Decreased generation of procoagulant platelets detected by flow cytometric analysis in patients with bleeding diathesis.

BACKGROUND: A clinically relevant bleeding diathesis is a frequent diagnostic challenge, which sometimes remains unexplained despite extensive investigations. The aim of our work was to evaluate the diagnostic utility of functional platelet testing by flow cytometry in this context. METHODS: In case of negative results after standard laboratory workup, flow cytometric analysis (FCA) of platelet function was done. We performed analysis of surface glycoproteins Ibα, IIb, IIIa; P-selectin expression and PAC-1 binding after graded doses of ADP, collagen, and thrombin; content/secretion of dense granules; and ability to generate procoagulant platelets. RESULTS: Of 437 patients investigated with standard tests between January 2007 and December 2011, we identified 67 (15.3%) with high bleeding scores and nondiagnostic standard laboratory workup including platelet aggregation studies. Among these patients, FCA revealed some potentially causative platelet defects: decreased dense granule content/secretion (n = 13); decreased α-granule secretion induced by ADP (n = 10), convulxin (n = 4), or thrombin (n = 3); decreased fibrinogen receptor activation induced by ADP (n = 11), convulxin (n = 11), or thrombin (n = 8); and decreased generation of COAT platelets, that is, highly procoagulant platelets induced by simultaneous activation with collagen and thrombin (n = 16). CONCLUSION: Our work confirms that storage pool defects are frequent in patients with a bleeding diathesis and normal coagulation and platelet aggregations studies. Additionally, FCA is able to identify discrete platelet activation defects. In particular, we show for the first time that a relevant proportion of these patients has an isolated impaired ability to generate COAT platelets--a conceptually new defect in platelet procoagulant activity, which is missed by conventional laboratory workup.

Decreased generation of procoagulant platelets detected by flow cytometric analysis in patients with bleeding diathesis.

Background: A clinically relevant bleeding diathesis is a frequent diagnostic challenge, which sometimes remains unexplained despite extensive investigations. The aim of our work was to evaluate the diagnostic utility of functional platelet testing by flow cytometry in this context. Methods: In case of negative results after standard laboratory work-up, flow cytometric analysis (FCA) of platelet function was done. We performed analysis of surface glycoproteins (GP) Ibα, IIb, IIIa; P-selectin expression and PAC-1 binding after graded doses of ADP, collagen and thrombin; content/secretion of dense granules; ability to generate procoagulant platelets. Results: Out of 437 patients investigated with standard tests between January 2007 and December 2011, we identified 67 (15.3%) with high bleeding scores and non-diagnostic standard laboratory work-up including platelet aggregation studies. Among these patients FCA revealed some potentially causative platelet defects: decreased dense-granule content/secretion (n=13); decreased alpha-granule secretion induced by ADP (n=10), convulxin (n=4) or thrombin (n=3); decreased fibrinogen-receptor activation induced by ADP (n=11), convulxin (n=11) or thrombin (n=8); decreased generation of COAT-platelets, i.e. highly procoagulant platelets induced by simultaneous activation with collagen and thrombin (n=16). Conclusion: Our work confirms that storage pool defects are frequent in patients with a bleeding diathesis and normal coagulation and platelet aggregations studies. Additionally, flow cytometric analysis is able to identify discrete platelet activation defects. In particular, we show for the first time that a relevant proportion of these patients has an isolated impaired ability to generate COAT-platelets - a conceptually new defect in platelet procoagulant activity, that is missed by conventional laboratory work-up. © 2014 Clinical Cytometry Society.

Rapid exclusion or confirmation of heparin-induced thrombocytopenia: a single-center experience with 1,291 patients.

BACKGROUND: The current gold-standard for diagnosing heparin-induced thrombocytopenia is the detection of platelet-activating antibodies by means of functional assays which, since they are time consuming and not widely available, are not suited to guiding acute treatment decisions. The objective of our study was to assess the ability of more rapid immunoassays to predict the presence of functionally relevant anti-platelet factor 4/heparin-antibodies. DESIGN AND METHODS: We analyzed 1,291 of 1,383 (93.4%) patients consecutively evaluated for suspected heparin-induced thrombocytopenia at our institution. Clinical pre-test probability was defined by the 4T-score. Anti-platelet factor 4/heparin-antibodies were measured with three immunoassays (ID-H/PF4-PaGIA, Asserachrom-HPIA, and GTI-PF4) and their functional relevance was assessed by a two-point heparin-induced platelet aggregation test. Performance of the immunoassays was evaluated by receiver operating characteristic analysis. RESULTS: Among 1,291 patients, 96 (7.4%) had a positive heparin-induced platelet aggregation-test: 7 of 859 (0.8%) with a low, 50 of 358 (14.0%) with an intermediate, and 39 of 74 (52.7%) with a high 4T-score. Receiver operating characteristics analysis indicated that best immunoassay thresholds for predicting a positive platelet aggregation test were: Titer of 4 or more (ID-H/PF4-PaGIA), optical density more than 0.943 (Asserachrom-HPIA) and more than 1.367 (GTI-PF4). A 100% negative predictive value was observed at the following thresholds: Titer of 1 or under (ID-H/PF4-PaGIA), optical density less than 0.300 (Asserachrom-HPIA) and less than 0.870 (GTI-PF4). A 100% positive predictive value was reached only by ID-H/PF4-PaGIA, at titers of 32 or over. Positive and negative likelihood ratios were calculated for results between the thresholds with 100% negative or positive predictive value. CONCLUSIONS: We show that: i) negative and weak positive results of immunoassays detecting anti-platelet factor 4/heparin-antibodies exclude heparin-induced thrombocytopenia; ii) anti-platelet factor 4/heparin-antibody titers of 32 or over (ID-H/PF4-PaGIA) have a 100% positive predictive value for functionally relevant antibodies; iii) combining the clinical pre-test probability with the likelihood ratio of intermediate immunoassay results allows assessment of post-test probability for heparin-induced thrombocytopenia in individual patients.

Stability of coagulation assays performed in plasma from citrated whole blood transported at ambient temperature.

Many preanalytical variables affect the results of coagulation assays. A possible way to control some of them would be to accept blood specimens shipped in the original collection tube. The aim of our study was to investigate the stability of coagulation assays in citrated whole blood transported at ambient temperature for up to two days after specimen collection. Blood samples from 59 patients who attended our haematology outpatient ward for thrombophilia screening were transported at ambient temperature (outdoor during the day, indoor overnight) for following periods of time: <1 hour, 4-6, 8-12, 24-28 and 48-52 hours prior to centrifugation and plasma-freezing. The following coagulation tests were performed: PT, aPTT, fibrinogen, FII:C, FV:C, FVII:C, FVIII:C, FIX:C, FX:C, FXI:C, VWF:RCo, VWF:Ag, AT, PC activity, total and free PS antigen, modified APC-sensitivity-ratio, thrombin-antithrombin-complex and D-dimer. Clinically significant changes, defined as a percentage change of more than 10% from the initial value, were observed for FV:C, FVIII:C and total PS antigen starting at 24-28 hours, and for PT, aPTT and FVII:C at 48-52 hours. No statistically significant differences were seen for fibrinogen, antithrombin, or thrombin-antithrombin complexes (Friedman repeated measures analysis of variance). The present data suggest that the use of whole blood samples transported at ambient temperature may be an acceptable means of delivering specimens for coagulation analysis. With the exception of factor V and VIII coagulant activity, and total PS antigen all investigated parameters can be measured 24-28 hours after specimen collection without observing clinically relevant changes.