DAT and Elution

Hello.

A sample that is sent to the Red Cell Reference Laboratory for red cell alloantibody identification would have a case history, where it would include the results of the tests that have been previously performed by the requesting hospital blood bank laboratory. Such tests include positive antibody screening or positive Direct Antiglobulin Test (DAT), where further investigation is needed.

A positive DAT may indicate that there are antibodies coating the RBCs. This is usually the case in patients suspected to have haemolytic transfusion reactions, haemolytic disease of the newborn or drug-induced haemolytic anaemia. In such cases, the Red Cell Reference Laboratory staff would have to perform a DAT test to double confirm the result and the strength of the graded reaction submitted by the requesting hospital blood bank laboratory. The stronger the DAT, the more likely it is to be clinically significant.

Test: DAT
Specimen: Blood collected in EDTA tube (purple cap). A clotted specimen collected in a plain tube (red cap) can result in a false positive reaction for the presence of complement due to in-vitro activation of the complement cascade.
DAT test is performed using the gel card technology, where 50 μl of the patient’s 0.8% red cell suspension is added to the microtube column in the ID-DiaMed LISS/Coombs gel card and spun at 910 rpm for 10 minutes. This gel card contains polyspecific AHG reagent to detect IgG and also contains anti-C3d to detect complement. If DAT were confirmed positive, next we would have to find out whether is it IgG antibody or complement C3d or both that is coating the RBCs. We would use an ID-DiaMed DC-Screening II gel card to differentiate the reaction and the steps are the same as the one above. This gel card contains monospecific AHG reagents such as anti-IgG and -C3d. If only C3d were present, an eluate is not likely to yield any useful information and should not be performed in most cases. If IgG were present, we would proceed to do an acid elution.

Elution allows the dissociation of the antibodies from the RBCs cell surface to allow for identification. Acid elution is a relatively quick and easy method and is most suitable for the detection of warm reactive antibody. The washed antibody-coated cells are mixed with a glycine acid solution at a pH of 3.0. The antigen-antibody bond is disrupted and the antibody is released into the acidic supernatant. The supernatant is separated and the pH is neutralised to allow for antibody identification.

The acid elution kit used is called DiaCidel Kit that consists of ready-to-use reagents offering an easy working procedure for the elution of most common antibodies.

Reagents

1. DiaCidel wash solution (concentrated) containing Glycine-NaCl buffer. The working solution can be prepared by diluting the concentrated wash solution with distilled water in 1:10 (i.e. 1ml concentrated wash solution and 9 ml distilled water).
2. DiaCidel elution solution containing a low pH glycine buffer solution with a colour indicator.
3. DiaCidel buffer solution containing Tris buffer with bovine albumin (1.2%).
Test Procedure

1. Wash the red cells which are DAT positive once with isotonic 0.9% saline solution. At least 1ml of packed cells are required.
2. Wash 1.0 of packed cells 4 times with DiaCidel working wash solution.
3. Decant completely after the last wash and keep part of supernatant to test for the presence of irregular antibodies.
4. Add to the 1ml of wash packed cells with 1.0 ml of DiaCidel elution solution. Mix well.
5. Centrifuge immediately for 1 minute at 3000 rpm.
6. Transfer the eluate into a clean tube.
7. Add 5 drops (250 μl) of DiaCidel Buffer Solution to the eluate and mix well. Observe the forming of a blue colour that indicates a neutral pH of 6.5-7.5 us reached. If the blue colour is not obtained, add more buffer 1 drop at a time while mixing.
8. Centrifuge the eluate for 1 minute at 3000 rpm to completely remove any residual cells.
9. Eluate is now ready for testing.

Perform the normal procedure used for antibody identification with the eluate. Use the supernatant solution kept form the last wash in parallel as a negative control.

When you perform the antibody identification with the eluate, you would run an 11-cell panel without including the own patient’s red cells. Confirm the presence of an antibody if the pattern of reactivity matches a pattern of antigen-positive cells. If your DAT is positive but after doing elution and running on the 11-cell panel, the result is negative for all common antibody, we would not proceed on with the investigation. This could be due to many underlying reasons such as hypergammaglobulinemia or drug reactions.

Some links to check out the gel cards and the elution kit that I was referring to:
http://www.diamed.com/product_detail.aspx?id=123&navvis=
http://www.diamed.com/product_detail.aspx?id=122&navvis=
http://www.diamed.com/product_detail.aspx?id=494&navvis=

Thank you.
Indah.

Bone marrow culture set-up

Hi everyone, I’m Justin here again. Today I’m going to talk about bone marrow culture set-up. This step involves the initiation of cultures by inoculating cells into the media. The specimen is bone marrow aspirate in sodium heparin anti-coagulant (the green top). Other types of anti-coagulants should not be used as they will affect cell viability. The purpose is to get viable cells that are dividing.

The specimen is centrifuged at 1500rpm for 10 minutes and the buffy layer is obtained. The buffy layer is the layer in between the red cells and the plasma portion. The amount of buffy layer to inoculate depends on its thickness and the WBC count. It is very important to inoculate the correct amount of cells. Adding too much cells will cause nutrients to be depleted fast, resulting in cell death too early or stopping of mitotic activity. Adding too little cells will result in not enough metaphases for analysis. As a guide from the technical procedures manual, if the layer is more than 1 mm thick, use ½-1 drop. If it is about 1 mm thick, use 2 to 3 drops.

The duration of culture depends on the suspected diagnosis. The purpose of using 2 durations is because certain abnormal clones can be found immediately while others need more time to grow. For most cases, setting a 24 and 48 hour culture will be appropriate to yield the abnormality present. Only abnormal clones show the specific cytogenetic abnormality.

For conditions such as AML (acute myeloid leukemia); CML (chronic myeloid leukemia); (MPD) myeloproliferative diseases eg, polycythemia vera, a 24 and 48 hour culture will be set up.

For cases of chronic lymphocytic leukemia (CLL), mitogens are added to stimulate specific clones as clones are dividing very slowly. TPA (Tetradecanoylphorbol acetate) and PHA (phytohemagglutinin) are growth factors specific for B-cells and T-cells respectively. For new cases of CLL the 2 cultures are 72 hours with TPA and PHA so as to determine the specific cause, whether the CLL is due to B-cells or T-cells. For follow up cases, a 48 hour culture is set up and another culture is the 72 hour with the specific mitogen.

For cases of Multiple myeloma (MM), a direct harvest is done and a 72 hour culture with Il-6 is set. The direct harvest is done so as to get abnormal clones characteristic of MM present that are aggressively dividing. They will die off fast, thus the need for the direct harvest. The 72 hour culture is set up and because other clones divide very slowly. Il 6 is a cytokine which would help abnormal clones proliferate.

The culture media used is the RPMI 1640 complete culture media. The media is supplemented with L-glutamine, antibiotics, and fetal bovine serum. L-glutamine is an essential amino acid. Fetal bovine serum provides proteins and growth factors. Antibiotics such as penicillin and streptomycin inhibit the growth of bacteria in the culture.

Cultures are incubated for their respective durations in a 37ºC incubator in 5% CO2. The CO2 allows the pH of cultures to be in equilibrium. The temperature of incubation at 37 ºC is the physiological body temperature which is optimal for growth of human tissues.

That’s all for now. Take care everyone ! All the best for MP and the remaining SIP !

Ng Tze Yang Justin
0703747F

Clinical Chemistry

Cobas is an analyzer used in biochemistry. Some of the common tests include Troponin-T, CKMB, HbA1c, and total PSA etc. The tests may use one of the two principles, competition or sandwich.

An example of a test that uses the competition principle is cortisol. Firstly, the sample will be incubated with a cortisol specific biotinylated antibody and a ruthenium complex labelled cortisol derivative. Depending on the concentration of cortisol in the sample and formation of respective immune complex, the biotinylated antibody binding site will be occupied partly by cortisol from the sample and the ruthenium complex labelled cortisol derivative.

Next, streptavidin coated microparticles are added. The mixture undergoes a second incubation whereby the immune complexes bind to the solid phase through interaction between the biotin and streptavidin.

Subsequently, the mixture will be added into the measuring cell. The microparticles will be magnetically attracted onto the surface of the electrode. Unbound substances are removed with Procell. Voltage will be applied onto the electrode and induce chemiluminescent emission that will be measured by a photomultiplier.

An example of test that uses sandwich principle is CKMB. The difference would be the use of two antibodies; CKMB specific antibody labelled with ruthenium complex and biotinylated anti-CKMB antibody. Therefore, during the first incubation, it would form a sandwich complex whereby the CKMB present in the sample will occupy the binding site of the CKMB specific antibody and the biotinylated anti-CKMB antibody will bind to the CKMB specific antibody. The subsequent process that takes place is similar to that mentioned above.

The clinical significance of cortisol is for diagnosis of Cushing's syndrome (overproduction of cortisol) and Addison's disease (underproduction of cortisol). The reference range of cortisol is 5-25ug/dL at 8am and 2-17 ug/dL at 4pm. This is due to significant diurnal variation in cortisol levels, whereby it is highest in the morning and lowest at night.

The clinical significance of CKMB is for diagnosis of myocardial ischaemia, e.g. in acute myocardial infarction and myocarditis. However, CKMB may also be present in stroke and rhabdomyolysis. Therefore, total CK and Troponin-T levels should also be tested to differentiate between the two clinical implications. In addition, CKMB will be present 3-8 hours after onset of cardiac symptoms. Therefore, sensitivity of CKMB test is dependent on the time the sample was collected. The reference range of CKMB is 0-5ng/mL.

Liyana
0703827F

Procedures involving documentation in Admin

Hello everyone, sorry for the late posting. Once again, I'm back to the administrative department. Since the ISO audition just passed yesterday, I'll talk more about the procedures involved in the department that requires documentation.

Firstly, as the administrative department (admin) is the first to come in contact with the different specimens dispatched to us, we will reject any urine or stool specimens that have leakage. If a unsuitable specimen was sent for a test, we will either call the clinic and request for a new specimen and put the one that we have on hold or reject the specimen. This will be dependent on where the specimen is received from, whether it is from the hospital ward or external clinic. Sometimes, after the plain tube is spun, the technicians will reject the blood due to haemolysed specimen. If EDTA or Sodium Citrate tube is clotted, the technicians will also reject the specimen. All the rejected specimens requires a rejection form, which will either be passed to the admin staffs by the technicians or be written by the admin staffs. After a rejection form is written, the admin staff is to call the respective clinic to inform them of the rejection and note down the name of the person that we spoke to. This will than be commented in the system and the respective test affected will be deleted from the patients' record unless the patient is requesting for a package (a series of tests billed under the same code). For eg, "EDTA clotted (Bld Grp) - infrm __". The rejection form will be faxed over to the respective clinic and be photocopied. The original rejection form will be dispatch to the clinic on the following day while the photocopied copy will be filed in the file label 'Rejection of Specimen'. Then, we will find the request form for that patient and comment on the form as what we have commented in the system.

If an extra specimen is received or as mention previously, a wrong specimen is sent by the specimen is put on hold while waiting for the new specimen to arrive, we are to record the patient's particular, lab request number and type of specimen in the EXTRA BOOK. A comment both in the system and on the request form will look like this, "extra plain", and we are to state that the specimen is an extra by writing the word 'extra' on the specimen label that we printed.

If a further confirmation is required for a particular patient's test result, the technician will inform the admin staffs who will then call and inform the clinic. While informing the clinic, we are also supposed to inform them of the delayed test report the turnaround time for the send-out tests differ. The name of the person that we spoke to will be noted and a comment will be noted in the system like this, "HIV added - inf__" when the HIV antigen and antibody level is tested reactive in our laboratory.

Often, we received specimens that are wrongly labelled (with a different patient's name or a spelling error in the patient's name). We are required to call the clinic and request for a staff to come to our laboratory to label the specimen with the correct label and also give us a memo that tells us that the specimen belongs to the particular patient. The patient's particulars will then be recorded in a blue coloured file with the name of the person we spoke to from the clinic and clinic that the patient belongs to.

Any memo that is faxed over or passed to us will be photocopied. The memo must contain the signature of the person who wrote the memo and any amendment required must be stated clearly. The original copy will be filed in the monthly memo file while the copied one will be stapled to the request form. If a name or ID amendment is made, the technician must be informed to re-approve the result so that the clinic is able to view the result via the system.

Finally, regarding the pictures of the VDRL, I am still trying to get the permission from my lab manager to post them (We are very busy during work, so there is no chance for me to ask...). Hope my explanation is clear enough to let you guys have an idea of how the admin department work! XD

Hui Juan

0702012F