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CCR5 Δ32 homozygous cord blood allogeneic transplantation in a patient with HIV: a case report

BACKGROUND Allogeneic donor CCR5 Δ32 homozygous haemopoietic cell transplantation (HCT) provides the only evidence to date of long-term control of HIV infection. However, availability of conventional CCR5 Δ32 homozygous donors is insufficient to develop this as a therapeutic strategy further. METHODS We present a 37-year-old patient with HIV-1 infection and aggressive lymphoma who had disease progression after five lines of radiochemotherapy including an autologous HCT, and in the absence of matched sibling donors, received an allogeneic HCT with four of six HLA-matched CCR5 Δ32 homozygous cord blood cells (StemCyte, Covina, CA), supported with purified CD34+ cells from a haploidentical sibling. Blood or tissue samples were obtained before and weekly after HCT to monitor transplant and HIV infection, including chimerism analysis, CCR5 genotyping and viral tropism, viral isolation and sequence, viral reservoir analysis, immune activation and proliferation, and ex-vivo cell infectivity assays. Combined antiretroviral therapy continued during the procedure. FINDINGS The patients HIV was CCR5-tropic by genotypic and phenotypic analyses. Baseline latent reservoir tests showed HIV DNA copies in bulk and resting CD4 T cells and in gut-associated lymphoid tissue, CD4 T-cell-associated HIV RNA, replication competent viral size of 2·1 copies per 10(7) CD4 T cells, and single copy assay of 303 copies per mL. After HCT, plasma HIV DNA load was undetectable by ultrasensitive analyses. Upon cord blood full chimerism, the patients CCR5 Δ32 homozygous CD4 T cells responded to proliferation and activation stimuli and became resistant to infection by the patients viral isolate and by laboratory-adapted HIV-1 strains. Death related to lymphoma progression regretfully prevented long-term monitoring of the patients viral reservoir. INTERPRETATION CCR5 Δ32 homozygous cord blood reconstitution can successfully eliminate HIV-1 and render the allogeneic graft recipients T lymphocytes resistant to HIV infection. Thus, they build on the evidence available to strongly support the use of cord blood as a strategic platform for a broader application of non-functional CCR5 transplantation to other infected individuals. FUNDING Spanish Secretariat of Research, the American Foundation for AIDS Research (amfAR).

HNA-1a, HNA-1b and HNA-1c gene frequencies in Argentineans.

Human neutrophil antigens (HNA) are polymorphic structures located in the neutrophil membrane. The neutrophil-specific antigens HNA-1a (NA1), 1b (NA2) and 1c (SH) are well-recognized allotypic forms of FcgammaRIIIb and the most frequent targets of neutrophil alloantibodies. The aim of this study was to determine the gene frequencies of the neutrophil-specific antigens belonging to the HNA-1 system in blood donors and Toba Amerindians from Rosario, Argentina. Two hundred and eighteen unrelated healthy Argentinean blood donors and Toba Amerindians from Rosario were typed for HNA-1a, HNA-1b and HNA-1c using polymerase chain reaction with sequence-specific primers. For the Argentinean blood donors, the HNA-1a and HNA-1b gene frequencies were 0.44 and 0.56 and for the Amerindians Toba were 0.77 and 0.23, respectively. The HNA-1c antigen is present in 4.7% (gene frequency=0.023) of the blood donors but in none of the Amerindian individuals. The present data showed that the HNA-1 allele frequencies in the major population and the Toba Amerindians from Rosario are similar to those described in European and others distant Amerindians populations, respectively.

Rh-null phenotype caused by a complete RHAG deletion

R hD and RhCE proteins on the red blood cell (RBC) membrane are part of a multiprotein complex. RhAG, a member of the Rh protein family, is one of the key components of that complex. Mutations in RHAG can lead to reduced expression (Rhmod phenotype) or no expression (Rhnull phenotype) of Rh antigens on the RBC membrane. Reported RHAG mutations that cause a Rhnull phenotype include missense substitutions, altered splicing substitutions, and small (oneto two-nucleotide) deletions. Here we report a Rhnull case caused by a complete deletion of the RHAG gene.

Molecular structures identified in serologically D– samples of an admixed population

The D– phenotype is mainly caused by the complete deletion of the RHD gene in Caucasians. However, a plethora of allelic variants have been described among D– individuals from different ethnic groups.

A simple approach to confirm the presence of anti-D in sera with presumed anti-D+C specificity.

The G antigen belongs to the Rh system and is present on most D-positive red blood cells (RBC) and on most C-positive RBC1. For this reason, the true specificity of serum that apparently has anti-D and anti-C specificity when tested with the usual panels for the identification of irregular antibodies could correspond to one of the five possible combinations: anti-D and anti-C (D+C), anti-D and anti-G (D+G), anti-D and anti-C and anti-G (D+C+G), anti-C and anti-G (C+G), and anti-G2. The correct identification of the specificity and, in particular, the determination of whether anti-D is present or not are of paramount clinical importance in different clinical settings. In pregnant women, the presence of anti-D excludes the need for the administration of prophylactic anti-D immunoglobulin (RhIG). In addition, the exclusion of the presence of anti-D in samples from D-negative women with D-negative partners or from D-negative recipients of D-negative blood components can avoid potential social or medico-legal complications2. We describe here a simple approach that enables us to determine whether anti-D is present or not in serum presumed to contain anti-D and anti-C specificity. This protocol was employed in the study of 32 samples with presumed anti-D+C specificity confirming the utility of this simple and less time-consuming strategy.

A new mutation in the platelet GPIbα gene interfering with HPA‐2 genotyping approaches: A case report

1. Guillygomarc’h A, Mendler MH, Moirand R, Laine F, Quentin V, David V, Brissot P, Deugnier Y. Venesection therapy of insulin resistance-associated hepatic iron overload. J Hepatol 2001;35:344-9. 2. Valenti L, Fracanzani AL, Dongiovanni P, Bugianesi E, Marchesini G, Manzini P, Vanni E, Fargion S. Iron depletion by phlebotomy improves insulin resistance in patients with nonalcoholic fatty liver disease and hyperferritinemia: evidence from a case-control study. Am J Gastroenterol 2007;102:1251-8. 3. Brissot P, Ball S, Rofail D, Cannon H, Jin VW. Hereditary hemochromatosis: patient experiences of the disease and phlebotomy treatment. Transfusion 2011;51:1331-8. 4. Dzepina I, Unusic J, Mijatovic D, Bulic K. Pseudoaneurysms of the brachial artery following venipuncture in infants. Pediatr Surg Int 2004;20:594-7. 5. Popovsky MA, McCarthy S, Hawkins RE. Pseudoaneurysm of the brachial artery: a rare complication of blood donation. Transfusion 1994;34:253-4.

Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells.

Background The identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs), which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories. Methods We generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua). High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment. Results TransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies. Conclusions Both stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs.

Foetal-neonatal alloimmune thrombocytopenia due to anti-HPA-2b antibodies present in the serum of a mother initially mistyped as HPA-1a negative

Thrombocytopenia is one of the commonest haematological problems observed in neonates, with a prevalence of 1 to 5% in all neonates but reaching 20 to 30% among neonates admitted to intensive care units1. The timing of onset, together with the accompanying clinical data, are useful for predicting its underlying cause. Foetal-neonatal alloimmune thrombocytopenia (FNAIT) arises from the destruction of foetal platelets induced by maternal alloantibodies directed against specific platelet antigens. The degree of thrombocytopenia is variable, but can be extremely severe and may result in major bleeding, particularly intracranial haemorrhage. Relevant criteria for the clinical diagnosis of FNAIT include unexplained thrombocytopenia in the first day of life, particularly if it is severe (<50×109/L), in term infants and without any apparent cause. The diagnosis is confirmed if maternal sensitisation against specific platelet antigens is found. Human platelet antigens (HPA) result from polymorphisms in the genes encoding the major platelet membrane glycoproteins (GP). More than 20 platelet-specific alloantigens have been defined, of which 12 are grouped in six biallelic systems2. Antibodies against HPA-1a antigen are implicated in about 80% of cases of FNAIT and those against HPA-5b in nearly 15%. Other specificities are infrequently identified3. The identification of anti-HPA antibodies in the maternal serum requires the combination of several techniques, such as immunofluorescence tests, enzyme-linked immunosorbent assay (ELISA) or monoclonal antibody immobilisation platelet antigens (MAIPA). Laboratory investigations include HPA genotyping to establish the existence of foeto-maternal incompatibilities. Finally, a cross-match against paternal platelets should be performed to ensure the detection of antibodies against low frequency antigens. It is important to proceed with all investigations in order to confirm the diagnosis of FNAIT because, if verified, the mother must be counselled on the high risk of recurrence in subsequent pregnancies. Furthermore, when FNAIT has been diagnosed in a previous sibling, the administration of antenatal treatment can reduce the risk of thrombocytopenia and haemorrhagic complications in the foetus4. We present a case of FNAIT in which alloimmunisation against the HPA-2b antigen was identified. Reaching an accurate diagnosis in this case was hampered by initial misleading results, which highlights the need to combine suitable techniques during the investigation of suspected cases of FNAIT.

The AQP1 mutation c.601delG causes the Co-negative phenotype in four patients belonging to the Romani (Gypsy) ethnic group

BACKGROUND The Colton blood group antigens Co(a), Co(b) and Co3 are encoded by the AQP1 gene which produces a water channel forming integral protein. The extremely rare Co-deficiency enables immunisation against the Co3 isoantigen. MATERIALS AND METHODS Four patients from different regions of Europe who belong to the ethnic minority of Romani (Gypsy) presented with irregular antibodies against a high frequency red blood cell antigen. Positive cross-matches with all red blood cells tested were reported. An Anti-Co3 antibody was identified as the cause of incompatibility in the four cases. The genetic background was determined by polymerase chain reaction typing with sequence-specific primers and by DNA sequencing. RESULTS The Co(a-b-) phenotype was confirmed in the four patients despite the fact that genotyping revealed the CO*01 allele of the AQP1 gene. A homozygous AQP1 c.601delG mutation, leading to a frame shift and producing a premature stop in the next codon, was responsible for the Co-negative phenotype in all four cases. While one patient was successfully transfused with blood from his sibling with the identical mutation, another case, a baby affected by haemolytic disease of the newborn, recovered without transfusion. DISCUSSION Despite the difficulties in undertaking a population study to determine the prevalence of this AQP1 c.601delG allele in the ethnic minority of Romani, the observations described in this report clearly suggest an accumulation of this mutation, which causes the Co(a-b-) phenotype, in Romani (Gypsy) patients. Further studies are necessary to prove such an accumulation.