Optimization of cytotoxicity assay by real-time, impedance-based cell analysis.

This paper presents an optimized procedure for assessing an immune-mediated cytotoxicity, produced after the addition of human and baboon serum to transgenic porcine fibroblasts. This procedure is performed with the xCELLigence Real-Time Cell Analyzer (RTCA). The xCELLigence system measures the impedance variations in the culture media of a 96-well microelectronic plate, and shows the changes in cell number and morphology in a real-time plot. However, different factors need to be optimized before developing an RTCA assay. Thus, we studied the influence of several variables, such as the number of cells seeded, the time the cells were allowed to grow before the tests, the serum concentration and the addition of rabbit complement. The findings were confirmed by the WST-1 classical cytotoxicity test. The results showed that 7.5 × 10(3) cells seeded per well produced the adequate CI in 10 h. The area under the curve and the CImin versus concentration values showed a very high correlation index (r(2) = 0.966 and r(2) = 0.92 for the first 50 h after challenge, respectively), proving that CI variations are directly proportional to the quantity of serum added. The addition of complement resulted in lower CImin values. Therefore, both the cytolysis level with and without exogenous complement addition had to be assessed. There was a high correlation between the relative cytotoxicity assessed by WST-1 and the CI obtained by RTCA when exogenous complement was not added (r(2) = 0.827; p < 0.001). The correlation was average when rabbit complement was added (r(2) = 0.523; p = 0.046). In conclusion, culture conditions have an important influence on RTCA cytotoxicity assays.

Presence of Pig IgG and IgM in Sera Samples From Baboons After an Orthotopic Liver Xenotransplantation.

INTRODUCTION: The immunorejection in xenotransplantation has mostly been studied from the host's immune system activation point of view and there is very little information about the graft-vs-host reaction. OBJECTIVES: To validate an enzyme-linked immunosorbent assay (ELISA) test for porcine IgM and IgG quantitation, the assessment of porcine IgG and IgM in sera samples from baboons after liver orthotopic xenotransplantation or in human plasma after xenotransfusion through pig organs, and to assess the presence of porcine immunoglobulin in a baboon after plasmapheresis to a complete change of plasma after 4 passages through pig liver. MATERIALS AND METHODS: Two commercial ELISA kits for pig IgG and IgM quantitation were evaluated for cross reactivity with samples from baboons, Rhesus monkeys, squirrel monkeys, and humans. Then, samples from 18 baboons after orthotopic liver xenotransplantation were studied for porcine IgG and IgM. To understand the phenomenon, human plasma samples after xenotransfusion 1, 2, 3, or 4 times through liver or kidney were assessed for porcine IgG presence and finally, the porcine IgG were quantified in sera samples obtained during more than 4 years from a baboon after plasmapheresis with baboon plasma after xenotransfusion 4 times through a pig liver. RESULTS: Porcine IgG and IgM were found in samples from xenotransplanted baboon during all survival. The quantity of porcine IgG in plasma after xenotransfusion correlated with the number of passages through the pig liver, and the IgG were completely cleared from the baboon 16 days after plasmapheresis and complete substitution of plasma after 4 xenotransfusions through a pig liver.

Influence of harvest bacterial contamination on autologous peripheral blood progenitor cells post-transplant.

Microbiological contamination of manipulated blood products, including hematopoietic progenitors obtained from peripheral blood, is an infrequent but persistent problem in transplant units. The relevance of such contamination in causing patient infection has been reported as insignificant, but the effect on the post-transplant course has not been well documented. We studied the incidence of bacterial contamination in autologous peripheral blood progenitor cell transplants in two of the bench processing steps, as well as the repercussions in the post-transplant course affecting incidence of infections, transfusion requirements and time to engraftment. A total of 365 aphereses performed on 152 patients were cryopreserved in 617 bags. In 31 of these bags (5.0%), bacterial cultures were positive for Coagulase-negative Staphylococcus (31.1%), S. epidermidis (21.9%), Corynebacterium sp. (6.3%), S. warneri (6.3%), Stenotrophomonas maltophilia (6.3%), Streptococcus sp. (9.4%), Viridans group Streptococcus (3.1%) and more than one bacteria (Coagulase-negative Staphylococcus plus Corynebacterium) (15.6%). Half of the bags were contaminated at the time of freezing and the others at the time of thawing. The 31 contaminated bags were infused into 17 patients. In five of these the same contaminating bacteria was found. No difference between the two groups of patients (contaminated and non-contaminated) was found on the day the fever started, length of fever, blood transfusion requirements and engraftment, but length of hospitalization was significantly greater in patients receiving contaminated transplants.

Second mobilization of peripheral blood progenitor cells in patients with poor first mobilization.

The aim of this study was to analyse the efficacy of 2 second mobilization (MB) protocols in 2 groups of patients who failed to obtain enough peripheral blood progenitor cells (PBPC) in the first MB. In 1 group (8 patients), 10 microg/kg of G-CSF was administered, and in the other group (8 patients), a double dosage (10 microg/kg twice a day) was administered. Both groups of patients received Cyclophosphamide (1.5 g/kg) 10 days before the apheresis. No difference was found among both groups of patients in diagnosis, previous chemotherapy, and time elapsed after the first MB. Administration of higher doses of G-CSF decreased the number of apheresis needed in the second MB to complete 2 x 10(6)/kg of CD34+ cells. It also increased the number of patients who achieved sufficient CD34+, namely, 75% versus 50%.

Non-ABO blood group systems phenotyping in non-human primates for blood banking laboratory and xenotransplantation.

Some biomedical research procedures, such as organ xenotransplantation, usually require intensive hemotherapy. Knowledge of the whole phenotype of blood donor and graft could be useful in the field of xenotransplantation. Human and simian-type categories of blood groups have been established and they can be tested by standard methods used for human blood grouping. The aim of this work was to study the incidence of non-ABO blood group systems in different species of non-human primates, which are employed in biomedical research. The phenotype of Rh, Lewis, Kidd, Kell, MNSs, Lutheran, P and Duffy antigens was investigated in olive baboon (n = 48), chacma baboon (n = 9), Guinea baboon (n = 14), Rhesus macaque (n = 38) and squirrel monkey (n = 30) by using commercial microtyping cards. Kell, Lutheran, Kidd and Duffy antigens have been detected in all species, Rh in squirrel monkey, MNSs in rhesus macaque and squirrel monkey, and Lewis in baboon and rhesus macaque. There were differences in frequency and haemagglutination scores between species regardless of their gender and age. The main differences were found in squirrel monkey when compared with baboons and macaques. This typing system provides a tool to assess the presence of antigens in animals used for experimental procedures, such as xenotransplantation and xenotransfusion.

Assessment of in vitro heparin complement regulation capacity during real-time cell analyzer antibody-mediated cytolysis assay: compatibility studies for pig-to-baboon xenotransplantation.

OBJECTIVE: To assess the effect of sodium heparin concentrations on antibody- and complement-mediated cytolysis by means of a real-time cell analyzer system (RTCA) investigating the complement regulation ability of heparin to reduce or prevent hyperacute in an in vitro model of pig-to-baboon xenotransplantation. MATERIALS AND METHODS: Fibroblasts isolated from the skin of two transgenic pigs were cultured in microelectronic 96-well plates for 9 hours. Then, we added 20 µL of normal sera from two healthy adult olive baboons (Papio anubis) or two volunteer healthy humans. Simultaneous cultures had added heparin at 3.5, 5, 7.5, 15, and 30 IU. Moreover, rabbit complement was added for the exogenous complement group (ExC) versus the other group only with the complement present in the sera as an endogenous complement group (EnC). Cellular cultures were monitored over 150 hours after challenge. With cellular index (CI) data recorded by the xCELLigence software system, we calculate area under the curve versus concentration (AUC) and minimum CI (CImin) versus concentration. RESULTS: All cultures showed decreased CI after challenge with human or baboon sera. There was a high correlation for AUC (r(2) > 0.90) and CImin versus concentration (r(2) > 0.970) during the first 40 hours postchallenge among the EnC group, regardless of human or baboon sera. However, there was no correlation for AUC and CImin for the ExC group. There was a reduction of CImin related to increased heparin concentrations. CONCLUSIONS: The addition of heparin did not reduce antibody- and complement-mediated cytolysis assessed in vitro by RTCA in pig-to-baboon compatibility assays.

Generation of human-to-pig chimerism to induce tolerance through transcutaneous in utero injection of cord blood-derived mononuclear cells or human bone marrow mesenchymals cells in a preclinical program of liver xenotransplantation: preliminary results.

OBJECTIVE: Using a percutaneous ecoguided injection system to obtain chimeric piglets through a less invasive and traumatic technique than previously reported. MATERIALS AND METHODS: The two types of human cells included umbilical cord blood mononuclear elements and mesenchymal stem cells cultured from bone marrow. Four sows at gestational day 50 were anesthetized. A needle was inserted through the skin and uterine wall to reach the peritoneal cavity of the fetuses under continuous ultrasound guidance. Fourteen piglets were injected with various cell concentrations. RESULTS: All sows carried pregnancies to term yielding 69 piglets, among which 67 were alive and two mummified. Two piglets died during the first 48 hours of life. Chimerism was detected using flow cytometry and by quantitative polymerase chain reaction (q-PCR) to detect Alu gene in blood or tissues samples. The analysis detected blood chimerism in 13 piglets (21%) by flow cytometry and the presence of the human Alu gene in 33 (51%) by q-PCR. The results suggest cell trafficking between littermates after in utero injection. CONCLUSIONS: Transcutaneous echo-guided injection succeeded to produce chimeric piglets without disadvantages to the sow or the fetuses and avoiding abortions or fetal death.

Donor-graft compatibility tests in pig-to-primate xenotransplantation model: serum versus plasma in real-time cell analyzer trials.

INTRODUCTION: Various strategies have been designed to assess in vitro donor-graft compatibility in pig-to-primate xenotransplantation models. Most of them are based on a cytolysis assessment by exposing donor tissue to host serum with investigations by flow cytometry, and photocolorimetric levels. The aim of this study was to analyze the difference in cytolysis produced by sera and plasma obtained using various anticoagulants, or containing high versus low levels of platelets. METHODS: The cytolysis trials were performed using an xCELLigence real-time cell analyzer (RTCA) in a cell model involving transgenic pig fibroblasts exposed to sera (S) or plasma obtained using EDTA, Li-heparin, or Na-heparin in combination with plasma containing high versus low content of platelets. Samples were obtained from two baboons and five volunteer human donors. Evolution of fibroblast cell growth was assessed by RTCA as the cell index (CI). After 9 hours of growth, cells were exposed to 20 µL of each sample. The minimum CI (CImin), time to CImin (TCImin), and time to reach the CI observed before compound addition (Trec) were recorded for each microwell. RESULTS: The lowest CImin, highest TCImin, and Trec observed for EDTA plasma showed significant differences from other samples (P < .001). DISCUSSION: On the basis of this study, using the RTCA assay, heparinized plasma produced complement inhibition and with undervaluation of the cytolysis reaction. EDTA plasma produced total death of most of cultures. The most accurate sample matrix seems to be serum.

ABO and RH1 blood group phenotyping in pigs (Sus scrofa) using microtyping cards.

Transplantation or transfusion with ABO disparity is a cause for rejection or for severe hemodynamic alterations. ABO groups in pigs are commonly an unknown variable, which has been previously assessed by means of hemagglutination tests or immunohistochemical procedures on tissues. Herein, we have reported a simple method using commercial microcards for human ABO typing. However, the reagents directly derived from human sera included in these cards can result in false determinations due to alpha-gal interference. The ABO groups of 19 wild-type pigs (Landrace x Large White) were assessed using 2 commercial cards: Human sera-based and monoclonal antibody-based cards. The human sera cards determined that 8 pigs belonged to the AB group and 11 to the B group. The monoclonal antibody cards determined that 8 pigs belonged to the A group and 11 to the O group. None of the pigs showed reactions to Rh1 antibodies. Because the B group has not been described in pigs, the reaction in human sera cards represented an interference with alpha-gal antigen, a molecule structurally similar to the B blood antigen. Thus, microtyping cards based on monoclonal antibodies provided simple, quick way to assess ABO groups in pigs used for xenotransplantation. ABO concordance should always be investigated for these types of procedures.

Design of a real-time quantitative polymerase chain reaction to assess human complement regulatory protein gene expression in polytransgenic xenograft pigs.

OBJECTIVE: To design a real-time quantitative polymerase chain reaction (q-PCR) to assess gene expression for hCD55, hCD59, and hCD46 in polytransgenic (PT) pigs used as xenograft donors for orthotopic liver xenotransplantation using a pig-to-baboon model. MATERIALS AND METHODS: Three pairs of primers were designed using PrimerBlast and mRNA of hCD55, hCD59, and hCD46 sequences. Blood samples from five PT pigs (two males and three females) were used to isolated peripheral blood mononuclear cells (PBMCs) by means of Ficoll gradients. After DNAase digestion of isolated mRNA, we synthesized cDNA. Using SYBR-Green chemistry of q-PCR, we constructed a standard curve. Two wild-type (WT) pigs were used as negative controls, and PBMCs from two healthy human volunteers as positive controls. The amplicon length was assessed by means of agarose gel electrophoresis and PCR products, sequenced. RESULTS: We observed amplification for hCD55, hCD59, and hCD46 in all samples from the five PT pigs except for hCD55 and hCD46 in one male PT pig. Neither the human samples nor the negative controls showed amplification. The expected amplicon length was confirmed; sequencing showed high homology with human mRNA for the three proteins and no match with any known pig sequence. CONCLUSIONS: The q-PCR allowed detection of animals with the highest gene expression for hCD55, hCD59, and hCD46 for xenograft donors in transplantation experiments.

Validation of xCELLigence real-time cell analyzer to assess compatibility in xenotransplantation with pig-to-baboon model.

OBJECTIVE: To validate the use of a microelectronic real-time cell analyzer system (RTCA) we developed a complement-mediated antibody cytotoxicity assay to investigate the compatibility of a graft and a recipient in pig-to-baboon xenotransplantation. MATERIALS AND METHODS: Fibroblasts isolated from the skin of five hCD55, hCD59, and hCD46 transgenic pigs (TP) were cultured in 96 microelectronic well plates for 17 hours. Then, we added to each microwell 20 µL of normal sera from nine healthy adult olive baboons (Papio anubis)-three males and six females. The evolution of the cell culture was assessed every 3 minutes during the pretreatment period, at 11 hours postaddition, and every 30 minutes from 12 to 96 hours. Simultaneously, we performed a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Fibroblasts from wild-type (WT) pigs were used as positive controls and microwells without serum addition from each TP as negative controls. The RTCA results were expressed as a normalized cellular index (NCI). RESULTS: Differences were observed between the five TP fibroblasts and the WT fibroblasts, with greater cytotoxicity on WT cells. Among TP, a higher cytolytic level was observed in males than females. The MTT results correlated with NCI at different times, with the minimum NCI and with the time to for NCI recovery before serum addition. The correlation was lower than that previously reported in environmental toxicity assays. CONCLUSIONS: RTCA allows a long-term assessment of the immunocytotoxic effect of baboon sera on pig cells, providing a suitable tool to perform compatibility tests for xenotransplantation.

[3 cases of postpartum acquired factor VIII inhibitor]. / Tres casos de inhibidor del factor VIII adquirido postparto.

Three cases of postpartum acquired factor VIII inhibitors were seen in our hospital between 1981 and 1989. The clinical picture, which was mild in one patient and severe in two, began several months after delivery (four to nine). After treatment with methylprednisolone, good clinical response was obtained in the three cases. However, normal values of factor VIII:C were obtained more easily with the higher doses of steroids. A new pregnancy and delivery in one of our patients did not induce the reappearance of the inhibitor after several years of follow-up.

[Appearance of a chronic myeloid leukemia in Waldenström's macroglobulinemia]. / Aparición de una leucemia mieloide crónica en el curso de una macroglobulinemia de Waldenström.

Philadelphia chromosome-positive chronic myeloid leukaemia (CML) developed three years after the diagnosis of Waldenström macroglobulinemia (WM) in a patient who had received chlorambucil and prednisone during this time. Treatment with busulphan was started and two and a half years later the died in blastic crisis. Six months before the blastic transformation of the leukaemia the amount of IgM, which remains stable (above normal values) along the chronic phase, initiates a decrease, reaching normal values in the phase of acute transformation. CML is a very rare second neoplasia in patients receiving anticancer drugs. The correlation between the decrease of IgM and the blastic crisis is very interesting. We do not find an explanation for this finding although similar observations have been described heralding the appearance of second neoplasias in WM, and one might speculate with a possible role of IgM in the control of chronic phase of the myeloid leukaemia or if the overgrowth of blastic cells could overwhelm the development of lymphoplasmacytoid cells.