T-cell depletion of bone marrow transplants: assessment of standard immunological methods of quantification.

The efficacy of bone marrow transplant (BMT) T-cell depletion for the prevention of acute graft-versus-host disease (GVHD) has been demonstrated in animal models and in clinical studies. The importance of T-cell depletion has to be evaluated with standardized methods suitable for routine purposes. We report herein an in vitro mature T-cell depletion using a cocktail of three monoclonal antibodies (CD2, CD5, and CD7) and baby rabbit complement in 38 histocompatibility leucocyte antigen (HLA)-identical BMT with no more than grade II acute GVHD. The T-cell depletion was quantified using three prestandardized immunological methods: immunofluorescence (IF) analysis, SRBC-rosetting assay, and PHA proliferation assay. A mean of 97.5% IF-assessed T-cell depletion was achieved in the 38 BMT. The immediate IF analysis using three distinct sets of anti-T-cell monoclonal antibodies allowed us to detect a mean of 1.2% residual T cells. The SRBC-rosetting assay was not useful to quantify T-cell depletion because no residual SRBC-rosette-forming cells could be detected in every case. The results of a prestandardized PHA-induced proliferation assay gave a mean 96.7% inhibition of proliferation, and they were correlated with the IF results although the IF threshold of detection was higher. From these data we conclude that our in vitro T-cell-depletion procedure is reproducible and that standardized simple immunological methods such as immediate immunofluorescence analysis and PHA proliferation assay provide good tools to assess a T-cell depletion effective in the prevention of acute GVHD.

Detection of malignant B cells in peripheral blood stem cell collections after chemotherapy in patients with multiple myeloma.

Autologous transplantation after high-dose chemo or radiotherapy is now frequently used for the treatment of patients with multiple myeloma (MM). The collection of peripheral blood stem cells (PBSC) has a theoretical advantage over autologous bone marrow collection as the malignant plasmacytic contamination is believed to be lower. However, the extent of B cell contamination in PBSC has not been extensively investigated. Using an immunoglobulin heavy chain gene 'fingerprinting' technique at diagnosis and during apheresis after one cycle of chemotherapy we detected a monoclonal population in 44% of PBSC samples (9 positives in 22 studied). There was no correlation between contamination and sex, age, Durie and Salmon classification, C-reactive protein and albumin. A significant correlation was observed with beta 2 microglobulin serum level (P = 0.02). Twenty one patients were grafted and up to the present, with a mean follow-up of 12 months, 6 patients have relapsed including 4 patients with contaminating B cells. Our results suggest that PBSC contamination, defines a 'poor risk' group of patients, with poor prognosis. However, we could not exclude reinitiation of the disease by plasmacyte stem cells after grafting.

Contamination of peripheral blood by monoclonal B cells following treatment of multiple myeloma by high-dose chemotherapy.

Peripheral blood samples obtained from five patients with multiple myeloma, after high-dose chemotherapy, were studied for monoclonal B plasma cell contamination. We used the technique of immunoglobulin heavy chain gene 'fingerprinting' at the time of diagnosis and during apheresis. The level of sensitivity of this technique is between 0.01% and 0.001% in two patients in whom a monoclonal population was detected in peripheral blood.

In vitro depletion of clonogenic cells in adult acute lymphoblastic leukemia with a CD10 (anti-cALLA) monoclonal antibody.

A semi-solid medium colony assay was used in common acute lymphoblastic leukemia (cALL) to test growth inhibition of leukemic progenitors (CFU-L) after exposure to monoclonal antibodies (MoAbs) directed against CD10 and CD9 antigens. Peripheral or bone marrow cells from 15 patients were plated after exposure to various concentrations of ALB2, a CD10 cytotoxic MoAb, followed by complement lysis. CFU-L inhibition was complete (no residual colony) in 5 cases (33%), marked (greater than or equal to 95%) in 4 cases (27%), but only moderate (64% +/- 28) in 6 cases (40%). This inhibition was not related to the percentage of cALLA positive cells before exposure to MoAb. In addition, cells of 5 patients were exposed to BA1 (CD24) + complement. In these cases, the proportion of CFU-L inhibition was equal to or higher than with ALB2. In 3 cases, cells were exposed to an association of ALB2 and SB4 (CD19) MoAbs followed by complement lysis, with a marked inhibition (greater than or equal to 99%) in 2/3 cases. These observations give supplementary support to the use of several MoAbs directed against various antigens present at early stages of B differentiation.

[Value of plasma exchange in the management of acute immunoallergic thrombocytopenic purpura (author's transl)]. / Intérêt des échanges plasmatiques dans le traitement des purpuras thrombopéniques immuno-allergiques aigus.

Plasma exchanges were performed in 5 patients with acute immunoallergic thrombocytopenic purpura (ITP) because of severe haemorrhages and/or inefficient or badly tolerated corticosteroid therapy. Plasma exchanges were carried out with cell separators and were usually well tolerated. They resulted in very rapid increase in platelet levels with disappearance of auto-antibodies and dramatic reduction of haemorrhages, and they brought about, or accelerated, recovery in 3 patients. One patient was slightly improved. In the 5th patient, who had meningeal haemorrhage, plasma exchange was effective in increasing platelet levels but was unable to prevent a fatal outcome. The main indications for plasma exchange seems to be acute ITP with severe, life-threatening haemorrhages.