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.

Five-headed superior omohyoid.

The omohyoid is an infrahyoid muscle with two bellies. It is responsible for lowering and positioning of the hyoid bone. It is morphologically variable in the origin, insertion and morphology of its bellies. Quantitative variations of the superior belly of the omohyoid muscle are not common. We present a case of a five-headed superior omohyoid, and a short clinical review related to this muscle. All the bellies had their origin in an intermediate tendon and were attached to the hyoid bone. The volume of its superior part was greater than usual. Knowledge of the anatomy of this muscle is important, especially for surgeons operating in the anterolateral neck region.

Molecular identification of T cells that respond in a primary bulk culture to a peptide derived from a platelet glycoprotein implicated in neonatal alloimmune thrombocytopenia.

Neonatal alloimmune thrombocytopenia induced by the human platelet alloantigen 1a (HPA1a) is characterized by generation of alloantibodies by a mother who is homozygous for the HPA1b alloantigen and almost always HLA-DRB3*0101. The disease is viewed as B cell mediated but the linkage with HLA is indicative of a role for T cells. The HPA1a and HPA1b allotypes are defined, respectively, by Leu and Pro at amino acid 33 of the beta-chain of the platelet integrin GPIIbIIIa (alpha(IIb)beta3). Under the assumption that the same polymorphism may control both the B cell epitope and constitute the MHC-bound peptide, we restimulated PBMC from a woman with an affected child with a synthetic peptide from this polymorphic region. Molecular analysis of the responding T cell repertoire identified two T cells which predominated in cultures stimulated with the alloantigen peptide and which were absent in cultures with the autoantigen peptide. In spite of the use of different V families, sequence of the CDR3 region of the T cell receptor (TCR) beta-chain revealed the presence of a shared motif, L-P-S/T. Oligonucleotide probes specific for the CDR3 sequence indicated that these T cells were present in the PBMC at the highest levels immediately after delivery of the affected infant and their frequency dropped at later times.

HIV antibody status and immunological abnormalities in Polish haemophiliacs.

Sera of 520 multitransfused haemophiliacs were examined for antibody to HIV; 447 patients had haemophilia A and 73 had haemophilia B. In 382 patients with haemophilia A and in 62 with haemophilia B solely Polish-made blood products were used for replacement therapy. The remaining haemophiliacs had also received imported clotting factor concentrates prior to the investigation. Only 8 patients (haemophilia A - 7, haemophilia B - 1) developed anti-HIV and all of them had been exposed to commercial concentrates. The analysis of T-cell subsets demonstrated an inverted T4/T8 ratio (less than 1.0) in 7 (30%) of the 23 haemophiliacs treated solely with domestic cryoprecipitate and in 3 (37%) of the 8 seropositive recipients of commercial concentrates. The most frequent alteration in both subgroups was a reduced ratio with either normal absolute numbers or an increase in T8 cells. Increased serum IgG levels were found in 82% of the users of cryoprecipitate and in 75% of the seropositive patients. Serum beta-2-microglobulin level was elevated in 69 and 62% of each subgroup, respectively. The observed immunological abnormalities, at least in the cryoprecipitate treated subgroup, may be causally related to factors other than HIV infection.

Molecular analysis of T cell repertoires. Spectratypes generated by multiplex polymerase chain reaction and evaluated by radioactivity or fluorescence.

The analysis of T-cell repertoires has been facilitated by the introduction of methods in which the length heterogeneity of the third complementarity region (CDR3) is used to further refine V-family-specific PCR. We call our implementation of this technique T-cell spectratyping. This method is especially important in analysis of specific expansion or retention of T cells in human immune system function. Current methodologies are cumbersome in the number of PCR reactions and gels needed for complete analysis of TCR BV repertoires. We describe here the optimized conditions for using 11 TCR BV primer pairs in multiplex PCR which allow for a more compact analysis. In addition, the two primers act as controls for each other in the PCR. The use of these primers is shown using either fluorescent or radiolabeled constant primers. The two labeling methods give comparable results. Fluorescent primers avoid the difficulties associated with use of radioactivity. Autoradiography with 32P-labeled primers is simpler, requiring less instrumentation.

Trials of rosette test application for determination of immune response to antigen D from the Rh system in blood donors immunized with human erythrocytes.

Periodic control examinations of Rh-negative blood donors sensitized with human Rh-positive erythrocytes were performed to establish the differences in the levels of T and B lymphocytes and their correlation with the production of anti-Rh D antibodies. The investigations were carried out on 10 healthy blood donors in the initial period of immunization and 8 blood donors who had received multiple injections of these erythrocytes and who were producing anti-Rh antibodies. The control group included 10 non-immunized blood donors. The percentage of T and B lymphocytes was calculated on the basis of the rosette tests (E, EA, EAC) while the level of antibodies produced in serum was determined by serological methods. During immunization of blood donors no significant differences were observed in the level of T lymphocytes, while the level of B lymphocytes increased, especially in the 3rd week after the 1st or 2nd injection of erythrocytes and this was not correlated with the production of anti-Rh D antibodies in later period.

Shedding of Fc receptor from mononuclear cells and their ability of binding IgG1 and IgG3 anti-Rh/D antibodies.

Fc receptors for IgG1 and IgG3 on peripheral blood lymphocytes and monocytes were studied before and after temperature shift from 4-37 degrees C. The investigations were performed in the EA test using human erythrocytes sensitized with anti-Rh/D/antibodies of IgG1 (EA IgG1) and IgG3 (EA IgG3) subclasses. It occurred that lymphocytes and monocytes were able to bind IgG1 and IgG3 antibodies before and after shedding, however, lower percentage of rosette was observed after temperature shift. This decrease was similar in the EAIgG1 and EAIgG3 tests. The supernatants obtained during shedding occurred to contain active Fc receptors since the inhibition of rosette formation was obtained after the incubation of sensitized erythrocytes with these supernatants. IgG1 as well as IgG3 myeloma proteins inhibited rosette formation in both EAIgG1 and EAIgG3 tests. Our data might suggest that IgG1 and IgG3 anti-D antibodies are able to bind to the same Fc receptor on lymphocytes as well as on monocytes.

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.

Sensitivity of automated red cell antibody detection.

The principle of detecting red cell antibodies by automated technique using the two-channel Technicon Auto-Analyser with some modification is described. The sensitivity of automated methods in detecting antibodies of different specificities was determined. The study was carried out with 76 diagnostic human sera produced by the Institute of Hematology, Transfusion Centers and commercial firms: anti-RhD, -C, -E, -CW, -c, -e, anti-K, -k, Kpa, anti-Fya, -Fyb, anti-Jka, -Jkb, anti-M, -S, -s, anti-P1, anti-Lua, anti-Lea, -Leb, anti-H, -A1, anti-I, anti-Lua and anti-Vel. A majority of the antibodies were detected by both automated methods with greater sensitivity than by manual methods. Greatest sensitivity was observed in the detection of anti-S, -Vel and antibodies of the Rh system. Anti-Leb and anti A1 antibodies were not detected. The studies are the basis for introduction of the automated technique into routine use for antibodies in blood donors and recipients.

T cell immunobiology of alloimmune thrombocytopenias.

Allorecognition and generation of alloantisera depend on T cell help. Here we summarize our recent work on identifying the T cells that recognize a specific platelet alloantigen generated by a Pro to Leu polymorphism in beta-chain of the platelet integrin alphaIIb beta3. The ability to generate alloantibodies is restricted by HLA-DRB3*0101. By measuring peptide binding to HLA-DRB3*0101, we have shown that the polymorphism controls the generation of the T cell epitope. This is not a result of the polymorphism changing a T cell contact residue, but rather by its generating a peptide anchor. The result is a directional antibody response in which only the beta-chain that is antigenic is the one that produces a peptide that binds to the HLA-DR. This mechanism of generating alloantibodies may be a paradigm for a whole class of responses.

Automatic Marsh method for detection and identification of erythrocyte antibodies in patients and blood donors.

Using a Technicon autoanalyzer method of Marsh 752 sera of patients, Rh-negative subjects in the initial stage of immunization with Rh-positive blood, and healthy blood donors were investigated. In 56 sera (8.7%) positive reactions with standard erythrocytes were found. In 21 of these cases (36%) specificity of antibodies was established. They were mostly alloantibodies or autoantibodies against Rh antigens. In 1 case anti-Ika alloantibody and in 1 case anti-K autoantibody were demonstrated. Antibodies with undetermined specificity belonged to the so called "analyzer antibodies", which are detected only by the automatic methods, and probably have no clinical importance. The possibility of recording by the autoanalyzer of subthreshold levels of alloantibodies undetected by other methods may be of great practical importance of patients receiving multiple transfusions of blood and for immunized blood donors.

Disturbances of cell-mediated immunity in ornithosis.

27 cases of ornithosis were observed during an epidemia in 1980 in Kielce and subsequently followed with respect to immunological characteristics of peripheral blood lymphocytes. Blastic transformation of these cells was tested after stimulation in vitro with three different mitogens. Identification of peripheral blood T and B lymphocytes was done using rosette tests (E,EA,EAC) and the occurrence of surface immunoglobulins was determined by the immunofluorescent method with polyvalent anti-immunoglobulin serum. The counts of T and B lymphocytes in the peripheral blood were normal throughout the whole period of the observation, but from the 3rd week on a significant impairment of 3H-thymidine incorporation into the cells stimulated with Con A was observed, and from the 10th week on, this impairment appeared also in cells stimulated with PHA and PWM. These observations revealed considerable disturbances in cell-mediated reactivity in patients with ornithosis and seem to be connected with chronic infection with Chlamydia psittaci.

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.

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.

[Feto-maternal incompatibility due to the platelet specific antigens: frequency, diagnosis and general rules of management]. / Konflikt matczyno-plodowy w zakresie swoistych antygenów krwinek plytkowych--wystepowanie, diagnostyka i zasady postepowania.

In this review actual problems of the frequency, diagnosis and general rules of management of the neonatal alloimmune thrombocytopenia (NAIT) due to platelet antigens are presented.

[Autoimmune thrombocytopenia in pregnant women: diagnosis, treatment and prevention of complications in children]. / Autoimmunologiczna maloplytkowosc u kobiet w ciazy: rozpoznawanie, leczenie, zapobieganie powiklaniom u dzieci.

9 pregnancies (in 6 women) complicated by autoimmune thrombocytopenia were studied. Corticosteroids and/or azathioprine and/or intravenous immunoglobulins (IgG i.v.) were administered to 5 women during pregnancy. 1 complete remission (CR) was achieved after 3 mo. of treatment by corticosteroids and azathioprine. In peripartum period the treatment was administered in 7 cases (corticosteroids, IgG i.v., platelet transfusion). There were no haemorrhagic complications during peripartum period (8 vaginal delivery, 1 cesarean section). In 3 of 5 examined cases IgG antiplatelet antibody was detected. Study group was to small to define the correlation between level of antiplatelet antibodies and neonatal thrombocytopenia. 10 infants were born. 4 infants (including siblings) were found to have normal platelet counts immediately after delivery. They mothers were treated by IgG i.v. in 2 cases and 1 women in CR at the time of delivery. 4 infants were thrombocytopenic but required no treatment. Duration time of thrombocytopenia was 1 to 4 weeks. In these cases mothers were treated only by corticosteroids or had no therapy.

[Immunology of blood platelets]. / Immunologia krwinki plytkowej.

Platelet specific antigen systems, their molecular structure and localization in cell membrane is presented. New techniques for the detection of platelet antibodies are discussed.

[Platelet antigens]. / Antygeny krwinek plytkowych.

Platelet specific antigens and platelet antigens shared with other peripheral blood cells are presented. The molecular characteristics of membrane glycoproteins which are platelet specific determinants is discussed.

Analysis of 15 cases with platelet EDTA-dependent antibodies.

In 15 patients with thrombocytopenia EDTA-dependent platelet antibodies (IgM agglutinins active in 20 degrees C) were detected. These antibodies were found in patients with autoimmune diseases, infections and neoplasma as well as in healthy persons (including pregnant woman). In 10 persons pseudothrombocytopenia (PTCP) was diagnosed since the low platelet counts were found only in EDTA-blood, and the patients did not have bleeding symptoms. In 5 other cases EDTA-dependent antibodies were not the only cause of thrombocytopenia since the low platelet counts were also observed in the citrate-blood and occassionally the bleeding symptoms occurred; in 3 of them in addition autoantibodies were suspected. Due to the proper diagnosis of PTCP, in five patients unnecessary corticotherapy was discontinued, one person avoided splenectomy, in two patients previously postponed surgery was performed and one person could be passed for employment.