Bone Marrow Transplantation, (1999) 23 , 779–781  1999 Stockton Press All rights reserved 0268–3369/99 $12.00 http:/ / www.stockton-press.co.uk/ bmt Monitoring anti-thymocyte globulin (ATG) in bone marrow recipients TH Eiermann, P Lambrecht and AR Zander Department of Transfusion Medicine and Bone Marrow Transplantation Unit, Department of Internal Medicine, University Hospital Eppendorf, Hamburg, Germany Summary: The present study was undertaken to acquire a ration- ale for clinical dose adjustment of anti-thymocyte globu- lin (ATG) to improve cost effectiveness and safety of graft-versus-host disease prophylaxis. The concen- tration of rabbit ATG in the serum of 12 patients was measured by ELISA and by the inhibitory effect on phy- tohaemagglutinin-induced blastogenesis. At 10 mg/ml ATG, 3 H-thymidine incorporation was effectively blocked. Serial two-fold dilution of ATG showed that this effect decreased in a concentration-dependent manner and was lost at 10 ng/ml ATG. One hundred microlitres serum taken at day 1 to 22 post trans- plant effected significant inhibition of the phytohaemag- glutinin-response with 49  12% c.p.m. (x  s.d.) on day 1 post transplant compared to 93  13% c.p.m. on day 1(P  0.001, unpaired one-sided t-test). The rabbit-IgG was maximal at a concentration of 907  187 l/ml at day 0. Subsequently, it decreased with time. While rabbit-IgG was detectable for a long period (eg 160 g/ml at day 22 in patient MD), the effect on the phytohaemagglutinin-response of normal mononuclear cells lasted up to 4 days post transplant. We conclude that 90 mg/kg body weight ATG-Fresenius given prior to marrow transplant leads to sustained T cell immunosuppression post transplant. Keywords: graft-versus-host disease; immunotherapy; T lymphocytes; transplantation Anti-thymocyte globulin (ATG) preparations have been used successfully in transplantation, aplastic anaemia and graft-versus-host disease (GVHD). 1–3 ATG-Fresenius is a polyclonal serum raised in rabbits against the Jurkat T cell line. 4 As indicated by previous reports, standardisation of in vitro and clinical activity of polyclonal ATG is a complex issue. 5–8 The present study was undertaken to acquire a rationale for clinical dose adjustment to improve cost effectiveness and safety of GVHD prophylaxis. Rabbit ATG con- centration was measured in the serum by ELISA and by inhibition of phytohaemagglutinin-induced blastogenesis. Correspondence: Dr TH Eiermann, University Hospital Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany Received 10 July 1998; accepted 28 October 1998 Patients and methods Clinical protocol Twelve patients (nine chronic myelogenous leukaemia, one acute myeloid leukaemia, one acute lymphoblastic leu- kaemia, one plasmacytoma) were studied, average age 41 (range 21–55 years). The average body weight was 79  16 kg (x  s.d.; range 52–106). Six patients had received marrow from a matched unrelated donor and six from an HLA-identical sibling. Conditioning therapy con- sisted of 1200 cGy total body irradiation and 60 mg of cyclophosphamide per kg of body weight per day for 2 days, or busulphan, cyclophosphamide and etoposide. ATG-Fresenius (30 mg/kg) was administered intravenously to 12 subsequent patients daily in three doses on days -3 to -1. Additional GVHD prophylaxis consisted of cyclo- sporine, methothrexate, mitromidazole and pentaglobulin, an IgM-enriched immunoglobulin preparation. 9 Mitogen assay Peripheral blood mononuclear cells (PBMC) from healthy volunteers were isolated from 20 ml heparinized human peripheral blood by centrifugation on Ficoll–Hypaque (Lymphoflot; Biotest, Dreieich, Germany) using standard methods. Quadruplicate cultures of 1 × 10 5 PBMC were plated in 96-well round-bottom microtitre plates (Greiner 650180, Frickenhausen, Germany) in 100 l RPMI 1640 supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin, Germany) and 100 U/ml penicillin/streptomycin (GibcoBRL, Life Technologies, Eggenstein, Germany). Sera of patients (100 l) treated with rabbit ATG (Fresenius, Bad Homburg, Germany) at various time points before or after bone marrow transplantation, serial dilution of ATG (range 10 mg/ml–2.4 g/ml), or media control were added. The cells were stimulated for 48 h at 37°C with 5 g/ml phytohaemagglutinin (PHA-L; Boehringer Mannheim, Mannheim, Germany). The proliferative response was measured by adding 1 Ci 3 H- thymidine/well (5 Ci/mmol; Amersham Life Science, Buckinghamshire, UK) approximately 12 h before har- vesting the cells. Thymidine incorporation (c.p.m.) was determined on a standard scintillation counter (Beckman LS 5000 TD, Fullerton, CA, USA). ELISA Rabbit ATG was determined in microtitre plates coated with anti-rabbit IgG antibody (Boehringer Mannheim