Moderator Prof. Dr.

ANIL KAPOOR
Presenter Dr. SOURABH MANDWARIYA

INTRODUCTION
 Oldest laboratory procedure  Mainly performed for two purpose
1. 2. To find out metabolic or endocrine disturbance To detect disorders of urinary tract or the kidneys

 Glomerular filtration rate (GFR) - 120ml/min. - 180 liter/day Nearly 99% reabsorbed

URINE FORMATION
 By summation of three processes
1. Filtration of blood plasma at the glomeruli 2. Selective reabsorption of threshold substance 3. Secretion of substances by tubules

 1500 ml to 1800 ml urine excreted per day  Minimum volume of urine required for excretion of waste products 500 ml/day

URINARY SYSTEM

COMPOSITION OF NORMAL URINE

 Volume 600 - 2500 ml/day  Specific gravity 1.003 1.030  Reaction Acidic (4.7 7.5)  Total solids 30 70 g/liter

Constituent 1. Urea 2. Creatinine 3. Uric acid 4. Creatine 5. Sodium 6. Potassium 7. Chlorides 8. Calcium 9. Ketone bodies

Quantity excreted/day 25 -30 g 1 1.8 g 0.3 1.0 g 60 150 mg 34g 1.5 2.0 g 9 16 g 0.1 o.3 g 3 15 mg

COLLECTION OF SPECIMEN
 Type of specimen
First voided midstream morning urine Random urine (for urgent examination)

 Clean and dry wide mouth glass or plastic bottles  Disposable collection apparatus for infants and young children

URINE COLLECTION CONTAINER

COLLECTION OF SPECIMEN
 Should be examined with in one hour of the collection  For quantitative measurements 24 hr urine sample is preferred

PRESERVATION
 Preservatives used
1. Toluene 2ml/100ml of urine 2. Formalin 3 drops/100ml of urine 3. Thymol One small crystal/100ml of urine 4. Chloroform 5 ml/100ml of urine

 Should be stored at 2 8C

CHANGES AT ROOM TEMPERATURE

 Lysis of red blood cells due to hypotonic urine  Decomposition of casts  Bacterial multiplication  Decrease in glucose level  Formation of ammonia from urea by the action of bacteria

CONTENTS
 Physical examination  Chemical examination  Microscopic examination

PHYSICAL EXAMINATION
A. Volume B. Color C. Appearance D. Sediment formation E. Odor F. Specific gravity (Sp. Gr.) G. Osmolality

VOLUME
 Normal average adult : 600 to 2000 ml/day  More urine formation during day time  Reversal of diurnal variation occurs in pregnancy.  Polyuria - >2000 ml of urine in 24 hours - >500 ml in first voided urine sample  Nocturia - >500ml of urine at night

VOLUME
 Causes of polyuria 1. Physiological : a. Polydipsia
b. Diuretic effect of drugs - Caffeine - Diuretic drugs c. Intra venous solution d. Increase salt intake e. High protein diet - Alcohol

2. Pathologic causes :
a) Defective hormonal regulation of water homeostasis - Diabetes insipidus - Central/Pituitary type - Hormonal deficiency - Nephrogenic type Renal unresponsiveness b) Defective renal salt/water absorption - Renal tubular abnormality - Progressive chronic renal failure c) Osmotic diuresis - Diabetes mellitus with hyperglycemia

VOLUME
 Oliguria - less than 500ml of urine/ day - <20 ml in first voided urine sample  Causes of oliguria 1. Prerenal Loss of intravascular volume
- Hemorrhage - Vomiting - Sever burns - Prolonged diarrhea - Excess sweating

b. Decrease renal blood flow - Congestive heart failure - Renal artery embolism - Sepsis - Anaphylaxis

2. Postrenal a. Obstruction of urinary tract

- Prostatic hyperplasia - Urethral stricture - Prostatic carcinoma - Urethral valve

- Stones, clots and sloughed tissue

3. Renal parenchymal disease

a. Acute renal failure i. Acute glomerulonephritis ii. Interstitial nephritis iii. Acute tubular necrosis - Heart failure - Antibiotics - Hypotension - Mercury

- Carbon tetrachloride (CCl4) - Glycerol - Myoglobinuria - Hemoglobinuria

b. Chronic renal failure i. Hypertension ii. Diabetes associated nephrosclerosis iii. Chronic glomerulonephritis iv. Polycystic kidney disease

 Anuria Complete absence of urine formation

COLOR
 Normally , pale yellow  Due to urochrome, urobilins and uroerythrin  Rough indictors of hydration and urine concentration

Abnormal color :
1. Yellow , dark yellow, brownish yellow to orange Causes : a. Pathological Bile pigment ( bilirubin) - Fever - Starvation b. Physiological Drugs - Vit. - B Complex - Serotonin - Thyrotoxicosis - Dehydration Nitrofurantoin - Senna - Pyridium

DARK YELLOW

GREEN

Whitish

RED COLOR

2. Whitish Causes : a. Pathological Chyle b. Physiological Phosphate 3. Pink to red color Causes : a. Pathological - Hemoglobinuria - Hematuria - Myoglobinuria - Pus

- Porphyria cutanea tarda - Congenital erythropoietic porphyria

b. Physiological - Bilifuscin

- Aniline dyes - Beets

- Menstrual contamination - Muscle relaxant Chlorzoxazone - Chelating agents- Deferoxamine 4. Brownish Black Causes a. Pathological - Melanin (Malignant melanoma) - Homogentisic acid (Alkaptonuria) - Rhabdomyolysis (Cola colored urine)

b. Physiological - Iron compounds - Levodopa - Quinine - Nitrofurantoin 5. Blue to green Causes : a. Pathological

Chloroquine - Hydroquinone - Metronidazole - Resorcinol

- Biliverdin - Pseudomonas infection

b. Physiological - Methylene blue - Phenyl salicylate - Indicans - Acriflavine - Chlorophyll

APPEARANCE
 Normally, clear  Abnormal appearance :
1. Cloudy urine - Causes: a. Physiological - Amorphous phosphates - Ammonium urate - Carbonate - Uric acid - Urates In acidic urine In alkaline urine

- Menstrual discharge - Powders or antiseptic contamination b. Pathological - Bacterial overgrowth - Leukocytes - Spermatozoa

- Red blood cells (may be smoky) - Epithelial cells - Blood clots - Fecal material - Mucus - Small calculi

Cloudy urine

Milky urine

2. Milky (Soap water) Causes a. Chyluria - Obstruction of lymph flow - Rupture of lymphatic vessels - Parasitic infection ( Wuchereria bancrofti ) - Enlarged abdominal lymph node - Tumors - Nephrotic syndrome - Fracture of bones b. Pseudochyluria Paraffinn-based vaginal cream

SEDIMENT FORMETION
 Present if urine contains
- Amorphous phosphate - Amorphous urate - Large numbers of leukocytes, epithelial cells

ODOR
 Normally Faint, aromatic odor  Ammoniacal fetid odor Bacterial overgrowth  Sweaty feet odor Isovaleric and glutaric acidemia  Maple syrup Maple syrup urine disease (MSUD)  Cabbage like odor Methionine malabsorption

 Mousy odor Phenylketonuria  Rotting fish - Trimethylaminuria  Rancid like odor - Tyrosinemia

SPECIFIC GRAVITY (Sp. Gr.)

 At constant temperature weight of volume of urine weight of same volume of distilled water  Measure the diluting and concentrating power of kidneys  Reliable indicator of body hydration status

 Depends upon the number and weight of particles in urine  Normal value - 1.003 to 1.035 (For random sample) - 1.015 1.030 (For 24 hr sample)  Normal contributors 1. Urea (20%) 3. Sulfate 2. Sodium chloride ( 25%) 4. Phosphates

 Hypersthenuria High specific gravity

Causes 1. Diabetes mellitus 3. Eclampsia 5. Nephrotic syndrome 7. Acute nephritis 9. Hepatic disease

2. Dehydration 4. Proteinuria 6. Fever 8. Adrenal insufficiency

10. Congestive heart failure

 Hyposthenuria Low specific gravity (<1.007)

Causes 1. Pyelonephritis 2. Hypertension 3. Diabetes insipidus 4. Protein malnutrition 5. Glomerulonephritis 5. Diuretics drugs 6. Natural diuretics - Alcohol - Coffee

 Isosthenuria

- Fixed specific gravity (1.010)

- Due to disruption of both concentrating and diluting abilities

 Measurement of specific gravity

1. Reagent strip 2. Refractometer 3. Urinometer 4. Falling drop method

REAGENT STRIP METHOD

 An indirect method  Ingredients
- Polyelectrolyte - Buffer - Indicator substance

 Principle Change in pKa of pretreated polyelectrolyte

in relation of ionic concentration of the urine.

 Not affected by glucose, protein, radiographic contrast medium.

REFRACTOMETER
 An indirect method
Velocity of light in air Velocity of light in a solution

 Requires only few drops of urine  It should read zero with distilled water  Copper sulfate solution To monitor high Sp. Gr.

 Procedure
- Apply a drop of urine at notched bottom of the cover slip - Point the instrument toward a light source - Rotate the eye piece until the scale is in focus - Read on sp. Gr. scale at the sharp line dividing between light and dark contrast

URINOMETER
 A direct hydrometer method  Temperature correction add or subtract 0.001
respectively for every 3C above or below the calibration temperature

 Protein correction 0.003 for every 1g/dl of protein  Glucose correction 0.004 for every 1g/dl of glucose

 Procedure- Fill the urinometer vessel 3/4th with urine - Insert the urinometer in spinning motion - Urinometer should not touch the sides or the bottom of cylinder - Avoid surface bubbles - Read the bottom of the meniscus

FALLING DROP METHOD

 Direct method  Procedure
- A measured drop of urine is introduced in specially designed colum filled with water immiscible oil. - As a drop falls, it encounter two beam of lights

- Start timer at breaking of first beam and off it while breaking the second beam - Falling time is measured and expressed as a Sp. Gr.

OSMOLALITY
 Number of particles of solute per unit of solution  Normal adult 500 to 850 mOsm/kg  In dehydration 800 to 1400 mOsm/kg  In water diuresis 40 to 80 mOsm/kg  Method - Freezing point depression method
- Solution containing 1000 mOsm water decrease in freezing point 1.86.C below the freezing point of water.

CHEMICAL EXAMINATION
A. Urine PH B. Protein C. Glucose D. Ketone bodies E. Occult blood F. Bile pigment G. Bile salts H. Urobilinogen

URINE PH
 Normal PH - 4.6 to 8 (Average 6 slightly acidic)  Lower PH - Acid urine A. Physiological causes
1. High protein diet 2. Fruits Cranberries 3. Mild respiratory acidosis of sleep

4. Drugs used to acidify the urine - Ammonium chloride - Methionine - Metheanamine mandelate

B. Pathological causes
1. Metabolic acidosis - Uremia - Diabetic ketoacidosis

- Starvation - Severe diarrhea

2. Paradoxical aciduria - Prolonged use of diuretics - Hypercorticism - Prolonged vomiting 3. Respiratory acidosis 4. Fever 5. Urinary Escherichia coli infection

 Higher PH- Alkaline urine A. Physiological causes

1. Drugs used to induce alkaline urine : - Sodium bicarbonate - Potassium citrate - Acetazolamide
also used in salicylate poisoning and in some urinary tract infection

2. Citrus fruits 3. High vegetables diet

4. Alkaline tide Urine become less acidic following a meal

B. Pathological causes
1. Metabolic alkalosis - Sever vomiting

- Gastric outlet obstruction 2. Respiratory alkalosis Hyperventilation 3. Proximal renal tubular acidosis (Fanconis syndrome) 4. Urinary infection (Urea splitting organism) - proteus - pseudomonas

 Methods to measure urinary pH

1. Litmus paper 2. Reagent strip 3. pH electrode 4. Titratable acidity of urine

LITMUS PAPER
Red color Blue litmus No color change Reaction : acidic Reaction : alkaline

REAGENT STRIP
 Indicator - Methyl red
- Bromothymol blue

 Should be measure immediately  On standing - pH rises due to los of carbon dioxide and bacterial overgrowth
(Produces ammonia)

pH ELECTRODE
 Procedure
- Measure accurately with a pH meter - Standardized with three buffers of known pH - Spray the electrode with distilled water, clean and dry with tissue paper - Immerse the electrode in urine sample and report the pH of urine

TITRATABLE ACIDITY OF URINE

 Normal range - 20 to 40 mEq/24 h  Depends largely on the amount of mono and dibasic phosphate

PROTEIN
 Normal - Less than 150mg/24 hours - 2mg to 10mg/dl  More then 200 Urinary protein detected  Derived from plasma protein and urinary tract  Plasma protein Albumin (33%)
- , , globulins

- Retinol binding protein - Lysozyme

 Urinary protein - Tamm-Horsfall glycoprotein (uromucoid)

- Ig A - Tubular epithelial enzymes and protein - Leukocytes - Desquamated cells

 Proteinuria types
A. Functional proteinuria Usually < 0.5g/day Causes 1. Dehydration 2. Strenuous exercise 3. Cold exposure

4. Fever 5. Congestive heart failure B. Intermittent / transient proteinuria 1. Normal pregnancy 2. Hypertension This pt. should follow every six months C. Postural proteinuria - 3 to 5 % apparently healthy young adults - Proteinuria during day time

- May develop persistent proteinuria - Rarely exceeds 1 g/day - Causes 1. Exaggerated lordotic position 2. Renal congestion or ischemia  To evaluate - Ask pt. to empty the bladder upon going to bed - Collect the first sample in morning immediately after raising

- Collect the next sample again after two hours of standing or walking - Assess for protein

First negative sample

Second positive sample

May have postural proteinuria

D. Proteinuria in elderly - Three to four fold greater incidence of glomerulonephritis - Occult malignancy

PROTEINURIA QUANTIFICATION
 Heavy proteinuria (>4g/day) Causes 1. Nephrotic syndrome
2. Congestive heart failure 3. Constrictive pericarditis 4. Renal vein thrombosis 5. Acute glomerulonephritis 6. Rapidly progressive glomerulonephritis

7. Chronic glomerulonephritis 8. Diabetes mellitus 9. Lupus erythematosus 10. Malaria 11. Malignant hypertension 12. Toxemia of pregnancy 13. Heavy metals (gold, mercury) 14. Drugs (penicillamine) 15. Neoplasia

16. Amyloidosis 17. Sickle cell disease 18. Renal transplant rejection 19. Primary antiphospholipid antibody syndrome

 Moderate proteinuria 1.0 to 4.0 g/day Causes1. All above mentioned causes 2. Nephrosclerosis 3. Multiple myeloma

4. Toxic nephropathies 5. Degenerative, malignant and inflammatory conditions of lower urinary tract 6. Calculi

 Minimal proteinuria (<1.0 g/day) Causes 1. Chronic pyelonephritis 2. Nephrosclerosis 3. Chronic interstitial nephritis

4. Polycystic kidney disease 5. Medullary cystic disease 6. Renal tubular disease 7. Postural proteinuria 8. Transient proteinuria

QUALITATIVE CATEGORIES OF PROTEINURIA

 Requires electrophoretic separation  Two types 1. Glomerular proteinuria:
- Damaged glomerular basement membrane but tubular function is normal - Heavy proteinuria (>4g/day)

Causes A. Proliferative glomerulonephritis

B. Membranous nephropathy

2. Tubular proteinuria
Low molecular weight protein excreted like a. 1 microglobulin b. -globulin

c. 2-microglobulin (Normally<100 g/day) d.Llight chain immunoglobulins e.Lysozyme - Moderate proteinuria (1.0 to 2.0 g/day )

- May missed by reagent strip Causes :

A. Cystinosis B. Fanconis syndrome C. Pyelonephritis D. Renal transplant rejections E. Wilsons disease

3. Overflow proteinuria - Excess levels of a protein in the circulation

Causes :

A. Hemoglobinuria B. Myoglobinuria C. Multiple myeloma

4. Bence jones proteinuria - First detected by henry bence jones(1847) and light chain

- Very small (mol. Wt. 44,000) protein - May be missed by reagent strip test

- Best method - Electrophoresis and Immunofixation method - Large amount may cause
a. Inclusion in tubular epithelial cells b. Desquamation of cells c. Casts formation

- Causes
a. Multiple myeloma (50-80% of pt. ) b. Macroglobulinemia c. Malignant lymphoma

5. Microalbuminuria - 20 t0 200 mg/liter - Not detectable by dipstick method Causes 1. Diabetes mellitus
2. Hypertension

METHODS FOR PROTEIN DETERMENATION

1. Reagent strip 2. Sulfosalicylic acid method 3. Heat test

REAGENT STRIP
 Principle : Impregnated with tetrabromphenol blue buffered or tetrachlorophenol-tetrabromosulfophthalein 30 to 60 second urine application Variable sheds of green color formed

 False positive results 1. Alkaline urine 2. Highly buffered urine 3. Quarternary ammonium compounds 4. Amidoamines in fabric softeners 5. Chlorhexidine 6. Excessive wetting of stripe

 False negative results

1. High salt level in urine 2. Protein other then albumin

 Not affected by1. Urine turbidity 2. Radiographic media 3. Drugs and there metabolite

SULFOSALICYLIC ACID METHOD

 Principle Precipitation of protein by acid  All types of protein are detected  False positive Radiographic contrast media  False negative High level of detergent

PROCEDURE
Mix well and Wait for 10 minutes

Transfer about 5ml urine to a centrifuge tube

Centrifuge

Transfer 3.0 ml of supernatant urine in a clean test tube

Add equal amount of 3% sulfosalicylic acid

Observe the degree of turbidity and flocculation

 Observation
Negative No turbidity (~5mg/dl or less) Trace Perceptible turbidity (~20 mg/dl) 1+ - Distinct turbidity but no discrete granulation(~50mg/dl) 2+ - Turbidity with granulation but no flocculation(~200mg/dl) 3+ - Turbidity with granulation and flocculation(~500mg/dl) 4+ - Clumps of precipitated protein, or solid precipitate (~1.0g/dl or more)

HEAT TEST
 Principle Precipitation of protein by acid  All types of protein are detected  Not affected by radiographic contrast media

PROCEDURE

Phosphates will clear

Transfer 5.0 ml of supernatant urine in a clean test tube

Boil the upper portion

If turbidity develops add 1 to 2 drops of 10% acidic acid

Reboil the specimen

Observation : No turbidity Proteins absent Presence of turbidity Proteins present

BENCE JONES PROTEINURIA DETERMINATION METHODS

1. Heat Precipitation test- Precipitate when heated to40-60C - Soluble again when boiled - Reappears after cooling

2. Precipitation in cold with salt, ammonium sulfate and acids

- False positive precipitation test

- Presence of other globulins

- False negative precipitation test

Very concentrated Bence Jones protein

3. Modified Coomassie brilliant blue stain

4. Protein electrophoresis
Single sharp peak in the globulin region

 Other tests to measure protein in urine 1. Trichloroacetic acid Biuret test 2. Colorimetric tests
- Pyrogallol red- molybdate method - Benzethonium chloride method - Coomassie blue method - Ponceau S turbidity method

GLUCOSE AND OTHER SUGARS

 Glucose :
- Small amount (2-20mg/dl) may be present

 Glycosuria Presence of detectable amount of

glucose in urine

 Factors affecting urine sugar

1. Blood glucose level 2. Glomerular filtration rate 3. Degree of tubular reabsorption

 Normal renal threshold for glucose 180-200 mg/dl  Causes of glycosuria1. Diabetes mellitus 3. Cushing's syndrome 5. Functioning or 2. Acromegaly 4. Hyperadrenocorticism
H Y P E R G L Y C E M I A

cell pancreatic tumors 7. Pheochromocytoma 9. Cystic fibrosis 11. Obesity

6. Hyperthyroidism 8. Pancreatitis 10. Brain tumors

12. Cerebral hemorrhage 13. Hypothalamic disease 14. Asphyxia 16. Infection 18. Myocardial infection 15. Burns 17. Fracture 19. Uremia
H Y P E R G L Y C E M I A

20. Glycogen storage disorder 21. Feeding after starvation 22. Drugs Thiazides - Corticosteroids

- Adrenocorticotropic hormone - Birth control pills

23. Galactosemia 25. Lead poisoning 27. Fanconi syndrome 28. Sever sprue

24. Cystinosis 26. Myeloma

Tubular dysfunction

29. Acute enteritis

30. Pregnancy lower renal threshold 31. Stress 32. Anxiety

 Fructosuria : Causes - Benign essential fructosuria

- Parenteral feeding that include fructose

 Galactosuria : Causes - Galactose-1-phophate uridyl

transferase or galactokinase deficiency

 Pentosuria : L-xylulose and L-arabinose Causes - Benign essential pentosuria

- Large amount of fruit intake

 Sucrose : Causes - Sucrase deficiency

- -dextrinase (isomaltase) deficiency - Sprue

 Lactose Causes - Pregnancy later trimester

- During lactation - 3 to 5 day old infants - Intestinal lactase deficiency

 Methods for sugar determination 1. Reagent strip 2. Benedict's test

 Reagent strip method : - Based on specific glucose oxidase and peroxidase method - Specific for glucose  Principle Glucose + O2
Glucose oxidase

Gluconic acid + H2 O 2

H2O2 + Chromogen Peroxidase Oxidized chromogen + H2O

 False positive :
- Oxidizing cleaning agent in urine container - Low specific gravity

 False negative
- High specific gravity - Ascorbic acid - Sodium fluoride

 Benedicts test - Based on copper reduction method - Detect any reducing sugar in urine  Principle Cu 2+
Hot alkaline solution

Cu + CuOH

Cu + + OH 2CuOH
Heat

Cu2O + H2O

 Preparation of Benedict's reagent Sodium citrate + Sodium carbonate (173 g) (100 g)

900 ml distilled water boil for 2 to 3 minutes

Cupric sulfate (17.3 g) Dissolve and make the final volume one liter

 Procedure

Take 5.0ml of Benedicts reagent

Add 8 drops of urine

Boil for 2 to 3 min

Cool

Observe

 Observations
Color Conclusion : Sugar

1. Blue 2. Green and slight yellow precipitate 3. Green and thick yellow precipitate 4. Yellow and orange precipitate 5. Orange and orange to red precipitate

Absent Present, trace

Present 1+ to 2+

Present 3+

Present 4+

 False positive test

1. Ascorbic acid 3. Creatine 5. Homogentisic acid 7. Radiographic media 2. Salycylates 4. Uric acid 6. Cephalosporins

 Determination of lactose A. Osazone test  Principle Phenylhydrazine hydrochloride

maltose /lactose/ monosaccharides Acidic ph and boil

Phenylhydrazone crystals

 Procedure

Take 5 ml of urine

Few drops of Glacial acetic acid (To make it acidic)

Sodium acetate (2 part) + Phenyl hydrazine hydrochloride (1part)One gram

Boiling water Observe collected bath - 30 minutes Then cool deposit under microscope

LACTOSAZONE

B. Lactose test  Procedure 15 ml of Urine + 3 gm Lead acetate

Shake and filter

Boil filtrate + 2ml concentrated NH4OH

Boil

Brick red color

 Determination of galactose Test Orthotoludine test ( Only if lactose and

glucose are absent)

 Principle Orthotoludine + Galactose

Acidic medium

Green color

 Procedure 5 ml of orthotoludine reagent + 0.5 ml urine

boil for 5 min.

Green color  Other tests for estimation of sugar 1. Resorcinol test Fructose 2. Thin layer chrometography

 Determination of ketones Ketone bodies Three types

1. Acetone (2%) 2. Acetoacetic acid (20%) 3. 3-hydroxybutyrate (78%)

Causes of ketonuria
1. Diabetic ketonuria(Type I>Type II)

2. Non diabetic ketonuria - Acute febrile diseases - Inherited metabolic diseases - Hyperemesis of pregnancy - Following anesthesia - Cold exposure - Sever exercise - Low carbohydrate diet for weight reduction - Cachexia - Toxic states

3. Lactic acidosis Shock - Liver failure - Sever infections - Drugs

- Phenformin - Salicylate poisoning

- Renal failure - Diabetes mellitus

 Methods to detect ketone bodies

1. Reagent strip 2. Rotheras test 3. Dumn and shipleys method 4. Gerhardt ferric chloride test

 Reagent strip - Based on nitroprusside reaction  Principle Sodium nitroprusside + Glycine

acetoacetic acid and aceton in alkaline medium

Violet color

 False positive
1. Large amount of phenylketones 2. Preservatives 8-hydroxyquinoline 3. L-dopa metabolite 4. Acetylcysteine 5. Methyldopa 6. Captopril 7. Dyes

 False negative
1. Loss of reagent activity 2. Loss of acetoacetic acid by bacterial action 3. Loss of acetone at room temperture

 Rotheras test - Based on nitroprusside reaction - Principal same as reagent strip  Procedure
Take 5.00 ml urine + 1.0 g of Rotheras powder
( Sodium nitroprusside : 0.75 g Ammonium sulfate : 20 g)

Concentrated ammonium hydroxide(1-2ml) by the side of test tube then observe for pink-purple ring

 Dumn and shipleys method  Procedure T C T C T C

Take Two test tube Add a pinch of powder mixture (sodium nitroprusside : 1.0 g Ammonium sulfate : 20 g Anhydrous sodium carbonate:20g) T- One drop of urine C- One drop of distilled water

T- Violet color C- no color change

 Determination of occult blood

- Hematuria Presence of abnormal number of red blood cells in urine - Hemoglobinuria Presence of free hemoglobin in urine

Hematuria Causes
1. Membranous nephropathy 2. Ig A nephropathy 3. Non - Ig A mesangioproliferative glomerulonephritis 4. Focal glomerulosclerosis

5. Mild glomerular abnormality 6. Trauma 7. Neoplastic disease of kidney or urinary bladder 8. Bleeding disorder 9. Anticoagulant use 10. Cyclophosphamide 11. Giant cell arteritis 12. Marathon runners 13. Renal tuberculosis

14. Nephrotic syndrome 15. Malignant hypertension 16. Renal calculi 17. Acute cystitis 18. Sickle cell disease 19. scurvy

 Hemoglobinuria Causes
1. Intravascular hemolysis 2. Sever exertion 3. Prosthetic cardiac valves 4. Extensive burns 5. Malaria 6. Bartonella 7. Clostridium welchii toxin 8. Spider and snack bite

9. Hemolytic uremic syndrome 10. Thrombotic thrombocytopenic purpura 11. Incompatible blood transfusions 12. Warm and cold antibodies 13. Paroxysmal nocturnal hemoglobinuria 14. Drugs Penicillins - Phenacetine - Sulfonamides - Nitrofurantoin

- Quinidine - -methyldopa - Sulfones

15. G6PD pt. exposed to - Antimalarial - Fava beans - Infections - Oxidant drugs - Diabetic acidosis

 Hemosiderinuria Causes 1. Hemochromatosis

2. 2 to 3 days after hemoglobinuria

 Myoglobinuria Causes 1. Myocardial infarction

2. Dermatomyositis 3. Strenuous exercises 4. Muscle phosphofructokinase deficiency 5. Adenosine monophosphate deaminase deficiency 6. Mitochondrial trifunctionl protein deficiency 7. Crush injury

8. Heat stroke 9. Electric shock 10. Convulsions

 Methods
1. Reagent strip 2. Benzidine test

 Reagent strip  Principle H2O2 + Chromogen

Oxidized chromogen + H2O2 (Color change)

 False negative
1. Ascorbic acid 2. Formalin (Preservative) 3. Nitrite Delay the reactions

 False positive 1. Oxidizing compounds Hypochlorites 2. Urinary tract infection Microbial peroxidase

 Benzidine test  Principle Same as regent strip  Procedure

A pinch of Benzidine powder

Glacial Hydrogen acetic acid peroxide 2 to 3 2.0 ml drops

Transfer 1.00 ml of supernatant

Urine 0.5 ml

Observe for color change

 Observation Color Faint green Green Greenish blue Blue Deep blue Report trace 1+ 2+ 3+ 4+

 Test for myoglobin

1. Urine color Fresh urine Red On standing Brown 2. Urine 1.0 ml + 3% Sulfosailcylic acid (3.0ml) - If pigment precipitated Its a protein 3. Urine 5.0 ml + Ammonium sulfate 2.8 g filter or centrifuge Supernatant Normal color Hemoglobin Supernatant Colored Myoglobin

4. Immunological tests Human antisera 5. Capillary electrophoresis 6. End point and rate nephelometric methods

 Test for Hemosiderin Prussian blue reaction

BILE PIGMENT
 Normally 0.02mg bilirubin/dl
Clinical condition Pre-hepatic Hepatic Post-hepatic Bile pigment Bile salts Absent Present Trace to 4+ Present 2+ to 4+ Absent Present Present Urobilinogen Very high 3+ to 4+ Increased 2+ Present or may be absent

 Pre hepatic jaundice ( Hemolytic jaundice) Causes :

1. Sickle cell disease 2. Thalassemia major 3. Acquired hemolytic anaemias 4. Incompatible blood transfusion

 Hepatic condition Causes :

1. Viral hepatitis 2. Liver cirrhosis 3. Chemical intoxication

4. Drug reaction 5. Dubin-johnson syndrome 6. Crigler-Najjar syndrome

 Post hepatic jaundice (Common bile duct obstruction) Causes :

1. Common bile duct stones 2. Carcinoma of the head of pancreas 3. Pancreatitis 4. Enlarged lymph nodes