Dear Colleagues,
As the year 2000 comes to a close, we want to take this opportunity to provide you with a brief overview of the status of our project, as well as provide feedback on the RLS QC testing process. Some of this information was presented at the Anthropology Component meeting at ASHI this past October, but much of it has been generated in the months since that meeting.
IHWG Status
The QC typings are currently being carried out for the RLS, SSOP and SBT typing systems. The generation of population data via the RLS method has begun for the Anthropology Component, and the first results will be submitted in January, 2001. In addition, microsatellite data are being generated by the HLA and Disease Components. The Hematopoietic Cell Transplantation Component is generating mostly SBT donor-recipient data. The Null Alleles and Serological Equivalents Component is submitting data to the central database.
Anthropology Project Progress
The Anthropology / Human Genetic Diversity project has grown to include sixty-seven laboratories in twenty eight countries; these laboratories are contributing samples from one hundred four different populations sampled in thirty-four countries, representing every region of the world, except for Australia. A list of these populations can be found on the Anthropology Component pages of the IHWG web site, at http://www.ihwg.org/components/populations.htm. The sample sizes number approximately 200 individuals on average, so on the order of twenty thousand samples will be typed for the Workshop and Conference in 2002.
Of the sixty-seven laboratories participating in the Anthro Component, thirty-eight are using the Reverse Line Strips for class I typing. Twenty-eight of these have received the RLS QC typing reagents, and one-third have submitted their results for evaluation. Similarly, approximately nineteen participating Anthropology labs are using the SSOP system for class I typing and seven of these have received SSOP QC reagents. Seven laboratories are using SBT methods, and have received SBT QC reagents.
Quality Control Typings
Reverse Line Strip System. Thanks to the QC process, we now have feedback on how well the Reverse Line Strips perform at each locus, as well as some additional suggestions as to how to get the most out of the system. We want to share this information with those of you who are using the strips, or who may still not have decided which system they would like to use to carry out class I typing. So far, of the ten laboratories who have submitted their QC results for all three loci, nine have passed at the HLA-A locus, six have passed at the HLA-B locus, and three have passed at the HLA-C locus.
In our evaluation, we have found that the probe reactivity patterns on most developed Reverse Line Strips are easy to evaluate using the Strip Interpretation software program. However, due to variation in the intensity with which some probe signals develop, there may be a few strips at each locus that will require closer inspection before alleles can be accurately determined. We found that most labs which had problems typing at a particular locus were not using the tables provided in the appendices, as described below. In those cases where a lab failed at a particular locus, we would like you to take the opportunity to re-evaluate the fifteen probe patterns for the locus in question in light of the information in this section, and to re-submit your QC results for that locus. We hope that, in most cases, this will allow us to avoid re-shipment of QC reagents, and will permit rapid re-evaluation of your QC results.
In some of these cases, the pattern in question is the result of false-positive lines and weak probe signals. Information on identifying false positives and weak probe signals, and instructions on how to account for them can be found in section IV.E, Undetermined Alleles, of the Reverse Lineblot Typing Protocol. The protocol is available on the IHWG website as a pdf download, in the IHWG Technical Manual, as well as on the IHWG CD-ROM disk sent out with each Quality Control package.
In addition, some probes may react in a problematic fashion with particular alleles at a given locus. Such probe-allele combinations are listed in Appendix B, Allele Associated Faint Probe Signal Tables, of the Reverse Lineblot Typing Protocol. In particular, careful attention must be paid to probes 14 and 18 on the C-Locus strip in order to obtain accurate allele calls. It is imperative that the Allele Associated Faint Probe Signal Tables section of the protocol be consulted before data is submitted to the workshop.
Because the Strip Interpretation Software is the most facile method for determining alleles, the output file and software should be consulted first to provide clues to the correct allele assignment. Subsequently, difficult-to-call Reverse Line Strips should be compared to the locus specific hit tables, faint probe tables in the Appendix, and then retyped if necessary.
These additional steps will address most potential errors, allowing accurate allele calls to be made at each HLA locus. However, the QC typings have revealed a few probes that result in faint false positives in conjunction with certain alleles, as well as a few additional probe-allele combinations that need to be included on the Allele Associated Faint Probe Signal Tables. These probe-allele combinations will be incorporated into the Appendix section of the Protocol, but we are also including them here (Tables 1 & 2 below) so that everyone will have access to them for the purpose of making accurate allele calls in their QC Tests.
Table 1: Additions to Appendix B, Allele Associated Faint Probe Signal Tables
( New information is emphasized in bold-faced red type below. )
( New information is emphasized in bold-faced red type below. )
HLA-A
- After compiling the current data, there are no additional probes for HLA-A that appear to be miscalled on a consistent basis as falsely negative. Please refer to the protocol for the Allele Associated Faint Probe Signal Tables to obtain data on weak signals for HLA-A.
HLA-B
| LINE POS. |
PROBE MOTIF |
COMMENTS |
| 25 | IF-TNT | Weak with *08xx & *18xx, *0702x & *3503, *0802 & *2708, *3508, and *15xx & *35xx, Weak with *5101, *0801, *3901, *35xx |
HLA-C
| LINE POS. |
PROBE MOTIF |
COMMENTS |
| 6 | ASPRGE(a) | Weak with alleles *03031 and *03041, *0302 and weak with *14xx |
| 7 | ASPRGE(b) | Weak with *0501, 0701/06 and 0803 |
| 8 | PRAPW(a) | Very faint cross-hyb with *0602 |
| 10 | PREPW | Weak with *0401, *0304 |
| 11 | AQTDR | Weak with *04xx, *03xx |
| 12 | AQADR | Weak with *0803 |
| 13 | RNLRG | Weak with *08xx, *12xx |
| 14 | RKLRG(a) | Probe is faint on this batch (with all alleles)*** |
| 16 | SEAGE | Weak with *0602, *0701 |
| 18 | QRMYG(a) | Weak with *03031 and *0302/6, *0304 |
| 19 | QRMYGB | Weak with *03xx |
| 22 | QRMSG | Weak with *0501, *0702/3 |
| 31 | EQRRAY | Weak with *0501, *0802 |
| 33 | EQWRAY | Weak with *02024 and *06xx |
***The Allele Associated Faint Probe Signal Table for HLA-C indicates that probe 14 is faint on this batch of Reverse Line Strips. Regardless of the availability of this information, probe 14 was still miscalled a total of 13 times by IHWG lab participants to date. Please be aware of this caveat and pay careful attention to probe 14 during strip interpretation.
Table 2: False Positive Faint Probe Signal Information
HLA-A
- Probe 41 appears as an occasional faint signal with the 2402 allele. However, under these circumstances, it should be considered to be a false positive.
HLA-B
- After compiling the current data, there are no additional probes for HLA-B that appear to be miscalled on a consistent basis as falsely positive.
HLA-C
Probe 15
Probe 15 has consistently shown up as a faint signal. It appears that, unless probe 15 is quite dark, this probe should be called negative. As shown on the C-locus hit table, this probe should only be positive for C*03031/032/11 and C*0312 alleles. The weak nature of probe 14 on this lot of strips, may make it difficult to accurately call probe 15. Please consider this probe carefully while interpreting strips.
Probe 19
Probe 19 was miscalled by lab participants and has been identified above as a possible false negative call. However, probe 19 has also been called falsely positive. Please consider this probe carefully during future strip interpretations and consult the hit table for HLA-C, when necessary, to help make a determination on probe 19.
Well, that wraps it up for the Anthropology / Human Genetic Diversity Component for the year 2000. We hope that your participation in this project has been rewarding so far, and we look forward with enthusiasm to working with you in 2001.
Happy Holidays and have a healthy New Year!
Henry Erlich, Steve Mack, and Laura Geyer
Anthropology / Human Genetic Diversity Component
