Volker Kretschmer

Influence of ABO blood groups on primary hemostasis

BACKGROUND: Recently, the in vitro bleeding time test (IVBT) was proved to be a very sensitive screening method for the detection of vWD, showing rather good correlation between the closure time and the level of vWF. The vWF levels have been found to be significantly lower in healthy humans who are group O than in those who belong to the other ABO blood groups (non‐group O). The aim of this study was to detect whether these differences in vWF levels in normal persons correspond to differences in nonvascular primary hemostasis when investigated by the IVBT.

Diagnostic performance of the platelet function analyzer (PFA-100) for the detection of disorders of primary haemostasis in patients with a bleeding history-a systematic review and meta-analysis.

The Platelet Function Analyzer (PFA-100®) is increasingly being used in the workup of patients with a bleeding diathesis. A profound knowledge of the possible diagnostic performance of this test is essential in order to make sound clinical decisions based on its results. It was the aim of this study to systematically review the published literature and provide valid estimates of the diagnostic performance of the PFA-100® for detecting disorders of primary haemostasis in newly presenting patients with a bleeding diathesis. A comprehensive literature search was performed for studies published between January 1994 and February 2006. Studies were eligible for the systematic review if they provided data supposed to be applicable to the determination of the diagnostic performance of the PFA-100®. Furthermore, they were included in a meta-analysis if study reporting allowed calculation of sensitivity and specificity and if study quality ensured minimized biases of these estimates for the described clinical setting. Pooled weighted sensitivity, specificity and diagnostic odds ratio were calculated applying random effects modelling and constructing summary operator characteristic curves. This was done separately for the available test modifications using either collagen/epinephrine (PFA-EPI) or collagen/adenosine-diphosphate (PFA-ADP) for platelet activation. Thirty-six articles were included in the systematic review. Six studies met our eligibility criteria for a meta-analysis. The major reason for exclusion from the meta-analysis was a case-control design. A total of 1486 and 1259 patients were included in the meta-analysis of the diagnostic performance of the PFA-EPI and PFA-ADP, respectively. Pooled weighted sensitivity and specificity of the PFA-EPI/PFA-ADP in detecting a disorder of primary haemostasis were: 82.5/66.9% (95%-confidence interval (95%-CI): 76.0–88.9%/57.9–75.9%), and 88.7/85.5% (95%-CI: 84.3–93.1%/82.0–89.1%). 83/75% of patients with a positive PFA-EPI/PFA-ADP result do have a disorder of primary haemostasis whereas 88/79% with a negative PFA-EPI/PFA-ADP result do not. The PFA-EPI appeared to have a higher sensitivity and better predictive values than the PFA-ADP in detecting disorders of primary haemostasis, although a rigorous gold standard definition for a disorder of primary haemostasis, particularly for platelet disorders, was not applied in most studies. The majority of the studies lacked important requirements for quality and reporting, precluding a more precise and definitive characterization of the clinical utility of the PFA-100®. This emphasizes the need for an evidence-based critical appraisal of diagnostic studies in haemostasis research in order to promote the conducting of studies that produce clinically relevant results.

Influence of Different Hydroxyethyl Starch (HES) Formulations on Fibrinogen Measurement in HES-Diluted Plasma

Background: Fibrinogen is the first coagulation factor becoming critical in dilution coagulopathy. Volume replacement in major blood loss is performed with large volumes of crystalloid and colloid solutions. The latter has been shown to compromise accurate photo-optical measurement of fibrinogen. This study determined the influence of different hydroxyethyl starch (HES) formulations. Methods: Citrated plasma samples of 8 healthy volunteers were diluted by 30% or 50% with either HES 10% (200/0.5; HES-200), HES 6% (70/0.5; HES-70), or HES 6% (450/0.7; HES-450). Fibrinogen concentrations were determined by photo-optical measurement (Behring coagulation system [BCS]: derived fibrinogen, or Clauss fibrinogen, calibrated for high [CLS] or low fibrinogen concentrations [CLS-low]) as well as mechanical end point determinations (KC4: CLS-KC4). Measured values were compared with calculated values. Results: On average and across all photo-optical methods, fibrinogen concentrations were overestimated, particularly with HES-200. Hydroxyethyl starch-70 and HES-450 did not differ much from each other. Overestimation was relatively greater for 50% dilutions with all HES formulations. Surprisingly, overestimation was most prominent with CLS-low, the method supposed to most reliably measure low fibrinogen concentrations; overestimation amounted to 92% and 120% with HES-200, 54% and 73% with HES-70, and 51% and 79% with HES-450, for 30% and 50% dilutions, respectively. In contrast, CLS-KC4 always yielded sufficiently accurate results. Conclusions: The study showed that all HES solutions more or less impaired the fibrinogen measurement with the photo-optical method. In particular, overestimation with CLS-low may prevent timely fibrinogen replacement in major blood loss. Hydroxyethyl starch concentration appears to be more relevant for this effect than its molecular size.

Automated blood component collection with the MCS 3p cell separator: evaluation of three protocols for buffy coat-poor and white cell- reduced packed red cells and plasma

BACKGROUND: Automated collection of blood components with a cell separator (MCS 3p, Haemonetics), was performed according to three protocols. STUDY DESIGN AND METHODS: The first protocol provided 2 units of fresh‐frozen plasma (FFP); and one buffy coat‐poor red cell (RBC) concentrate in additive solution. The second protocol included an additional in‐line filtration of the RBC in a closed system after storage at 4 degrees C for 24 hours. In the third protocol, an additional platelet concentrate (PC) was recovered from the buffy coat. Cell counts and biochemical characterization of the RBCs (n=20 each) were determined on Days 0, 1, 14, 28, and 49. RESULTS: The RBC volume was 336 +/− 9 mL (first protocol), 337 +/− 7 mL (second protocol) and 293 +/− 12 mL (third protocol) with a hematocrit of 59 +/− 2, 53 +/− 3, and 61 +/− 5, percent respectively. On Day 49, hemolysis was 0.24 +/− 0.1 percent (first protocol), 0.33 +/− 0.32 percent (second protocol), and 0.38 +/− 0.1 percent (third protocol). The filtered RBC concentrate met the international standards for white cell‐reduced RBCs. Filtration resulted in a clinically irrelevant increase of hemolysis. The in vitro RBC values (lactate dehydrogenase, 2‐hydroxybutyrate dehydrogenase, hemolysis, potassium, 2,3 DPG, ATP) were at least equal to those in RBCs collected by conventional whole‐blood donation. There is a trend toward extended preservation of 2,3 DPG in RBCs collected by apheresis. Two units of FFP could be collected with each donation (first protocol: 420 +/− 55 mL, 5.4 +/− 7 WBCs/microL, 6.5 +/− 5 × 10(3) platelets/microL; second protocol: 440 +/− 33 mL, 3 +/− 5.2 WBCs/microL, 32 +/− 12 × 10(3) platelets/microL; third protocol: 398 +/− 32 mL, 5 +/− 12 WBCs/microL; 3.4 +/− 3.5 × 10(3) platelets/microL). PCs prepared from the buffy coat collected by the third protocol contained 90 +/− 30 × 10(9) platelets in 88 +/− 14 mL of plasma. In vitro test results in these PCs were superior to those in PCs collected by conventional whole‐blood donation. The procedure was well tolerated by all donors. No adverse reactions appeared. CONCLUSION: Erythroplasmapheresis with the MCS 3p cell separator is a useful alternative to conventional whole‐blood donation and separation.

Leukodepletion of autologous whole blood has no impact on perioperative infection rate and length of hospital stay

BACKGROUND: Several mechanisms have been proposed as possible causes of transfusion‐related immunomodulation (TRIM) after allogeneic transfusion. If one of these mechanisms, the release of mediators of immunity and inflammation (“biologic response modifiers”[BRMs]) from disintegrating blood cells during storage of blood products, really causes TRIM, it should in principle also occur after autologous transfusion. As a consequence, prestorage leukoreduction of autologous blood should be able to prevent the clinical consequences of TRIM after autologous transfusion.

Collection of WBC-reduced single-donor PLT concentrates with a new blood cell separator: results of a multicenter study†

BACKGROUND: A new cell separator (COM.TEC, Fresenius) was recently developed aimed at efficient collection of WBC‐reduced single‐donor PLT concentrates (SDPs).

Photo-Optical Methods Can Lead to Clinically Relevant Overestimation of Fibrinogen Concentration in Plasma Diluted With Hydroxyethyl Starch

Background: Adequate fibrinogen concentration is a crucial component of sufficient perioperative/posttraumatic hemostasis. In major blood loss, large volumes of fluids are being administered, which have been shown to interfere with valid determination of fibrinogen concentration. This may lead to wrong treatment decisions. We studied the variables that cause the discrepancies between measured and true fibrinogen concentrations in samples diluted with volume replacement fluids. Methods: Citrated plasma samples of healthy volunteers were diluted by 30% and 50% with phosphate buffered saline (PBS), hydroxyethyl starch (HES) 10% (200/0.5), or gelatine (GEL). Fibrinogen concentrations of diluted samples were derived from the prothrombin time (PT) and the Clauss method (CLS) was applied. With the latter, several modifications and combinations of detection principles and thrombin reagents were investigated. Values were compared with ‘‘true,’’ that is, calculated values based on the results of undiluted samples for each method. Results: Photo-optical methods resulted in significant overestimation of the fibrinogen concentration in blood diluted with HES, depending on the thrombin reagent used. This was particularly true for modifications of the CLS aimed at measuring low fibrinogen concentrations. Use of another thrombin reagent gave satisfactory results for this modification. The validity of mechanical end point determination methods was considered sufficient and was not influenced by the use of different thrombin reagents. Conclusions: Fibrinogen determination methods used in situations of major blood loss need to be validated with samples containing significant amounts of volume replacement fluids, particularly colloids. Only some combinations of test principle, detection method, and reagents will give valid results.

Deformability of Red Blood Cells and Correlation with ATP Content during Storage as Leukocyte-Depleted Whole Blood.

Background: Storage duration of red cells has been associated with increased morbidity and mortality following transfusion. This association has been attributed to the loss of deformability of stored red cells leading to deterioration of microvascular perfusion. ATP content is considered a critical determinant of the deformability of stored red cells. Methods: ATP content and deformability were determined after storage for up to 49 days in 40 leukocyte-depleted whole blood units. Red cell deformability was determined using a laser-assisted optical rotational cell analyzer (LORCA®) employing shear stress (SS) ranging from 0.3 to 30 Pa. Deformability was expressed as the elongation index (EI). EI was correlated with ATP content. Results: ATP content decreased from 3.5 to 1.7 mmol/g hemoglobin. EI increased from 0.03 to 0.05 at an SS of 0.3 Pa, and decreased from 0.62 to 0.59 at an SS of 30 Pa. Correlation coefficient (r) of ATP vs. EI at 0.3 Pa ranged from –0.17 to +0.15 during storage. At 30 Pa, r ranged from –0.03 to +0.45. Correlation increased with storage irrespective of SS, and increased with SS irrespective of storage. Conclusions: ATP content is not a valid surrogate marker for red cell deformability and may not reflect in vivo survival of stored red cells.

Periodic alternating interface positioning to lower WBC contamination of apheresis platelet concentrates: a multicenter evaluation.

BACKGROUND: A new software version of a cell separator (AS TEC 204, Fresenius) providing WBC‐reduced single‐donor plateletpheresis concentrates was tested.

Flow-Cytometric Determination of Survival Time and 24-Hour Recovery of Transfused Red Blood Cells

Introduction: We tested the feasibility of the determination of 24-hour recovery and survival time of transfused red blood cells (RBCs) by flow cytometry to evaluate a new RBC preparation in a clinical application study. Patients and Methods: Eleven patients received 22 inline filtered RBC concentrates from apheresis (mean storage time of transfused RBCs 25.6 ± 12 days). Recovery and survival were calculated based on flow-cytometric determination of autologous and transfused RBCs by measuring antigens differing between donor and recipient. Human polyclonal erythrocyte antibodies (IgG) were used as primary and FITC-conjugated rabbit antihuman IgG antibody (F(ab’)2) fragments as secondary antibodies. Results: The mean (± SD), 24-hour recovery determined, using anti-c, anti-C, anti-D, anti-Jka and anti-Fya as primary antibodies, was 78 ± 12%, the mean RBC survival time was 70.1 ± 26.5 days. Discussion: Flow cytometry using blood group antigen differences between donor and recipient appears to be suitable for the determination of RBC 24-hour recovery and survival time. This method requires no pretransfusional labeling of the RBCs, thus avoiding any bias by manipulation of the transfused RBCs. However, this method is applicable only to allogeneous transfusion.