Laboratory diagnosis of heparin-induced thrombocytopenia: advantages of a functional flow cytometric test in comparison to the heparin-induced platelet-activation test.

Nearly one third of patients with heparin-induced thrombocytopenia (HIT) will progress to overt thrombosis. Owing to the severity of HIT, a reliable prompt diagnosis is mandatory. In this study 248 consecutive samples from patients referred to our laboratory for HIT diagnosis and 97 specimens from normal controls were prospectively evaluated in parallel using the heparin-induced platelet aggregation (HIPA) test and a flow cytometric (FC) test. The HIPA test resulted in 214 negative, 17 indeterminate and 17 positive samples of patients. The FC method detects activated platelets induced by heparin-immune complexes using the highly sensitive recombinant probe annexin V and pooled platelets from multiple donors. The criteria for positive FC test results included an increase in platelet activiation of at least 11% at 0.3 IU/mL heparin concentration in the tube, and a ratio of more than 1.5 between platelet activation at 0.3 and 200 IU/mL heparin. According to the cut-off level 17 patients who showed indeteminate HIPA test results had 14 negative and 3 indeterminate corresponding FC test results. Only one of these patients (HIPA test indeterminate, FC test indeterminate) had no other obvious medical cause for thrombocytopenia than HIT. Infections or inflammations did not show any association with the FC test results, whereas thromboembolic events displayed a significant patelet activation at pharmacological heparin concentration. Therefore the FC test is associated to the complications of HIT. In conclusion, the FC test, which is fast and practical, showed a good agreement with the HIPA test and may be an accurate and useful test for HIT.

Changes in platelet membrane glycoproteins before bone marrow transplantation and after engraftment--a pilot study.

Thrombotic complications are observed in patients undergoing bone marrow transplantation despite thrombocytopenia and impaired coagulation due to liver function disturbances. Endothelial cell damage which is involved in the pathogenesis of major transplant related complications like graft-versus-host disease, veno-occlusive disease, sepsis or microangiopathy may be a contributing factor. Little is known about platelet function in bone marrow transplant recipients. In order to study functional alterations in circulating platelets we investigated unstimulated and ADP-stimulated platelets of 10 bone marrow transplant recipients ex vivo by flow cytometry in a pilot study using a panel of monoclonal antibodies to characterize changes in membrane glycoproteins. Samples were collected before and during conditioning and at three timepoints after engraftment. 10 healthy volunteers served as controls. Platelets of bone marrow transplant recipients showed partly a significant, higher expression of surface bound fibrinogen, activated fibrinogen receptor, and glycoprotein Ib as compared to controls. P-selectin, a marker of platelet degranulation was significantly elevated after ADP-induced stimulation at all timepoints compared to controls. Only marginal differences were found for GP IIb/IIIa surface expression. The data point to an increased platelet activation state in bone marrow transplant recipients which might contribute to the thrombotic phenomena observed in these patients.

Molecular defects of the RHCE gene in Rh-deficient individuals of the amorph type.

The deficiency of Rh proteins on the red blood cells from individuals of the Rhnull amorph type may be the result of homozygosity for a silent allele at the RH locus. This phenotype is also associated with the lack or reduced expression of glycoproteins (Rh50, CD47, LW, and glycophorin B), which interact with Rh polypeptides to form the multisubunit Rh membrane complex. In this study, we describe two molecular alterations affecting the RHCE gene in two unrelated Rhnull amorph individuals bearing Rh50 and CD47 normal transcripts. The first type of mutation, located at the donor splice-site in intron 4, induced the activation of two cryptic splice-sites within this intron and one such site in exon 4 that all generated aberrant transcripts. The second type of mutation affected the coding region and introduced a frameshift and a premature stop codon resulting in a shorter predicted protein (398 v 417 residues), including a completely different C-terminus of 76 amino acids. This suggests that protein folding and/or protein-protein interaction mediated by the C-terminal domain of the Rh proteins may play a role in the routing and/or stability of the Rh membrane complex.

Prognostic influence of blood transfusion on minimal residual disease in resected gastric cancer patients.

BACKGROUND: The effect of blood transfusion on prognosis of resected cancer patients has been debated controversially. Therefore, we raised the hypothesis that transfusion-associated immunomodulation affects minimal residual disease after curative tumour resection, an unknown and uncontrolled phenomenon in all former studies which might significantly influence long-term prognosis. PATIENTS AND METHODS: 104 patients of a prospective study with curatively resected gastric cancer were stratified according to the immunocytochemical detection of disseminated tumour cells in bone marrow and the prognostic impact of allogeneic blood transfusion was tested. Multiple sequential bone marrow aspirations during follow-up were performed in 74 patients to investigate the blood transfusion effect on long-term development of this systemic disease component. RESULTS: Whereas in patients with tumour cell detection in bone marrow a significant association of blood transfusion and survival was seen (P = 0.048; relative risk 2.91; 95% CI 1.51-5.61), this was not found in patients without disseminated tumour cells (P = 0.129). Quantitative development of tumour cells in bone marrow during follow-up demonstrated a significant quantitative increase of tumour cells in transfused patients only (P = 0.028). CONCLUSION: These findings might explain the contradictory results of recent studies and suggest that the prognostic effect of transfusion is mediated through an impact on minimal residual disease in resected cancer patients.

The Rhesus D-negative phenotype is an independent predictor of poor prognosis in curatively (RO) resected gastric cancer patients.

Among gastric cancer patients, the Rhesus D-negative phenotype correlated with increased tumour recurrence [all patients, n = 83, P = 0.026; curatively (R0) resected patients, n = 51, P = 0.093] and reduced overall survival time (all patients, log-rank P = 0.0028; R0 patients, log-rank P = 0.0003) and was identified in multivariate analysis as the most important independent prognostic marker in the R0 patient group (relative risk 9.1, P = 0.0013).

Perisurgical erythropoietin application in anemic patients with colorectal cancer: A double-blind randomized study.

BACKGROUND: Blood transfusions are associated with higher postoperative morbidity and tumor recurrence rates in colorectal cancer surgery, To reduce the need for transfusions in patients with tumor-induced anemia who are not suitable for autologous blood donation, it was tested whether perisurgical erythropoietin application would be able to stimulate hematopoiesis adequately. METHODS: In a double-blind randomized study 150 IU/kg body weight erythropoietin was given subcutaneously every 2 days beginning 10 days before operation and continuing until postoperative day 2. Twenty patients were randomized into the erythropoietin group with three observed dropouts and 10 patients into the placebo group. RESULTS: In the erythropoietin group two episodes of hypertension and one deep venous thrombosis were observed. Preoperative hemoglobin response in the erythropoietin group (p = 0.069) was paralleled by a highly significant reticulocyte increase (p = 0.0004). However, frequency of blood transfusion was not different between both study groups (erythropoietin, 1.82 +/- 0.80 units/ patient; placebo, 1.80 +/- 0.97 units/patient). If iron availability was analyzed, a strong correlation between ferritin blood levels and transferrin iron saturation with hemoglobin response was observed in regression analysis (p < 0.001). CONCLUSIONS: These results indicate that hematopoiesis in anemic patients with colorectal cancer can be stimulated by erythropoietin; however, clinical efficacy is to be expected only in selected patients with high iron availability, which calls for further studies combining erythropoietin and parenteral iron application.

[Post-transfusion rise in thrombocytes: observations in a hematologic-oncologic patient sample]. / Der posttransfusionelle Thrombozytenanstieg: Beobachtungen an einem hämatologisch-onkologischen Patientengut.

OBJECTIVE: Aim of the present analysis was the evaluation of clinical conditions and product-specific parameters influencing posttransfusion platelet increment. DESIGN: 400 single-donor platelet transfusions were analyzed for patient- and concentrate-specific factors influencing posttransfusion platelet increment. Statistical analysis was performed by the General Mixed Model Analysis of Variance. SETTING: Department of hematology and oncology at a university hospital. PATIENTS: 46 patients (24 male, 22 female; age 17-80 years). INTERVENTIONS: Single-donor platelet transfusions. RESULTS: As demonstrated earlier, splenomegaly, body temperature, and bone marrow transplantation could be proven as factors reducing posttransfusion platelet increment. In addition, hepatomegaly and application of antibiotics had negative effects on platelet increment. Among the product-specific parameters leukocyte contamination and pretransfusion storage time reduced transfusion success significantly. CONCLUSIONS: Clinical factors influencing posttransfusion platelet increment can hardly be controlled. In contrast, concentrate-specific parameters can be influenced by preparation technique and storage procedure. Therefore, high value should be set on low leukocyte contamination and short pretransfusion storage time of platelet concentrates.

Cryopreservation of human platelets with dimethyl sulfoxide: changes in biochemistry and cell function.

BACKGROUND: The shelf life of liquid-stored platelet concentrates is limited to 5 days. Therefore, much work has been carried out in an attempt to establish the optimum method for cryopreservation. Among the various cryoprotectants, dimethyl sulfoxide (DMSO) has been shown to be the most effective. However, DMSO-frozen platelets are characterized by a number of cell lesions. This report describes metabolic and functional changes that should give rise to some concern about the functional integrity of these cells. STUDY DESIGN AND METHODS: Single-donor platelet concentrates were frozen in liquid nitrogen by use of DMSO (5%). After thawing, the cells were washed and resuspended in autologous plasma. Before, during, and after the freezing process, samples for analysis of metabolic measures (e.g., pH; calcium, potassium, and lactate dehydrogenase concentrations; plasma complement factors) and functional measures (e.g., aggregometry, in vitro bleeding time, alpha-granule membrane protein-140 expression) were taken. RESULTS: Mean platelet volume increases during the deep-freezing process. Potassium, calcium, and lactate dehydrogenase are released from the intracellular space to the extracellular space. A strong activation of complement, which is mainly due to the addition of DMSO, is observed. Platelets become activated as indicated by the expression of alpha-granule membrane protein-140. Accordingly, decreased platelet function can be observed. CONCLUSION: DMSO-frozen platelets are characterized by several metabolic and functional changes. Although these cells have been shown to exert hemostatic effects in vivo, it is conceivable that those effects could be improved by further development of platelet-freezing techniques.

Infections and immunological hazards of allogeneic bone transplantation.

Allogeneic transplantation of human cancellous and cortical bone is a controversially discussed concept in trauma and orthopaedic surgery. Biological and immunological arguments support transplantation of autologous material whenever this is technically possible. On the other hand, synthetic alloplastic materials for bone substitution are available free of immunological and hygienic hazards. In this context the value of allogeneic bone grafts is discussed, especially considering the problem of AIDS. If autologous corticospongious bone is to be used its supply is limited. On the other hand, alloplastic synthetic artificial bone does not meet all the requirements demanded for substitution of large osseous defects up to now. The problems of geometric and mechanical stability of these alloplastic materials still remain. Therefore, no alternative to allografting of large, stable, corticospongious fragments exists in some cases. Bone transplantation is performed without vital indication in nearly every case. Thus an optimum of hygienic security has to be claimed for recipients of allogeneic bone. The "Munich model" for bone transplantation is presented and discussed.

[Preoperative autologous blood donation and disposition. Possibilities and prerequisites]. / Präoperative Eigenblutspende und-bereitstellung. Möglichkeiten und Voraussetzungen.

Preoperative blood deposit can help to avoid the risks of homologous blood transfusions. However, it is necessary that prerequisites concerning medicine standard, organisation, technical questions and administrative aspects have to be fulfilled.

Blood transfusion-modulated tumor recurrence: first results of a randomized study of autologous versus allogeneic blood transfusion in colorectal cancer surgery.

PURPOSE: Allogeneic blood transfusions have reportedly been associated with a poor prognosis in patients with curatively resected cancer. To control for immunosuppression induced by a speculatively causal allogeneic blood transfusion, we designed a randomized study in which the control group received autologous blood transfusions not related to any condition of immunosuppression. PATIENTS AND METHODS: One hundred twenty patients with potentially curative resectable colorectal cancer and the capability to predeposit autologous blood were randomly selected to receive either standard allogeneic blood transfusion or predeposited autologous blood. RESULTS: In curatively resected cancer patients, the number who needed allogeneic blood transfusions was reduced from 60% in the allogeneic blood group to 33% in the autologous blood group (P = .009). After a median follow-up duration of 22 months (range, 8 to 48) tumor recurrence was observed in 28.9% of the allogeneic blood group and 16.7% of the autologous blood group. Life-table analysis established a tendency toward a shorter tumor-free survival for the allogeneic blood group (log-rank P = .11). The problem with this analysis was the strong association of allogeneic blood transfusions with tumor recurrence, which interfered in 33% of patients in the autologous blood group who required additional allogeneic blood transfusions. Multivariate analysis of established risk factors for tumor recurrence and surgery-related variables reflecting potential immunosuppressive conditions showed that only pT stage (relative risk, 6.61; 95% confidence interval [CI], 1.82 to 23.99; P = .004), pN stage (relative risk, 8.39; 95% CI, 3.15 to 22.33; P < .001), and the need for allogeneic blood (relative risk, 6.18; 95% CI, 2.20 to 17.37; P < .001) were independent predictors of tumor recurrence. Subgroup analysis of patients who received a transfusion of < or = 2 U blood found a significantly higher risk of tumor recurrence in the allogeneic blood group (relative risk, 5.16; 95% CI, 1.13 to 23.62; P = .034), which was reduced to borderline significance (relative risk, 3.54; 95% CI, 0.76 to 16.51; P = .107) by adjustment for tumor (T) and node (N) stage. CONCLUSION: As indicated by these first results, the blood transfusion modality has a significant effect on tumor recurrence after surgical treatment of colorectal cancer. A change in the practice of blood transfusion might thus potentially surpass the impact of any recent adjuvant treatment strategies.

Single-donor platelet concentrates: changes of surface glycoproteins during storage.

Storage of single-donor platelet concentrates is currently limited to 5 days. During this period, however, numerous morphologic and biochemical changes have been observed. These changes result in functional impairment of stored platelets. The present study describes increased binding of a monoclonal antibody against GMP 140 on the surface of stored single-donor platelets revealing an activation process. In contrast, binding of monoclonal antibodies directed against glycoprotein complex (GP) IIb-IIIa and ligand-induced binding site (LIBS1) is slightly diminished during storage. When platelets are stimulated with ADP GMP 140, GP IIb-IIIa, and LIBS1 are expressed to a higher extent than on the surface on nonstimulated platelets. The quantity exposed, however, depends upon the storage time. It is significantly reduced when platelets are stored for longer than 1-2 days. The present data indicate that storage of single-donor platelet concentrates affects fibrinogen binding, cell to cell cohesion, and release reaction. The results are in good agreement with conventional aggregation and in vitro bleeding time measurements.

Complement activation during storage of single-donor platelet concentrates.

Single-donor platelets are stored up to 5 days prior to transfusion. Since contact of plasma to plastic surfaces may lead to complement activation, we investigated whether there is any increase in the complement factors C3a, C4a and C5a in routinely stored single-donor platelet concentrates. C3a levels increased about 40-fold during a 7-day storage. C4a levels also increased with storage time but to a lesser extent. By contrast, C5a levels remained stable throughout this period. ADP- and collagen-induced aggregation was impaired after storage of platelets, indicating severe functional injury. In platelet-poor plasma stored under identical conditions a comparable increase in C3a and C4a concentrations was observed. The loss of platelet function during storage might at least in part be due to the excessive anaphylatoxin concentrations observed.

[Quality control of stored platelet concentrates by means of flow cytometry]. / Qualitätskontrolle gelagerter Thrombozytenkonzentrate mittels Durchflusszytometrie.

Storage of single-donor platelets is currently limited to 5 days. During this period, however, numerous morphologic and biochemical changes have been observed. The present study describes increased binding of anti-GMP 140 to stored single-donor platelet concentrates; this reveals a progressive activation process. In contrast, when stored platelets are stimulated with ADP, GMP 140, expression is reduced indicating a diminished release reaction. Additionally, the GP IIb/IIIa receptor complex and its fibrinogen-induced binding site (LIBS1) are reduced by storage time, demonstrating a diminished fibrinogen binding. Further experiments have to clarify whether these changes observed in vitro translate into a reduced hemostatic capacity after transfusion in vivo.

[Post-transfusional platelet increment: effect of clinical factors]. / Der posttransfusionelle Thrombozytenanstieg: Einfluss klinischer Faktoren.

The results of platelet transfusions depend upon a variety of different conditions; besides alloimmunization, a lot of clinical factors are responsible for transfusion success. The present paper tries to work out clinical findings, which are related to successful or unsuccessful platelet transfusions. The following criteria could be identified to influence posttransfusion platelet increment: hepatomegaly, splenomegaly, diagnosis, antibiotics, number and time of previous platelet transfusions. AB0 compatibility, pretransfusion storage time and leucocyte contamination. Therefore, in platelet transfusion therapy a high value should be set on AB0 compatibility, brief storage time and low leucocyte contamination, since these parameters--in contrast to the other influencing factors--can easily be controlled.

[Previous activation of platelets during storage: possible causes]. / Aktivierungsvorgänge von Thrombozyten während der Lagerung: mögliche Ursachen.

Stored single-donor platelets are characterized by a progressive cell activation. The data presented indicate that activation of plasma coagulation and complement system could contribute to this storage lesion process.

Effects of recombinant human erythropoietin on autologous blood donation before open heart surgery.

We studied the effects of four different doses of recombinant human erythropoietin (rhEPO) on the amount of preoperative autologous blood donation. 43 patients prior to open heart surgery were randomized into 5 groups (100, 200, 400, 800 U/kg rhEPO i.v. or placebo) and treated twice weekly over a period of 4 weeks. Autologous blood was taken at a hemoglobin of 13 g/dl and a hematocrit of 34% respectively. Application of low-dose rhEPO (100 and 200 U/kg) did not increase the amount of autologous blood donated, only 400 and 800 U/kg produced a significant increase by 27% and 39% respectively (p < 0.01) In addition RBC showed a reduced decline of hemoglobin level (p < 0.01). Reticulocytes increased by 2.5 times the baseline in the placebo and treatment groups up to 200 U/kg. Again, only 400 and 800 U/kg produced a significantly higher increase of 3.2 and 3.6 times respectively (p < 0.05 and p < 0.01). Although iron was supplied orally, ferritin levels declined in all groups whereas serum iron and transferrin levels remained unchanged. No influence could be detected on WBC, thrombocyte count, or arterial blood pressure. Mild and reversible side effects were observed in 8 patients (19%). Perioperatively 34 patients (81%) received exclusively autologous blood, only 8 patients (19%) needed additional homologous blood transfusions. Administration of high-dosed rhEPO (400-800 U/kg) seems to be an effective treatment with only slight side effects for increasing erythropoiesis during autologous blood donation.