and renal calculi
 CARBOHYDRATE DISORDERS
MELITURIA
• Increased urinary sugar

GALACTOSURIA
• Presence of Galactose in urine

LACTOSURIA
• Pregnancy and lactation

FRUCTOSURIA
• Ingestion of large amount of fruits
• Parenteral feeding
OTHER BODY FLUIDS
CEREBROSPINAL FLUID (CSF)
 CSF

• A major body fluid

• Approximately 20 mL of fluid is produced every hour in the choroid plexus
and reabsorbed by the arachnoid villi.
• Functions:
1. Supply nutrients to the nervous tissue.
2. Remove metabolic wastes.
3. Mechanical barrier to cushion the brain and spinal cord against trauma.
 TOTAL ADULT: 90 to 150 mL
VOLUME: NEONATE: 10 to 60 mL

COLLECTION Routinely collected by lumbar puncture between 3rd to 5th lumbar

OF CSF: vertebra; 20 mL of CSF may normally be removed.

THREE COLLECTION TUBES: Specimens are collected in three sterile tubes which are labeled as 1, 2 and 3
in the order in which they are withdrawn.
TUBE 1 Chemical and Serology tests Frozen
TUBE 2 Microbiology Room Temperature
TUBE 3 Cell count and Physical exam Refrigerated
• TUBE 4 may be drawn for Microbiology (better exclude skin contamination).
• If ONLY ONE TUBE is collected: Microbiology, Hematology, Chemistry and other tests.
 APPEARANCE:
CRYSTAL CLEAR • Normal CSF
HAZY, TURBID, • WBC count over 200 𝜇/L
CLOUDY, MILKY • RBC count over 400 𝜇/L
• Microorganisms
• Proteins
GROSSLY • RBC count greater than 6, 000 𝜇/L
BLOODY CSF
CLOTTED, • Protein
PELLICLE • Clotting factors
• Tubercular Meningitis
 OILY • Radiographic contrast media
• Supernatant is pink, orange or yellow.
• Presence of RBC degradation products.
• Other causes include elevated serum bilirubin, presence
of pigment carotene, markedly increased protein
concentration and melanoma pigment.

XANTHOCHROMIC
CSF PINK • Very slight amount of Oxyhemoglobin.

YELLOW • Conversion of Oxyhemoglobin to

Unconjugated bilirubin.
ORANGE • Heavy hemolysis
TRAUMATIC TAP VS/ INTRACRANIAL HEMORRHAGE
TRAUMATIC TAP INTRACRANIAL
HEMORRHAGE
DISTRIBUTION OF Uneven Even
BLOOD (1>2>3) (1=2=3)
CLOT FORMATION + -
SUPERNATANT Clear Xanthochromic
ERYTHROPHAGES - +
D-DIMER - +
 CELL COUNT:
• Any cell count must be performed immediately because WBCs
(particularly granulocytes) and RBCs tend to lyse within 1 hour and
40% of WBCs disintegrate after 2 hours.
• If specimens cannot be analyzed immediately, it should be refrigerated.
• Normal adult CSF contains 0 to 5 WBCs/𝜇L
• Normal in newborn: >30 mononuclear cells/𝜇L
DIFFERENTIAL COUNT ON CSF:
• Performed on stained smear.
• Methods available for specimen concentration include sedimentation,
filtration, centrifugation and cytocentrifugation.
▪ Sedimentation and filtration are not routinely used in clinical
laboratory (less cellular distortion).
• Normal cells in CSF:
▪ Adults: Lymphocytes and Monocytes (70: 30)
▪ Children: Reverse
• Increased in number of normal cells - PLEOCYTOSIS
• ADULT: Predominance of Lymphocytes
• CHILDREN: Predominance of Monocytes
LYMPHOCYTES • Normal
• Viral, tubercular and fungal meningitis
• Multiple Sclerosis

MONOCYTES • Normal
• Viral, tubercular and fungal meningitis
• Multiple Sclerosis

NEUTROPHILS • Bacterial meningitis

• Early stages of viral, tubercular and fungal
meningitis
• Cerebral hemorrhage
 • RBC in the spinal fluid
MACROPHAGES • Contrast media

BLASTS • Acute Leukemia

PLASMA CELLS • Multiple Sclerosis
• Lymphocyte reaction
EPENDYMAL, • Diagnostic procedures
CHOROIDAL AND
SPINDLE-SHAPED
CELLS
MALIGNANT • Metastatic CA
CELLS • Primary CNS CA
 CHEMISTRY TEST:
Most frequently performed test in CSF.

Normal CSF contains small number of proteins.

Reference values of proteins: 15 to 45 mg/dL

ELEVATED Meningitis, hemorrhage, primary CNS tumors, MS, Guillain-Barre

Syndrome, Neurosyphilis, Myxedma.....
RESULTS:
DECREASED CSF leakage/ trauma, recent puncture, rapid CSF production and
water intoxication
RESULTS:
CHEMISTRY TESTS
• Major CSF protein: Albumin
• 2nd prevalent: (Prealbumin) Transthyretin
• Alpha-globulins: Haptoglobin, Ceruloplasmin
• Beta-globulins: Transferrin, TAU – separate carbohydrate-
deficient transferrin; seen in CSF not in serum
• Gamma-globulins: Primarily IgG and some IgA
• Not in normal CSF: IgM, Fibrinogen and 𝜷-LPP
 METHODS (TOTAL PROTEIN):
1. TURBIDIMETRIC a. TRICHOLORACETIC ACID (TCA)
• Reagent of choice
• It precipitates both Albumin and globulin
b. SULFOSALYCYLIC ACID (SSA)
• Combined with Sodium Sulfate, Albumin will contribute to the
turbidity more.
2. DYE-BINDING a. COOMASSIE BRILLIANT BLUE (CBB)
• Protein binds to the dye (red to blue)

ELECTROPHORESIS:
• Detection of oligoclonal bands: Electrophoretic bands migrating in the gamma region that
are present in CSF and serum.
• 2 or more oligoclonal bands present in CSF: MULTIPLE SCLEROSIS
• Other causes: Encephalitis, Neurosyphilis, Guillain-Barre Syndrome and Neoplastic
disorders.
 CSF GLUCOSE
Reference CSF glucose: 60% to 70% of plasma conc.

Elevated plasma glucose values are always a result of plasma elevations.

Low CSF value can be considerable of diagnostic value in determining the

causative agent in Meningitis.

DECREASED CSF
Bacterial, Tubercular and Fungal Meningitis
GLUCOSE:
NORMAL CSF GLUCOSE: Viral Meningitis
 CSF LACTATE
Lactate levels can be a valuable aid in diagnosing and managing Meningitis.

Bacterial, Tubercular and Fungal Meningitis: >25 mg/dL

Viral Meningitis: <25mg/dL

INCREASED CSF LACTATE: Bacterial, Tubercular and Fungal Meningitis

NORMAL CSF LACTATE: Viral Meningitis

CSF GLUTAMINE
• Produced from Ammonia and 𝛼 − ketoglutarate .
• Normal concentration: 8 – 18 mg/dL
• Elevated levels are associated with liver disorders that result in increased blood and CSF
Ammonia.
• Disturbance of consciousness is almost seen when Glutamine levels are >35 mg/dL.
▪ Glutamine test is requested for patients with coma of unknown origin.
• REYE SYNDROME
▪ 75% of children has elevated CSF Glutamine levels
▪ Acute encephalopathy and liver infiltration seen in children after viral infections.
 MICROBIOLOGY TESTS
BACTERIAL MENINGITIS TUBERCULAR MENINGITIS
• ↑ WBC count • ↑ WBC count
• Neutrophils present • Lymphocytes and monocytes present
• Markedly ↑ Protein • Moderate to marked protein elevation
• Markedly ↓ Glucose • Decreased glucose level
• Lactate level: >35 mg/dL • Lactate level: >25 mg/dL
• (+) Gram stain and culture • Pellicle formation
VIRAL MENINGITIS FUNGAL MENINGITIS
• Elevated WBC count • Elevated WBC count
• Lymphocytes present • Lymphocytes and monocytes present
• Moderate protein elevation • Moderate to marked protein elevation
• Normal glucose level • Decreased glucose level
• Normal lactate level • Lactate level: >25 mg/dL
• (+) India ink
• (+) Immunologic test with C. neoformans
OTHER BODY FLUIDS
SEMINAL FLUID
PHYSIOLOGY:
• Semen is composed of four fractions that are contributed by the
testes, epididymis, seminal vesicles, prostate glands and
bulbourethral glands.
1. TESTES AND EPIDIDYMIS SPERMATOZOA – 5%
• Stages of cellular maturation from youngest to adult form:
1. Spermatogonia
2. Spermatocytes
3. Spermatids
4. Spermatozoa
• In complete spermatogenesis, the immature sperm (nonmotile) will enter the
epididymis, the sperm will mature and develop flagella. It will take 90 days.
2. SEMINAL VESICLES SEMINAL FLUID – 60 to 70%
• Contains high concentrations of fructose and flavin.
• Spermatozoa metabolizes the fructose for energy for the flagella to propel through the
female reproductive tract.

3. PROSTATE PROSTATE FLUID – 20 to 30%

• Milky acidic fluid contains high concentration of ACP, citric acid, zinc, and proteolytic
enzymes responsible for both liquefaction and coagulation of the semen following
ejaculation.

4. BULBOURETHRAL GLANDS ALKALINE MUCUS – 5%

• Alkaline mucus that helps neutralization the acidity from prostate secretions and
vagina.
COLLECTION:
1. Specimens are collected following a period of sexual
abstinence (at least 2 days to not more than 7 days).
2. Prolonged abstinence (↑ volume, ↓ motility).
3. Warm sterile glass/ plastic containers.
4. Analysis should be kept at 37℃.
5. Collected by masturbation (only nonlubricant-
containing rubber/ polyurethane condoms should be
used.
LIQUEFACTION:
1. Fresh specimen is clotted and should liquefy within 30 to 60 minutes
after collection.
2. Failure of liquefaction to occur within 60 minutes may be caused a
prostatic enzyme deficiency; reported.
3. If after 2 hours the specimen has not yet liquefied, equal volume of
Dulbecco’s Phosphate-buffered saline or proteolytic enzymes ( 𝛼 -
chymotrypsin or bromelain) may be added to induce liquefaction, thus
can perform other tests.
 SEMEN ANALYSIS:
APPEARANCE:
GRAY-WHITE/ TRANSLUCENT • Normal appearance
↑ WHITE TRUBIDITY • Infection
RED/ BROWN • Presence of blood
• Urine contamination
YELLOW • Prolonged abstinence
• Medications
VOLUME
NORMAL VOLUME: • 2 to 5 mL
↑ VOLUME: • Prolonged abstinence
↓ VOLUME: • Infertility
• Incomplete specimen collection
 VISCOSITY:
Consistency of the fluid and related to liquefaction.

Incomplete liquefaction – clumped, highly viscous

Droplets forming threads (2 cm) – abnormal

Rating/ reporting: 0 (watery) to 4 (gel-like)

Can also be reported as low, normal or high.

NORMAL: • Pour in droplets and will not appear clumped/ stringy.

• Increased viscosity and incomplete liquefaction affects sperm motility,
INCREASED: concentration, anti-sperm Ab-detection, and measurement of
biochemical fluids.
 pH
NORMAL: • pH 7.2 to 8.0
INCREASED: • Infection
DECREASED: • Increased prostatic fluid
• Ejaculatory duct obstruction
• Poorly developed seminal vesicles
 SPERM CONCENTRATION:
Concentrations between 10M and 20M/ mL are considered borderline.
Reference value: 20M to 250M sperms/ mL

• Counting undiluted specimens

MAKLER • Sperms are immobilized by heating prior to
COUNTING charging the chamber.
CHAMBER • Sperm motility using the unheated portion of
the specimen.
 • Sperm are counted in the 4 corner and center squares
of the large center square (similar to manual RBC
count).
• Both sides are loaded and allowed to settle for 3 to 5
NEUBAUER
mins; then they are counted – agreeing within 10%.
COUNTING • Performed using Phase/ Bright microscopy
CHAMBER • Fully developed sperm cells are counted.
• WBCs and immature sperms (round cells) are not
counted.

• Most common dilution is 1:20 using • Traditional dilution fluid: Sodium

mechanical pipette. Bicarbonate and Formalin.
SPERM COUNT:
• >40M per ejaculate – normal
• Only fully developed sperms should be counted.
• Immature sperms/ WBCs “round cells”
▪ >1M leukocytes/ mL – infection/ infertility
▪ >1M spermatids/ mL – disruption in spermatogenesis
 SPERM MOTILITY:
• Assessed using a well-mixed,
liquefied specimen within 1 hour of
collection.
• Motility is evaluated by both speed
and direction.
• Minimum motility of 50% with a rating
of 2.0 after 1 hour considered normal.
 CASA:
COMPUTER-ASSISTED SEMEN ANALYSIS

Determination of:
• Sperm velocity
• Trajectory (direction/ motion)
• Sperm concentration
• Morphology

Found in laboratories that specialize in Andrology and performs high volume of

semen analysis.
 SPERM MORPHOLOGY:
NORMAL SPERM HEAD • Oval-shaped
• 5 𝜇m long and 3 𝜇m wide
FLAGELLAR TAIL • 45𝜇m long
ACROSOMAL CAP • Critical to ovum penetration
• Half of the head; 2/3 of sperm nucleus

ABNORMALITIES IN HEAD • Poor ovum penetration

MORPHOLOGY
NECKPIECE, MIDPIECE AND TAIL • Affects motility
ABNORMALITIES
SPERM VIABILITY/ VITALITY:
• Abnormal result: ↓ motility with normal count
• MODIFIED BLOOM’S TEST: Vitality is evaluated by mixing the
specimen with an Eosin-nigrosin stain, preparing a smear, and
counting the number of dead cells in 100 sperm using a Bright-
field or Phase contrast microscope.
• Living cells are not infiltrated by the dye and remain bluish white;
dead cells stain red against the purple background.
SEMINAL FLUID FRUCTOSE:
• Low sperm concentration may be caused by lack of the support medium
produced in the seminal vesicles, which can be indicated by a low to
absent fructose level in the semen.
• Specimens can be screened for the presence of fructose using the
Resorcinol test that produces an orange color when fructose is present.
• Normal quantitative level of fructose is ≥13 μmol/ ejaculate.
• Specimens for fructose levels should be tested within 2 hours of collection
or frozen to prevent fructolysis.
MICROBIAL TESTING:
• Presence of 1M WBCs/ mL indicates infection, frequently the prostate.
• Routine aerobic and anaerobic cultures and tests: Chlamydia trachomatis,
Mycoplasma hominis and Ureaplasma urealyticum are frequently performed.

POSTVASECTOMY SEMEN ANALYSIS:

• Presence or absence of spermatozoa.
• Specimens are routinely tested at monthly intervals, starting at 2 months post
vasectomy and continuing until 2 consecutive monthly specimens show no
spermatozoa.
SPERM FUNCTION TEST:
• Advances in assisted reproduction and IVF have resulted in a need for more
sophisticated semen analysis to assess not only the characteristics of sperm but also
the functional ability.
FERTILITY TESTING

Varicocele
▪ Most common cause of male infertility.
▪ Hardening of veins that drain the testes; causes blood from
adrenal vein to flow to the spermatic vein.
▪ 2 samples are collected not less than 7 days or 3 weeks apart,
with two abnormal samples considered significant.
POST-VASECTOMY ANALYSIS
• Surgical removal of all or part of the vas deferens for the
purpose of male sterilization.
• Presence or absence of spermatozoa.

FORENSIC ANALYSIS
• For suspected rape cases
MEDICO-LEGAL (SUSPECTED RAPE CASE)
• Microscopically examining the specimen for the presence of sperm.
• Best results is obtained by enhancing the specimen with Xylene and examining under
Phase Microscopy.
• Positive – high conc. of prostatic ACP
• Positive – detection of seminal gp30, PSA
• Perform ABO Blood grouping and DNA Analysis
• Note:
1. Motile sperm can be detected 24 hours after intercourse.
2. Nonmotile sperm can persist for 3 days.
3. As sperm die off, only heads remain and may be present for 7 days after intercourse.
FLORENCE TEST Test for Choline Iodine, KI (+) Dark rhombic
crystals
BARBIERO’S TEST Test for Spermine Picric acid, TCA (+) Yellow leaf-shaped
crystals
OTHER BODY FLUIDS:
SYNOVIAL FLUID
 Located in the cavities between the moveable
joints.
Synoviocytes secrete hyaluronic acid, which
produces the viscosity of the fluid.
SYNOVIAL Color: Colorless to pale yellow
FLUID:
Viscosity:

• Mucin clot test detects hyaluronic acid.

• Addition of Acetic acid forms a mucin clot
surrounded by clear liquid.
 SYNOVIAL FLUID:
CELL COUNTS: PATHOLOGY: COLOR:
• Do not use Acetic acid; use Normal Osteoarthritis Clear, yellow
Saline.
Autoimmune Cloudy, yellow
• Normal: <200 cell/L disorders
DIFFERENTIAL COUNT: Gout/ Pseudogout Clear or milky

• Incubate with Hyaluronidase before Infection Cloudy, yellow-green

slide preparation.
• Primary cells: Monocytes and MACs Trauma Cloudy, red
 SYNOVIAL FLUID:
CRYSTAL IDENTIFICATION: MICROBIOLOGY:
▪ Gout – Monosodium urate ▪ Gram stains and culture
oNeedle-shaped, birefringent ▪ Culture requires enriched agar
oElevated serum Uric acid (CAP) for detection of:
levels oHaemophilus spp.
▪ Pseudogout – Calcium oNeisseria gonorrheae
Pyrophosphate Dihydrate
• Rhombic
 SEROUS FLUID:
Located between the parietal and visceral membranes that
lines the closed body cavities.

Cavities: Pleural, Pericardial, and Peritoneal

Normally produced and reabsorbed at a constant rate;

disruption produces an Effusion.
TRANSUDATES • Effusions caused by systemic disorders are Transudates.
VS. EXUDATES: • Effusions caused by membrane disorders are Exudates.

TESTS: TRANSUDATES: EXUDATES:

APPEARANCE Clear Cloudy
FLUID-TO-SERUM PROTEIN RATIO <0.5 >0.5
FLUID-TO-SERUM LD RATIO <0.6 >0.6
WBC COUNT <1000/L >1000/L
SPONTANEOUS CLOTTING No Yes
 PLEURAL • Collected by Thoracentesis
• Microbiology:
FLUID ▪ Acid-fast staining

APPERANCE: CLINICAL SIGNIFICANCE:

Milky Thoracic duct leakage (Chylous effusion), chronic infection
(pseudochylous effusion)
Bloody Hemothorax, hemorrhagic effusion (Embolus, TB,
malignancy)
Viscous Malignant mesothelioma
 PLEURAL FLUID:
DIFFERNTIAL COUNT: CLINICAL SIGNIFICANCE:
Neutrophils Pneumonia, Pancreatitis
Lymphocytes TB, Viral infections
Eosinophils Pneumothorax
Mesothelial cells Norma;  with TB
Plasma cells TB
Malignant cells Small cell CA and adenocarcinoma

CHEMISTRY:
pH <7.0 Chest tube drainage
pH <6.0 Esophageal rupture
 PERICARDIAL FLUID:
• Collected by pericardiocentesis
APPEARANCE:
Cloudy, bloody-streaked Infection, malignancy
Grossly bloody Cardiac puncture, AC medications

HEMATOLOGY:
 Neutrophils Bacterial Endocarditis
Malignant cells Cytologic examination
PERITONEAL • Ascitic fluid, effusion in ascites
• Effusions are caused by liver disorders (Cirrhosis),
FLUID: Intestinal Infection (Peritonitis) and malignancy.

APPEARANCE:
Turbid Infection
Green Gallbladder or pancreatic disorder
Blood-streaked Trauma, infection, malignancy
Milky Lymphatic trauma or blockage
 HEMATOLOGY:
Normal WBC Count: <350/L
>250/L Peritonitis
CHEMISTRY:
Glucose  In infection and malignancy
Amylase  In Pancreatitis and GI perforations
ALP  In Intestinal Perforations
BUN and Creatinine Bladder puncture and rupture
Tumor markers CEA and CA125
MICROBIOLOGY:
Gram stain and culture Aerobic and anaerobic organisms
Blood cultures Detection of anaerobes
 Hemolytic
Disease of the
Collected by Newborn (HDN)
Amniocentesis specimens must

AMNIOTIC
be protected
from light.

FLUID: Specimens for

FLM – delivered
Specimens for
cytogenic
on ice,
testing – RT;
refrigerated or
delivered ASAP
frozen
 AMNIOTIC FLUID:
APPEARANCE:
Colorless Normal
Blood-streaked Trauma, traumatic tap
Yellow Bilirubin, HDN
Dark green Meconium
Dark-red/ brown Fetal death
 TEST FOR FETAL DISTRESS:
• HDN: Spectrophotometric analysis at OD between 365 to 550 nm
• NEURAL TUBE DEFECTS:
•  maternal serum -fetoprotein (AFP)
• AF are measured first between 12 to 15 gestational weeks; compared to maternal serum levels
• Positive results → measure Acetylcholinesterase

TEST FOR FETAL LUNG MATURITY (FLM):

• RDS is caused by  lung surfactant
• Primary lung surfactants: Lecithin, Sphingomyelin, and Phosphatidylglycerol
• AMINOSTAT-FLM
• Immunologic Agglutination test for Phosphatidylglycerol
• Not affected by meconium and blood
• LAMELLAR BODIES
• Storage form of phospholipids in the lungs; secreted by Type II Pneumocytes; enters the amniotic
fluid at 26 weeks gestation.
REFERENCES:
Urinalysis and Other Body Fluids, Strasinger (8th ed)

Elsevier’s Medical Laboratory Science, Graeter (1st ed)

Success in Clinical Laboratory Science, Ciulla (4th ed)

Quick Review Cards for Medical Laboratory Science (2nd ed)