Erythrocyte Sedimentation Rate and Haemoglobin Electrophoresis
AIM; The ESR is useful as a screening test for the presence of any chronic or acute condition which is marked by alteration in plasma protein concentration serial estimation of ESR can be used to monitor disease progression or treatment. SPECIMEN: Venous blood collected directly into sodium citrate or EDTA anti-coagulated blood diluted in sodium citrate can be used.
EQUIPMENT: Westergren ESR pipette, Westergren ESR stand with leveling device, Timer capable of timing accurately 1 hour REAGENT: Tii- sodium citrate.
PRINCIPLE: When citrate blood in a vertically positioned westergren pipette is left undisturbed, red cells aggregate, stack together to form rouleaux and sediment through the plasma. The rate at which this sedimentation occurs in 1 hour as indicated by the length of the cocluirm of clear plasma above the red cells, measured in mm.
- Pipette 0.5ml of sodium citrate against 2rnl of blood in a small container.
- Mix the two avoiding clothing of the blood.
- Remove the cap of the container and place the sample in the ESR stand making a knot of the number in the patient's notes.
- With the aid of westergren pipette, avoiding air bubbles, pipette the mixture to zero mark.
- Stand it on the werstergren stand making sure that ESR stand is vertical and on a leveled, vibration free environment.
- Set the tinier for complete one hour. Ensure that the ESR stand and pipette were not exposed to direct sunlight.
- After exactly one hour, read the level at which the plasma meets the red cells in mm.
- After reading the ESR, return the blood to its container, remove carefully the pipette and soak it in sodiumhypochlorite (JIK)ESR is expressed in millimeter per hour
(mrn/hr) and its reference range varies as follows Men 3-5mm/hrWomen 4-7mm/hrsignificant raised ESR could be as a result of Anarnia due to any cause. HIVTuberculosis Pelvic inflammatory disease etc.
Reduced ESR could be as a result of Dehydration
Abnormally shaped red cells as in sickle cells etc
GENOTYPE (HAEMOGLOBIN ELECTROPHORESIS) AIM: Genotype is used to separate and identify the different haemoglobin by their migration within electric field haemoglobin variants separate at different rate due to differences in their surface electrical charge as determined by their ammo acicl structure.
SPECIMEN: Capillary blood or EDTA anti-coagulated venous blood can be used.
EQUIPMENT: An electrophoresis chamber (tank), cellulose acetate paper.
REAGENT: This -EDTA borate buffer
CONTROL: Well known genotype "AS” that is free HIV and Hepatitis
Collect an EDTA anti-coagulated blood or capillary blood. -. Add a drop of it on a clean white laboratory tile.
- Add equal volume of water to lyze the cells and mix well
- Equally acid a drop of control (As genotype) and equal volume of water on the laboratory white tile separately and mix well
- Moisten the cellulose acetate paper making sure that the application, is thin, line
- Place the acetate paper in the electrophoresis tank and cover to prevent evaporation making sure that the face of
the blood sample was applied faces upward.
- Plug the tank to an electric power source and allow for about 10minutes.
If the patient's genotype is "AA", it shows or remains a single thin line at the anode (positive pole).
If the patient's genotype is "AS", it shows a double line with the "S" haemoglobin moving in separation toward the cathode (negative pole).
If the patient's genotype is "S3", it shows a single which shifted from the anode (positive pole) toward the cathode (negative pole)
BLOOD GROUPING AND RHESUS FACTOR
The ABO blood grouping is used to ensure that the correct ABO group has been determined and should always is performed as part of a compatibility test prior to transfusion because transfusion of ABO incompatible blood frequently is fatal.
The D-antigen is the most antigenic and therefore the most clinically significant red cell antigen after the A and B antigen of the ABO system this means that introduction of the D-antigen in an individual lacking that antigen is more likely to stimulate antibody production than other Rhesus antigen. Thus, blood for transfusion is routinely tested for ABO and Rhesus only.
ABO blood grouping system is carried out using monoclonal blood grouping antisera. Monoclonal blood grouping antisera is commercially available containing preservative, which gave it a long shelf life from the date of manufacture. Anti-A antiserum is color coded blue; Anti-Bantisera are mixed with blood, antigens in the red blood cells react with the monoclonal antibodies causing agglutination.
SPECIMEN: EDTA anti-coagulated venous blood or capillary blood can be used,
EQUIPMENT: Laboratory white tiles, Applicator stick cotton wool
REAGENTS: Anti-sera A, B and D - Add a drop of a patient's blood on 3 different spaces on a white laboratory tile.
Add equal volume of Anti A antiserum, Anti B antiserum and Anti-D antiserum on the 3 different drops of blood respectively
Mix each drop of the blood and the antiserum added to it with the applicator stick cleaning at each mixing interval.
- Rock the tile by tilting gently from side to side
- Look out for agglutination and record the result.
If agglutination occurs in the space containing anti-B antiserum, he patient belongs to the blood group "B"
- If agglutination occur in both "A" and "B" space, the patient belongs to the blood group "AB" (the Universal recipient).
- If there is no agglutination on both "A" and "Bw, the patient belongs to the blood group "O" (the Universal Donor).
-If there is no agglutination on the "D" the patient is rhesus negative.
The purpose of compatibility testing (cross-matching) is to prevent a transfusion reaction; It is a final check on the compatibility of blood for a patient, particularly ABO compatibility. Compatibility testing is essential before transfusion to confirm and individual or patient who is eligible to donate or receive blood for transfusion.
- Centrifuge the recipient's blood to obtain the serum.
- Add equal quantity of normal saline to the donor's blood and centrifuge to wash the red cells.
- Decant the supernatant
- Add 2 drops of recipient's serum to 2 drops of the sediment got from that of the donor on a clean microscope slide.
- Mix 40 x objectives.
If agglutination occurs, they are not compatible.
If no agglutination occurs, they are compatible.
When there is rouleaux formation, apply a drop of normal saline and. mix very well and re-view.
The technique of collecting blood from a donor is as follows:
- Suspend bag below donor's arm, at a minimum of 60cm between bag and the donor's arm
- Apply a tourniquet to the upper arm of the donor above the elbow.
- Disinfect the puncture site with mentholated spirit.
- Perform the venipunctrure with the needle directed upwards in the line of the vein.
Secure the needle in place with a strip of adhesive tape.
- As soon as the blood flow starts, repeatedly mix the blood with the anti-coagulant by gently shaking the bag.
- Collect up to 450ml as the donor pumps his arm by opening and closing of his palm.
- Release the tourniquet and withdraw the needle.
- Knot firm after collection and immediately invert bag up and down gently for at least two times to thoroughly mix blood and anticoagulant.
- Label the blood bag correctly giving the required information on it.
- Refrigerate the blood after allowing time for it to cool and for natural bactericidal activity of white cells
NOTE: Before a donor's blood is giving to a recipient, the following test must be carried out on the donor's blood.
- HIV Screening test 1& 2
HCV and Hbs test.