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    • HLA Analysis Dpt.
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HLA Analysis Department

Administrational and personal data

Guideline for taking primary samples (valid from 1.2.2007):

• HLA serology [PDF]
• HLA genotyping [PDF]

Submission forms (valid from 1.2.2007):

• HLA serology [PDF]
• HLA genotyping [PDF]

Serology Division of the HLA Laboratory

Focuses on

• Detection of surface antigens HLA (main histocompatibility complex in human)
• Detection and identification of HLA antibodies

Basic protocol - standard micro-lymphocyte-toxicity test (LCT-NIH)

Typing of surface HLA antigens used for:

• HLA typing of bone marrow donors (cooperation with National Registry of Bone Marrow Donors in Prague)
• HLA typing of blood donors
• typing of particular HLA antigens – especially B27
• monitoring of correlation between HLA markers and diseases
• monitoring of frequency of HLA antigens in population

Detection of HLA antibodies by lymphocyte-toxicity test used for:

• detection of development of alloimunization after transfusion (in cooperation with Department of Immune-hematology)
• during testing suitable donor of thrombocytes – using lymphocyte cross-match assay

Testing of soluble HLA antigens is available in cooperation with Department of Biochemistry.

Laboratory of HLA Genotyping

This laboratory is accredited within NRL for DNA diagnosticsby Czech Institute for Accreditation (CIA).

Determination of HLA Genotype - Major Human Histocompatibility System

HLA system (Human Leukocyte System) is the main histocompatibility complex in humans. It is located within the short arm of chromosome 6 (6p21.31 - 6p21.33). Products of its extremely polymorph genes (probably hundreds of alleles, but exact number has not yet been determined) are antigens (trans-membrane glycoprotein), which recognize organism-unfamiliar structures and thus start a cascade of immune responses.

Determination of exact HLA genotype in a patient and his donor is one of the basic requirements for successful allogenous transplantation of hematopoetic stem cells. Incompatibility of the HLA system between patient and donor might lead to graft rejection or even cause GvHD (Graft Versus Host Disease), which is a serious, in the worst case, fatal complication.

Some diseases (rheumatoid arthritidis, celiac, narcolepsy, insulin dependent diabetes mellitus) are known to be associated with distinct alleles of the HLA system. There is higher risk of disease development in the presence of that particular allele. Nevertheless, none of these relationships are to be absolute.

Our laboratory has provided HLA genotyping since 1993, as the first place in the Czech Republic. It serves patients from IHBT and the Clinic of Child Hematology and Oncology Motol, as well as other clinical submitters.

As of recently, the laboratory provides routine genotyping of the most important transplantation loci HLA I. and II. class: HLA -A, -B, -C, -DRB1, -DQB1 in patients indicated for allogenous transplantation of hematopoetic stem cells and their related donors; as well as confirmation analysis for unrelated donors from registers from the Czech Republic and abroad. Genotyping of chosen loci HLA system, as additional information for diagnostics of HLA-associated diseases, is available by agreement.

Methods used in genotyping:

• Polymerase chain reaction with sequence-specific primers (PCR-SSP) - method validated by CIA, Prague (Fig. 1)

Fig. 1 Example of HLA typing by PCR-SSP method

• Direct sequencing, SBT (sequence based typing) – method validated by CIA, Prague (Fig. 2)

Fig. 2 Part of sequence of HLA-B (typing by SBT)

• Indirect dot blot (alternative method)

Evaluation of results is obtained by software analysis connected to the international database of HLA alleles www.ebi.ac.uk/imgt/hla; and is regularly actualized upon that database. It ensures the maximal credibility of outgoing results.