A comparison of continuous blood gas monitors during cardiopulmonary bypass LivaNova B-Capta, Terumo CDI 500, spectrum medical M4.

INTRODUCTION: Accurate and precise management of blood gas parameters during cardiopulmonary bypass (CPB) is crucial to patient care and outcome. This study compares the data provided by Livanova B-Capta, Terumo CDI500, and Spectrum Medical M4 with the results from a gold standard blood gas analyzer to test accuracy. METHODS: All three continuous blood gas monitoring (CBGM) devices were used simultaneously during CPB on one dedicated HLM. Arterial and venous blood samples of 40 adult patients who underwent elective cardiac surgery with CPB were taken from the CPB circuit. RESULTS: Pre- and post-alignment deviation in percentages are compared with CLIA guidelines. B-Capta data reveals that the deviation pre-alignment is small and within the CLIA threshold for all parameters. Pre-alignment data for CDI 500 is within CLIA threshold for SvO2 and PaO2. The pre-alignment data for the M4 exceeds the CLIA thresholds for all parameters. Post-alignment data for B-Capta and CDI 500 reveals an accurate agreement for Hb and Hct and strong agreement for PaO2. All values for B-Capta and CDI 500 are within CLIA threshold values except for SvO2. Post-alignment the M4 exceeded the CLIA threshold value only for PaO2. CONCLUSION: B-Capta is the only CBGM device that operates within the CLIA guidelines and is in agreement with laboratory values prior to alignment.

Acute isotonic hyponatremia after single dose histidine-tryptophan-ketoglutarate cardioplegia: an observational study.

INTRODUCTION: Histidine-tryptophan-ketoglutarate cardioplegia is used for prolonged myocardial protection in complex cardiac surgery. Administration leads to acute hyponatremia in a majority of patients, because of its low sodium concentration (15 mmol/L). However, histidine-tryptophan-ketoglutarate solution's osmolality is slightly hypertonic (310 mOsm/kg). Hypothesized was that acute isotonic hyponatremia will be induced, which does not need to be corrected with hypertonic saline. METHODS: Cardiac surgery patients who received histidine-tryptophan-ketoglutarate cardioplegia were included in this prospective single center study. Serial blood samples were taken from each patient at five different time points: after induction of anesthesia (T1) and 10 minutes (T2), 6 hours (T3), 12 hours (T4), and 18 hours (T5) after administration of histidine-tryptophan-ketoglutarate cardioplegia, respectively. Blood samples were analyzed for sodium concentration, osmolality, and acid-base balance. RESULTS: Twenty-five patients were included. Median blood sodium levels decreased from 140 [138-141] at T1 to 128 [125-130] mmol/L at T2 (p < 0.001). At T3, T4, and T5, median blood sodium concentrations were 136 [134-138], 139 [137-140], and 140 [137-142] mmol/L, respectively. Median osmolality was 289 [286-293] at T1 and increased to 296 [291-299] mOsm/kg (p < 0.001) at T2. At T3, T4, and T5, osmolality was 298 [292-302], 298 [294-304], and 300 [297-306] mOsm/kg, respectively. Median pH decreased from 7.38 [7.36-7.40] at T1 to 7.30 [7.27-7.32] at T2 (p < 0.001). CONCLUSION: Administration of histidine-tryptophan-ketoglutarate cardioplegia during cardiac surgery leads to acute moderate to severe isotonic hyponatremia, which resolves spontaneously in the first 18 hours perioperatively. Correction with hypertonic saline is not necessary.

Diabetes mellitus at the time of diagnosis of cirrhosis is associated with higher incidence of spontaneous bacterial peritonitis, but not with increased mortality.

DM (diabetes mellitus) is present in 20-40% of patients with liver cirrhosis, but its prognostic impact is unclear. Therefore, in the present study, we investigated whether the presence of DM in patients with cirrhosis was associated with increased mortality, and/or with increased incidence of SBP (spontaneous bacterial peritonitis). We reviewed medical and laboratory data of 230 patients with cirrhosis from the period 2001-2011, for whom data were complete in n=226. Follow-up for the outcomes mortality and SBP was performed until May 2012, with only 13 patients lost to follow-up. DM was present at baseline in 78 patients (35%). Median follow-up was 6.2 (interquartile range, 3.1-9.3) years, during which 118 patients died [47 out of 78 with DM (60%), and 71 out of 148 without DM (48%)]. The presence of DM at baseline was not associated with increased mortality after adjustment for age {HR (hazard ratio), 1.00 [95% CI (confidence interval), 0.67-1.50]}. Further adjustment for sex, aetiology of cirrhosis, platelet count and the Child-Pugh or MELD (model for end-stage liver disease) score did not change this finding. During follow-up, 37 patients developed incident SBP (19 with DM and 18 without DM). DM at baseline was associated with incident SBP, even after adjustment for age, sex, aetiology, platelet count and the Child-Pugh [HR, 2.39 (95% CI, 1.10-5.18)] or MELD score [HR, 2.50 (95% CI, 1.16-5.40)]. In conclusion, the presence of DM at baseline in patients with cirrhosis was associated with an increased risk of SBP, which may represent an increased susceptibility to infections. On the other hand, DM was not clearly associated with increased mortality in these patients.

Reference intervals of extended erythrocyte and reticulocyte parameters.

BACKGROUND: Optical analysis of erythrocytes can provide information on the hemoglobin concentration and content of reticulocytes and mature erythrocytes. Such parameters have proven clinical utility in anemia diagnosis and therapy monitoring. For interpretation, reliable reference ranges are needed. The aim of this study was to establish reference intervals for extended erythrocyte and reticulocyte parameters as measured with the Abbott CELL-DYN Sapphire hematology analyzer. Secondary aims were to study sample stability and to investigate gender- and age dependency of the reference ranges. METHODS: Extended RBC parameters were measured in routine samples of a primary health care laboratory. The study cohort included 8161 samples of unique individuals, which were analyzed using Bhattacharya statistics. As a comparison, reference intervals were calculated in a subset of individuals without iron depletion. RESULTS: The majority of erythrocyte and reticulocyte parameters were normally distributed, allowing calculation of reference intervals. Only for hypo- and hyperchromic erythrocytes non-parametric statistics had to be used. The reference range for mean cellular hemoglobin content of reticulocytes (MCHr) was 28.5-34.5 pg (1.77-2.14 fmol) in the entire study group and 26.0-35.1 pg (1.60-2.17 fmol) in the non iron-depleted subgroup. No differences between sexes were found. Most parameters showed significant age effects in children and adolescents. CONCLUSIONS: Reference intervals have been established for extended RBC and reticulocyte parameters for the CELL-DYN Sapphire. Gender effects could not be demonstrated and age effects were of limited size, except for individuals younger than 18 years. Extended RBC parameters are stable for at least 6 h after blood collection.

Trueness in the measurement of haemoglobin: consensus or reference method?

BACKGROUND: For the measurement of haemoglobin a reference method exists: the haemiglobincyanide method. However, a Dutch external quality assessment organization does not use this method in the evaluation of trueness of results. The aim of this work was to assess whether trueness was compromised by the use of a consensus value. METHODS: Five Cell Dyn Sapphires (Abbott) in three independent locations were used to measure haemoglobin concentration. Results were compared to the reference method (haemiglobincyanide). Patient samples with a distribution over clinically relevant concentrations (Hb 2.5-10.2 mmol/L) were used next to samples from external quality assessment rounds. Passing and Bablok regression analysis and Bland-Altman plots were used to evaluate any systematic deviation. RESULTS: Results measured on the Cell Dyn Sapphires deviated significantly from the results obtained with the reference method. Remarkably, consensus results from external quality control samples also deviated significantly from the reference method. CONCLUSIONS: A significant negative bias exists in the measurement of haemoglobin on Cell Dyn Sapphires. Additionally, the consensus value as reported in external quality control assessment also shows an even greater significant negative bias compared to the reference method. As a reference method is available, external quality assessment would benefit from using this method instead of a consensus value to evaluate trueness.

Comparison of different immunoassays for the detection of antibodies against Intrinsic Factor and Parietal Cells.

OBJECTIVES: In the diagnostic work up of autoimmune gastritis several immunological methods are available for the detection of antibodies against Intrinsic Factor (IF) and Parietal Cells (PC). However, there are no recent reports directly comparing all the available assays and methods. The objective of this study was to compare the performance of several commercially available anti-IF and anti-PC antibody assays from different manufacturers in a multi-center multi-cohort setting. METHODS: Sera were used from 5 different cohorts consisting of samples from 25 healthy elderly, 20 HCV or HIV positive patients and 150 patients positive for anti-IF or anti-PC antibodies or in whom these antibodies were requested. These cohorts were tested for anti-IF antibodies with 6 different assays (IIF, ELISA, DIA and EliA) and for anti-PC antibodies with 7 different assays (IIF, ELISA, DIA and EliA). Performance was evaluated by calculating the concordance and relative sensitivity and specificity. RESULTS: Good concordance was found between the assays for both antibody specificities, ranging from 81 to 100% and 91-100% for anti-IF and anti-PC antibodies, respectively. Highest relative sensitivity was found with the (automated) ELISA based methods. However, all assays had a relative sensitivity between 85 and 100% for anti-IF antibodies and between 95 and 100% for anti-PC antibodies. The relative specificity ranged between 76 and 100% for anti-IF antibodies and between 96 and 100% for anti-PC antibodies. CONCLUSIONS: We conclude that most assays perform well and are concordant to each other, despite the methodological differences and the different sources of antigen used. However, the method used affects the sensitivity and specificity. The (automated) ELISA based assays have the highest relative sensitivity and relative specificity. Care should be taken in the interpretation of positive results by IIF and negative results by the Blue Diver when testing for anti-IF antibodies.

Analytical Performance and Clinicopathologic Correlation of Four Fecal Calprotectin Methods.

OBJECTIVES: Calprotectin is a noninvasive biomarker that can distinguish inflammatory bowel disease from irritable bowel syndrome. We investigated four automated fecal calprotectin methods on five different platforms for their preanalytical process, analytical performance, and clinicopathologic correlation. METHODS: Four calprotectin methods (Bühlmann, EliA CN, EliA CN2, and DiaSorin) were performed on five platforms (Cobas 8000 E502, Phadia Immunocap 100 and 250, and Liaison and Liaison XL) in two hospital laboratories. RESULTS: Overall variation for the different extraction devices was less than 19% when feces were of normal consistency. Freeze-thawing of samples resulted in comparable results compared with fresh samples. The different methods had a good analytic correlation (R = 0.83-0.95). Their clinicopathologic correlation was comparable, but the Bühlmann method showed significantly higher calprotectin values in every patient category. CONCLUSIONS: The automated calprotectin methods showed a good performance and comparable clinicopathologic correlation. Due to lack of standardization, the numerical values differ for the various methods.

Frequencies and clinical associations of myositis-related antibodies in The Netherlands: A one-year survey of all Dutch patients.

Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of connective tissue diseases, collectively known as myositis. Diagnosis of IIM is challenging while timely recognition of an IIM is of utter importance considering treatment options and otherwise irreversible (severe) long-term clinical complications. With the EULAR/ACR classification criteria (2017) considerable advancement has been made in the diagnostic workup of IIM. While these criteria take into account clinical parameters as well as presence of one autoantibody, anti-Jo-1, several autoantibodies are associated with IIM and are currently evaluated to be incorporated into classification criteria. As individual antibodies occur at low frequency, the development of line blots allowing multiplex antibody analysis has improved laboratory diagnostics for IIM. The Euroline myositis line-blot assay (Euroimmun) allows screening and semi-quantitative measurement for 15 autoantibodies, i.e. myositis specific antibodies (MSA) to SRP, EJ, OJ, Mi-2α, Mi-2ß, TIF1-γ, MDA5, NXP2, SAE1, PL-12, PL-7, Jo-1 and myositis associated antibodies (MAA) to Ku, PM/Scl-75 and PM/Scl-100. To evaluate the clinical significance of detection and levels of these autoantibodies in the Netherlands, a retrospective analysis of all Dutch requests for extended myositis screening within a 1 year period was performed. A total of 187 IIM patients and 632 non-IIM patients were included. We conclude that frequencies of MSA and MAA observed in IIM patients in a routine diagnostic setting are comparable to cohort-based studies. Weak positive antibody levels show less diagnostic accuracy compared to positive antibody levels, except for anti-NXP2. Known associations between antibodies and skin involvement (anti-MDA5, anti-TIF1-γ), lung involvement (anti-Jo-1), and malignancy (anti-TIF1-γ) were confirmed in our IIM study population. The availability of multiplex antibody analyses will facilitate inclusion of additional autoantibodies in clinical myositis guidelines and help to accelerate diagnosing IMM with rare but specific antibodies.

Increased thrombin generation and fibrinogen level after therapeutic plasma transfusion: relation to bleeding.

In a clinical setting, fresh frozen plasma (FFP) is transfused to diluted patients with complicated surgery or trauma, as guided by prolonged conventional coagulation times or low fibrinogen levels. However, the limited sensitivity of these coagulation tests may restrict their use in measuring the effect of transfusion and hence predicting the risk of perioperative bleeding. We used the more sensitive, calibrated automated thrombogram (CAT) method to evaluate the result of therapeutic FFP transfusion to 51 patients with dilutional coagulopathy. Thrombin generation was measured in pre- and post-transfusion plasma samples in the presence of either platelets or phospholipids. For all patients, the transfusion led to higher plasma coagulation factor levels, a shortened activated partial thromboplastin time, and a significant increase in thrombin generation (peak height and endogenous thrombin potential). Interestingly, thrombin generation parameters and fibrinogen levels were higher in post-transfusion plasmas from patients who stopped bleeding (n = 32) than for patients with ongoing bleeding (n = 19). Plasmas from 15 of the 19 patients with ongoing bleeding were markedly low in either thrombin generation or fibrinogen level. We conclude that the thrombin generation method detects improved haemostatic activity after plasma transfusion. Furthermore, the data suggest that thrombin generation and fibrinogen are independent determinants of the risk of perioperative bleeding in this patient group.

Hemostatic and signaling functions of transfused platelets.

Metabolic studies have revealed a gradual impairment in platelet integrity during storage, a process termed the platelet storage lesion. Recent evidence shows that stored platelets also lose signaling responses to physiological agonists with impaired integrin activation, secretion, and aggregation of the cells. On the other hand, storage leads to a gain in platelet activation properties, such as release of microparticles and appearance of surface epitopes for their clearance by macrophages. New techniques for measuring flow-induced thrombus formation and platelet-dependent coagulation provide evidence that the hemostatic activity of platelets decreases during storage. Besides pharmacological inhibition, novel storage strategies, like metabolic suppression, should be considered to better preserve platelet functionality while limiting the expression of clearance markers. Understanding the changes that occur in association with the platelet storage lesion and the use of updated storage methods will help to generate platelets for transfusion with optimal hemostatic function and a long circulation time after transfusion.

Short article: The effect of implementation of a treatment algorithm for infliximab on remission rates and drug costs in inflammatory bowel disease patients.

INTRODUCTION: The effective, but expensive, drug infliximab is used in patients with inflammatory bowel disease (IBD). Monitoring infliximab trough levels and anti-infliximab antibody (ATI) formation can lead to a more cost-effective use of infliximab therapy. The aim of our study was to investigate the effect of implementation of a treatment algorithm for infliximab in a single-centre IBD cohort, focussing on remission rates and drug costs. METHODS: IBD patients aged 18 years or older treated with infliximab were asked to participate in this study. Remission rates were assessed using faecal calprotectin levels and a validated questionnaire. Infliximab trough levels and ATIs were determined at baseline and at the third infliximab infusion. According to the advice given by the treatment algorithm, infliximab dosage adjustments were performed at the second infliximab infusion. RESULTS: Between January and December 2015 a total of 62 IBD patients in our centre were treated with infliximab, of whom 33 (53%) patients agreed to participate in this study. The number of patients in remission was 28 (85%) at baseline and there were 13 dose adaptations suggested by the treatment algorithm for the successive second infusion. Four patients possessed undetectable infliximab levels and positive ATI status at baseline. After the second infusion, there were 29 (88%) patients in remission at the third infusion. All of this resulted in an annual drug cost reduction of &OV0556;47 026 (7.4%). CONCLUSION: Our developed treatment algorithm of infliximab led to optimization of infliximab therapy in IBD patients by increasing remission rates and reducing drug costs.

Analytical evaluation of a new point of care system for measuring cardiac Troponin I.

OBJECTIVES: Point-of-care cardiac troponin testing with adequate analytical performances has the potential to improve chest pain patients flow in the emergency department. We present the analytical evaluation of the newly developed Philips Minicare cTnI point-of-care immunoassay. DESIGN & METHODS: Li-heparin whole blood and plasma were used to perform analytical studies. The sample type comparison study was performed at 4 different hospitals. The 99th percentile upper reference limit (URL) study was performed using Li-heparin plasma, Li-heparin whole blood and capillary blood samples from 750 healthy adults, aging from 18 to 86years. RESULTS: Limit of the blank, limit of detection and limit of quantitation at 20% coefficient of variation (CV) were determined to be 8.5ng/L, 18ng/L and 38ng/L respectively without significant differences between whole blood and plasma for LoQ. Cross-reactivity and interferences were minimal and no high-dose hook was observed. Total CV was found to be from 7.3% to 12% for cTnI concentrations between 109.6 and 6135.4ng/L. CV at the 99th percentile URL was 18.6%. The sample type comparison study between capillary blood, Li-heparin whole blood and Li-heparin plasma samples demonstrated correlation coefficients between 0.99 and 1.00 with slopes between 1.03 and 1.08. The method comparison between Minicare cTnI and Beckman Coulter Access, AccuTnI+3 demonstrated a correlation coefficient of 0.973 with a slope of 1.09. The 99th percentile URL of a healthy population was calculated to be 43ng/L with no significant difference between genders or sample types. CONCLUSIONS: The Minicare cTnI assay is a sensitive and precise, clinical usable test for determination of cTnI concentration that can be used in a near-patient setting as an aid in the diagnosis of acute myocardial infarction.

Shedding of procoagulant microparticles from unstimulated platelets by integrin-mediated destabilization of actin cytoskeleton.

Platelet activation by potent, Ca(2+)-mobilizing agonists results in shedding of microparticles that are active in coagulation. Here we show that platelets under storage produce procoagulant microparticles in the absence of agonist. Microparticle formation by resting platelets results from alphaIIbbeta3 signaling to destabilization of the actin cytoskeleton in the absence of calpain activation. Integrin-mediated spreading of platelets over fibrinogen similarly results in microparticle formation. After transfusion of stored platelet preparations to thrombocytopenic patients, the microparticles contribute to coagulant activity in vivo.

Effects of hereditary and acquired risk factors of venous thrombosis on a thrombin generation-based APC resistance test.

BACKGROUND: Several hereditary and acquired risk factors for venous thromboembolism (VTE) are associated with impaired down-regulation of thrombin formation via the protein C pathway. To identify individuals at risk, functional tests are needed that estimate the risk to develop venous thrombosis. METHOD: We determined the effects of hereditary and acquired risk factors of venous thrombosis on an APC resistance test that quantifies the influence of APC on the time integral of thrombin formation (the endogenous thrombin potential, ETP) initiated in plasma via the extrinsic coagulation pathway. APC sensitivity ratios (APCsr) were determined in plasma from carriers of factor V(Leiden) (n = 56) or prothrombin G20210A (n = 18), of individuals deficient in antithrombin (n = 9), protein C (n = 7) or protein S (n = 14) and of women exposed to acquired risk factors such as hormone replacement therapy (n = 49), oral contraceptive use (n = 126) or pregnancy (n = 35). We also analysed combinations of risk factors (n = 60). RESULTS: The thrombin generation-based APC resistance test was sensitive for the factor V(Leiden) and prothrombin G20210A mutation, to protein S deficiency, hormone replacement therapy, oral contraceptive use and pregnancy. The assay was not influenced by antithrombin or protein C deficiency. The presence of more than one risk factor of venous thrombosis resulted in more pronounced APC resistance. The APCsr of individuals with a single or combined risk factors of VTE correlated well with reported risk increases. INTERPRETATION: The thrombin generation-based APC resistance test identifies individuals at risk for venous thrombosis due to acquired risk factors and/or hereditary thrombophilic disorders that affect the protein C pathway.

Effects of (pre-)analytical variables on activated protein C resistance determined via a thrombin generation-based assay.

The normalized activated protein C sensitivity ratio (nAPC-sr) determined with an assay that quantifies the effect of APC on thrombin formation initiated via the extrinsic coagulation pathway identifies hereditary and acquired defects of the protein C system. We investigated the influence of assay conditions (analytical variables) and plasma handling (pre-analytical variables) on nAPC-sr obtained with this APC resistance test. The effect of the analytical variables (CaCl2, phospholipid and APC concentrations and the concentration and source of tissue factor) was determined in pooled normal plasma. Inhibition of thrombin formation by APC was dependent on the APC concentration and was also affected by the tissue factor, Ca2+ and phospholipid concentrations. Thus, strict standardization of reactant concentrations is required to obtain reproducible nAPC-sr. Three different tissue factor preparations were compared by determining nAPCsr in plasma samples obtained from 90 healthy individuals. nAPC-sr were similar for all three tissue factor preparations although, compared with the noncommercially available tissue factor used in earlier studies, values determined with commercial tissue factor preparations showed larger variation. Pre-analytical variables, investigated in plasma of nine volunteers (3 normal individuals and 6 individuals with an APC-resistant phenotype) were: concentration of anticoagulant (3.2% vs. 3.8% trisodiumcitrate), time before processing of blood (0, 4 and 24 h), centrifugation speed, storage temperature of plasma (-20 degrees C vs. -80 degrees C) and sample thawing. Multiple linear regression analysis showed that only the citrate concentration affected the nAPC-sr, which was higher in samples collected in 3.2% trisodiumcitrate than in samples collected in 3.8% trisodiumcitrate.

Reference intervals of reticulated platelets and other platelet parameters and their associations.

CONTEXT: Reticulated platelets are platelets recently released from the bone marrow, and they can serve as a noninvasive indicator of recent megakaryopoietic activity. Widespread clinical use has been hampered by laborious methods and lack of standardization. Recently, a fully automated method was released on the Abbott CELL-DYN Sapphire hematology analyzer. OBJECTIVE: To establish reference ranges for reticulated platelets. Secondary aims were to investigate associations between reticulated platelets and other platelet parameters like mean platelet volume, plateletcrit, and platelet distribution width. DESIGN: Reticulated platelets and other platelet parameters were measured in an unselected cohort of 8089 subjects visiting a primary health care laboratory. The reticulated platelet data were analyzed using the Bhattacharya technique. In addition, a nonparametric method was used in selected subjects with normal platelet counts for providing reference ranges. RESULTS: Reticulated platelets ranged from 0.4% to 6.0% or from 1 to 18 × 10(3)/µL. Reticulated platelets increased significantly with the subjects' age. Statistically, males had slightly higher values than females, but the differences were negligible. Reticulated platelets were positively correlated with platelet count and negatively with mean platelet volume. CONCLUSIONS: Reference ranges have been established for reticulated platelets as measured on the CELL-DYN Sapphire hematology analyzer. There were no relevant differences between the sexes, but there was a clear effect of age. An individual's reticulated platelets are associated with the platelet count as well as mean platelet volume.

Measuring direct thrombin inhibitors with routine and dedicated coagulation assays: which assay is helpful?

The use of direct thrombin inhibitors (DTIs) for prophylactic or therapeutic anticoagulation is increasing because of the predictable bioavailability and short half-life of these DTIs. However, in certain situations, indication of the concentration is warranted. We investigated the effects of 3 DTIs (lepirudin, argatroban, and bivalirudin) in 6 pooled plasma specimens on routine coagulation assays (activated partial thromboplastin time [aPTT], prothrombin time [PT], and thrombin time [TT]) and dedicated DTI assays (Hemoclot, HemosIL, the ecarin clotting time, and a chromogenic ecarin clotting time) on 2 coagulation analyzers. We found routine tests to be nondiscriminative between concentrations of different DTIs in the aPTT. Moreover, for PT and TT, the responses for different DTIs differed. This was similar for ecarin clotting assays. The Hemoclot and HemosIL assays showed identical linear increases for all 3 DTIs. We conclude that dedicated calibrated assays based on a diluted TT (Hemoclot and HemosIL) appear to be the most suitable for monitoring purposes.

Evaluation of the Ves-Matic Cube 200 erythrocyte sedimentation method: comparison with Westergren-based methods.

The erythrocyte sedimentation rate (ESR) is still a widely used parameter for acute phase inflammation. Recently, new methods based on direct undiluted measurement of ESR in a standard EDTA tube have been developed. We evaluated the analytic performance of one of these new methods, the Ves-Matic Cube 200 (Diesse Diagnostica Senese, Siena, Italy), and compared it with several established Westergren-based diluted methods. The Ves-Matic Cube 200 showed a poor correlation (r = 0.83) with the International Council for Standardization in Haematology Westergren reference method, mainly caused by a considerable negative bias at low ESR levels. Moreover, a random bias was found at higher ESR levels that correlated with hematocrit levels, suggesting a differential influence of packed cell volume on the Ves-Matic Cube 200 results compared with Westergren results. We conclude that the Ves-Matic Cube 200 method is not interchangeable with Westergren-based diluted methods and generates ESR results that are too deviant to be clinically acceptable.

Flow cytometric measurement of CD62P (P-selectin) expression on platelets: a multicenter optimization and standardization effort.

BACKGROUND: One of the variables to determine the quality of platelets (PLTs) in vitro is measurement of CD62P expression. Different protocols are in use, however, making comparison of results virtually impossible. It was our aim to develop a uniform CD62P protocol that would yield comparable results in various laboratories. STUDY DESIGN AND METHODS: The effects of fixation, source and dilution of CD62P antibody, source of immunoglobulin G (IgG) isotypic antibody, and analysis of results were investigated. Once the optimal variables were defined, comparative studies were performed at five participating centers. In the final comparative study, eight split PLT concentrates were shipped to the centers, where samples were stained and fixed according to the uniform protocol. Analyses were performed using commercially available flow cytometers (BD Biosciences and Beckman Coulter). RESULTS: Uniformity between centers could be achieved by using a single clone for CD62P and IgG monoclonal antibody. A protocol was selected using fixation with 0.5 percent methanol-free formaldehyde. To increase conformity between flow cytometers, in the analysis of electronic data the thresholds of the isotypic control were set at 0.5 percent for the BD Biosciences and 2 percent for the Beckman Coulter flow cytometers. In the final comparative study, the 95 percent confidence intervals (CIs) for CD62P ranged between 8 and 21 percent in fresh and 20 to 40 percent in 8-day-old PLT concentrates. CONCLUSION: A uniform CD62P staining protocol and subsequent analysis can be used at multiple centers using different flow cytometers, yielding comparable results with acceptable 95 percent CIs.