Using the mean platelet volume MPV for the quality assessment of apheresis procedures.

INTRODUCTION: Referring to current standards the quality of an apheresis procedure is estimated by the quantity of collected cells. Nowadays a new kind of quality measurement could be found in the detection of cell volumina. Recent diagnostics have shown that stem cells and platelets - when separated - are likely to appear in a higher volume inside the cell product. Therefore, in this study the question should be discussed wether platelets of higher volume are more likely to be separated than platelets showing a lesser volume. METHODS: Blood samples of three different apheresis procedures could be observed: allogenic platelet donations (n = 7) (Trima, Terumo), autologous (n = 5) and allogenic stem cell donations (n = 5) (Cobe Spectra, Terumo). To examine the blood samples the Sysmex hematology analyser (XT-2000) has been used. RESULTS: During stem cell apheresis, the volume of the separated platelets was 1.2 fold increased compared to the platelet volume in the peripheral blood before separation. Before apheresis the mean platelet volume in the peripheral blood was found to be 6,21 fl, after apheresis 6,09 fl and inside the platelet concentrate 7,42 fl. The platelet number in the peripheral blood was also significantly decreased (before separation 180.1/nl and after separation 133.5/nl). In the blood products the concentration of platelets was nearly 8 fold higher than in the peripheral blood before separation. CONCLUSION: Overall, the observed apheresis procedures are more likely to separate platelets showing a higher voulme than common in the peripheral blood. This might indicate that not only the amount of separated cells reflects the quality of the apheresis procedure but also that the volume of the separated cells can be used as a parameter for quality assessment.

Soluble CD40L release as test for functional platelet loss.

BACKGROUND: During platelet storage, alterations of the platelet function, 'platelet storage lesion', can be observed resulting in a reduced platelet viability. The release of soluble CD40 ligand (sCD40L) by platelets reflects different aspects of platelet metabolism and activity. Therefore, we used the sCD40L release to test for functional platelet loss in platelet products during storage in comparison to the formation of thromboxane (TXB2). METHODS: On day 1, 3, and 5 in single donor apheresis platelet products (n = 8) under routine storage conditions, sCD40L (measured by ELISA) and TXB2 (measured by RIA) were determined after platelet stimulation (recalcification and clot formation). Results were related to a therapeutic unit (TU = 2 x 10*11 platelets). RESULTS: In platelet-rich plasma of the donors, sCD40L release was 42.5 +/- 7.1 ng/TU and TXB2 formation 2,183 +/- 576 ng/TU. On day 1, 3, and 5 sCD40L release was reduced to 95%, 64%, and 57% and TXB2 formation to 92%, 80%, and 65% of the respective control values. CONCLUSIONS: In single donor apheresis PCs, sCD40L release and TXB2 formation showed a comparable course over storage time and were reduced to about 60% of the respective control values after a storage period of 5 days. These findings are in line with literature data indicating that a functional platelet loss of about 30% will occur after 5 days of storage. Overall, sCD40L release could be easily induced by recalcification and clot formation and can be used as a marker for functional platelet loss.

Platelet transfusion alters CD40L blood level and release capacity in patients suffering from thrombocytopenia.

BACKGROUND: Platelet (PLT)-derived cytokines, such as soluble CD40 ligand (sCD40L), play an important role in the development of adverse transfusion reactions associated with the administration of PLT products. In this study, we determined sCD40L concentration and release capacity in patients with thrombocytopenia before and after receiving a PLT transfusion. STUDY DESIGN AND METHODS: The study included 12 patients suffering from chemotherapy-induced thrombocytopenia. sCD40L levels and release capacity were measured in plasma samples of the patients before and after PLT administration as well as in the respective plateletpheresis concentrates by enzyme-linked immunosorbent assay. Sixteen healthy blood donors served as a control group. RESULTS: In PLT concentrates, elevated sCD40L levels (2567±134 pg/mL) were observed in comparison to plasma sCD40L levels in controls (238.4±35.3 pg/mL). sCD40L plasma concentration of patients with thrombocytopenia was significantly reduced (86.3±16.7 pg/mL) before transfusion and increased to nearly normal levels (204.4±24.8 pg/mL) after PLT administration. In parallel, the sCD40L release capacity per PLT showed no significant difference between controls and patients with thrombocytopenia before transfusion (33.3±2.6 and 29.3±4.6 ag/PLT, respectively) but was significantly reduced after PLT transfusion (22.4±2.7 compared to 29.3±4.6 ag/PLT). CONCLUSIONS: In patients with thrombocytopenia, sCD40L levels were clearly influenced by PLT transfusions: PLT administration led to a normalization of sCD40L plasma concentration. Nevertheless, adverse transfusion reactions did not occur in these patients. The sCD40L release capacity was enhanced by PLT administration dependent on the increase in the amount of PLT count.

Determination of thromboxane formation, soluble CD40L release and thrombopoietin clearance in apheresis platelet concentrates.

All deleterious changes in platelet morphology, structure and function that occur in platelet concentrates (PC) during storage are titled as the 'platelet storage lesion'. No single in vitro test currently available is sufficient in assessing these changes of platelet quality. The release of soluble CD40 Ligand (sCD40L), the formation of thromboxane (TXB2) and the thrombopoietin (TPO) clearance reflect different aspects of platelet metabolism and activitiy, and were used to examine platelet quality in apheresis platelet products. At days 1, 3 and 5, in single-donor apheresis platelet products (n = 10) under routine storage conditions, sCD40L (measured by ELISA) and TXB2 (measured by RIA) were determined after platelet stimulation (recalcification and clot formation). TPO (measured by ELISA) was determined after an incubation time of 5 h at 37°C with platelet-rich plasma (adjusted initial TPO concentration of about 500 pg/mL). Results were related to a therapeutic unit (TU = 2 × 10(11) platelets). Immediately after platelet preparation, sCD40L release was 41 ± 7.6 ng/TU, TXB2 formation 1688 ± 374 ng/TU and TPO clearance 1.22 ± 0.32 ng/h/TU. At days 1, 3 and 5, sCD40L was reduced to 89 ± 7%, 71 ± 12% and 57 ± 9%, TXB2 release to 91 ± 6%, 74 ± 12% and 58 ± 9% and TPO clearance to 90 ± 15%, 84 ± 5% and 79 ± 10% of the respective control values. In conclusion, in single-donor apheresis PC, sCD40L release and TXB2 formation as well as TPO clearance by the platelets were dependent on storage duration and reduced to about 60% to 80% of the respective control values after a storage period for 5 days. These findings are in line with literature data, indicating that a loss of platelet functionality of about 30% will occur after 5 days of storage.

Comparison of soluble CD40L concentrations and release capacities in apheresis and prestorage pooled platelet concentrates.

INTRODUCTION: Soluble CD40L (sCD40L) is expressed by platelets and is involved in the stabilization of arterial thrombi. Additionally, it was shown that sCD40L accumulation occurred in stored blood products triggering adverse transfusion reactions like TRALI. To study the influence of the preparation technique on sCD40L accumulation and platelet function we examined CD40L concentrations in prestorage pooled platelet concentrates compared to apheresis products. In addition, sCD40L release capacity was determined as a marker for platelet viability. MATERIAL AND METHODS: sCD40L concentrations were determined in prestorage pooled platelet concentrates (n = 8) and in platelet apheresis concentrates (n = 8) before and after platelet stimulation (recalcification and clot formation) at day 1, 3 and 5 under routine storage conditions. sCD40L concentrations were determined by a commercially available ELISA kit. RESULTS: sCD40L concentrations in storage medium increased over time in prestorage pooled platelet concentrates (from 1,185 pg/mL ± 87 pg/mL at day 1 to 4,464 pg/mL ± 212 pg/mL at day 5) as well as in apheresis products (from 581 pg/mL ± 124 pg/mL at day 1 to 2,718 pg/mL ± 154 pg/mL at day 5) in a hyperbolic manner. Recalcification and clot formation caused an increase in sCD40L concentrations (for example 3,842 pg/mL ± 769 pg/mL before platelet activation to 31,219 pg/mL ± 2,063 pg/mL after platelet activation at day 3), and we observed comparable release capacities for both preparation techniques, however, decreasing over storage time up to 50% (day 5) of the respective control value (day 1). CONCLUSIONS: Amounts of sCD40L accumulation and release capacity during storage of platelet concentrates were dependent on storage duration, but showed no relevant differences regarding the preparation technique. After 5 days of storage, CD40L basal levels were increased, in contrast sCD40L release capacity was decreased. By recalcification and clot formation sCD40L release capacity could be easily induced and is assumed to be used as a marker for platelet viability.

Reducing costs in flow-cytometric counting of residual white blood cells in blood products: utilization of a single-platform bead-free flow-rate calibration method.

BACKGROUND: Commercial flow-cytometric methods for counting residual white blood cells (rWBCs) in leukoreduced blood products use calibration beads for estimation of the measured sample volume. A bead-free flow-rate calibration method is developed and validated. STUDY DESIGN AND METHODS: The analyzed volume was calculated by acquisition time (ACQ). Twenty-nine spiking series of red blood cell (RBC) or platelet (PLT) products were prepared containing levels ranging from 0.08 × 10(6) up to 2048 × 10(6) WBCs/L. Nearly WBC-free triple-leukofiltered RBCs or PLT concentrates (PCs) served as background. Propidium iodide (PI) was used to identify rWBCs. Five RBC series were compared against a commercially available kit (LeukoSure, Beckman Coulter). Routine capabilities were tested on 41 RBC and 92 PC samples of two independent transfusion services. RESULTS: The lower detection limit in RBC was 0.08 × 10(6) rWBCs/L for ACQ and 0.16 for LeukoSure. Criteria for linearity, accuracy, and precision were fulfilled within the range of 0.5 × 10(6) to 512 × 10(6) WBCs/L. For PCs, all these criteria were fulfilled between 0.5 × 10(6) and 32 × 10(6) rWBCs/L (lower detection limit of 0.25) for PI. ACQ and LeukoSure agreed sufficiently (81%) when tested on routine RBCs or PCs. CONCLUSION: A residual WBC count of fewer than 0.5 × 10(6) WBCs/L can be accurately counted using the ACQ approach at a total reagent cost of less than 0.5€ per sample.

Transient appearance of postoperative EDTA-dependent pseudothrombocytopenia in a patient after gastrectomy.

Ethylenediaminetetraacetic acid-dependent pseudothrombocytopenia (EDTA-PTCP) is a well known phenomenon. Antiplatelet antibodies cause platelet clumping in EDTA anticoagulated blood samples, and blood count analysers calculate a spurious low platelet count. We describe a case of a transient appearance of EDTA-PTCP in a patient after gastrectomy. A 58-year-old man underwent partial gastrectomy in for gastric cancer. Preoperatively, his platelet count was in a normal range, and the surgical procedure was performed without bleeding complications. At day 10 after surgery the patient showed a low platelet count, which could be identified as EDTA-PTCP. The phenomenon disappeared in a following postoperative time interval of 2 months. In cases of recently occurring thrombocytopenias EDTA-PTCP should always be considered as a possible cause of low platelet count, in particular in cases of inconspicuous clinical findings. Appropriate laboratory analysis should be applied.

Alterations of serum erythropoietin and thrombopoietin levels in patients undergoing Coronary Artery Bypass Grafting (CABG).

INTRODUCTION: The hormones erythropoietin (EPO) and thrombopoietin (TPO) are main regulators of erythro- and thrombopoiesis. Cell loss caused by operative procedures may alter serum levels of the hormones, resulting in well known phenomenons like reactice thrombocytosis. MATERIAL AND METHODS: Blood samples from 10 patients (mean age 63 ± 9 years) were obtained before and at day 1, 5 and 10 after coronary artery bypass grafting (CABG). EPO and TPO levels were determined by commercially available ELISA-Kits (R&D Systems, Germany). In addition, platelet count (PC) and hemoglobin concentration (Hb) were determined. RESULTS: Prior to CABG, EPO (13.2 ± 8.2 mU/mL), TPO (189 ± 52 pg/mL), Hb (8.8 ± 1.1 mmol/L) and PC (254 ± 121/nL) were within a normal range. At day 1 after surgery, Hb and PC were significantly decreased to 6.6 ± 0.9 mmol/L and 138 ± 70/nL. In contrast, EPO and TPO were significantly elevated to 32 ± 18 mU/mL and 336 ± 96 pg/mL, respectively, in spite of hemodilution. In particular, TPO elevation was followed by a significant increase in PC (342 ± 144/nL) at day 10 after surgery compared to preoperative values. CONCLUSIONS: Appropriate to the decrease in hemoglobin concentration and platelet count, clear alterations of serum erythropoietin and thrombopoietin levels could postoperatively be observed. EPO levels showed an inverse correlation to hemoglobin concentrations, whereas a disturbed thrombopoietin feedback mechanism resulted in the phenomenon of reactive thrombocytosis.

Fetal alloimmune thrombocytopenia and maternal intravenous immunoglobulin infusion.

BACKGROUND: Different therapeutic approaches have been used in fetal-neonatal alloimmune thrombocytopenia, but many centers administer immunoglobulin G infusions to the pregnant woman. We studied the effect of maternal antenatal immunoglobulin infusions on fetal platelet counts in pregnancies with fetal alloimmune thrombocytopenia. DESIGN AND METHODS: We retrospectively analyzed the clinical courses of fetuses with fetal alloimmune thrombocytopenia whose mothers were treated with immunoglobulin G infusions in a single center between 1999 and 2005. In a center-specific protocol, weekly maternal immunoglobulin G infusions were given to 25 pregnant women with previously affected neonates and four women with strong platelet antibodies, but no previous history of fetal alloimmune thrombocytopenia; before each infusion diagnostic fetal blood sampling was performed to determine fetal platelet counts and immunoglobulin G levels. RESULTS: There were 30 fetuses with fetal alloimmune thrombocytopenia, confirmed by initial fetal blood sampling showing fetal platelet counts between 4×10(9)/L and 130×10(9)/L and antibody-coated fetal platelets using a glycoprotein specific assay. Despite weekly antenatal maternal immunoglobulin G infusions fetal platelet counts did not change significantly. Maternal and fetal immunoglobulin G levels, measured before every infusion, increased significantly with the number of maternal immunoglobulin G infusions. CONCLUSIONS: In this group of fetuses with fetal alloimmune thrombocytopenia no consistent increase of fetal platelets was achieved as a result of regular maternal immunoglobulin G infusions.

Alteration of serum thrombopoietin levels in patients with chronic hepatitis C under interferon therapy.

Thrombocytopenia is commonly observed during interferon-alpha (IFN-alpha) therapy in patients with chronic hepatitis C. Since thrombopoietin (TPO) is the main regulator of thrombopoiesis, thrombocytopenia may partially be due to a reduced TPO generation. Because of the developments of the second generation of TPO mimetic drugs patients with reduced TPO levels should be identified possibly having a benefit by medicinal stimulation of thrombopoiesis. Therefore, platelet count and serum TPO concentration of patients receiving an interferon-alpha therapy were determined.Twelve patients treated with IFN-alpha (daily 10 x 106 IU s.c. for four weeks) in cause of chronic hepatitis C were examined during the first month of therapy. Serum TPO concentration significantly decreased from 80.8+/-48.0 to 34.6+/-24.5 pg/ml (p<0.05, Mann-Whitney test), and platelet count declined from 214,417+/-48,292 to 151,333+/-44,726 platelets/microl. During the following three weeks platelet count remained at a low level, while serum TPO increased to normal range.In conclusion, an interferon treatment causes reduced serum TPO level during the first week of therapy accompanied by decreased platelet count. The reduction in TPO synthesis contributes to the development of thrombocytopenia in patients during interferon therapy.