Clinical Microscopy & Urinalysis
Master urine analysis, body fluid examination, and clinical microscopy for the MedTech board exam.
Table of Contents
1. Urine Formation & Specimen Collection
Kidney Physiology Review
- • Nephron: Functional unit of kidney (~1 million per kidney)
- • Glomerular filtration: 180 L/day filtrate (99% reabsorbed)
- • Normal urine output: 1-2 L/day (600-2000 mL)
- • GFR: ~125 mL/min (measured by creatinine clearance)
- • Filtration barrier: Endothelium, basement membrane, podocytes
Urine Composition
Normal Components
- • Water: 95%
- • Urea: Major nitrogenous waste
- • Creatinine: Muscle metabolism
- • Uric acid: Purine metabolism
- • Electrolytes: Na, K, Cl, phosphates
- • Hormones, pigments
Abnormal Components
- • Protein (>150 mg/day)
- • Glucose
- • Blood/hemoglobin
- • Bilirubin
- • Ketones
- • Bacteria, WBCs
Specimen Collection Types
| Specimen Type | Description | Best For |
|---|---|---|
| Random | Any time, no preparation | Routine screening |
| First Morning | Most concentrated, 8+ hours in bladder | Pregnancy test, protein, nitrite, microscopic |
| Second Void | Fresh specimen after first morning | Glucose monitoring (diabetes) |
| Clean-Catch Midstream | After cleansing, midstream portion | Urine culture, reduces contamination |
| 24-Hour Collection | All urine over 24 hours | Creatinine clearance, protein quantitation |
| Catheterized | Sterile via catheter | Bedridden patients, sterile culture |
| Suprapubic Aspiration | Needle through abdominal wall | Infants, anaerobic culture |
Specimen Handling
Critical Points
- • Analyze within 2 hours or refrigerate (4°C for up to 24 hours)
- • Preservatives: Formalin, thymol, boric acid, refrigeration
- • Delays cause: Bacterial overgrowth, increased pH, decreased glucose, cast dissolution
- • Minimum volume: 12 mL (routine UA), 50 mL (culture)
2. Physical Examination of Urine
Color
| Color | Cause | Significance |
|---|---|---|
| Pale/Colorless | Dilute, diabetes insipidus | Low specific gravity |
| Yellow (normal) | Urochrome pigment | Normal |
| Dark Yellow/Amber | Concentrated, bilirubin | Dehydration, liver disease |
| Orange | Bilirubin, phenazopyridine (Pyridium) | Liver disease, medication |
| Red/Pink | Blood, hemoglobin, myoglobin, beets | Hematuria, hemolysis, rhabdomyolysis |
| Brown/Black | Myoglobin, methemoglobin, melanin | Rhabdomyolysis, alkaptonuria |
| Green | Biliverdin, Pseudomonas, medications | Infection, drugs |
| Milky/White | Pyuria, chyluria, lipiduria | UTI, lymphatic obstruction |
Clarity/Turbidity
Normal: Clear
- • Freshly voided urine should be clear
- • May cloud on standing (normal)
Causes of Turbidity
- • Cells: RBCs, WBCs, epithelial
- • Bacteria, yeast
- • Crystals (urates, phosphates)
- • Mucus, lipids, spermatozoa
- • Contrast media, chyle
Specific Gravity
- • Normal range: 1.005 - 1.030
- • First morning: Should be >1.023 (kidney concentrating ability)
- • Isosthenuria: Fixed at 1.010 (renal failure)
- • Methods: Urinometer, refractometer (gold standard), reagent strip
Increased SG (>1.030)
- • Dehydration
- • Glucosuria, proteinuria
- • IV contrast media
- • SIADH
Decreased SG (<1.005)
- • Diabetes insipidus
- • Excessive fluid intake
- • Renal tubular damage
- • Diuretics
Odor
- • Normal: Aromatic (faint, not offensive)
- • Ammonia: Old specimen, UTI (urea-splitting bacteria)
- • Fruity/Sweet: Ketones (diabetic ketoacidosis)
- • Maple syrup: Maple syrup urine disease (branched-chain amino acids)
- • Mousy/Musty: Phenylketonuria (PKU)
- • Foul/Putrid: Bacterial infection
3. Chemical Examination of Urine
pH
- • Normal range: 4.5 - 8.0 (average 6.0)
- • Method: Double indicator (methyl red + bromthymol blue)
- • Acidic urine: High protein diet, metabolic acidosis, diabetes
- • Alkaline urine: UTI (Proteus), vegetarian diet, old specimen, renal tubular acidosis
- • Clinical use: Stone management, drug therapy monitoring
Protein
Principle: Protein Error of Indicators
- • Normal: Negative (<10 mg/dL)
- • Trace to 4+ grading
- • Most sensitive to albumin (may miss Bence Jones protein)
- • False positive: Highly alkaline urine, quaternary ammonium compounds
- • False negative: Dilute urine, non-albumin proteins
Types of Proteinuria
- • Glomerular: Albumin, high molecular weight (nephrotic syndrome)
- • Tubular: Low molecular weight proteins (Fanconi syndrome)
- • Overflow: Bence Jones protein (multiple myeloma)
- • Post-renal: Inflammation, bleeding in urinary tract
- • Functional: Exercise, fever, orthostatic
Glucose
Glucose Oxidase Method
- • Normal: Negative (<30 mg/dL)
- • Renal threshold: ~160-180 mg/dL blood glucose
- • Glucosuria causes: Diabetes mellitus, renal glucosuria, pregnancy
- • Specific for glucose (not other sugars)
- • False negative: Ascorbic acid (vitamin C), high specific gravity
- • False positive: Oxidizing cleaning agents
Ketones
Legal Nitroprusside Reaction
- • Detects: Acetoacetic acid > Acetone
- • Does NOT detect: Beta-hydroxybutyrate (most abundant in DKA)
- • Ketonuria causes: Diabetic ketoacidosis, starvation, vomiting, high fat diet
- • False positive: Phthalein dyes, some drugs
Blood
Peroxidase-like Activity
- • Detects: Hemoglobin, myoglobin, intact RBCs
- • Hematuria: Intact RBCs (confirm microscopically)
- • Hemoglobinuria: Free hemoglobin (hemolysis)
- • Myoglobinuria: Muscle damage (rhabdomyolysis)
- • False positive: Oxidizing agents, bacterial peroxidases
- • False negative: High specific gravity, ascorbic acid, formalin
Other Chemical Tests
Bilirubin
- • Diazo reaction
- • Only conjugated bilirubin in urine
- • Hepatocellular disease, obstruction
- • Light sensitive - test promptly
Urobilinogen
- • Ehrlich's aldehyde reaction
- • Normal: 0.2-1.0 EU/dL
- • Increased: Hemolytic anemia, hepatitis
- • Absent: Biliary obstruction, broad-spectrum antibiotics
Nitrite
- • Detects nitrate-reducing bacteria (most GNRs)
- • Requires 4+ hours incubation in bladder
- • First morning specimen ideal
- • Not all bacteria produce nitrite (Enterococcus, Staph)
Leukocyte Esterase
- • Enzyme from granulocytes (neutrophils)
- • Indicates pyuria
- • False negative: High glucose, protein, ascorbic acid
- • Combined with nitrite = presumptive UTI
4. Microscopic Examination of Urine
Specimen Preparation
- Mix well, pour 10-15 mL into conical tube
- Centrifuge at 400g (1500-2000 RPM) for 5 minutes
- Decant supernatant, resuspend sediment (0.5 mL)
- Place drop on slide with coverslip
- Examine under reduced light (lower condenser)
- Report per low-power field (LPF) or high-power field (HPF)
Cells in Urine
| Cell Type | Size/Appearance | Normal | Significance |
|---|---|---|---|
| RBCs | 7-8 µm, biconcave, no nucleus | 0-2/HPF | Glomerulonephritis, stones, trauma, tumors |
| WBCs | 10-12 µm, granular, lobed nucleus | 0-5/HPF | UTI, pyelonephritis, inflammation |
| Squamous epithelial | Largest cell, flat, small nucleus | Few | Vaginal/urethral contamination |
| Transitional epithelial | Pear-shaped or round, large nucleus | Few | Renal pelvis, ureter, bladder |
| Renal tubular epithelial | Round, large eccentric nucleus | 0-1/HPF | Acute tubular necrosis, nephrotoxicity |
Dysmorphic RBCs
Irregular shapes, membrane blebs - indicate glomerular origin (glomerulonephritis). Isomorphic RBCs suggest lower urinary tract bleeding.
Urinary Casts
Formation: Formed in renal tubules, Tamm-Horsfall protein matrix. Indicates renal pathology!
| Cast Type | Appearance | Significance |
|---|---|---|
| Hyaline | Colorless, transparent | Normal (0-2/LPF), exercise, dehydration |
| RBC | Orange-red, RBCs embedded | Glomerulonephritis (pathognomonic) |
| WBC | WBCs in matrix | Pyelonephritis, interstitial nephritis |
| Renal tubular epithelial | RTE cells in matrix | Acute tubular necrosis, nephrotoxicity |
| Granular (coarse) | Large granules, dark | Degraded cellular casts, renal disease |
| Granular (fine) | Fine granules | Further degeneration, chronic renal disease |
| Waxy | Waxy, sharp edges, cracked | End-stage degeneration, chronic renal failure |
| Fatty | Fat globules, Maltese cross | Nephrotic syndrome |
| Broad | Wide diameter, any type | Renal failure, tubular dilation |
Crystals
Acidic Urine Crystals
- • Uric acid: Yellow-brown, rhombic, rosettes
- • Calcium oxalate: Envelope/dumbbell, birefringent
- • Amorphous urates: Yellow-brown, granular
- • Cystine: Hexagonal, colorless (pathologic)
- • Tyrosine: Fine needles (liver disease)
- • Leucine: Yellow spheres, concentric circles
Alkaline Urine Crystals
- • Triple phosphate: Coffin lid, prism
- • Ammonium biurate: Thorny apple (yellow-brown)
- • Calcium phosphate: Prisms, rosettes
- • Amorphous phosphates: White, granular
- • Calcium carbonate: Dumbbell, small
Always Pathologic Crystals
- • Cystine: Cystinuria (hexagonal, cyanide-nitroprusside +)
- • Cholesterol: Nephrotic syndrome (notched plates)
- • Tyrosine/Leucine: Severe liver disease
- • Sulfonamide: Drug crystals (sheaves, rosettes)
Other Microscopic Findings
- • Bacteria: Rods or cocci, >10⁵ CFU/mL significant
- • Yeast: Oval, budding (Candida)
- • Trichomonas vaginalis: Motile, flagellated protozoan
- • Spermatozoa: Head and tail, post-ejaculation
- • Mucus: Threads, ribbons (not significant)
- • Oval fat bodies: RTE cells with lipid, Maltese cross under polarized light
5. Clinical Correlations
Urinary Tract Infection (UTI)
Typical Findings
- • Turbid urine, foul odor
- • Positive leukocyte esterase
- • Positive nitrite (if GNR)
- • WBCs >10/HPF (pyuria)
- • Bacteria present
- • WBC casts if pyelonephritis
Glomerulonephritis
Typical Findings
- • Smoky/cola-colored urine
- • Proteinuria
- • Hematuria (dysmorphic RBCs)
- • RBC casts (pathognomonic)
- • Decreased GFR
Nephrotic Syndrome
Typical Findings
- • Heavy proteinuria (>3.5 g/day)
- • Oval fat bodies
- • Fatty casts
- • Free fat droplets
- • Maltese cross under polarized light
- • Waxy casts (chronic)
Diabetes Mellitus
- • Glucosuria (when blood glucose >180 mg/dL)
- • Ketonuria (DKA)
- • High specific gravity
- • Microalbuminuria (early nephropathy)
- • Increased susceptibility to UTI
Acute Tubular Necrosis (ATN)
- • Renal tubular epithelial cells
- • RTE casts
- • Granular casts ("muddy brown")
- • Isosthenuria (fixed SG ~1.010)
- • Increased FENa (>2%)
6. Cerebrospinal Fluid (CSF) Analysis
Normal CSF Values
- • Volume: 90-150 mL (adults)
- • Appearance: Crystal clear, colorless
- • Opening pressure: 60-180 mm H₂O
- • Protein: 15-45 mg/dL (lumbar)
- • Glucose: 40-70 mg/dL (60-70% of blood glucose)
- • WBC: 0-5/µL (lymphocytes)
- • RBC: 0
CSF in Meningitis
| Parameter | Bacterial | Viral | TB/Fungal |
|---|---|---|---|
| Appearance | Turbid/purulent | Clear/slightly hazy | Slightly turbid, fibrin web |
| WBC count | >1000/µL | 100-500/µL | 100-500/µL |
| Cell type | Neutrophils (PMNs) | Lymphocytes | Lymphocytes |
| Protein | Very high (>150 mg/dL) | Normal to mild increase | High (100-500 mg/dL) |
| Glucose | Very low (<40 mg/dL) | Normal | Low |
Xanthochromia
- • Yellow discoloration of CSF supernatant
- • Due to oxyhemoglobin → bilirubin (takes 2-4 hours)
- • Indicates subarachnoid hemorrhage (vs traumatic tap)
- • Traumatic tap: RBCs decrease tube 1 to tube 4, no xanthochromia
7. Serous Fluids Analysis
Types of Serous Fluids
- • Pleural fluid: Around lungs (thoracentesis)
- • Pericardial fluid: Around heart (pericardiocentesis)
- • Peritoneal fluid (ascites): Abdominal cavity (paracentesis)
Transudate vs Exudate
| Parameter | Transudate | Exudate |
|---|---|---|
| Cause | Hydrostatic/oncotic pressure imbalance | Inflammation, infection, malignancy |
| Appearance | Clear, pale yellow | Cloudy, may be bloody |
| Protein | <3 g/dL | >3 g/dL |
| Fluid/serum protein ratio | <0.5 | >0.5 |
| LDH | <200 IU/L | >200 IU/L |
| Fluid/serum LDH ratio | <0.6 | >0.6 |
| WBC | <1000/µL | >1000/µL |
| Specific gravity | <1.015 | >1.015 |
Light's Criteria: One or more of protein ratio, LDH ratio, or LDH value indicates exudate
Special Markers
Pleural Fluid
- • Low pH (<7.2): Empyema, malignancy, TB
- • Amylase: Pancreatitis, esophageal rupture
- • ADA: Adenosine deaminase (TB)
- • Chylous: Triglycerides >110 mg/dL
Peritoneal Fluid (Ascites)
- • SAAG: Serum-Ascites Albumin Gradient
- • SAAG ≥1.1 g/dL: Portal hypertension
- • SAAG <1.1 g/dL: Non-portal hypertension
- • SBP: PMNs >250/µL
8. Other Body Fluids
Synovial Fluid
Normal Values
- • Appearance: Clear, pale yellow, viscous
- • Volume: <3.5 mL
- • WBC: <200/µL
- • Glucose: Near serum levels
| Condition | Appearance | WBC | Crystals |
|---|---|---|---|
| Normal | Clear, yellow | <200 | None |
| Osteoarthritis | Clear to slightly turbid | 200-2000 | None |
| Gout | Turbid | 2000-100,000 | Monosodium urate (needle, negative birefringent) |
| Pseudogout | Turbid | 2000-100,000 | CPPD (rhomboid, positive birefringent) |
| Septic arthritis | Turbid/purulent | >50,000 | None |
Seminal Fluid Analysis
WHO Reference Values (2021)
- • Volume: ≥1.4 mL
- • pH: ≥7.2
- • Sperm concentration: ≥16 million/mL
- • Total sperm count: ≥39 million per ejaculate
- • Progressive motility: ≥30%
- • Total motility: ≥42%
- • Normal morphology: ≥4%
- • Vitality: ≥54% live
Amniotic Fluid
- • L/S ratio: Lecithin/sphingomyelin ≥2.0 indicates fetal lung maturity
- • PG (phosphatidylglycerol): Present = mature lungs
- • Foam stability index: Surfactant assessment
- • Bilirubin (ΔOD 450): Hemolytic disease of newborn
- • AFP (alpha-fetoprotein): Elevated in neural tube defects
Fecal Analysis
- • Occult blood: Guaiac-based (diet restriction) or immunochemical (FIT - specific for human Hb)
- • Fecal fat: Sudan III stain, 72-hour quantitative
- • Muscle fibers: Pancreatic insufficiency
- • WBCs (fecal leukocytes): Invasive bacterial diarrhea
- • Reducing substances: Carbohydrate malabsorption
- • Calprotectin: IBD marker
- • Elastase-1: Pancreatic function
Key Takeaways
- ✓First morning specimen is most concentrated - best for microscopy
- ✓RBC casts are pathognomonic for glomerulonephritis
- ✓Maltese cross under polarized light indicates lipids (nephrotic)
- ✓Cystine crystals are always pathologic (hexagonal)
- ✓Bacterial meningitis: Low glucose, high protein, PMN predominant
- ✓Light's criteria differentiates transudate from exudate
- ✓MSU crystals: Needle-shaped, negative birefringent (gout)
- ✓L/S ratio ≥2.0 indicates fetal lung maturity