An immunoperoxidase procedure is described for staining intracellular leucocyte antigens in peripheral blood and bone marrow smears. Brief exposure of cell smears to a buffered formol acetone mixture was found to give optimal fixation, combining good cellular morphology with preservation of antigenic reactivity. The immunoperoxidase method is superior to immunofluorescence in that it provides a permanent preparation which can be counterstained with orthodox reagents and viewed by conventional light microscopy. In addition the technique is considerably more sensitive than immunofluorescence procedures. Immunoglobulin was demonstrated in plasma cells, Türk cells and a minority of peripheral blood lymphocytes. Lysozyme was found in cells of the neutrophil series from promyelocytes to mature granulocytes. Monocytes stained for lysozyme but the reaction was less intense than in neutrophils and some monocytes were devoid of activity. Lactoferrin stained strongly in mature neutrophil polymorphs and metamyelocytes, but was weak or absent in earlier myeloid cells. These reaction patterns are in keeping with previous reports on the distribution of these antigens in human leucocytes. In the case of immunoglobulin and lysozyme it was possible to abolish leucocyte staining by incubation of the specific antisera with the appropriate purified antigen, providing additional proof of the specificity of the reactions. Anti-ferritin antisera stained granulocytes and myeloid precursors strongly, and reached weakly with a minority of monocytes. These latter observations are not entirely in accordance with published data on the leucocyte distribution of ferritin and may be attributed to antibody activity of unknown specificity in the anti-ferritin antiserum.

Summary. We evaluated 27 factors for their influence on acute graft‐versus‐host disease (GVHD) in 40 recipients of HLA‐identical sibling marrow transplants. These factors included the doses of mononuclear cell subpopulations present in the donor marrow inoculum quantitated using a panel of monoclonal antibodies. Female donors were associated with increased severity of acute GVHD, and the older the female donor the greater this effect. Increasing donor parity was also associated with an increased risk of acute GVHD. The number of T cells, T cells subsets, natural killer cells and monocytes infused did not influence the incidence or severity of acute GVHD in this study, and we could not explain the influence of female donors and of female donor age on acute GVHD by the cellular content of their marrow inocula. We postulate that non‐HLA histocompatibility antigen disparity is a more important determinant for acute GVHD than the number of infused donor T cells, especially when female donors are used. The association between acute GVHD and increasing parity suggests that some female marrow donors have been pre‐sensitized to their respective recipients by preceding pregnancies.

This study suggests that together with molecular typing the HTLp frequency should be considered when selecting the most suitable sibling donor for bone marrow transplantation.

No abstract

SUMMARY It has recently been reported that HLA‐DP antigens may play an important role in the development of graft‐versus‐host disease (G VHD) following transplantation of haploidentical bone marrow as a treatment for haematological malignancies. Mixed lymphocyte culture (MLC) is routinely performed prior to bone marrow transplantation to assess the suitability of the donor, and we have therefore examined the role of HLA‐DP in this test. One‐way MLC chequerboard experiments were performed between 17 HLA‐Dw3 homozygous typing cells (HTC) with a range of HLA‐DP antigens represented, including HLA‐DPw1, w2, w3, w4 and CP63. The experiments were performed on multiple occasions and each time a highly significant difference (P= <0.001) was observed between the Relative Responses (RR) in the HLA‐DP matched responder/stimulator pairs and the HLA‐DP mismatched pairs. There was, however, considerable overlap in these results with ranges in the HLA‐DP‐matched group RRs of 0–17%, and 0–62% in the mismatched group. Only 3.1% of the HLA‐DP‐matched group had a RR>5%, while 48% of the HLA‐DP mismatched group had a RR>5%. From these results it was calculated that a positive response (> 5%) has a 96% chance of being due to an HLA‐DP disparity of one or two antigens. Conversely, with a negative MLC the chance of their being no HLA‐DP antigen disparity was only 65%. From these results we conclude, firstly, that HLA‐DP has a significant influence on the MLC. Secondly, it is clear that a negative MLC does not necessarily indicate identity at the HLA‐DP locus. An increased incidence of bone marrow transplants mismatched at HLA‐DP would be expected from increased use of phenotypically identical, but unrelated, donors and related donors sharing only one haplotype with the recipient. It would thus seem important to consider the HLA‐DP type of all patients and potential donors prior to bone marrow transplantation, since a negative MLC is not sufficient to eliminate HLA‐DP disparity.