Quality of factor XI activity testing in North American Specialized Coagulation Laboratories.

INTRODUCTION: The performance of factor XI activity (FXI) by laboratories in the North American Specialized Coagulation Laboratory Association proficiency testing program was analyzed. METHODS: Over 10 years (2003-2013), 80 samples were distributed; 33-55 laboratories participated per exercise providing 3833 total responses. Analysis was performed on numeric results and qualitative classification of results. RESULTS: The sample FXI levels ranged from 3.8 to 154.0 IU/dL. The overall interlaboratory average coefficient of variation (CV%) was 17.5%; the CV was higher for a sample with low (3.8 IU/dL) FXI. Results were correctly classified as abnormal (100%) for a sample with 3.8 IU/dL FXI and normal/borderline normal (97.7%) for 45 samples with 80 to < 140 IU/dL FXI. The classification was heterogeneous for samples with FXI of 50 to < 80 IU/dL. Six specimens were repeat-tested from 2007 to 2013. The mean FXI was not significantly different in laboratories using the same method on both exercises, suggesting good intralaboratory precision over time. Univariate analysis of data from 2011 to 2012 did not find a consistent significant difference among the activators, analyzers, calibrators, and FXI-deficient plasmas. CONCLUSION: Laboratories generally performed well in assessment of FXI based on interlaboratory precision when FXI >30 IU/dL and on classification of samples with very low or normal FXI.

Factor VII assay performance: an analysis of the North American Specialized Coagulation Laboratory Association proficiency testing results.

The performance of factor VII (FVII) assays currently used by clinical laboratories was examined in North American Specialized Coagulation Laboratory Association (NASCOLA) proficiency tests. Data from 12 surveys conducted between 2008 and 2010, involving 20 unique specimens plus four repeat-tested specimens, were analyzed. The number of laboratories per survey was 49-54 with a total of 1224 responses. Numerous reagent/instrument combinations were used. For FVII > 80 or <40 U/dL, 99.5% of results (859/863) were correctly classified by laboratories as normal/abnormal. Classification of specimens with 40-73 U/dL FVII was heterogeneous. Interlaboratory precision was better for normal specimens (coefficient of variation (CV) 10.7%) than for FVII<20 U/dL (CV 33.1%), with a mean CV of 17.2% per specimen. Intralaboratory precision for repeated specimens demonstrated no significant difference between the paired survey results (mean absolute difference 2.5-5.0 U/dL). For specimens with FVII >50 U/dL, among commonly used methods, one thromboplastin and one calibrator produced results 5-6 U/dL higher and another thromboplastin and calibrator produced results 5-6 U/dL lower than all other methods, and human thromboplastin differed from rabbit by +7.6 U/dL. Preliminary evidence suggests these differences could be due to the calibrator. For FVII <50 U/dL, differences among the commonly used reagents and calibrators were generally not significant.

Haemolysis after treatment with intravenous immunoglobulin due to anti-A.

BACKGROUND: Intravenous immunoglobulin (IVIG) is used to treat an increasing number of conditions. IVIG contains immunoglobulin G (IgG) directed against many targets, including red blood cell (RBC) antigens. METHODS/MATERIALS: We report on three patients identified within a 7-month period in a single institution who developed haemolysis because of passively transferred anti-A. RESULTS: The patients were a 34-year-old A (non-A1) D-positive male with aplastic anaemia, a 61-year-old A1 D-negative female with myasthenia gravis and a 57-year-old AB D-positive female lung transplant recipient. The haemoglobin decreased from 11.1 to 5.3 g dL(-1) over 2 days, 12.8 to 7.8 g dL(-1) over 6 days and 7.8 to 6.0 g dL(-1) over several hours, respectively. All three patients had a negative antibody screen, positive direct antiglobulin test for IgG only and an elution containing anti-A1 reactivity. The patients were transfused with O RBC with an appropriate rise in haemoglobin. CONCLUSION: These cases illustrate the potential severity of haemolysis after IVIG because of passively transferred antibodies to blood group antigens. Lack of recognition of IVIG as a cause for haemolysis by clinicians may be further confounded if routine testing fails to detect the passively transferred ABO blood group antibodies.

Equivalence of spray-dried K2EDTA, spray-dried K3EDTA, and liquid K3EDTA anticoagulated blood samples for routine blood center or transfusion service testing.

We compared the results of routine blood tests for 102 blood donors' samples and 100 patients' samples collected in spray-dried K2EDTA, spray-dried K3EDTA, and liquid K3EDTA blood collection tubes to evaluate the impact of changes in formulation of the anticoagulant (K2EDTA vs.K3EDTA), its application (liquid vs. spray-dried), and tube material (glass vs. plastic). Methods for ABO/D testing, antibody screening, and antibody identification included direct hemagglutination/microplate (Olympus(R) PK 7200) and gel column methods (Ortho ID-Micro Typing System/Gel Test). Additional studies on blood donors' samples included time delayed antigen testing and antibody identification and half-draw/half-evacuated collections. Also, we compared the results of routine ABO/D testing and antibody screening for 50 patients' samples collected in spray-dried K2EDTA and spray-dried K3EDTA and for an additional 50 patients' samples collected in spray-dried K2EDTA tubes from two different manufacturers. All patients' samples were tested in parallel by solid phase/microplate method (Immucor ABS 2000) and the standard manual tube method. All test results for routine blood bank tests on donors' and patients' samples were concordant, demonstrating the equivalence of spray-dried K2EDTA, spray-dried K3EDTA, and liquid K3EDTA blood collection tubes for routine donor center or transfusion service testing.

MCF-10A-NeoST: a new cell system for studying cell-ECM and cell-cell interactions in breast cancer.

PURPOSE: There is a continuing need for genetically matched cell systems to model cellular behaviors that are frequently observed in aggressive breast cancers. EXPERIMENTAL DESIGN: We report here the isolation and initial characterization of a spontaneously arising variant of MCF-10A cells, NeoST, which provides a new model to study cell adhesion and signal transduction in breast cancer. RESULTS: NeoST cells recapitulate important biological and biochemical features of metastatic breast cancer, including anchorage-independent growth, invasiveness in three-dimensional reconstituted membranes, loss of E-cadherin expression, and increased tyrosine kinase activity. A comprehensive analysis of tyrosine kinase expression revealed overexpression or functional activation of the Axl, FAK, and EphA2 tyrosine kinases in transformed MCF-10A cells. CONCLUSIONS: MCF-10A and these new derivatives provide a genetically matched model to study defects in cell adhesion and signaling that are relevant to cellular behaviors that often typify aggressive breast cancer cells.

EphA2 overexpression causes tumorigenesis of mammary epithelial cells.

Elevated levels of protein tyrosine phosphorylation contribute to a malignant phenotype, although the tyrosine kinases that are responsible for this signaling remain largely unknown. Here we report increased levels of the EphA2 (ECK) protein tyrosine kinase in clinical specimens and cell models of breast cancer. We also show that EphA2 overexpression is sufficient to confer malignant transformation and tumorigenic potential on nontransformed (MCF-10A) mammary epithelial cells. The transforming capacity of EphA2 is related to the failure of EphA2 to interact with its cell-attached ligands. Interestingly, stimulation of EphA2 reverses the malignant growth and invasiveness of EphA2-transformed cells. Taken together, these results identify EphA2 as a powerful oncoprotein in breast cancer.

E-cadherin regulates the function of the EphA2 receptor tyrosine kinase.

EphA2 is a member of the Eph family of receptor tyrosine kinases, which are increasingly understood to play critical roles in disease and development. We report here the regulation of EphA2 by E-cadherin. In nonneoplastic epithelia, EphA2 was tyrosine-phosphorylated and localized to sites of cell-cell contact. These properties required the proper expression and functioning of E-cadherin. In breast cancer cells that lack E-cadherin, the phosphotyrosine content of EphA2 was decreased, and EphA2 was redistributed into membrane ruffles. Expression of E-cadherin in metastatic cells restored a more normal pattern of EphA2 phosphorylation and localization. Activation of EphA2, either by E-cadherin expression or antibody-mediated aggregation, decreased cell-extracellular matrix adhesion and cell growth. Altogether, this demonstrates that EphA2 function is dependent on E-cadherin and suggests that loss of E-cadherin function may alter neoplastic cell growth and adhesion via effects on EphA2.