Basic Reagents and Solutions for Haematological and Blood Transfusion Tests
Haematological and Blood Transfusion tests are group of tests in the Laboratory used study blood, it is constituents and blood sharing from one person to another person. Example of Haematological test is Sickle cell anaemia, and one example of Blood Transfusion test is ABO blood group testing.
In this article you will lean:
- Definition of terms used in Haematological and Blood Transfusion Tests: Haematology, and blood transfusion.
- Significant of Haematological Tests
- Basic Reagents and solutions for Haematological and Blood Transfusion Tests
- Principle, uses, advantages and disadvantages of each Reagents
Definition of terms used in Haematological and Blood Transfusion Tests
- Haematology is a medical science that deals with the study of blood and blood-forming organs.
- It is concerned with the study, diagnosis, treatment, and prevention of diseases related to blood.
- Blood transfusion is the transfer of blood or blood components from one person (the donor) into the bloodstream of another person (the recipient).
- It is a lifesaving maneuver to replace blood cells or blood products lost through bleeding or due to depression of the bone marrow.
Blood-related Diseases (Haematological diseases)
- Clotting disorders.
- Anemia . Example sickle cell anemia, iron deficiency anemia, megaloblastic anemia etc.
Significant of Haematological Tests and Blood Transfusion Tests
- Blood film test – This investigates anemia, leukemia & hemo-parasites like Plasmodium falciparum.
- Coagulation tests – This investigates the clotting disorders.
- Bone marrow aspirate test – This investigates anemia & leukemia.
- Complete blood count – this investigates anemia, leukemia & clotting disorders.
- Erythrocyte Sedimentation Rate – This investigate infections that affect ESR like TB.
- Hemoglobin estimation – This investigates Anemia.
- Sickle Cell Screening test – This investigates sickle cell anemia.
- Manual white blood cell differential – This investigates the type of white blood cells affected during leukocytosis or leukopenia.
- ABO blood grouping tests- used to investigate type of blood groups to human. Example a certain patient have blood group A.
- Cross matching- used to investigate blood donor and blood patient ABO compatibility or incompatibility. example Blood group B is compatible with AB and B.
Basic Reagents and Solutions for Haematological Tests.
- Drabkin’s solution
- CuSO4 solution
- Turk’s solution
- Leishman stain
- Sodium metabisulphite
- Buffer solution pH 7.2
- 0.85%Normal saline
- 3.8% Sodium citrate
- This solution is used in hemoglobin estimation test.
Components of Drabkin’s solution are :
- Potassium ferricyanide = 200mg
- Potassium cyanide = 50mg
- Potassium dihydrogen phosphate = 140mg
- Non-ionic detergent = 1ml
- Distalled water = Make up to 1000 ml (1 L)
Principle of Drabkin’s solution
- This is based on oxidation of Haemoglobin(Hb) by Potassium ferricyanide to form methemoglobin in alkaline medium.
- The methemoglobin reacts with potassium cyanide to form a very stable compound, called cyanmethemoglobin.
- Then, absorbance of the cyanmethemoglobin is measured at 540 nm in a colorimeter or spectrophotometer.
- The absorbance of this complex is directly proportional to the Hb concentration.
- The concentration of Hb of the sample is then obtained by comparing the absorbance of sample to that of standard solution.
Hb concentration of sample = (absorbance of sample/absrobance of standard) x concentration of standard.
The Hb concentration may also be obtained directly from the already calibration curve.
Application of Drabkin’s solution
- Used for the quantitative colorimetric determination of blood hemoglobin.
Procedures for estimating Hb by Drabkin’s solution
- Take 20 microlit. of blood + Drabkin 4 mL = 1 : 200 dilution or take 20 microliter of blood + Drabkin 5 mL = 1 : 250 dilution.
- Now mix well.
- Read within 6 hours of mixing on filter 540.
- Read against blank of drabkin solution (Drabkin solution can be used as blank).
- Also read the standard solution (12 G/dL) with the same dilution like test sample.
Advantages of Drabkin’s Technique
- Error due to subjective visual matching is avoided as spectrophotometer is used and hence reading is precise and reliable.
- Measures all forms of Haemoglobin except sulphaemoglobin.
- Single step procedure using single reagent.
- Cyanmethemoglobin formed produces broad absorbent band at 530 nm.
- Good stable Haemoglobin standards are available.
Disadvantages of Drabkin’s Technique
- Drabkin’s solution is toxic.
- It used in screening of Hb concentration of blood donors.
The principle of CuSO4 Method
- CuSO4 solution has a specific gravity of 1.055, which is equivalent to 12.5 g/dl.
- Blood drop is observed for a short time to determine whether it sinks or swims on CuSO4 solution.
- Blood drop from a person with Hb above 12.5g/dL sinks.
- Blood drop from a donor with Hb below 12.5g/dL swims.
- So when a blood drop sinks indicates that Hb concentration is greater than 12.5 g/dl.
- And when it swims indicates that Hb is less than 12.5 g/dl.
- Only people with Hb above 12.5 g/dl are allowed to donate blood.
Application of CuSO4 Method
- It used in screening of Hb concentration of blood donors.
Advantage of CuSO4 Method
- Simplest way to estimate Hb.
- It is inexpensive.
Disadvantage of CuSO4 Method
- Many false-low and high Hb (hemoglobin is not <12.5 g/dL) are produced, because the test is just an estimate.
- Therefore, hemoglobin may be checked in another more accurate manner.
- Components of Turk’s solution
It is a composed of;
- a stain called Gentian violet and 1-2% acetic acid.
- The 1-2% acetic acid destroys red blood cells within a blood sample.
- And the gentian violet stains the nuclei of the white blood cells, making them easier to see and count.
The principle of Turk’s solution
- Turk’s solution ruptures red blood cells and leaves white blood cells(Wbc) and platelets intact and stained.
- This makes white blood cells to be easier seen and counted in the Hemocytometer.
Application of Turk’s solution
- Turk’s Solution is intended for use in counting leukocytes in a defined volume of blood.
- Erythrocytes are hemolysed while leukocytes are stained for easy visualization.
Preparation of Turk’s solution
- 20 ml of glacial acetic acid, 980 ml 0f distilled water and 2-3 drops of 0.5% gentian violet.
Advantages of Turk’s solution
- Eliminates the obscuring effect of RBC during white cell total and differential count.
- Leishman stain is named after its inventor, the Scottish pathologist William Boog Leishman.
- It is similar to and partially replaceable with Giemsa stain and Wright’s stain.
- Leishman’s stain, also Leishman stain, is used in microscopy for staining blood smears.
- It provides excellent stain quality.
- It is based on a methanolic mixture of “polychromed” methylene blue and eosin.
- The methanolic stock solution is stable and also serves the purpose of directly fixing the smear eliminating a prefixing step.
- If a working solution is made by dilution with an aqueous buffer the resulting mixture is very unstable and cannot be used for long.
Application/use of Leishman stain
In hematology, it is used to differentiate and identify leucocytes. (differential white blood cells count)
Advantages of Leishman stain
- Leishman’s is also a simple method which is especially suitable when a stained blood film is required urgently or the routine stain is not available (e.g. at night).
- Fairly good contrast: Leishman stain generally shows the brilliant violet color of the nucleus and the neutrophil granules for which differential count becomes convenient and makes the quality of staining better than the stains that are simple methylene blue and Eosin based which does not produce enough contrast between the cytoplasm and the nucleus.
Disadvantages of Leishman stain
- Difficulty controlling molar ratio: Composition of polychromed methylene blue mixed with Eosin is never as good as the directly weighed and mixed proportions in Giemsa type stains.
- Instability: The Leishman stain if reconstituted with buffer becomes very unstable (in contrast with Giemsa which is relatively more stable due to Glycerol) and starts precipitating and needs repeated filtering.
- Cytoplasmic contrast good but nuclear contrast not as good as H&E.
- It is a reducing agent.
- It acts by removing oxygen molecules in a given environment.
- It is very useful in screening of HbS molecules, a characteristic hemoglobin in sickle cell patients.
Application/use of Sodium metabisulphite
- It is used as reducing agent in sickle cell screening test .
The principle of Sickle cell screening test by 2% sodium metabisulphite solution
- If red cells containing HbS are mixed with 2% sodium metabisulphite solution and allowed to stand at room temperature or incubated at 37⁰C in the environment with limited oxygen, the HbS precipitates and the cells become sickle-shaped.
- 2% Sodium metabisulphite solution should be prepared immediately before use.
- This is because it cannot be stored because it is readily oxidized by the air and loses its reducing activity.
Advantages of 2% sodium metabisulphite solution in screening of sickle cell.
- Simple to perform
- Allows direct visualization of sickle cells.
- Cheaper if compared to other techniques like electrophoresis and PCR.
Disadvantages of 2% sodium metabisulphite solution in screening of sickle cell.
- False negative results may be produced due loss of its reducing ability when exposed to air.
- It is Ethylenediaminetetraacetic acid.
- It is an anticoagulant, it prevents blood from clotting.
Application of EDTA
- Used as an anticoagulant.
The Principle of EDTA
- EDTA strongly and irreversibly binds calcium ions in the blood sample, this prevents the coagulation proteins from initiating the coagulation cascade.
Advantage of using EDTA
- It has an excellent preserving power and recommended for use in routine blood examination.
- Does not alter the erythrocyte size when employed in the recommended concentration.
- Leukocytes staining is excellent.
- EDTA blood was used in the determination of creatinine, urea nitrogen, glucose, phosphorus and uric acid.
Disadvantage of using EDTA
- Higher concentration of salt withdraws water from red cells and reduces PCV values.
- Not advisable for estimation of alkaline phosphatase activity because it can combine with magnesium ion which are needed for activation of ALP.
- Chloride estimation in EDTA blood was always higher than with other anticoagulants.
3.8% Sodium citrate
- It is also an anticoagulant.
- It is employed in blood transfusion because citrate is metabolized and excreted rapidly.
The principle of sodium citrate
- Binds calcium ions in the blood sample, this prevents the coagulation proteins from initiating the coagulation cascade.
Application of sodium citrate
Advantage of using sodium citrate
- It is the best anticoagulant that preserves cellular constituents.
- used in biochemical constituents .
- keep cell morphology.
Disadvantage of using sodium citrate
- Interfere with many biochemical tests.
- Prevents clotting for only few hours.
- Increased concentration may cause shrinkage of cells.
Buffer solution pH 7.2
- Used as diluent.
- Consists of Disodium hydrogen phosphate powder 0.7g, potassium dihydrogen, phosphate powder 0.3g and 1,00 ml distilled water.
Preparation of Buffer solution pH 7.2
- Dissolve 10 buffer tablets, pH 7.2 in 1,000 ml distilled water.
- F.J. Baker, R.E. Silverton (2001): Introduction to Medical Laboratory Technology, 7thEdition, Oxford University Press; Oxford, England.
- Monica Cheesbrough (1981): Medical Laboratory Manual for Tropical Countries Volume I, 2nd Edition Butterworth & Co. (Publishers) Ltd;
- Monica Cheesbrough (2002): District Laboratory Practice in Tropical Countries Part 1 & 2, Cambridge University Press; Cambridge, England.