Analysis of leucocyte antibodies, cytokines, lysophospholipids and cell microparticles in blood components implicated in post-transfusion reactions with dyspnoea.

BACKGROUND AND OBJECTIVES: Post-transfusion reactions with dyspnoea (PTR) are major causes of morbidity and death after blood transfusion. Transfusion-related acute lung injury (TRALI) and transfusion-associated circulatory overload (TACO) are most dangerous, while transfusion-associated dyspnoea (TAD) is a milder respiratory distress. We investigated blood components for immune and non-immune factors implicated in PTR. MATERIAL AND METHODS: We analysed 464 blood components (RBCs, PLTs, L-PLTs, FFP) transfused to 271 patients with PTR. Blood components were evaluated for 1/antileucocyte antibodies, 2/cytokines: IL-1ß, IL-6, IL-8, TNF-α, sCD40L, 3/lysophosphatidylcholines (LysoPCs), 4/microparticles (MPs) shed from plateletes (PMPs), erythrocytes (EMPs) and leucocytes (LMPs). RESULTS: Anti-HLA class I/II antibodies or granulocyte-reactive anti-HLA antibodies were detected in 18.2% of blood components (RBC and FFP) transfused to TRALI and in 0.5% of FFP transfused to TAD cases. Cytokines and LysoPCs concentrations in blood components transfused to PTR patients did not exceed those in blood components transfused to patients with no PTR. Only EMPs percentage in RBCs transfused to patients with TRALI was significantly higher (P < 0.05) than in RBCs transfused to patients with no PTR. CONCLUSION: Immune character of PTR was confirmed mainly in 1/5 TRALI cases. Among non-immune factors, only MPs released from stored RBCs are suggested as potential mediators of TRALI. Our results require further observations in a more numerous and better defined group of patients.

Further characterization of the thrombasthenia-related idiotype OG. Antiidiotype defines a novel epitope(s) shared by fibrinogen B beta chain, vitronectin, and von Willebrand factor and required for binding to beta 3.

A patient (OG) with Glanzmann thrombasthenia became refractory to platelet transfusion after the production of an immunoglobulin G (IgG) isoantibody (Ab1) specific for the integrin subunit beta 3. To determine the frequency at which the OG idiotype is found in the general population and in immune-mediated disease states, we developed a rabbit polyclonal antibody (Ab2) specific for affinity-purified OG anti-beta 3 Fab. The binding of Ab2 to Ab1 is inhibited by purified alpha IIb beta 3. Ab2 als binds to IgG specific for alpha IIb beta 3 obtained from one nonrelated Glanzmann thrombasthenia patient ES who has developed isoantibodies of similar specificity. On the other hand, Ab2 does not recognize alpha IIb beta 3-specific antibodies produced by two Glanzmann thrombasthenia patients, AF and LUC, who have developed isoantibodies with specificities distinct from that of the OG isoantibody. Moreover, Ab2 does not recognize alpha IIb beta 3-specific antibodies developed by three representative patients with (autoimmune) thrombocytopenic purpura or six representative patients with alloimmune thrombocytopenias, nor does it bind to IgG from any of 13 nonimmunized individuals. We have found that Ab2 also binds to selected protein ligands of alpha IIb beta 3 namely, fibrinogen, vitronectin, and von Willebrand factor, but not to other protein ligands or control proteins, such a fibronectin, type I collagen, and albumin. The epitope(s) recognized by Ab2 on each adhesive protein are either very similar or identical since each protein can inhibit the binding of Ab2 to any of the other proteins. The epitope on fibrinogen recognized by Ab2 resides in the B beta chain, and is likely contained within the first 42 amino acids from the NH2 terminus. Since OG IgG inhibits fibrinogen binding to alpha IIb beta 3, the specificity of the OG idiotype defines a novel binding motif for the integrin alpha IIb beta 3 that is shared by fibrinogen, vitronectin, and von Willebrand factor, but distinct from previously described RGD-containing sites on the fibrinogen, A alpha chain or the fibrinogen gamma chain COOH-terminal decapeptide site. Our findings reported here represent an excellent example of molecular mimicry in which an antigen-selected, IgG inhibitor of alpha IIb beta 3 function shares a novel recognition sequence common to three physiologic protein ligands of that receptor.

Molecular and serological characterization of hepatitis B virus genotype A and D infected blood donors in Poland.

Hepatitis B virus (HBV) genotypes have distinct geographical distributions and influence severity of clinical outcome and response to antiviral therapies. HBV polymorphism in HBV surface antigen (HBsAg) positive first time blood donors from Poland was examined. HBV serological markers and HBV DNA were tested in 170 samples. Whole genome (n = 53) or specific region sequences: pre-S/S and basic core promoter/precore (BCP/PC) region (91 and 154 samples, respectively) were phylogenetically analyzed. The median age of infected donors was 21 years. Anti-HBs, anti-HBe and hepatitis B e antigen were detected in 5%, 92.4% and 10.5% of tested donors, respectively. The HBV DNA load ranged between unquantifiable and 3.1 x 10(10) IU/mL (median: 4.10 x 10(3) IU/mL). Genotypes A2 (81.2%) and D (18.8%) co-circulated. Phylogenetic analyses revealed differences between the genotypes. Viral load and level of HBsAg tended to be lower in genotype D. The median HBsAg/HBV DNA ratio expressed in IU/mL was one for both genotypes, but very low or very high ratios appeared more frequent in genotype D infections. Higher amino acid variability in the surface proteins (median: 4%vs 1.5%; P = 0.01) and in the major hydrophilic region was observed in genotype D (P = 0.01). BCP/PC region analysis revealed the double mutation 1762T/1764A in 49/125 (39.2%) genotype A2 and 6/29 (20.7%) genotype D strains (P = 0.08). Mutations in PC and BCP regions correlated neither with HBsAg nor HBV DNA levels. HBV genotype A2 is dominant in HBsAg positive donors in Poland. Minority genotype D strains are significantly more substituted than genotype A2 strains potentially affecting the course of infection.

Repertoire cloning of a human IgG inhibitor of alpha IIB beta 3 function. The OG idiotype.

A patient (OG) with Glanzmann thrombasthenia became refractory to platelet transfusion following the production of IgG antibodies (Ab1) specific for the integrin subunit beta 3. We generated recombinant VH and VL cDNA libraries using IgG-specific mRNA isolated from OG peripheral blood B-lymphocytes that had been selected for binding to antigen (alpha IIb beta 3 adsorbed to plastic dishes). These antigen-specific B-lymphocytes contain rearranged VH DNA segments that belong exclusively to the VH4 gene family. Recombinant Fab were expressed on the surface of filamentous phage coinfected with VH and VL segments cloned into the phagemid pHEN1 or the phage fd-tet-DOG1. To facilitate selection of the desired recombinant Ab1 Fab, we developed a rabbit polyclonal antibody specific for affinity-purified OG anti-beta 3 Fab (Ab2). Ab2 reacts specifically with Ab1, and this interaction is inhibited by purified alpha IIb beta 3. Following three rounds of phage selection on Ab2 adsorbed to plastic dishes and random reassociation of heavy and light chains, we isolated Ab1 Fab and tested their binding to alpha IIb beta 3. Five Id-positive Fab were selected for further characterization. These Fab use one of two VH genes (H21 or H23) complexed with one of three V lambda genes. Subsequent sequence data demonstrated that all three lambda genes are the same clone L22 which uses a germline V lambda gene segment. Fab using H23 bind to alpha IIb beta 3, while those using H21 do not. Based on sequence homology, both H21 and H23 use VH gene segments belonging to the VH4 gene family. Thus, the idiotype OG is restricted to the VH4 gene family and is the first sequenced prototype of human antibodies that bind close to or at a functional epitope(s) of alpha IIb beta 3.

Critical evaluation of the ADCC test.

The activity of monocytes in the ADCC test against human red blood cells sensitized with anti-Rh antibodies is unquestionable, however, a participation of lymphocytes in this reaction is still controversial. The aim of this paper was to test the cytotoxic activity of monocytes and lymphocytes isolated from human peripheral blood against erythrocytes coated with anti-C + D antibodies of Rh system. Due to some individual changes in the activity of mononuclears observed in the ADCC test, it was of interest to test how this activity changes with time, in individual healthy subjects. Both types of mononuclears were observed to damage the sensitized non-treated as well as papain-treated red cells. The cytotoxic activity of these cells, however, showed substantial deviation in the same person.

Evaluation of human blood monocytes in erythrophagocytosis assay with anti-Rh antibodies.

Results of the phagocytosis assay with monocytes and erythrocytes sensitized with subclass specific anti-Rh(D) antibodies (IgG1, IgG3, IgG1 + 3) are dependent on the selection of sera used for sensitization. Phagocytic abilities expressed as the percentage of active monocytes as well as the number of interacting red cells requires a certain degree of the sensitization of erythrocytes and is higher if serum contains IgG3 antibodies.

Lymphocyte subpopulations in healthy newborns and in immunohemolytic anemia due to Rh incompatibility.

The subpopulations of lymphocytes in cord blood were analyzed in 50 healthy newborns and in 25 with hemolytic disease of the newborns (HDN) using E, EA, EAC rosette tests with sheep erythrocytes, EA rosette test with human erythrocytes and SmIg+ test. A statistically significant decrease of the percent of T lymphocyte and increase in the absolute values of all lymphocyte subpopulations were found in healthy newborns as compared with adults. In the newborns with HDN a correlation was observed between the severity of the disease and the results of rosette tests. Three groups were distinguished: 1) very low values of all rosette tests, severe anemia in newborns, and high titer of antibodies in their mothers, 2) low values of EA rosette tests, less severe anemia antibody titer in the mothers lower than in the first group, 3) rosette tests within normal range, newborns usually without anemia, low titer of maternal antibodies.

DRB polymerase chain reaction fingerprinting, DQA oligotyping and mixed lymphocyte culture results in related bone marrow donors and recipients.

Donor and recipient are usually regarded as well matched for bone marrow transplantation when they are compatible in HLA class I and II antigens and in mixed lymphocyte culture (MLC). However, results of serological typing of class II antigens may be unreliable. Hence, polymerase chain reaction fingerprinting of HLA DRB (PCR FP) was introduced for the screening of related donors for 29 patients awaiting bone marrow transplantation. In addition, the sequence-specific oligonucleotide (SSO) typing of DQA alleles was performed. In 18 pairs the results of DNA analysis methods were compared with the results of MLC. 72% of pairs were HLA DQA compatible and 59% showed compatibility in PCR FP. MLC compatibility was found in 61%. A higher correlation of PCR FP and MLC results was observed.

Immune condition in patients with Hodgkin's disease.

In 40 cases of Hodgkin's disease (24 untreated patients and 16 patients in remission) the immunological humoral and cell-mediated competence was studied using test of hypersensitivity of DNCB and PPD, blastic transformation and the ability of 3H-DNA synthesis in lymphocytes stimulated with PHA and PWM, and in some cases, with PPD. In all patients the concentration of serum immunoglobulins was determined as well. Impairment of immunological reactivity was found in about 40% of patients, on the average, in various stages of the disease and during remissions following treatment with cytostatic agents and radiotherapy. The most frequent abnormality found in 52.5% of cases was impairment of blastic transformation of lymphocytes stimulated with PHA in vitro. In 60% of cases negative results of intradermal test with PPD and in 75% of patients the DNCB test was negative. No correlation could be found between impairment of the immune competence in this disease and the clinical stage and/or type of histological changes in lymph nodes.

Preliminary evaluation of immunotherapy in patients with Hodgkin's disease.

The clinical course of 38 patients with Hodgkin's disease in the stage of remission was evaluated. The material consisted of two groups of patients: 1. submitted to immunotherapy and 2. control group. In 16 cases immunostimulation was performed with BCG vaccine, including four cases treated with BCG vaccine and levamisole and 3 patients were given levamisole only. In some patients, immunotherapy restored normal immunologic reactivity, as shown by the reversion of negative tests of delayed hypersensitivity. In all patients treated with levamisole caused normal blastic transformation of PHA-stimulated lymphocytes. In the group of patients treated with immunotherapy relapses of malignant lymphogranulomatosis were observed in 10.6% of cases, and in the control group--in 15.7%. The patients of both groups remain under continuous clinical surveillance.

[Diagnosis of human parvovirus B19 infection in nonimmune hydrops fetalis]. / Diagnostyka zakazenia ludzkim parwowirusem B19 w nieimmunologicznym obrzeku plodu.

Parvovirus B19 (PV B19) infection was investigated in 29 pregnant women with fetal hydrops, after exclusion of feto-maternal incompatibility within red blood cell antigens, TORCH infections, feto-maternal hemorrhage and genetics reasons. The active viral infection was detected in 9 women (31%) by PCR amplification of DNA B19; in 2 of them IgM and IgG, in 1 IgM and in 4 IgG antibodies were also present. In 6 women (20%) IgG antibodies were only found, but not IgM and DNA B19, which confirmed infection in the past. In addition in 9 cases DNA B19 was evaluated in the fetal blood. The results in the mothers and their fetuses were concordant (4 positive, 5 negative). Our conclusion is that in nonimmune hydrops fetalis, PV B19 infection should be based on the viral DNA evaluation in the blood of mother (or fetus). IgM antibodies, in time of fetal disorders, might not be detected.

[The assessment of the treatment of viral hepatitis C with interferon alpha in children with leukemias and lymphomas]. / Ocena leczenia wirusowego zapalenia watroby typu C interferonem alfa u dzieci z chorobami rozrostowymi ukladu krwiotwórczego.

Interferon alfa in dose 3-6 MU IM was administered during six months 3 times per week in 30 children with chronic hepatitis C with leukaemias and lymphomas. Chronic hepatitis was diagnosed basing on anti-HCV, RNA HCV by PCR, high activity of ALT and histopathologic examination of the liver. The patients were followed-up for 12 months after IFN therapy. Sustained normalization of ALT levels, together with serum HCV RNA elimination was considered as a complete response and achieved in 33% children. Partial response (ALT normalization without serum HCV RNA elimination) was recorded in 57% patients. Anti HCV antibody was detected in all children during the time of observation.

[The polymerase chain reaction (PCR) and its application in transfusion medicine and hematology]. / Reakcja lancuchowa polimerazy (PCR) i jej zastosowanie w transfuzjologii i hematologii.

The new technique, polymerase chain reaction, used for in vitro DNA fragments amplification is described. The conditions of the reaction and methods of data analysis are discussed as well as the usefulness of this technique in research and medical practice.

[Evaluation of the microplate enzyme-linked assay with isolation of glycoprotein IIb/IIIa from platelet membranes: comparison with immunofluorescent tests and monoclonal antibodies]. / Ocena wykrywalnosci przeciwcial przeciwplytkowych przy uzyciu testu enzymatycznego z wyizolowana glikoproteina IIb/IIIa blony krwinki plytkowej: porównanie z testem immunofluorescencyjnym oraz testem enzymatycznym z uzyciem przeciwcial monoklonalnych.

The enzyme test with GPIIb/IIIa has been used to detect antibodies in 500 sera from patients with thrombocytopenia and mothers of infants with thrombocytopenia. The results were compared with antibody detection in the platelet suspension immunofluorescence test (PIFT) and in the monoclonal antibody immobilization of platelet antigens (MAIPA). Platelet antibodies were detected in 125 sera (25%). In 46.2% the results were positive in all three tests. However, in 38.4% the ELISA was negative while the PIFT and MAIPA were positive. On the other hand, in 14.4% the ELISA was positive while the PIFT and/or the MAIPA were negative. The ELISA appears to be useful in the detection of anti-HPA-1a antibodies, which are the main cause of AIMN and PTP. This test, however, seems to be less useful in the detection of autoantibodies. The significance of the ELISA in the detection of platelet alloantibodies responsible for refractoriness to platelet transfusions is not clear yet and requires further investigations.

[A relationship between the breakpoint of the bcr gene and some hematologic parameters in patients with chronic myelogenous leukemia]. / Zaleznosc miedzy miejscem zlamania genu bcr a niektórymi parametrami hematologicznymi u chorych na przewlekla bialaczke szpikowa.

We examined the hybrid bcr/abl mRNA present in 59 patients with chronic myeloid leukemia, using the reverse transcription method and polymerase chain reaction. Bcr/abl gene was found in 98% of patients. 60% of patients had b3a2 type of translocation, 40% type b2a2. Patients with b3a2 type had higher platelet count at diagnosis presentation than patients with b2a2. Other hematological data were similar in both groups.

[Diagnostic evaluation of HLA sera obtained as a result of cooperation between the Institute of Hematology and blood donation stations]. / Ocena diagnostyczna surowic ukladu HLA uzyskiwanych we wspólpracy Instytutu Hematologii ze stacjami krwiodawstwa.

The aim of the cooperation between the Laboratory of Leucocyte and Platelet Immunology, Institute of Haematology and Blood Donation Stations was typing of anti-HLA sera from the blood of pregnant women. During 18 months of this cooperation 477 sera were studied. In 255 sera (53.1%) lymphocytotoxic antibodies were demonstrated and their specificity was determined. The types anti-HLA sera will be used for determination of HLA antigens in blood donors donating blood in the Stations. The results of these determinations, technical problems and conclusions drawn from this cooperation are discussed. Continuation of this cooperation will contribute to further limitation of the import of anti-HLA sera.