- Email: [email protected]
Symposium S29 Transfusion transmitted infections
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Communications
S28-002
S28-004
TRANSFUSION MANAGEMENT OF NEONATES WITH T ACTIVATION
SEVERE HEMOLYTIC DISEASE OF THE NEWBORN WITH ANTIKEL2, ANTI-YT1, ANTI-FY1 AND ANTI-JK2 ANTIBODIES
BORALESSA H.*, MODI N.**, I ROBERTS.**, COCKBURN H*, LETSKY E.***, MALDE R.*, EDWARDS M.** *NBS LONDON - SOUTH EAST, LONDON ** IMPERIAL COLLEGE OF SCIENCE, TECHNOLOGY - MEDICINE, HAMMERSMITH HOSPITAL, LONDON ***QUEEN CHARLOTTES HOSPITAL, NEONATAL UNIT, LONDON [email protected] Background : Reports of severe haemolysis following the transfusion of blood products to infants with ‘T’ activated red cells have resulted in the delay or withholding of transfusion. Aims : We conducted a prospective cohort study aiming to establish the prevalence of T activation in a neonatal intensive care population and the incidence of T activation associated with haemolysis following transfusion of blood products. Methods : During a 12 month period, blood samples from 379 infants admitted to a tertiary referral Neonatal Unit were screened for T activation using a peanut lectin, arachis hypogaea. ‘T’ was distinguished from ‘Tk’ activation on the basis of a reaction with the lectin glycine soya. Evidence of haemolysis was sought on the basis of pre and post transfusion haemoglobins, bilirubin and red cell morphology. Results : Forty-eight neonates had a positive T activation screen and further testing showed ‘T’ activation in only one baby with the other 47 having ‘Tk’ activation. Twenty-two of the 48 received transfusions of blood products, but none showed evidence of post transfusion haemolysis. Three hundred random donor samples were screened for anti-’Tk’ and anti-’T’. No anti’Tk’ was detected in any sample, but anti-’T’ was present in all samples. Conclusion : A positive T activation screen in sick neonates is not unusual and the majority have ‘Tk’ rather than ‘T’ activated cells. All samples positive on a T activation screen should be tested against a lectin panel to distinguish ‘T’ from ‘Tk’ activation. It is safe to transfuse standard blood products to neonates with ‘Tk’ activated red cells.
S28-003 ANTI-D PROPHYLAXIS IN THE EAST ANGLIA REGION, UK.
LE PENNEC P.Y., NOIZAT-PIRENNE F., *LARSEN M., **MONSAINGEON-LION A., ***AUDIBERT F., ROUGER P., ANSARTPIRENNE H.
CNRGS AND INTS, PARIS, FRANCE *CHP, PARIS, FRANCE **EFS, ILE DE FRANCE, FRANCE ***AP-HP ANTOINE BÉCLÈRE [email protected] The frequency of KEL :-2 and YT :-1 phenotypes is about 2/1000. AntiKEL2 has been held responsible for hemolytic transfusion reaction (HTR) and hemolytic disease of the newborn (HDN). One anti-YT1 has been associated with moderate HTR but not with HDN. The mother (Caucasian, O, RH1(D) positive) received 5 blood units 5 years ago (delivery hemorrhage). Blood samples were sent to the laboratory for antibody identification. Anti-KEL2 and anti-FY1 were identified. A program of blood donations in the rare blood bank was recommended. A new blood sample was sent 4 months later at the beginning of a pregnancy (10 weeks). A new antibody (anti-JK2) was detected. Because of the antiKEL2 titer (256), a strict follow up (echography) of the pregnancy was set. At 20 weeks, a 4th antibody (anti-YT1) was identified, later associated with an autoantibody. Five intrauterine transfusions were needed at 22, 23, 25, 28 and 32 weeks of gestation. Because of the fetus blood group (O, RH1(D) negative), frozen blood units (phenotype : RH :-1,-2,-3, KEL :-2, FY :-1, JK :-2, YT :1) were transfused. On the first transfusion, the Hb level was 2,5 g/100ml ; the direct antiglobulin test (DAT) was positive (IgG + C). The compatibility tests were performed after absorptions of the anti-YT1 and the autoantibody. At birth (34 weeks), there was a discrete jaundice ; only phototherapy was needed. The DAT was positive (IgG + C) with a double-population. The 4 alloantibodies were present in the eluate. A last transfusion was performed 21 days after delivery because of post-natal anemia. We report a successful delivery of a baby with severe hemolytic disease of the newborn from a mother lacking 2 high incidence antigens, with the corresponding antibodies, two other alloantibodies and an autoantibody. Intrauterine and post-natal transfusions were needed.
BURGESS G.J., SIMPSON E., FLEETWOOD P., BORALESSA H.
DEPARTMENT OF IMMUNOHAEMATOLOGY, NBS, EAST ANGLIA CENTRE, CAMBRIDGE, UK. [email protected] The National Blood Service provides an antenatal screening service for the East Anglia region of the United Kingdom. Currently, ‘routine antenatal prophylaxis’ is not offered to Rh D negative women, but is administered for potentially sensitising events. To optimise management of these cases, we collected data relating to the serology, frequency and dosage of RhD immunoprophylaxis within the region over a 12-month period. Of 22,000 pregnancies, 3152 (14.3 %) were found to be RhD negative. Of these 3152 cases, 224 (7.6 %) were confirmed to have received prophylaxis and had anti-D in their serum as determined by the routine DiaMed™ IAT. All samples were referred for quantitative antibody assessment using the Autoanalyser. In all cases, anti-D did not persist beyond 12 weeks from the date of last injection. Despite no instances of seroconvertion, 9 (4 %) cases were assessed to have anti-D levels in excess of 0.2 iu/ml (0.04mg/ml). However, we would recommend that this group continue to receive prophylaxis and would further recommend that ; Known prophylaxis or prophylaxis uncertain within previous 12 weeks and anti-D levels below 0.2 iu/ml, monitor anti-D level 4 weekly up to 12 weeks post injection and give prophylactic anti-D where indicated. Known prophylaxis or prophylaxis uncertain within previous 12 weeks and anti-D level is 0.2 iu/ml or greater. Monitor anti-D level 4 weekly up to 12 weeks post injection and decide whether antibody is immune, passive or non-specific. Continue to give prophylactic anti-D when indicated until this decision is made. Where there is definitely no history of prophylaxis then treat any presence of anti-D as immune.
Transfusion Transmitted Infections Chair: Philippe De Micco, FRA – Dirk De Korte, NLD S1246782001001719/MIS S29-001 DIVERSION OF FIRST VOLUME REDUCES THE DEGREE OF BACTERIAL CONTAMINATION FOR WHOLE BLOOD COLLECTIONS DE KORTE D., MARCELIS J., SOETERBROEK A.M.
CLB AND BLOODBANK DE MEIERIJ, SANQUIN BLOOD SUPPLY FOUNDATION, AMSTERDAM D—de—[email protected] Objective. After determination of the prevalence of bacterial contamination in whole blood collections, we investigated the effect on this prevalence of removal of the first 10-ml of the collected blood unit. Method. Whole blood was collected under bloodbank conditions in special designed 5-bag systems (NPBI), equipped with a Composampler. The Composampler was used to fill a Vacutainer tube with the first 10-ml of the donation, followed by collection of a normal blood unit. The additional 5th bag allowed sampling in a closed system for BacT Alert (Organon Teknika)
Communications anaerobic and aerobic culture flasks. Whole blood samples were taken after overnight storage at 20°C and subsequent mixing. Culture flasks were incubated in BacT Alert cabinets for 7 days, positive samples were ented on agar plates for confirmation and determination. Results. The prevalence in whole blood collections was 0.39 % with a 95 % confidence interval of 0.28 - 0.55 % (9000 units were tested). For the units collected after removal of the first 10-ml, the prevalence was significantly lower (p < 0.05), i.e. 0.21 % (3700 units tested, with 8 positive samples). The majority of bacteria were Propionibacterium species. Conclusions. Removal of the first 10-ml of blood, containing the skin-plug eventually contaminated with bacteria, was shown to reduce the prevalence of bacteral contamination of collected whole blood units.
S29-002 MATHEMATICAL MODELING OF INFECTION RISK BY BLOOD COMPONENTS CONTAINING UNDETECTABLE LEVELS OF HBV, HCV OR HIV. WEUSTEN J.J.A.M., VAN DRIMMELEN A.A.J. AND LELIE P.N.
ORGANON TEKNIKA, BOXTEL, THE NETHERLANDS, SANQUIN-CLB, AMSTERDAM, THE NETHERLANDS H—van—[email protected] Background : Blood transfusion centres around the world are introducing more sensitive screening methods to reduce the risk of virus transmission by blood donations drawn in the infectious window period before seroconversion. Methods : A mathematical model is developed to measure risk reduction by introduction of NAT screening options. The model builds on time dependency of two probabilities in the window phase : 1) probability of detection of increasing HBV, HCV and HIV load, which doubles in 2.8, 0.74 and 0.90 days according to Busch et al. and 2) probability of infectivity of transfused amounts of virus (minimum infectious dose in chimpansees is 1-20 geq for HBV/ HCV and 1000-10.000 geq for HIV). Results : In seroconversion panels the HBsAg, HCV-Ag and HIV-Ag tests become positive at concentrations of around 2000 geq/ml of HBV-DNA, 100.000 geq/ml of HCV-RNA and 10.000 geq/ml of HIV-RNA. With the latter two antigen assays the infectious window period can be shortened 6and 2-fold respectively. The probability of detecting VQC standard dilutions (Pelicheck, CLB) in for example the TMA assay showed 50 % detection limits of 8 geq/ml for HCV-RNA and 4 geq/ml for HIV-RNA. Using the mathematical model we calculated 24-fold and more than 100-fold reduction of infection risk by single donation testing with HCV TMA and HIV-TMA respectively. These risk reduction factores were 13 and 20 fold if HCV and HIV TMA is used on pools of 16 donations. Assuming similar sensitivities for HBV-TMA the risk reduction factor is 2-fold with minipool testing and 3-fold when single donations are screened. Conclusion : A proper mathematical model for calculating residual infection risk helps understanding the impact of new blood safety procedures.
S29-003 CHARACTERISATION OF HCV INFECTION IN GHANAIAN BLOOD DONORS CANDOTTI D., SARKODIE F.*, ACHEAMPONG J.W.*, ALLAIN J-P.**
NATIONAL BLOOD SERVICE, UK, *KATH BLOOD BANK, DPT MEDICINE, KOMFO ANOKYE TEACHING HOSPITAL, GHANA **DIV. TRANSFUSION MEDICINE, UNIVERSITY OF CAMBRIDGE, UK [email protected] Background : The prevalence of hepatitis C virus (HCV) in West Africa ranges from 0.5 % in Niger to 6.6 % in Gabon and 2.8 % in Ghana. HCV infection induces humoral and cellular immune responses which fail to eradicate the virus in a high proportion of infected hosts, thus leading to chronic infection. In order to evaluate the HCV seroprevalence and the distribution between chronic and recovered infections among Ghanaian blood donors, 4,984 blood donations from Kumasi were screened for antiHCV.
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Methods : Serums were screened for anti-HCV using EIA from Murex. Repeatably reactive samples were tested with an alternative assay by SANOFI and two confirmatory assays : Chiron RIBA HCV 3.0 and Murex developmental HCV confirmatory assay. HCV RNA was detected by Transcriptionmediated amplification (TMA). Positive samples were confirmed by RT-PCR and genotyped using sequences from E1/E2 and NS5 regions. Results : 151 donations (3 %) were found positive with two screening assays. 30 of 142 samples tested contained HCV RNA (21 %). All RNA positive samples were confirmed anti-HCV positive with both confirmatory assays. Among 44 RNA negative samples, 9 were confirmed, 10 were indeterminate and one was negative with both assays. 12 samples, indeterminate by RIBA-3, were confirmed by Murex. 52 % HCV RNA negative samples were confirmed by one or more assays, strongly suggesting that at least 40 % anti-HCV positive donors have recovered infection. Preliminary phylogenetic analyses of E1/E2 sequences showed that the most common genotype was a non a, b, c subtype of genotype 2. Conclusions : HCV infection is frequent in Ghanaian blood donors and anti-HCV should be screened. However, a large proportion of donors with HCV markers had recovered from the infection. The West Africa-specific genotype 2 subtype needs further characterisation.
S29-004 HIGH TITER LEUKOCYTE INACTIVATION IN INTERCEPT RED BLOOD CELLS (RBC)
HANSON D., PROPST M., DUPUIS K.*, SCHOTT M.A. AND STASSINOPOULOS A.* JOHNSON K.M
CERUS CORPORATION, CONCORD CA USA BAXTER HEALTHCARE CORPORATION ROUND LAKE IL USA [email protected] INTERCEPT RBC utilize a nucleic acid targeted compound (S-303) for inactivating pathogens (Helinx™ technology). S-303 inactivates a broad spectrum of viruses and bacteria while preserving RBC function in vitro and recovery in vivo. In addition, the S – 303 treatment is predicted to inactivate leukocytes present in RBC and may curtail other leukocyte associated transfusion reactions. Leukocyte inactivation is important for the prevention of transfusion associated graft vs host disease (TA GVHD). Currently gamma irradiation is used to achieve leukocyte inactivation in high risk patient groups. We report data on leukocyte inactivation in INTERCEPT RBC. RBC (HCT \\ 126\\60 %), prepared from whole blood, were divided in two identical parts. The Control was left untreated. The Test was treated with S-303, and incubated at room temperature (20 h). After Ficoll separation, T-cells from Control and Test were tested for proliferation through a limiting dilution assay (LDA). Serial T-cell dilutions were cultured in the presence of allo-stimulator cells, and monitored for clonal expansion and DNA synthesis through the uptake of 3H-thymidine. After 21 days of culture, the Control T-cells underwent clonal expansion at a frequency of more than 1 :100. The Test T-cells, even when cultured in titers as high as 1× 106 (24 wells), showed no signs of growth. Allo-stimulator cells alone showed no indication of clonal expansion. These results demonstrate = 5 log leukocyte inactivation in INTERCEPT RBC. S – 303 treatment should eliminate the need for gamma irradiation of RBC to prevent TA GVHD, as well as the 14 day limitation on storage associated with the use of RBC gamma irradiation.
S29-005 SEROLOGICAL COURSE OF HUMAN PARVOVIRUS B19 INFECTION IN BLOOD DONORS HITZLER W.E., RUNKEL S.
TRANSFUSION CENTRE OF JOHANNES GUTENBERG UNIVERSITY, MAINZ, GERMANY [email protected] Backgrounds : The relevance of human Parvovirus B19 for blood and blood products is discussed intensively. Although Parvovirus B19 infection goes along with severe complications in some cases, donor screening is not yet
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mandatory. Persistence of B19 DNA and serological status in blood donors was to be monitored retrospectively and prospectively. Method : Prevalence of Parvovirus B19 was investigated by hemagglutination assay. 28,972 donations from 15,660 donors were tested Results were confirmed by PCR. HPV B19 DNA, antibody and antigen were determined from sequential serum samples of infected donors. Findings : 4 donations/donors detected by hemagglutination assay were positive for B19 DNA. Thus we have a frequency of 1 :7,243 B19-positive donations and 1 :3,915 positive donors. Specificity was determined to be 99.1 %. Follow up showed a long persistence of viremia in low concentrations. The serological course of the HPV B19-infected donors was monitored. In 3 donors high titers (> 1012 Geq/mL) led to a positive reaction in the hemagglutination assay. No antibody was detected in these cases. Follow-up samples confirmed acute B19 infection by appearance of antibody against parvovirus. In all three cases low concentrations of DNA (< 104 Geq/mL) were still detectable after at least 1 year. In one case we still detected DNA 18 months after the positive antigen result. Conclusion : Human Parvovirus B19 can be detected in blood donations with high viremia by hemagglutination assay. Viremia can persist for more than one year in a healthy blood donor in low concentrations.
S29-006 CONTINUOUS-FLOW UVC IRRADIATION INACTIVATES HUMAN PARVOVIRUS B19, AS SHOWN BY INHIBITION OF VIRUS EXPRESSION AND AMPLIFICATION IN TOLERANT CELLS
CAILLET-FAUQUET P.*, DRAPS M.L.*, DI GIAMBATTISTA M.**, BRANCKAERT T.H. **, LAUB R.**
*UNIVERSITÉ LIBRE, BRUSSELS, BELGIUM **RED CROSS, BRUSSELS, BELGIUM [email protected] Background : Parvovirus B19 (B19) is a small, non-enveloped virus. B19 infection can be asymtomatic but can also cause a variety of mild to severe clinical manifestations. It can be present in plasma pools to be used in fractionation, despite efforts to select only healthy donors. Furthermore, B19 is resistant to most physical and chemical treatments applied at dosages preserving the activity of blood constituents. Its transmission through administration of solvent/detergent- or dry-heat-treated blood products has been documented. Objective : To validate continuous-flow UVC irradiation as a means of inactivating B19-like viruses, using (1) a murine model ; (2) the human B19 model. Results : The murine model chosen was the Minute Virus of Mice MVMp. Continuous-flow UVC irradiation at low dosage drastically reduced infectivity (by approximately 7 log10). To confirm these promising results, we infected the chronic myelogenous leukaemia cell line KU812F, described as permissive to B19, with plasmas shown by PCR to contain B19 DNA. The plasmas were subjected or not to continuous-flow UVC irradiation, and then incubated for different times with KU812F cells. Measurement of virus amplification and virus expression confirmed that UVC irradiation totally inhibits parvovirus B19 infectivity at dosages low enough to preserve the activity of blood products. Conclusion : This inactivation method can considerably enhance the safety of biological products potentially contaminated by parvoviruses.
S29-007 INACTIVATION OF BABESIA MICROTI IN HUMAN RBC BY HELINX (TM) TECHNOLOGY
KJEMTRUP A.M., CONRAD P.A. *, LOUI C., PACKHAM A.E. STASSINOPOULOS A.*Y DEPARTMENT OF PATHOLOGY, MICROBIOLOGY AND IMMUNOLOGY, UNIVERSITY OF CALIFORNIA, DAVIS CA, USA CERUS CORPORATION Y, CONCORD CA, USA [email protected]
No approved method for pathogen inactivation in packed red blood cells (RBC) exists. An RNA/DNA targeted method (Helinx™) for inactivating pathogens in INTERCEPT RBC was developed using anchor linker effector compounds (ALE). S-303, a frangible ALE, inactivates a broad spectrum of viruses and bacteria while preserving RBC function in vitro and recovery in vivo. Parasites are a problem in blood transfusion, since they are not tested for, and infected donors may be asymptomatic. B. microti is an obligate intracellular parasite with documented transmission through blood transfusion. There is no treatment for babesiosis, which in some patients may be fatal. We report the inactivation of the parasite B. microti by S – 138, an ALE, in a hamster infectivity model. B. microti (MN-1 strain) infected blood was collected from Syrian hamsters (parasitemia1˜ -5 % ; 4.2× 108 parasites/mL). It was diluted 1 :100, 1 :104 and 1 :106 – fold in human leukoreduced RBC (HCT 60 %). Half of the human RBC preparations and the undiluted hamster blood were treated with S – 138. Half were left untreated. After RT incubation, 200 µL of each group was inoculated i.p. in 5 hamsters per group. Four treated and four control groups were generated with 7.9, 5.9, 3.9 and 1.9 log10 parasites/mL. The first three control groups became parasitemic within 6 weeks. The last group never developed parasitemia. No treated groups became parasitemic after 6 weeks of infection demonstrating a = 6 log inactivation. Helinx™ technology is efficacious in the inactivation of B. microti in human RBC in an infectivity model. The same treatment may be effective in the inactivation of other intracellular parasites such as plasmodia.
S29-008 HEPATITIS A TRANSMISSION BY PLATELETS TO BONE MARROW TRANSPLANT PATIENT MOORE C., REGAN F., TRELEAVEN J.*, HEWITT P.
NATIONAL BLOOD SERVICE, NORTH LONDON CENTRE, LONDON, UK *ROYAL MARSDEN HOSPITAL, SURREY, UK [email protected] A 26 year old male repeat donor reported feeling unwell with jaundice 6 days after donating blood. His AST was grossly abnormal and the provisional diagnosis of acute hepatitis A (HAV) infection was confirmed by a positive anti-HAV IgM result on a sample 10 days after donation. Platelets from his donation had been transfused to a 32 year old female who had undergone an unrelated bone marrow transplant (BMT) for acute lymphoblastic leukaemia two weeks previously. She had no IgG anti-HAV antibodies detectable prior to the BMT, so following notification of the illness in the donor, the patient was given intravenous immunoglobulin (IVIg) for passive immunisation against HAV. Ten weeks after the platelet transfusion and the administration of IVIg, the recipient developed both IgM and IgG anti-HAV antibodies, indicating recent HAV infection. Her liver function tests became abnormal 12 weeks after the transfusion and she became mildly unwell ; drugs and mild graftversus-host-disease may also have contributed to the illness. Transfusion transmitted HAV is rare : 4 previous cases have been reported in the UK in the last 25 years. The viraemic phase before becoming unwell is short ; therefore blood is rarely donated in the viraemic phase. Although the majority of recipients may either be immune or suffer a self-limiting illness, immunosuppressed patients are most at risk of severe morbidity. In our case, treatment with IVIg prolonged the incubation time and almost certainly attenuated the course of the HAV infection This case illustrates the importance of encouraging donors to report illness after donation, as this information may be relevant for the clinical management of the recipient(s).
Communications
Immunology of blood transfusion and transplantation Chair: Wolfgang Mayr, AUT – Dominique Charron, FRA S1246782001001720/MIS
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ments was at 1.0 %, depending on the individual STR loci analysed. The combined discrimination power of 0.99 with a corresponding probability of match (pM) of > 1.0 x 109 makes this set of markers also highly suitable for family-related transplantation, where genetic differences are limited. The analysis can be performed overnight, which facilitates therapeutic decisions.
S30-001
S30-003
CORD BLOOD CONTAINS BOTH MYELOID AND LYMPHOID DENDRITIC CELLS : IMPLICATIONS FOR TRANSPLANTATION
INCREASED INCIDENCE OF ANTI-HLA IMMUNIZATION AFTER G-CSF MOBILIZED PERIPHERAL BOOD STEM TRANSPLANTATION
BORRÀS F.E.*, GOMEZ J.*, RAJANANTHANAN P.*, AND NAVARRETE C.V.* +
*DEPT. OF HISTOCOMPATABILITY - IMMUNOGENETICS,NATIONAL BLOOD SERVICE, NORTH LONDON CENTRE, LONDON + DEPT. OF IMMUNOLOGY, ROYAL FREE - UNIVERSITY COLLEGE MEDICAL SCHOOL, LONDON [email protected] Background : Dendritic cells (DCs) are the most potent antigen presenting cells described so far. In human peripheral blood, both myeloid and lymphoid subsets of DCs have been identified. In contrast, cord blood (CB) DCs have been recently described as being exclusively of the immature CD11c- lymphoid DC subset. Methods : An alternative method of enrichment, based on a negative selection system with specific antibodies, was set up. The cells were then passed trough a column where labelled cells were retained. Phenotypical and functional characteristics of the cells contained in the effluent (enriched cells) were analysed by flow cytometry and MLC assays. Results : Both lymphoid (HLA-DR+CD123++ + CD11c- CD33-) and myeloid (HLA-DR++ CD123+CD11c+CD33+) DCs were identified in CB. The majority of CB DCs showed a lymphoid phenotype. However, a significant number of CD11c+ myeloid DCs (25.6 % ± 14.5 %, n = 13) were also present. Other markers, such as CD80 and CD83 were negative in both subsets. Analyses of the allostimulatory capacity showed that freshly isolated CB lymphoid DCs failed to induce a potent allostimulation of naive CB T cells. Conclusion :These features are therefore consistent with previous work reporting an immature phenotype for lymphoid DCs in adult blood. The significance of the high numbers of lymphoid DCs and the reduced GvHD observed following CB transplantation should be considered.
S30-002 DETECTION OF MIXED HAEMATOPOIETIC CHIMERISM AFTER ALLOGENEIC STEM CELL TRANSPLANTATION
ARNOLD C.*, BASARA N.**, KIEHL M.**, BECKER S.*, TONN T.*, FAUSER A.**, SEIFRIED E.* AND SEIDL C.*
*INSTITUTE FOR TRANSFUSION MEDICINE AND IMMUNOHAEMATOLOGY, RCBDS HESSEN, FRANKFURT ; AND ** CLINIC FOR BONE MARROW TRANSPLANTATION, HAEMATOLOGY AND ONCOLOGY, IDAR-OBERSTEIN, GERMANY [email protected] Molecular detection of the status of hematopoetic chimerism between donor and recipient cells after stem cell transplantation has been used in recent years to facilitate monitoring of engraftment or disease relapse. In particular, high-frequent sequential analysis of chimerism can be used to define the clinical progress of disease and is suitable to subsidize therapeutic interventions, e.g. donor lymphocyte infusion. Sensitivity and the time frame for analysis are however important criteria for the clinical suitability of chimerism analysis. We have established a quantitative and highly sensitive method to determine stem cell engraftment using 10 different short tandem repeat (STR) loci (TH01, CYP19, D8S639, D11S488, D19S246, D21S11, ACPP, VWA, FGA, and ACTBP2). Individual STR profiles of donor and recipient are determined before stem cell transplantation. Semi-quantitative analysis is achieved by PCR-amplification of STR-Loci using fluorescent labelled primers and subsequent measurement of fluorescence intensity on polyacrylamid gelelectrophoresis (373A) or capillary electrophoresis (Prism 310) (both Perkin Elmer, Applied Biosystems). The sensitivity in mixing experi-
LAPIERRE V., TAYEBI H., AUPERIN A., CHABOD J., TRAMALLONI D., OUBOUZAR N., BLAISE D., KUENTZ M., ROBINET E., TIBERGHIEN P POUR LA SOCIÉTÉ FRANÇAISE DE GREFFE DE MOELLE (SFGM).
INSTITUT GUSTAVE ROUSSY, VILLEJUIF, FRANCE ETABLISSEMENT FRANÇAIS DU SANG BOURGOGNE-FRANCHE-COMTÉ, BESANÇON, FRANCE INSTITUT PAOLI CALMETTES, MARSEILLE, FRANCE HÔPITAL HENRI MONDOR, CRÉTEIL, FRANCE [email protected] Background : We have recently shown that the use of an allogeneic G-CSFmobilized peripheral blood hematopoietic stem cell transplantation (PBHSCT) compared with bone marrow transplantation (BMT) is associated with an increased titers of antibodies (Ab) directed against red blood cell ABO antigens (Ag). To further evaluate the influence of a G-CSF mobilized PBHSC graft on anti-allo-Ab responses, we have examined the incidence of anti-HLA Ab immunization after transplantation, in the setting of a randomized study comparing allogeneic BMT to allogeneic PBHSCT in adults. Methods : The presence of anti-HLA Ab were determined by microlymphocytotoxicity (LCT) and by flow-cytometry. Anti-HLA Ab detection were performed in the donor as well as the recipient before and 30 days after transplantation. Findings : The use of an allogeneic PBHSCT, when the donor and the recipient were anti-HLA Ab negative before transplant, was associated with an increased incidence of anti-HLA immunization as determined by LCT 30 days after the transplantation. This increase concerned both IgG (PBHSCT : 4/23 vs BMT : 0/26, p = 0.04) as well as IgM isotype Ab (8/24 vs 0/26, p = 0.001). Flow-cytometric anti-HLA detection further confirmed these results. IgG anti-HLA Ab after PBHSCT were directed against both class I (7/20 vs 0/20 after BMT, p = 0.008) and/or class II HLA Ag (1/19 vs 0/22, p = 0.46). Importantly, the PBHSCT-associated increase in anti-HLA immunization was observed despite a reduction in the median number of platelet transfusion episodes/patient in PBHSCT versus BMT recipients (3[ 1-21] vs 6 [3-33], p = 0.02). Moreover, in PBHSCT recipients, IgG Class I and IgM HLA immunization detected at day 30 was significantly associated with an higher frequency of number of CD3 + CD25 + in the graft. Conclusion : Our study strongly suggests that G-CSF-mobilized PBHSCT results in an increased incidence of anti-HLA-immunization and further confirms that the use of such a graft alters allo-immune Ab responses.
S30-004 RED BLOOD CELL TRANSFUSION ARE ASSOCIATED WITH ALTERATIONS IN SELF-REACTIVE ANTIBODY REPERTOIRES OF PLASMA IGM AND IGC, INDEPENDENT OF THE PRESENCE OF A SPECIFIC IMMUNE RESPONSE TOWARD RBC ANTIGENS
STAHL D., LACROIX-DESMAZES S., SIBROWSKI W., KAZATCHKINE M.D., KAVERI S.V. INSERM U430 AND UNIVERSITÉ PIERRE ET MARIE CURIE, HÔPITAL BROUSSAIS, PARIS, FRANCE WESTFÄLISCHE WILHELMS-UNIVERSITÄT MÜNSTER, INSTITUTE FOR TRANSFUSION MEDICINE, MÜNSTER, GERMANY [email protected] Background : Immunization to allogeneic RBC antigens occurs in transfused patients, and may be associated with the development of RBCdestructive antibodies directed against autologous RBC. The present study
Communications
S28-002
S28-004
TRANSFUSION MANAGEMENT OF NEONATES WITH T ACTIVATION
SEVERE HEMOLYTIC DISEASE OF THE NEWBORN WITH ANTIKEL2, ANTI-YT1, ANTI-FY1 AND ANTI-JK2 ANTIBODIES
BORALESSA H.*, MODI N.**, I ROBERTS.**, COCKBURN H*, LETSKY E.***, MALDE R.*, EDWARDS M.** *NBS LONDON - SOUTH EAST, LONDON ** IMPERIAL COLLEGE OF SCIENCE, TECHNOLOGY - MEDICINE, HAMMERSMITH HOSPITAL, LONDON ***QUEEN CHARLOTTES HOSPITAL, NEONATAL UNIT, LONDON [email protected] Background : Reports of severe haemolysis following the transfusion of blood products to infants with ‘T’ activated red cells have resulted in the delay or withholding of transfusion. Aims : We conducted a prospective cohort study aiming to establish the prevalence of T activation in a neonatal intensive care population and the incidence of T activation associated with haemolysis following transfusion of blood products. Methods : During a 12 month period, blood samples from 379 infants admitted to a tertiary referral Neonatal Unit were screened for T activation using a peanut lectin, arachis hypogaea. ‘T’ was distinguished from ‘Tk’ activation on the basis of a reaction with the lectin glycine soya. Evidence of haemolysis was sought on the basis of pre and post transfusion haemoglobins, bilirubin and red cell morphology. Results : Forty-eight neonates had a positive T activation screen and further testing showed ‘T’ activation in only one baby with the other 47 having ‘Tk’ activation. Twenty-two of the 48 received transfusions of blood products, but none showed evidence of post transfusion haemolysis. Three hundred random donor samples were screened for anti-’Tk’ and anti-’T’. No anti’Tk’ was detected in any sample, but anti-’T’ was present in all samples. Conclusion : A positive T activation screen in sick neonates is not unusual and the majority have ‘Tk’ rather than ‘T’ activated cells. All samples positive on a T activation screen should be tested against a lectin panel to distinguish ‘T’ from ‘Tk’ activation. It is safe to transfuse standard blood products to neonates with ‘Tk’ activated red cells.
S28-003 ANTI-D PROPHYLAXIS IN THE EAST ANGLIA REGION, UK.
LE PENNEC P.Y., NOIZAT-PIRENNE F., *LARSEN M., **MONSAINGEON-LION A., ***AUDIBERT F., ROUGER P., ANSARTPIRENNE H.
CNRGS AND INTS, PARIS, FRANCE *CHP, PARIS, FRANCE **EFS, ILE DE FRANCE, FRANCE ***AP-HP ANTOINE BÉCLÈRE [email protected] The frequency of KEL :-2 and YT :-1 phenotypes is about 2/1000. AntiKEL2 has been held responsible for hemolytic transfusion reaction (HTR) and hemolytic disease of the newborn (HDN). One anti-YT1 has been associated with moderate HTR but not with HDN. The mother (Caucasian, O, RH1(D) positive) received 5 blood units 5 years ago (delivery hemorrhage). Blood samples were sent to the laboratory for antibody identification. Anti-KEL2 and anti-FY1 were identified. A program of blood donations in the rare blood bank was recommended. A new blood sample was sent 4 months later at the beginning of a pregnancy (10 weeks). A new antibody (anti-JK2) was detected. Because of the antiKEL2 titer (256), a strict follow up (echography) of the pregnancy was set. At 20 weeks, a 4th antibody (anti-YT1) was identified, later associated with an autoantibody. Five intrauterine transfusions were needed at 22, 23, 25, 28 and 32 weeks of gestation. Because of the fetus blood group (O, RH1(D) negative), frozen blood units (phenotype : RH :-1,-2,-3, KEL :-2, FY :-1, JK :-2, YT :1) were transfused. On the first transfusion, the Hb level was 2,5 g/100ml ; the direct antiglobulin test (DAT) was positive (IgG + C). The compatibility tests were performed after absorptions of the anti-YT1 and the autoantibody. At birth (34 weeks), there was a discrete jaundice ; only phototherapy was needed. The DAT was positive (IgG + C) with a double-population. The 4 alloantibodies were present in the eluate. A last transfusion was performed 21 days after delivery because of post-natal anemia. We report a successful delivery of a baby with severe hemolytic disease of the newborn from a mother lacking 2 high incidence antigens, with the corresponding antibodies, two other alloantibodies and an autoantibody. Intrauterine and post-natal transfusions were needed.
BURGESS G.J., SIMPSON E., FLEETWOOD P., BORALESSA H.
DEPARTMENT OF IMMUNOHAEMATOLOGY, NBS, EAST ANGLIA CENTRE, CAMBRIDGE, UK. [email protected] The National Blood Service provides an antenatal screening service for the East Anglia region of the United Kingdom. Currently, ‘routine antenatal prophylaxis’ is not offered to Rh D negative women, but is administered for potentially sensitising events. To optimise management of these cases, we collected data relating to the serology, frequency and dosage of RhD immunoprophylaxis within the region over a 12-month period. Of 22,000 pregnancies, 3152 (14.3 %) were found to be RhD negative. Of these 3152 cases, 224 (7.6 %) were confirmed to have received prophylaxis and had anti-D in their serum as determined by the routine DiaMed™ IAT. All samples were referred for quantitative antibody assessment using the Autoanalyser. In all cases, anti-D did not persist beyond 12 weeks from the date of last injection. Despite no instances of seroconvertion, 9 (4 %) cases were assessed to have anti-D levels in excess of 0.2 iu/ml (0.04mg/ml). However, we would recommend that this group continue to receive prophylaxis and would further recommend that ; Known prophylaxis or prophylaxis uncertain within previous 12 weeks and anti-D levels below 0.2 iu/ml, monitor anti-D level 4 weekly up to 12 weeks post injection and give prophylactic anti-D where indicated. Known prophylaxis or prophylaxis uncertain within previous 12 weeks and anti-D level is 0.2 iu/ml or greater. Monitor anti-D level 4 weekly up to 12 weeks post injection and decide whether antibody is immune, passive or non-specific. Continue to give prophylactic anti-D when indicated until this decision is made. Where there is definitely no history of prophylaxis then treat any presence of anti-D as immune.
Transfusion Transmitted Infections Chair: Philippe De Micco, FRA – Dirk De Korte, NLD S1246782001001719/MIS S29-001 DIVERSION OF FIRST VOLUME REDUCES THE DEGREE OF BACTERIAL CONTAMINATION FOR WHOLE BLOOD COLLECTIONS DE KORTE D., MARCELIS J., SOETERBROEK A.M.
CLB AND BLOODBANK DE MEIERIJ, SANQUIN BLOOD SUPPLY FOUNDATION, AMSTERDAM D—de—[email protected] Objective. After determination of the prevalence of bacterial contamination in whole blood collections, we investigated the effect on this prevalence of removal of the first 10-ml of the collected blood unit. Method. Whole blood was collected under bloodbank conditions in special designed 5-bag systems (NPBI), equipped with a Composampler. The Composampler was used to fill a Vacutainer tube with the first 10-ml of the donation, followed by collection of a normal blood unit. The additional 5th bag allowed sampling in a closed system for BacT Alert (Organon Teknika)
Communications anaerobic and aerobic culture flasks. Whole blood samples were taken after overnight storage at 20°C and subsequent mixing. Culture flasks were incubated in BacT Alert cabinets for 7 days, positive samples were ented on agar plates for confirmation and determination. Results. The prevalence in whole blood collections was 0.39 % with a 95 % confidence interval of 0.28 - 0.55 % (9000 units were tested). For the units collected after removal of the first 10-ml, the prevalence was significantly lower (p < 0.05), i.e. 0.21 % (3700 units tested, with 8 positive samples). The majority of bacteria were Propionibacterium species. Conclusions. Removal of the first 10-ml of blood, containing the skin-plug eventually contaminated with bacteria, was shown to reduce the prevalence of bacteral contamination of collected whole blood units.
S29-002 MATHEMATICAL MODELING OF INFECTION RISK BY BLOOD COMPONENTS CONTAINING UNDETECTABLE LEVELS OF HBV, HCV OR HIV. WEUSTEN J.J.A.M., VAN DRIMMELEN A.A.J. AND LELIE P.N.
ORGANON TEKNIKA, BOXTEL, THE NETHERLANDS, SANQUIN-CLB, AMSTERDAM, THE NETHERLANDS H—van—[email protected] Background : Blood transfusion centres around the world are introducing more sensitive screening methods to reduce the risk of virus transmission by blood donations drawn in the infectious window period before seroconversion. Methods : A mathematical model is developed to measure risk reduction by introduction of NAT screening options. The model builds on time dependency of two probabilities in the window phase : 1) probability of detection of increasing HBV, HCV and HIV load, which doubles in 2.8, 0.74 and 0.90 days according to Busch et al. and 2) probability of infectivity of transfused amounts of virus (minimum infectious dose in chimpansees is 1-20 geq for HBV/ HCV and 1000-10.000 geq for HIV). Results : In seroconversion panels the HBsAg, HCV-Ag and HIV-Ag tests become positive at concentrations of around 2000 geq/ml of HBV-DNA, 100.000 geq/ml of HCV-RNA and 10.000 geq/ml of HIV-RNA. With the latter two antigen assays the infectious window period can be shortened 6and 2-fold respectively. The probability of detecting VQC standard dilutions (Pelicheck, CLB) in for example the TMA assay showed 50 % detection limits of 8 geq/ml for HCV-RNA and 4 geq/ml for HIV-RNA. Using the mathematical model we calculated 24-fold and more than 100-fold reduction of infection risk by single donation testing with HCV TMA and HIV-TMA respectively. These risk reduction factores were 13 and 20 fold if HCV and HIV TMA is used on pools of 16 donations. Assuming similar sensitivities for HBV-TMA the risk reduction factor is 2-fold with minipool testing and 3-fold when single donations are screened. Conclusion : A proper mathematical model for calculating residual infection risk helps understanding the impact of new blood safety procedures.
S29-003 CHARACTERISATION OF HCV INFECTION IN GHANAIAN BLOOD DONORS CANDOTTI D., SARKODIE F.*, ACHEAMPONG J.W.*, ALLAIN J-P.**
NATIONAL BLOOD SERVICE, UK, *KATH BLOOD BANK, DPT MEDICINE, KOMFO ANOKYE TEACHING HOSPITAL, GHANA **DIV. TRANSFUSION MEDICINE, UNIVERSITY OF CAMBRIDGE, UK [email protected] Background : The prevalence of hepatitis C virus (HCV) in West Africa ranges from 0.5 % in Niger to 6.6 % in Gabon and 2.8 % in Ghana. HCV infection induces humoral and cellular immune responses which fail to eradicate the virus in a high proportion of infected hosts, thus leading to chronic infection. In order to evaluate the HCV seroprevalence and the distribution between chronic and recovered infections among Ghanaian blood donors, 4,984 blood donations from Kumasi were screened for antiHCV.
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Methods : Serums were screened for anti-HCV using EIA from Murex. Repeatably reactive samples were tested with an alternative assay by SANOFI and two confirmatory assays : Chiron RIBA HCV 3.0 and Murex developmental HCV confirmatory assay. HCV RNA was detected by Transcriptionmediated amplification (TMA). Positive samples were confirmed by RT-PCR and genotyped using sequences from E1/E2 and NS5 regions. Results : 151 donations (3 %) were found positive with two screening assays. 30 of 142 samples tested contained HCV RNA (21 %). All RNA positive samples were confirmed anti-HCV positive with both confirmatory assays. Among 44 RNA negative samples, 9 were confirmed, 10 were indeterminate and one was negative with both assays. 12 samples, indeterminate by RIBA-3, were confirmed by Murex. 52 % HCV RNA negative samples were confirmed by one or more assays, strongly suggesting that at least 40 % anti-HCV positive donors have recovered infection. Preliminary phylogenetic analyses of E1/E2 sequences showed that the most common genotype was a non a, b, c subtype of genotype 2. Conclusions : HCV infection is frequent in Ghanaian blood donors and anti-HCV should be screened. However, a large proportion of donors with HCV markers had recovered from the infection. The West Africa-specific genotype 2 subtype needs further characterisation.
S29-004 HIGH TITER LEUKOCYTE INACTIVATION IN INTERCEPT RED BLOOD CELLS (RBC)
HANSON D., PROPST M., DUPUIS K.*, SCHOTT M.A. AND STASSINOPOULOS A.* JOHNSON K.M
CERUS CORPORATION, CONCORD CA USA BAXTER HEALTHCARE CORPORATION ROUND LAKE IL USA [email protected] INTERCEPT RBC utilize a nucleic acid targeted compound (S-303) for inactivating pathogens (Helinx™ technology). S-303 inactivates a broad spectrum of viruses and bacteria while preserving RBC function in vitro and recovery in vivo. In addition, the S – 303 treatment is predicted to inactivate leukocytes present in RBC and may curtail other leukocyte associated transfusion reactions. Leukocyte inactivation is important for the prevention of transfusion associated graft vs host disease (TA GVHD). Currently gamma irradiation is used to achieve leukocyte inactivation in high risk patient groups. We report data on leukocyte inactivation in INTERCEPT RBC. RBC (HCT \\ 126\\60 %), prepared from whole blood, were divided in two identical parts. The Control was left untreated. The Test was treated with S-303, and incubated at room temperature (20 h). After Ficoll separation, T-cells from Control and Test were tested for proliferation through a limiting dilution assay (LDA). Serial T-cell dilutions were cultured in the presence of allo-stimulator cells, and monitored for clonal expansion and DNA synthesis through the uptake of 3H-thymidine. After 21 days of culture, the Control T-cells underwent clonal expansion at a frequency of more than 1 :100. The Test T-cells, even when cultured in titers as high as 1× 106 (24 wells), showed no signs of growth. Allo-stimulator cells alone showed no indication of clonal expansion. These results demonstrate = 5 log leukocyte inactivation in INTERCEPT RBC. S – 303 treatment should eliminate the need for gamma irradiation of RBC to prevent TA GVHD, as well as the 14 day limitation on storage associated with the use of RBC gamma irradiation.
S29-005 SEROLOGICAL COURSE OF HUMAN PARVOVIRUS B19 INFECTION IN BLOOD DONORS HITZLER W.E., RUNKEL S.
TRANSFUSION CENTRE OF JOHANNES GUTENBERG UNIVERSITY, MAINZ, GERMANY [email protected] Backgrounds : The relevance of human Parvovirus B19 for blood and blood products is discussed intensively. Although Parvovirus B19 infection goes along with severe complications in some cases, donor screening is not yet
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mandatory. Persistence of B19 DNA and serological status in blood donors was to be monitored retrospectively and prospectively. Method : Prevalence of Parvovirus B19 was investigated by hemagglutination assay. 28,972 donations from 15,660 donors were tested Results were confirmed by PCR. HPV B19 DNA, antibody and antigen were determined from sequential serum samples of infected donors. Findings : 4 donations/donors detected by hemagglutination assay were positive for B19 DNA. Thus we have a frequency of 1 :7,243 B19-positive donations and 1 :3,915 positive donors. Specificity was determined to be 99.1 %. Follow up showed a long persistence of viremia in low concentrations. The serological course of the HPV B19-infected donors was monitored. In 3 donors high titers (> 1012 Geq/mL) led to a positive reaction in the hemagglutination assay. No antibody was detected in these cases. Follow-up samples confirmed acute B19 infection by appearance of antibody against parvovirus. In all three cases low concentrations of DNA (< 104 Geq/mL) were still detectable after at least 1 year. In one case we still detected DNA 18 months after the positive antigen result. Conclusion : Human Parvovirus B19 can be detected in blood donations with high viremia by hemagglutination assay. Viremia can persist for more than one year in a healthy blood donor in low concentrations.
S29-006 CONTINUOUS-FLOW UVC IRRADIATION INACTIVATES HUMAN PARVOVIRUS B19, AS SHOWN BY INHIBITION OF VIRUS EXPRESSION AND AMPLIFICATION IN TOLERANT CELLS
CAILLET-FAUQUET P.*, DRAPS M.L.*, DI GIAMBATTISTA M.**, BRANCKAERT T.H. **, LAUB R.**
*UNIVERSITÉ LIBRE, BRUSSELS, BELGIUM **RED CROSS, BRUSSELS, BELGIUM [email protected] Background : Parvovirus B19 (B19) is a small, non-enveloped virus. B19 infection can be asymtomatic but can also cause a variety of mild to severe clinical manifestations. It can be present in plasma pools to be used in fractionation, despite efforts to select only healthy donors. Furthermore, B19 is resistant to most physical and chemical treatments applied at dosages preserving the activity of blood constituents. Its transmission through administration of solvent/detergent- or dry-heat-treated blood products has been documented. Objective : To validate continuous-flow UVC irradiation as a means of inactivating B19-like viruses, using (1) a murine model ; (2) the human B19 model. Results : The murine model chosen was the Minute Virus of Mice MVMp. Continuous-flow UVC irradiation at low dosage drastically reduced infectivity (by approximately 7 log10). To confirm these promising results, we infected the chronic myelogenous leukaemia cell line KU812F, described as permissive to B19, with plasmas shown by PCR to contain B19 DNA. The plasmas were subjected or not to continuous-flow UVC irradiation, and then incubated for different times with KU812F cells. Measurement of virus amplification and virus expression confirmed that UVC irradiation totally inhibits parvovirus B19 infectivity at dosages low enough to preserve the activity of blood products. Conclusion : This inactivation method can considerably enhance the safety of biological products potentially contaminated by parvoviruses.
S29-007 INACTIVATION OF BABESIA MICROTI IN HUMAN RBC BY HELINX (TM) TECHNOLOGY
KJEMTRUP A.M., CONRAD P.A. *, LOUI C., PACKHAM A.E. STASSINOPOULOS A.*Y DEPARTMENT OF PATHOLOGY, MICROBIOLOGY AND IMMUNOLOGY, UNIVERSITY OF CALIFORNIA, DAVIS CA, USA CERUS CORPORATION Y, CONCORD CA, USA [email protected]
No approved method for pathogen inactivation in packed red blood cells (RBC) exists. An RNA/DNA targeted method (Helinx™) for inactivating pathogens in INTERCEPT RBC was developed using anchor linker effector compounds (ALE). S-303, a frangible ALE, inactivates a broad spectrum of viruses and bacteria while preserving RBC function in vitro and recovery in vivo. Parasites are a problem in blood transfusion, since they are not tested for, and infected donors may be asymptomatic. B. microti is an obligate intracellular parasite with documented transmission through blood transfusion. There is no treatment for babesiosis, which in some patients may be fatal. We report the inactivation of the parasite B. microti by S – 138, an ALE, in a hamster infectivity model. B. microti (MN-1 strain) infected blood was collected from Syrian hamsters (parasitemia1˜ -5 % ; 4.2× 108 parasites/mL). It was diluted 1 :100, 1 :104 and 1 :106 – fold in human leukoreduced RBC (HCT 60 %). Half of the human RBC preparations and the undiluted hamster blood were treated with S – 138. Half were left untreated. After RT incubation, 200 µL of each group was inoculated i.p. in 5 hamsters per group. Four treated and four control groups were generated with 7.9, 5.9, 3.9 and 1.9 log10 parasites/mL. The first three control groups became parasitemic within 6 weeks. The last group never developed parasitemia. No treated groups became parasitemic after 6 weeks of infection demonstrating a = 6 log inactivation. Helinx™ technology is efficacious in the inactivation of B. microti in human RBC in an infectivity model. The same treatment may be effective in the inactivation of other intracellular parasites such as plasmodia.
S29-008 HEPATITIS A TRANSMISSION BY PLATELETS TO BONE MARROW TRANSPLANT PATIENT MOORE C., REGAN F., TRELEAVEN J.*, HEWITT P.
NATIONAL BLOOD SERVICE, NORTH LONDON CENTRE, LONDON, UK *ROYAL MARSDEN HOSPITAL, SURREY, UK [email protected] A 26 year old male repeat donor reported feeling unwell with jaundice 6 days after donating blood. His AST was grossly abnormal and the provisional diagnosis of acute hepatitis A (HAV) infection was confirmed by a positive anti-HAV IgM result on a sample 10 days after donation. Platelets from his donation had been transfused to a 32 year old female who had undergone an unrelated bone marrow transplant (BMT) for acute lymphoblastic leukaemia two weeks previously. She had no IgG anti-HAV antibodies detectable prior to the BMT, so following notification of the illness in the donor, the patient was given intravenous immunoglobulin (IVIg) for passive immunisation against HAV. Ten weeks after the platelet transfusion and the administration of IVIg, the recipient developed both IgM and IgG anti-HAV antibodies, indicating recent HAV infection. Her liver function tests became abnormal 12 weeks after the transfusion and she became mildly unwell ; drugs and mild graftversus-host-disease may also have contributed to the illness. Transfusion transmitted HAV is rare : 4 previous cases have been reported in the UK in the last 25 years. The viraemic phase before becoming unwell is short ; therefore blood is rarely donated in the viraemic phase. Although the majority of recipients may either be immune or suffer a self-limiting illness, immunosuppressed patients are most at risk of severe morbidity. In our case, treatment with IVIg prolonged the incubation time and almost certainly attenuated the course of the HAV infection This case illustrates the importance of encouraging donors to report illness after donation, as this information may be relevant for the clinical management of the recipient(s).
Communications
Immunology of blood transfusion and transplantation Chair: Wolfgang Mayr, AUT – Dominique Charron, FRA S1246782001001720/MIS
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ments was at 1.0 %, depending on the individual STR loci analysed. The combined discrimination power of 0.99 with a corresponding probability of match (pM) of > 1.0 x 109 makes this set of markers also highly suitable for family-related transplantation, where genetic differences are limited. The analysis can be performed overnight, which facilitates therapeutic decisions.
S30-001
S30-003
CORD BLOOD CONTAINS BOTH MYELOID AND LYMPHOID DENDRITIC CELLS : IMPLICATIONS FOR TRANSPLANTATION
INCREASED INCIDENCE OF ANTI-HLA IMMUNIZATION AFTER G-CSF MOBILIZED PERIPHERAL BOOD STEM TRANSPLANTATION
BORRÀS F.E.*, GOMEZ J.*, RAJANANTHANAN P.*, AND NAVARRETE C.V.* +
*DEPT. OF HISTOCOMPATABILITY - IMMUNOGENETICS,NATIONAL BLOOD SERVICE, NORTH LONDON CENTRE, LONDON + DEPT. OF IMMUNOLOGY, ROYAL FREE - UNIVERSITY COLLEGE MEDICAL SCHOOL, LONDON [email protected] Background : Dendritic cells (DCs) are the most potent antigen presenting cells described so far. In human peripheral blood, both myeloid and lymphoid subsets of DCs have been identified. In contrast, cord blood (CB) DCs have been recently described as being exclusively of the immature CD11c- lymphoid DC subset. Methods : An alternative method of enrichment, based on a negative selection system with specific antibodies, was set up. The cells were then passed trough a column where labelled cells were retained. Phenotypical and functional characteristics of the cells contained in the effluent (enriched cells) were analysed by flow cytometry and MLC assays. Results : Both lymphoid (HLA-DR+CD123++ + CD11c- CD33-) and myeloid (HLA-DR++ CD123+CD11c+CD33+) DCs were identified in CB. The majority of CB DCs showed a lymphoid phenotype. However, a significant number of CD11c+ myeloid DCs (25.6 % ± 14.5 %, n = 13) were also present. Other markers, such as CD80 and CD83 were negative in both subsets. Analyses of the allostimulatory capacity showed that freshly isolated CB lymphoid DCs failed to induce a potent allostimulation of naive CB T cells. Conclusion :These features are therefore consistent with previous work reporting an immature phenotype for lymphoid DCs in adult blood. The significance of the high numbers of lymphoid DCs and the reduced GvHD observed following CB transplantation should be considered.
S30-002 DETECTION OF MIXED HAEMATOPOIETIC CHIMERISM AFTER ALLOGENEIC STEM CELL TRANSPLANTATION
ARNOLD C.*, BASARA N.**, KIEHL M.**, BECKER S.*, TONN T.*, FAUSER A.**, SEIFRIED E.* AND SEIDL C.*
*INSTITUTE FOR TRANSFUSION MEDICINE AND IMMUNOHAEMATOLOGY, RCBDS HESSEN, FRANKFURT ; AND ** CLINIC FOR BONE MARROW TRANSPLANTATION, HAEMATOLOGY AND ONCOLOGY, IDAR-OBERSTEIN, GERMANY [email protected] Molecular detection of the status of hematopoetic chimerism between donor and recipient cells after stem cell transplantation has been used in recent years to facilitate monitoring of engraftment or disease relapse. In particular, high-frequent sequential analysis of chimerism can be used to define the clinical progress of disease and is suitable to subsidize therapeutic interventions, e.g. donor lymphocyte infusion. Sensitivity and the time frame for analysis are however important criteria for the clinical suitability of chimerism analysis. We have established a quantitative and highly sensitive method to determine stem cell engraftment using 10 different short tandem repeat (STR) loci (TH01, CYP19, D8S639, D11S488, D19S246, D21S11, ACPP, VWA, FGA, and ACTBP2). Individual STR profiles of donor and recipient are determined before stem cell transplantation. Semi-quantitative analysis is achieved by PCR-amplification of STR-Loci using fluorescent labelled primers and subsequent measurement of fluorescence intensity on polyacrylamid gelelectrophoresis (373A) or capillary electrophoresis (Prism 310) (both Perkin Elmer, Applied Biosystems). The sensitivity in mixing experi-
LAPIERRE V., TAYEBI H., AUPERIN A., CHABOD J., TRAMALLONI D., OUBOUZAR N., BLAISE D., KUENTZ M., ROBINET E., TIBERGHIEN P POUR LA SOCIÉTÉ FRANÇAISE DE GREFFE DE MOELLE (SFGM).
INSTITUT GUSTAVE ROUSSY, VILLEJUIF, FRANCE ETABLISSEMENT FRANÇAIS DU SANG BOURGOGNE-FRANCHE-COMTÉ, BESANÇON, FRANCE INSTITUT PAOLI CALMETTES, MARSEILLE, FRANCE HÔPITAL HENRI MONDOR, CRÉTEIL, FRANCE [email protected] Background : We have recently shown that the use of an allogeneic G-CSFmobilized peripheral blood hematopoietic stem cell transplantation (PBHSCT) compared with bone marrow transplantation (BMT) is associated with an increased titers of antibodies (Ab) directed against red blood cell ABO antigens (Ag). To further evaluate the influence of a G-CSF mobilized PBHSC graft on anti-allo-Ab responses, we have examined the incidence of anti-HLA Ab immunization after transplantation, in the setting of a randomized study comparing allogeneic BMT to allogeneic PBHSCT in adults. Methods : The presence of anti-HLA Ab were determined by microlymphocytotoxicity (LCT) and by flow-cytometry. Anti-HLA Ab detection were performed in the donor as well as the recipient before and 30 days after transplantation. Findings : The use of an allogeneic PBHSCT, when the donor and the recipient were anti-HLA Ab negative before transplant, was associated with an increased incidence of anti-HLA immunization as determined by LCT 30 days after the transplantation. This increase concerned both IgG (PBHSCT : 4/23 vs BMT : 0/26, p = 0.04) as well as IgM isotype Ab (8/24 vs 0/26, p = 0.001). Flow-cytometric anti-HLA detection further confirmed these results. IgG anti-HLA Ab after PBHSCT were directed against both class I (7/20 vs 0/20 after BMT, p = 0.008) and/or class II HLA Ag (1/19 vs 0/22, p = 0.46). Importantly, the PBHSCT-associated increase in anti-HLA immunization was observed despite a reduction in the median number of platelet transfusion episodes/patient in PBHSCT versus BMT recipients (3[ 1-21] vs 6 [3-33], p = 0.02). Moreover, in PBHSCT recipients, IgG Class I and IgM HLA immunization detected at day 30 was significantly associated with an higher frequency of number of CD3 + CD25 + in the graft. Conclusion : Our study strongly suggests that G-CSF-mobilized PBHSCT results in an increased incidence of anti-HLA-immunization and further confirms that the use of such a graft alters allo-immune Ab responses.
S30-004 RED BLOOD CELL TRANSFUSION ARE ASSOCIATED WITH ALTERATIONS IN SELF-REACTIVE ANTIBODY REPERTOIRES OF PLASMA IGM AND IGC, INDEPENDENT OF THE PRESENCE OF A SPECIFIC IMMUNE RESPONSE TOWARD RBC ANTIGENS
STAHL D., LACROIX-DESMAZES S., SIBROWSKI W., KAZATCHKINE M.D., KAVERI S.V. INSERM U430 AND UNIVERSITÉ PIERRE ET MARIE CURIE, HÔPITAL BROUSSAIS, PARIS, FRANCE WESTFÄLISCHE WILHELMS-UNIVERSITÄT MÜNSTER, INSTITUTE FOR TRANSFUSION MEDICINE, MÜNSTER, GERMANY [email protected] Background : Immunization to allogeneic RBC antigens occurs in transfused patients, and may be associated with the development of RBCdestructive antibodies directed against autologous RBC. The present study