Advertisement

Bile salts are salts of four different types of bile acids: cholic, deoxycholic, chenodeoxycholic, and lithocholic. These bile acids combine with glycine or taurine to form complex salts or acids. Bile salts enter the small intestine through the bile and act as detergents to emulsify fat and reduce the surface tension on fat droplets so that enzymes (lipases) can breakdown the fat. In the terminal ileum, bile salts are absorbed and enter in the blood stream from where they are taken up by the liver and re-excreted in bile (enterohepatic circulation).

Bile salts along with bilirubin can be detected in urine in cases of obstructive jaundice. In obstructive jaundice, bile salts and conjugated bilirubin regurgitate into blood from biliary canaliculi (due to increased intrabiliary pressure) and are excreted in urine. The test used for their detection is Hay’s surface tension test. The property of bile salts to lower the surface tension is utilized in this test.

Take some fresh urine in a conical glass tube. Urine should be at the room temperature. Sprinkle on the surface particles of sulphur. If bile salts are present, sulphur particles sink to the bottom because of lowering of surface tension by bile salts. If sulphur particles remain on the surface of urine, bile salts are absent.

Thymol (used as a preservative) gives false positive test.

Bilirubin is converted to non-reactive biliverdin on exposure to light (daylight or fluorescent light) and on standing at room temperature. Biliverdin cannot be detected by tests that detect bilirubin. Therefore fresh sample that is kept protected from light is required. Findings associated with bilirubinuria are listed below.

Methods for detection of bilirubin in urine are foam test, Gmelin’s test, Lugol iodine test, Fouchet’s test, Ictotest tablet test, and reagent strip test.

  1. Foam test: About 5 ml of urine in a test tube is shaken and observed for development of yellowish foam. Similar result is also obtained with proteins and highly concentrated urine. In normal urine, foam is white.
  2. Gmelin’s test: Take 3 ml of concentrated nitric acid in a test tube and slowly place equal quantity of urine over it. The tube is shaken gently; play of colors (yellow, red, violet, blue, and green) indicates positive test (Figure 823.1).
  3. Lugol iodine test: Take 4 ml of Lugol iodine solution (Iodine 1 gm, potassium iodide 2 gm, and distilled water to make 100 ml) in a test tube and add 4 drops of urine. Mix by shaking. Development of green color indicates positive test.
  4. Fouchet’s test: This is a simple and sensitive test.
    i. Take 5 ml of fresh urine in a test tube, add 2.5 ml of 10% of barium chloride, and mix well. A precipitate of sulphates appears to which bilirubin is bound (barium sulphate-bilirubin complex).
    ii. Filter to obtain the precipitate on a filter paper.
    iii. To the precipitate on the filter paper, add 1 drop of Fouchet’s reagent. (Fouchet’s reagent consists of 25 grams of trichloroacetic acid, 10 ml of 10% ferric chloride, and distilled water 100 ml).
    iv. Immediate development of blue-green color around the drop indicates presence of bilirubin (Figure 823.2).
  5. Reagent strips or tablets impregnated with diazo reagent: These tests are based on reaction of bilirubin with diazo reagent; color change is proportional to the concentration of bilirubin. Tablets (Ictotest) detect 0.05-0.1 mg of bilirubin/dl of urine; reagent strip tests are less sensitive (0.5 mg/dl).
Figure 823.1 Positive Gmelins test for bilirubin showing play of colors
Figure 823.1 Positive Gmelin’s test for bilirubin showing play of colors

Figure 823.2 Positive Fouchets test for bilirubin in urine
Figure 823.2 Positive Fouchet’s test for bilirubin in urine

The proportion of ketone bodies in urine in ketosis is variable: β-hydroxybutyric acid 78%, acetoacetic acid 20%, and acetone 2%.

No method for detection of ketonuria reacts with all the three ketone bodies. Rothera’s nitroprusside method and methods based on it detect acetoacetic acid and acetone (the test is 10-20 times more sensitive to acetoacetic acid than acetone). Ferric chloride test detects acetoacetic acid only. β-hydroxybutyric acid is not detected by any of the screening tests.

Methods for detection of ketone bodies in urine are Rothera’s test, Acetest tablet method, ferric chloride test, and reagent strip test.

1. ROTHERA’S’ TEST (Classic Nitroprusside Reaction)

Acetoacetic acid or acetone reacts with nitroprusside in alkaline solution to form a purple-colored complex (Figure 822.1). Rothera’s test is sensitive to 1-5 mg/dl of acetoacetate and to 10-25 mg/dl of acetone.

article continued below
Figure 822.1 Principles of Rothera Test in Urine
Figure 822.1 Principles of Rothera’s test and reagent strip test for ketone bodies in urine. Ketones are detected as acetoacetic acid and acetone but not β-hydroxybutyric acid

Method

  1. Take 5 ml of urine in a test tube and saturate it with ammonium sulphate.
  2. Add a small crystal of sodium nitroprusside. Mix well.
  3. Slowly run along the side of the test tube liquor ammonia to form a layer.
  4. Immediate formation of a purple permanganate colored ring at the junction of the two fluids indicates a positive test (Figure 822.2).

False-positive test can occur in the presence of L-dopa in urine and in phenylketonuria.

Figure 822.2 Rotheras tube test and reagent strip test for ketone bodies in urine
Figure 822.2 Rothera’s tube test and reagent strip test for ketone bodies in urine

2. ACETEST TABLET TEST

This is Rothera’s test in the form of a tablet. The Acetest tablet consists of sodium nitroprusside, glycine, and an alkaline buffer. A purplelavender discoloration of the tablet indicates the presence of acetoacetate or acetone (≥ 5 mg/dl). A rough estimate of the amount of ketone bodies can be obtained by comparison with the color chart provided by the manufacturer.

The test is more sensitive than reagent strip test for ketones.

3. FERRIC CHLORIDE TEST (Gerhardt’s)

Addition of 10% ferric chloride solution to urine causes solution to become reddish or purplish if acetoacetic acid is present. The test is not specific since certain drugs (salicylate and L-dopa) give similar reaction. Sensitivity of the test is 25-50 mg/dl.

4. REAGENT STRIP TEST

Reagent strips tests are modifications of nitroprusside test (Figures 822.1 and 822.2). Their sensitivity is 5-10 mg/dl of acetoacetate. If exposed to moisture, reagent strips often give false-negative result. Ketone pad on the strip test is especially vulnerable to improper storage and easily gets damaged. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.

1. HEAT AND ACETIC ACID TEST (BOILING TEST)
 
This test is based on the principle that proteins get precipitated when boiled in an acidic solution.
 
Method
 
Urine should be clear; if not, filter or use supernatant from a centrifuged sample.
 
Urine should be just acidic (check with litmus paper); if not, add 10% acetic acid drop by drop until blue litmus paper turns red.
 
A test tube is filled 2/3rds with urine. The tube is inclined at an angle and the upper portion is boiled over the flame. (Only the upper portion is heated so that convection currents generated by heat do not disturb the precipitate and the upper portion can be compared with the lower clear portion). Compare the heated part with the lower part. Cloudiness or turbidity indicates presence of either phosphates or proteins (Figure 821.1). A few drops of 10% acetic acid are added and the upper portion is boiled again. Turbidity due to phosphates disappears while that due to proteins does not.
 
Figure 821.1 Principle of heat test for proteins
Figure 821.1 Principle of heat test for proteins
 
False-positive test occurs with tolbutamide and large doses of penicillins.
 
2. REAGENT STRIP TEST
 
The reagent area of the strip is coated with an indicator and buffered to an acid pH which changes color in the presence of proteins (Figures 821.2 and 821.3). The principle is known as “protein error of indicators”.
 
Figure 821.2 Principle of reagent strip test for proteins
Figure 821.2 Principle of reagent strip test for proteins. The principle is called as ‘protein error of indicators’ meaning that one color appears if protein is present and another color if protein is absent. Sensitivity is 5-10 mg/dl. The test does not detect Bence Jones proteins, hemoglobin, and myoglobin
 
The reagent area is impregnated with bromophenol blue indicator buffered to pH 3.0 with citrate. When the dye gets adsorbed to protein, there is change in ionization (and hence pH) of the indicator that leads to change in color of the indicator. The intensity of the color produced is proportional to the concentration of protein. The test is semi-quantitative.
 
Figure 821.3 Grading of proteinuria with reagent strip test
Figure 821.3 Grading of proteinuria with reagent strip test (above) and sulphosalicylic acid test (below)
 
Reagent strip test is mainly reactive to albumin. It is false-negative in the presence of Bence Jones proteins, myoglobin, and hemoglobin. Overload (Bence Jones) proteinuria and tubular proteinuria may be missed entirely if only reagent strip method is used. This test should be followed by sulphosalicylic acid test, which is a confirmatory test. Highly alkaline urine, gross hematuria, and contamination with vaginal secretions can give false-positive reactions. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.
 
3. SULPHOSALICYLIC ACID TEST
 
Addition of sulphosalicylic acid to the urine causes formation of a white precipitate if proteins are present (Proteins are denatured by organic acids and precipitate out of solution).
 
Take 2 ml of clear urine in a test tube. If reaction of urine is neutral or alkaline, a drop of glacial acetic acid is added. Add 2-3 drops of sulphosalicylic acid (3 to 5%), and examine for turbidity against a dark background (Figure 821.3).
 
This test is more sensitive and reliable than boiling test.
 
False-positive test may occur due to gross hematuria, highly concentrated urine, radiographic contrast media, excess uric acid, tolbutamide, sulphonamides, salicylates, and penicillins.
 
False-negative test can occur with very dilute urine.
 
The test can detect albumin, hemoglobin, myoglobin, and Bence Jones proteins.
 
Comparison of reagent strip test and sulphosalicylic acid test is shown in Table 821.1.
 
Table 821.1 Comparison of two tests for proteinuria
Parameter Reagent strip test Sulphosalicylic acid test
1. Principle Colorimetric Acid precipitation
2. Proteins detected Albumin All (albumin, Bence Jones proteins, hemoglobin, myoglobin)
3. Sensitivity 5-10 mg/dl 20 mg/dl
4. Indicator Color change Turbidity
5. Type of test Screening Confirmatory
 
QUANTITATIVE ESTIMATION OF PROTEINS
 
Indications for quantitative estimation of proteins in urine are:
 
  • Diagnosis of nephrotic syndrome
  • Detection of microalbuminuria or early diabetic nephropathy
  • To follow response to therapy in renal disease
 
Proteinuria >1500 mg/ 24 hours indicates glomerular disease; proteinuria >3500 mg/24 hours is called as nephrotic range proteinuria; in tubular, hemodynamic and post renal diseases, proteinuria is usually < 1500 mg/24 hours.
 
Grading of albuminuria is shown in Table 821.2. There are two methods for quantitation of proteins:
 
  1. Estimation of proteins in a 24-hour urine sample, and
  2. Estimation of protein/creatinine ratio in a random urine sample.
 
Table 821.2 Grading of albuminuria
Condition mg/24 hr mg/L mg/g creatinine μg/min μg/mg creatinine g/mol creatinine
Normal < 30 < 20 < 20 < 20 < 30 < 2.5
Microalbuminuria 30-300 20-200 20-300 20-200 30-300 2.5-25
Overt albuminuria > 300 > 200 > 300 > 200 > 300 > 25
 
1. Quantitative estimation of proteins in a 24-hour urine sample: Collection of a 24-hour sample is given earlier. Adequacy of sample is confirmed by calculating expected 24-hour urine creatinine excretion. Daily urinary creatinine excretion depends on muscle mass and remains relatively constant in an individual patient. In adult males creatinine excretion is 14-26 mg/kg/24 hours, while in women it is 11-20 mg/kg/24 hours. Various methods are available for quantitative estimation of proteins: Esbach’s albuminometer method, turbidimetric methods, biuret reaction, and immunologic methods.
 
2. Estimation of protein/creatinine ratio in a random urine sample: Because of the problem of incomplete collection of a 24-hour urine sample, many laboratories measure protein/creatinine ratio in a random urine sample. Normal protein/creatinine ratio is < 0.2. In low-grade proteinuria it is 0.2-1.0; in moderate, it is 1.0-3.5; and in nephrotic- range proteinuria it is > 3.5.
 
MICROALBUMINURIA
 
This is defined as urinary excretion of 30 to 300 mg/24 hours (or 2-20 mg/dl) of albumin in urine.
 
Significance of Microalbuminuria
 
  1. Microalbuminuria is considered as the earliest sign of renal damage in diabetes mellitus (diabetic nephropathy). It indicates increase in capillary permeability to albumin and denotes microvascular disease. Microalbuminuria precedes the development of diabetic nephropathy by a few years. If blood glucose level and hypertension are tightly controlled at this stage by aggressive treatment then progression to irreversible renal disease and subsequent renal failure can be delayed or prevented.
  2. Microalbuminuria is an independent risk factor for cardiovascular disease in diabetes mellitus.
 
Detection of Microalbuminuria: Microalbuminuria cannot be detected by routine tests for proteinuria. Methods for detection include:
 
  • Measurement of albumin-creatinine ratio in a random urine sample
  • Measurement of albumin in an early morning or random urine sample
  • Measurement of albumin in a 24 hr sample
 
Test strips that screen for microalbuminuria are available commercially. Exact quantitation can be done by immunologic assays like radioimmunoassay or enzyme linked immunosorbent assay.
 
BENCE JONES PROTEINURIA
 
Bence Jones proteins are monoclonal immunoglobulin light chains (either κ or λ) that are synthesized by neoplastic plasma cells. Excess production of these light chains occurs in plasma cell dyscrasias like multiple myeloma and primary amyloidosis. Because of their low molecular weight and high concentration they are excreted in urine (overflow proteinuria).
 
Bence Jones proteins have a characteristic thermal behaviour. When heated, Bence Jones proteins precipitate at temperatures between 40°C to 60°C (other proteins precipitate between 60-70°C), and precipitate disappears on further heating at 85-100°C (while precipitate of other proteins does not). When cooled (60-85°C), there is reappearance of precipitate of Bence Jones proteins. This test, however, is not specific for Bence Jones proteins and both false-positive and -negative results can occur. This test has been replaced by protein electrophoresis of concentrated urine sample (Figure 821.4).
 
Figure 821.4 Urine protein electrophoresis showing heavy Bence Jones proteinuria
Figure 821.4 Urine protein electrophoresis showing heavy Bence Jones proteinuria (red arrow) along with loss of albumin and other low molecular weight proteins in urine
 
Further evaluation of persistent overt proteinuria is shown in Figure 821.5.
 
Figure 821.5 Evaluation of proteinuria
Figure 821.5 Evaluation of proteinuria.
Note: Quantitation of proteins and creatinine clearance are done in all patients with persistent proteinuria

1. COPPER REDUCTION METHODS

A. Benedict’s qualitative test:

When urine is boiled in Benedict’s qualitative solution, blue alkaline copper sulphate is reduced to red-brown cuprous oxide if a reducing agent is present (Figure 820.1). The extent of reduction depends on the concentration of the reducing substance. This test, however, is not specific for glucose.

Figure 820.1 Principle of Benedict’s qualitative test for sugar in urine
Figure 820.1 Principle of Benedict’s qualitative test for sugar in urine. Sensitivity is 200 mg of glucose/dl

Other carbohydrates (like lactose, fructose, galactose, pentoses), certain metabolites (glucuronic acid, homogentisic acid, uric acid, creatinine), and drugs (ascorbic acid, salicylates, cephalosporins, penicillins, streptomycin, isoniazid, para-aminosalicylic acid, nalidixic acid, etc.) also reduce alkaline copper sulphate solution.

Method

  1. Take 5 ml of Benedict’s qualitative reagent in a test tube (composition of Benedict’s qualitative reagent: copper sulphate 17.3 gram, sodium carbonate 100 gram, sodium citrate 173 gram, distilled water 1000 ml).
  2. Add 0.5 ml (or 8 drops) of urine. Mix well.
  3. Boil over a flame for 2 minutes.
  4. Allow to cool at room temperature.
  5. Note the color change, if any.

Sensitivity of the test is about 200 mg reducing substance per dl of urine. Since Benedict’s test gives positive reaction with carbohydrates other than glucose, it is also used as a screening test (for detection of galactose, lactose, fructose, maltose, and pentoses in urine) for inborn errors of carbohydrate metabolism in infants and children.

article continued below

For testing urine only for glucose, reagent strips are preferred (see below).

The result is reported in grades as follows (Figure 820.2):

  • Nil: no change from blue color
  • Trace: Green without precipitate
  • 1+ (approx. 0.5 grams/dl): Green with precipitate
  • 2+ (approx. 1.0 grams/dl): Brown precipitate
  • 3+ (approx. 1.5 grams/dl: Yellow-orange precipitate
  • 4+ (> 2.0 grams/dl): Brick- red precipitate.
Figure 820.2 Grading of Benedicts test
Figure 820.2 Grading of Benedict’s test (above) and reagent strip test (below) for glucose

B. Clinitest tablet method (Copper reduction tablet test):

This is a modified form of Benedict’s test in which the reagents are present in a tablet form (copper sulphate, citric acid, sodium carbonate, and anhydrous sodium hydroxide). Sensitivity is 200 mgs/dl of glucose.

2. REAGENT STRIP METHOD

This test is specific for glucose and is therefore preferred over Benedict’s and Clinitest methods. It is based on glucose oxidase-peroxidase reaction. Reagent area of the strips is impregnated with two enzymes (glucose oxidase and peroxidase) and a chromogen. Glucose is oxidized by glucose oxidase with the resultant formation of hydrogen peroxide and gluconic acid. Oxidation of chromogen occurs in the presence of hydrogen peroxide and the enzyme peroxidase with resultant color change (Figure 820.3). Nature of chromogen and buffer system differ in different strips. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.

The strip is dipped into the urine sample and color is observed after a specified time and compared with the color chart provided (Figure 820.2).

Figure 820.3 Principle of reagent strip test for glucose in urine
Figure 820.3 Principle of reagent strip test for glucose in urine. Each mole of glucose produces one mole of peroxide, and each mole of peroxide reduces one mole of oxygen. Sensitivity is 100 mg glucose/100 ml

This test is more sensitive than Benedict’s qualitative test and specific only for glucose. Other reducing agents give negative reaction.

Sensitivity of the test is about 100 mg glucose/dl of urine.

False-positive test occurs in the presence of oxidizing agent (bleach or hypochlorite used to clean urine containers), which oxidizes the chromogen directly.

False-negative test occurs in the presence of large amounts of ketones, salicylates, ascorbic acid, and severe Escherichia coli infection (catalase produced by organisms in urine inactivates hydrogen peroxide).

The parameters to be examined on physical examination of urine are listed below.

  • Volume
  • Color
  • Appearance
  • Odor
  • Specific Gravity
  • pH

VOLUME

Volume of only the 24-hr specimen of urine needs to be measured and reported. The average 24-hr urinary output in adults is 600-2000 ml. The volume varies according to fluid intake, diet, and climate. Abnormalities of urinary volume are as follows:

  • Polyuria means urinary volume > 2000 ml/24 hours. This is seen in diabetes mellitus (osmotic diuresis), diabetes insipidus (failure of secretion of antidiuretic hormone), chronic renal failure (loss of concentrating ability of kidneys) or diuretic therapy.
  • Oliguria means urinary volume < 400 ml/24 hours. Causes include febrile states, acute glomerulonephritis (decreased glomerular filtration), congestive cardiac failure or dehydration (decreased renal blood flow).
  • Anuria means urinary output < 100 ml/24 hours or complete cessation of urine output. It occurs in acute tubular necrosis (e.g. in shock, hemolytic transfusion reaction), acute glomerulonephritis, and complete urinary tract obstruction.

COLOR

Normal urine color in a fresh state is pale yellow or amber and is due to the presence of various pigments collectively called urochrome. Depending on the state of hydration urine may normally be colorless (over hydration) or dark yellow (dehydration). Some of the abnormal colors with associated conditions are listed in Table 819.1.

Table 819.1 Different colors of urine
Colors Conditions
Colorless Dilute urine (diabetes mellitus, diabetes insipidus, overhydration)
Red Hematuria, Hemoglobinuria, Porphyria, Myoglobinuria
Dark brown or black Alkaptonuria, Melanoma
Brown Hemoglobinuria
Yellow Concentrated urine
Yellow-green or green Biliverdin
Deep yellow with yellow foam Bilirubin
Orange or orange-brown Urobilinogen/Porphobilinogen
Milky-white Chyluria
Red or orange fluorescence with UV light Porphyria
Note: Many drugs cause changes in urine color; drug history should be obtained if there is abnormal coloration of urine

APPEARANCE

Normal, freshly voided urine is clear in appearance. Causes of cloudy or turbid urine are listed in Table 819.2. Foamy urine occurs in the presence of excess proteins or bilirubin.

article continued below
Table 819.2 Causes of cloudy or turbid urine
Cause Appearance Diagnosis
1. Amorphous phosphates White and cloudy on standing in alkaline urine Disappear on addition of a drop of dilute acetic acid
2. Amorphous urates Pink and cloudy in acid urine Dissolve on warming
3. Pus cells Varying grades of turbidity Microscopy
4. Bacteria Uniformly cloudy; do not settle at the bottom following centrifugation Microscopy, Nitrite test

ODOR

Freshly voided urine has a typical aromatic odor due to volatile organic acids. After standing, urine develops ammoniacal odor (formation of ammonia occurs when urea is decomposed by bacteria). Some abnormal odors with associated conditions are:

  • Fruity: Ketoacidosis, starvation
  • Mousy or musty: Phenylketonuria
  • Fishy: Urinary tract infection with Proteus, tyrosinaemia.
  • Ammoniacal: Urinary tract infection with Escherichia coli, old standing urine.
  • Foul: Urinary tract infection
  • Sulfurous: Cystinuria.

SPECIFIC GRAVITY (SG)

This is also called as relative mass density. It depends on amount of solutes in solution. It is basically a comparison of density of urine against the density of distilled water at a particular temperature. Specific gravity of distilled water is 1.000. Normal SG of urine is 1.003 to 1.030 and depends on the state of hydration. SG of normal urine is mainly related to urea and sodium. SG increases as solute concentration increases and decreases when temperature rises (since volume expands with rise in temperature).

SG of urine is a measure of concentrating ability of kidneys and is determined to get information about this tubular function. SG, however, is affected by proteinuria and glycosuria.

Causes of increase in SG of urine are diabetes mellitus (glycosuria), nephrotic syndrome (proteinuria), fever, and dehydration.

Causes of decrease in SG of urine are diabetes insipidus (SG consistently between 1.002-1.003), chronic renal failure (low and fixed SG at 1.010 due to loss of concentrating ability of tubules) and compulsive water drinking.

Methods for measuring SG are urinometer method, refractometer method, and reagent strip method.

1. Urinometer method:

This method is based on the principle of buoyancy (i.e. the ability of a fluid to exert an upward thrust on a body placed in it). Urinometer (a hydrometer) is placed in a container filled with urine (Figure 819.1A). When solute concentration is high, upthrust of solution increases and urinometer is pushed up (high SG). If solute concentration is low, urinometer sinks further into the urine (low SG).

Figure 819.1 A. Urinometer method and B. Reagent strip method for measuring specific gravity of urine
Figure 819.1 (A) Urinometer method and (B) Reagent strip method for measuring specific gravity of urine

Accuracy of a urinometer needs to be checked with distilled water. In distilled water, urinometer should show SG of 1.000 at the temperature of calibration. If not, then the difference needs to be adjusted in test readings taken subsequently.

The method is as follows:

  1. Fill a measuring cylinder with 50 ml of urine.
  2. Lower urinometer gently into the urine and let it float freely.
  3. Let urinometer settle; it should not touch the sides or bottom of the cylinder.
  4. Take the reading of SG on the scale (lowest point of meniscus) at the surface of the urine.
  5. Take out the urinometer and immediately note the temperature of urine with a thermometer.

Correction for temperature: Density of urine increases at low temperature and decreases at higher temperature. This causes false reading of SG. Therefore, SG is corrected for difference between urine temperature and calibration temperature. Check the temperature of calibration of the urinometer To get the corrected SG, add 0.001 to the reading for every 3°C that the urine temperature is above the temperature of calibration. Similarly subtract 0.001 from the reading for every 3°C below the calibration temperature.

Correction for dilution: If quantity of urine is not sufficient for measurement of SG, urine can be appropriately diluted and the last two figures of SG are multiplied by the dilution factor.

Correction for abnormal solute concentration: High SG in the presence of glycosuria or proteinuria will not reflect true kidney function (concentrating ability). Therefore it is necessary to nullify the effect of glucose or proteins. For this, 0.003 is subtracted from temperature-corrected SG for each 1 gm of protein/dl urine and 0.004 for every 1 gm of glucose/dl urine.

2. Refractometer method:

SG can be precisely determined by a refractometer, which measures the refractive index of the total soluble solids. Higher the concentration of total dissolved solids, higher the refractive index. Extent of refraction of a beam of light passed through urine is a measure of solute concentration, and thus of SG. The method is simple and requires only 1-2 drops of urine. Result is read from a scale or from digital display.

3. Reagent strip method:

Reagent strip (Figure 819.1B) measures the concentration of ions in urine, which correlates with SG. Depending on the ionic strength of urine, a polyelectrolyte will ionize in proportion. This causes a change in color of pH indicator (bromothymol blue). Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.

REACTION AND pH

The pH is the scale for measuring acidity or alkalinity (acid if pH is < 7.0; alkaline if pH is > 7.0; neutral if pH is 7.0). On standing, urine becomes alkaline because of loss of carbon dioxide and production of ammonia from urea. Therefore, for correct estimation of pH, fresh urine should be examined.

There are various methods for determination of reaction of urine: litmus paper, pH indicator paper, pH meter, and reagent strip tests.

  1. Litmus paper test: A small strip of litmus paper is dipped in urine and any color change is noted. If blue litmus paper turns red, it indicates acid urine. If red paper turns blue, it indicates alkaline urine (Figure 819.2A).
  2. pH indicator paper: Reagent area (which is impregnated with bromothymol blue and methyl red) of indicator paper strip is dipped in urine sample and the color change is compared with the color guide provided. Approximate pH is obtained.
  3. pH meter: An electrode of pH meter is dipped in urine sample and pH is read off directly from the digital display. It is used if exact pH is required.
  4. Reagent strip test: The test area (Figure 819.2B) contains polyionic polymer bound to H+; on reaction with cations in urine, H+ is released causing change in color of the pH-sensitive dye. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.
Figure 819.2 A. Testing pH of urine with litmus paper and B. with reagent strip test
Figure 819.2 Testing pH of urine with litmus paper (A) and with reagent strip test (B)

Normal pH range is 4.6 to 8.0 (average 6.0 or slightly acidic). Urine pH depends on diet, acid base balance, water balance, and renal tubular function.

Acidic urine is found in ketosis (diabetes mellitus, starvation, fever), urinary tract infection by Escherichia coli, and high protein diet. Alkaline urine may result from urinary tract infection by bacteria that split urea to ammonia (Proteus or Pseudomonas), severe vomiting, vegetarian diet, old ammoniacal urine sample and chronic renal failure.

Determining pH of urine helps in identifying various crystals in urine. Altering pH of urine may be useful in treatment of renal calculi (i.e. some stones form only in acid urine e.g. uric acid calculi; in such cases urine is kept alkaline); urinary tract infection (urine should be kept acid); and treatment with certain drugs (e.g. streptomycin is effective in urinary tract infection if urine is kept alkaline). In unexplained metabolic acidosis, measurement of urine pH is helpful in diagnosing renal tubular acidosis; in renal tubular acidosis, urine pH is consistently alkaline despite metabolic acidosis.

Fresh urine sample should be used because on standing urobilinogen is converted to urobilin, which cannot be detected by routine tests. A timed (2-hour postprandial) sample can also be used for testing urobilinogen.

Methods for detection of increased amounts of urobilinogen in urine are Ehrlich’s aldehyde test and reagent strip test.

1. EHRLICH’S ALDEHYDE TEST

Ehrlich’s reagent (pdimethylaminobenzaldehyde) reacts with urobilinogen in urine to produce a pink color. Intensity of color developed depends on the amount of urobilinogen present. Presence of bilirubin interferes with the reaction, and therefore if present, should be removed. For this, equal volumes of urine and 10% barium chloride are mixed and then filtered. Test for urobilinogen is carried out on the filtrate. However, similar reaction is produced by porphobilinogen (a substance excreted in urine in patients of porphyria).

Fig. 818.1 Ehrlichs aldehyde test for urobilinogen
Figure 818.1 Ehrlich’s aldehyde test for urobilinogen

article continued below

Method

Take 5 ml of fresh urine in a test tube. Add 0.5 ml of Ehrlich’s aldehyde reagent (which consists of hydrochloric acid 20 ml, distilled water 80 ml, and paradimethylaminobenzaldehyde 2 gm). Allow to stand at room temperature for 5 minutes. Development of pink color indicates normal amount of urobilinogen. Darkred color means increased amount of urobilinogen (Figure 818.1).

Since both urobilinogen and porphobilinogen produce similar reaction, further testing is required to distinguish between the two. For this, Watson-Schwartz test is used. Add 1-2 ml of chloroform, shake for 2 minutes and allow to stand. Pink color in the chloroform layer indicates presence of urobilinogen, while pink coloration of aqueous portion indicates presence of porphobilinogen. Pink layer is then decanted and shaken with butanol. A pink color in the aqueous layer indicates porphobilinogen (Figure 818.2).

Figure 818.2 Interpretation of Watson Schwartz test
Figure 818.2 Interpretation of Watson-Schwartz test

False-negative reaction can occur in the presence of (i) urinary tract infection (nitrites oxidize urobilinogen to urobilin), and (ii) antibiotic therapy (gut bacteria which produce urobilinogen are destroyed).

2. REAGENT STRIP METHOD

This method is specific for urobilinogen. Test area is impregnated with either p-dimethylaminobenzaldehyde or 4-methoxybenzene diazonium tetrafluoroborate. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.

Porphyrias (from Greek porphura meaning purple pigment; the name is probably derived from purple discoloration of some body fluids during the attack) are a heterogeneous group of rare disorders resulting from disturbance in the heme biosynthetic pathway leading to the abnormal accumulations of red and purple pigments called as porphyrins in the body. Heme, a component of hemoglobin, is synthesized through various steps as shown in Figure 817.1. Each of the steps is catalyzed by a separate enzyme; if any of these steps fails (due to hereditary or acquired cause), precursors of heme (porphyrin intermediates) accumulate in blood, get deposited in skin and other organs, and excreted in urine and feces. Depending on the site of defect, different types of porphyrias are described with varying clinical features, severity, and the nature of accumulated porphyrin.
 
Porphyria has been offered as a possible explanation for the medieval tales of vampires and werewolves; this is because of the number of similarities between the behavior of persons suffering from porphyria and the folklore (avoiding sunlight, mutilation of skin on exposure to sunlight, red teeth, psychiatric disturbance, and drinking of blood to obtain heme).
 
Porphyrias are often missed or wrongly diagnosed as many of them are not associated with definite physical findings, screening tests may yield false-negative results, diagnostic criteria are poorly defined and mild disorders produce an enzyme assay result within ‘normal’ range.
 
Heme is mainly required in bone marrow (for hemoglobin synthesis) and in liver (for cytochromes). Therefore, porphyrias are divided into erythropoietic and hepatic types, depending on the site of expression of disease. Hepatic porphyrias mainly affect the nervous system, while erythropoietic porphyrias primarily affect the skin. Porphyrias are also classified into acute and nonacute (or cutaneous) types depending on clinical presentation (Table 817.1).
 
Table 817.1 Various classification schemes for porphyrias
Classification based on predominant clinical manifestations
Classification based on site of expression of disease
Classification based on mode of clinical presentation
Neuropsychiatric
Hepatic
Acute
1. Acute intermittent porphyria
1. ALA-dehydratase porphyria
1. ALA-dehydratase porphyria (Plumboporphyria)
2. ALA-dehydratase porphyria (Plumboporphyria)
2. Acute intermittent porphyria
2. Acute intermittent porphyria
Cutaneous (Photosensitivity)
3. Hereditary coproporphyria
3. Hereditary coproporphyria
1. Congenital erythropoietic porphyria
4. Variegate porphyria
4. Variegate porphyria
2. Porphyria cutanea tarda
Erythropoietic porphyria
Non-acute (cutaneous)
3. Erythropoietic protoporphyria
1. Congenital erythropoietic porphyria
1. Porphyria cutanea tarda
Mixed (Neuropsychiatric and cutaneous)
2. Erythropoietic protoporphyria
2. Congenital erythropoietic porphyria
1. Hereditary coproporphyria
Hepatic/Erythropoietic
3. Erythropoietic protoporphyria
2. Variegate porphyria
1. Porphyria cutanea tarda
 
 
Inheritance of porphyrias may be autosomal dominant or recessive. Most acute porphyrias are inherited in an autosomal dominant manner (i.e. inheritance of one abnormal copy of gene). Therefore, the activity of the deficient enzyme is 50%. When the level of heme falls in the liver due to some cause, activity of ALA synthase is stimulated leading to increase in the levels of heme precursors up to the point of enzyme defect. Increased levels of heme precursors cause symptoms of acute porphyria. When the heme level returns back to normal, symptoms subside.
 
Accumulation of porphyrin precursors can occur in lead poisoning due to inhibition of enzyme aminolevulinic acid dehydratase in heme biosynthetic pathway. This can mimick acute intermittent porphyria.
 
CLINICAL FEATURES
 
Clinical features of porphyrias are variable and depend on type. Acute porphyrias present with symptoms like acute and severe abdominal pain/vomiting/constipation, chest pain, emotional and mental disorders, seizures, hypertension, tachycardia, sensory loss, and muscle weakness. Cutaneous porphyrias present with photosensitivity (redness and blistering of skin on exposure to sunlight), itching, necrosis of skin and gums, and increased hair growth over the temples (Table 817.2).
 
Table 817.2 Clinical characteristics of porphyrias
Porphyria Deficient enzyme Clinical features Inheritance Initial test
1. Acute intermittent porphyria (AIP)* PBG deaminase Acute neurovisceral attacks; triggering factors+ (e.g. drugs, diet restriction) Autosomal dominant Urinary PBG; urine becomes brown, red, or black on standing
2. Variegate porphyria Protoporphyrinogen oxidase Acute neurovisceral attacks + skin fragility, bullae Autosomal dominant Urinary PBG
3. Hereditary coproporphyria Coproporphyrinogen oxidase Acute neurovisceral attacks + skin fragility, bullae Autosomal dominant Urinary PBG
4. Congenital erythropoietic porphyria Uroporphyrinogen cosynthase Onset in infancy; skin fragility, bullae; extreme photosensitivity with mutilation; red teeth and urine (pink red urinestaining of diapers) Autosomal recessive Urinary/fecal total porphyrins; ultraviolet fluorescence of urine, feces, and bones
5. Porphyria cutanea tarda* Uroporphyrinogen decarboxylase Skin fragility, bullae Autosomal dominant (some cases) Urinary/fecal total porphyrins
6. Erythropoietic protoporphyria* Ferrochelatase Acute photosensitivity Autosomal dominant Free erythrocyte protoporphyrin
Disorders marked with * are the three most common porphyrias. PBG: Porphobilinogen
  
Symptoms can be triggered by drugs (barbiturates, oral contraceptives, diazepam, phenytoin, carbamazepine, methyldopa, sulfonamides, chloramphenicol, and antihistamines), emotional or physical stress, infection, dieting, fasting, substance abuse, premenstrual period, smoking, and alcohol. Autosomal dominant porphyrias include acute intermittent porphyria, variegate porphyria, porphyria cutanea tarda, erythropoietic protoporphyria (most cases), and hereditary coproporphyria. Autosomal recessive porphyrias include: congenital erythropoietic porphyria, erythropoietic protoporphyria (few cases), and ALAdehydratase porphyria (plumboporphyria).
 
LABORATORY DIAGNOSIS
 
Porphyria can be diagnosed through tests done on blood, urine, and feces during symptomatic period. Timely and accurate diagnosis is required for effective management of porphyrias. Due to the variability and a broad range of clinical features, porphyrias are included under differential diagnosis of many conditions. All routine hospital laboratories usually have facilities for initial investigations in suspected cases of porphyrias; laboratory tests for identification of specific type of porphyrias are available in specialized laboratories.
 
INITIAL STUDIES
 
In suspected acute porphyrias (acute neurovisceral attack), a fresh randomly collected urine sample (10-20 ml) should be submitted for detection of excessive urinary excretion of porphobilinogen (PBG) (see Figure 817.2). In AIP, urine becomes red or brown on standing (see Figure 817.3). In suspected cases of cutaneous porphyrias (acute photosensitivity without skin fragility), free erythrocyte protporphyrin or FEP in EDTA blood (for diagnosis of erythrocytic protoporphyria) and for all other cutaneous porphyrias (skin fragility and bullae), examination of fresh, random urine (10-20 ml) and either feces (5-10 g) or plasma for excess porphyrins are necessary (see Figure 817.4 and Table 817.2).
 
Figure 817.2 Evaluation of acute neurovisceral porphyria
 Figure 817.2 Evaluation of acute neurovisceral porphyria
 
Figure 817.3 Red coloration of urine on standing in acute intermittent porphyria
Figure 817.3 Red coloration of urine on standing in acute intermittent porphyria
 
Figure 817.4 Evaluation of cutaneous porphyrias
Figure 817.4 Evaluation of cutaneous porphyrias
 
Apart from diagnosis, the detection of excretion of a particular heme intermediate in urine or feces can help in detecting site of defect in porphyria. Heme precursors up to coproporphyrinogen III are water-soluble and thus can be detected in urine. Protoporphyrinogen and Protoporphyrin are insoluble in water and are excreted in bile and can be detected in feces. All samples should be protected from light.
 
Samples required are
 
  1. 10-20 ml of fresh random urine sample without any preservative;
  2. 5-10 g wet weight of fecal sample, and
  3. blood anticoagulated with EDTA.
 
Test for Porphobilinogen in Urine
 
Ehrlich’s aldehyde test is done for detection of PBG. Ehrlich’s reagent (p-dimethylaminobenzaldehyde) reacts with PBG in urine to produce a red color. The red product has an absorption spectrum with a peak at 553 nm and a shoulder at 540 nm. Since both urobilinogen and porphobilinogen produce similar reaction, further testing is required to distinguish between the two. Urobilinogen can be removed by solvent extraction. (See Watson-Schwartz test). Levels of PBG may be normal or near normal in between attacks. Therefore, samples should be tested during an attack to avoid false-negative results.
 
Test for Total Porphyrins in Urine
 
Total porphyrins can be detected in acidified urine sample by spectrophotometry (Porphyrins have an intense absorbance peak around 400 nm). Semiquantitative estimation of porphyrins is possible.
 
Test for Total Porphyrins in Feces
 
Total porphyrins in feces can be determined in acidic extract of fecal sample by spectrophotometry; it is necessary to first remove dietary chlorophyll (that also absorbs light around 400 nm) by diethyl ether extraction.
 
Tests for Porphyrins in Erythrocytes and Plasma
 
Visual examination for porphyrin fluorescence, and solvent fractionation and spectrophotometry have now been replaced by fluorometric methods.
 
Further Testing
 
If the initial testing for porphyria is positive, then concentrations of porphyrins should be estimated in urine, feces, and blood to arrive at specific diagnosis (Tables 817.3 and 817.4).
 
Table 817.3 Diagnostic patterns of concentrations of heme precursors in acute porphyrias
Porphyria Urine Feces
Acute intermittent porphyria PBG, Copro III
Variegate porphyria PBG, Copro III Proto IX
Hereditary coproporphyria PBG, Copro III Copro III
PBG: Porphobilinogen; Copro III: Coproporphyrinogen III; Proto IX: Protoporphyrin IX
 
Table 817.4 Diagnostic patterns of concentrations of heme precursors in cutaneous porphyrias
Porphyria Urine Feces Erythrocytes
Congenital erythropoietic porphyria Uro I, Copro I Copro I
Porphyria cutanea tarda Uroporphyrin Isocopro
Erythropoietic protoporphyria Protoporphyrin
Uro I: Uroporphyrinogen I; Copro I: Coproporphyrinogen I; Isocopro: Isocoproporphyrinogen
 
In latent porphyrias and in patients during remission, porphyrin levels may be normal; in such cases, enzymatic and DNA testing is necessary for diagnosis.
 
If porphyria is diagnosed, then it is necessary to investigate close family members for the disorder. Positive family members should be counseled regarding triggering factors.
This is done by flow cytometric analysis for detection of lack of GpIb/IX in Bernanrd Soulier syndrome (deficiency of CD42), and lack of GpIIb/IIIa in Glanzmann’s thrombasthenia (deficiency of CD41, CD61).
 
What is the best protocol for platelet glycoprotein (GPIIb/IIIa) analysis using flow cytometry?
 
Fresh platelets should always be used. Storing platelets dramatically changes the level of transmembrane proteins. The best way is to follow one of standardized protocols defined in: Immunophenotypic analysis of platelets. Krueger LA, Barnard MR, Frelinger AL 3rd, Furman MI, Michelson AD.Curr Protoc Cytom. 2002 Feb;Chapter 6:Unit 6.10

TEST FOR D-DIMER

  • 03 Aug 2017
D-dimer is derived from the breakdown of fibrin by plasmin and D-dimer test is used to evaluate fibrin degradation. Blood sample can be either serum or plasma. Latex or polystyrene microparticles coated with monoclonal antibody to D-dimer are mixed with patient’s sample and observed for microparticle agglutination. As the particle is small, turbidometric endpoint can be determined in automated instruments. D-dimer and FDPs are raised in disseminated intravascular coagulation, intravascular thrombosis (myocardial infarction, stroke, venous thrombosis, pulmonary embolism), and during postoperative period or following trauma. D-dimer test is commonly used for exclusion of thrombosis and thrombotic tendencies.
 
Further Reading:
 
FDPs are fragments produced by proteolytic digestion of fibrinogen or fibrin by plasmin. For determination of FDPs, blood is collected in a tube containing thrombin (to remove all fibrinogen by converting it into a clot) and soybean trypsin inhibitor (to inhibit plasmin and thus prevent in vitro breakdown of fibrin). A suspension of latex particles linked to antifibrinogen antibodies (or fragments D and E) is mixed with dilutions of patient’s serum on a glass slide. If FDPs are present, agglutination of latex particles occurs (see Figure 814.1). The highest dilution of serum at which agglutination is detected is used to determine concentration of FDPs. Increased levels of FDPs occur in fibrinogenolysis or fibrinolysis. This occurs in disseminated intravascular coagulation, deep venous thrombosis, severe pneumonia, and recent myocardial infarction.
Because diagnoses and treatment plans are made based on laboratory findings, it is imperative that the equipment utilized in the lab be in excellent working order, serviced at regular intervals, calibrated and cleaned as recommended by the manufacturer, and used properly. In addition to properly functioning equipment, there are things the technician can do to improve the accuracy of their test results:
 
  1. Follow manufacturer directions precisely.
  2. Become familiar with normal and abnormal findings.
  3. Log all activity of equipment, including daily, weekly, and monthly servicing.
  4. Save enough sample to perform tests more than once to verify accuracy of findings.

 

Remember, all laboratory equipment and its results are only as reliable as the human operating the equipment!
Routine care and proper maintenance of microscope will ensure good performance over the years. In addition to this, a properly maintained and clean microscope will always be ready for use at any time. Professional cleaning and maintenance should be considered when routine techniques fail to produce optimal performance of the microscope.
 
Cleaning and maintenance supplies
 
Dust cover: When not in use, a microscope should be covered to protect it from dust, hair, and any other possible sources of dirt. It is important to note that a dust cover should never be placed over a microscope while the illuminator is still on.
 
Lens tissue: Lint-free lens tissues are delicate wipes that would not scratch the surface of the oculars or objective. Always ensure that you are using these types of tissues. Never substitute facial tissue or paper towel, as they are too abrasive.
 
Lens cleaner: Lens cleaning solution assists in removing fingerprints and smudges on lenses and objectives. Apply the lens cleaner to the lens tissue paper and clean/polish the surface.
 
Compressed air duster: Using compressed air to rid the microscope of dust particles is far superior to using your own breath and blowing onto the microscope. Compressed air is clean, and avoids possible contamination of saliva particles.
 
Maintenance tips
 
  1. Whenever the microscope is not in use, turn off the illuminator. This will greatly extend the life of the bulb, as well as keep the temperature down during extended periods of laboratory work.
  2. When cleaning the microscope, use distilled water or lens cleaner. Avoid using other chemicals or solvents, as they may be corrosive to the rubber or lens mounts.
  3. After using immersion oil, clean off any residue immediately. Avoid rotating the 40× objective through immersion oil. If this should occur, immediately clean the 40× objective with lens cleaner before the oil has a chance to dry.
  4. Do not be afraid to use many sheets of lens tissue when cleaning. Use a fresh piece (or a clean area of the same piece) when moving to a different part of the microscope. This avoids tracking dirt/oil/residue to other areas of the microscope.
  5. Store the microscope safely with the stage lowered and the smallest objective in position (4× or 10×). This placement allows for the greatest distance between the stage and the objective. If the microscope is bumped, the likelihood of an objective becoming damaged by the stage surface will be greatly minimized.
Automated hematology analyzers work on different principles:
 
  • Electrical impedance
  • Light scatter
  • Fluorescence
  • Light absorption
  • Electrical conductivity.
 
Most analyzers are based on a combination of different principles.
 
(1) Electrical impedance: This is the classic and timetested technology for counting cellular elements of blood. As this method of cell counting was first developed by Coulter Electronics, it is also called as Coulter principle (see Figure 811.1). Two electrodes placed in isotonic solutions are separated by a glass tube having a small aperture. A vacuum is applied and as a cell passes through the aperture, flow of current is impeded and a voltage pulse is generated.
 
Figure 811.1 Coulter principle of electrical impedance
Figure 811.1 Coulter principle of electrical impedance
 
The requisite condition for cell counting by this method is high dilution of sample so that minimal numbers of cells pass through the aperture at one point of time. There are two electrodes on either side of the aperture; as the solution in which the cells are suspended is an electrolyte solution, an electric current is generated between the two electrodes. When a cell passes through this narrow aperture across which a current is flowing, change in electrical resistance (i.e. momentary interruption of electrical current between the two electrodes) occurs. A small pulse is generated due to a temporary increase in impedance. This pulse is amplified, measured, and counted. The height of the pulse is proportional to cell volume. The width of the pulse corresponds with the time required for the cell to traverse the aperture. Cells that do not pass through the center of the aperture generate a distorted pulse that is not representative of the cell volume. Some analyzers use hydrodynamic focusing to force the cells through the central path so that all cells take the same path for volume measurement.
 
An anticoagulated whole blood sample is aspirated into the system, divided into two portions, and mixed with a diluent. One dilution is passed to the red cell aperture bath (for red cell and platelet counting), and the other is delivered to the WBC aperture bath (where a reagent is added for lysis of red cells and release hemoglobin; this portion is used for leukocyte counting followed by estimation of hemoglobin). Particles between 2-20 fl are counted as platelets, while those between 36-360 fl are counted as red cells. Hemoglobin is estimated by light transmission at 535 nm.
 
(2) Light scatter: Each cell flows in a single line through a flow cell. A laser device is focused on the flow cell; as the laser light beam strikes a cell it is scattered in various directions. One detector captures the forward scatter light (forward angle light scatter or FALS) that is proportional to cell size and a second detector captures side scatter (SS) light (90°) that corresponds to the nuclear complexity and granularity of cytoplasm. This simultaneous measurement of light scattered in two directions is used for distinguishing between granulocytes, lymphocytes, and monocytes.
 
(3) Fluorescence: Cellular fluorescence is used to measure RNA (reticulocytes), DNA (nucleated red cells), and cell surface antigens.
 
(4) Light absorption: Concentration of hemoglobin is measured by absorption spectrophotometry, after conversion of hemoglobin to cyanmethemoglobin or some other compound. In some analyzers, peroxidase cytochemistry is used to classify leukocytes; the peroxidase activity is determined by absorbance.
 
(5) Electrical conductivity: Some analyzers use conductivity of high frequency current to determine physical and chemical composition of leucocytes for their classification.
 
Further Reading:
 
AUTOMATED HEMOTOLOGY ANALYZER
 
Automation is a process of replacement of tasks hitherto performed by humans by computerized methods.
 
Until recently, hematological tests were performed only by manual methods. These methods, though still performed in many peripheral laboratories, are laborintensive, and involve use of hemocytometers (counting chambers), centrifuges, Wintrobe tubes, photometers, and stained blood smears. Hematology cell analyzers can generate the blood test results rapidly and also perform additional tests not possible by manual technology.
 
Both manual and automated laboratory techniques have advantages and disadvantages, and it is unlikely that one will completely replace the other.
 
Advantages of Automated Hematology Analyzer
 
  • Speed with efficient handling of a large number of samples.
  • Accuracy and precision in quantitative blood tests.
  • Ability to perform multiple tests on a single platform.
  • Significant reduction of labor requirements.
  • Invaluable for accurate determination of red cell indices.
 
Disadvantages of Automated Hematology Analyzer
 
  • Flags: Flagging of a laboratory test result demands labour-intensive manual examination of a blood smear.
  • Comments on red cell morphology cannot be generated. Abnormal red cell shapes (such as fragmented cells) cannot be recognized.
  • Erroneously increased or decreased results due to interfering factors.
  • Expensive with high running costs.
 
Automated hematology analyzers are of two main types:
 
  • Semi-automated: Some steps like dilution of blood sample are performed by the technologist; can measure only a few parameters.
  • Fully automated: Require only anticoagulated blood sample; measure multiple parameters.
CAUSES OF ERRONEOUS RESULTS (INTERFERENCES CAUSING ABNORMAL RESULT)
 
These are listed in Table 809.1
 
Table 809.1 Causes of erroneous results with hematology analyzer
Parameter Interfering factors
  Erroneous increase Erroneous decrease
0. All parameters
  • Clotted sample
1. WBC count
  • Nucleated red cells
  • Large platelet clumps
  • Unlysed red cells (some abnormal red cells resist lysing)
  • Cryoglobulins
  • Clotted sample
2. RBC count
  • Very high WBC*
  • Large numbers of giant platelets
  • Clotted sample
  • Microcytic red cells
  • Autoagglutination
3. Hemoglobin
  • Clotted sample
4. MCV
  • Very high WBC
  • Hyperglycemia
  • Autoagglutination (cold agglutinins)
  • Cryoglobulins
5. MCHC
  • Hyperlipidemia
  • Autoagglutination (cold agglutinins)
  • Very high WBC
6. Platelets
  • Microcytic red cells
  • WBC fragments
  • Cryoglobulins
  • Platelet satellitism
  • Platelet clumping
*: WBCs are counted along with RBCs, but normally their number is statistically insignificant
 
 
FLAGGING
 
‘Flags’ are signals that occur when an abnormal result is detected by the analyzer. Flags are displayed to reduce false-positive and false-negative results by mandating a review of blood smear examination.
Parameters measured by hematology analyzers and their derivation are shown in Tables 808.1 and 808.2. Most automated hematology analyzers measure red cell count, red cell indices (mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration), hemoglobin, hematocrit, total leukocyte count, differential leukocyte count (three-part or five-part), and platelet count.
 
Table 808.1 Parameters measured by hematology analyzers
Parameters measured by most analyzers Parameters measured by some analyzers
  • RBC count
  • Hemoglobin
  • Mean cell volume
  • Mean cell hemoglobin
  • Mean cell hemoglobin concentration
  • WBC count
  • WBC differential
  • Platelet count
  • Red cell distribution width
  • Reticulocyte count
  • Reticulocyte hemoglobin content
  • Mean platelet volume
  • Platelet distribution width
  • Reticulated platelets
 
Table 808.2 Parameters reported by hematology analyzers
Parameters measured directly or derived through histogram Parameters measured through calculation
  • RBC count
  • Mean cell volume (Derived from RBC histogram)
  • Red cell distribution width (Derived from RBC histogram)
  • Hemoglobin
  • Reticulocyte count
  • WBC count
  • Differential WBC count (Derived through WBC histogram)
  • Platelet count
  • Mean platelet volume (Derived from platelet histogram)
  • Hematocrit
  • Mean cell hemoglobin
  • Mean cell hemoglobin concentration
 
Estimation of Hemoglobin
 
Hemoglobin is measured directly by a modification of cyanmethemoglobin method (all hemoglobins are converted to cyanmethemoglobin by potassium ferricyanide; cyanmethemoglobin has a broad absorbance peak at 540 nm). Some analyzers use a nonhazardous reagent such as sodium lauryl sulphate. A non-ionic detergent is added for rapid red cell lysis and to minimize turbidity caused by cell membranes and plasma lipids.
 
Estimation of Red Blood Cell Count and Mean Cell Volume (MCV)
 
Red cell count and cell volume are directly measured by aperture impedance or light scatter analysis. In a red cell histogram, cell numbers are plotted on Y-axis, while cell volume is indicated on Xaxis (see Figure 808.1). The analyzer counts those cells as red cells volume of which ranges between 36 fl and 360 fl. MCV is used for morphological classification of anemia into microcytic, macrocytic, and normocytic types.
 
Figure 808.1 Diagrammatic representation of red cell histogram obtained by aperture impedance
Figure 808.1 Diagrammatic representation of red cell histogram obtained by aperture impedance. The analyzer counts cells between 36 fl and 360 fl as red cells. Although leukocytes are present and counted along with red cells in the diluting fluid, their number is not statistically significant. Only if leukocyte count is markedly elevated (>50,000/μl), histogram and the red cell count will be affected. Area of the peak between 60 fl and 125 fl is used for calculation of mean cell volume and red cell distribution width. Abnormalities in red cell histogram include: (1) Left shift of the curve in microcytosis, (2) Right shift of the curve in macrocytosis, and (3) Bimodal peak of the curve in double (dimorphic) population of red cells
 
Estimation of MCH, MCHC, and Hematocrit (HCT/PCV)
 
These parameters are obtained indirectly through calculations.
 
 
MCH (pg) = Hemoglobin (g/l)
                     RBC count (10⁶/μl)
 
 
MCHC (g/dl) = Hemoglobin (g/dl)
                         Hematocrit (%)
 
 
Hematocrit (%) = Mean Cell Volume (fl)
                              RBC count (10⁶/μl)
 
 
Estimation of Red Cell Distribution Width (RDW)
 
RDW is a quantitative measure of variation in sizes of red cells and is expressed as coefficient of variation of red cell size distribution. It is equivalent to anisocytosis observed on blood smear. It is derived from red cell histogram in some analyzers. RDW is usually elevated in iron deficiency anemia, but not in β-thalassemia minor and anemia of chronic disease (other causes of microcytic anemia). However, this distinction is not absolute and there is a significant overlap between values among patients. Raised RDW requires examination of blood smear.
 
Among the red cell values generated by the analyzer (red cell count, hemoglobin, hematocrit, MCV, MCH, MCHC, and RDW), most important for decision-making are hemoglobin, hematocrit, and MCV.
 
WBC Differential
 
Difference between 3-part and 5-part hemotology analyzer...
 
Hematology analyzers can either generate a 3-part differential (differential count reported as lymphocytes, monocytes, and granulocytes) or a 5-part differential (lymphocytes, monocytes, neutrophils, eosinophils, and basophils). The 3-part differential counting is based on electrical impedance volume measurement of leukocytes. In volume histogram for WBCs, approximate numbers of cells are plotted on Y-axis and cell size on X-axis. Those cells with volume 35-90 fl are designated as lymphocytes, cells with volume 90-160 fl as mononuclear cells, and cells with volume 160-450 fl as neutrophils (see Figure 808.2). Any deviation from the expected histogram is flagged by the analyzer, mandating review of blood smear. A large proportion of 3-part differential counts are ‘flagged’ to avoid missing abnormal cells.
 
Instruments measuring a 5-part differential work on a combination of different principles, e.g. light scatter, impedance, and electrical conductivity, a combination of light scatter, peroxidase staining, and resistance of basophils to lysis in acid buffer, etc.
 
Figure 808.2 Diagrammatic representation of WBC histogram
Figure 808.2 Diagrammatic representation of WBC histogram. WBC histogram analysis shows relative numbers of cells on Y-axis and cell size on X-axis. The lytic agent lyses the cytoplasm that collapses around the nucleus causing differential shrinkage. The analyzer sorts the WBCs according to the nuclear size into 3 main groups (3-part differential): Cells with 35-90 fl volume are designated as lymphocytes, cells with 90-160 fl volume are designated as monocytes, and cells with 160-450 fl volume are designated as neutrophils. Abnormalities in WBC histogram include: (1) Peak to the left of lymphocyte peak: Nucleated red cells, (2) Peak between lymphocytes and monocytes: Blast cells, eosinophilia, basophilia, plasma cells, and atypical lymphocytes, and (3) Peak between monocytes and neutrophils: Left shift
 
Platelet Count
 
Platelets are difficult to count because of their small size, marked variation in size, tendency to aggregation, and overlapping of size with microcytic red cells, cellular fragments, and other debris. In hematology analyzers, this difficulty is addressed by mathematical analysis of platelet volume distribution so that it corresponds to lognormal distribution. Platelets are counted by electrical impedance method in the RBC aperture, and a histogram is generated with platelet volume on X-axis and relative cell frequency on Y-axis (see Figure 808.3). Normal platelet histogram consists of a right-skewed single peak. Particles greater than 2 fl and less than 20 fl are classified as platelets by the analyzer.
 
Figure 808.3 Diagrammatic representation of normal platelet histogram
Figure 808.3 Diagrammatic representation of normal platelet histogram: Counting and sizing of platelets by electrical impedance method occurs in the RBC aperture. The counter designates particles between sizes 2 fl and 20 fl as platelets. Abnormalities in platelet histogram result from interferences such as cytoplasmic fragments (peak at left end of histogram) or severely microcytic red cells and giant platelets (peak at right end of histogram)
 
Two other platelet parameters can be obtained from platelet histogram using computer technology: mean platelet volume (MPV) and platelet distribution width (PDW). Some analyzers can generate another parameter called as reticulated platelets.
 
MPV refers to the average size of platelets and is obtained from mathematical calculation. Normal MPV is 7-10 fl. Increased MPV (> 10 fl) results from presence of immature platelets in circulation; peripheral destruction of platelets stimulates megakaryocytes to produce such platelets (e.g. in idiopathic thrombocytopenic purpura). Decreased MPV (< 7 fl) is due to presence of small platelets in circulation (in conditions associated with reduced production of platelets in bone marrow).
 
PDW is analogous to RDW and is a measure of variation in size of platelets (normal <20%). Increased PDW is observed in megaloblastic anemia, chronic myeloid leukemia, and after chemotherapy.
 
Some analyzers measure reticulated platelets or young platelets that contain RNA (similar to reticulocytes). Increased numbers of reticulated platelets are seen in thrombocytopenia due to peripheral destruction of platelets.

Reticulocyte Count
 
Various fluorescent dyes can combine with RNA of reticulocytes; the fluorescence then is counted in a flow cytometer. More immature reticulocytes fluoresce more strongly as they contain more RNA.
 
Reticulocyte hemoglobin content is a parameter that estimates hemoglobinization of most recently produced red cells. It is a predictor of iron deficiency.
 
WBC Cytogram (Scattergram)
 
In the scattergram, each dot represents a cell of a given volume and density, and the positions of dots in the graph are determined by the degree of side scatter, degree of forward scatter, light absorption by the cell, and cytochemical staining (if used). The forward angle light scatter (FALS) is represented on Y-axis, and the side scatter (SS) is represented on X-axis. Low FALS and low SS are indicative of lymphocytes; with subsequent increasing FALS and SS, monocytes, neutrophils, and lastly eosinophils are designated in the graph. Counting of basophils is based on a different technology.
 
Further Reading:
 

FLOW CYTOMETRY

  • 29 Jul 2017
FLOW CYTOMETRY
 
Box 807.1 Properties of a cell measured by a flow cytometerFlow cytometry is a procedure used for measuring multiple cellular and fluorescent properties of cells when they flow as a single cell suspension through a laser beam by a specialized instrument called as a flow cytometer. Flow cytometry can analyze numerous cells in a short time and multiple parameters of a single cell can be analyzed simultaneously. From the measured parameters, specific cell populations are defined. Cells or particles with size 0.2-150 μm are suitable for flow cytometer analysis.
 
Flow cytometry can provide following information about a cell (Box 807.1):
 
  • Cell size (forward scatter)
  • Internal complexity or granularity (side scatter)
  • Relative fluorescence intensity.
 
A flow cytometer consists of three main components or systems: fluidics, optics, and electronics.
 
(1) Fluidics: The function of the fluidics system is to transport cells in a stream to the laser beam for interrogation. Cells (fluorescence-tagged) are introduced into the cytometer (injected into the sheath fluid within the flow chamber) and made to flow in a single file past a laser (light amplification by stimulated emission of radiation) beam. The stream transporting the cells should be positioned in the center of the laser beam. The portion of the fluid stream where the cells are located is called as the sample core. Only a single cell or particle should pass through the laser beam at one time. Flow cytometers use the principle of hydrodynamic focusing (process of centering the sample core within the sheath fluid) for presenting cells to the laser.
 
(2) Optics: This system consists of lasers for illumination of cells in the sample, and filters to direct the generated light signals to the appropriate detectors.
 
The light source used in most flow cytometers is laser.
 
The laser most commonly used in flow cytometry is Argon-ion laser. The light signals are generated when the laser beam strikes the cell, which are then collected by appropriately positioned lenses. A system of optical mirrors and filters then directs the specified wavelengths of light to the designated detectors. Two types of light signals are generated when the laser beam strikes cells tagged with fluorescent molecules: fluorescence and light scatter. The cells tagged with fluorescence emit a momentary pulse of fluorescence; in addition, two types of light scatter are measured: forward scatter (proportional to cell diameter) and side scatter (proportional to granularity of cell).
 
(3) Electronics: The optical signals (photons) are converted to corresponding electronic signals (electrons) by the photodetectors (photodiodes and photomultiplier tubes). The electronic signal produced is proportional to the amount of light striking a cell. The electric current travels to the amplifier and is converted to a voltage pulse. The voltage pulse is assigned a digital value representing a channel by the Analog-to Digital Converter (ADC). The channel number is then transferred to the computer which displays it to the appropriate position on the data plot.
 
Further Reading:
 
  1. Leukemias and lympomas: Immunophenotyping (evaluation of cell surface markers), diagnosis, detection of minimal residual disease, and to identify prognostically important subgroups.
  2. Paroxysmal nocturnal hemoglobinuria: Deficiency of CD 55 and CD 59.
  3. Hematopoietic stem cell transplantation: Enumeration of CD34+ stem cells.
  4. Feto-maternal hemorrhage: Detection and quantitation of foetal hemoglobin in maternal blood sample.
  5. Anemias: Reticulocyte count.
  6. Human immunodeficiency virus infection: For enumeration of CD4+ lymphocytes.
  7. Histocompatibility cross matching.
Platelet aggregation tests are carried out in specialized hematology laboratories if platelet dysfunction is suspected. These tests are usually indicated in patients presenting with mucocutaneous type of bleeding and in whom screening tests reveal normal platelet count, prolonged bleeding time, normal prothrombin time, and normal activated partial thromboplastin time. Platelet aggregation studies are carried out on platelet-rich plasma using aggregometer. When a platelet aggregating agent is added to platelet-rich plasma, platelets form aggregates and optical density falls (or light transmission increases); this is recorded by a chart recorder on a strip chart. Commonly used platelet aggregating agents are ADP (adenosine 5-diphosphate), epinephrine (adrenaline), collagen, arachidonic acid, and ristocetin. ADP (low dose) and epinephrine induce primary and secondary waves of aggregation (biphasic curve). Primary wave is due to the direct action of aggregating agent on platelets. Secondary wave is due to thromboxane A2 synthesis and secretion from platelets. Collagen, arachidonic acid and ristocetin induce a single wave of aggregation (monophasic curve) Normal aggregation curve is shown in Figure 804.1. Aggregation patterns in various platelet function defects are shown in Figures 804.2 to 804.4, and in Table 804.1.
 
Figure 804.1 Normal platelet aggregation curves
Figure 804.1 Normal platelet aggregation curves
 
Figure 804.2 Platelet aggregation curves in von Willebrand disease and Bernard Soulier syndrome absent aggregation with ristocetin normal aggregation with ADP epinephrine and arachidonic acid
Figure 804.2 Platelet aggregation curves in von Willebrand disease and Bernard-Soulier syndrome (absent aggregation with ristocetin, normal aggregation with ADP, epinephrine, and arachidonic acid)
 
Figure 804.3 Platelet aggregation curves in storage pool defect absent second wave of aggregation with ADP and epinephrine absent or greatly diminished aggregation with collagen and normal ristocetin aggregation
Figure 804.3 Platelet aggregation curves in storage pool defect (absent second wave of aggregation with ADP and epinephrine, absent or greatly diminished aggregation with collagen, and normal ristocetin aggregation)
 
Figure 804.4 Platelet aggregation curves in Glanzmanns thrombasthenia absent aggregation with all agonists except ristocetin
Figure 804.4 Platelet aggregation curves in Glanzmann’s thrombasthenia (absent aggregation with all agonists except ristocetin)
A blood smear is examined for:
 
 
A peripheral blood smear has three parts: Head, body, and tail. Also read: PREPARATION OF BLOOD SMEAR BY WEDGE METHOD.
 
A blood smear should be examined in an orderly manner. Initially, blood smear should be observed under low power objective (10×) to assess whether the film is properly spread and stained, to assess cell distribution, and to select an area for examination of blood cells. Best morphologic details are seen in the area where red cells are just touching one another. Low power view is also helpful for the identification of Rouleaux formation, autoagglutination of red cells, and microfilaria. High power objective (45×) is suitable for examination of red cell morphology and for differential leukocyte count. A rough estimate of total leukocyte count can be obtained which also serves to crosscheck the total leukocyte count done by manual counting or automated method. Oil-immersion objective (100×) is used for more detailed examination of any abnormal cells.
 
Further Reading:
 
Box 802.1 Role of blood smear in thrombocytopeniaPlatelets are small, 1-3 μm in diameter, purple structures with tiny irregular projections on surface. In blood films prepared from non-anticoagulated blood (i.e. direct fingerstick), they occur in clumps. If platelet count is done on automated blood cell counters using EDTA-anticoagulated blood sample, about 1% of persons show falsely low count due to the presence in them of EDTA dependent antiplatelet antibody. Examination of a parallel blood film is useful in avoiding the false diagnosis of thrombocytopenia in such cases. Occasionally, platelets show rosetting around neutrophils (platelet satellitism) (see Figure 802.1). This is seen in patients with platelet antibodies and in apparently normal persons. Blood smear examination can be helpful in determining underlying cause of thrombocytopenia such as leukemia, lymphoma, or microangiopathic hemolytic anemia (Box 802.1).
 
Also Read:
 
For meaningful interpretation, absolute count of leukocytes should be reported. These are obtained as follows:
 
Absolute Leukocyte Count = Leukocyte% × Total Leukocyte Count/ml
 
 
Neutrophilia:
 
An absolute neutrophil count greater than 7500/μl is termed as neutrophilia or neutrophilic leukocytosis.
 
Causes
 
  1. Acute bacterial infections: Abscess, pneumonia, meningitis, septicemia, acute rheumatic fever, urinary tract infection.
  2. Tissue necrosis: Burns, injury, myocardial infarction.
  3. Acute blood loss
  4. Acute hemorrhage
  5. Myeloproliferative disorders
  6. Metabolic disorders: Uremia, acidosis, gout
  7. Poisoning
  8. Malignant tumors
  9. Physiologic causes: Exercise, labor, pregnancy, emotional stress.
 
Leukemoid reaction: This refers to the presence of markedly increased total leukocyte count (>50,000/cmm) with immature cells in peripheral blood resembling leukaemia but occurring in non-leukemic disorders (see Figure 801.2). Its causes are:
 
  • Severe bacterial infections, e.g. septicemia, pneumonia
  • Severe hemorrhage
  • Severe acute hemolysis
  • Poisoning
  • Burns
  • Carcinoma metastatic to bone marrow Leukemoid reaction should be differentiated from chronic myeloid leukemia (Table 801.1).
 
Table 801.1 Differences between leukemoid reaction and leukemia
Table 801.1 Differences between leukemoid reaction and leukemia
 
Figure 801.2 Leukemoid reaction in blood smear
Figure 801.2 Leukemoid reaction in blood smear
 
 
Absolute neutrophil count less than 2000/μl is neutropenia. It is graded as mild (2000-1000/μl), moderate (1000-500/μl), and severe (< 500/μl).
 
Causes
 
I. Decreased or ineffective production in bone marrow:
 
  1. Infections 
    (a) Bacterial: typhoid, paratyphoid, miliary tuberculosis, septicemia
    (b) Viral: influenza, measles, rubella, infectious mononucleosis, infective hepatitis.
    (c) Protozoal: malaria, kala azar
    (d) Overwhelming infection by any organism
  2. Hematologic disorders: megaloblastic anemia, aplastic anemia, aleukemic leukemia, myelophthisis.
  3. Drugs:
    (a) Idiosyncratic action: Analgesics, antibiotics, sulfonamides, phenothiazines, antithyroid drugs, anticonvulsants.
    (b) Dose-related: Anticancer drugs
  4. Ionizing radiation
  5. Congenital disorders: Kostman's syndrome, cyclic neutropenia, reticular dysgenesis.
 
II. Increased destruction in peripheral blood:
 
  1. Neonatal isoimmune neutropaenia
  2. Systemic lupus erythematosus
  3. Felty's syndrome
 
III. Increased sequestration in spleen:
 
  1. Hypersplenism
 
Eosinophilia:
 
This refers to absolute eosinophil count greater than 600/μl.
 
Causes
 
  1. Allergic diseases: Bronchial asthma, rhinitis, urticaria, drugs.
  2. Skin diseases: Eczema, pemphigus, dermatitis herpetiformis.
  3. Parasitic infection with tissue invasion: Filariasis, trichinosis, echinococcosis.
  4. Hematologic disorders: Chronic Myeloproliferative disorders, Hodgkin's disease, peripheral T cell lymphoma.
  5. Carcinoma with necrosis.
  6. Radiation therapy.
  7. Lung diseases: Loeffler's syndrome, tropical eosinophilia
  8. Hypereosinophilic syndrome.
 
Basophilia:
 
Increased numbers of basophils in blood (>100/μl) occurs in chronic myeloid leukemia, polycythemia vera, idiopathic myelofibrosis, basophilic leukemia, myxedema, and hypersensitivity to food or drugs.
 
Monocytosis:
 
This is an increase in the absolute monocyte count above 1000/μl.
 
Causes
 
  1. Infections: Tuberculosis, subacute bacterial endocarditis, malaria, kala azar.
  2. Recovery from neutropenia.
  3. Autoimmune disorders.
  4. Hematologic diseases: Myeloproliferative disorders, monocytic leukemia, Hodgkin's disease.
  5. Others: Chronic ulcerative colitis, Crohn's disease, sarcoidosis.
 
Lymphocytosis:
 
Box 801.1 Differential diagnosis of LymphocytosisThis is an increase in absolute lymphocyte count above upper limit of normal for age (4000/μl in adults, >7200/μl in adolescents, >9000/μl in children and infants) (Box 801.1).
 
Causes
 
  1. Infections: 
    (a) Viral: Acute infectious lymphocytosis, infective hepatitis, cytomegalovirus, mumps, rubella, varicella
    (b) Bacterial: Pertussis, tuberculosis
    (c) Protozoal: Toxoplasmosis
  2. Hematological disorders: Acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, lymphoma.
  3. Other: Serum sickness, post-vaccination, drug reactions.
Approximate idea about total leukocyte count can be gained from the examination of the smear under high power objective (40× or 45×). A differential leukocyte count should be carried out. Abnormal appearing white cells are evaluated under oil-immersion objective.
 
Morphology of normal leukocytes (see Figure 800.1):
 
  1. Polymorphonuclear neutrophil: Neutrophil measures 14-15 μm in size. Its cytoplasm is colorless or lightly eosinophilic and contains multiple, small, fine, mauve granules. Nucleus has 2-5 lobes that are connected by fine chromatin strands. Nuclear chromatin is condensed and stains deep purple in color. A segmented neutrophil has at least 2 lobes connected by a chromatin strand. A band neutrophil shows non-segmented U-shaped nucleus of even width. Normally band neutrophils comprise less than 3% of all leukocytes. Majority of neutrophils have 3 lobes, while less than 5% have 5 lobes. In females, 2-3% of neutrophils show a small projection (called drumstick) on the nuclear lobe. It represents one inactivated X chromosome.
  2. Eosinophil: Eosinophils are slightly larger than neutrophils (15-16 μm). The nucleus is often bilobed and the cytoplasm is packed with numerous, large, bright orange-red granules. On blood smears, some of the eosinophils are often ruptured.
  3. Basophils: Basophils are seen rarely on normal smears. They are small (9-12 μm), round to oval cells, which contain very large, coarse, deep purple granules. It is difficult to make out the nucleus since granules cover it.
  4. Monocytes: Monocyte is the largest of the leukocytes (15-20 μm). It is irregular in shape, with oval or clefted (kidney-shaped) nucleus and fine, delicate chromatin. Cytoplasm is abundant, bluegray with ground glass appearance and often contains fine azurophil granules and vacuoles. After migration to the tissues from blood, they are called as macrophages.
  5. Lymphocytes: On peripheral blood smear, two types of lymphocytes are distinguished: small and large. The majority of lymphocytes are small (7-8 μm). These cells have a high nuclearcytoplasmic ratio with a thin rim of deep blue cytoplasm. The nucleus is round or slightly clefted with coarsely clumped chromatin. Large lymphocytes (10-15 μm) have a more abundant, pale blue cytoplasm, which may contain a few azurophil granules. Nucleus is oval or round and often placed on one side of the cell.
 
Figure 800.1 Normal mature white blood cells in peripheral blood
Figure 800.1 Normal mature white blood cells in peripheral blood
 
Morphology of abnormal leukocytes:
 
  1. Box 800.1 Role of blood smear in leukemiaToxic granules: These are darkly staining, bluepurple, coarse granules in the cytoplasm of neutrophils. They are commonly seen in severe bacterial infections.
  2. Döhle inclusion bodies: These are small, oval, pale blue cytoplasmic inclusions in the periphery of neutrophils. They represent remnants of ribosomes and rough endoplasmic reticulum. They are often associated with toxic granules and are seen in bacterial infections.
  3. Cytoplasmic vacuoles: Vacuoles in neutrophils are indicative of phagocytosis and are seen in bacterial infections.
  4. Shift to left of neutrophils: This refers to presence of immature cells of neutrophil series (band forms and metamyelocytes) in peripheral blood and occurs in infections and inflammatory disorders.
  5. Hypersegmented neutrophils: Hypersegmentation of neutrophils is said to be present when >5% of neutrophils have 5 or more lobes. They are large in size and are also called as macropolycytes. They are seen in folate or vitamin B12 deficiency and represent one of the earliest signs.
  6. Pelger-Huet cells: In Pelger-Huet anomaly (a benign autosomal dominant condition), there is failure of nuclear segmentation of granulocytes so that nuclei are rod-like, round, or have two segments. Such granulocytes are also observed in myeloproliferative disorders (pseudo-Pelger-Huet cells).
  7. Atypical lymphocytes: These are seen in viral infections, especially infectious mononucleosis. Atypical lymphocytes are large, irregularly shaped lymphocytes with abundant cytoplasm and irregular nuclei. Cytoplasm shows deep basophilia at the edges and scalloping of borders. Nuclear chromatin is less dense and occasional nucleolus may be present.
  8. Blast cells: These are most premature of the leukocytes. They are large (15-25 μm), round to oval cells, with high nuclear cytoplasmic ratio. Nucleus shows one or more nucleoli and nuclear chromatin is immature. These cells are seen in severe infections, infiltrative disorders, and leukemia. In leukemia and lymphoma, blood smear suggests the diagnosis or differential diagnosis and helps in ordering further tests (see Figure 800.2 and Box 800.1).
 
Figure 800.2 Morphological abnormalities of white blood cells
Figure 800.2 Morphological abnormalities of white blood cells: (A) Toxic granules; (B) Döhle inclusion body; (C) Shift to left in neutrophil series; (D) Hypersegmented neutrophil in megaloblastic anemia; (E) Atypical lymphocyte in infectious mononucleosis; (F) Blast cell in acute leukemia
 
Further Reading:
 
Role of blood smear in anemiasRed cells are best examined in an area where they are just touching one another (towards the tail of the film). Normal red cells are 7-8 μm in size, round with smooth contours, and stain deep pink at the periphery and paler in the center. Area of central pallor is about 1/3rd the diameter of the red cell. Size of a normal red cell corresponds roughly with the size of the nucleus of a small lymphocyte. Normal red cells are described as normocytic (of normal size) and normochromic (with normal staining intensity i.e. hemoglobin content).
 
Morphologic abnormalities of red cells in peripheral blood smear can be grouped as follows:
 
  • Red cells with abnormal size (see Figure 799.1)
  • Red cells with abnormal staining
  • Red cells with abnormal shape (see Figure 799.1)
  • Red cell inclusions (see Figure 799.2)
  • Immature red cells (see Figure799.3)
  • Abnormal red cell arrangement(see Figure 799.4).
 
Red cells with abnormal size:
 
Mild variation in red cell size is normal. Increased variation in red cell size is called as anisocytosis. This is a feature of most anemias and is non-specific. Anisocytosis is due to the presence of microcytes, macrocytes, or both in addition to red cells of normal size.
 
Microcytes are red cells smaller in size than normal. They are seen when hemoglobin synthesis is defective i.e. in iron deficiency anemia, thalassemias, anemia of chronic disease, and sideroblastic anemia.

Macrocytes are red cells larger in size than normal. Oval macrocytes (macro-ovalocytes) are seen in megaloblastic anemia, myelodysplastic syndrome, and in patients being treated with cancer chemotherapy. Round macrocytes are seen in liver disease, alcoholism, and hypothyroidism.
 
Red cells with abnormal staining (hemoglobin content):

Staining intensity of red cells depends on hemoglobin content. Red cells with increased area of central pallor (i.e. containing less hemoglobin) are called as hypochromic. They are seen when hemoglobin synthesis is defective, i.e. in iron deficiency, thalassemias, anaemia of chronic disease, and sideroblastic anemia.
 
In dimorphic anemia, there are two distinct populations of red cells in the same smear. An example is presence of both normochromic and hypochromic red cells seen in sideroblastic anemia, iron deficiency anemia responding to treatment, and following blood transfusion in a patient of hypochromic anemia. In myelodysplastic syndrome, dimorphic picture results from admixture of microcytic hypochromic cells and macrocytes.
 
Red cells with abnormal shape:
 
Increased variation in red cell shape is called as poikilocytosis and is a feature of many anemias. A red cell that is abnormal in shape is called as a poikilocyte.
 
Sickle cells are narrow and elongated red cells with one or both ends pointed. Sickle form is assumed when a red cell containing hemoglobin S is deprived of oxygen. Sickle cells are seen in sickle cell disorders, particularly sickle cell anemia. Sickle cells are not seen on blood smear in neonates with sickle cell disease because high percentage of fetal hemoglobin in red cells prevents sickling.
 
Spherocytes are red cells, which are slightly smaller in size than normal, round, stain intensely, and do not have central area of pallor. The surface area of spherocytes is less as compared to the volume. They are seen in hereditary spherocytosis, autoimmune hemolytic anemia (warm antibody type), and ABO hemolytic disease of newborn.
 
Schistocytes are fragmented red cells, which take various forms like helmet, crescent, triangle, etc. and usually have surface projections or spicules. They are seen in microangiopathic hemolytic anemia, cardiac valve prosthesis, and severe burns.
 
Target cells are red cells with bull's eye appearance. These red cells show a central stained area and a peripheral stained rim with unstained cytoplasm in between. They are seen in hemoglobinopathies (e.g. thalassemias, hemoglobin disease, sickle cell disease), obstructive jaundice, and following splenectomy.disease, sickle cell disease), obstructive jaundice, and following splenectomy.
 
Burr cells or echinocytes are small red cells with regularly placed small projections on surface. They are seen in uremia.
 
Acanthocytes are red cells with irregularly spaced sharp projections of variable length on surface. They are seen in spur cell anemia of liver disease, McLeod phenotype, and following splenectomy.
 
Teardrop cells or dacryocytes have a tapering droplike shape. Numerous teardrop red cells are seen in myelofibrosis and myelophthisic anemia.
 
Blister cells or hemi ghost cells are irregularly contracted cells in which hemoglobin is contracted and condensed away from the cell membrane. This is seen in glucose-6-phosphate dehydrogenase defici-ency during acute hemolytic episode.
 
Bite cells result from removal of Heinz bodies by the pitting action of the spleen (i.e. a part of red cell is bitten off by the splenic macrophages). They are seen in glucose-6-phosphate dehydrogena-se deficiency and unstable hemoglobin disease.
 
Red cell inclusions:
 
Those inclusions that can be visualized on Romanowsky-stained smears are basophilic stippling, Howell-Jolly bodies, Pappenheimer bodies, and Cabot's rings.

Basophilic stippling or punctate basophilia refers to the presence of numerous, irregular basophilic (purple-blue) granules which are uniformly distributed in the red cell. These granules represent aggregates of ribosomes. Their presence is indicative of impaired erythropoiesis and they are seen in thalassemias, megaloblastic anemia, heavy metal poisoning (e.g. lead), and liver disease.cell. These granules represent aggregates of ribosomes. Their presence is indicative of impaired erythropoiesis and they are seen in thalassemias, megaloblastic anemia, heavy metal poisoning (e.g. lead), and liver disease.
 
Red cell inclusions
Figure 799.2 Red cell inclusions: (A) Basophilic stippling; (B) Howell-Jolly bodies; (C) Pappenheimer bodies; (D) Cabot’s ring
 
Howell-Jolly bodies are small, round, purple-staining nuclear remnants located peripherally in red cells. They are seen in megaloblastic anemia, thalasse-mias, hemolytic anemia, and following splenectomy.

Pappenheimer bodies are basophilic, small, ironcontaining granules in red cells. They give positive Perl's Prussian blue reaction. Unlike basophilic stippling, Pappenheimer bodies are few in number and are not distributed throughout the red cell. They are seen following splenectomy and in thalassemias and sideroblastic anemia.

Cabot's rings are fine, reddish-purple or red, ring-like structures. They appear like loops or figure of eight structures. They indicate impaired erythropoiesis and are seen in megaloblastic anemia and lead poisoning.
 
Immature red cells:
 
Polychromatic cells are young red cells containing remnants of ribonucleic acid. These cells are slightly larger than normal red cells and have a diffuse bluishgrey tint. (They represent reticulocytes when stained with a supravital stain like new methylene blue). Polychromasia is due to the uptake of acid stain by hemoglobin and basic stain by ribonucleic acid. Presence of polychromatic cells is indicative of active erythropoiesis and are increased in hemolytic anemia, acute blood loss, and following specific therapy for nutritional anemia.and are increased in hemolytic anemia, acute blood loss, and following specific therapy for nutritional anemia.
 
Nucleated red cells are red cell precursors (erythroblasts), which are released prematurely in peripheral blood from the bone marrow. They are a normal finding in cord blood of newborns. Large number of nucleated red cells in blood smear is seen in hemolytic disease of newborn, hemolytic anemia, leukemias, myelophthisic anemia, and myelofibrosis.
 
Immature red cells
Figure 799.3 Immature red cells: (A) Polychromatic red cell; (B) Nucleated red cell
 
Abnormal red cell arrangement:
 
Rouleaux formation refers to alignment of red cells on top of each other like a stack of coins. It occurs in multiple myeloma, Waldenström's macroglobulinemia, hypergammaglobulinemia, and hyper fibrinogenemia.
 
Abnormal red cell arrangement
Figure 799.4 Abnormal red cell arrangement: (A) Rouleaux formation; (B) Autoagglutination

Autoagglutination refers to the clumping of red cells in large, irregular groups on blood smear. It is seen in cold agglutinin disease. Role of blood smear in anemia is shown in Box 799.1 and Figures 799.5 to 799.7.
 
Figure 799.5 Differential diagnosis of macrocytic anemia on blood smear
Figure 799.5 Differential diagnosis of macrocytic anemia on blood smear: (A) Megaloblastic anemia; (B) Hemolytic anemia; (C) Liver disease; (D) Myelodysplastic syndrome
 
Figure 799.6 Differential diagnosis of microcytic anemia on blood smear
Figure 799.6 Differential diagnosis of microcytic anemia on blood smear: (A) Iron deficiency anemia; (B) Thalassemia minor; (C) Thalassemia major; (D) Sideroblastic anemia
 
Figure 799.7 Differential diagnosis of hemolytic anemia on blood smear
Figure 799.7 Differential diagnosis of hemolytic anemia on blood smear. (A) Microangiopathic hemolytic anemia showing fragmented red cells, (B) Hereditary spherocytosis showing spherocytes and a polychromatic red cell, and (C) Glucose-6-phosphate dehydrogenase deficiency showing a blister cell and a bite cell
 
Further Reading:
 

The microscope is the most important piece of equipment in the clinic laboratory. The microscope is used to review fecal, urine, blood, and cytology samples on a daily basis (see Figure). Understanding how the microscope functions, how it operates, and how to care for it will improve the reliability of your results and prolong the life of this valuable piece of equipment.

Parts and functions of a compound microscope

(A) Arm

Used to carry the microscope.

(B) Base

Supports the microscope and houses the light source.

(C) Oculars (or eyepieces)

The lens of the microscope you look through. The ocular also magnifies the image. The total magnification can be calculated by multiplying the objective power by the ocular power. Oculars come in different magnifications, but 10× magnification is common.

article continued below

(D) Diopter adjustment

The purpose of the diopter adjustment is to correct the differences in vision an individual may have between their left and right eyes.

(E) Interpupillary adjustment

This allows the oculars to move closer or further away from one another to match the width of an individual’s eyes. When looking through the microscope, one should see only a single field of view. When viewing a sample, always use both eyes. Using one eye can cause eye strain over a period of time.

(F) Nosepiece

The nosepiece holds the objective lenses. The objectives are mounted on a rotating turret so they can be moved into place as needed. Most nosepieces can hold up to five objectives.

(G) Objective lenses

The objective lens is the lens closest to the object being viewed, and its function is to magnify it. Objective lenses are available in many powers, but 4×, 10×, 40×, and 100× are standard. 4× objective is used mainly for scanning. 10× objective is considered “low power,” 40× is “high power” and 100× objective is referred to as “oil immersion.” Once magnified by the objective lens, the image is viewed through the oculars, which magnify it further. Total magnification can be calculated by multiplying the objective power by the ocular lens power.

For example: 100× objective lens with 10× oculars = 1000× total magnification.

Compound Microscope

(H) Stage

The platform on which the slide or object is placed for viewing.

(I) Stage brackets

Spring-loaded brackets, or clips, hold the slide or specimen in place on the stage.

(J) Stage control knobs

Located just below the stage are the stage control knobs. These knobs move the slide or specimen either horizontally (x-axis) or vertically (y-axis) when it is being viewed.

(K) Condenser

The condenser is located under the stage. As light travels from the illuminator, it passes through the condenser, where it is focused and directed at the specimen.

(L) Condenser control knob

Allows the condenser to be raised or lowered.

(M) Condenser centering screws:

These crews center the condenser, and therefore the beam of light. Generally, they do not need much adjustment unless the microscope is moved or transported frequently.

(N) Iris diaphragm

This structure controls the amount of light that reaches the specimen. Opening and closing the iris diaphragm adjusts the diameter of the light beam.

(O) Coarse and fine focus adjustment knobs

These knobs bring the object into focus by raising and lowering the stage. Care should be taken when adjusting the stage height. When a higher power objective is in place (100× objective for example), there is a risk of raising the stage and slide and hitting the objective lens. This can break the slide and scratch the lens surface. Coarse adjustment is used for finding focus under low power and adjusting the stage height. Fine adjustment is used for more delicate, high power adjustment that would require fine tuning.

(P) Illuminator

The illuminator is the light source for the microscope, usually situated in the base. The brightness of the light from the illuminator can be adjusted to suit your preference and the object you are viewing.

What is Kohler illumination?

Kohler illumination is a method of adjusting a microscope in order to provide optimal illumination by focusing the light on the specimen. When a microscope is in Kohler, specimens will appear clearer, and in more detail.

Process of setting Kohler

Materials required

  • Specimen slide (will need tofocus under 10× power)
  • Compound microscope.

Kohler illumination

  1. Mount the specimen slide onthe stage and focus under 10×.
  2. Close the iris diaphragm completely.
  3. If the ball of light is not in the center, use the condenser centering screws to move it so that it is centered.
  4. Using the condenser adjustment knobs, raise or lower the condenser until the edges of the field becomes sharp (see Figure 797.1 and Figure 797.2).
  5. Open the iris diaphragm until the entire field is illuminated.
Note the blurry edges of the unfocused light
Figure 797.1 Note the blurry edges of the unfocused light

Adjusting the condenser height sharpens the edges of the ball of light
Figure 797.2 Adjusting the condenser height sharpens the edges of the “ball of light.”

When should you set/check Kohler?

  • During regular microscope maintenance
  • After the microscope is moved/transported
  • Whenever you suspect objects do not appear as sharp as they could be.

Further Reading:

COLLECTION OF BLOOD

It is necessary to follow a standard procedure for specimen collection to get the most accurate and trustworthy results of the laboratory test. The blood sample can be collected from the venipuncture or skin puncture for the hematological investigations.

SKIN PUNCTURE

This method is most common and mostly used in infants and small children and if the small amount of blood is required. This method is suitable for the estimation of hemoglobin, cell counts, determination of hematocrit (HCT) or packed cell volume (PCV) by micro method and preparation of blood films. Blood obtained by this method is also called as capillary blood. However, it is the mixture of blood from arterioles, venules, and capillaries. It also contains small amount of tissue fluid. In infants, blood is collected from the heel (the medial or lateral aspect of plantar surface or great toe). In adults, it is collected from the side of a middle or ring finger (distal digit) or from the earlobe. (see Figure 796.1).

A. Blood lancet and sites of B. finger puncture cross and C. heel puncture shaded areas
Figure 796.1 (A) Blood lancet and sites of (B) finger puncture (cross) and (C) heel puncture (shaded areas)

The puncture site is cleansed with the 70% ethanol or another suitable disinfectant. After drying, a puncture is made with a sterile, dry, disposable lancet, in deep to allow free flow of blood. The first drop of blood is wiped away with the dry and sterile cotton as it contains tissue fluid. After wiping the first drop of blood, next few drops of blood are collected. Excessive pressing should be avoided, as it may dilute the blood with the tissue fluid. After collection of blood, a piece of dry and sterile cotton is pressed over the puncture site till the bleeding ends. Hemoglobin, red cell count and hematocrit (HCT) or packed cell volume (PCV) are moderately higher in the blood collected from skin puncture, as compared to the venous blood. The reason behind this scenario is that platelets adhere to the puncture site and cause the lower count of platelet, and due to small sample size, instant repeat testing is not possible if the result is abnormal or unusual.

Avoid collecting blood from cold, cyanosed skin since the false elevation of values of red blood cells, white blood cells and hemoglobin will be obtained.

article continued below

VENOUS BLOOD COLLECTION

Venous blood is obtained when the larger quantity of blood is needed to perform multiple tests. Different test tubes are filled with blood as per requirement of anticoagulant and blood ratio for the test. Anticoagulant is not required for the test performed by the serum.

Method

  1. Common sites of venepuncture in antecubital fossaThe best site for obtaining blood is the veins of antecubital fossa. A rubber tourniquet is applied to the upper arm (see Figure; Common sites of venepuncture in antecubital fossa (red circles)). It should not be too much tight and should not remain in a place for more than 120 seconds. To get veins more palpable and prominent, the patient is asked to make a fist.
  2. The puncture site is cleansed with the 70% ethanol or other suitable disinfectant and allowed to dry.
  3. The preferred vein is anchored by squeezing and pulling the soft tissues below the prick site with the left hand.
  4. Sterile, dry, disposable needles and syringes should be used for the collection of blood. Needle size should be 23-gauge in children and 19- to 21-gauge in adults. Venepuncture is made along with the direction of the vein and with the bevel of the needle up. Blood is withdrawn slowly. Pulling the piston quickly can cause hemolysis and collapse the vein. The tourniquet should be released as soon as the blood begins to flow into the syringe.
  5. When the required blood is collected, the patient is asked to open his/her fist. The needle is removed from the vein. A sterile alcohol swab is pressed over the puncture site. The patient is asked to press the alcohol swab over the site till the bleeding ends.
  6. The needle is removed from the syringe and the required amount of blood is carefully transferred into the test tube containing anticoagulant as per requirement of the laboratory test. If the blood is forced through the syringe without removing the needle, hemolysis can occur. Containers may be glass bottles or disposable plastic tubes with corks and flat bottom.
  7. Blood is mixed with the anticoagulant in the container thoroughly by gently inverting the container several times. The container should not be shaken strenuously as it can cause hemolysis and fizzing.
  8. Check whether the patient is dizzy and bleeding has stopped. Cover the site of puncture with a sticky bandage strip. Recapping the needle by hand can cause needle-prick injury. After the usage of disposable syringe, needles are crashed by the syringe needle destroyer and the syringe is disposed into the biohazard box. The blood container is labeled properly with the patient’s name, age, gender and the time of collection. The sample should be sent without delay to the laboratory with accompanying properly filled laboratory requisition form.

Precautions

  1. The tourniquet should not be too tight and should not be applied for more than 120 seconds as it will cause hemoconcentration and variation of test results.
  2. The tourniquet should be released before removing the needle from the vein to prevent the formation of a hematoma.
  3. Blood is never collected from the arm being used for the intravenous line since it will dilute the blood sample.
  4. Blood is never collected from an area with hematoma and from a sclerosed vein.
  5. A small bore needle should not be used, blood is withdrawn gradually and the needle is removed from the syringe before transferring blood into the container to avoid hemolysis.
  6. Proper precautions should be noticed while collecting blood either from a skin or a vein puncture since all blood samples are considered as infectious.
  7. The anticoagulated blood sample should be tested within 1-2 hours of collection. If this is not possible, the sample can be stored for 24 hours in a refrigerator at 4-6° C. After the sample is taken out of the refrigerator, it should be allowed to return to room temperature, mixed properly, and then laboratory test is performed.

Complications

  1. Failure to obtain blood: This is very common and usually painful for the patient. This happens if the vein is missed, or excessive pull is applied to the piston causing collapse of the vein.
  2. Formation of hematoma, abscess, thrombosis, thrombophlebitis, or bleeding.
  3. Transmission of infection like human immunodeficiency virus (HIV) or hepatitis B virus (HBV) if reusable syringes and needles, which are not properly sterilized, are used.

Further Reading:

SEQUENCE OF FILLING OF TUBES
 
Following order of filling of tubes should be followed after withdrawal of blood from the patient if multiple investigations are ordered:
 
  1. First tube: Blood culture.
  2. Second tube: Plain tube (serum).
  3. Third tube: Tube containing anticoagulant (EDTA, citrate, or heparin).
  4. Fourth tube: Tube containing additional stabilizing agent like fluoride.
 
Further Reading:
 
Plasma is the supernatant liquid obtained after centrifugation of anticoagulated whole blood.
 
Serum is the liquid obtained after clotting of whole blood sample collected in a plain tube.
 
Some of the differences between the two are as follows:
 
  1. Plasma contains fibrinogen as well as all the other proteins, while serum does not contain fibrinogen.
  2. Plasma can be obtained immediately after sample collection by centrifugation, while minimum of 30 minutes are required for separation of serum from the clotted blood.
  3. Amount of sample is greater with plasma than with serum for a given amount of blood.
  4. Use of anticoagulant may alter the concentration of some constituents if they are to be measured like sodium, potassium, lithium, etc.
Page 3 of 17

Dictionary:

Our Sponsors

We use cookies to improve our website. By continuing to use this website, you are giving consent to cookies being used. More details…