Potentials and prospects of using a confocal microscopy for the HLA-Crossmatch result evaluation in bone marrow transplantation patients
Currently, it has become progressively ostensible that the role of the anti-donor antibody against HLA class I and II antigens is not limited to their participation in the development of hyperacute rejection of allografts. An increasing number of researchers are inclined to believe that the formation of anti-donor HLA antibody in the posttransplant period leads to the development of hyperacute, acute or chronic rejection. The immunological compatibility test between donor and recipient for transplantation is the critical factor for deciding whether to perform a transplant. Antibodies to HLA founded in more than 30% of the population; their detection indicates a prior immunization with HLA. It is possible with multiple transfusion of whole blood, during pregnancy and after transplantation. A number of authors making a point that the donor non-specific antibodies to HLA are one of the factors not only of the risk of acute but also chronic rejection of the transplant and are considered as a poor prognostic indicator [P.I. Terasaki, 2006; M. Morrel, 2011; M. Crespo, 2011].
The purpose of the lymphocyte crossmatch is to detect the lymphocytotoxic antibodies specific to a potential donor (i.e., allogeneic crossmatch) or self (i.e., autologous crossmatch.) A forward crossmatch used with serum from a prospective recipient (or donor for reverse crossmatch) and the target cells are the mononuclear cells of the potential donor (or recipient for reverse crossmatch). The target cells used may be unseparated or separated into specific subsets such as T cell lymphocytes, B cell lymphocytes, monocytes, etc. Evaluation of the results of the HLA-Crossmatch reaction is highly subjective by simple optical microscopy because it is impossible to accurately calculate the number of dead cells at the Terasaki camera view field and subtract the background (the results of negative control). It is also problematic to estimate to which class of HLA (I or II) antibodies belong.
Materials and methods
We developed a methodology for evaluating the results of HLA-crossmatch using a confocal microscope of the bone marrow transplantation patients. The entire HLA-crossmatch reaction performed by a standard method, except for the last two steps: staining and evaluation of the viability of lymphocytes in the wells. We used Acridine Orange (AO emission 525 nm) and Propidium Iodide (PI – 620 nm) as vital dyes. To stain B-lymphocytes, anti-CD19 monoclonal antibodies conjugated to PE-Cyanine7 (775 nm) were used. The images taken with a ZEISS confocal microscope, followed by image analysis with the ImageJ software (National Institutes of Health), and then calculating the results with the program.
The advantages of the proposed method of evaluation are as follows: (1) In one well, a single serum sample of the recipient is analyzed for the presence of antibodies against donor HLA antigens simultaneously at both I and II classes. 2) The method is fast, accurate and high-performance (in one Terasaki plate, up to 72 sera samples can be analyzed in one run). 3) The high sensitivity of fluorescent label reading. (4) Presence of a multilevel system of various controls. (5) The ability to archive the results obtained in the program database for interpretation and PDF reports. (6) Subjective biases of the data analysis are minimized by statistical processing of the data by software.
HLA, HLA-crossmatch, bone marrow transplantation, confocal microscopy.