MTAP 1: CLINICAL MICROSCOPY

CONSTITUENTS OF URINE
1. Water- the largest component of urine.
2. Solids
➔ 35 grams (organic)
❖ Urea- major organic.
➔ 25 grams (inorganic)
❖ Chloride- major inorganic.

Principal Salt: Sodium chloride (NaCl)

TYPES OF URINE SPECIMEN

● FIRST MORNING
- Most concentrated; most acidic.
- Commonly required or requested by patients.
- Use for identification or determination of orthostatic proteinuria.

● THREE- GLASS TECHNIQUE

- First sample: slightly contaminated with WBCs and bacteria.
- 2nd or Mid-stream sample: sterile; no WBCs and bacteria content. Serve as the
control of the three-glass technique.
- 3rd sample: have a lot of content of WBCs and bacteria; about 10x more
compared to the first sample.

TAKE NOTE: If the second specimen or mid-stream sample has a content of WBCs and
Bacteria, the result for the third specimen would be INVALID.

If the second specimen or mid-stream sample is unsterile, the patient has a possible UTI.

● DRUG TEST
- The volume of urine specimen: 30- 45 mL
- The temperature should be 32.5- 37.7°C within 4 minutes.
- Upon collection, there should be no adulteration. To avoid adulteration, put the
blueing agent into a bowl.

CHANGES IN UNPRESERVED URINE

Protein- least affected by alkaline urine.
INCREASED DECREASED

pH Clarity

Bacteria Glucose
 Odor Ketones

Nitrite Bilirubin

Urobilinogen

RBC/ WBC/ Cast

PHYSICAL EXAMINATION OF URINE

URINE VOLUME
● Normal daily volume (24 hours): 600- 2000 mL.
● Normal day-to-night ratio: 2:1- 3:1
● Normal night-to-day ratio: 1:2- 1:3
● Diuresis- indicates the increase in urine production.
● Routine UA: 10- 15 mL (required volume) for reagent strip, and urinometer; 12mL
average urine volume.

URINE COLOR
● Should correlate with urine-specific gravity.
● Rough indicator of the degree of hydration.
● The normal color of urine: colorless to deep yellow.
● The abnormal color of urine: red or red-brown (most common).

↑ Fluid intake ↓ SG = Pale Yellow urine.

↓ Fluid intake ↑ SG = Dark Yellow urine.

PIGMENTS
1. UROCHROME
● Major pigment.
● ↑ urochrome: dehydrated.
● Yellow pigment.
● ↑ in thyrotoxicosis, fever, starvation.

2. UROERYTHRIN
● Pink or red (sometimes) pigment.
● ↑ uroerythrin: red.

3. UROBILIN
● Common in drug test samples, when it is in the refrigerator.
● Dark yellow or orange.
● Imparts an orange-brown color to urine which is not fresh.

LABORATORY CORRELATIONS
● Red or red-brown
 - Most commonly seen is abnormal urine color.
- RBC: cloudy or smoky red; the patient has hematuria.
- Hemoglobin: clear red; intravascular hemolysis.
- Myoglobin: clear red or reddish-brown; muscle damage.

TAKE NOTE: If the patient is female, you have to know if the specimen has menstrual
contamination. You should consider that there is the possibility that the urine specimen is
reddish because the patient has menstruation.

● Dark brown to black

- Alkaptonuria or malignant melanoma.
- Alkaptonuria: a rare disorder, caused by the lack of a specific enzyme which is
the homogentisic acid oxidase.

Homogentisic Acid Oxidase

➢ Required for the catabolism of tyrosine and phenylalanine.
➢ Excreted in urine because there is a lot of homogentisic acid oxidase, once excreted
there are changes in the color of urine.
➢ At first, normal color when freshly voided, upon long-standing urine specimen or when it
becomes alkaline, the color turns into black.

- Malignant Melanoma: releases melanogen.

Melanogen
➢ Chromogen.
➢ Excreted in urine patients.
➢ Once excreted and upon light exposure, melanogen will be converted into melanin,
which is black.
● Yellow-brown to yellow-green
- Result from the excretion of bilirubin or bile pigments into the urine.
- It is released when the patient has obstructive jaundice.

● Colors ranging from green to blue

- Typically, indication for the presence of a drug metabolite or possibly a dye.
- Blue: Due to the product or the abnormal breakdown of Tryptophan, which is
Indican.
- Green: Pseudomonas
- A possible example of a drug metabolite that causes green to blue color is
propofol.
➢ Propofol is a type of drug metabolite that could cause the production of
green to blue color of urine.
- Dye: Methylene blue is used as a contrast dye or treatment, it can cause remnants and
causes green to blue coloration of urine.

URINE CLARITY
 - To visualize the correct urine clarity, you have to view it against newspaper print.
- To visualize the correct urine color, you have to view it against a white background.
- Freshly voided: Clear
● Refrigerated normal urine.
➔ White turbidity:
- Alkaline pH.
- Amorphous phosphate, and carbonates.
➔ Pink turbidity:
- Acid pH
- Amorphous urates.

URINE ODOR
Aromatic/ Odorless Normal

Ammoniacal, foul Urea - Urease -> NH3

Ex. UTI (Proteus: ↑ urease)

Fruity, sweet Ketones (Diabetes Mellitus, starvation,

vomiting)

Rotten fish/ Galunggong Trimethylaminuria

Sweaty feet Isovaleric acidemia, glutaric acidemia

Mousy Phenylketonuria

Cabbage hops Methionine malabsorption

Caramelized sugar, curry, maple syrup Maple Syrup Urine Disease (MSUD)

Bleach Contamination (Semen)

Sulfur Cystine disorder

Rancid butter Tyrosinemia

Pungent/ Asparagus Ingestion of onions, garlic, and asparagus

(Methylmercaptan)

Swimming pool Hawkinsinuria; related to abnormal tyrosine

metabolites.

Cat Hydroxymethylglutaricaciduria; correlated

with HMG-CoA lyase deficiency.

Tom Cat Multiple carboxylase deficiency.

CHEMICAL EXAMINATION OF URINE

 READING TIME URINE PARAMETER PRINCIPLE POSITIVE COLOR

30 seconds Glucose The double sequential Green to brown

enzyme reaction

Bilirubin Diazole reaction Tan or pink to violet

40 seconds Ketones Sodium nitroprusside Purple

reaction

45 seconds Specific gravity pKa change of Blue (SG 1.000) to yellow (SG
polyelectrolyte 1.030)

60 seconds Protein Protein error of indicator Blue

pH Double indicator system Orange (pH 5.0) to blue (pH 9.0)

Blood Pseudoperoxidase Uniform green/ blue (Hgb/ Mgb)

activity of hemoglobin Speckled/ spotted (intact RBCs)

Urobilinogen Ehrlich reaction Red

Nitrite Greiss reaction Uniform pink

120 seconds Leukocytes Leukocyte esterase Purple

A. SPECIFIC GRAVITY (SG)

● Screening test for renal tubular reabsorption of any elements being filtered by our
glomerulus.
● The glomerular filtrate is 1.010.omparison of the destiny of urine to the destiny of
distilled water (1.000).
● Influenced by the number and size of particles in solution.

Normal SG 1.003- 1.035 Isothenuria 1.010

(random)

When SG <1.003 Not urine, possible water Hyposthenuria <1.010: diluted specimen
Exemption: Diabetes
Insipidus

When SG >1.040 Due to the presence of Hypersthenuria >1.010: specimen is concentrated

radiographic dye or
radiographic contrast
media.

1. URINOMETRY (URINOMETER/ HYDROMETER)

● Urine Volume: 10- 15 mL
● Calibration:
 - SG: 1.015, always the SG upon the calibration
- Potassium sulfate (K2SO4) solution.
- 20.29 g K2SO4 to 1L H2O.

TAKE NOTE:
Temperature correction.
➢ Subtract 0.001 to SG for every 3°C ↓ calibration temperature (20°C).
➢ Addition of 0.001 to SG for every 3°C ↑ calibration temperature (20°C).

Glucose and Protein correction.

➢ 1 g/dL of Glucose- subtract 0.004 in SG.
➢ 1 g.dL of Protein- subtract 0.003 in SG.

2. REFRACTOMETRY (REFRACTOMETER/ Ts METER)

● Calibration:
- Distilled water: 1.001 SG
- 5% NaCl: 1.022 ± 0.001 SG
- 9% sucrose: 1.034 ± 0.001SG
● NO temperature correction.
Glucose and Protein correction.
➢ 1 g/dL of Glucose- subtract 0.004 in SG.
➢ 1 g.dL of Protein- subtract 0.003 in SG.

Sample Problem
● Urine SG reading by refractometer is 1.030
➔ With 1g/ dl glucose
➔ With 1g/ dl protein
● Urine temperature is 18°C

What is SG?
● 1.023 SG- FINAL ANSWER
● N/A

3. REAGENT STRIP REACTION

● Principle: change in pKa (dissociation constant) of a polyelectrolyte.
● ↑ urine concentration = ↑ hydrogen ions
● Color reaction: blue (↓ hydrogen ions) → green (↑ hydrogen ions) → yellow (↑↑
hydrogen ions).
● Reagents:
➔ Multistix: poly (methyl vinyl ether/ maleic anhydride) bromothymol blue.
➔ Chemstrip: ethylene glycol diaminoethylether tetraacetic acid,
bromothymol blue.

4. HARMONIC OSCILLATION DENSITOMETRY

 ● The frequency of sound waves entering a solution will change in proportion to the
density (SG) of the solution.
● Example: yellow IRIS (International Remote Imaging System).
- IRIS Slideless microscope- automated microscopic urinalysis.
- IRIS Gravity meter- used for SG determination; it uses HOD.

B. pH
● Important in the identification of crystals and determination of unsatisfactory
specimens.
● Normal pH:
- Random: 4.5- 8.0
- First: 5.0- 6.0
● 9.0 pH is an indication of an old specimen or an unpreserved specimen.

REAGENT STRIP REACTION

● Principle: double indicator system.
● pH ----------> pH

Methyl Red Bromothymol Red

pH 4.0- 6.0 (↑ H+) pH 6.0- 9.0 (↓H+)

red to yellow yellow to blue

● Reagents: methyl red (acidic), bromothymol blue (alkaline).

C. PROTEIN
● Albumin- major serum protein found in urine.
● Most indicative of renal disease.
● 30 mL or greater is indicative of clinical proteinuria caused by the glomerular or
tubular disorder.
● Normal values:
➔ <10 mg/ dl or <100 mg/ 24hrs (Strasinger).
➔ Up to 150 mg/24 hrs (Henry).

CLINICAL SIGNIFICANCE
A. Pre-renal Proteinuria
● Caused by conditions affecting the plasma before it reached the kidney and,
therefore, is not indicative of actual renal disease.
Reabsorbed in plasma but some are excreted in urine:
1. Intravascular hemolysis (Hgb)- increase level of hemoglobin.
2. Muscular injury (Mgb)- increase level of myoglobin.
3. Severe inflammation (APRs)- increases the level of APRs.
4. Multiple myeloma- Ig plasma cells; they are Bence Jones Proteins. The identification of
Bence Jones Proteins: Urine precipitates at 40-60°C; dissolves at 100°C (clear).
 - Identification: serum electrophoresis, immunofixation, electrophoresis.

B. Renal Proteinuria
● “True Renal Disease”
● Glomerular proteinuria
1. Diabetic nephropathy
- May lead to renal failure.
- ↓ Glomerular filtration.
- INDICATOR: Microalbuminuria; detected urine micral test.

2. Orthostatic Proteinuria
- Also known as “Cadet” or “Postural Proteinuria”, due to standing
for a long period.
- Type of proteinuria when standing, due to increased pressure to
the renal veins.
URINE SPECIMEN ORTHOSTATIC PROTEINURIA CLINICAL PROTEINURIA

FIRST MORNING NEGATIVE POSITIVE

2 HRS AFTER POSITIVE POSITIVE

STANDING

● Tubular proteinuria
- Normally filtered albumin can no longer be reabsorbed.
- Cause of tubular dysfunction:
➔ Fanconi Syndrome
➔ Presence of toxic agents or heavy metals.
➔ Cases of severe viral infections.

C. Post-renal Proteinuria
● “After”
● Protein can be added to a urine specimen as it passes through the structures of
the lower urinary tract (ureters, bladder, urethra, prostate, and vagina).
● Cause of post-renal proteinuria:
➔ Lower UTI/ Inflammation
➔ Injury/ Trauma
➔ Menstrual contamination
➔ Spermatozoa
➔ Vaginal secretions

REAGENT STRIP REACTION

● Indicator: Sensitive to Albumin
● Principle: protein error of indicators.
● Correlations with other tests:
 - Blood
- Nitrite
- Leukocytes
- Microscopic examination
● Reagents:
➔ Multistix: tetrabromophenol blue
➔ Chemstrip: 3’, 3”, 5’, 5” -tetrachlorophenol; 3, 4, 5, 6 -tetrabromosulfophthalein.

SULFOSALICYLIC ACID PRECIPITATION TEST

- Reacts equally to all forms of protein.

REPORTING SSA TURBIDITY

GRADE TURBIDITY PROTEIN RANGE (mg/ dL)

Negative No increase in turbidity. Less than 6

Trace Noticeable turbidity. 6- 30

1+ Distinct turbidity, no granulation. 30- 100

2+ Turbidity, granulation, no 100- 200

flocculation.

3+ Turbidity, granulation, flocculation. 200- 400

4+ Clumps of protein. Greater than 400

D. GLUCOSE (Dextrose)
● Frequently performed due to its value in monitoring Diabetes Mellitus.
● Early diagnosis of DM provides an improved prognosis (the prospect of
recovery).
● Tubular reabsorption of glucose is by active transport in response to the body’s
need to maintain an adequate concentration of glucose.
● Renal threshold
- The plasma concentration at which the tubular reabsorption stops.
- 160- 180 mg/ dL.

REAGENT STRIP REACTION

● Multistix: glucose oxidase, peroxidase, potassium iodide.
● Chemstrip: glucose oxidase, peroxidase, tetramethylbenzidine.

COPPER REDUCTION TEST (Clinitest)

● Non-specific test.
● False (+) = increase level of ascorbic acid
● False (-) = “pass through phenomenon”; color production passes through to orange/ red
color then goes back to green color.
 ● Blue → Green → Yellow → Orange → Red
● Principal: copper reduction.
CLINITEST PROCEDURE PASS-THROUGH COMPOSITION OF TABLET
5 gtts + 10 gtts H2O + PHENOMENON ● CuSO4
Clinitest tablet ● Occurs if >2 g/ dL sugar ● NaCO3
is present in urine. ● Sodium Citrate
● NaOH
● Prevention of pass-through phenomenon: change the gtts (2 gtts).

E. KETONES
● Result from increased fat metabolism due to inability to metabolize
carbohydrates.
● In normal urine there are no ketones, when metabolized by fat it is completely
broken down.
● Ketones appear in urine when fat reserves are needed for energy; needed when
we can't metabolize carbohydrates.

KETONE BODIES
78% Β-hydroxybutyric acid: major ketone but not detected in reagent strip.

20% Acetoacetic acid (AAA); Diacetic acid: 1st produced by liver cells.

2% Acetone: being detected only when glycine is present.

F. BLOOD
● Hematuria
- Microscopically: Intact RBCs
● Hemoglobinuria
- Seen in cases of intravascular hemolysis.
● Myoglobinuria
- Seen in muscle injury or Rhabdomyolysis.

HEMOGLOBIN vs. MYOGLOBIN

Test Hemoglobin Myoglobin

1. Plasma Examination - Red or Pink Plasma - Pale yellow plasma

- Decrease haptoglobin - Increase CK
- Increase aldolase

2. Blondheim’s Test - Precipitated by - Not precipitated by

(Ammonium SO4) ammonium sulfate or NH4SO4.
NH4SO4. - (+) Blood
Procedure: - (-) Blood
Urine + 2.8 g NH4SO4 (80%
Saturated) ---- (Filter/
 Centrifuge) -----> Supernatant

G. BILIRUBIN
● An early indication of liver disease.
● Conjugated bilirubin enters the urine as a result of leakage from a damaged liver.
Conditions related:
- Cirrhosis
- Hepatitis
- Bile duct obstruction
● “TEA-COLORED”
● Amber urine with yellow foam.

REAGENT STRIP REACTION

● Principle: diazo reaction
● Reagents:
- Multistix: 2,4-dicholoroaniline diazonium salt
- Chemstrip: 2,6-dicholorobenzenediazonium salt

H. UROBILINOGEN
● Bile pigment is a result of hemoglobin degradation.
● Conjugated bilirubin converted to urobilinogen in the intestine.
● Small amount in normal urine (afternoon: 2-4:00 PM) (<1 mg/dL or Ehrlich unit).

URINE BILIRUBIN and UROBILINOGEN in JAUNDICE

Urine Bilirubin Urobilinogen

Bile Duct Obstruction +++ Normal

Liver Damage +|- ++

Hemolytic Disease (-) +++

WATSON- SCHWARTZ TEST

● Uses extraction with organic solvents.
- Chloroform
- Butanol
● Soluble to both chloroform and butanol: UROBILINOGEN
● Insoluble to both chloroform and butanol: PORPHOBILINOGEN
● Soluble to butanol and insoluble to chloroform: OTHER EHRLICH’S COMPOUNDS
● For differentiating urobilinogen and porphobilinogen from other Ehrlich’s reactive
compounds.

I. NITRITE
● Specimen: first morning or 4hr urine.
 ● Rapid screening test for UTI/ bacteriuria.
● (+) for nitrite: 100,000 organisms/ mL.
● Pink spots or edges: (-) NEGATIVE

REAGENT STRIP REACTION

● Multistix: p-arsanilic acid tetrahydrobenzo(h)-quinolin-3-ol
● Chemstrip: sulfanilamide, hydroxytetrahydro benzoquinoline

J. LEUKOCYTES
● Screening test of urine culture specimen.

REAGENT STRIP REACTION

● Principle: leukocyte esterase
● (+): Neutrophils, Eosinophils, Monocytes, Basophils, Histocytes, Trichomonas; detected
because they contain esterase.
● Lymphocytes- not detected because there’s no esterase.
● Reagents:
- Multistix: derivatized pyrrole amino acid ester diazonium salt
- Chemstrip: indoxylcarbonic acid ester diazonium salt

K. ASCORBIC ACID
● A reducing agent that causes false-negative reactions.
● Affected are Blood, Bilirubin, Leukocytes, Nitrite, and Glucose (“BB LNG”).
● 11th reagent pad: ascorbic acid (≥5 mg/dL) + phosphomolybdate ---> (+)
molybdenum blue
● GC- MS is a more accurate quantitative method.

SEDIMENT CONSTITUENTS

RBCs (Hematuria)
● Biconcave disk, about 7 μm, smooth, non-nucleated.
● Hypertonic: crenated or shrink.
● Hypotonic: swell or hemolyzed (ghost cell).
● Glomerular damage/ injury: dysmorphic with projections, and fragmented.
● NV = 0-2 or 0-3/hpf.
● Sources of error in the microscopic examination: “COYA”
- CaOX
- Oil droplets
- Yeasts
- Air bubbles

WBCs (Pyuria)
● Increased WBCs indicate the presence of infection or inflammation.
 ● Hypertonic solution: granules from neutrophils will swell and will undergo Brownian
movement that will produce a sparkly appearance, which is called GLITTER CELLS
(non-pathogenic).
● >1% Eosinophils = significant; eosinophils should be <1% only.
● NV = 0-5 or 0-8/ hpf
● Larger than RBCs and contain a nucleus,
● Neutrophils are predominant.

EPITHELIAL CELLS
A. SQUAMOUS EPITHELIAL CELLS
● The largest cell in the urine sediment.
● Considered as a point of reference in focusing a microscope.
● An increase of # in female urine indicates specimen is not collected using
midstream clean catch.
● Variation: clue cells.
- An epithelial cell that is studded with bacteria (bacterial vaginosis) caused
by Gardnerella vaginalis.
- To detect, a Whiff or Sniff test is performed.
- Vaginal discharge + 10% KOH = Fishy- amine like odor.

B. TRANSITIONAL EPITHELIAL CELLS (UROTHELIAL CELLS)

● Three different forms: spherical, caudate, and polyhedral.
● Normally seen after catheterization (seen in clumps) procedures.
● ↑ # transitional epithelial cells indicate malignancy.
● ↓ # indicates NO CLINICAL SIGNIFICANCE.

C. RENAL TUBULAR EPITHELIAL CELLS

● Clinical Significance: Tubular injury/ necrosis (often seen in poisoning or viral
infection).
● >2 RTE/ hpf, an indication of tubular injury/ necrosis.
● RTE cell variation:
➢ Oval fat body
- Lipid containing RTE cells.
- Identified using the lipid stains (triglycerides and neutral fats) and
polarizing microscope (cholesterol: “Maltese Cross” appearance).
➢ Bubble Cells
- Seen in acute tubular necrosis.
- RTE cell with the nonlipid-filled vacuole.

YEASTS
● Oval structures with buds or mycelia.
● True yeast infection, there is the presence of yeasts and WBC.

PARASITES
● T. vaginalis
 - The most frequent parasite encountered.
- Agent of Pingpong disease.

● S. haematobium
- Associated with bladder cancer.
- Causes hematuria.
- Marker: Nuclear Matrix Protein.
- To detect, 24 hr unpreserved urine.

BACTERIA
● Small spheres (cocci) and rod-shaped organisms.
● Clinical significance: UTI (presence of bacteria and WBCs).
➔ Enterobacteriaceae- most common cause of UTI.
➔ Staphylococcus
➔ Enterococcus

MUCUS THREADS
● Major protein: Tamm-Horsfall Protein (Uromodulin)
● No Clinical Significance

SPERMATOZOA
● Occasionally, it is found in both male and female urine specimens after the following
intercourse; for males, it could be due to masturbation, or it is possible in nocturnal emission.

CASTS (Cylindruria)
● Major constituent: Tamm- Horsfall Protein (Uromodulin)- produced by RTE cells.
● Detect along the edges of the coverslip with subdued light.
● Formed in the distal and collecting tubules.
● Only elements found in the urinary sediment are unique to the kidney.
● Sequence: Hyaline cast → Cellular cast → Coarse granular cast → Fine granular cast →
Waxy cast.

1. HYALINE CASTS
- NV: 0-2/ lpf
- Prototype cast; the beginning of all types of the cast.
- Cellular cast.
2. RBC CASTS
- Cellular cast
- Indication of bleeding within the nephron.
3. WBC CASTS
- Indication of inflammation within the nephron.
- To differentiate upper and lower UTIs:
Upper UTI Lower UTI

Pyelonephritis with Cystitis with No

 Cast Cast
- To differentiate from epithelial cell cast:
1. Phase Contrast Microscopy
2. Supravital Stain

4. COARSE GRANULAR CASTS

- Being formed from the disintegration of the cellular cast.
5. FINE GRANULAR CASTS
- Being formed from the disintegration of the cellular cast.
- Smooth
6. FATTY CASTS
- To detect cast, Sternheimer Malbin is used to stain cast, however, it cannot stain
fatty casts.
- High # of fats.
7. WAXY CASTS
- Final degenerative form of all types of casts.
8. BROAD CASTS
- Formed when there is renal failure.
- “Renal Failure Cast”
- Widening and destruction in tubular walls.

CRYSTALS
● Formed by the precipitation of urine solutes, including inorganic salts, organic
compounds, and medications (iatrogenic compounds).
● Extremely abundant in refrigerated urine.
● Factors: “TEMPS”
- Temperature
- pH
- Solute concentration

NORMAL CRYSTALS
● Amorphous urates
- Frequently encountered in the refrigerated specimens; pink sediment (due to
presence of uroerythrin).

● Uric acid
- Rhombic, wedge, rosette, hexagonal, 4-sided plate, or lemon-shaped.
- Mistaken as cystine.
- Clinical significance:
➔ Found in cases of ↑ Lesch-Nyhan Syndrome- orange sand in a diaper.
➔ ↑ Gout
➔ ↑ Chemotherapy
 ● Calcium oxalate
- Two types: Weddellite and Whewellite.
➔ Weddellite- dihydrate CaOx; envelope or pyramidal.
➔ Whewellite- monohydrate CaOx; oval or dumbbell shape.
➔ ↑ Ethylene glycol poisoning.
- Most renal stones consist of CaOx.

● Amorphous phosphates
- White precipitation in a urine specimen.
- ↑ after a meal due to alkaline tide.

● Ammonium biurate
- Indication of an old specimen due to the presence of urea-splitting bacteria.

● Triple phosphate
- Magnesium ammonium phosphate.
- Struvite, or Staghorn appearance- large structure with multiple branches.

● Calcium phosphate
- Flat rectangular plates are often in rosette forms.
- Rosette forms resembles sulfonamides.

● Calcium carbonate
- Spherical
- (+) Effervescence

ABNORMAL CRYSTALS
● Cystine
- Hexagonal plates of which are seen in cases of cystinuria.

● Cholesterol
- Staircase pattern; seen in cases of lipiduria (Nephrotic Syndrome).
- Resembles Radiographic Contrast Media

To differentiate RCM from Cholesterol:

1. Know the patient history.
2. Correlate with other UA results.
3. RCM: ↑ SG ≥ 1.040

● Tyrosine
- Needle-like structure.
- Most commonly seen in liver disease.
- (+) in Nitrosonaphthol test.
 ● Leucine
- Present in liver disease.
- Yellow-brown concentric circles with radial striations.

● Bilirubin
- Present in cases of liver disease.
- Clump-needles or granules.
- (+) Diazo reaction

● Sulfonamide
- Deposits in nephrons.
- Indication of possible tubular damage.
- Needles, sheaves of wheat, and rosette.

To differentiate Sulfonamide and Calcium phosphate: LIGNIN TEST

1. Newspaper
2. Urine
3. + 25% HCL
POSITIVE: YELLOW (Sulfonamide)

● Ampicillin
- Massive doses of ampicillin are being taken by patients.

GLOMERULAR DISORDERS
DISORDER ETIOLOGY CLINICAL COURSE

Acute glomerulonephritis Deposition of immune Rapid onset of hematuria and

complexes, formed in edema, permanent renal
conjunction with group A damage seldom occurs.
Streptococcus infection,
on the glomerular
membranes.

Rapidly progressive Deposition of immune Rapid onset with glomerular

glomerulonephritis complexes from systemic damage and possible
immune disorders on the progression to end-stage
glomerular membrane. renal failure.

Goodpasture syndrome Attachment of a cytotoxic Hemoptysis and dyspnea

antibody formed during viral followed by hematuria.
respiratory infections to
glomerular and alveolar Possible progression to
basement membranes. end-stage renal failure.

Wegener granulomatosis Antineutrophil cytoplasmic Pulmonary symptoms

autoantibody including hemoptysis
develop first followed
 binds to neutrophils in by renal involvement and
vascular walls producing possible progression to
damage to small vessels in end-stage renal failure.
the lungs and glomerulus.

Henoch-Schönlein Occurs primarily in children The initial appearance of

purpura following viral respiratory purpura
infections; a decrease in followed by blood in sputum
platelets disrupt vascular and stools and eventual renal
integrity. involvement.

Complete recovery is
common but may progress to
renal failure.

Membranous Thickening of the glomerular A slow progression to the

glomerulonephritis membrane following IgG nephrotic syndrome or
immune complex deposition possible remission.
associated with systemic
disorders.

Membranoproliferative Cellular proliferation affecting A slow progression to chronic

glomerulonephritis the capillary walls or the glomerulonephritis or
glomerular basement nephrotic syndrome.
membrane, possibly
immune-mediated.

Chronic glomerulonephritis A marked decrease in renal Noticeable decrease in renal

function resulting from function progressing to renal
glomerular damage failure.
precipitated by other renal
disorders.

IgA nephropathy or Berger Deposition of IgA on the Recurrent macroscopic

Disease glomerular membrane hematuria following exercise
resulting from increased with slow progression to
levels of serum IgA. chronic glomerulonephritis.

Nephrotic syndrome Disruption of the shield of Acute onset following

negativity and damage to the systemic shock.
tightly fitting podocyte barrier
resulting in massive loss of Gradual progression from
protein and lipids. other glomerular disorders
and then to renal failure.

Minimal change disease Disruption of the podocytes Frequent complete remission

occurring primarily in children following corticosteroid
following allergic reactions treatment.
and immunizations.
 Focal segmental Disruption of podocytes in May resemble nephrotic
glomerulosclerosis certain areas of glomeruli syndrome or minimal change
associated with heroin and disease.
analgesic abuse and AIDS.

Alport syndrome Genetic disorder showing A slow progression to

lamellated and thinning nephrotic syndrome and
glomerular basement end-stage renal disease.
membrane.

NEPHROTIC SYNDROME
↓ Oncotic Pressure
↑ Fluid Loss into Spaces
- There are spaces in the glomerulus that allow constituents (albumin) to go out.
- Albumin: a major protein that can maintain oncotic pressure.
- Maintaining oncotic pressure prevents leakage of the fluids.
- It causes edema.

Normal Glomerulus:
● Tightly fit- no constituents or fluids can go out of the glomerulus.

TUBULAR DISORDERS
DISORDER ETIOLOGY CLINICAL COURSE

Acute tubular necrosis Damage to renal tubular cells Acute onset of renal
caused by ischemia or toxic dysfunction is usually
agents. resolved when the underlying
cause is corrected.

Fanconi syndrome Inherited in association with Generalized defect in renal

cystinosis and Hartnup tubular reabsorption requiring
disease or acquired through supportive therapy.
exposure to toxic agents.

Uromodulin-associated An inherited defect in the Continual monitoring of renal

kidney disease production of normal function for progression to
uromodulin by the renal renal failure and possible
tubules and increased uric kidney transplantation.
acid causing gout.

Nephrogenic diabetes Inherited defect of tubular Requires supportive therapy

insipidus response to ADH or acquired to prevent dehydration
from medications.

Renal glucosuria Inherited autosomal Benign disorder.

recessive trait.
Neurogenic Diabetes- synthesis and release of ADH are decreased.
Nephrogenic Diabetes- normal synthesis and release of ADH, however, there’s a defect in
tubular response to ADH.
Dehydration- life-threatening to diabetes insipidus patients, because the plasma becomes
hypertonic which makes the patient shocked.

INTERSTITIAL DISORDERS
DISORDER ETIOLOGY CLINICAL COURSE

Cystitis Ascending bacterial infection Acute onset of urinary

of the bladder. frequency and burning
resolved with antibiotics.

Acute pyelonephritis Infection of the renal tubules Acute onset of urinary

and interstitium is related to frequency, burning, and
the interference of urine flow lower back pain resolved with
to the bladder, reflux of urine antibiotics.
from the bladder, and
untreated cystitis.

Chronic pyelonephritis Recurrent infection of the Frequently diagnosed in

renal tubules and interstitium children; requires correction
caused by structural of the underlying structural
abnormalities affecting the defect.
flow of urine.
Possible progression to renal
failure.

Acute interstitial nephritis Allergic inflammation of the Acute onset of renal

renal interstitium in response dysfunction is often
to certain medications. accompanied by a skin rash.

Resolves following
discontinuation of medication
and treatment with
corticosteroids.

RENAL CALCULI/ RENAL LITHIASIS

● “Kidney Stones”
● Conditions favoring the formation of renal calculi.
● Primary urinalysis finding: Microscopic Hematuria
● Factors that contribute to the formation of calculi: “CPU”
- Chemical Concentration
- pH
- Urinary Stasis
Renal Calculi Description
 Calcium Oxalate (CaOx) Major constituents of renal calculi.
Very hard, dark in color with a rough surface.

Uric Acid Associated with increased intake of foods

with high purine content.
Yellowish to browning red and moderately
hard.

Cystine Seen in hereditary disorders of cysteine

metabolism.
Yellow-brown, greasy, and resembles an old
soap.
Least common calculi (1-2%)

Phosphate Pale and friable.

Magnesium Ammonium Phosphate Accompanied by a urinary infection involving

(Triple Phosphate/ Struvite) urea-splitting bacteria.

CEREBROSPINAL FLUID
- 3rd major body fluid.
- Production through filtration and active transport secretion.
● Functions:
1. Supply nutrients to the nervous tissue.
2. Remove metabolic waste.
3. Provide a mechanical barrier to cushion the brain and spinal cord against trauma.

Choroid Plexus
● Produce CSF at approximately 20 ml/ hr.

PRODUCTION OF CSF:
- CSF is composed of water and a small amount of organic and inorganic substances.
- RBC, WBC(not filtered), Protein, Sodium flows in capillary and will be filtered to the
choroid plexus.

Arachnoid Villi/ Granulations

● Reabsorbs CSF.
● From arachnoid villi to capillary.
● Prevents reflux of the fluid.
● If arachnoid villi cannot reabsorb CSF, CSF will accumulate into the brain that will cause
Hydrocephalus.

BLOOD-BRAIN BARRIER
● Functions:
1. Protects the brain from organisms.
 2. Shields the brain from hormones and neurotransmitters.
3. Maintains homeostasis for the brain.

Disruption in Blood-Brain Barrier:

- It will allow WBCs, proteins, and other chemicals to enter the CSF.
- Examples: Meningitis, Multiple Sclerosis

SPECIMEN COLLECTION
● Method: Lumbar puncture (Physician)
● Collected between 3rd, 4th, and 5th lumbar vertebrae.
● 3 sterile CSF tubes.
1st Tube: Chemistry/ Serology; FROZEN
2nd Tube: Microbiology; ROOM TEMPERATURE
3rd Tube: Hematology/ Cell Count; REFRIGERATED
4th tube (optional): Microbiology/ Serology- exclusion of skin contamination.

What if 1 tube was only collected?

1. Microbiology- to avoid contamination.
2. Hematology- cells disintegration.
3. Chemistry/ Serology- least affected.

CSF APPEARANCE:
● NORMAL
➔ Crystal clear

● HEMOLYZED/ BLOODY
➔ Traumatic tap- an indication of puncture of blood vessels.
➔ Intracranial hemorrhage- an indication of bleeding within the braincase.

● XANTHOCHROMIA- the appearance of CSF that has different colors.

➔ Pink- slight amount of oxyhemoglobin.
➔ Orange- heavy hemolysis.
➔ Yellow- oxyhemoglobin into bilirubin.
➔ RBC degradation products- most common cause of xanthochromia.

TRAUMATIC TAP vs. INTRACRANIAL HEMORRHAGE

Traumatic Tap Intracranial Hemorrhage

Distribution of blood on 3 1>2>3 1= 2= 3

tubes UNEVEN EVEN

Clot formation (+) due to plasma fibrinogen (-)

Supernatant Clear Xanthochromic

 Erythrophage (macrophages (-) (+)
with ingested RBCs)

CSF CELL COUNT

- Must be performed immediately.
- RBCs and WBCs lyse within 1 hour.

1. WBC COUNT
● Routinely performed in CSF.
● Diluting Fluid: 3% acetic acid with methylene blue.
● WBC Count = (no. of cells) (dilution factor) / (no. of sq. ctd) (vol. of 1 sq.)
● The volume of 1 sq.- 0.1 uL

2. RBC COUNT
● To correct for WBC count and total protein concentration.
➔ Subtract 1 WBC for every 700 RBCs seen.
➔ Subtract 8 mg/ dl total protein concentration for every 10,000 RBCs/ ul
(Henry)
➔ Subtract 1 mg/ dl total protein concentration for every 1, 200 RBCs/ ul
(Strasinger)

PREDOMINANT CELLS IN CSF- lymphocytes and monocytes; occasionally, neutrophils.

● Adult
- 70 (lymph):30 (mono)
- 90%: Pleocytosis- an indication of the increased number of normal cells in CSF.

● Neonates
- Up to 80% of monocytes are considered normal.
- Monocytes are greater than lymphocytes.
- Inversed ratio.

MEASUREMENT OF TOTAL CSF PROTEIN:

1. TURBIDIMETRIC
● Principle: Precipitation of protein.
● TCA- reagent of choice; precipitates albumin and globulin.
● SSA- precipitates albumin; to precipitates, sodium sulfates are needed.

2. DYE-BINDING TECHNIQUE
● Principle: Protein error indicator.
● Increase the level of protein, increase the intensity of the blue color (more protein
content).
● Coomassie brilliant blue
- The protein binds to dye = red to blue
MEASUREMENT OF PROTEIN FRACTIONS:
1. CSF/ SERUM ALBUMIN INDEX
● Increase CSF/ Serum Index, Increase CSF Albumin
● Assess the integrity of the blood-brain barrier.
a. Index <9- indicates an intact blood-brain barrier (normal).
b. Index ≥9- indicates that the blood-brain barrier is damaged.
c. Index 100- indicated complete damage to BBB.

2. IgG INDEX
● Increase IgG index, Increase CSF IgG
● Assess conditions with IgG production within the CNS (ex: multiple sclerosis)
a. Index >0.70- indicates IgG production within the CNS or the Central
Nervous System.
b. Index <0.70- indicates that there’s no active IgG production within CNS.

CSF ELECTROPHORESIS
● Elevated IgG indices and presences of 2 or more oligoclonal bands.
● 2 or more oligoclonal bands- complementary findings useful for the diagnosis of multiple
sclerosis.
● Oligoclonal bands- bands found in CSF but not in the serum.

CSF GLUCOSE
● Normal Value: 60- 70% of plasma glucose concentration.
● Example: Blood- 100 mg/dL CSF- 60 to 70 mg/dL
● Plasma glucose must also be run for comparison.
● Specimens for blood glucose should be drawn 2 hours prior to the spinal tap.

CSF LACTATE
● Frequently used to monitor Severe Head Injuries.
● Valuable in managing meningitis.
● Inversely proportional to glucose.

CSF GLUTAMINE
● Requested for patients with coma of unknown origin.
● Indirect test for the presence of excess ammonia in CSF.
● Product of ammonia and α-ketoglutarate.

CSF ENZYMES:
LACTATE DEHYDROGENASE (LDH)
● LD 1 and 2 = for brain tissues.
● LD 2 and 3 = for lymphocytes.
● LD 4 and 5 = for neutrophils.

SERUM LDH
 ● Normal - LD 2> 1> 3> 4> 5
● AMI- LD 1>2 “flipped pattern”

CSF LDH
● Normal- LD 1> 2> 3> 4> 5
● Neurological abnormalities- LD 2>1
● Bacterial meningitis- LD 5> 4> 3> 2> 1

AGENTS OF BACTERIAL MENINGITIS

Streptococcus agalactiae Neonates- 1 month

Haemophilus influenzae 1 month- 5 years old

Neisseria meningitidis 5- 29 years old

Streptococcus pneumoniae >29 years old

Listeria monocytogenes Infants, the elderly, immunocompromised

patients

SEMINAL FLUID
STRUCTURE FUNCTION

Seminiferous tubules of testes Spermatogenesis.

● Sertoli cells - nurse cells.

Epididymis Sperm maturation.

● Sperm becomes motile.

Ductus deferens Propel sperm to ejaculatory ducts

Seminal vesicles Provide nutrients for sperm and fluid.

● Major contributor in semen.
● Increase in fructose.

Prostate Gland Provide enzymes and proteins for coagulation

and liquefaction.

Bulbourethral glands Add alkaline mucus to neutralize prostatic

acid and vaginal acidity.

SPECIMEN COLLECTION
● Abstinence
- 2 to 3 days but not more than 7 days.
- Increase abstinence, increase semen volume, decrease sperm motility.
● Delivery of specimens should be within 1 hour (room temperature).
● Analysis should be done after liquefaction (30 to 60 minutes).
METHODS OF COLLECTION
● Masturbation- the best method of collection.
● Coitus interruptus- withdrawal method.
● Condom method- non-lubricant containing rubber or silastic type of condom.

SEMEN ANALYSIS
● Liquefaction Time
- Not liquified after 2 hours use α-chymotrypsin or bromelain.
● Volume
- Decrease: Incomplete collection/ infertility.
- Increase: Prolonged abstinence.
● Yellowish Semen
- Prolonged abstinence.
- Medication.
- Urine contamination.
● White Turbidity
- Increase (WBCs): Infection.
● Red/ Brown Coloration
- (+) RBCs
● Viscosity
- Reporting:
0 - Watery
4 - Gel-like
● pH
- Too basic: Infection
- Too acidic: Increase prostatic fluid.

SPERM CONCENTRATION:
IMPROVED NEUBAUER COUNTING CHAMBER

● Diluting Fluids- immobilize sperms

➔ Distilled water
➔ Sodium bicarbonate (NaHCO3)
➔ Formalin
➔ Saline
● Alternative: Cold Tap Water
● Dilution: 1:20

SHORTCUT METHOD FOR SPERM CONCENTRATION COMPUTATION:

1) 2 WBC SQUARES = # of sperms counted x 100,000
● Answer: sperms in million/ mL

2) 5 RBC SQUARES = # of sperms counted x 1,000,000

 ● Answer: sperms in million/ mL

LONG METHOD FOR SPERM CONCENTRATION

● SPERM CONCENTRATION = (# sperms counted) (dilution) / (# of squared counted)
(depth)

Example:
60 Sperm Counted
5 RBC Squares

(60)(20) / (5/25)(0.1) = 60,000/ uL

60,000 uL x 1000 = 60,000, 000 mL

SPERM COUNT
● FORMULA = sperm concentration x volume of specimen

Compute for the sperm count:

Sperm conc. = 60M sperms/ mL
Sample volume = 4mL/ ejaculate

60,000,000 sperms/ mL x 4 mL/ ejaculate = 240, 000, 000/ ejaculate.

SPERM MOTILITY
- View the sperm into microscopes.
- Read 20 Hpf.
- Quality: ≥ 2.0 (to be qualified normal).

SPERM MOTILITY GRADING

Grade WHO Criteria Sperm Motility Action

4.0 a Rapid, straight-line motility.

3.0 b Slower speed, some lateral

movement.

2.0 b Slow forward progression,

noticeable lateral movement.

1.0 c No forward progression.

0 d No forward.

SPERM MORPHOLOGY
- At least 200 sperms were evaluated for the morphology.
1. Routine Criteria- >30% Normal Morphology
2. Kruger’s Strict Criteria- >14% Normal Morphology.
 - Head, neck, tail (not routinely used but recommended by WHO).

REFERENCE SEMEN CHEMICAL VALUES

Neutral α-glucosidase ≥ 20 mU/ejaculate
- The decrease indicates epididymis
disorder.

Zinc ≥ 2.4 umol/ ejaculate

- The decrease indicates a decrease in
prostatic fluid.

Citric acid ≥52 umol/ ejaculate

- The decrease indicates a decrease in
prostatic fluid.

Acid phosphatase ≥200 Units/ ejaculate

- The decrease indicates a decrease in
prostatic fluid.

MICROBIAL TESTING
● ROUNDS CELLS
- WBCs and spermatids (immature sperm cells).
- Normal value: <1 million/ ml
- >1 million WBCs/ ml= indicates INFECTION.
- >1 million spermatids/ ml= indicates DISRUPTION IN SPERMATOGENESIS.

TERMINOLOGY
● ASPERMIA = No ejaculate; complete absence of semen.
● AZOOSPERMIA = Absence of sperm cells.
● NECROSPERMIA = Immotile/ dead sperm cells.
● OLIGOSPERMIA = Decreased sperm concentration.