- 10 Aug 2017
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.
- 09 Aug 2017
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.
- 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.
- 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).
- 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.
- 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).
- 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).
- 07 Aug 2017
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.
- Take 5 ml of urine in a test tube and saturate it with ammonium sulphate.
- Add a small crystal of sodium nitroprusside. Mix well.
- Slowly run along the side of the test tube liquor ammonia to form a layer.
- 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.
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.
- 07 Aug 2017
|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|
- Diagnosis of nephrotic syndrome
- Detection of microalbuminuria or early diabetic nephropathy
- To follow response to therapy in renal disease
- Estimation of proteins in a 24-hour urine sample, and
- Estimation of protein/creatinine ratio in a random urine sample.
|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|
|Overt albuminuria||> 300||> 200||> 300||> 200||> 300||> 25|
- 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.
- Microalbuminuria is an independent risk factor for cardiovascular disease in diabetes mellitus.
- 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
- 05 Aug 2017
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.
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.
- 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).
- Add 0.5 ml (or 8 drops) of urine. Mix well.
- Boil over a flame for 2 minutes.
- Allow to cool at room temperature.
- 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.
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.
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).
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).
- 05 Aug 2017
The parameters to be examined on physical examination of urine are listed below.
- Specific Gravity
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.
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.
|Colorless||Dilute urine (diabetes mellitus, diabetes insipidus, overhydration)|
|Red||Hematuria, Hemoglobinuria, Porphyria, Myoglobinuria|
|Dark brown or black||Alkaptonuria, Melanoma|
|Yellow-green or green||Biliverdin|
|Deep yellow with yellow foam||Bilirubin|
|Orange or orange-brown||Urobilinogen/Porphobilinogen|
|Red or orange fluorescence with UV light||Porphyria|
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.
|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|
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).
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:
- Fill a measuring cylinder with 50 ml of urine.
- Lower urinometer gently into the urine and let it float freely.
- Let urinometer settle; it should not touch the sides or bottom of the cylinder.
- Take the reading of SG on the scale (lowest point of meniscus) at the surface of the urine.
- 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.
- 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).
- 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.
- 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.
- 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.
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.
- 05 Aug 2017
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).
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).
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.
- 03 Aug 2017
Classification based on predominant clinical manifestations
Classification based on site of expression of disease
Classification based on mode of clinical presentation
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
3. Hereditary coproporphyria
3. Hereditary coproporphyria
1. Congenital erythropoietic porphyria
4. Variegate porphyria
4. Variegate porphyria
2. Porphyria cutanea tarda
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
3. Erythropoietic protoporphyria
2. Variegate porphyria
1. Porphyria cutanea tarda
|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|
- 10-20 ml of fresh random urine sample without any preservative;
- 5-10 g wet weight of fecal sample, and
- blood anticoagulated with EDTA.
|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|
|Congenital erythropoietic porphyria||Uro I, Copro I||Copro I||–|
|Porphyria cutanea tarda||Uroporphyrin||Isocopro||–|
|Uro I: Uroporphyrinogen I; Copro I: Coproporphyrinogen I; Isocopro: Isocoproporphyrinogen|
- 03 Aug 2017
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.
- 03 Aug 2017
- 02 Aug 2017
- Follow manufacturer directions precisely.
- Become familiar with normal and abnormal findings.
- Log all activity of equipment, including daily, weekly, and monthly servicing.
- 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!
- 31 Jul 2017
- 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.
- 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.
- 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.
- 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.
- 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.
- Electrical impedance
- Light scatter
- Light absorption
- Electrical conductivity.
- 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.
- 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.
- 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.
- 29 Jul 2017
|Erroneous increase||Erroneous decrease|
0. All parameters
1. WBC count
2. RBC count
|*: WBCs are counted along with RBCs, but normally their number is statistically insignificant|
- 29 Jul 2017
|Parameters measured by most analyzers||Parameters measured by some analyzers|
|Parameters measured directly or derived through histogram||Parameters measured through calculation|
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.
- 29 Jul 2017
- Cell size (forward scatter)
- Internal complexity or granularity (side scatter)
- Relative fluorescence intensity.
The light source used in most flow cytometers is laser.
- Leukemias and lympomas: Immunophenotyping (evaluation of cell surface markers), diagnosis, detection of minimal residual disease, and to identify prognostically important subgroups.
- Paroxysmal nocturnal hemoglobinuria: Deficiency of CD 55 and CD 59.
- Hematopoietic stem cell transplantation: Enumeration of CD34+ stem cells.
- Feto-maternal hemorrhage: Detection and quantitation of foetal hemoglobin in maternal blood sample.
- Anemias: Reticulocyte count.
- Human immunodeficiency virus infection: For enumeration of CD4+ lymphocytes.
- Histocompatibility cross matching.
- 28 Jul 2017
- 27 Jul 2017
- Red cells: Morphology, immature forms, inclusion bodies, arrangement of cells.
- White cells: Differential count, abnormal or immature forms.
- Platelets: Adequacy, abnormal forms.
- Parasites: Malaria, filaria.
- 26 Jul 2017
- 26 Jul 2017
- Acute bacterial infections: Abscess, pneumonia, meningitis, septicemia, acute rheumatic fever, urinary tract infection.
- Tissue necrosis: Burns, injury, myocardial infarction.
- Acute blood loss
- Acute hemorrhage
- Myeloproliferative disorders
- Metabolic disorders: Uremia, acidosis, gout
- Malignant tumors
- Physiologic causes: Exercise, labor, pregnancy, emotional stress.
- Severe bacterial infections, e.g. septicemia, pneumonia
- Severe hemorrhage
- Severe acute hemolysis
- Carcinoma metastatic to bone marrow Leukemoid reaction should be differentiated from chronic myeloid leukemia (Table 801.1).
(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
- Hematologic disorders: megaloblastic anemia, aplastic anemia, aleukemic leukemia, myelophthisis.
(a) Idiosyncratic action: Analgesics, antibiotics, sulfonamides, phenothiazines, antithyroid drugs, anticonvulsants.
(b) Dose-related: Anticancer drugs
- Ionizing radiation
- Congenital disorders: Kostman's syndrome, cyclic neutropenia, reticular dysgenesis.
- Neonatal isoimmune neutropaenia
- Systemic lupus erythematosus
- Felty's syndrome
- Allergic diseases: Bronchial asthma, rhinitis, urticaria, drugs.
- Skin diseases: Eczema, pemphigus, dermatitis herpetiformis.
- Parasitic infection with tissue invasion: Filariasis, trichinosis, echinococcosis.
- Hematologic disorders: Chronic Myeloproliferative disorders, Hodgkin's disease, peripheral T cell lymphoma.
- Carcinoma with necrosis.
- Radiation therapy.
- Lung diseases: Loeffler's syndrome, tropical eosinophilia
- Hypereosinophilic syndrome.
- Infections: Tuberculosis, subacute bacterial endocarditis, malaria, kala azar.
- Recovery from neutropenia.
- Autoimmune disorders.
- Hematologic diseases: Myeloproliferative disorders, monocytic leukemia, Hodgkin's disease.
- Others: Chronic ulcerative colitis, Crohn's disease, sarcoidosis.
(a) Viral: Acute infectious lymphocytosis, infective hepatitis, cytomegalovirus, mumps, rubella, varicella
(b) Bacterial: Pertussis, tuberculosis
(c) Protozoal: Toxoplasmosis
- Hematological disorders: Acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, lymphoma.
- Other: Serum sickness, post-vaccination, drug reactions.
- 26 Jul 2017
- 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.
- 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.
- 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.
- 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.
- 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.
- Toxic granules: These are darkly staining, bluepurple, coarse granules in the cytoplasm of neutrophils. They are commonly seen in severe bacterial infections.
- 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.
- Cytoplasmic vacuoles: Vacuoles in neutrophils are indicative of phagocytosis and are seen in bacterial infections.
- 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.
- 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.
- 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).
- 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.
- 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).
- 25 Jul 2017
- 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).
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.
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.
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.
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.
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.
- 25 Jul 2017
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
Used to carry the microscope.
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.
(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.
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.
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.
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.
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.
- 25 Jul 2017
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
- Specimen slide (will need tofocus under 10× power)
- Compound microscope.
- Mount the specimen slide onthe stage and focus under 10×.
- Close the iris diaphragm completely.
- If the ball of light is not in the center, use the condenser centering screws to move it so that it is centered.
- 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).
- Open the iris diaphragm until the entire field is illuminated.
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.
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.
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).
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.
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.
- The 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.
- The puncture site is cleansed with the 70% ethanol or other suitable disinfectant and allowed to dry.
- The preferred vein is anchored by squeezing and pulling the soft tissues below the prick site with the left hand.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- The tourniquet should be released before removing the needle from the vein to prevent the formation of a hematoma.
- Blood is never collected from the arm being used for the intravenous line since it will dilute the blood sample.
- Blood is never collected from an area with hematoma and from a sclerosed vein.
- 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.
- Proper precautions should be noticed while collecting blood either from a skin or a vein puncture since all blood samples are considered as infectious.
- 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.
- 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.
- Formation of hematoma, abscess, thrombosis, thrombophlebitis, or bleeding.
- 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.
- First tube: Blood culture.
- Second tube: Plain tube (serum).
- Third tube: Tube containing anticoagulant (EDTA, citrate, or heparin).
- Fourth tube: Tube containing additional stabilizing agent like fluoride.
- Plasma contains fibrinogen as well as all the other proteins, while serum does not contain fibrinogen.
- 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.
- Amount of sample is greater with plasma than with serum for a given amount of blood.
- Use of anticoagulant may alter the concentration of some constituents if they are to be measured like sodium, potassium, lithium, etc.