The application of three statistical methods for reference intervals establishment of coagulation assays during pregnancy / 中华检验医学杂志

Objective:To compare the practical value of three statistical methods in establishing reference intervals by the indirect method.Methods:This is a methodological evaluation study. The data of coagulation parameters were obtained from laboratory information system, which were from pregnant women who had done prenatal examination in Peking University First Hospital from January 2017 to December 2019. The test results from 32 401 pregnant women were collected. Those healthy pregnant women were divided into three groups: early pregnancy group(n=11 151), middle pregnancy group(n=4 872) and late pregnancy group(n=16 378). Statistical analysis was performed for the result of PT, APTT, FIB, TT, D-D and FDP, and the necessity of stratification based on age in different periods of pregnancy was analyzed. Stratification based on age was necessary in three pregnancy groups for PT and APTT, in late pregnancy group for FIB and TT, and in early pregnancy group for D-D. The reference intervals of coagulation parameters were calculated by three statistical methods: non-parametric method, Hoffmann method and Q-Q plot method. Forty-two healthy pregnant women from October 2019 to January 2020 were enrolled as reference individuals for the validation of the reference intervals.The proportions of test results outside the reference intervals in the reference population calculated and compared.Results:The levels of the six coagulation assays vary significantly during the three periods of pregnancy, stratification based on age was necessary in three pregnancy groups for PT and APTT, in late pregnancy group for FIB and TT, and in early pregnancy group for D-D. If the number of test results was large, non-parametric and Hoffmann method provided more similar results, while the reference intervals calculated with Q-Q plot method was slightly wider than Hoffmann method. If the number of test results was small, reference intervals calculated with Hoffmann and Q-Q plot method were more reliable. For pregnant women during early pregnancy under the age of 35, the reference intervals of PT, APTT, FIB and TT calculated by this method were (10.44-13.11)s, (25.29-35.88)s, (2.61-4.64)g/L and (11.53-15.58)s.Conclusion:When establishing the reference interval, stratification according to pregnancy period and age was needed. Hoffmann method can be used as an alternative to the direct method.

Mutational analysis of 3 Glanzmann's thrombasthenia pedigrees / 中华检验医学杂志

Objective To explore the molecular pathogenesis of 3 Glanzmann's thrombasthenia pedigree by using bioinformatics software and provide evidence for in vitro experiments. Methods The genetic analysis of 3 pedigree diagnosed as Glanzmann's thrombasthenia was carried out. Clustalx-2.1 win software was used to analyze the conservatism of mutant sites in homologous sequences. Bioinformatics software such as PolyPhen-2, PROVEAN, SIFT and Mutationtaster was used to analyze the biological effect of mutation. SPDBV software constructed the molecular structure model of mutant protein and evaluated the influence of mutation on protein structure. Results The "new mutations" found in 3 Glanzmann's thrombasthenia pedigree were ITGA2B:c. 814G>C (p. Val272Leu), ITGA2B:c. 432G>A (p. Trp144Ter) and ACTN1:c. 2458A>G (p. Ile820Val). All three mutations were highly conserved among homologous species. Mutationtaster software showed that 3 new mutations were likely pathogenic. PolyPhen-2 and PROVEAN software showed ITGA2B p.Val272Leu and ACTN1 p.Ile820Val were benign and SIFT software showed that ITGA2B p. Val272Leu were likely pathogenic, while ACTN1 p. Ile820Val is benign. The result of SPDBV software showed that the Val272 of ITGA2B was transformed to Leu, neutralizing all the original hydrogen bond. The Trp144 of ITGA2B is transformed to Ter, resulting in the truncated proteins with only 113 amino acid residues. All these mutations affected the molecular structure of GPⅡb, resulting in a decrease ofGPⅡb/Ⅲa expression. When the Ile820 of ACTN1 is transformed to Val, onlyretained the hydrogen bond of Ile820 and Asp822, neutralized the rest hydrogen bond, whichaffected the molecular structure and protein function of ACTN1. Conclusion The mutations of ITGA2B:c.814G>C (p.VAL272LEU), ITGA2B:c.432G>A (p.Trp144Ter) and ACTN1:c.2458A>G (p.Ile820Val) are pathogenic.

Study on molecular pathogenesis for a case of Glanzmann′s thrombasthenia / 临床检验杂志

Objective@#To investigate the molecular pathogenesis for a patient with Glanzmann thrombasthenia (GT). @*Methods@#The peripheral blood of a patient with Glanzmann′s thrombasthenia was collected, and the genetic mutations were detected by gene sequencing technology. The mutant plasmids were prepared by PCR site-directed mutagenesis and transfected into CHO-K1 cells of Chinese hamster ovary to construct in vitro eukaryotic expression system. The expressions of αⅡb and β3 protein subunits in CHO-K1 cells were detected by western blot. The expression levels of αⅡb and β3 in cellular membrane and cytoplasm of CHO-K1 cells were detected by flow cytometry. The expression and distribution of αⅡb and β3 in CHO-K1 cells were observed by immunofluorescent labeling under microscope. @*Results@#This patient was diagnosed with type Ⅱ GT. Gene sequencing revealed two mutations in ITGB3 gene which has not been reported in the literature. ITGB3 c.1495 T＞C missense mutation resulted in replacement of cysteine no.499 by arginine (p.C499R). ITGB3 c.1728 delC code shift mutation resulted in a change in the amino acid synthesis initiated by the β3 protein subunit serine no.577 and terminated by the 92nd amino acid following these changes. The results of western blotting showed that the synthesis and expression of primary structures of αⅡb and β3 were detectable in the lysates of mutant CHO-K1 cells. The results of flow cytometry showed that no expression of β3 on the surface and intracellular of mutant CHO-K1 cells was observed. Under fluorescence microscopy no distribution of β3 protein subunit was displayed in mutant CHO-K1 cells. @*Conclusion@#The mutation of ITGB3 c.1728 del C or ITGB3 c.1495 T＞C should be relevant to the cause of GT in this patient. The mutation of ITGB3 c.1728 del C and ITGB3 c.1495 T＞C seems not to affect the formation of the primary structure of β3 protein subunit, but did affect the formation of its high-level structure.

Monitoring of antiplatelet therapy and changes of mean platelet volume after percutaneous coronary intervention in the patients with coronary heart disease / 临床检验杂志

Objective To compare the consistency of thrombelastography (TEG) and light transmittance aggregometry (LTA) in monitoring the antiplatelet therapy of the patients with coronary heart disease (CHD) after percutaneous coronary intervention (PCI),and observe the changes of mean platelet volume (MPV) of the patients treated with aspirin and clopidogrel after PCI.Methods A total of 177 patients undergoing PCI and the treatment of aspirin and clopidogrel in Peking University First Hospital during March 2014 and May 2015 were enrolled in the study.Their adenosine diphosphate (ADP) or arachidonic acid (AA) induced platelet inhibition rates determined by TEG,MPV before and after antiplatelet therapy,and the maximum platelet aggregation rates measured by LTA from 99 patients were retrospectively analyzed.Results There was no any correlation between the maximum aggregation rates measured by LTA and the platelet inhibition rates determined by TEG regardless of using ADP or AA as agonist (all P ＞ 0.05).The detection rates of clopidogrel hyporesponsiveness determined by LTA and TEG were 30.3％ and 45.5％,respectively,while those of aspirin hyporesponsiveness were 19.2％ and 31.3％,respectively.The detection rate of hyporesponsiveness determined by LTA was significant lower than that by TEG (P ＜ 0.05).The MPVs after antiplatelet therapy were significant lower than that before treatment (all P ＜ 0.01) regardless of clopidogrel hyporesponsive or sensitive and aspirin hyporesponsive or sensitive.The MPVs in clopidogrel hyporesponsive group before and after treatment were significantly lower than that in clopidogrel sensitive group (all P ＜ 0.05).The PLT counts in clopidogrel or aspirin hyporesponsive groups after treatment were significantly higher than that before treatment (all P ＜ 0.05).Conclusion There is poor correlation between LTA and TEG.It should be noted that the incidence rate of antiplatelet drug hyporesponsiveness is high in clinical practice.The MPVs of the patients significantly decrease after antiplatelet therapy.The patients with a significant increase of PLT after antiplatelet therapy are more likely to become drug hyporesponsiveness,while the patients with lower MPV are more likely to have clopidogrel hyporesponsiveness.

Light transmittance aggregometry with non-adjusted PRP is more suitable for clinical laboratory =monitoring antiplatelet therapy / 中华检验医学杂志

Objective In this study,maximum platelet aggregation rate of Light transmittance aggregometry (LTA) for coronary heart disease(CHD) patients taking antiplatelet drug and patients without antiplatelet therapy was measured in non-adjusted and platelet count-adjusted platelet-rich plasma (PRP).The aim of this study is to compare which method is superior in evaluation of antiplatelet drug effect.Methods This is a methodology comparative research.560 CHD outpatients and inpatients that visited Beijing Anzhen Hospital of the Capital University of Medical Sciences from May to June,2012 were chosen,who were treated with aspirin monotherapy,or patients on combination therapy with aspirin and clopidogrel,as well as patients without antiplatelet therapy.LTA was performed in non-adjusted (improved method) and platelet count (200 × 109/L)-adjusted PRP (original method),using 6 μmol/L adenosine diphosphate (ADP) and 0.5 mmol/L arachidonic acid (ARA) as agonists.The maximum aggregation rates in 5 min were detected,and consistency and differences of the two methods were compared.Results There is no statistically significant correlation between maximum aggregation rate and platelet count in PRP with 6 μmol/L ADP or 0.5 mmol/L ARA as agonists in all subgroups including aspirin monotherapy,combination therapy with aspirin and clopidogrel and patients without antiplatelet therapy (-0.21 ≤ r ≤0.111,P ＞ 0.05).The maximum aggregation rate using ADP as agonists in original method is decreased compared with improved method,there is statistically significant difference in all subgroups including patients without antiplatelet therapy,aspirin monotherapy,combination therapy with aspirin and clopidogrel less than one week and more than one week.The variability of platelet aggregation rate using ADP as agonists with improved method is lower than that with original method in all subgroups.Yet the maximum aggregation rates using ADP as agonists with improved method and original method correlate well with each other in all subgroups (r =0.78,0.73,0.40,0.71,P ＜0.01).In the subgroup of subjects without antiplatelet therapy using ARA as agonist,platelet aggregation rate is decreased in original method compared with improved method,there is statistically significant difference,and the variability of the aggregation rate with improved method is also lower than that with original method,ranging from 62％-98％ relative to 5％-89％.The decrease of aggregation rate using ARA as agonist for patients taking antiplatelet drug compared with patients without antiplatelet therapy can be detected both with improved method and original method.Conclusion Non-adjusted PRP in LTA is more convenient and time-saving,and it also means less effects on platelet in vitro.Therefore,non-adjusted PRP is more suitable for monitoring efficacy of antiplatelet therapy in clinical laboratory.