Haematology Dept (week 4-6)

Done By: Janice Yeh
Dept: Haematology
Section: ABO Grouping and Du Typing (only done by 1 person)

This test is very simple and i believe it is definitely not foreign to all of us. However, i decided to post it because there are slight differences of how it is conducted in a lab as compared to our sch lab sessions. There are also many steps before doing the actual testing, such as clerical checking and preparation of reagent. Precision and good judgement, as well as speed is required in producing the results.

P.S. All pictures taken are of APPROVAL from my department supervisor. There are no barcodes or patient information present.

Preparation: (all done in the morning by me :D)Procedure: Aliquot about 1ml of saline into the tube. It does not have to be at precision as is main purpose is just to dilute the blood.
Purpose: If blood is not well diluted, then it may cause a higher frequency of false-positive results.

Procedures: Remove the needed reagents from the fridge and allow it to cool for awhile. Arrange them in order so as to not confuse the order. Check if there are sufficient reagents left in the fridge, if there isn't, med tech has to go to the store room and get new packs. New packs are sent to the lab monthly.

Procedure: Aliquot one drop of reagent by using the pipette attached to the cap of the reagent, onto a microplate. Each microplate allows 12 samples to be tested. I usually like to prepare about 20 plates every morning when the samples are not in yet so as to the ease the work load of the person in-charge of doing the ABO grouping.

Purpose: Early preparation of plates helps to saves time and allows results to be produced in a shorter time. Using a microplate is also a faster method as compared to what we do in the lab, which is to use test tubes. It also saves reagent and cost as only 1 drop is needed.

ABO Grouping

Procedures: When samples first arrive, we will scan the barcodes to allocate to which test they are suppose to undergo. The samples that are in the 'ABO'-labelled rack, will be first placed in the centrifuge and spin for 50s at 3000rpm. Machine used- Diacent 12

Purpose: It is to separate the serum from the blood. Blood is needed for forward grouping, whereas the plasma is needed for reverse grouping.

Procedures: After scanning the barcodes of the samples, a worklist is printed with the patient's ID and barcode no. Before doing the test, check against the EDTA tubes against the information present on the worklist.
Purpose: This is to ensure that the results are written and recorded for the correct patient. Sometimes, problems such as a wrong worklist may be printed (as multiple worklist can be printed at one time; the printed is also shared by various departments)

Procedures: Using a pasteur pipette, the plasma is carefully taken out from the tube. The plasma should not be mixed with the blood. Plasma should be clear-yellowish colour. There are times when the plasma is reddish in colour and it is acceptable as the samples could have been stored for a long time and has haemolysed. One drop of plasma is dropped into the last 3 columns (for Reverse grouping). 1 drop of the commercial A, B, O cells are placed into the wells respectively. After which, the blood is taken (approximately 3 drops) and mixed with the saline. after which, 1 drop is dropped into the columns labelled - A, B, AB, D

Procedure: The plate is spin this centrifuge before reading the results for 15minutes. Machine used - Diacent MP 2
Purpose: This to ensure proper mixing of reagent with the sample for reaction as well as to encourage clumping.

Procedure: after which, the plate is place on this some sort of vortex machine. The speed is constantly changed and the machined is swivelled.

Purpose: It is to dislodge any "false" buttons and agglutinations. As the plate may have settled down at the bottom after a long period of centrifuging.

Procedures: Using a thin wooden stick, check on any of the buttons to see if they will dislodge. Usually, agglutination without clear background would most probably be a negative result.
After confirming the results, the results are written on the worklist manually. The forward results will always be opposite of the reverse grouping results. If the result is blood group A, D postive, it will be written as ' AP'. Results and the samples are place at on the table to validation by another staff. In total, 2 validations are done and signed upon the worklist before the results are released. Usually, validations are done immediately so as not to delay the release of results.

Purpose: This is because the results are written based on visual observation and there could be human error and careless overlooks.

Procedure: this is a confirmation test. It's very brief and does not require any incubation or centrifuging. When results is suppose to be negative but is postive or when there is discrepancies with the forward and reverse grouping, then this is done. It is just seemly placing a drop of the different reagents on the plate and placing one drop of the patient's sample and mixing it.

Du typing- Confirmation Test

Preparation for Du typing is when there is a negative results for the D. Meaning there no D antigen present on the patient's RBC. This is more uncommon and needs to be confirm in order to ensure that results were not due to pre-analytical or analytical variations, causing a possible false negative result.

2 tubes are prepared, 1 with 1 ml of saline and the other is an empty tube. A 2-5% cell suspension is done. This is also a dilution method to ensure and lower the chances of any false-positive results. 2 drops of the suspension is aliquot into the other empty tube together with 2 drops of anti-D commercially prepared reagent. The tube is spin down in the centrifuge at 3000rpm, for 15 s. After which, a slide is made to observe for agglutinations of RBC under 10x magnification of a light microscope. if there is, then Du typing test can stop here.

If there are no clumps present, the tube of sample is incubated in a water bath at 37 degrees for 15 mins. This is to encourage clumping of antigens to antibody as the reagent anti-D is IgG, a wamr antibody and clumps best at body temperature (37 degrees). The samples is then washed 3 times with saline (like how we do it in school). This step is crucial as to remove as much plasma and proteins present in the samples. Removing proteins and plasma will prevent the false-positive clumping with AHG reagent. After which, 2 drops of AHG is added and the sample is spin down at low speed of 2000rpm, for 50s. A slide is made to check for clumps under the microscope. If there are no clumps, the results are confirmed negative. To ensure that AHG reagent is having it's proper function of encouraging the clumping of Ag and Ab, check cells are added to the sample.

Check cells is a pool of group O red vlood cells that have been sensitised with IgG antibodies. They are contained in preservative solutions with adenosine and adenine to retard hemolysis during the storage period. It produces a positive clumping result in the presence of AHG. If there is no clumping --> it means that either AHG has been used up in the test or it has been inactive

I hope you've learnt as much as I did. :D happy SIP-ing~!