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U.S. Department of Health and Human Services

Vaccines, Blood & Biologics

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Establishment of Reference Panels for Blood Group Genotyping Tests

Principal Investigator: Zhugong "Jason" Liu, MD, PhD
Office / Division / Lab: OBRR / DBA / DRB


General Overview

The determination of surface characteristics (blood groups) of red blood cells (RBCs) is important for the proper preparation of blood for transfusion. Hemagglutination, the original serological method to determine blood groups, is useful for typing major blood types such as ABO, but is limited in more complex scenarios. For example, hemagglutination may fail to identify donor RBCs in a recently transfused patient when these RBCs are coated with IgG, or to identify RBC antigens or antibodies that have weak reactivities. Also, the antibodies necessary for hemagglutination assays for some clinically significant blood types are rare, expensive or unavailable.

Molecular assays offer a good alternative for problems encountered by serology. The International Society of Blood Transfusion (ISBT) currently recognizes 30 major blood group systems including more than 300 inherited RBC antigens. The molecular bases of almost all clinically significant blood group antigen systems have been determined. Different molecular blood group testing methods have been developed. In addition, high-throughput technologies open the possibility for large-scare donor screening. DNA testing is expected to provide more informative, accurate and cost effective services than serological assays.

While blood group genotyping is becoming increasingly used in many areas of transfusion medicine, typing errors have been documented. For example, in the 2008 ISBT workshop on molecular blood group genotyping, only 24 of 33 laboratories obtained completely correct results. As it is possible that a genotyping test needs to be done only once in a person's lifetime, the consequence of errors can be substantial. Well validated DNA reference panels are required to ensure the quality of the tests. Reference materials are critical for the development and validation of new assays, for test calibration and for monitoring of assay performance. They are also used as proficiency testing samples. Currently, limited reference materials are available for blood group genotyping. These materials often do not include all variants tested in clinical assays. Laboratories and device manufactures may use uncharacterized, non-renewable clinical materials for test development, validation and quality control. Therefore, the quality of genotyping results is potentially compromised.

In order to address this challenge, CBER has initiated a project to develop comprehensive reference panels for blood group genotyping for international accreditation and distribution. Our goal is to include all clinically significant blood group antigens in the panels. Specifically, blood samples will be collected from consenting and well-characterized donors. Blood group genotypes are examined using at least two molecular methods. To ensure a continuing supply of DNA reference materials, cell lines are created from these samples. DNA extracted from cultured cell lines will be freeze-dried to ensure greater stability for easy storage over time. A collaborative study will be initiated to test suitability of the materials as reference panels, and to secure consensus results from collaborating laboratories.

CBER regulates blood typing devices, including the novel molecular typing devices. This initiative contributes to the regulatory and research efforts of CBER to support development of safe and effective products that improve public health.


Scientific Overview

Blood group genotyping has advantages over traditional serological tests. There are useful applications of blood group genotyping in transfusion medicine, including fetal DNA typing, extensive blood typing of donors for alloimmunized patients, determining the blood group of a recently transfused patient and screening donors for rare blood types. Reference materials are required for the development and validation of these tests, and for their optimal use in routine practice.

The AABB publication, "Standards for molecular testing for red cell, platelet, and neutrophil antigens" has recommended alleles from 17 blood groups for reference standards. However, the reference materials that are under development often do not include all recommended genotype variants. For example, the proficiency exchanges from the Consortium for Blood Group Genes (CBGG) are currently restricted to 16 alleles from 5 blood group systems. At the recent ISBT international workshop on genotyping, 46 laboratories reported test results on alleles from 11 blood group systems. Proposed WHO reference reagents include 4 samples from Caucasians and Blacks typed for 7 blood group systems. The lack of comprehensive reference panels poses a significant challenge to the development and validation of molecular testing devices that CBER regulates.

We plan to develop reference panels that are designed to include about 90 genotypes of the blood group systems that recommended by AABB. Whenever possible, we will include samples from 3 donors for each of these genotypes. These donors have been historically typed by both serological and at least one molecular method. Blood samples will be collected from consenting donors as starting materials. Part of each blood sample will be used for genomic DNA extraction to confirm the blood group genotypes by another molecular method. Red cells will be cryopreserved in aliquots for further serological typing if necessary.

To make reference panels we will first establish lymphoblastoid cell lines by Epstein-Barr virus (EBV) transformation so that we can have a supply of genomic DNA from the same source. The remainder of each blood sample will undergo B-lymphocytes isolation for EBV transformation. We will create master and working cell banks to ensure long-term survival of the cell line. Cells from the working cell bank will be cultured for genomic DNA extraction. Isolated DNA will be freeze-dried and sealed in glass ampoules, which has been demonstrated to be critical for greater stability of the reference reagents. We will carry out an international study with collaborating laboratories to obtain consensus typing results for each genotype, and to test the suitability of these panels as reference material. Coded samples will be provided to participating laboratories. Participants will perform molecular blood typing using their routine method(s) for the required genes. We will analyze the results reported by the participants, and seek their opinion on the suitability of the materials for use as reference panels. The materials found to be suitable will be made available to blood typing device manufactures and testing laboratories.

 

Contact FDA

(800) 835-4709
(301) 827-1800
Consumer Affairs Branch (CBER)

Division of Communication and Consumer Affairs

Office of Communication, Outreach and Development

Food and Drug Administration

1401 Rockville Pike

Suite 200N/HFM-47

Rockville, MD 20852-1448