Published in Clinical Pathology
Saturday, 05 August 2017 15:23
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.
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
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).
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.


Published in Clinical Pathology
Thursday, 03 August 2017 18:15
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
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
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 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).
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.
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.


Published in Hemotology
Thursday, 03 August 2017 16:55
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


Published in Hemotology
Thursday, 03 August 2017 16:33
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:


Published in Hemotology
Thursday, 03 August 2017 13:02
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.


Published in Biomedical Engineering
Wednesday, 02 August 2017 09:18
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!


Published in Microbiology
Monday, 31 July 2017 18:06
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.


Published in Hemotology
Monday, 31 July 2017 17:06
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:


Published in Hemotology
Sunday, 30 July 2017 18:20
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.


Published in Hemotology
Saturday, 29 July 2017 19:18
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
  • 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
‘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.




CHOLERA is a specific infectious disease that affects the lower portion of the intestine and is char...

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