[When A is not A anymore: problems and pitfalls with blood group typing]. / Wenn A nicht mehr A ist: Schwierigkeiten und Fallstricke bei der Blutgruppenbestimmung.

Correct blood group typing is a prerequisite for transfusion. In most cases blood group determination is without problems; however, in individual cases various factors can complicate blood group determination and sometimes lead to confusing findings. For a better understanding the clinician should have basic knowledge of blood typing. Blood group determination usually covers the AB0 blood groups, Rhesus and Kell systems; in addition, a direct Coombs test and an antibody screening test for the detection of irregular antibodies in the recipient are performed. Confusion of patients, blood samples, results or preparations can lead to severe consequences due to incompatible transfusion and must be prevented. In this context, bedside blood type testing before transfusion is of utmost importance. Problems in laboratory analysis as well as patient-related factors, such as the existence of irregular antibodies against red blood cells can complicate the immunohematology diagnostics. Certain medications, such as daratumumab, lead to a significantly increased complexity in laboratory analyses. Massive transfusions can lead to chimerism with more than one population of circulating red blood cells. Hematopoetic stem cell transplantation can also lead to a change in blood groups as well as chimerism. In addition, there are various other rare causes that can result in difficulties in blood group determination, such as rare blood groups or rare disease-associated phenomena. In the case of problems in blood group determination, early and close cooperation with transfusion medicine is essential for the clinician.

CD20-Specific Immunoligands Engaging NKG2D Enhance γδ T Cell-Mediated Lysis of Lymphoma Cells.

Human γδ T cells are innate-like T cells which are able to kill a broad range of tumour cells and thus may have potential for cancer immunotherapy. The activating receptor natural killer group 2 member D (NKG2D) plays a key role in regulating immune responses driven by γδ T cells. Here, we explored whether recombinant immunoligands consisting of a CD20 single-chain fragment variable (scFv) linked to a NKG2D ligand, either MHC class I chain-related protein A (MICA) or UL16 binding protein 2 (ULBP2), could be employed to engage γδ T cells for tumour cell killing. The two immunoligands, designated MICA:7D8 and ULBP2:7D8, respectively, enhanced cytotoxicity of ex vivo-expanded γδ T cells against CD20-positive lymphoma cells. Both Vδ1 and Vδ2 γδ T cells were triggered by MICA:7D8 or ULBP2:7D8. Killing of CD20-negative tumour cells was not induced by the immunoligands, indicating their antigen specificity. MICA:7D8 and ULBP2:7D8 acted in a dose-dependent manner and induced cytotoxicity at nanomolar concentrations. Importantly, chronic lymphocytic leukaemia (CLL) cells isolated from patients were sensitized by the two immunoligands for γδ T cell cytotoxicity. In a combination approach, the immunoligands were combined with bromohydrin pyrophosphate (BrHPP), an agonist for Vδ2 γδ T cells, which further enhanced the efficacy in target cell killing. Thus, employing tumour-directed recombinant immunoligands which engage NKG2D may represent an attractive strategy to enhance antitumour cytotoxicity of γδ T cells.

Direct volumetric flow cytometric quantitation of CD34+ stem and progenitor cells.

OBJECTIVES: In this study, we compared a classic single-platform (SP) method applying beads for enumeration of CD45+ or CD34+ cells with a new device allowing direct volumetric measurements of stem and progenitor cells. BACKGROUND: Following apheresis and cyropreservation, the precise enumeration of CD34+ cells as key parameter of graft quality is mandatory for the clinical course after transplantation. Currently, flow cytometry with SP technique represents the 'gold standard' for such determinations. METHODS/MATERIALS: Fresh samples, 14 from mobilised peripheral blood (PB), 9 from apheresis products (AP) and 13 samples from frozen-thawed (FT) haematopoietic progenitor cell grafts, were analysed for CD34+ cells, CD45+ cells, and in frozen-thawed samples for viability by a bead-based flow cytometric method and in parallel by a direct, volumetric flow cytometric method. RESULTS: Comparison of CD34+ analyses revealed a significant correlation (P < 0·01) for each material between both techniques with r = 0·95 (PB), r = 0·933 (AP) and r = 0·929 (FT). Also, for analysis of CD45+ cells µL(-1) , the measured numbers evaluated with the different techniques did not significantly differ for all three materials analysed. In frozen-thawed samples, the analysis of viability was comparable for both techniques. CONCLUSIONS: The results of this study demonstrate that a direct volumetric analysis of CD34+ cells µL(-1) or CD45+ cells µL(-1) is feasible. This technique represents a simple and economical approach for standardisation of progenitor and stem cell analyses.

Evidence for gene recombination in FCGR3 gene variants.

BACKGROUND AND OBJECTIVES: The genes encoding the Fcgamma receptors (FcgammaR) IIIa and IIIb (FCGR3A and FCGR3B) are clustered on chromosome 1 band q23-24 and exhibit allelic polymorphism. We investigated the molecular basis of additional new FCGR3 genomic variation. MATERIALS AND METHODS: A segment shared by FCGR3A and FCGR3B containing the polymorphic nucleotide positions 141, 147, 227, 266, and 277 in exon 3 was cloned and sequenced from genomic DNA of 30 donors and 3 bacterial artificial chromosome (BAC) clones. A mixture consisting of isolated FCGR3B*2- and FCGR3A- plasmids was cloned and sequenced as well. Additionally, nucleotide databases were screened for clones with variant FCGR3 sequences. RESULTS: A total of 12 FCGR3 variants defined by the polymorphic positions were detected in whole blood genomic DNA from 23 of 24 FCGR3B*2 and/or FCGR3B*3 positive donors, the DNA from two of three BAC clones and in the DNA mixture of isolated FCGR3B*2- and FCGR3A- plasmids. CONCLUSION: Nucleotide exchanges of the variants were non-random and resulted from two alternative nucleotides present in one of the polymorphic position of the basic FCGR3 forms. Polymerase chain reaction (PCR) artefacts cannot be excluded as origin of new variants, but there is strong evidence that at least two variants are the result of a somatic recombination.

Re-transplantation from the same unrelated donor in three adolescents with severe aplastic anemia after graft rejection.

Hematopoietic stem cell transplantation (HSCT) from a matched unrelated donor (MUD) has become the accepted salvage treatment for patients with severe aplastic anemia (SAA) lacking a matched sibling donor and failing immunosuppressive treatment. However, non-engraftment and early rejection remain main reasons for treatment related morbidity and mortality. We report on three adolescents who were grafted from MUD, rejected their graft and were re-grafted 47-51 days after first HSCT from the same donor. For conditioning, fludarabine, cyclophosphamide, ATG and/or OKT3 in combination with total lymphoid irradiation was used. Unmanipulated peripheral blood stem cells at a minimum dose of 8 x 10(6)/kg CD34+cells were infused. Acute toxicity was low. Two patients are alive and well for more than 3 years, one patient developed extended chronic graft-versus-host disease from which he died 34 months after second HSCT. Re-transplantation from MUD in the case of non-engraftment or rejection from the same donor is possible following immunoablation combined with intensive serotherapy in young patients with SAA.

Proliferation-based T-cell selection for immunotherapy and graft-versus-host-disease prophylaxis in the context of bone marrow transplantation.

Graft-versus-host disease (GvHD) caused by alloreactive T cells within the graft is a major drawback of allogeneic BMT, but depletion of T cells leads to higher rates of relapse, opportunistic infections and graft failure. Therefore, selective removal of GvHD-inducing alloreactive T cells might be beneficial. We describe here the separation of alloresponsive T cells, based on carboxyfluorescein succimidyl ester labeling, in vitro allostimulation and FACS-sorting. In vivo effects of the separated cell populations were investigated in the context of allogeneic BMT in murine models: in vitro resting T cells were shown to survive in the allogeneic host and retain immunoreactivity against 'third-party' antigens. As demonstrated in two different transplantation models, elimination of proliferating cells significantly reduces GvHD but offers no advantages to using T-cell-depleted bone marrow alone concerning engraftment and tumor control. Transplanting T cells that proliferate in response to tumor antigens in vitro may narrow down the spectrum of antigens recognized by T cells and therefore reduce GvHD while maintaining graft-facilitating function and tumor control. Therefore, selecting tumor-reactive T cells on the basis of their proliferative response in vitro may be beneficial for the recipient, less time consuming than T-cell cloning and still reduce the extent of GvHD.

Successful autologous stem cell transplantation in a severely immunocompromised patient with relapsed AIDS-related B-cell lymphoma.

There is now evidence that the tolerability and response to systemic chemotherapy in HIV-infected patients with AIDS-related lymphoma (ARL) is significantly improved by highly active antiretroviral therapy. Here we report an severely immunocompromised AIDS patient with recurrent ARL who was successfully treated with autologous stem cell transplantation (ASCT). We also review the current literature of ASCT in HIV-infected patients.

Hepatitis C virus transmission through quarantine fresh-frozen plasma.

In 1994, quarantine fresh-frozen plasma (Q-FFP) was introduced in Germany in order to reduce the risk of HIV and HCV transmission. In 1998, an acute HCV infection of a patient was reported to us. The look-back revealed that this patient had received two Q-FFP from a donor who had seroconverted for HCV in the meantime. Recipients of further plasma donations from this donor were identified. Back-up specimens of these donations were investigated in several laboratories. A total of 25 additional HCV-PCR positive plasma units had been transfused to 12 further patients. HCV infections were diagnosed in seven of these recipients, three patients had already been deceased. One of the remaining two recipients was already HCV positive prior to transfusion, in the other patient, no HCV infection was detectable. This patient had received three units of an "early" plasma donation , which was tested negative by PCR in one laboratory, but positive in the other. The subsequent, clinically infectious donation had the same discrepant PCR results. Thus, eight cases of HCV transmission were revealed and classified as "certain" with regard to causality, also due to an identical HCV genotype, i.e. 3e. Some of these infections would have been prevented by application of a different anti-HCV assay. The assay used in the respective plasmapheresis station was in-sensitive in this individual case for more than 400 days after the first PCR positive donation. This caused the release of the above mentioned infectious units. Upon re-testing the backups, three of four other anti-HCV assays revealed a positive result already 104 days after the first PCR-positive donation. The donor had increased ALAT levels (> 23 IU/L) at nine of 28 donations, two of these were higher than 2.5 times the upper normal limit, and two were higher than 68 IU/L, which is the cut-off value for male blood donors in Germany. The results of these (look-back) studies arouse several queries, i.e. differences in the diagnostic sensitivity between current anti-HCV and PCR tests, the accuracy of risk-estimates (especially when based on hemovigilance studies for Q-FFP), the value of ALAT testing, and currently practised release algorithms for Q-FFP.

Early intensive and myeloablative adjuvant chemotherapy in women with high-risk breast cancer.

BACKGROUND: Women with breast cancer and > 10 positive lymph nodes have an unfavorable prognosis. The optimal combination and intensity of adjuvant chemotherapy is uncertain. Between July 1994 and December 1996 we treated 19 patients with early intensive followed by high-dose chemotherapy and autologous peripheral blood stem cell transplantation. PATIENTS AND METHODS: Patients were initially diagnosed with breast cancer and multiple positive lymph nodes. Induction chemotherapy consisted of two courses VP16, ifosphamide, cisplatin and epirubicin (VIPE) and one course of mitoxantrone, cyclophosphamide and thiotepa (MCT). Peripheral blood stem cells were mobilized after the first or second course of VIPE and retransfused two days after high dose chemotherapy. RESULTS: Stem cells were successfully collected in all patients. Major toxicities (WHO grade III and IV) were neutropenia, thrombocytopenia, alopecia, nausea, infections and mucositis. Hematopoietic recovery occurred in all patients with a median of 10 days for leukocytes and 13 days for platelets. No patient died of therapy-induced complications. The median observation time is 24 months. Two patients have relapsed, one with locoregional disease. The projected rate of patients with disease-free survival after three years is 88%. CONCLUSIONS: Early intensive and myeloablative chemotherapy followed by peripheral blood stem cell transplantation is a highly efficient and feasible protocol for high risk patients with breast cancer.

The novel tribody [(CD20)(2)xCD16] efficiently triggers effector cell-mediated lysis of malignant B cells.

Bispecific antibodies (bsab) offer a promising approach for optimizing antibody-based therapies. In the present study, [(CD20)(2)xCD16], a recombinant CD20- and CD16-directed bsab in the tribody format, was designed to optimize recruitment of FcγRIII (CD16)-positive effector cells. [(CD20)(2)xCD16] retained the antigen specificities of the parental monoclonal antibodies and binding to FcγRIIIa was not compromised by the F/V polymorphism at amino-acid position 158. [(CD20)(2)xCD16] mediated potent lysis of lymphoma cell lines and freshly isolated tumor cells from patients, even at low picomolar concentrations (∼10 pM). Irrespective of the CD16a allotype, potency as well as efficacy of lysis obtained with the tribody was significantly higher than lysis triggered by rituximab. Tumor cell killing also occurred when autologous NK cells were used as effector cells. Compared with rituximab, the tribody demonstrated depletion of autologous B cells in ex vivo whole blood assays at 100-fold lower antibody concentration. In mice with a reconstituted humanized hematopoietic system, established by transplantation of human CD34-positive cord blood cells, this novel tribody significantly depleted autologous human B cells. Thus, tribodies such as [(CD20)(2)xCD16], recruiting CD16-positive effector cells, may represent promising candidates for clinical development.

Quantitative MRD monitoring identifies distinct GVL response patterns after allogeneic stem cell transplantation for chronic lymphocytic leukemia: results from the GCLLSG CLL3X trial.

The purpose of this study was to prospectively analyze minimal residual disease(MRD) kinetics after reduced-intensity allogeneic stem cell transplantation (allo-SCT) in high-risk chronic lymphocytic leukemia (CLL). Subjects were the first 30 consecutive patients from a prospective clinical trial, and seven pilot patients treated identically. Using real-time quantitative-PCR (RQ-PCR) and/or flow-based MRD monitoring (sensitivity >or=10(-4)), five distinct patterns of MRD kinetics could be identified: patients who promptly achieved durable MRD negativity without direct evidence of graft-versus-leukemia (GVL) effects (Group 1) (n=4; no clinical relapse); patients with complete and sustained MRD response after GVL induced by immunosuppression tapering (Group 2) or donor lymphocyte infusions (Group 3) (n=18; one relapse); patients without MRD response due to lack of GVL (Group 4) (n=2; two relapses); patients with incomplete and transient MRD response to GVL (Group 5) (n=4; three relapses). In summary, this study provides a comprehensive map of possible MRD courses and their prognostic implications after T-replete allo-SCT in high-risk CLL, indicating that effective GVL activity is induced virtually in all patients who develop chronic GVHD. However, in a significant proportion of cases, this does not translate into sustained disease control due to development of secondary GVL resistance.

Myelodysplastic syndrome of donor origin subsequent to successful treatment of myeloid/NK-cell precursor leukaemia with allogeneic PBSCT: two very rare conditions in one patient.

We report a 36-year-old male with myeloid/natural killer (NK)-cell precursor acute leukaemia with a complex aberrant karyotype, who was treated according to an acute-myeloid-leukaemia (AML) treatment protocol (idarubicine, cytarabine, and etoposide) followed by high-dose cytarabine consolidation and achieved complete remission. He underwent allogeneic matched unrelated donor (MUD) peripheral blood stem-cell transplantation (PBSCT) and remained in remission throughout his remaining life. Seven months posttransplantation, a myelodysplastic syndrome (MDS) with (20q-) of donor origin was diagnosed causing severe thrombocytopenia and finally leading to infection and death. This patient represents one of the few cases published achieving remission for a significant period of time after being diagnosed with myeloid/NK-cell precursor acute leukaemia, a very rare malignant disease. We conclude, despite the fatal outcome due to infection, that allogeneic PBSCT is a therapeutic option for patients with this entity. In addition, the development of a myelodysplastic syndrome of donor origin is extremely rare and only very few cases are published worldwide.

A prospective, randomized, sequential crossover trial of large-volume versus normal-volume leukapheresis procedures: effects on serum electrolytes, platelet counts, and other coagulation measures.

BACKGROUND: LVL procedures with the administration of heparin as an additional anticoagulant are increasingly performed because of the potentially higher yield of autologous peripheral blood HPCs. A prospective, randomized crossover trial was performed to evaluate the influence of leukapheresis volume-that is, large versus normal-on serum electrolytes, platelet count, and other coagulation measures in 25 patients with breast cancer and 14 patients with non-Hodgkin's lymphoma. STUDY DESIGN AND METHODS: Patients were randomly assigned to start either with an LVL on Day 1 followed by a normal-volume leukapheresis (NVL) on Day 2 or vice versa. In LVL, heparin was administered in addition to ACD-A. Bleeding complications, transfusion support, whole-blood counts, and several coagulation measures as well as plasma heparin levels were evaluated. RESULTS: Although the duration, the infused amount of ACD-A, the flow rate, the drop in platelet count, and the drop in potassium were significantly greater in LVL, and although LVL patients also received heparin, there was no significant difference in clinical tolerance or bleeding complications. After LVL, patients exhibited a significantly longer activated partial thromboplastin time (APTT), with a median of 70 seconds (range, 44-100 sec), and a median anti-factor Xa activity of 0.69 IU per mL (range, 0.10-1.29 IU/mL). The value of the APTT after LVL correlated with anti-factor Xa activity (r = 0.37, p<0.05), but not with platelet count or heparin infusion rate. Markers for coagulation activation did not increase during NVL or LVL. CONCLUSION: LVL with heparin as an additional anticoagulant seems to be a safe procedure in patients with low preleukapheresis platelet counts. No activation of coagulation occurred after NVL or LVL procedures.