Automatisierte HLA-Klasse-II-Typisierung an Bead-separierten B- und unseparierten mononukleären Zellen mit einem PC-gesteuerten Mikroskop-Photometersystem

Abstract

By means of automation of the serological working steps and automated reading of typing trays by a PC-based microscope photometer System, 34000 voluntary unrelated bone marrow donors were been HLA-class l (A, B)-typed from March 199 to November 1992 in the laboratory of the Düsseldorf Center of the Bone Marrow Donor Registry of ' lab.med. 17: 95 (1993) 95 Brought to you by | University of Manchester Authenticated Download Date | 5/26/15 6:06 PM Originalle North Rhine Westfalia. This high frequency of HLA-class l typing (2000-2500/month) correlates with the increase in the number of requests for HLA-class II (DP, DQ) typing. In order to ensure rapid and accurate DB, DQ typing, either isolated B-cell preparations or unseparated mononuclear cells were employed in the dual color fluorescent cytotoxicity test. The advantage of B/T cell Separation with immunomagnetic beads lies in the extreme purity ofthe isolated B lymphocyte subset (95-100%) and in a noticeable saving of time in comparison to nylon wool-enriched B-lymphocyte preparations. In particular, automated reading of the HLA class II typing trays using a PC-based microscope photometer System results in superior precision äs well a$ further considerable saving of time. Thus the effective reading time has been reduced from 5-75 min per tray (visual) to 20 sec. This, furthermore, provides the prerequisite for the high frequency oftypings per day. The direct comparison of HLA-DR, DQ typing between automated and visual reading shows a very high degree of reliability (99%, basis 1600 DR, DQ typings). The concordance ofboth readouts (visual and by automatic microscope) is 93.9%. This calculation is based on the comparison of the serological scores of 100 defined DR72 well trays equaling 72000 wells, which were examined for consistency on a score-by-score basis, and by cross-tabulation ofthese data. The fact that unseparated B and T cells, i.e. mononuclear cell can also be employed directly for the automated reading of class II typing trays, is of particular importance in regard to infectious cell material, e.g. from HIV positive individuals. The automatic microscope sets an electronic tray specific sensitivity Standard with respect to HLA-DR and -DQ typing. This methodological Variation is particularly effective when separate trays are used for the DR and DQ reading. Reading by the automatic microscope, however, becomes complicated because of the different sera reactions for DR and DQ, if DR and DQ specific sera are on the same tray, Sharing their positive and negative control reactions.

Rapid method for successful HLA class I and II typing from cadaveric blood for direct matching in cornea transplantation

“…Isolation of living B-cells for HLA class II typing from aged blood of cadaveric donors via Dynabeads Ficoll-isolated BMNCs from cadaveric donors were resuspended in 0.6% sodium citrate and incubated with B-cell beads (Dynal, Hamburg) as described previously [7]. Supernatant was removed, centrifuged and used for the isolation of living B: MNCs for HLA class I typing via Lymphokwik MN (see below).…”

“…Supernatant was removed, centrifuged and used for the isolation of living B: MNCs for HLA class I typing via Lymphokwik MN (see below). The HLA class II typing was performed on a DR 72-well HLA class I tray (One Lambda, Los Angeles, Calif. BMT, Krefeld, Germany) as described previously [7].…”

“…The cell pellet was resuspended and washed two times in PBS. A double-fluorescence cytotoxicity assay with carboxy-fluorescein diacetate and propidium iodide was carried out as described previously [7]. HLA-typing trays (260 wells or 172 wells) were obtained from One Lambda (German distributor BMT).…”