[Postmortem serologic and biochemical HLA typing with cultivated retinal pigment epithelium cells].
A quick and reliable method for HLA typing of human cadavers has been established. Retinal pigment epithelial cells (RPE) were obtained from cadaver bulbi 8-36 h post mortem (n = 24). After 24 hours, cell growth correlated inversely with the time lapse between the donor's death and bulbus explantation. Cultivated human RPE were stimulated by different Y-interferon (IFN) concentrations and examined by FACS analysis for the expression of MHC class I and II molecules. A concentration of 100 units/ml human Y-IFN was sufficient to induce maximal class I antigen expression by about 90% of cells after 3 days. At 50 units/ml Y-IFN, the cells showed maximal class II antigen expression for HLA-DR, whereas 100 units/ml I-IFN was required for maximal HLA-DQ expression. Higher concentrations of Y-IFN induced a further HLA-DP expression. However, serological HLA typing was performed after cell stimulation with 250 units/ml Y-IFN for 3 days. A concentration of 500 units/ml Y-IFN yielded equivalent results, but 750 units/ml Y-IFN led to poor identification of class I MHC specificities. Unclear serological results on class I specificities were clarified by 1D-isoelectric focusing (1D-IEF), thus allowing full HLA typing of all donor bulbi examined. In summary, successful HLA typing of cultivated RPE is dependent on both the Y-IFN concentration and the time lapse between stimulation and typing of the RPE. The method provides a much improved basis for cadaver cornea grafting.
Nölle
B
B
Klinik für Ophthalmologie, Universitäts-Augenklinik, Kiel 1, Bundesrepublik Deutschland.
Halene
M
M
Zavazava
N
N
Westphal
E
E
Duncker
G
G
Müller-Ruchholtz
W
W
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002478:Cells, Cultured; D016039:Corneal Transplantation / Q000473:pathology*; D005260:Female; D015395:Histocompatibility Antigens Class I / Q000032:analysis*; D000949:Histocompatibility Antigens Class II / Q000032:analysis*; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000473:pathology*; D011180:Postmortem Changes
D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II
Postmortale serologische und biochemische HLA-Typisierung mit kultivierten retinalen Pigmentepithelzellen (RPE).