International Histocompatibility Working Group (IHWG)
May 22, 2000
Dear colleagues,
The Anthropology/Human Diversity Component of the 13th workshop is now ready to move into its next phase of activity. In this phase, typing reagents and QC panels will be sent to participating laboratories, laboratories will become qualified for the generation and submission of HLA data, and the HLA typing of workshop samples will begin. Subsequent to the distribution of our March 2000 update, many of you have asked questions pertaining to these topics; I apologize for the delayed response, and hope that you will find all of your questions and concerns addressed here.
This update is intended to serve as a complement for materials that are already available on the IHWG web site. If you have not already done so, or have not visited the site for a while, please take the opportunity to familiarize yourself with the web pages of the Anthropology Component, as well as other pages on the web site for the appropriate typing technologies.
Ordering Reagents
Approved standard SSOP and Reverse Line Strip SSOP reagents can now be ordered using these forms on the IHWG web site. Standard SSOP reagents are available in amounts sufficient for typing 500 samples at a single class I or class II locus, and may be ordered from Lifecodes at prices ranging from $1100 to $2170 for 500 class I locus typings and $200 to $2100 for 500 class II locus typings (pricing depends on probe and primer labeling options).
Reverse Line Strip SSOP reagents are available in sets of 50 tests for each class I locus (A, B, and C), and may be ordered through the Children's Hospital Oakland Research Institute (CHORI) for a price of $1750 for each set of 50 tests. The A and C loci tests use a single reverse line strip for each sample, while the B locus test uses two strips, one for each exon. Therefore, each set of 50 tests includes 50 A locus and 50 C locus strips, and 100 B locus strips. Sets of 15 tests for the A, B and C loci, to be used for QC typing (see below), must be ordered initially. Once the laboratory has successfully typed the QC samples, additional Reverse Line Strip reagents may be ordered.
In addition to the strips and associated PCR amplification, hybridization, and detection reagents, pattern interpretation software, permitting facile allele assignment based on probe reactivity patterns, will be included as well. Because the Reverse Line Strip tests are being produced exclusively for the research purposes of the IHWG, participating laboratories are asked to affirm that the tests will be used solely for that purpose.
SBT reagents are not yet available.
Internal Validation
IHWG SBT and SSOP typing methods should be established in the laboratory by using positive control samples of known type to monitor the amplification and hybridization of IHWG reagents. Once the laboratory can confidently generate accurate typing results with the new reagents, the QC cell panel should be ordered from the IHWG Coordinating Center in Seattle (see below). QC test cells (N = 15) for the Reverse Line Strip assay will be distributed through the IHWG Technology Development Laboratory (Bethesda) and Reference Cell Panels will be distributed through the IHWG Cell and Gene Bank (Seattle).
A set of 10 Internal Validation Reverse Line Strip tests will be included with the initial set of 15 QC typing tests. These Internal Validation strips are marked with a red line, and are to be used only for a given laboratory's Internal Validation of the Reverse Line Strip typing system. The Internal Validation strips are NOT to be used for QC typing or the typing of IHWG samples.
Ordering QC and Reference Cell Panels
QC panels and Reference Cells for the standard SSOP, SBT and Reverse Line Strip typing systems can be ordered using these forms. All participating laboratories must type the panel of QC cells (25 cells for the SSOP system, 15 cells for the Reverse Line Strip system) at all loci being studied before their data will be "qualified" for entry into the central database (see following section). The initial set of QC cells will be provided at no charge. However, only two sets of QC cells will be distributed to a given lab. While we recognize that many labs have extensive experience with both standard SSOP and Reverse Line Strip typing systems, this validation process is necessary both because the SSOP and Reverse Line Strip reagents and their associated data interpretation methods may be new to your group, and because the probe reactivity patterns generated using these typing systems are the focus of the central database. If any consistent experimental variations should arise, the central database will need to be aware of them.
In order to assess the specificity of probes and primers over the course of the data generation phase of the workshop, laboratories will need to amplify and type Reference Cells along with their samples (see the Cell and Gene Bank). The Reference Panel will provide positive controls for sensitivity and specificity. These cells will be distributed by the IHWG Cell and Gene Bank, and should be included as positive controls for the routine typing of workshop samples, as required by the locus being typed. A 51 cell set of SSOP reference DNA is available for $250 and a 32 cell set of SBT reference DNA is available $135. Each aliquot will consist of 10 micrograms of DNA. Laboratories using Reverse Line Strip method only need to run a single reference cell (SSOP reference cell #4, designated G085). This reference cell will be distributed along with the 15 cell Reverse Line Strip QC cell panel at no charge.
Evaluation of QC Panel Typing
The evaluation of standard SSOP and Reverse Line Strip testing of QC panel cells will depend upon the accuracy of positive and negative probe hybridizations as well as correct allele assignments. For the Reverse Line Strip system, the pattern interpretation software output file will contain all the information necessary to evaluate typing results. Evaluation of SBT results will require submission of histograms and allele assignments.
All QC panel typing data should be submitted via email to the central database at database@ihwg.org. In addition, photographs of the developed Reverse Line Strips will aid in the diagnosis of any consistent procedural errors that might arise. Photographs of the strips may be mailed to:
- Dr. Steven J. Mack
Anthropology Component/IHWG
Department of Human Genetics
Roche Molecular Systems
1145 Atlantic Avenue
Alameda, CA 94501-1145, USA
Please include your Lab Code, as well as a list describing which QC panel cell corresponds to which strip, and which strips pertain to what loci.
Submission of Available HLA Data
Many participating laboratories have already typed samples using other reagents than those provided by the IHWG. These data, termed "Available Data", will be reported to the 13th Workshop Database. The Database and the Biostatistics Core will accept both phenotype data generated with serological reagents and high resolution DNA typing data generated with local or commercial reagents for analysis by the Anthropology Component.
A data submission template can be downloaded from the IHWG web site for this purpose. The data template is an excel file that presents all of the required datafields in an organized, easy-to-fill-in fashion. The first worksheet of this excel template serves as documentation for the data format required by the template. In addition, a sample data file on the data submission web page provides an example of a properly filled in data template. Please follow the guidelines provided in the documentation file, and submit all data generated with non-13th-IHWG typing methods via email attachment to database@ihwg.org.
Submission of lymphocytes, cell lines or DNA from selected volunteer donors to the IHWG Cell & Gene Bank
Participating laboratories will be asked in consultation with the Anthropology/Human Diversity project leaders to submit samples from selected volunteer donors to the IHWG Cell and Gene Bank. The purpose of this is to accumulate samples from individuals that are characteristic and unique for a given population. This repository will function as a shared resource for the scientific community to facilitate future research. If there are no restrictions on submitted samples, they will eventually be available (with the agreement of the submitting investigator and project leaders) to all investigators. Submission of these samples should be accompanied by a Letter of Certification insuring that acquisition of the sample has been made under appropriate human subjects guidelines (see below).
Letters of Certification for Human Subjects Research
In order that the various projects of the IHWG proceed in accordance with Federal and international regulations pertaining to the protection of human subjects, each participating laboratory has been asked to provide documentation to the effect that their use of human subjects is in compliance with all applicable laws and regulations at their local institution in the forms of letters of certification. A sample version of this letter (Microsoft Word format download) may be obtained here. Please mail this letter to:
- Henry Erlich, Anthropology Component/IHWG
Department of Human Genetics
Roche Molecular Systems
1145 Atlantic Avenue
Alameda, CA 94501-1145, USA
This letter should be sent when the participating laboratory first submits data to the Anthropology Component.
We look forward to working with you over this coming year.
Best regards,
Henry A. Erlich, Ph.D., Chair and
Steven J. Mack, Ph.D.
Anthropology/Human Diversity Component
International Histocompatibility Working Group
ROCHE MOLECULAR SYSTEMS
CHILDREN'S HOSPITAL OAKLAND RESEARCH INSTITUTE
