Clinical laboratory professional specialized to external quality assessment (proficiency testing) schemes for Laboratory medicine and clinical pathology. Author/Writer/Blogger
Hemoglobin is composed of heme (iron + protoporphyrin) and globin polypeptide chains. It is present in the red blood cells of all vertebrates except Channichthyidae (the family of fish: white-blooded fish also called crocodile fish found in southern South America and the Southern Ocean around Antarctica). It carries oxygen from the lungs to the tissues and carbon dioxide from tissues to the lungs.
In humans, hemoglobin is not homogeneous and normally different variants and derivatives exist. Normal hemoglobin variants are fetal hemoglobin (Hb F), adult hemoglobin (Hb A), Hb A2 and embryonic hemoglobins (Gower I, Gower II and Portland). They differ from each other on the basis of the structure and the type of polypeptide chains.
There are different methods for estimation of hemoglobin. These are:
(1) Colorimetric methods: In these methods, the color comparison is made between the standard and the test sample, either visually or by colorimetric methods.
(2) Gasometric method: In this method, oxygen-carrying capacity of red blood cells (RBCs) 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 methemoglobin, sulfhemoglobin, and carboxyhemoglobin. Also, this method is expensive and time-consuming, and the result is about 2% less than other methods.
(3) Chemical method: In this method, iron-content of hemoglobin is first evaluated. The value of hemoglobin is then derived indirectly from the formula that 100 grams of hemoglobin contain 374 mg of iron. This method is tiresome and time-consuming.
(4) Specific gravity method: In this method, an approximate value of hemoglobin is estimated from the specific gravity of blood as determined from copper sulfate technique. This method is simple and rapid. This method is useful and most common in mass screening like the 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%. In this method, a drop of blood obtained by finger puncture is placed on a piece of absorbent paper. The color produced is matched against the color on the chart and the corresponding reading is taken. The room of error is 20-50%. Although this method is very cheap and simple.
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, 2000⁻¹⁵
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.
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.
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.
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:
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:
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.
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.
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.
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.
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:
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.
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.
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.
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.