Moderator – Prof. Dr. ANIL KAPOOR

Presenter – Dr. SOURABH MANDWARIYA

INTRODUCTION

Oldest laboratory procedure

Mainly performed for two purpose

1. To find out metabolic or endocrine disturbance

2. 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 Quantity excreted/day

1. Urea

2. Creatinine

3. Uric acid

4. Creatine

5. Sodium

6. Potassium

7. Chlorides

8. Calcium

9. Ketone bodies

25 -30 g

1 – 1.8 g

0.3 – 1.0 g

60 – 150 mg

3 – 4 g

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 – 8°C

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 - Alcohol

- Diuretic drugs

c. Intra venous solution

d. Increase salt intake

e. High protein diet

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 - Prolonged

diarrhea

- Vomiting - Excess sweating

- Sever burns

b. Decrease renal blood flow

- Congestive heart failure - Sepsis

- Renal artery embolism -

Anaphylaxis

2. Postrenal – a. Obstruction of urinary tract

- Prostatic hyperplasia - Prostatic

carcinoma

- Urethral stricture - 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 - Hypotension

- Antibiotics - Mercury

- Carbon tetrachloride (CCl4)

- Glycerol - Hemoglobinuria

- Myoglobinuria

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 - Thyrotoxicosis

- Starvation - Dehydration

b. Physiological – Drugs – Nitrofurantoin

- Vit. - B Complex - Senna

- Serotonin - Pyridium

DARK YELLOW

Whitish

RED COLOR

GREEN

2. Whitish – Causes :

a. Pathological -

– Chyle - Pus

b. Physiological – Phosphate

3. Pink to red color – Causes :

a. Pathological - - Hematuria

- Hemoglobinuria - Myoglobinuria

- Porphyria cutanea tarda

- Congenital erythropoietic porphyria

b. Physiological - - Aniline dyes

- Bilifuscin - 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 – Chloroquine

- Iron compounds - Hydroquinone

- Levodopa - Metronidazole

- Quinine - Resorcinol

- Nitrofurantoin

5. Blue to green – Causes :

a. Pathological - Biliverdin

- Pseudomonas infection

b. Physiological

- Methylene blue - Acriflavine

- Phenyl salicylate -

Chlorophyll

- Indicans

APPEARANCE Normally, clear

Abnormal appearance :

1. Cloudy urine - Causes:

a. Physiological - Amorphous phosphates

- Ammonium urate

- Carbonate

- Uric acid

- Urates

In alkalineurine

In acidic urine

- Menstrual discharge

- Powders or antiseptic contamination

b. Pathological -

Leukocytes

- Bacterial overgrowth -

Spermatozoa

- Red blood cells (may be smoky)

- Epithelial cells - Mucus

- Blood clots - Small

calculi

- Fecal material

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%) 2. Sodium chloride ( 25%)

3. Sulfate 4. Phosphates

Hypersthenuria – High specific gravity

Causes – 1. Diabetes mellitus

2. Dehydration 3. Eclampsia

4. Proteinuria 5. Nephrotic syndrome

6. Fever 7. Acute nephritis

8. Adrenal insufficiency 9. Hepatic disease

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 - Indicator substance

- Buffer

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 methodVelocity 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 3°C 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 G. Bile salts

B. Protein H. Urobilinogen

C. Glucose

D. Ketone bodies

E. Occult blood

F. Bile pigment

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

(Fanconi’s 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

Blue litmus

Red color Reaction : acidic

No color change 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%)

- α, β, γ 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

Causes- 1. 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. α1 – microglobulin b. β-

globulin

c. β2-microglobulin

(Normally<100μg/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. Fanconi’s syndrome

C. Pyelonephritis

D. Renal transplant rejections

E. Wilson’s 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)

- κ and λ 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 by-

1. 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

Transfer about 5ml urine to a centrifuge tube

CentrifugeTransfer 3.0 ml of supernatant urine in a clean test tube

Add equal amount of 3% sulfosalicylic acid

Mix well and Wait for 10 minutes

Observe the degree of turbidity and flocculation

PROCEDURE

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

Transfer 5.0 ml of supernatant urine in a clean test tube

Boil the upper portion

PROCEDURE

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

Phosphates will clear

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 glycosuria-

1. Diabetes mellitus 2. Acromegaly

3. Cushing's syndrome 4. Hyperadrenocorticism

5. Functioning α or β cell pancreatic tumors

6. Hyperthyroidism 7. Pheochromocytoma

8. Pancreatitis 9. Cystic fibrosis

10. Brain tumors 11. Obesity

HYPERGLYCEMIA

12. Cerebral hemorrhage 13. Hypothalamic

disease

14. Asphyxia 15. Burns

16. Infection 17. Fracture

18. Myocardial infection 19. Uremia

20. Glycogen storage disorder

21. Feeding after starvation

22. Drugs – Thiazides - Corticosteroids

- Adrenocorticotropic hormone

- Birth control pills

HYPERGLYCEMIA

23. Galactosemia 24. Cystinosis

25. Lead poisoning 26. Myeloma

27. Fanconi syndrome

28. Sever sprue 29. Acute enteritis

30. Pregnancy – lower renal threshold

31. Stress 32. Anxiety

Tubular

dysfunction

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

- α-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 Gluconic acid + H2O2

H2O2 + Chromogen Oxidized

chromogen + H2O

Glucose oxidase

Peroxidase

False positive :

- Oxidizing cleaning agent in urine container

- Low specific gravity

False negative

- High specific gravity

- Ascorbic acid

- Sodium fluoride

Benedict’s test

- Based on copper reduction method

- Detect any reducing sugar in urine

Principle

Cu 2+ Cu +

Cu + + OH - CuOH

2CuOH Cu2O + H2O

Hot alkaline solution

Heat

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

Add 8 drops

of urine

Boil for 2

to 3 min

CoolTake 5.0ml of

Benedict’s

reagent

Observe

Observations

Color Conclusion : Sugar

1. Blue Absent

2. Green and slight yellow

precipitate

Present, trace

3. Green and thick yellow

precipitate

Present 1+ to 2+

4. Yellow and orange

precipitate

Present 3+

5. Orange and orange to red

precipitate

Present 4+

False positive test

1. Ascorbic acid 2. Salycylates

3. Creatine 4. Uric acid

5. Homogentisic acid 6. Cephalosporins

7. Radiographic media

Determination of lactose

A. Osazone test

Principle

Phenylhydrazine hydrochloride

maltose /lactose/ Acidic ph and boil

monosaccharides

Phenylhydrazone crystals

Procedure

Sodium acetate

(2 part) + Phenyl

hydrazine

hydrochloride

(1part)One gram

Take 5 ml

of urine

Few drops of

Glacial acetic

acid (To

make it

acidic)

Boiling water

bath - 30

minutes

Then cool

Observe

collected

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 - Toxic

states

- Inherited metabolic diseases

- Hyperemesis of pregnancy - Cachexia

- Following anesthesia

- Cold exposure

- Sever exercise

- Low carbohydrate diet for weight reduction

3. Lactic acidosis

– Shock - Renal failure

- Liver failure - Diabetes mellitus

- Sever infections

- Drugs - Phenformin

- Salicylate poisoning

Methods to detect ketone bodies

1. Reagent strip

2. Rothera’s test

3. Dumn and shipley’s 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

Rothera’s test

- Based on nitroprusside reaction

- Principal same as reagent strip

Procedure

Take 5.00 ml urine + 1.0 g of Rothera’s 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 shipley’s method

Procedure

Take Two test tube

Add a pinch of powder mixture

(sodium nitroprusside : 1.0 g

Ammonium sulfate : 20 g

Anhydrous sodium carbonate:20g)

T C T C

T- One drop of urine

C- One drop of distilled

water

T C

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

- Quinidine - Phenacetine

- α-methyldopa - Sulfonamides

- Sulfones - Nitrofurantoin

15. G6PD pt. exposed to

- Antimalarial - Oxidant drugs

- Fava beans - Diabetic acidosis

- Infections

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 acetic acid 2 to 3 drops

Hydrogen peroxide 2.0 ml

Transfer 1.00 ml of supernatant

Urine 0.5 ml

Observe for color change

Observation

Color Report

Faint green trace

Green 1 +

Greenish blue 2 +

Blue 3 +

Deep blue 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 – It’s 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

Bile pigment Bile salts Urobilinogen

Pre-hepatic Absent Absent Very high 3+ to 4+

Hepatic Present – Trace to 4+

Present Increased 2 +

Post-hepatic Present – 2+ to 4+

Present 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