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.

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.

The relevance of HPA-15 antigen expression for anti-HPA-15 antibody detection.

INTRODUCTION: The HPA-15 antigen system is characterized by a low antigen expression on platelets. The antibodies against this antigen are implied in fetal/neonatal alloimmune thrombocytopenia (F/NAIT), post-transfusion purpura, and refractoriness to platelet transfusions. Detection of these antibodies appears to be related to the level of HPA-15 expression on the platelets used in the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay. METHODS: We performed genotyping of 300 healthy blood donors for HPA-15 by TaqMan real-time PCR technology, and the HPA-15 antigen expression was investigated in 13 HPA-15aa and 19 HPA-15bb individuals. We also investigated the relevance of HPA-15 antigen expression on donor platelets used in MAIPA for antibody detection in 223 multitransfused hematological patients and 271 women with suspected F/NAIT. RESULTS: In Polish donors, the HPA-15a allele frequencies were lower than the HPA-15b (0.480 vs. 0.515). We identified three HPA-15 expression groups: high (36.7 ± 8.36 MFI - eight cases), medium (19.5 ± 6.2 MFI - 21 cases), and low (6.5 ± 5.9 MFI - three cases). The HPA-15 expression was stable over time. The HPA-15aa and HPA-15bb platelets with high antigen expression were used for anti-HPA-15 antibody detection; anti-HPA-15 antibodies were detected in 4/223 (1.8%) patients receiving multiple transfusions but in none of the 271 women with suspected F/NAIT. Further examination of the four sera by MAIPA with various platelets revealed the optical density in the assay to be closely related to the level of HPA-15 antigen expression. CONCLUSION: Anti-HPA-15 antibody detection should be based on carefully selected platelets with high HPA-15 expression level.

Reticulated platelets as a marker of platelet recovery after allogeneic stem cell transplantation.

Reticulated platelets (RP) are the youngest forms of platelets in blood and reflect the rate of bone marrow platelet production. In the present study, we used flow cytometric analysis to determine the percentage of RPs in patients undergoing allogeneic stem cell transplantation. We investigated 10 patients after transplantation from HLA identical siblings: five with acute myeloid leukemia (AML), four with chronic myeloid leukemia (CML), and one patient with myelodysplastic syndrome (MDS). Of the patients examined, four patients underwent allogeneic bone marrow transplantation and six patients underwent peripheral blood stem cell transplantation. It was observed that the initially reduced percentage of RPs (2.9 +/- 1.7%; mean +/- SD) was significantly higher (P = 0.0109) in all patients (13.6 +/- 6.4%) in the following 10-26 days. The RP percentage peak preceded the recovery of peripheral platelet count up to 45.6 x 10(9)/l on average by 3 days. We found no difference in RP% between the AML and CML patients but we did observe that in CML patients the RP percentage increased on average 7 days earlier than in AML patients. The elevated RP percentage reflects increased bone marrow regeneration and can be considered an additional marker of thrombopoietic recovery in the patients undergoing allogeneic stem cell transplantation.

The effect of platelet autoantibodies on the course of the disease and clinical response of patients with idiopathic thrombocytopenic purpura.

In this study, we evaluated the response to treatment of 409 idiopathic thrombocytopenic purpura (ITP) patients who were tested for the presence of platelet-associated autoantibodies by direct-platelet immunofluorescence test (PIFT) and for the presence of plasma antibodies directed against the GPIIb/IIIa, GPIb and GPIa/IIa by monoclonal antibody immobilization of platelet antigens (MAIPA). In patients with platelet autoantibodies in comparison with patients without antibodies more frequently were observed the chronic form of disease (83.5%vs. 68.5%) and severe symptoms of haemorrhage diathesis (17.3%vs. 6.9%). Evaluation of the treatment response (to corticosteroids, immunosuppressive drugs and splenectomy) referred to patients with complete response, e.g. complete remission defined as platelet count of >100 x 10(9)/l for at least 2 years. The percentage of complete response in the whole population of ITP patients, both with and without autoantibodies regardless of the method of treatment, was similar (about 54%). However, the presence of platelet autoantibodies had effect on patients treated with corticosteroids: complete response approximately 71% (36/51) of patients with autoantibodies and in 60% (72/120) of patients without antibodies, as well as in patients treated with immunosuppressive drugs (cyclophosphamide, azathioprine, vincristin and vinblastin); complete response approximately 51% (11/21) of patients with autoantibodies and in 34.8% (6/17) of patients without autoantibodies. The presence of autoantibodies had no effect on the response of splenectomy patients.

Transfusion-related acute lung injury and leucocyte-reacting antibodies.

BACKGROUND AND OBJECTIVES: Transfusion-related acute lung injury (TRALI) is underdiagnosed and underreported. This is why we present cases suspected for TRALI, in which leucocyte antibodies were examined. MATERIAL AND METHODS: We analysed 44 patients with respiratory insufficiency, related to transfusion, who met criteria of acute lung injury (ALI). Lymphocyte and granulocyte antibodies were examined in donors and patients by six methods. RESULTS: Based on recent trends, we divided patients into two groups: TRALI (without risk factors for ALI) and possible TRALI (with probable risk factors). The incidence of antibodies was 68.2%, the majority were human leucocyte antigen (HLA) class I and/or II, the minority were non-specific granulocyte antibodies; half of all detected antibodies, however, reacted with granulocytes. Antibodies were found in 17 donors (more often in TRALI than in possible TRALI) and in 19 patients (in four - suspected to be of the donor origin, which would diminish the number of antibodies to 15). In seven available cases, we observed cognate antigen and/or positive cross-match. In the majority of patients, TRALI occurred after transfusion of red cells, in 56.2%- stored above 14 days; all the units were non-leucoreduced. Lookback in two donors showed that transfusions in 20 patients did not result in reported TRALI, even in the patient with cognate antigen. CONCLUSIONS: Our clinical observations suggest that to distinguish between TRALI and possible TRALI is difficult and the results are equivocal - it is worth considering whether it can be omitted. We have confirmed that antibodies are involved in TRALI, although their role is very complex. The role of stored red blood cells in the development of TRALI requires further observations in comparison with a control group of patients without TRALI.

Leucocyte antibodies in blood donors and a look back on recipients of their blood components.

BACKGROUND AND AIM: The role of leucocyte antibodies in donors is poorly understood in pathogenesis of transfusion-related acute lung injury (TRALI). We examined antibodies in donors and traced recipients transfused with their blood components. MATERIAL AND METHODS: Antibodies were examined in 1043 donors by five methods, look back performed in 26 recipients. RESULTS: Anti-human leucocyte antigen detected by enzyme-linked immunosorbent assay in 9.8% women but none in men. Specificities identified using FlowPRA, antibodies detected after several months. TRALI reported in one recipient from immunized donor. In 11 of 26 recipients without TRALI, cognate antigens were identified. CONCLUSION: Detection of antibodies in donors cannot predict TRALI, even in recipients with cognate antigen(s).

The risk of antibody formation against HNA1a and HNA1b granulocyte antigens during pregnancy and its relation to neonatal neutropenia.

The evaluation of immunization by the HNA1a and 1b antigens during pregnancy was based on (i) their genotyping in 1038 unselected mothers and newborns of homozygous mothers, (ii) granulocyte counting in all born infants and (iii) examination of granulocyte antibodies in maternal sera if an HNA1 incompatibile child was born. A total of 548 (52.8%) mothers were heterozygous--thus further examinations were not done. Four hundred and ninety (47.2%) were homozygous, of whom 203 (41.3%) delivered an incompatible child, i.e. 19.6% of all the infants. Among available sera from 195 mothers with feto-maternal incompatibility, the granulocyte-specific antibodies were found in nine (4.5%); six of these (3%) were HNA1 (four anti-1a, two anti-1b), and in three others the specificity was not determined. In the remaining 28 sera, the only antibodies detected were HLA. Hence, six out of 1000 pregnant women can be expected to develop anti-HNA1. In none of the newborns was the cord neutrophil count below 1.5 x 109 L-1 and signs of infection found, thus the incidence of NAIN seems to be lower than 1 per 1000 infants. A comparison with our previous, unpublished data suggests that the incidence of severe NAIN is roughly 1 per 6000 (four cases among 24101 newborns).

Isoimmune neonatal neutropenia due to anti-Fc(gamma) RIIIb antibody in a mother with an Fc(gamma) RIIIb deficiency.

A rare case of neutropenia in a newborn due to anti-Fc(gamma) RIIIb antibody is described. The newborn, born from the 5th pregnancy, had severe infection and no neutrophils. Full clinical and neutrophil count recovery was observed when the child was 5 weeks old. In maternal serum, panreactive granulocyte alloantibodies were detected. The mother's and her two sisters' granulocytes appeared to be Fc(gamma) RIIIb deficient as found using pheno- and genotyping methods. All of them were healthy. The anti-Fc(gamma) RIIIb specificity of antibodies was identified by the monoclonal antibody immunobilization of neutrophil antigen assay. Such antibodies were not found in both sisters with the Fc(gamma) RIIIb deficiency, although they were pregnant, one of them on the seventh occasion.

[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.

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.

Neonatal thrombocytopenia: incidence, serological and clinical observations.

In this study, platelet counts were determined from the cord blood of consecutive 9142 newborns. Neonates with known autoimmune thrombocytopenia were not included. The platelet count < 100 x 10(9)/L was found in 64 newborns. In five of them, neonatal alloimmune thrombocytopenia (NAIT) was diagnosed. The overall incidence of neonatal thrombocytopenia was 0.7%, the incidence of NAIT was about 10 times less. Serological and clinical observations are summarized from 238 thrombocytopenic newborns (54 from the above group and 184 previously referred to serological investigations). All of the newborns were divided into two groups: NAIT (46 cases) and other thrombocytopenias (192 cases). Among platelet-specific antibodies in NAIT, 91.4% were anti-HPA-1a, the rest were anti-HPA-1b and anti-HPA-5b. In the majority of the cases, antibodies were detectable by the platelet suspension immunofluorescence test (PSIFT) and monoclonal antibody immobilization of platelet antigens (MAIPA) assay. In 19.6% cases, antibodies were detectable by MAIPA only. In 10.9% of these cases, antibodies were undetectable. Thrombocytopenia < 50 x 10(9)/L and hemorrhagic diathesis were more often observed in NAIT than in other thrombocytopenias, whereas associated disorders that could contribute to thrombocytopenia, here observed almost only in the latter group. We also report certain other observations, such as the presence of anti-HLA antibodies, a rise in the anti-HPA-1 a antibody titer after infection without pregnancy, and a higher incidence of petechiae in nonimmune thrombocytopenia as compared with the incidence of low platelet counts.

An integrin polymorphism that defines reactivity with alloantibodies generates an anchor for MHC class II peptide binding: a model for unidirectional alloimmune responses.

Polymorphic proteins are often the targets of T and B lymphocytes in alloimmune responses. The polymorphic residue 33 of integrin beta3 is responsible for both a B cell response and a T cell response. Leu at position 33 controls the epitopes recognized by alloantibodies generated in individuals homozygous for Pro at 33. The alloantibody response shows a tight class II MHC restriction, and T cells have been identified that respond to the alloantigen. As part of the T cell response, the alloantigen could be recognized as foreign, and the autoantigen would be ignored due to self-tolerance. Alternatively, the polymorphism could change the ability of the peptide to bind. We tested these two possibilities using synthetic peptides corresponding to the polymorphic region and insect cell-derived class II MHC. The Leu33-containing peptide bound to the restricting MHC allele, whereas the Pro33 peptide did not. Thus, the presence of Leu at position 33 generates an anchor specific for the restricting MHC allele and helps define the peptide binding motif of this allele, HLA-DRB3*0101 (DR52a). These data indicate that certain alloresponses can be viewed entirely as foreign because the polymorphism generates a functional anchor residue in a peptide that otherwise would not bind. On the basis of these results, we would predict a unidirectional alloantibody response as the lack of binding of the Pro33 peptide would preclude T cell help. This could explain the observation that alloantibodies to the Pro33 allele are very rare.

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.

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.