Tissue Typing (HLA) Laboratory

I was attached to the tissue typing (HLA) laboratory for a week and had fun learning new things. It was a bit stressful at times, as the procedures performed in this laboratory requires critical attention with much understanding of the HLA system.

Human leukocyte antigen (HLA) is a specialised category of immunology that deals with human histocompatibility testing. This laboratory discipline supports clinical specialities in transplantation, transfusion and immunogenetics. Hence, a laboratory officer must be specialised in this field to handle the heavy responsibilities. All of the preliminary result is double-checked by senior laboratory officers for confirmation. The HLA laboratory in BSG handles samples that are mainly for organ and bone marrow transplantation. We also receive samples from regional countries such as Vietnam and Malaysia.

It is necessary to determine the HLA antigen composition in an organ or bone marrow recipient to match with the prospective donor, who may be living family-related or unrelated deceased. For transfusion cases, patients that underwent platelet therapy may develop platelet refactoriness that can result in poor platelet survival following random donor platelet infusion. Hence, it is important to identify HLA alloantibodies in the recipient serum before transplantation or transfusion. HLA alloantibodies are majority IgG.

The testing services offered by HLA laboratory in BSG are HLA antigen typing, HLA antibody identification (i.e. determining the specificity of the anti-HLA class I and II antibodies) and lymphocyte crossmatching between donor cells and recipient serum for compatibility. I got the opportunity to view and do both serological and molecular techniques in a HLA typing test. Since it is very lengthy to explain both, I will focus on explaining the serology part.

For serological procedures, the very commonly requested test is the HLA-B27 typing. This tissue antigen is associated with ankylosing spondylitis. Samples are received in acid-citrate-dextrose (ACD) tube or heparin tube (green cap). We will do layering of the blood sample with Ficoll-Paque in the ratio 3:1 and centrifuged the tube at 2000 rpm for 20 minutes. This condition is ideal to create a density gradient so that we can separate out the lymphocytes. 4 layers are obtained: bottom – red cells, Ficoll-Paque, lympocytes and monocytes, and plasma – top. Discard the plasma and carefully transfer the lymphocytes and monocytes layer into a clean tube without taking so much Ficoll-Paque using Pasteur pipette. Wash 3X this layer with cold PBS for purification.

Next, we want to harvest T cells. Why T cells? This is because HLA-B27 is categorised as HLA Class I molecule and we don’t want B cells that have both Class I and II molecules. We will use the magnetic particle concentrator (MPC), where we add the Class I beads (they are actually monoclonal antibodies specific against Class I antigens). This will positively select the T cells. We will dilute the cells to the optimal concentration with prepared solution that contains Hank’s balance salt solution (HBSS), 2% fetal calf serum (FCS) and acridine orange (AO).

Next, we will perform the complement-dependent cytotoxicity (CDC) assay. Before doing so, we will check if we got the optimum cell concentration and cell viability (graded with score of 1 i.e. 0-10% dead cell) by adding 2μl of cells to an empty well of a microtiter plate and 2μl of Propodium Iodine (PI). This will stain the cells under the fluorescent microscope. Green means cells are alive while red means cells are dead.

We use a 60-well commercial microtiter plate. The plate has 1μl of known antisera with mineral oil layer on top to protect the antisera from evaporation. We will add 2μl of the patient T cells and incubate at room temperature for 40-50 minutes. Then we add 5-6μl of rabbit complement to each well and incubate for 60 minutes in the dark (we just put the plate in a metal plate with a lid). After that we add 2μl of PI in 5% EDTA solution to each well for 15 minutes at room temperature. This is for staining and to also stop the cytotoxic reaction. Lastly, we will remove excess fluid and add paraffin oil to prevent evaporation when viewing under the fluorescent microscope (10X objective). If the 81-100% of the cells are dead (i.e. graded with a score of 8), the cells are strongly positive for the HLA-B27 antigen.

Indah.
0705361D