T-cell death, phosphatidylserine exposure and reduced proliferation rate to validate extracorporeal photochemotherapy.

BACKGROUND AND OBJECTIVES: Extracorporeal photochemotherapy (ECP) is an established therapy in various diseases, such as cutaneous T-cell lymphoma and graft-versus-host disease. This study was performed to investigate the practicability of a flow cytometric T-cell evaluation after ECP as a tool to validate the quality of ECP procedures and to enable the comparability of treatments with different ECP devices. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMNCs) of healthy volunteer blood donors were treated by offline ECP. To quantify the effect of ECP on T cells in vitro, phosphatidylserine exposure and 7-aminoactinomycin D (7-AAD) reactivity as well as the proliferative activity of phytohaemagglutinin-induced, viable CD3(+) lymphocytes were analysed by flow cytometry. RESULTS: The expected T-cell death after ECP was confirmed by 7-AAD measurements. Phosphatidylserine exposure gradually increased between 20 and 70 h after ECP. Treatment-related inhibition of T-cell proliferation was 92.6 ± 1.4%. CONCLUSION: The combination of viability, phosphatidylserine exposure and T-cell division analyses by flow cytometry in a single-platform system provides a valuable tool to validate ECP procedures.

Influence of multicomponent apheresis on donors' haematological and coagulation parameters, iron storage and platelet function.

BACKGROUND AND OBJECTIVES: Multicomponent collection (MCC) enables production and processing of various blood components during one apheresis session. In this prospective crossover study, the effects of donating platelets (PLTs) and packed red blood cells (PRBCs) on donor's blood cell count, coagulation, PLT function and iron state were analysed. MATERIALS AND METHODS: Forty-eight MCCs were performed using two different cell separators (Fenwal Amicus(®), CaridianBCT Trima Accel(®)). Two units of platelet concentrates and one unit of PRBCs were collected during each session. Full blood cell count and iron status were obtained on day 0 before and after apheresis, day 2, day 14 and day 42. PLT function was analysed by aggregometry and rotation thromboelastometry in parallel with coagulation tests before and after MCC and at day 2. RESULTS: Multicomponent collection was well tolerated without adverse side effects. Blood cell count and iron parameters declined and most of them (haemoglobin, haematocrit, transferrin, transferrin saturation and ferritin) were significantly below baseline values until at least day 42 after donation. Absent iron stores were seen in 31·3% of the donors. In contrast, PLTs significantly exceeded pre-donation values after 14 days and remained significantly increased for 42 days. After 2 days, coagulation parameters were only slightly (P > 0·05) altered, whereas PLT function was significantly reduced. CONCLUSION: Multicomponent collection is an obviously safe procedure; however, the significant long-term impact on the donor's blood count and iron store, as well as impaired PLT function, has to be considered in regard to donor safety.

Function and activation state of platelets in vitro depend on apheresis modality.

BACKGROUND AND OBJECTIVES: In multicomponent collection, various blood components are prepared during one apheresis process. The aim of this prospective crossover study was to compare the function, metabolic parameters and activation state of fresh and stored platelets (PLTs) collected by two different cell separators. MATERIALS AND METHODS: Twenty-four donors underwent apheresis on each of two cell separators (Fenwal Amicus(®) and CaridianBCT Trima Accel(®)) with an interval of at least 2 months between donations. Per donation, one double dose of PLT concentrate (PC) and one unit of packed red-blood-cells were collected. In total, 48 single unit PCs were tested for pH, glucose, bicarbonate, lactate, potassium and LDH concentration during 7 days of storage. PLT function was analysed by aggregometry, rotation thrombelastometry and hypotonic shock response. The PLT surface expression of P-selectin (CD62P) and LAMP-3 (CD63) was estimated by flow cytometry. RESULTS: During storage, metabolic parameters were well maintained in both groups, but levels of glucose and pH were significantly lower, while lactate and LDH were significantly higher in Amicus(®)-PCs. Amicus(®)-derived PLTs were significantly more activated as evidenced by higher CD62P and CD63 expression. In parallel, the in vitro function of Amicus(®)-PLTs was significantly reduced compared to Trima(®)-PLTs. CONCLUSION: In multicomponent apheresis, standardized PLT collection is effective and well tolerated. The higher activation of Amicus(®)-derived PLTs may be because of the divergent centrifugation modalities during collection. Possible consequences for the clinical outcome of thrombocytopenic patients will be evaluated in further trials.

Thrombin generation before and after multicomponent blood collection.

The development of apheresis technology has increased efficiency in donor blood use by collecting specific blood components in several combinations. The question of donor safety raised by the contact of donor blood with foreign, only in part biocompatible surfaces remains. The aim of this study was to estimate the effect of multicomponent blood collection on thrombin generation performing an overall function test of coagulation. DONORS, METHODS: 26 blood donors were included. Per apheresis two units of platelets and one unit of RBCs were collected by two cell separators (Amicus and Trima Accel). Each donor underwent the procedure on both apheresis systems. Samples were collected before, immediately after, and 48 hours after apheresis. Thrombin generation was measured by means of calibrated automated thrombography (CAT). RESULTS: CAT-data changed only slightly and no significant changes were seen before, immediately after, and 48 hours after apheresis. The parameters did not differ significantly between the two different apheresis devices. CONCLUSION: No change in parameters of continuous thrombin generation occurred, suggesting that apheresis did not lead to severe alterations in the haemostatic system.

C242T polymorphism of the p22 phox gene is not associated with peripheral arterial occlusive disease.

Formation of reactive oxygen metabolites is vital for the microbicidal activity of phagocytes. As an unwanted side effect, these metabolites may contribute to oxidative stress in the vasculature and thus lead to arteriosclerosis. p22 phox, a component of the NADH/NADPH oxidase in phagocytes and vascular smooth muscle cells, is essential for production of reactive oxygen metabolites. Recently, a C/T polymorphism at position 242 of the p22 phox gene has been associated with coronary artery disease (CAD), suggesting a protective effect of the 242 T allele on the vasculature. In the present study, we analysed the relation of this polymorphism to peripheral arterial occlusive disease (PAOD). C242T polymorphism was determined by restriction fragment polymorphism (RFLP) analysis in 324 patients with documented PAOD and 295 control subjects without any known arterial disease. p22 phox 242 T allele frequencies and genotype distributions were not significantly different between patients and controls; the adjusted relative risk associated with the 242 T allele was 1.14 (95% CI 0.84-1.54, P=0.39), assuming an additive effect of the T allele. C242T polymorphism was not associated with the age of patients at the onset of the disease. Our data indicate that C242T polymorphism of the p22 phox gene is not associated with PAOD.

Factor II G20210A and factor V G1691A gene mutations and peripheral arterial occlusive disease.

BACKGROUND: G to A mutations at positions 20210 of the prothrombin gene (F2) and 1691 of the factor V gene (F5) are established risk factors for venous thrombosis. Several factors associated with coagulation and/or fibrinolysis have been associated with arterial occlusive disease, but the role of F2 20210A and F5 1691A for arterial occlusive disease remains unclear. OBJECTIVE: To investigate if F2 20210A and F5 1691A are associated with peripheral arterial occlusive disease (PAOD). METHODS AND RESULTS: We analyzed the prevalence of F2 20210A and F5 1691A alleles in 336 patients with documented PAOD at Fontaine stage II-IV and 300 controls without vascular disease. Allele frequencies in patients and controls were 0.013 and 0.022 for F2 20210A, and 0.042 and 0.045 for F5 1691, respectively, both differences being not statistically significant. CONCLUSION: Our data suggest that mutations F2 G20210A and F5 G1691A are not associated with PAOD.

Prothrombin G20210A, factor V Leiden, and factor XIII Val34Leu: common mutations of blood coagulation factors and deep vein thrombosis in Austria.

Mutations in the gene for prothrombin (F2 20210A) and factor V (F5 1691A, factor V Leiden) are established risk factors for deep venous thrombosis (DVT). Recently, a mutation in the gene for factor XIII (F13 100T) leading to a Valine-Leucine exchange at amino acid position 34 has been reported to be protective against DVT. To analyze the role of these mutations for DVT in Austria, we analyzed their prevalence in 154 patients with documented DVT and 308 sex- and age-matched control subjects. Allele frequencies of F2 20210A, F5 1691A, and F13 100T were 0.018, 0.039, and 0.274 among controls, and 0.045, 0.120, and 0.211 among patients, respectively. Odds ratios for DVT associated with F2 20210A, F5 1691A, and F13 100T alleles were 2.5 (95% CI: 1.1-5.7), 3.4 (95% CI: 1.9-5.8), and 0.7 (95% CI: 0.5-1.0). We conclude that F2 20210A, F5 1691A, and F13 100T are common mutations in the Austrian population. F2 20210A and F5 1691 increase the risk for DVT, whereas F13 100T is associated with a decreased risk for DVT. Routinely, analysis of these mutations may help to analyze the individual risk for DVT.

Reticulocyte hemoglobin content allows early and reliable detection of functional iron deficiency in blood donors.

BACKGROUND: Frequent blood donations may lead to a depletion of body iron stores resulting in manifest anemia. Reticulocyte hemoglobin content (CHr) - a marker for impaired hemoglobinisation (IH) caused by functional iron deficiency (FID) - was investigated regarding its value as a routine screening parameter in frequent whole blood donors. METHODS: In a prospective study, 917 frequent blood donors and 688 first time or reactivated donors were tested for iron status and red blood cell count, including CHr. The ferritin index as a marker to indicate absent iron stores (AIS) was calculated. RESULTS: Depending on the number of donations during the preceding 12 months, AIS were detected in up to 21.4% of male and 27.8% of female donors, respectively. IH was present in up to 6.4% male and 16.7% female donors with 2 and 4 preceding donations, respectively. The defined CHr cut-off value was 28.0 pg to detect IH in frequent whole blood donors with AIS, leading to a test specificity of 98.2% (positive predictive value, PPV: 57.7%) in male and of 97.8% (PPV: 82.9%) in female donors. CONCLUSION: Determination of CHr is feasible to detect FID resulting in IH in frequent blood donors. It may help to prevent the development of anemia in frequent blood donors and also can help to decide whether donor deferral or even iron substitution need to be recommended.

Specificities of platelet autoantibodies and platelet activation in lupus anticoagulant patients: a relation to their history of thromboembolic disease.

Lupus anticoagulants (LA) prolong in vitro phospholipid-dependent coagulation tests, but are associated with thromboembolic disease (TE). However, a subgroup of individuals with LA has no TE, and it is therefore desirable to distinguish those at risk for TE from those without. Whether platelets have a primary role in the development of TE is not clear yet. We determined platelet autoantibodies to identify a specific platelet target which is associated with platelet activation in 97 patients with a long history of detectable LA, 65 patients with TE (LA/TE+), and 32 individuals without TE (LA/TE-). Thrombocytopenia was more common in the LA/TE- than in the LA/TE+ group (P < 0.05). Both groups had platelet antibodies, but the frequency of antibodies was lower in LA/TE+ than LA/TE- patients (P < 0.01), who had higher antibody titres against glycoprotein IIb/IIIa and glycoprotein Ib/IX (P < 0.05). Also, their platelets were more activated, as determined by PAC-1 binding (P < 0.01). These differences were also noted if patients with arterial thrombosis were evaluated separately. These findings in LA/TE- individuals were similar to those in patients with chronic autoimmune thrombocytopenia. However, there was no autoantibody target identifiable to distinguish between LA/TE- from LA-TE+ individuals. We therefore conclude that the presence of platelet antibodies, even if associated with platelet activation, is not sufficient to dispose LA patients to thromboembolic disease.

Severe thrombocytopenia due to host-derived anti-HPA-1a after non-myeloablative allogeneic haematopoietic stem cell transplantation for multiple myeloma: a case report.

BACKGROUND AND OBJECTIVES: Host- or donor-derived alloimmune thrombocytopenia can develop after non-myeloablative allogeneic haematopoietic stem cell transplantation (HSCT). We report the first case of host-derived HPA-1a antibodies. CASE REPORT: A 52-year-old male patient received HSCT from his human leucocyte antigen (HLA)-A, -B, -C, -DR identical brother after reduced intensity conditioning. Bilinear engraftment around day 12 was accompanied by a continuous decrease of platelet counts. We investigated for platelet antibodies because of a progressive decline of platelet counts and refractoriness to platelet transfusions. METHODS: The patient's serum was tested by enzyme-linked immunosorbent assay (ELISA), a solid phase assay and monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay. Recipient's DNA from the time before HSCT and donor's DNA were genotyped for human platelet antigens. RESULTS: Serum obtained on day 15 after HSCT reacted strongly with the donor's platelets due to host-derived anti-HPA-1a- and anti-HLA I antibodies. Serum samples from days 39, 45 and 65 after HSCT contained only anti-HLA I; no antibodies were detectable on day 149. Platelet counts increased on day 20 spontaneously. The decrease of the antibodies accompanied by the increase of the platelet counts suggests progressive elimination of residual host cells. CONCLUSIONS: The HPA-1a antibodies affected thrombopoietic engraftment and the success of platelet transfusions.

[Statins--attaining goal values--with reference to recent studies]. / Statine--Zielwerte erreichen--im Lichte neuer Studien.

Recent lipid intervention studies led to the implementation of lipid lowering therapy in the cardiovascular risk management. These secondary as well as primary prevention studies share the effect of HMG-CoA-reductase inhibition. Despite varying product properties there seem to be no major differences in risk reduction between the drugs. One could argue the main action of the statins, lipid lowering, is the most prominent mode of action. A new study (AirForce/Texas Coronary Atherosclerosis Prevention Study) extended that to patients with relatively low total cholesterol levels (mean 221 mg/dl with a slightly lowered LDL-C of 37 mg/dl, LDL-C of 150 mg/dl) successfully treated with lovastatin in primary prevention. That supports the concept to reach lipid levels as low as possible in the longer term, even if the absolute clinical event reduction in primary prevention is not so impressive. On the contrary, in secondary prevention: in the AVERT-study in patients with a 82% mean stenosis in one vessel PTCA-treated patients with conventional drug therapy were compared to such without PTCA and aggressive lipid lowering therapy (resulting LDL-C 77 mg/dl). One could be surprised that the "intervention group" was not better, though representing the usual clinical procedere. Interestingly, borderline significant (p < 0.046 at a level of significance of p < 0.045), results were in favor for the drug treated group. Such data could, if confirmed in further investigations, change cardiovascular disease management to aggressive lipid lowering prior to or instead invasive management, especially in initial therapy of CVD and diabetes mellitus type II.

[Eating behavior of patients with metabolic diseases and metabolically healthy probands in Austria. Results of a questionnaire survey at the Graz Autumn Fair 1991]. / Essverhalten Stoffwechselkranker und -gesunder in Osterreich. Ergebnisse einer Fragebogenaktion bei der Grazer Herbstmesse 1991.

Malnutrition as the cause of developing atherosclerosis is undoubtedly of major importance. For that reason, proper nutrition and eating habits among the population is of specific significance in preventive medicine. In order to establish a more pronounced food consciousness among the population of Styria, a questionnaire was issued to 1.354 persons attending the Graz Autumn Fair in 1991. The results showed above all that approximately 40% of the subjects investigated presented a disease due to malnutrition and metabolic disorder, mainly hyperlipidemia. The choice of various foods varied according to male and female tastes; roasted pork was more often a men's favourite dish (p < 0.001) while women had a prediction for vegetarian food (p < 0.001). There was, however, no difference in the choice of eating habits in persons with or without metabolic disorders. Thus, women in general do pursue a healthier consciousness was not so pronounced in man. Yet, it could not be established by means of the questionnaire that subjects with metabolic disorders showed different eating habits with respect to their disease.

Lack of association of the Glu298Asp polymorphism of endothelial nitric oxide synthase with manifest coronary artery disease, carotid atherosclerosis and forearm vascular reactivity in two Austrian populations.

OBJECTIVE: Conflicting data exists about the possible contribution of the homozygous Asp/Asp genotype of the Glu298Asp polymorphism of endothelial nitric oxide synthase to human atherosclerotic vascular disease. We investigated the polymorphism in two independent study populations: a case-control study including patients with angiographically verified coronary artery disease (CAD) on the one hand and a cross-sectional epidemiological study on the other hand. METHODS: The Glu298Asp polymorphism was determined by PCR-RFLP as established. In the case-control study (240 patients and 248 controls) a possible association between the polymorphism and CAD, and age of onset of CAD and myocardial infarction was investigated. In the cross-sectional epidemiological study (932 subjects) intima-media thickness (IMT) of the carotid artery as well as morphological plaque burden and forearm vascular reactivity (peak postischemic reactive hyperaemia, determined by venous occlusion plethysmography) were measured. RESULTS: In the case-control study genotype distribution (Glu/Glu; Glu/Asp; Asp/Asp) was not different between the CAD patients (43/46/11%) and the controls (49/41/10%, P = NS). No association of the polymorphism with age of onset of CAD or myocardial infarction was found. In the epidemiological study no influence of the genetic variant on IMT was observed after correction for classical determinants of IMT (average IMT: Asp/ Asp: 0.077 +/- 0.011 mm; Glu/Glu and Glu/Asp: 0.080 +/- 0.012 mm, P = NS). Forearm vascular reactivity was also not different between homozygous Asp/Asp subjects and Glu/Glu and Glu/Asp subjects (peak-reactive hyperaemia 20.1 +/- 7.3 mL min-1 100 mL-1 vs. 20.0 +/- 6.5 mL min-1 100 mL-1, P = NS). CONCLUSIONS: Our results suggest that there is no association of the Glu298Asp polymorphism with coronary or carotid atherosclerosis or forearm vascular reactivity in these populations recruited in a country with a rather high risk for atherosclerosis. We suggest additional investigations to be performed in populations at different risk for coronary events to further elucidate the possible contribution of this polymorphism to vascular disease.

[Evaluation of a newly discovered LDL receptor mutation (exon 10, GAC>AAC, D271N, "FH Graz-1") in familial hypercholesterolemia-- a familystudy]. / Evaluation einer neu entdeckten LDL-Rezeptor-Mutation (Exon 10, GAC>ACC, D471N, "FH Graz-1") bei familiärer Hypercholesterinämie--eine Familienanalyse.

Heterozygous familial hypercholesterolemia (FH, prevalence 1:500) is a major cause of early atherosclerotic disease. Little is known about possible co-factors influencing individual patient's risk. We investigated this question in a large family carrying a new LDL-receptor-mutation. Genetic analysis of all exons of the LDL-receptor gene in the index case using polymerase chain reaction (PCR) and Denaturing Gradient Gel Electrophoresis (DGGE) revealed a previously unknown mutation in exon 10 (GAC > ACC, D471N, "FH Graz-1"). Investigation of 21 family members (15 females, 6 males), aged 17 to 86 years, revealed 9 female and 4 male carriers of the mutation. 7 female carriers aged 17 to 58 years show no clinical signs of macrovascular disease. An 86-year old female patient, who was asymptomatic until 85, recently suffered a transient cerebral ischemic attack. All these females were normotensive. The only hypertensive 76-year old patient (ex-smoker with a history of 15 pack years) suffers from angina pectoris. 2 male carriers of the mutation (32 and 38 years old) are asymptomatic. A 65-year old patient suffers from cardiovascular disease. A 49-year old patient had a coronary artery bypass graft after a myocardial infarction at the age of 37. Additionally he has a history of bilateral thrombendarterectomy of the carotid arteries and suffers from bilateral peripheral artery disease. This patient also carries the apoE-genotype 4/3, which might be responsible for his poor response to stain therapy, and needs extracorporal lipid elimination (LDL-C > 200 mg/dl under drug therapy). Both of his daughters are homozygous for the apoE-allele 3 and and responded well to stain therapy. Genetic analysis in patients with FH assures diagnosis, but is not sufficient to determine the individual patient's risk. A precise clinical examination remains the gold standard for individual risk evaluation.