Chemical Examination

CHEMICAL EXAMINATION

(For abnormal constituents)

Chemical tests can be done by manual methods, using reagent strips and by automated methods.

Urine Reagent Strips for Urinalysis: These are firm plastic strips to which several different reagent areas are affixed. Depending on the product being used, the urine reagent strips provide

 tests for Glucose, Bilirubin, Ketone (Acetoacetic acid), Specific Gravity, Blood, pH, Protein, Urobilinogen, Nitrite, Leukocytes, and Ascorbic Acid in Urine. Test results provide information regarding the status of carbohydrate metabolism, kidney and liver function, acid-base balance, and bacteriuria.

Reagent strip test procedure:

• • • Reagent strips for tests should be stored in bottles with a tight cap.

    • Strips removed from the bottle should be used immediately 

    • Completely immerse the reagent areas of the strip in fresh, well-mixed urine.

    • Remove the strip immediately to avoid dissolving out the reagent areas. 

    • While removing, touch the side of the strip against the rim of the urine container to remove excess urine. 

    • Blot the lengthwise edge of the strip on an absorbent paper towel to further remove excess urine and avoid running over (contamination from adjacent reagent pads.) 

7.  Compare each reagent area to its corresponding color blocks on the color chart and read at the times specified. Proper read time is critical for optimal results. 

8.  Obtain results by direct color chart comparison. The color blocks represent nominal values; actual values will vary around the nominal values. 

Quality control:  

For best results, performance of reagent strips should be confirmed by testing known negative and positive specimens or controls whenever a new bottle is first opened. Each laboratory should establish its own goals for adequate standards of performance, and should question handling and testing procedures if these standards are not met. 

Limitations of procedure:

Comparison to the color chart is dependent on the interpretation of the individual. It is therefore, recommended that all laboratory personnel interpreting the results of these strips be tested for color blindness. 

1. Glycosuria: Signifies presence of glucose in the urine. Usually, glucose will not be present in the urine until the blood level exceeds 160–180 mg/dL, which is the normal renal threshold for glucose.

a. Benedict’s test: Semi quantitative test for reducing substances.

Principle: In an alkaline medium, in the presence of heat, reducing sugars (carbohydrate possessing either a free ketone or aldehyde group) will reduce cupric ions to cuprous oxide. The reaction produces a color change of the blue Benedict’s reagent through green to orange depending upon the amount of reducing substances present in the urine.

Benedict’s reagent is composed of cupric sulphate (173g), sodium citrate (173g) and sodium carbonate (100g) in 1000ml of distilled water.

Procedure: If albumin is present in any considerable quantity, it interferes with reduction of copper in the test and should be removed by acidifying with acetic acid or by boiling and filtering. To 5 ml of Benedict’s reagent add 8 drops (0.5ml) of protein free urine. The mixture is boiled for 2 minutes and allowed to cool. Depending on the amount of reducing substance being present a precipitate appears which progresses from green to yellow and finally orange to brick red. 

 b. Reagent strip method: 

This test is based on a double sequential enzyme reaction. One enzyme, glucose oxidase, catalyzes the formation of gluconic acid and hydrogen peroxide from the oxidation of glucose. A second enzyme, peroxidase, catalyzes the reaction of hydrogen peroxide with potassium iodide chromogen to oxidize the chromogen to colors ranging from blue-green to greenish-brown through brown and dark brown. 

• 1) Catalysed by glucose oxidase

Glucose + O2 → D-glucono-δ-lactone + H2O2

• 2) Catalysed by peroxidase

H2O2 + Chromogen → oxidised chromogen (coloured) + H2O

False positivity could result due to the presence of oxidising agents or peroxide from disinfectants used on laboratory instruments. 

Causes of glycosuria with hyperglycemia:

1. Diabetes mellitus

2. Endocrine disorders-Acromegaly, Cushing’s syndrome, Hyperthyroidism, Pheochromocytoma

3. Pancreatic diseases – cystic fibrosis, hemochromatosis, severe chronic pancreatitis, carcinoma of pancreas

4. Metabolic derangements- severe burns, uremia, sepsis, advanced liver disease, cardiogenic shock

5. CNS dysfunctions – Asphyxia, tumors, haemorrhage

Causes of glycosuria without hyperglycemia:

1. Renal glycosuria

2. Advanced glomerulonephritis

False-Positive Results: Other reducing substances which may be present in urine that may give a false positive result include 

• Other sugars - fructose, maltose and lactose 

• Homogentisic acid (alkaptonuria), 

• Excess of mucin,

• Drugs - Vitamin C (ascorbic acid), salicylates, antibiotics - nalidixic acid, cephalosporins  

• Urinary preservatives - formalin and formaldehyde. 

2. Proteinuria: Signifies presence of excess proteins in the urine. It may be a sign of renal  damage indicating either an insufficiency of absorption or impaired filtration. There are also few physiologic conditions such as exercise and fever that can lead to increased protein excretion in the urine in the absence of renal disease.

a. Heat and acetic acid test 

Principle: In the presence of acid or heat proteins loses their structure and denature to form a coagulum that precipitates.

 Procedure: Tests for presence of protein are carried out on a clear specimen of urine which should be filtered or centrifuged to remove any turbidity.

1. Three quarters of a test tube is filled with clear urine. 

2. Heat the upper 1/3rd of the column for about 2 minutes. The lower 2/3rd serve as control.

3. Watch for the appearance of turbidity in the heated part of urine. Turbidity may be due to proteins or phosphates. 

4. Add 3-5 drops of 10% acetic acid. If turbidity persists, it is because of proteins; if it dissolves then it is due to amorphous phosphates.

b. Sulphosalicylic acid test:  Few drops of sulphosalicylic acid is added to 2ml of urine. Turbidity indicates presence of protein.

c. Heller’s test: 2ml of Conc.HNO3 in a taken in a test tube (Kahn’s tube). With a pipette, a small quantity of urine is added along the side of the test tube to form a layer on the top. If albumin is present a white ring is formed at the junction. 

d. Reagent test strips: This is a colorimetric method based on the concept that the point of color change of some pH indicators is different in the presence of protein (“protein error of indicators”). This is because proteins act as hydrogen ion acceptors at a constant pH. Usually, the indicator changes from yellow to blue (or green) between pH 3 and pH 4, but in the presence of protein, the color change will occur between pH 2 and pH 3. Therefore, in the presence of protein an “error” occurs in the behavior of the indicator.

In the test strip an acid buffer is added to the reagent area to maintain a constant pH of 3, which in the absence of urine protein produces a yellow color. The development of any green to blue color indicates the presence of protein. The intensity of the color is proportional to the amount of protein that is present. The protein area is read at 60 seconds.

Esbach’s test: Quantitative test for albumin: Accurate estimation of the amount of albumin present in the urine is seldom necessary for routine clinical work. Ordinarily, information obtained from properly conducted qualitative tests will suffice.

The urine is made acidic by addition of 5% acetic acid, and poured into an Esbach’s tube upto the ‘U’ mark. Esbach’s reagent (picric acid, citric acid, water) is added upto the mark ‘R’. Tube is closed with a rubber cork and the contents mixed by inversion. The tube is allowed to stand for 24 hrs at room temperature and the results are read from the height of the protein precipitate in the scale at the bottom of the tube in grams of protein/litre of urine.

Bence Jones proteinuria: These proteins are the free light chains (either kappa or lambda) produced by plasma cells.  This protein precipitates between 50-60°C, redissolves on boiling and precipitates again on cooling. Presence of this is pathognomonic of multiple myeloma. It is also seen in leukaemia, lymphoma and amyloidosis.

Causes of proteinuria - Proteinuria can occur in varying severity. It is graded as follows

Minimal proteinuria <0.5g/day, Moderate proteinuria-0.5 to 3g/day, High proteinuria- >3g/day

1.  Physiologic conditions 

   1. Severe exercise

   2. Exposure to cold

   3. Orthostatic proteinuria

2. Mild proteinuria

3. Congestive heart failure

   1. Fever 

   2. Urinary tract infection

   3. Toxemia of pregnancy

   4. Polycystic kidney

   5. Hypertension

4. Moderate proteinuria 

   1. Chronic glomerulonephritis

   2. Chronic pyelonephritis

   3. Nephrosclerosis

   4. Multiple myeloma

5. Severe proteinuria

6. Nephrotic syndrome

7. Renal vein thrombosis

8. Severe diabetic nephropathy

3. Ketonuria: Implies presence of ketone bodies in the urine. The ketone bodies include: acetone, acetoacetic acid and β hydroxybutyric acid. They are formed as a result of incomplete fat metabolism as with 

• Defective carbohydrate metabolism

• Defective carbohydrate absorption

• Inadequate dietary intake

Ketone bodies are formed during the catabolism of fatty acids. One of the intermediate products of fatty acid breakdown is acetyl CoA. Acetyl CoA enters the Krebs cycle in the body if fat and carbohydrate degradation are appropriately balanced. The first step in the Krebs cycle is the reaction of acetyl CoA with oxaloacetate to yield citrate. When carbohydrate is not available or is not being properly utilized, all available oxaloacetate will be used to form glucose, and so there will be none available for condensation with acetyl CoA. So CoA cannot enter the Krebs cycle; and is diverted to the formation of ketone bodies. In certain conditions where metabolisms of lipids and carbohydrates are disturbed, the rate of ketogenesis in the liver is too high and ketone bodies accumulate in the blood and are excreted in the urine.

a. Rothera’s Test: This test detects diacetic acid and acetone

Principle: In alkaline medium, ketone bodies (isonitroamine derivative of the ketone bodies) react with Sodium Nitroprusside and form a purple colored complex (ferropentacyanide).

Procedure: 

5ml of urine is saturated with ammonium sulphate in a test tube

Few crystals of sodium nitroprusside are added and dissolved. 

Overlay with liquor ammonia. 

A gradually deepening purple ring shows the presence of acetone. 

b.Gerhardt’s test: Principle: It is based on the reaction of ferric chloride with diacetic acid to form a port wine or Bordeaux red color

Procedure: 

To 5cc of urine, add drop by drop 10% solution of ferric chloride.

A precipitate of ferric phosphate appears and then dissolves in an excess of reagent. 

Formation of a Bordeaux red color indicates presence of aceto-acetic acid

c. Reagent test strips: This test is based on the reaction of acetoacetic acid with sodium nitroprusside in a strongly basic medium. The colors range from beige or buff-pink color for a “Negative” reading to pink and pink-purple for a “Positive” reading. 

The ketone test area provides semi-quantitative results and reacts with acetoacetic acid in urine. This test does not react with beta-hydroxybutyric acid or acetone. The reagent area detects as little as 5 -10 mg/dl acetoacetic acid in urine.

False positive results may be obtained with urine specimens containing large amounts of phenylketones  or L-dopa metabolites.

Conditions associated with ketonuria: Normally, no ketones are present in urine. Detectable levels of ketone may occur in urine during physiological stress conditions such as fasting, pregnancy, and frequent strenuous exercise. In starvation diets, or in other abnormal carbohydrate metabolism situation, ketones appear in the urine in excessively large amounts before serum ketones are elevated. The conditions associated with ketosis include:

Diabetes mellitus

Prolonged vomiting/ diarrhea 

Starvation

High fat-low carbohydrate diet 

Prolonged febrile illness

 Hyperthyroidism

Following ether or chloroform anesthesia

Haemoglobinuria and Haematuria: Refers to presence of haemoglobin or blood in urine. Chemical tests can be used to detect the presence of RBCs or Hb in urine, but hematuria is best recognized by microscopy and haemoglobinuria by spectroscopy. 

a. Benzidine test: 

Principle: The peroxide activity of haemoglobin decomposes hydrogen peroxide and liberates nascent oxygen which in turn oxidizes benzidine to give a blue or green color.

Procedure: 

Mix 2ml of urine and 2ml of glacial acetic acid 

Add a pinch of benzidine powder.

Add 1ml of H 2 O2 slowly along the side of test tube. 

The appearance of blue color indicates the presence of Hb.

b. Reagent strip test: This test is based on the pseudoperoxidase action of hemoglobin and erythrocytes which catalyzes the reaction of 3, 3’, 5, 5’-tetramethyl-benzidine and buffered organic peroxide. The resulting colors range from orange to yellow-green and dark green. Very high blood concentration may cause the color development to continue to dark blue.

The sensitivity of the blood test is reduced in urine with high specific gravity and/or high ascorbic acid content. Microbial peroxidase, associated with urinary tract infection may cause false positive reactions. 

Any green spots or green color developing on the reagent area within 40 seconds is significant and the urine should be examined further. Blood is frequently, but not invariably found in the urine of menstruating females. 

Causes of occult blood in urine

a. Prerenal causes: due to intravascular hemolysis

Hemolytic anaemia

Incompatible blood transfusion

Paroxysmal nocturnal haemoglobinuria

Paroxysmal cold haemoglobinuria

b. Renal causes

Acute Glomerulonephritis

Nephrolithiasis

Nephrosclerosis

Tumors

Infections

Drugs –anticoagulants, penicillin and cephalosporins

c. Post renal: due to bleeding from the urinary tract

Infections – cystitis, urethritis

Tumors/ calculi –in ureter and bladder

Trauma

A false positive reaction may be given by hypochlorite (an oxidizing agent), high bacterial content (bacterial peroxidases) and contamination of urine with menstrual blood.

A false negativity could result because of high levels of ascorbic acid or when the urine sample is not mixed well before testing as red cells tend to settle at the bottom of the container.

Bilirubinuria (Bile Pigments): Refers to presence of bilirubin in the urine. Bilirubin is the breakdown product of haemoglobin and normally no bilirubin is passed in urine. If the blood level of conjugated bilirubin becomes elevated, e.g. due to hepatic disease, excess conjugated bilirubin is excreted in the urine, indicating a pathological process. Unconjugated bilirubin is not water-soluble and so is not excreted in the urine. Testing urine for both bilirubin and urobilinogen can help differentiate obstructive liver disease from other causes of jaundice. 

a. Fouchet’s Test:

Principle: Barium chloride reacts with urinary sulphate to form barium sulphate. If bilirubin is present in urine, it adheres to the barium sulphate precipitate and Ferric chloride oxidises bilirubin to green biliverdin in the presence of trichloroacetic acid.

Fouchet’s reagent

Trichloroacetic acid – 25%

10% ferric chloride - 10cc

Distilled water – 100cc

Procedure:

 To 5ml of urine add 5ml of 10% barium chloride and filter the mixture

Put one drop of Fouchet’s reagent on the precipitate on the filter paper

Green or blue color indicates presence of bilirubin.

Bilirubinuria is found in cases of hepatic and obstructive jaundice but not in hemolytic jaundice.

b. Reagent strip test: This test is based on the coupling of bilirubin with a diazotized dichloroaniline in a strongly acid medium. The colors range from light tan to reddish -brown. 

Reactions may occur with urine containing large doses of chlorpromazine or rifampicin that might be mistaken for positive bilirubin. Indican (indoxyl sulfate) and metabolites of Cortez may cause false positive or atypical color while ascorbic acid (25mg/dL or greater) may cause false negative results. 

Normally, no bilirubin is detectable in urine by even the most sensitive method. Even trace amounts of bilirubin are sufficiently abnormal to require further investigation. 

Bile Salts: 

Hay’s test

Principle: Bile salts if present in urine lowers the surface tension of the urine and the sulphur 

powder sprinkled over the surface of urine will sink to the bottom. 

 Procedure: 

      Take urine in a wide bore test tube and fill it with urine about 2/3rd -3/4th 

Sprinkle finely powdered sulphur powder over the urine

Sulphur powder will sink if bile salts are present, otherwise it floats.

Urobilinogen:

a.Chemical test: Ehrlichs aldehyde test

Principle: Ehrlichs aldehyde reagent is used to detect urobilinogen in urine. Urobilinogen is one of the bile pigments found in urine in case of liver defects, (epidemic icterus, cirrhosis) or as a result of excessive formation of bilirubin (haemolytic jaundice). Urobilinogen is normally present in urine at concentrations up to 1.0 mg/dL. The colourless urobilinogen reactes with Ehrlichs aldehyde reagent in an acidic medium to form pink-red condensing products. 

False positive results can be caused by medications.

 High nitrite concentrations can cause false negative reactions. 

Pigmented urine can interfere with detection of urobilinogen.

Procedure: To 10 ml of urine, add 1.0 ml Ehrlich's benzaldehyde reagent, mix and let it stand for about 10 minutes. Observe colour by looking down into the tube held over a white surface. Formation of pink-red colour indicates positivity.

b.Reagent strip test:

This test is based on a modified Ehrlich reaction in which p- diethylaminobenzaldehyde reacts with urobilinogen in a strongly acid medium. Colors range from light pink to bright magenta. 

In a healthy population, the normal urine urobilinogen range obtained is 0.2-1.0 Ehrlich Unit/dl. A result of 2.0 EU/dl is of clinical significance and the patient should be evaluated further

The test area will react with interfering substances known to react with Ehrlich’s reagent, such as porphobilinogen and p-aminosalicyclic acid. This test is not a reliable method for the detection of porphobilinogen. Drugs containing azo-dyes (Cortez) may give a masking golden color. The absence of urobilinogen cannot be determined with this test. 

Features of hepatic jaundice: 1.Positive urine bilirubin 2. Decreased fecal urobilinogen and 

3. Normal/ increased/ decreased urine urobilinogen. 

Causes of hepatic jaundice include: Viral hepatitis, cirrhosis, chemical intoxication

Features of obstructive jaundice: 1. Positive urine bilirubin 2. Negative urine urobilinogen  

3. Negative/ trace amounts of fecal urobilinogen

Causes of obstructive jaundice include: Obstruction of intrahepatic/common bile duct by gall stones/ carcinoma of gall blabber, carcinoma of head of pancreas, pancreatitis

Features of hemolytic jaundice: Negative urine bilirubin, increased urine and fecal urobilinogen.

Causes of hemolytic jaundice include: intravascular hemolysis as in hemolytic anaemia