Influence of age, sex and HCMV-serostatus on blood lymphocyte subpopulations in healthy adults.

Using a standardized immunophenotyping procedure we studied thirty-eight distinct subpopulations of T, B and NK lymphocytes in 253 healthy blood donors aged from 19 to 67. We analysed the influence of age, sex and HCMV seropositivity on each lymphocyte subpopulations and established reference ranges. We observed that aging influences the largest number of lymphocyte subpopulations with a slow increase of CD8+ EMRA T lymphocytes and of the numbers of circulating Tregs. The proportion of HLA-DR+ cells among Tregs increased with age and was correlated to the proportion of HLA-DR+ cells among effector T CD4+ lymphocytes. Sex had a major impact on absolute counts of CD4+ T cells which were higher in females. HCMV-seropositivity was associated with higher frequencies of CD8+ EMRA memory T lymphocytes while a high frequency of terminally differentiated EMRA CD4+ T cells was observed in 80% of HCMV-positive individuals and in none of the HCMV seronegative individuals.

A simple genotyping procedure without DNA extraction to identify rare blood donors.

BACKGROUND: Transfusion-induced alloimmunization has severe clinical consequences including haemolytic transfusion reactions, impaired transfused RBCs longevity and greater difficulty in finding compatible blood. Molecular analysis of genomic DNA now permits prediction of blood group phenotypes based on identification of single nucleotide polymorphisms. Implementation of molecular technologies in donor centres would be helpful in finding RBC units for special patient populations, but DNA extraction remains an obstacle to donor genotyping. MATERIALS AND METHODS: We propose a simple method compatible with high throughput that allows blood group genotyping using a multiplex commercial kit without the need for DNA extraction. The principle relies on pre-PCR treatment of whole blood using heating/cooling procedure in association with a recombinant hotstart polymerase. RESULTS: In a prospective analysis, we yielded 5628 alleles identification and designated 63 donors with rare blood, that is either negative for a high-frequency antigen or with a rare combination of common antigens. CONCLUSION: The procedure was optimized for simplicity of use in genotyping platform and would allow not only to supply antigen-matched products to recipients but also to find rare phenotypes. This methodology could also be useful for establishing a donor repository for human platelet antigens (HPA)-matched platelets since the same issues are involved for patients with neonatal alloimmune thrombocytopenia or post-transfusion purpura.

[Hypotension and adverse transfusion reactions: from the associated clinical signs to the hypotensive transfusion reaction]. / L'hypotension artérielle dans les effets indésirables receveurs: du signe clinique associé à la réaction transfusionnelle hypotensive.

OBJECTIVE: The first aim of this study was to confirm the presence of hypotension blood transfusion reactions and to assess the part of hypotension as a principal event, as defined by the literature but not characterized in French haemovigilance data. As well, recent series of several cases led us to consider a possible incidence increase. STUDY DESIGN: Using a retrospective observation, the haemovigilance data from 2000 to the end of 2007 of two French regions were reviewed. During this period, 1159657 blood units were transfused by nearly 100 hospitals and 3727 adverse reactions observed. RESULTS: One hundred and sixty-eight adverse reactions with hypotension were noticed and analyzed, representing 4.5% of all transfusion reactions and revealing an incidence of 14.5 for 100000 blood units transfused. It turned out to be mostly male recipients, severe reactions and appearing rather in the beginning of transfusions. Although platelets having greater incidence, all types of blood products may be involved. The clinical diagnosis was the following: 40 to 47% were classified as febrile reactions, 13 to 17% were allergic reactions, 8 to 9% were due to immunologic and/or haemolytic reactions, 5 to 7% resulted of cardiologic disorders, 5% resulted of hypovolemic contexts and 22% were unexplained hypotensive transfusion reactions. CONCLUSION: In about three cases out of four, transfusion-induced hypotension was associated with other clinical reactions. Indeed, hypotensive transfusion reactions were identified, having an incidence of 3.2 for 100000 blood units transfused. Furthermore, there was no explanation found for the incidence increase in our region during 2007. A national study was suggested to analyse the national data as well as a prospective study to clear out this type of transfusion reactions.

[Evolution of the blood products distribution in France - detailed analysis in a regional blood transfusion centre]. / Evolution de la distribution des produits sanguins labiles en France - analyse détaillée au sein d'un Etablissement français du sang interrégional.

OBJECTIVE: The present increase of blood products distribution raises some adaptation questions. To understand this evolution, it is necessary to assure patients needs satisfaction. STUDY DESIGN: The study focuses on years 1997 to 2007. All blood products and hospitals are taken into account. The possible impact of size and clinical specialties is analyzed for the main hospitals. RESULTS: The evolution varies according to blood product: continuous drop for autologous red cells and plasmas, drop and rise for homologous red cells and platelets with a turnaround in 2001-2002, reverse and more chaotic movement for homologous plasmas. These movements are the result of public hospitals, with an upsurge of the medium sized ones. Private hospitals go down for all blood products, with a concentration on the larger ones. Surgical hospitals fall from 3 to 4% for all blood products, while medical ones rise of 3% for homologous red cells, 27% for platelets and fall of 10% for homologous plasmas. Private mixed hospitals fall for all blood products, while public ones rise for homologous red cells and platelets. CONCLUSION: Evolution understanding of blood products distribution requires precisions about the kind of products and the hospitals status and specialties: medical, surgical, even obstetrical or urgency-related. The present trend is a rise for public hospitals and medical specialties, which are both the largest. Blood transfusion needs will thus go on rising in the years to come.

[Health care units in the French blood banks, why?]. / Les structures de soins dans les établissements de transfusion sanguine, pourquoi faire ?

In France for several years, many patients have been treated in Blood Transfusion Centers belonging to the EFS. This partnership between public hospitals and EFS is appreciated by the patients who find a competent staff in transfusion and apheresis process, in a more pleasant environment than in hospital. There is a total of 93 Health Care Units in Blood Transfusion Centers. Sixty-three of these Health Care Units perform only transfusions and bleeding. In the remaining 30 Health Care Units apheresis, peripheral blood hematopoietic stem, cell harvesting, plasmatic exchanges and extracorporeal photopheresis are also performed. Despite the perfect fit between hospital needs, comfort and easiness for patients, an economical problem remains. At the present time, the reimbursement rate by national health insurance is below the real cost. If unsolved, this discrepancy could force an end to this beneficial partnership.

[Transfusion protocols in hematopoietic stem cell allogeneic transplantation]. / Consignes transfusionnelles en cas d'allogreffe de cellules souches hématopoïétiques.

Hematopoietic stem cell (HSC) allogeneic transplantation is now commonly used as a therapeutic tool in patients with certain types of hematologic malignancies. Such patients, on account of severe pre-graft conditioning regimens, present with severe marrow aplasia justifying specific transfusion care. Given a complex immunological situation (immediately after transplantation, co-existence of two cell populations with different immunohematological characteristics), transfusion protocols must rest on clear and well-defined recommendations. Recent transfusion recommendations in settings of HSC allogeneic transplantation have defined criteria for the choice of blood products (red blood cell concentrates, plasma and platelet concentrates) depending on recipient and graft immunohematological characteristics (minor/major/mixed ABO compatibility/incompatibility and time of transplantation). Transfusion instructions are summarized in a synthesis document entitled : "Instructions for transfusion following HSC allogeneic transplantation". This document specifies the immunohematological characteristics of blood products and various transfusion protocols (systematic irradiation, negative CMV, etc.). This document is used by the teams who distribute blood products, for selection purposes, as well as by the medical transfusion team when they perform ultimate pre-transfusion control steps.

Reference values for T, B and NK human lymphocyte subpopulations in adults.

The data presented in this paper are reference ranges for frequencies of thirty-eight subpopulations of T, B and NK lymphocytes, established from a cohort of 253 healthy blood donors aged from 19 to 67. When relevant, the influence of age or sex was taken into account to calculate these reference values. This article is related to the research article entitled "Influence of age, sex and HCMV-serostatus on blood lymphocyte subpopulations in healthy adults" (Apoil et al., 2017) [1]. Immunophenotyping data obtained from each individual is made publicly available for extended analyses.

[Validation of antibody screening by indirect antigloblin test and ABO blood typing by filtration and microplate techniques: assessment of robustness]. / Maîtrise des tolérances métrologiques : application à la recherche des anticorps anti-érythrocytaires (RAI) en test indirect à l'antiglobuline (TIA) et au groupage sanguin ABO par les procédés de filtration et en microplaque.

According to requirements of the French Committee for Accreditation (Cofrac), it is essential to use validated and standardised methods in Immunohematology. This imposes first the knowledge of metrological tolerances for all the technics. Two multicenter studies were carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and tests cells for antibody screening using indirect antiglobulin test on one hand and for reverse grouping on another hand. All equipment used (temperature test chamber, chronometer, pipettes) were calibrated according to Cofrac standards. The antibody screenings were performed manually using 3 different filtration systems: ID Diamed, Biovue Ortho and Scangel Biorad, the same tests cells, a standard 20 ng/mL anti RH1, a positive control anti KEL1 and a negative control; the reverse blood grouping was performed manually using the above mentionned filtration systems and microplate technic with the same A1 and B test cells. These two studies showed that all the tests from the multiples combinations of the above parameters gave the same results and allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening and blood typing.

Influence of 12-O-tetradecanoylphorbol-13-acetate on proliferation and maturation of human breast carcinoma cells (MCF-7): relationship to cell cycle events.

Exposure of MCF-7 human mammary carcinoma cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) results in changes in cell morphology and arrest of cell growth. The inhibition of cell proliferation and the increase in cell volume are concentration dependent; these effects are reversible upon removal of the tumor promoting agent. Electron microscopic studies reveal that TPA increases endoplasmic reticulum and induces the appearance of secretory granules. MCF-7 cells treated by TPA therefore present morphological characteristics of secretory cells. These effects of TPA on MCF-7 cells are accompanied by specific disruption of cell cycle events, a block of cells in G1 at the expense of S base, and a delayed passage through G2. Studies in which a cell cycle lock in G1 is produced by tamoxifen show that exposure of such cells to PA produces cell morphological changes and an inability to progress through the cell cycle when estradiol is added.

[Feasibility study for Intercept platelets]. / Étude de faisabilité des plaquettes Intercept.

During 3 months, platelet concentrates prepared by "Établissement français du sang Pyrénées-Méditerranée" (Blood bank) were treated with the Intercept process (CERUS©). This study primarily aimed to measure the organizational impact of this technology on transfusion chain. The introduction of Intercept did not raise any major difficulties, but required some adaptations upstream from the deployment. Prior information of health care institutions and physician was essential to anticipate the practical changes, including the prescription of platelet concentrates (CMV negative, irradiation). This study allowed to analyze also the transfusion consequences for patients, in the form of observational studies. The patients transfused with platelet concentrates treated with Intercept received more platelet concentrates (+12.9%), less rich in platelets (-12.8%), the cumulated quantity of platelet being stable.

Variable region gene segment utilization in rhesus monkey hybridomas producing human red blood cell-specific antibodies: predominance of the VH4 family but not VH4-21 (V4-34).

Structural analyses of human immunoglobulin gene segments from monoclonal cell lines provide valuable information regarding the antibody repertoire. This information, in conjunction with a nearly complete knowledge of the human immunoglobulin germline repertoire, now allows further investigation into the underlying molecular basis responsible for some of the observed biases found in the expressed repertoire. One human heavy chain variable region gene segment, V4-34 (VH4-21), is one of the most prevalent gene segments in the expressed repertoire. The overwhelming presence of the V4-34 gene segment suggests that it may play an important role in immune responses. However, there is currently little information regarding its presence and potential importance in nonhuman primates. In order to determine if this gene segment is used by lower primates in a similar manner we determined the molecular structure of the variable region gene segments that are expressed by macaque monoclonal heterohybridomas that are specific for human red blood cell antigens. Eleven of the 12 hybridomas are derived from Rhesus monkeys (Macaca mulatta) and one is from a cynomologous monkey (Macaca fascicularis), all of which have been immunized with human red blood cells. The predominance of a VH4-like family and the specific absence of a VH4-21 equivalent led us to further characterize the macaque VH4 gene family at the germline level. Therefore, germline gene segments from the macaque equivalent to the human VH4 gene family are also described.

Effects of an anti HLA-DR immunotoxin on leukaemia cells and hematopoietic progenitors.

An anti HLA-class II immunotoxin has been prepared by coupling to ricin A-chain (RTA) and anti-DR-DP monoclonal antibody (2G5-MoAb). Protein synthesis inhibition assays showed that 2G5-RTA immunotoxin is: highly cytotoxic to B-cell lymphoid neoplastic cells, and variable so to ALL cells, while AML cell lines display a generally poor susceptibility. Toxicity of 2G5-RTA on normal hematopoietic cells (HPC) was found to be dose-dependent, and to increase significantly with the addition of NH4Cl, used as an activating agent. After 4 h of incubation with 2G5-RTA (10(-8) M), without NH4Cl, percentages of CFU-GM and CFU-GEMM (colony forming units-granulo-monocytes and -multipotent, respectively) progenitor cell recovery were in the order of 50% and 30% respectively. In the same treatment conditions, 2G5-RTA induced a 6 log kill on RAJI cells--measured by clonogenic assay. Finally, this study shows that anti-DR immunotoxins may represent an original, efficient, and relatively safe approach for bone marrow purging of DR positive malignant B-cell populations; while their clinical potential pertaining to ALL and AML remains uncertain.

Sensitivity of fresh leukemic cells to T101 ricin A-chain immunotoxin: a comparative study between Fab fragment and whole Ig conjugates.

We compared the cell killing potency of a whole Ig ricin A-chain immunotoxin (T101 IgG-RTA) against its Fab fragment counterpart (T101 Fab-RTA) on both CEM cells and fresh malignant lymphoid cells. A dye exclusion assay (DEA), was used to evaluate the kinetics of leukaemia cell viability mediated in vitro by each immunotoxin (IT). This study found that in the absence of ammonium chloride (NH4Cl), used as an enhancer agent, T101 Fab-RTA was significantly more toxic to both CEM and fresh leukaemia cells than T101 IgG-RTA. In the presence of NH4Cl (10(-2) M), while no differences could be found between the two IT on CEM cells, T101 Fab-RTA was clearly superior to T101 IgG-RTA on fresh leukaemia cells. These results suggest that T101 Fab-RTA may offer an excellent alternative to T101 IgG-RTA for IT treatment of CD5 positive leukaemia patients.

Local immunotherapy of recurrent glioblastoma multiforme by intracerebral perfusion of interleukin-2 and LAK cells.

A non randomized pilot study has been undertaken to evaluate the feasibility of local immunotherapy (IT) of recurrent glioblastoma multiforme (GM) by continuous intracerebral perfusion of recombinant interleukin-2 (rIL-2, Eurocetus) with and without lymphokine activated killer (LAK) cells. At time of surgical removal of the tumor, a catheter was implanted in the cavity left by tumor debulking allowing continuous perfusion of rIL-2. Five patients received 18 x 10(6) IU/day or rIL-2 for five days. At days 1, 3, and 5 after surgery, rIL-2 perfusion was briefly interrupted for the injection of LAK cells. Eight other patients received rIL-2 alone, either 24 x 10(6) IU/day (five patients) or 54 x 10(6) IU/day (three patients). Capillary leak syndrome, which is the main side effect of systemic infusion of rIL-2, was never observed, but local immunotherapy induced fever, confusion, and cerebral edema in all patients. Despite local IT, tumor progression was diagnosed by CT scan 4 to 12 weeks after the treatment.

Characterization of human anti-hrB-like monoclonal antibody.

To develop a blood typing reagent with Rh specificity, peripheral blood lymphocytes were isolated from a multiparous woman with apparent alloanti-Ce and fused with FIS heteromyeloma cells. One hybridoma cell line, FOR-2E3, secreted human IgG, which agglutinated all human red blood cells except R2R2, RZRZ, Dc-, D--, Rhnull, and e+ hrB-. The supernatant is useful as a reagent to screen for hrB- blood donors and to differentiate haplotypes with C and e in cis from haplotypes with C and e in trans.

Use of LOR-15C9 monoclonal anti-D to differentiate erythrocytes with the partial DvI antigen from those with either partial D antigens or weak D antigens.

Historically, red blood cells (RBCs) with partial D antigens have been defined serologically by their pattern of reactivity with polyclonal and monoclonal anti-D. Although numerous variants have been described in tests with well-characterized monoclonal anti-D, definition remains difficult to ascertain serologically. RBCs of known partial D type were tested with LOR-15C9 (a monoclonal anti-D) and commercial anti-D by the tube indirect antiglobulin test (IAT), by micro typing system IgG gel cards, and by immunoblotting. By IAT, LOR-15C9 reacted strongly with DIIIa, DIIIc, DVa, DVI, DVII, and DFR RBCs in addition to RBCs with common D antigens; weakly with DII, DNU, and DIIIb RBCs; and not at all with DIVa, DIVb, DBT, or R0 Har RBCs. Reactivity was variable (1+ to 4+), with RBCs classified as weak D (Du). As expected, the commercial anti-D agglutinated all D variants and weak D RBC samples by the IAT and by using IgG gel cards; however, the reactivity with DVI RBCs was weaker than with LOR- 15C9. By immunoblotting, LOR-15C9 detected a band with an apparent molecular mass of approximate Mr 30,000-34,000 in membranes prepared from D-positive, DIIIa, DIIIc, DVa, DVI, DVII, and DFR RBCs and an additional band of Mr 20,000-22,000 in membranes prepared from DVI RBCs. No band(s) was detected in membranes from DII, DNU, DIIIb, DIVa, DIVb, DBT, R0 Har, weak D, or D-negative samples. LOR-15C9 provides a useful tool to identify positively DVI samples and thereby differentiate this partial D from other D variants and from weak D samples.

IgG anti-D MAbs in tests by flow cytometry.

Seventy-two IgG anti-D Mabs were studied by indirect immunofluorescence and flow cytometry (FCM) with R1r, rr and various D variant phenotype red blood cells (RBCs) (DIIIb,DVI,DVII,Rh33 and uncategorized variant ISBT number 48). The results were compared with those obtained by indirect antiglobulin test (IAT).

[Detection of irregular anti-erythrocyte antibodies using the indirect antiglobulin test in a low-ionic-strength medium. Immunohematology Group of the French Blood Transfusion Society]. / Recherche d'anticorps irréguliers anti-érythrocytaires (RAI) en test indirect à l'antiglobuline en milieu de basse force ionique. Groupe Immunohématologie de la Société Française de Transfusion Sanguine.

The detection of irregular antibodies is usually performed with serum by the indirect antiglobulin test (IAT) with a polyspecific antiglobulin reagent. In a first study in 1996, we compared the results obtained with 3,264 blood samples of patients drawn with or without anticoagulant: no significant difference was observed among the 240 allo-antibodies detected and identified. In this paper we report the comparison of the results obtained by IAT on column of filtration with two kinds of reagents: polyspecific and anti-IgG antibodies. Respectively 2,927 (76 contained an antibody), and 643 (161 contained an antibody) sera of patients were tested with methods ID-Diamed and Ortho-Biovue. Titrations of 153 other antibodies were also performed with the two reagents. Results showed no significant difference using either polyspecific reagent or the anti-IgG antibodies. This study proves that it is possible to perform screening of irregular antibodies on uncoagulated blood samples. This possibility allows automation as blood typing and screening of irregular antibodies can be carried out with the same sample.

[Problem-solving in immunohematology: direct compatibility laboratory test ]. / Aide à la décision en immunohématologie: épreuve directe de compatibilité au laboratoire (EDC).

Cross-matching between the serum of a patient and the red blood cells to be transfused is most important for the prevention of hemolytic transfusion reactions in allo-immunized or new-born patients found positive with direct antiglobulin test. Cross-matching is a time-consuming and complex laboratory test. In order to obtain valid results, it is necessary to abide by some technical rules detailed in this article. The choice of the blood units to be cross-matched depends on the patient's clinical story and on the specificity of anti-erythrocyte antibodies present in the serum. The identification and the management of most frequent difficulties met by using the cross-match technique are discussed hereby.