Anti-donor DR103 Immunization in a DRB1*0101 kidney allograft recipient.

Posttransplant appearance of donor-specific anti-HLA antibodies is correlated with poor graft survival. Herein, we have provided evidence that an HLA-DRB1*0101 kidney allograft recipent developed anti-DR103 antibody after receiving a transplant from a HLA-DRB1*0103 cadaveric donor, resulting in graft loss. HLA-DRB1*0103 is a rare allele in Caucasian populations. It differs from DRB1*0101 only by three amino-acid substitutions and may play a central role in allorecognition. Nevertheless, our data showed that it induced alloimmunization in a DRB1*0101 recipient. Therefore, this new possibility of immunization must be taken into account before transplantation as well as after grafting.

Chimerism in lymphoid tissues and donor-specific antibody response after injection of allogenic splenic dendritic cells from Fischer rats to Lewis recipients.

The aim of this work was to study cellular chimerism achieved in lymphoid tissues and production of antidonor lymphocyte antibodies after injection of splenic dendritic cells (DCs) from Fischer F344 rats to Lewis recipients, a model of chronic rejection. DCs isolated from the spleen expressed OX62 (95%), CD80 (70%), and CD86 (80%). Two doses of these nonplasmacytoid splenic DCs from Fischer rats (2 x 10(6) and 5 x 10(6)), which had been labeled ex vivo with a TRITC fluorochrome (PKH26), were injected to Lewis recipients. Using fluorescence microscopy TRITC positive cells were localized at day 15 and day 45 in frozen sections from spleen, thymus, mesenteric lymph nodes, heart, liver, kidney, and skin (n = 5 per group). Donor-specific antibodies were sought with flow cytometric crossmatches in serum samples taken at 7, 15, 30, and 45 days. TRITC-positive DCs were essentially localized in the spleen, the thymus, and lymph nodes of Lewis recipients. The majority of DCs were detected in the spleen (14.9 +/- 3.3 and 14.3 +/- 0.9 DCs/per high power field respectively at day 45). A significant number of DCs was also detected in the thymus and mesenteric lymph nodes at both times. Only some scattered TRITC-positive cells were observed in other organs. The number of DCs was stable over time and did not depend on the injected dose. A positive flow cytometric cross-match was observed at day 30 in all recipients independent of the injected dose. These data showed that 2 x 10(6) mature, nonplasmocytoid DCs from F344 rats injected to Lewis recipients induced stable chimerism in primary and secondary lymphoid organs and a humoral response to donor antigens.

Induction of tissue factor expression on human umbilical vein endothelial cells by cell-specific HLA class I antibody: preliminary data.

Donor-specific antibodies may play an important role in the development of chronic allograft rejection process. However, the mechanisms leading to intimal vascular proliferation and fibrosis remain poorly understood. The aim of this study was to examine whether donor-specific HLA antibodies induce overexpression of tissue factor (TF) by endothelial cells. HLA typed human umbilical vein endothelial cells (HUVEC) were incubated for 1 to 12 hours with LPS (10 microg/mL), and increasing concentrations (1 to 500 microg/mL) of anti-HLA A1 antibody specific for an antigen expressed by HUVEC and of an anti-HLA A2 antibody for which A2 was not expressed by the HUVEC. Expression of TF mRNA transcripts was quantified using real time Q-RT PCR and TF activity was tested in cell lysates of cultured HUVEC using a chromogenic TF activity assay. HUVEC-specific anti-HLA A1 antibody at low concentrations (10 microg/mL) induced both a significant increase of TF mRNA transcripts after 1 hour of incubation and TF activity after 3 hours incubation compared to incubation with medium alone or with the nonspecific anti-HLA A2 antibody (n = 4 for all experiments, P < .05). These data show for the first time that specific anti-HLA antibody can induce overexpression of TF on endothelial cells. TF, a transmembrane glycoprotein involved not only in the onset of the coagulation cascade, but also in cell proliferation and anti-apoptotic processes, may play a role in the development of alloantibody-induced chronic rejection.

Plateletpheresis concentrates produced with the COMTEC cell separator: the French experience.

The latest generation of cell separators such as Trima (Gambro), Amicus (Baxter) and AS-TEC 204 (Fresenius), allow the collection of leucocyte-reduced platelet concentrates without secondary filtration. Fresenius has recently developed the COMTEC cell separator whose performance has been evaluated by several teams in France. This new cell separator is an improved version of the Fresenius AS-TEC 204 cell separator, designed to allow more efficient platelet collections. This study reports on the experience of six French teams (from Bordeaux, Clermont-Ferrand, Creteil, Dijon, Lille and Nancy) who obtained 696 leucocyte-reduced plateletpheresis concentrates in the course of collection using the new Fresenius COMTEC cell separator. All healthy volunteer donors fulfilled French selection criteria for platelet apheresis. Donors were eligible if they had suitable venous accesses, if their bodyweight was *50 kg and if their pre-apheresis platelet count was >150 x 10(9) l(-1). Between 4606 and 5229 ml of blood were processed. The mean volume of the platelet concentrates was between 439 and 493 ml (mean 460 +/- 63 ml). The platelet yield was of the order of 5.18 +/- 1.02 x 10(11) with only one platelet concentrate below the norm of 2 x 10(11) platelets (0.91 x 10(11)). No plausible explanation for this was found. The residual leucocyte levels conform to current norms. The platelet concentrates contained less than 1 x 10(6) leucocytes per concentrate (mean 0.233 +/- 0.150 x 10(6) leucocytes) in more than 97% of the components produced with >95% statistical confidence. The efficacy of the cell separator (52.44 +/- 7.35%) is comparable to that of other separators. The Fresenius COMTEC cell separator makes it possible to obtain leucocyte-reduced platelet concentrates which comply with current standards both in terms of platelet content and residual leucocyte level.

Hypertriglyceridaemia and Lewis (A-B-) phenotype in non-insulin-dependent diabetic patients.

A relationship between Lewis (a-b-) phenotype and the metabolic syndrome X has been suggested. We studied the frequency of Lewis (a-b-) phenotype in subjects with non-insulin-dependent diabetes mellitus (NIDDM) as well as the relationship between Lewis phenotype and lipid concentration in NIDDM patients. Lewis red blood cell phenotyping was done in 207 NIDDM subjects and 345 non-diabetic control subjects by immuno-agglutination with anti-Lewis a and b monoclonal antibodies. Among NIDDM patients, the proportion with the Lewis (a-b-) phenotype was significantly increased (23.6% vs 14.3%, p = 0.01), and this phenotype was associated with higher levels of triglycerides (2.40 +/- 2.58 vs 1.97 +/- 1.25, p = 0.03). This study shows a relationship between NIDDM and Lewis (a-b-) phenotype. Hypertriglyceridaemia in Lewis-negative NIDDM could suggest an increased risk of ischaemic heart disease for these subjects.

Elevated serum CA19-9 levels in poorly controlled diabetic patients. Relationship with Lewis blood group.

Many non-malignant diseases may be associated with elevated serum CA19-9 levels. Recent reports suggest that diabetes mellitus may also be responsible for some elevations. In this study we investigated the influence of the glycaemic level and Lewis phenotype on the serum CA19-9 levels in diabetic patients. In 15 out of 84 patients (17.8%) serum CA19-9 exceeded 100 kU/L (highest value: 208 kU/L). CA19-9 concentrations were significantly correlated with glycosylated haemoglobin levels. On the other hand, no correlation was found between CA19-9 levels and the type of diabetes, lipase levels, or the presence of anti-islet cell antibodies. Le(a-b-) patients had significantly lower serum CA19-9 levels. This study emphasizes the frequency of CA19-9 elevations in diabetic patients without cancer.

[Standardization trial of ABO-Rh(D) blood typing using a U-microplate]. / Etude pour un essai de standardisation du groupage ABO-D en microplaque à fond en U. Groupe de travail "Microméthodes et immuno-hématologie".

This study reports the results of ABO-Rh (D) typing in microplate according to a suggested protocol. 35,532 blood typings were performed by 13 laboratories, compared to usual technics. This work has proved the feasibility in routine of this protocol in order to identify the A, B, D and weak antigens. However the difficulties in detecting some weak variants reveal the interest of standards for immuno-haematology reagents, to apply in the microplate technology.

[Preparation of leukocyte-depleted human platelet concentrates by centrifugation and filtration of a pool of sterilely connected buffy-coats]. / Préparation de concentrés de plaquettes humaines déleucocytés par centrifugation et filtration d'un pool de buffy-coats connectés stérilement.

We describe a new method for the preparation of standardised therapeutic doses of leukocyte depleted platelets. The first step is to remove the buffy-coat from whole blood units drawn on triple Siamese ACD/SAGM bags (Maco-Pharma) by means of a Compomat (NPBI). The second step is to connect (SCD Haemonetics) six buffy-coats and one plasma to a special kit (Maco-Pharma) including a PALL PL 100 filter; after centrifugation, the supernatant platelet concentrate is extracted, filtered and recovered in a 2 litre TOTM PVC bag. The volume, the number of platelets and leukocytes of these pools are measured. A comparison of these parameters is made with therapeutic doses prepared in the same way without filtration. Besides, pH measurements up to the 6th day of storage and bacteriological checks are carried out. The results show: no platelet loss related to filtration; a synergy between the preparation process out of buffy-coats and the filtration: so each dose contains less than 10(6) leukocytes; a good pH level allowing the storage for five days as it is associated to the bacteriological safety of the functionally closed system. This technique makes it possible to transfuse only leukocyte depleted platelet concentrates. In addition, it offers new prospects for standardisation and quality improvement.

Serological characterization of murine monoclonal antibodies directed against acquired B red cells.

Balb/c mice were immunized with acquired B red cells. Twelve clones specific for acquired B red cells were obtained from two fusions. A detailed investigation of three clones is reported here. These antibodies appear to be directed to the B-like epitope since they are inhibited by galactosamine and fail to react after acetylation of red cells. E 231 is an example of a series of antibodies closely specific for acquired B red cells which can be useful in elucidating some AB0 typing problems. E 167 and F 47 showed a cross-reactivity with A1 red cells and a synthetic A trisaccharide. No affinity for B antigen could be demonstrated for any of the antibodies.