Harald Klüter

Rapid typing for human platelet antigen systems −1, −2, −3 and −5 by PCR amplification with sequence-specific primers

Typing for human platelet antigens (HPA) is useful in a variety of clinical situations. We developed a method for genotyping for HPA‐1, ‐2, ‐3 and ‐5 by means of the PCR amplification with sequence‐specific primers (PCR‐SSP) technique. Primer sets were designed to allow PCR amplification for all systems using the same assay conditions. Specificity and sensitivity of the method were assessed in a blind quality control study (n = 112). In 111 cases, results obtained by PCR‐SSP were identical as compared with PCR‐restriction fragment length polymorphism technique. One discrepancy was found to be due to a typing error in the data sheet. The results of the PCR‐SSP technique were available within 3 h. We conclude that genotyping based on PCR‐SSP enables rapid typing for HPA systems, which makes this technique feasible in most clinical settings where urgent HPA typing is required.

Chemokines in stored platelet concentrates

BACKGROUND: Platelets contain several mediators, belonging to a family of proinflammatory cytokines named chemokines, that are stored in the organelles. Release and accumulation of these chemokines during storage of platelet concentrates (PCs) might be responsible for nonhemolytic transfusion reactions.

Febrile and allergic transfusion reactions after the transfusion of white cell‐poor platelet preparations

BACKGROUND: Nonhemolytic transfusion reactions (NHTRs) frequently occur after platelet transfusions. White cell (WBC)‐derived inflammatory cytokines can cause these reactions, but they are rarely found in WBC‐poor platelet preparations. Transfusion reactions were investigated with regard to the residual WBC content in the stored platelet concentrate in two consecutive study periods.

Preparation of autologous platelets for the ophthalmologic treatment of macular holes.

BACKGROUND: Platelet concentrates were recently used for ophthalmologic treatment of macular holes. This strategy was investigated to define standardized blood bank components.

Platelets Stored in a Glucose‐Free Additive Solution or in Autologous Plasma – An Ultrastructural and Morphometric Evaluation

We evaluated a new glucose‐free citrate‐acetate‐NaCl platelet additive solution (PAS 2). In series I, platelet concentrates (PC) were prepared by apheresis and subsequently stored either in plasma (n =16) or in PAS 2 (n =15). In series II, PCs were prepared from pools of four buffy coats (BC) and stored in plasma (n =12) or in PAS 2 (n =11). By means of ultrastructural morphometry, the volume fractions of α‐granules, the open canalicular system (OCS) and the fraction of storage granules secreted into the OCS were analyzed during storage for up to 8 days. Additionally, we determined pH, glucose, lactate, pCO2, HCO3–, lactate dehydrogenase and platelet factor 4. Apheresis platelets stored in plasma showed no changes in their contents of α‐granules and in the fractions of the OCS. In contrast, apheresis platelets stored in PAS 2 displayed a decrease of their relative volume fraction of α‐granules from 9.1±1% on day 1 to 3.7±0.9% on day 5. The fraction of the OCS increased from 7.4±0.8% on day 1 to 17.1±1.4% on day 3. On day 8, 93±9% of all platelets were lysed. Levels of glucose were significantly lower in these preparations and after day 3 glucose consumption decreased to zero. Among PC derived from pooled BC, differences between storage in PAS 2 or plasma were less striking. Only the fraction of α‐granules secreted into the OCS was significantly greater in BC derived PC stored in PAS 2 on all days. These PCs stored in PAS 2 had a higher plasma carryover (30%) in comparison to apheresis PC stored in PAS 2 (10%). We conclude that plasma is superior to PAS 2 for storage of both apheresis and buffy coat platelets. For preservation of the structural integrity of platelets, the use of PAS 2 requires a minimum of 30% plasma carryover.

Evidence for de novo synthesis of cytokines and chemokines in platelet concentrates.

Background and Objectives Inflammatory cytokines in platelet concentrates (PC) may cause side‐effects such as febrile non‐haemolytic transfusion reactions. The maximum white blood cell (WBC) content tolerable to avoid the accumulation of cytokines, and whether these cytokines originate from degranulating leucocytes or de novo synthesis during storage, had not been investigated prior to this study.

Effects of amantadine treatment on in vitro production of interleukin-2 in de-novo patients with idiopathic Parkinson's disease.

An involvement of immunological events in the process of neurodegeneration has frequently been reported. We investigated the cytokine producing capacity for interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) in whole blood cultures of de-novo patients with idiopathic Parkinsons disease (PD) at the time of first diagnosis and after oral amantadine treatment. Before treatment, productions of IL-2 and IFN-gamma were markedly decreased in PD patients compared to patients with major depressive disorder and healthy controls. After amantadine treatment, the in vitro IL-2 secretion defect was corrected to normal levels in half of the patients, and the increase in IL-2 production was correlated with an increase in IFN-gamma secretion. Our findings suggest that immunological abnormalities occur in the course of PD and that a formerly unappreciated therapeutic potential of amantadine may arise from its immunomodulatory effects on altered T cell function in patients with PD.

Immunocytochemical colocalization of adhesive proteins with clathrin in human blood platelets: further evidence for coated vesicle-mediated transport of von Willebrand factor, fibrinogen and fibronectin

Coated membranes and vesicles play an important role in receptor-mediated endocytosis and intracellular trafficking in various cell types, and are also present in blood platelets. Platelets take up certain proteins from the blood plasma, such as von Willebrand factor and fibrinogen, and these substances are transferred to storage granules. The receptors for these plasma proteins on the platelet plasma membrane have been well characterized, but morphological evidence for their transport to the storage granules is not yet available. In an attempt to clarify this aspect, we employed postembedding immunocytochemistry on platelets embedded in the acrylic resin LR White. Clathrin as the major coat component of coated vesicles was localized in the cytoplasm, on the plasmic faces of α-granules and the open canalicular system, and on the plasmic face of the plasma membrane. Colocalizations of the adhesive proteins, von Willebrand factor, fibrinogen and fibronectin, with clathrin could be observed at the same typical locations as coated vesicles were seen in Araldite-embedded material. These colocalizations have not been reported to date and furnish further evidence for a coated vesicle-mediated transport of blood plasma-derived adhesive proteins from their receptors on the outer plasma membrane to the α-granules.

Cytokines in Platelet Concentrates Prepared from Pooled Buffy Coats

Platelet concentrates (PC) prepared from pooled buffy coat (BC‐PC) contain a variable number of leukocytes from different donors. We questioned whether storage of BC‐PC can lead to a lymphocyte activation in the sense of a mixed lymphocyte reaction. BC‐PC were prepared from four ABO‐identical buffy coats and we undertook leukocyte analyses and measurement of different cytokines on days 1, 3 and 5 of PC storage (n = 72). Cytokine content was also determined in freshly prepared plasma (n = 48) and PC prepared by thrombapheresis (SD‐PC) (n = 12). As control, we studied lymphoproliferation of pooled peripheral blood mononuclear cells from four individuals in 10 mixed lymphocyte cultures (MLCs) under optimal conditions. In the BC‐PC, whole blood count and lymphocyte analysis showed a mean leukocyte contamination of 64±28 times 106 per unit with a proportion of lymphocytes of 66.7±13%. In the MLC, levels of interleukin‐2 (IL‐2) and interferon‐γ (IFN‐γ) were increased on day 3 and 5 of storage (p<0.001). In a proportion of BC‐PC, tumor necrosis factor‐α (72.2%) and IL‐2 (43.1%) were detectable immediately after preparation, whereas IFN‐γ (4.2%), interleukin‐1β (4.2%) and interleukin‐8 (11.1%) were only found in some BC‐PC. In all cases, initial values of cytokines did not increase during storage. Cytokine measurement in FFP and SD‐PC showed similar results. The study demonstrates that cytokines are detectable in a variety of blood products immediately after preparation. Levels of cytokines did not increase in the preparations. BC‐PC can be stored for up to 5 days without any signs of lymphocyte activation.

Impact of buffy coat storage on the generation of inflammatory cytokines and platelet activation.

BACKGROUND: Whole blood‐derived buffy coat (BC) has become an alternative source from which to prepare random‐donor platelet concentrates. The influence of prolonged storage of BC prior to platelet concentrate preparation is a matter of controversy. The impact of BC storage on cytokine release was evaluated and the platelet activation quantified. STUDY DESIGN AND METHODS: BCs were prepared from whole‐blood donations after hard‐spin centrifugation. After 1, 3, 6, 12, and 24 hours of storage at 22 degrees C without agitation, samples were withdrawn for cell count and blood gas analysis and measurement of interleukin (IL)‐1beta, IL‐6, IL‐8, tumor necrosis factor alpha and platelet factor 4. Platelet surface markers CD41a, CD42b, CD62P, and CD63 were analyzed by flow cytometry, and the antibody‐binding sites were quantified by using microbeads. RESULTS: Inflammatory cytokines IL‐ 1beta, IL‐6, and tumor necrosis factor alpha were hardly detectable in stored BCs but levels of IL‐8 increased in 25 percent of BCs after 24 hours. A constant increase in platelet factor 4 was observed, which accelerated after 12 hours of storage. Analysis of platelet surface markers showed an initial decrease of platelet activation, followed by an increase after storage for 12 to 24 hours. CONCLUSION: Storage of BCs for up to 12 hours without agitation showed a good preservation of platelets but storage of BCs for 24 hours resulted in increased platelet activation and significantly higher release of platelet factor 4 and IL‐8. Stored BCs might well be suitable for platelet preparation, but their storage time should not greatly exceed 12 hours.