Urine routine and microscopy KEM Hospital,Mumbai.

Urinanalysis is fundamental test that should be performed in all urologic patients. It will help in conveying the structural and functional status of urinary system and associated abnormalities in it.

Collection of urine specimen- In the male patient, a midstream urine sample is obtained after retracting the prepuce. In female before obtaining sample clean the vulva and separate the labia.Mid stream urine is collected. To evaluate for a possible infection in a female, a catheterized urine sample should always be obtained. In infants sterile plastic bag with adhesive collar over genitalia can be used.Suprapubic aspirate can be used. Need to be examined within 1 hour

Clean Catch

Routine urine analysis includes- Physical examination Chemical examination Microscopic examination

Physical examination Urine volume Colour Turbidity PH Specific gravity (SG) & Osmolality

Urine dipstick analysis- Glucose Bilirubin Ketones Specific Gravity Blood pH Protein Urobilinogen Nitrite Leukocyte Esterase

Colour- Color : normally , pale to dark yellow (urochrome) Abnormal color : some drugs cause colour changes 1. red urine : causes: hematuria hemoglobinuria myoglobinuria 2. yellow-brown or green-brown urine: bilirubin cause : obstructive jaundice

Red Urine Microscopic Hematuria Urinary tract source Pseudohematuria (non-hematuria related red urine) Myoglobinuria Hemoglobinuria Phenolphthalein Laxatives Phenothiazines Porphyria Rifampin Pyridium Bilirubinuria Phenytoin Foods (Beets, Blackberries, Rhubarb) Microscopic Hematuria Urinary tract source Urethra or bladder Prostate Ureter or kidney Non-Urinary tract source Vagina Anus or rectum

Appearance Clarity: normally, clear Abnormal colour: cloudy urine/turbidity Causes: 1. crystals or nonpathologic salts phosphate, carbonate in alkaline urine (dissolve---add acetic acid) uric acid in acid urine (dissolve---warming to 60℃) 2. various cellular elements: leukocytes, RBCs, epithelial cells 3.Chyluria,Lipiduria 4.Hyperoxaluria,Hyperuricosuria

Odour has little diagnostic significance. Aromatic odour------> Normal urine due to aromatic acids. Ammonia odour------> On standing due to decomposition of urea. Fruity odour--------> Diabetes due to the presence of ketones. An infection with E. coli bacteria can cause a bad odor

Urine volume The average adult : 1000ml to 2000ml/24h Increase Polyuria---more than 2000ml of urine in 24 hr 1. physiological states: water intake, some drugs, intravenous solutions 2. pathologic states: diabetes mellitus, diabetes insipidus

Urine volume Decrease Oliguria---less than 400ml of urine in 24 hours Anuria---less than 100ml of urine in 24 hours 1. prerenal: hemorrhage, dehydration, congestive heart failure 2. postrenal: obstruction of the urinary tract (may be stones, carcinoma) 3. renal parenchymal disease: acute tubular necrosis, chronic renal failure

Specific gravity (SG) Reflect the density of the urine Ranges from 1.001 to 1.035 Increase(>1.020): Dehydration、Decreased fluid intake,Diabetes mellitus,SIADH. Decrease(<1.008): Increased water intake,diabetes insipidus,diuretics,decreased renal concentrating ability. Fixed specific gravity of 1.010 is seen in renal insufficiency either acute or chronic. Urine osmolality ranges from 50 to 1200 mosm/lit

The Urine Dipstick: Specific Gravity 1.000 Chemical Principle 1.005 1.010 1.015 1.020 1.025 1.030 Chemical Principle X+ + Polymethyl vinyl ether / maleic anhydride ---------------> X+-Polymethyl vinyl ether / maleic anhydride + H+ H+ interacts with a Bromthymol Blue indicator to form a colored complex. Read up to 2 minutes RR: 1.003-1.035

Urine PH- Normal PH- Ranges from 4.5 -8 The average ranges from 5.5 to 6.5 Higher PH---alkaline urine 1.drugs: sodium bicarbonate 2.Renal tubular acidosis type I 3.alkalosis (metabolic or respiratory) Lower PH---acid urine 1.drugs: ammonium chloride 2. acidosis (metabolic or respiratory)

Methyl Red (at high concentration; low pH) and The Urine Dipstick: pH 5.0 6.0 6.5 7.0 7.5 8.0 8.5 Chemical Principle H+ interacts with: Methyl Red (at high concentration; low pH) and Bromthymol Blue (at low concentration; high pH), to form a colored complexes (dual indicator system) Read up to 2 minutes R.R.: 4.5-8.0

Chemical examination Protein Glucose Ketones Occult blood Bilirubin Urobilinogen Nitrites & Leukocyte esterase.

The Urine Dipstick: Protein Chemical Principle “Protein Error of Indicators Method” Negative Trace + (30 mg/dL) ++ (100 mg/dL) +++ (300 mg/dL) ++++ (2000 mg/dL) Pr H Tetrabromphenol Blue (buffered to pH 3.0) H+ Pr Pr Pr Pr Pr Pr Read at 60 seconds RR: Negative

Proteins in urine Normal adult excrete less than 150mg of protein in urine within 24 hours. Urine proteins come from plasma protein(albumin,globulins) and Tamm-Horsfall (T-H) glycoprotein . More than 150mg proteins in urine in 24 hours is called as proteinuria. It can be quantified as- heavy proteinuria----＞4.0g/24 hours moderate proteinuria----1.0-4.0g/24 hours minimal proteinuria----＜1.0g/24 hours

Pathophysiological causes- 1)Glomerular proteinuria: damage to glomerular basement membrane but tubular function is normal It can be selective proteinuria-chiefly albumin or nonselective proteinuria heavy proteinuria acute glomerulonephritis,Diabetes

2)Tubular proteinuria- -Renal tubular disease damage tubular function but glomerular function is normal -Moderate proteinuria - disease: pyelonephritis 3)Overflow proteinuria- Excess levels of a protein in the circulation,hemoglobin, myoglobin, etc. The renal function is normal Hemoglobinuria Myoglobinuria Multiple Myeloma Amyloidosis

Glucose in urine- Normally all glucose filtered is absorbed in PCT. Renal threashold for serum glucose is 180mg/dl Glycosuria- 1.hyperglycemia: diabetes mellitus Cushing’s syndrom 2.without hyperglycemia: renal tubular dysfunction such as pyelonephritis

Horseradish Peroxidase Chemical Principle Negative Trace (100 mg/dL) + (250 mg/dL) ++ (500 mg/dL) +++ (1000 mg/dL) ++++ (2000+ mg/dL) Glucose Oxidase Glucose + 2 H2O + O2 ---> Gluconic Acid + 2 H2O2 Horseradish Peroxidase 3 H2O2 + KI ---> KIO3 + 3 H2O Read at 30 seconds RR: Negative

Limitations of Urine Glucose Detection Interference: reducing agents, ketones. Only measures glucose and not other sugars. Renal threshold must be passed in order for glucose to spill into the urine. Other Tests CuSO4 test for reducing sugars.

Acetoacetic Acid + Nitroprusside ------> Colored Complex The Urine Dipstick: Ketones Negative Trace (5 mg/dL) + (15 mg/dL) ++ (40 mg/dL) +++ (80 mg/dL) ++++ (160+ mg/dL) Chemical Principle Acetoacetic Acid + Nitroprusside ------> Colored Complex Read at 40 seconds RR: Negative

Uses and Limitations of Urine Ketone Detection Normally not seen in urine. Significance - Diabetic ketoacidosis - Prolonged fasting -pregnancy -rapid weight loss Limitations - Interference: expired reagents (degradation with exposure to moisture in air) - Only measures acetoacetate not other ketone bodies(acetone, beta hydroxy butyric acid)

The Urine Dipstick: Bilirrubin Negative Chemical Principle + (weak) ++ (moderate) +++ (strong) Chemical Principle Acidic Bilirubin + Diazo salt ---------> Azobilirubin Read at 30 seconds RR: Negative

Urobilinogen + Diethylaminobenzaldehyde -------> Colored Complex The Urine Dipstick: Urobilinogen 0.2 mg/dL 1 mg/dL 2 mg/dL 4 mg/dL 8 mg/dL Chemical Principle Urobilinogen + Diethylaminobenzaldehyde -------> Colored Complex (Ehrlich’s Reagent) Read at 60 seconds RR: 0.02-1.0 mg/dL

Significance - Increased direct bilirubin seen in obstructive jaundice. -Increased urobilinogen is seen in hemolysis and hepatocellular diseases. Limitations - Interference: prolonged exposure of sample to light - Only measures direct bilirubin--will not pick up indirect bilirubin - Serum test for total and direct bilirubin is more informative

The Urine Dipstick: Blood Negative Trace (non-hemolyzed) Chemical Principle Trace (non-hemolyzed) Lysing agent to lyse red blood cells Moderate (non-hemolyzed) Diisopropylbenzene dihydroperoxide + Tetramethylbenzidine ------------> Colored Complex Trace (hemolyzed) Heme + (weak) ++ (moderate) Read at 60 seconds RR: Negative Analytic Sensitivity: 10 RBCs +++ (strong)

Uses and Limitations of Urine Blood Detection Significance - Hematuria (nephritis, trauma, etc) - Hemoglobinuria (hemolysis, etc) - Myoglobinuria (rhabdomyolysis, etc) Limitations - Interference: reducing agents, microbial peroxidases - Cannot distinguish between the above disease processes

Types of hematuria- 1)Glomerular-dysmorphic RBCs with cast and proteinuria. e.g.-Glomerulonephritis all types. 2)Non glomerular-Normal RBCs no cast,proteinuria+ e.g.-tubulointerstitial diseases,renovascular HTN 3)Essential/surgical-Normal RBCs no cast,no proteinuria e.g.Tumors,stones,UTI

The Urine Dipstick: Nitrite Chemical Principle Negative Positive Acidic Negative Positive Nitrite + p-arsenilic acid -------> Diazo compound Diazo compound + Tetrahydrobenzoquinolinol ----------> Colored Complex Read at 60 seconds RR: Negative

Uses and Limitations of Nitrite Detection Significance - Gram negative bacteriuria(convert nitrate to nitrites) Limitations - Interference: bacterial overgrowth - Only able to detect bacteria that reduce nitrate to nitrite Other Tests - Correlate with leukocyte esterase and - Urine microscopic examination (bacteria) - Urine culture

The Urine Dipstick: Leukocyte Esterase Chemical Principle Derivatized pyrrole amino acid ester ------------> 3-hydroxy-5-phenyl pyrrole Negative Trace + (weak) ++ (moderate) +++ (strong) Esterases 3-hydroxy-5-phenyl pyrrole + diazo salt -------------> Colored Complex Read at 2 minutes RR: Negative Analytic Sensitivity: 3-5 WBCs

Indicates presence of WBCs in urine. Significance Leukocyte Esterase Detection Indicates presence of WBCs in urine. Significance - Pyuria - Acute inflammation - Renal calculus Both leucocyte esterase test and nitrite test used in conjuction to screen for UTI. If these tests are positive then microscopic analysis of urine should be done.

Microscopic Examination General Aspects Preservation - Cells and casts begin to disintegrate in 1 - 3 hrs. at room temp. - Refrigeration for up to 48 hours (little loss of cells). Specimen concentration - Ten to twenty-fold concentration by centrifugation.(3000 rpm for 5 minutes of 10 -15 ml urine sample) -Urinary sediment should be examined with both low power(100x) and high power field(400X) -Low power- RBC,WBC,cast,cystine crystal,trichomonas,schistosoma. -High power- Dysmorphic RBCs,other crystals,bacteria,yeast.

Types of microscopy - Phase contrast microscopy - Polarized microscopy - Bright field microscopy with special staining (e.g., Sternheimer-Malbin stain)

Microscopic Examination Abnormal Findings Per High Power Field (HPF) (400x) > 3 erythrocytes > 5 leukocytes > 2 renal tubular cells > 5 bacteria Per Low Power Field (LPF) (200x) > 3 hyaline casts or > 1 granular cast > 10 squamous cells (indicative of contaminated specimen) Any other cast (RBCs, WBCs) Presence of: Fungal hyphae or yeast, parasite, viral inclusions Pathological crystals (cystine, leucine, tyrosine) Large number of uric acid or calcium oxalate crystals

Microscopic Examination Bacteria & Yeasts Bacteria - Bacteriuria-More than 5 per HPF reflects CFU 1,00,000/ml Yeasts - Candidiasis-Most likely a contaminant but should correlate with clinical picture. Viruses CMV inclusions-Probable viral cystitis. Parasites- -Trichomonas,Schistosoma

Urinary casts- Urinary casts are cylindrical structures produced by the kidney and present in the urine in certain disease states. They are protein coagulum formed in the distal convoluted tubule (DCT) and collecting ducts of nephrons and traps any luminal contents within matrix then dislodge and pass into the urine, where they can detected by microscopy. -Urinary casts may be made up of cells (such as white blood cells, red blood cells, kidney cells) or substances such as Tamm-Horsfall mucoprotein.

Microscopic Examination Casts Erythrocyte Casts:Glomerular diseases Leukocyte Casts:Pyuria, glomerular disease Hyaline casts are composed primarily of a mucoprotein (Tamm-Horsfall proteins) secreted by tubule cells. Granular cast or waxy cast arise from further degeneration of cellular elements. Fatty cast- Nephrotic syndrome

Hyaline Casts appear Transparent

Red Cell Casts 45

White Cell Casts 46

Crystals in urine Normal-Cholesterol Crystals in acidic urine Uric acid Calcium oxalate Cystine Leucine Crystals in alkaline urine Ammonium magnesium phosphates(triple phosphate crystals) Calcium carbonate

Calcium Oxalate dihydrate Crystals-Envelope shaped

Calcium Oxalate monohydrate Crystals-Dumbell shaped Dumbbell Shape

Triple Phosphate Crystals-coffin lid shaped.

Urate Crystals-rhomboid

Microscopic Examination Leucine Crystals

Cystine Crystals-Hexagonal

Ammonium Biurate Crystals Microscopic Examination Ammonium Biurate Crystals

Microscopic Examination Cholesterol Crystals

Summary-Normal values Color – Yellow (light/pale to dark/deep amber) Clarity/turbidity – Clear or cloudy pH – 4.5-8 Specific gravity – 1.001-1.035 Glucose -Negative Ketones – None Nitrites – Negative Leukocyte esterase – Negative Bilirubin – Negative Urobilirubin – Small amount (0.5-1 mg/dL) Blood - ≤3 RBCs Protein - ≤150 mg/d RBCs - ≤3RBCs/hpf WBCs - ≤2-5 WBCs/hpf Squamous epithelial cells - ≤15-20 squamous epithelial cells/hpf Casts – 0-5 hyaline casts/lpf Crystals – Occasionally Bacteria – None

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