Sahli’s Acid Hematin Method for the Estimation of Hemoglobin

Published in Cell Biology
Friday, 29 July 2016 11:56
Principle
 
When blood is mixed with an acid solution, the hemoglobin converts into the brown-colored acid hematin. The acid hematin is then diluted with distilled water till the color of the acid hematin matches that of the brown glass standard. The hemoglobin is estimated by reading the value directly from the scale.

Equipments
 
  1. Sahli’s hemoglobinometer: This equipment consist of a comparator with a brown glass standard and  Sahli’s graduated hemoglobin tube which is marked in percent and gram.
  2. Hemoglobin pipette or Sahli’s pipette (marked at 0.02 ml or 20 μl).
  3. Stirrer (a small glass rod).
  4. Dropping pipette (dropper).
 
  1. N/10 hydrochloric acid
  2. Distilled water
 
Specimen
 
Blood is obtained directly by skin puncture or EDTA-anticoagulated venous blood.
 
Method
 
  1. The N/10 hydrochloric acid is place into Sahli’s graduated tube up to mark 2 grams.
  2. With the help of Sahli’s pipette, take blood sample exactly up to 20 μl mark. Blood adhering to the outer part of the pipette is wiped away with the help of tissue paper (absorbent paper) or cotton (gauze piece).
  3. Add blood sample to the N/10 hydrochloric acid solution which is placed into Sahli’s graduated tube, mix the mixture with the help of a glass stirrer, and allow the tube to stand for 10 minutes.
  4. Add distilled water drop by drop into the mixture placed in Sahli’s graduated tube, till the color of the solution matches that of the brown glass standard.
  5. Take the reading of the lower meniscus from the Sahli’s graduated tube in grams.
 
Note:
 
  1. All the Sahli’s graduated tube is marked in both percent and grams figures, this is because (a) different manufacturers of hemoglobinometers have different values as 100%, so that blood sample will yield different results on different instruments and (b) no single hemoglobin is can be evaluate as 100% since it is different according to the sex and age of the individual and altitude.
  2. Disadvantages of Sahli’s method:
    • It is impossible to match the color perfectly of the mixture into the Sahli’s graduated tube with the brown glass standard.
    • Minimum 1 hour is required for the maximum color development of acid hematin because 95% color of acid hematin is attained at the end of 10 minutes.
    • Sulfhemoglobin, methemoglobin and carboxyhemoglobin cannot be converted into acid hematin. Fetal hemoglobin is also not converted to acid hematin and therefore this technique is not appropriate for in small infants.
    • The acid hematin solution is not firm and stable, and the color development is slow.
    • Lights may affect the visual comparison of color.
    • Color of the brown glass standard dims with time.
    • Personal error in matching the color of the mixture in Sahli’s graduated tube with the brown glass standard is 10%.

What is Hemoglobin? Types, Indications and Methods

Published in Cell Biology
Wednesday, 20 July 2016 00:38
Hemoglobin

Hemoglobin carries oxygen from the lungs to the tissues and carbon dioxide from tissues to the lungs. It is composed of heme (iron + protoporphyrin) and globin polypeptide chains. Hemoglobin is not homogeneous and normally different variants and derivatives exist.
Normal hemoglobin variants are embryonic hemoglobins (Gower I, Gower II, and Portland), fetal hemoglobin (Hb F), adult hemoglobin (Hb A), and Hb A2.They differ from each other in the type of polypeptide chains.

INDICATION FOR HEMOGLOBIN ESTIMATION
 
(1) To determine presence and severity of anemia: Anemia refers to low hemoglobin concentration or oxygen-carrying capacity of blood. Clinical signs of anemia (pallor of skin, conjunctival vessels, or mucous membranes) are unreliable for diagnosis of anemia. Anemia is best assessed by estimation of hemoglobin or packed cell volume.
(2) Screening for polycythemia: Polycythemia refers to increased hemoglobin level above the normal range. It may be primary, secondary, or relative.
(3) To assess response to specific therapy in anemia.
(4) Estimation of red cell indices (along with packed cell volume and red cell count) i.e. mean cell hemoglobin and mean cell hemoglobin concentration.
(5) Selection of blood donors.

METHOD FOR ESTIMATION OF HEMOGLOBIN
 
There are various methods for estimation of hemoglobin. These are:

(1) Colorimetric methods: In these methods, color comparison is made between the standard and the test sample, either visually or by colorimetric methods.
• Visual methods: Tallqvist chart, Sahli’s acid hematin method, and WHO hemoglobin color scale.
• Photoelectric methods: Cyanmethemoglobin (hemiglobin-cyanide) method, oxyhemoglobin method, and alkaline hematin method.

(2) Gasometric method: Oxygen carrying capacity of blood is measured in a Van Slyke apparatus. The amount of hemoglobin is then derived from the formula that 1 gram of hemoglobin carries 1.34 ml of oxygen. However, this method measures only physiologically active hemoglobin, which can carry oxygen. It does not measure carboxyhemoglobin, sulfhemoglobin, and methemoglobin. Also, this method is time-consuming and expensive. The result is about 2% less than other methods.

(3) Chemical method: Iron-content of hemoglobin is first estimated. Value of hemoglobin is then derived indirectly from the formula that 100 grams of hemoglobin contain 374 mg of iron. This method is tedious and time-consuming.

(4) Specific gravity method: A rough estimate of hemoglobin is obtained from the specific gravity of blood as determined from copper sulphate technique. This method is useful in mass screening like selection of blood donors. See Procedure.
 
Tallqvist Hemoglobin Chart
Tallqvist hemoglobin chart consists of a series of lithographed colors said to correspond to hemoglobin values ranging from 10 to 100%. A drop of blood obtained by finger puncture is placed on a piece of absorbent paper. Color produced is matched against the color on the chart and corresponding reading is taken. Although this method is cheap and simple, margin of error is 20-50%.

Red Cell Indices

Published in Cell Biology
Tuesday, 19 July 2016 14:02
Red Cell Indices

Red cell indices are mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC). They are also called as “absolute values”. They are derived from the values of hemoglobin, packed cell volume (PCV or hematocrit), and red cell count. Red cell indices are accurately measured by automated hematology analyzers. Recently, a new parameter called red cell distribution width (RDW) has been introduced.
 
USES OF RED CELL INDICES
 
(1) Morphological classification of anemia: Based on values of red cell indices, anemia is classified into three main types: normocytic normochromic, microcytic hypo-chromic, and macrocytic normo-chromic. Calculation of red cell indices is especially helpful in mild or moderate anemia when red cell changes are subtle and often difficult to appreciate on stained blood smear.
(2) Differentiation of iron deficiency anemia from thalassemia trait: In iron deficiency, MCV, MCH, and MCHC are low, while in thalassemia trait, MCV and MCH are low and MCHC is normal.
 
MEAN CELL VOLUME
 
MCV is a measure of average size of the red cells. It is measured directly by automated instruments from the measurement of volume of each red cell. With semiautomated instruments and by manual method, it is obtained by dividing PCV by red cell count.
 
MCV =                PCV in%                  x 10
            RBC count in million/cmm
 
     MCV is expressed in femtoliters or fl (10⁻¹⁵ of a liter). It corresponds with red cell diameter on blood smear. Normal MCV is 80-100 fl.
 
Causes of Increased MCV
 
• Megaloblastic anemia
• Non-megaloblastic macrocytosis: Chronic alcoholism, liver disease, hypothyroidism, normal pregnancy, reticulocytosis
• Newborns.
 
Causes of Low MCV
 
• Microcytic hypochromic anemia
 
MCV is normal in normocytic normochromic anemia (acute blood loss, hemolysis, aplastic anemia).
     In the presence of large number of abnormal red cells like sickle cells, and in dimorphic anemia (e.g. mixed normocytic and microcytic), MCV may be normal (since it is an average value) and thus unreliable for morphological classification.
     Mentzer index is derived by dividing MCV with red cell count. Ratio of less than 13 is seen in thalassemia while ratio is more than 13 in iron deficiency anemia.
 
MEAN CELL HEMOGLOBIN (MCH)
 
MCH is the average amount of hemoglobin in a single red cell. It is obtained by dividing hemoglobin value by red cell count.
 
MCH =    Hemoglobin in grams/dl     x 10
           RBC count in millions/cmm
 
MCH is expressed in picograms or pg (10⁻¹² gram). Reference range is 27-32 pg.
     MCH is decreased in microcytic hypochromic anemia, and increased in macrocytic anemia and in newborns.
 
MEAN CELL HEMOGLOBIN CONCENTRATION (MCHC)
 
MCHC is obtained by dividing hemoglobin value by PCV and expressed in grams/dl or grams/liter. It refers to concentration of hemoglobin in 1 dl or 1 liter of packed red cells.
 
MCHC = Hemoglobin in grams/dl  x 100
                        PCV in %
 
Reference range is 30-35 grams/dl. MCHC is raised in hereditary spherocytosis, and is decreased in hypochromic anemia. MCHC corresponds with degree of hemoglobinization of red cells on a blood smear. If MCHC is normal, red cell is normochromic, and if low, red cell is hypochromic.
 
Red Cell Distribution Width (RDW)
 
Some automated analyzers measure red cell distribution width or RDW. It is a measure of degree of variation in red cell size (anisocytosis) in a blood sample. It is helpful in differential diagnosis of some anemias. Amongst microcytic anemias, RDW is low in ß thalassemia trait, high in iron deficiency anemia, and normal in anemia of chronic disease. Normal RDW is 9.0 to 14.5.
 
REFERENCE RANGES
 
• Mean cell volume: 80-100 fl
• Mean cell hemoglobin: 27-32 pg
• Mean cell hemoglobin concentra-tion: 30-35 g/dl
• Red cell distribution width: 9.0-14.5
 
REFERENCES
 
1. Henry JB. Clinical diagnosis and management by laboratory methods (20th Ed). Philadelphia: WB Saunders Company, 2001.
2. Wallach J. Interpretation of Diagnostic Tests (7th Ed). Philadelphia: Lippincott Williams and Wilkins, 2000¹⁵

Troubleshooting in Microscope

Published in Microbiology
Sunday, 03 July 2016 18:35

“I can focus my slide under 10×, but not under 40×.”

A common reason for this is that the slide is upside down. Double check which side the smear is on (may not be the same side as the label!) and try focusing again. Another cause could be dried immersion oil on the 40× objective that is obstructing your view. When switching from oil immersion (100×) to 40×, there is a good chance that the tip of the 40× objective could be dragged through some immersion oil. If it is not immediately cleaned off, it will dry, producing a thick haze. To fx: Use lens paper and lens cleaner to clean the end of the 40× objective. This may need to be repeated several times depending on how thick the dried oil is. After cleaning, use a dry piece of lens paper to polish the objective. To avoid the problem: Clean up oil immediately after use. Clean the end of the 100× objective and any heavy oil present on the slide before moving back down to 40× objective.

“In hematology, when I focus under 40×, my red blood cells appear shiny.”

This is most likely due to water artifact during the staining and drying process. To make visualization of the cells easier, add a small drop of immersion oil to your slide. Gently spread the drop of oil over the area you will be examining. Wipe of excess oil using the side of your finger. Be very gentle when doing this, and use a clean finger each time you wipe. Wiping too hard or rough will cause your smear to rub off. This technique will leave a very thin layer of oil on your smear. The film is thin enough that you can use the 40× objective without running the risk of the lens becoming contaminated with oil. Try focusing under 40× again, and the shininess should have been resolved.

“There’s no light coming from the illuminator.”

The first assumption is always that the bulb is burnt out, but it is a good idea to check a couple of other possibilities as well. If the iris diaphragm is closed and the brightness of the illuminator is at its lowest, the light may be so small that it appears as if there is no light present. Check to make sure the cord is fully plugged into the back of the microscope. This plug can become dislodged slightly during transport and microscope set up. If your microscope is the type that uses fuses, it may be the fuse—not the bulb—that needs replacing. When the microscope is not in use, be sure to turn it off. This will help prolong the life of the bulb.

Clean up
When the use of the microscope is complete, following proper clean up procedures will improve the quality of images that are viewed and extend the life of the microscope and its components:

(1) Remove the slide from the stage and dispose of it properly.
(2) Clean any oil residue or sample material that may have contaminated the stage surface.
(3) Lower the stage and move the smallest objective into place.
(4) Clean the objective lens and oculars after every use. The order in which they are cleaned is important. Cleaning the 100× objective first and then moving onto other parts will result in immersion oil being spread onto all other components. Using lens tissue and lens cleaner, begin with cleaning the oculars, then the 4× objective, the 10× objective, 40× objective, and finish with the 100× objective lens.

Cross Match Procedure in Blood Bank (Manual Method)

Published in Microbiology
Sunday, 26 June 2016 12:26
When the recipient’s ABO and Rh blood groups are determined, the donor blood unit that is ABO and Rh compatible is selected, and compatibility test is carried out. The purpose of compatibility test is to prevent the transfusion of incompatible red cell units and thus avoidance of hemolytic transfusion reaction in the recipient. Compatibility test detects (i) major ABO grouping error, and (ii) most clinically significant antibodies reactive against donor red cells.

There are two types of cross-match: major cross-match (testing recipient’s serum against donor’s red cells) and minor cross-match (testing donor’s serum against recipient’s red cells). However, minor cross-match is considered as less important since antibodies in donor blood unit get diluted or neutralized in recipient’s plasma. Also, if antibody screening and identification is being carried out, minor cross-matching is not essential. Therefore, only the red cells from the donor unit are tested against the recipient’s serum and the name compatibility test has replaced the term cross-matching.

For transfusion of platelets or fresh frozen plasma, cross-matching is not required. However, fresh frozen plasma should be ABO-compatible.
A full cross-matching procedure consists of:
 
  • Immediate spin cross-match at room temperature, and
  • Indirect antiglobulin test at 37°C.

IMMEDIATE SPIN CROSS MATCH
 
The purpose of this test is to detect ABO incompatibility. Equal volumes of 2% saline suspension of red cells of donor and recipient’s serum are mixed, incubated at room temperature for 5 minutes, and centrifuged. Agglutination or hemolysis indicates incompatibility.
 
Causes of False-negative Test
 
  1. A2B donor red cells and group B recipient serum.
  2. Rapid complement fixation of potent ABO antibodies with bound complement interfering with agglutination.
 
Causes of False-positive Test
 
  1. Rouleaux formation
  2. Cold-reactive antibodies: If agglutination disappears by keeping the tube at 37°C for 10 minutes, presence of cold agglutinins is confirmed.
 
INDIRECT ANTIGLOBULIN TEST
 
Saline-suspended red cells of the donor after being incubated in patient’s serum are washed in saline and antiglobulin reagent is added. Following re-centrifugation, examine for agglutination or hemolysis. This test detects most of the clinically significant IgG antibodies.

If agglutination or hemolysis is not observed in any of the above stages, donor unit is compatible with recipient’s serum. Agglutination or hemolysis at any stage is indicative of incompatibility.
 
EMERGENCY CROSS-MATCH
 
If blood is required urgently, ABO and Rh grouping are carried out by rapid slide test and immediate spin cross match (i.e. the first stage of cross match) is performed (to exclude ABO incompatibility). If the blood unit is compatible, then after issuing it, remaining stage of the cross-match is completed. If any incompatibility is detected, the concerned physician is immediately informed about the incompatibility detected.
 
ANTIBODY SCREENING AND IDENTIFICATION
 
Screening for unexpected or irregular antibodies is done during pre-transfusion testing in recipient’s serum and in donor’s blood. In this test, serum of the recipient is tested against a set of three group O screening cells of known antigenic type. If unexpected antibodies are detected, then they are identified and blood unit that lacks the corresponding antigen is selected for compatibility test.
 
REFERENCE
Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical Hematology (9th Ed). London: Churchill Livingstone, 2001.

Activated Partial Thromboplastin Time (APTT)

Published in Microbiology
Monday, 13 June 2016 22:01
APTT is a measure of coagulation factors in intrinsic pathway (F XII, F XI, high molecular weight kininogen, prekallikrein, F IX, and F VIII) and common pathway (F X, F V, prothrombin, and fibrinogen).
 
Principle
Plasma is incubated with an activator (which initiates intrinsic pathway of coagulation by contact activation). Phospholipid (also called as partial thromboplastin) and calcium are then added and clotting time is measured.
 
Equipment
This is same as for Prothrombin Time test. (Click here to see)
 
Reagents
(1) Kaolin 5 gm/liter: This is a contact activator.
(2) Phospholipid: Various APTT reagents are available commercially, which contain phospholipids.
(3) Calcium chloride 0.025 mol/liter.
 
Specimen
Method
(1) Mix equal volumes of phospholipid reagent and calcium chloride solution in a glass test tube and keep in a waterbath at 37°C.
(2) Deliver 0.1 ml of plasma in another test tube and add 0.1 ml of kaolin solution. Incubate at 37°C in the waterbath for 10 minutes.
(3) After exactly 10 minutes, add 0.2 ml of phospholipidcalcium chloride mixture, start the stopwatch, and note the clotting time.
 
Normal Range
30-40 seconds.
 
Causes of prolongation of APTT
(1) Hemophilia A or B.
(2) Deficiencies of other coagulation factors in intrinsic and common pathways.
(3) Presence of coagulation inhibitors
(4) Heparin therapy
(5) Disseminated intravascular coagulation
(6) Liver disease
 
Uses of APTT
(1) Screening for hereditary disorders of coagulation: Since deficiencies of F VIII (hemophilia A) and F IX (hemophilia B) are relatively common, APTT is the most important screening test for inherited coagulation disorders. APTT detects deficiencies of all coagulation factors except F VII and F XIII. PT is also performed along with APTT. Prolongation of both PT and APTT is indicative of deficiency of coagulation factors in common pathway. Normal PT with prolongation of APTT is indicative of intrinsic pathway deficiency (particularly of F VIII or IX).
(2) To monitor heparin therapy: Heparin potentiates the action of natural anticoagulant antithrombin III which is an inhibitor of thrombin and activated factors IX, X, and XI. Full dose heparin therapy needs monitoring by APTT to maintain the dose in the therapeutic range (1.5 to 2.5 times the upper reference limit of APTT).
(3) Screening for circulating inhibitors of coagulation: APTT is prolonged in the presence of specific inhibitors (which are directed against specific coagulation factors) and non-specific inhibitors (which interfere with certain coagulation reactions).
 
Mixing experiment for detection of inhibitors: Mixing studies are used to distinguish between factor deficiencies and factor inhibitors (specific coagulation factor inhibitor or non-specific inhibitor such as lupus anticoagulant). If APTT is prolonged, patient’s plasma is mixed with an equal volume of normal plasma (called as a 50:50 mix) and APTT is repeated. In coagulation factor deficiency, prolongation of APTT gets corrected by more than 50% of the difference between the clotting times of control and test plasma. In the presence of lupus anticoagulant, there is no such correction. With lupus anticoagulant, APTT remains prolonged after mixing and for 2 hours following incubation. With F VIII inhibitor (which is time- and temperature-dependent), prolong-ed APTT gets immediately corrected after mixing, but becomes prolonged after incubation.

Prothrombin Time (PT)

Published in Microbiology
Friday, 10 June 2016 01:14
PT assesses coagulation factors in extrinsic pathway (F VII) and common pathway (F X, F V, prothrombin, and fibrinogen).
 
Principle
Tissue thromboplastin and calcium are added to plasma and clotting time is determined. The test determines the overall efficiency of extrinsic and common pathways.
 
Equipment
(1) Water bath at 37°C
(2) Test tubes (75 × 12 mm)
(3) Stopwatch
 
Reagents
(1) Thromboplastin reagent: This contains tissue factor and phospholipids and is available commercially.
(2) Calcium chloride 0.025 mol/liter.
 
Specimen
Venous blood is collected from antecubital fossa with a plastic, siliconized glass, or polypropylene syringe and a large bore needle (20 or 21 G in adults, 22 or 23 G in infants). Blood should never be collected from indwelling intravenous lines, as these often contain heparin. Glass syringe should not be used for collection since it activates coagulation. The blood is drawn gently but quickly after a single, smooth venepuncture. The needle is detached from the syringe, and the sample is passed gently into the plastic container. After capping the container, the blood and the anticoagulant are mixed immediately by gentle inversion 5 times. The anticoagulant used for coagulation studies is trisodium citrate (3.2%), with anticoagulant to blood proportion being 1:9. Most coagulation studies require platelet poor plasma (PPP). To obtain PPP, blood sample is centrifuged at 3000-4000 revolutions per minute for 15-30 minutes. Coagulation studies are carried out within 2 hours of collection of sample.
 
Method
(1) Deliver 0.1 ml of plasma in a glass test tube kept in water bath at 37°C.
(2) Add 0.1 ml of thromboplastin reagent and mix.
(3) After 1 minute, add 0.1 ml of calcium chloride solution. Immediately start the stopwatch and record the time required for clot formation.
 
Normal Range
11-16 seconds.
 
Causes of prolongation of PT
(1) Treatment with oral anticoagulants
(2) Liver disease
(3) Vitamin K deficiency
(4) Disseminated intravascular coagulation
(5) Inherited deficiency of factors in extrinsic and common pathways.
 
Uses of PT
(1) To monitor patients who are on oral anticoagulant therapy: PT is the standard test for monitoring treatment with oral anticoagulants. Oral anticoagulants inhibit carboxylation of vitamin K-dependent factors (Factors II, VII, IX, and X) and make these factors inactive.
In patients receiving oral anticoagulants, PT should be reported as a ratio of PT of patient to PT of control; it should not be reported as percentage. Various types of thromboplastin reagents obtained from different sources (like ox brain, rabbit brain, or rabbit lung) are available for PT test. These differ in their responsiveness to deficiency of vit. K-dependent factors. Technique of PT is also different in different laboratories. For standardization and to obtain comparable results, it is recommended to report PT (in persons on oral anticoagulants) in the form of an International Normalized Ratio (INR).

INR =  PT of Patient ISI
          PT of Control
 
International Sensitivity Index (ISI) of a particular tissue thromboplastin is derived (by its manufacturer) by comparing it with a reference thromboplastin of known ISI.
INR should be maintained in the therapeutic range for the particular indication (INR of 2.0-3.0 for prophylaxis and treatment of deep venous thrombosis; INR of 2.5-3.5 for mechanical heart valves). Therapeutic range provides adequate anticoagulation for prevention of thrombosis and also checks excess dosage, which will cause bleeding.
(2) To assess liver function: Liver is the site of synthesis of various coagulation factors, including vitamin Kdependent proteins. Therefore PT is a sensitive test for assessment of liver function.
(3) Detection of vitamin K deficiency: PT measures three of the four vitamin K-dependent factors (i.e. II, VII, and X).
(4) To screen for hereditary deficiency of coagulation factors VII, X, V, prothrombin, and fibrinogen.

Bleeding Time (BT) and Clotting Time (CT)

Published in Microbiology
Wednesday, 08 June 2016 01:33
BLEEDING TIME
 
The bleeding time test is dependent on appropriate functioning of platelets blood vessels and platelets and evaluates earliest hemostasis (platelets components and vascular).
 
In this test, incision (a surgical cut made in skin) or a superficial skin puncture is made and the time is measured for bleeding to stop.
 
There are three methods most commonly used to measure bleeding time:
 
  1. Duke’s  method
  2. Ivy’s method
  3. Template method
 
In Duke’s method, ear lobe is puncture, and the time is measured for bleeding to stop.  This method is not recommended and cannot be standardized because it can cause a large local hematoma. In Ivy’s method, on the volar surface of the forearm, three punctures are made with a lancet (cutting depth 2-2.5 mm) under normal pulse pressure (between 30-40 mm Hg). A disadvantage of Ivy’s method is closure of puncture wound before stoppage of bleeding. In Template method, a special surgical blade is uses to make a larger cut of about 1 mm deep and 5 mm long. Although Template method is better than other methods, it may produce large scar and even form a keloid (irregular fibrous tissue formed at the site of a scar) in predisposed individuals. Ivy’s method for the measurement of bleeding time is described below.
 
Ivy’s Method
 
Principle: On the volar surface of forearm, three normal punctures are made with the help of a lancet under normal pulse pressure (between 30-40 mm Hg).  The average time is measured for bleeding to stop from the puncture sites.
 
Equipment
 
  1. Disposable sterile lancets
  2. Sphygmomanometer
  3. Filter paper
  4. Stopwatch
 
Method
 
  1. Blood pressure of the patient is measured with the help of sphygmomanometer. The blood pressure of the patient should be normal before going to the further process.
  2. The volar surface of the forearm is cleansed with ethanol 70% and allowed to dry.
  3. With the help of a lancet, in quick succession, three punctures are made about 5 cm apart. Note that scars and superficial veins should be avoided.
  4. Start the stopwatch as soon as puncture made on the volar surface of the forearm.
  5. With the help of the filter paper, blood oozing from the puncture wound is gently absorbed with intervals of 15 seconds.
  6. The timer is stopped when blood no more mark the filter paper.
  7. Time measured for bleeding to stop from all the three puncture wound is recorded. The average time is calculated and reported as the bleeding time.
 
Reference Ranges
 
  • Normal range: 2 -7 minutes.
  • The greater numbers of individuals have bleeding time less than 4 minutes. The bleeding time should be reported in minutes or nearest half minute. If the bleeding continues more than twenty minutes, the test is stopped and the bleeding time should be reported as >20 minutes (more than 20 minutes).
 
Cause of extend of duration of bleeding time
 
  1. Disorders of blood vessels
  2. Thrombocytopenia: This term is uses when the platelet count is less than its normal value. The bleeding time test should not be performed if the platelet count is less than 1,00,000/ml. It may be difficult to control the bleeding if the platelet count is very low.
  3. Von Willebrand disease
  4. Disorder of platelet function
  5. Afibrinogenemia
 
CLOTTING TIME
 
In this test, required time is measured for the blood to clot in a glass test tube, kept at 37° C. Extend of duration of clotting time occurs only if severe deficiency of a clotting factor exists and is normal in moderate or mild deficiency.

Thrombin Time (TT)

Published in Microbiology
Monday, 06 June 2016 18:58
Thrombin time assesses the final step of coagulation i.e. conversion of fibrinogen to fibrin by thrombin.
 
Principle
Thrombin is added to patient’s plasma and time required for clot formation is noted.
 
Equipment
(1) Water bath at 37°C
(2) Test tubes (75 × 12 mm)
(3) Stopwatch
 
Reagent
Thrombin solution.
 
Specimen
Venous blood is collected from antecubital fossa with a plastic, siliconized glass, or polypropylene syringe and a large bore needle (20 or 21 G in adults, 22 or 23 G in infants). Blood should never be collected from indwelling intravenous lines, as these often contain heparin. Glass syringe should not be used for collection since it activates coagulation. The blood is drawn gently but quickly after a single, smooth venepuncture. The needle is detached from the syringe, and the sample is passed gently into the plastic container. After capping the container, the blood and the anticoagulant are mixed immediately by gentle inversion 5 times. The anticoagulant used for coagulation studies is trisodium citrate (3.2%), with anticoagulant to blood proportion being 1:9. Most coagulation studies require platelet poor plasma (PPP). To obtain PPP, blood sample is centrifuged at 3000-4000 revolutions per minute for 15-30 minutes. Coagulation studies are carried out within 2 hours of collection of sample.
 
Method
Take 0.1 ml of buffered saline in a test tube and add 0.1 ml of plasma. Note clotting time after addition of 0.1 ml of thrombin solution.
 
Normal Range
± 3 seconds of control.
 
Causes of Prolongation of TT
(1) Disorders of fibrinogen: Prolongation of TT occurs in afibrinogenemia (virtual absence of fibrinogen), hypofibrinogenemia (fibrinogen less than 100 mgs/dl), and dysfibrinogenemia (dysfunctional fibrinogen).
(2) Heparin therapy: Heparin inhibits action of thrombin.
(3) Presence of fibrin degradation products (FDPs): These interfere with fibrin monomer polymeri-zation. TT is repeated using a mixture of normal plasma and patient’s plasma. If TT remains prolonged, then FDPs are present (provided patient is not receiving heparin).

Platelet Function Analyzer (PFA-100)

Published in Microbiology
Monday, 06 June 2016 15:16
This is a newly introduced screening test for platelet function that assesses both platelet adhesion and aggregation. This method uses an instrument called as PFA-100 in which anticoagulated whole blood is passed at a high shear rate through small membranes that have been coated with either collagen and epinephrine or collagen and ADP. Platelets adhere to each membrane and gradually occlude an aperture at the centre of the membrane. The time required for complete occlusion of the aperture is called as closure time. Normal closure time is 1-3 minutes. The PFA-100 test is performed initially with the collagen/epinephrine membrane; if closure time is normal, there is no significant platelet function defect. If closure time with collagen/epinephrine is prolonged, test with collagen/ADP is carried out; if normal, aspirin-induced platelet dysfunction is the probable cause; if prolonged, other platelet function defect (acquired or inherited) is likely. This test is more sensitive than bleeding time to assess primary hemostasis, sensitive for detection of von Willebrand disease and easy to perform. However, in the presence of thrombocytopenia and anemia, closure time is prolonged. Also, in the presence of a strong clinical suspicion of a platelet function defect and normal PFA-100 result, further testing is still necessary.
 
REFERENCES
1. Evatt BL, Gibbs WN, Lewis SM, McArthur JR. Fundamental Diagnostic Hematology: The Bleeding and Clotting Disorders (2nd ed), 1992. US Dept. of health and Human Services, Atlanta, Georgia and World Health Organization, Geneva, Switzerland.
2. Shrikhande AV, Warhadpande MS, Kawthalkar SM. A laboratory manual of coagulation (1994). Dept. of Pathology. Govt. Medical College, Nagpur.
3. Wallach J. Interpretation of Diagnostic Tests (7th ed). Philadelphia: Lippincott Williams and Wilkins, 2000.
4.  David J. Kuter, MD, DPhil. Laboratory Tests for Blood Disorders. The Merck™ Manuals, (2014).
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