Clinical significance of heparin/platelet factor 4 antibody in maintenance hemodialysis patients / 第二军医大学学报

Objective To investigate the positive rate of heparin/platelet factor 4 (H/PF4) antibody in maintenance hemodialysis (MHD) patients, and to explore its clinical significance. Methods Fifty-four MHD patients treated in the Department of Nephrology of Changzheng Hospital of Naval Medical University (Second Military Medical University) were selected. The dialysis duration (unfractionated heparin/low-molecular-weight heparin) of all patients was more than 3 months, with no infections or other active diseases. Serum samples were collected from the MHD patients before dialysis, and IgG H/PF4 antibody was detected by particle immunofiltration assay. The general condition, hemoglobin level, platelet count, anticoagulant method (unfractionated heparin/low-molecular-weight heparin), anticoagulant dosage, and dialysis mode (conventional hemodialysis/nocturnal extended hemodialysis) were compared between the H/PF4 antibody-positive group and H/PF4 antibody-negative group. After 3 years' follow-up, the change of platelets, the incidence of vascular access thrombosis, cardio-cerebral vascular events, hospitalization rates and mortality were compared between the two groups. Results The positive rate of H/PF4 antibody was 63.0% (34/54) in MHD patients. There were no significant differences in gender, age, dialysis age, hemoglobin level or platelet count between the H/PF4 antibody-positive group and H/PF4 antibody-negative group (P0.05). The positive H/PF4 antibody was not correlated with primary kidney disease, anticoagulant method, anticoagulant dosage, or dialysis mode (all P0.05). After 3 years' follow-up, there were no significant differences in the change of platelet, the incidence of vascular access thrombosis (14.7%5/34vs 25.0%5/20), cardiovascular events, cerebrovascular events, hospitalization rates, or mortality between the two groups (all P0.05). Conclusion The positive rate of H/PF4 antibody is high in MHD patients. The production of H/PF4 antibody is not related to the heparin type, heparin dosage, or dialysis mode. The positive H/PF4 antibody has no significant effect on platelet counts or adverse events, including thrombosis and cardiovascular events.

Diagnostic value of two immunoassays for detecting heparin/PF4 complex antibodies in heparin-induced thrombocytopenia / 中华血液学杂志

Objectives@#To assess the diagnostic values of latex immunoturbidimetric assay (LIA) and particle immunofiltration assay (PIFA) for heparin-induced thrombocytopenia (HIT) .@*Methods@#Samples from 94 patients with suspected HIT from May 2016 to July 2018 in our hospital were prospectively analyzed by the two immunoassays. Their medical records and further follow-up data were also collected and analyzed by our hematologists to make the 4Ts scores and confirm the diagnosis of HIT, respectively. Performance characteristics of the two immunoassays were assessed, including sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) . Their post-test probabilities (PTP) were also calculated based on the 4Ts score.@*Results@#Among 94 cases, 15 (16.0%) had a positive HIT, including 6 of 37 (16.2%) with an intermediate, and 9 of 15 (60.0%) with a high 4Ts score. PIFA operating characteristics were: sensitivity 100.0% (15/15) , specificity 51.9% (41/80) , PPV 28.3% (15/53) , NPV 100.0% (41/41) . The positive PTP in intermediate and high 4Ts score group were 28.7% and 75.7%, respectively, while negative PTP were all 0. At manufacturers’ cutoffs, LIA operating characteristics were: sensitivity 66.7% (10/15) , specificity 94.9% (75/79) , PPV 71.4% (10/14) and NPV 93.8% (75/80) . The positive and negative PTP in intermediate 4Ts score group were 71.8% and 6.3%, while 95.2% and 34.4% in high 4Ts score group, respectively. Receiver operating characteristic (ROC) analysis manifested that LIA was preferable than PIFA, and combining the 2 assays together was significantly better than single test.@*Conclusions@#4Ts score is still an important tool for the diagnosis of HIT. Combining clinical score with heparin/PF4 antibody assay can increase the accuracy of confirming or excluding HIT. Although PIFA is inferior to LIA in the diagnostic value, its user friendliness and 100% NPV have major advantages. Combining the 2 assays together can achieve a higher diagnostic value.

Expression of serum platelet factor 4 in high-grade intraepithelial neoplasia of gastric cardia and its clinical significance / 肿瘤研究与临床

Objective To investigate the expression of serum platelet factor 4 (CXCL4) in high-grade intraepithelial neoplasia (HGIN) of gastric cardia and its clinical significance. Methods Sixty-four HGIN patients and 29 patients with advanced cardiac cancer (ACC) from January 2010 to December 2011 in the Fourth Hospital of Hebei Medical University were selected as the research objects. Forty healthy people treated with endoscope screening were chosen as the control group. Enzyme linked immunosorbent assay (ELISA) was used to detect the expression of CXCL4 in serum. The expression differences were compared by using variance analysis and LSD-t. Results The CXCL4 detection showed that the expression levels of CXCL4 were(0.75±0.37)ng/ml,(0.87±0.33)ng/ml,(1.13±0.51)ng/ml respectively in HGIN group,ACC group and the control group (F = 10.77, P < 0.05). The CXCL4 expression levels in HGIN and ACC group were lower than that in the control group (t = 4.80, t = 2.67, both P < 0.05). There was no difference between the CXCL4 expression levels in HGIN and ACC group(t =1.27,P >0.05).Conclusions The expression level of CXCL4 in HGIN patients with gastric cardia is significantly decreased. CXCL4 may be related with the occurrence and angiogenesis of gastric cardia cancer, which can be considered as an important serum marker for early screening of gastric cardia cancer.

Diagnostic value of clinical significance of serum chemokine-4 detection in patients with systemic sclerosis complicated with interstitial lung disease / 中华风湿病学杂志

Objective To investigate the clinical significance of serum chemokine-4 (CXCL-4) in systemic sclerosis (SSc) patients complicated with SSc-interstitial lung disease (SSc-ILD).Methods Sixty-two patients with SSc wbo met the new classification criteria of American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) SSc in 2013 and were treated by rheumatology department of Jiangxi Provincial People's Hospital from September 2015 to July 2017 were divided into the SSc combined with ILD group (30 cases) and simple SSc group (32 cases) according to the results of HRCT test,in reference to the 2013 American Thoracic Society/European Respiratory Society (ATS/ERS) ILD diagnostic criteria.A certain number of healthy subjects were selected as healthy control group (35 cases).Serum concentrations of CXCL-4 were determined by enzyme-linked immunosorbent assay (ELISA),and the difference of CXCL-4 concentration among the 3 groups were analyzed.All data were analyzed by t test,x2 test,rank-sum test and Logistic regressive analysis.Results The level of CXCL-4 in the SSc-ILD group [(133±17) ng/ml] was higher than that of the SSc group [(122±19) ng/ml] and normal control group [(115±19) ng/ml] (t=11.414,P=0.012;t=17.917,P=0.000);there was no significant difference between the SSc group and the normal control group (t=6.504,P=0.130);binary Logistic regression analysis found that the level of CXCL4 was positively correlated with ILD [P=0.019,OR=1.035,95％CI(1.006,1.066)].Conclusion Elevated levels of CXCL-4 in patients with SSc may be associated with concurrent ILD,CXCL-4 is expected to be a new serological marker for early diagnosis and evaluation of patients with SSc-ILD.It may be used as a sensitive indicator for evaluating the condition and prognosis of patients with SSc-ILD and provide a new target for clinical guidance of SSc-ILD therapy.

Relationship between the level of human platelet factor IV and Plasmodium falciparum infection / 实用医学杂志

Objective To investigate the relationship between Plasmodium infection and platelet factor Ⅳ(PF4) and to provide the ideas for the treatment of malaria.Methods The serum PF4 concentration was determined with the Enzyme-linked immunosorbent assay (ELISA) in the malaria group (122 cases of malaria patients)and the control group (399 cases of normal).The average value (mean) and positive rate of PF4 were compared between two groups.Results The PF4 level of malaria group was significantly higher than that in the control group and the difference between two groups was statistical significance (P ＜ 0.05).There was no significant difference in Plasmodium falciparum density between the PF4 high level group and the PF4 normal group in the malaria group.Conclusions The expression of human PF4 level has definite relationship with the plasmodium infection of Equatorial Guinea.

Effect of sublingual immunotherapy on platelet activity in children with allergic rhinitis / Efeito da imunoterapia sublingual sobre a atividade plaquetária em crianças com rinite alérgica

Abstract Introduction: The role of platelet activation in allergic inflammation is receiving increasing attention. Sublingual immunotherapy for allergic rhinitis can modify the immunological process to an allergen, rather than simply treating symptoms. Objective: The aim of this study was to explore the role of platelet activation during sublingual immunotherapy in children with allergic rhinitis. Methods: Forty-two House Dust Mite - sensitized children with allergic rhinitis were enrolled and received House Dust Mite allergen extract for sublingual immunotherapy or placebo. Serum of different time points during treatment was collected and used for detection of Platelet Factor-4 and Beta-Thromboglobulin concentration by Enzyme-Linked Immuno Sorbent Assay. Results: Our data showed decreased expression of Platelet Factor-4 and Beta-Thromboglobulin protein after one year's sublingual immunotherapy. In addition, the decrease of symptom scores and serum Platelet Factor-4 and Beta-Thromboglobulin protein concentrations was positively related. Conclusion: During sublingual immunotherapy, platelet activation was inhibited significantly. Our results might indicate that inhibition of platelet activation within the systemic circulation is an important mechanism during sublingual immunotherapy.

Resumo Introdução: O papel da ativação de plaquetas na inflamação alérgica recebeu atenção crescente. A imunoterapia sublingual para rinite alérgica pode modificar o processo imunológico a um alérgeno, em vez de tratar os sintomas simplesmente. Objetivo: Explorar o papel da ativação plaquetária durante a imunoterapia sublingual em crianças com rinite alérgica. Método: Quarenta e duas crianças com rinite alérgica sensibilizadas por ácaros de poeira domiciliar (APD) foram inscritas e receberam extrato de alérgeno de APD para imunoterapia sublingual ou placebo. O soro de diferentes pontos no tempo durante o tratamento foi recolhido e usado para a detecção de fator 4 plaquetário e concentração de beta-tromboglobulina por ensaio imunoenzimático. Resultados: Nossos dados mostraram diminuição da expressão de fator 4 plaquetário e proteína beta-tromboglobulina após imunoterapia sublingual de um ano. Além disso, a diminuição dos escores de sintomas e o fator 4 plaquetário sérico e concentrações de proteína beta-tromboglobulina foram relacionados de maneira positiva. Conclusão: Durante imunoterapia sublingual, a ativação plaquetária foi inibida significativamente. Os nossos resultados podem indicar que a inibição da ativação de plaquetas dentro da circulação sistêmica é um mecanismo importante durante imunoterapia sublingual.

Changes of platelet rheological behavior and the interventional effects of ulinastatin in rats with high-voltage electrical burns / 中华烧伤杂志

Objective@#To explore the influence of high-voltage electrical burns on the number of platelet aggregation, β-thromboglobulin (β-TG) and platelet factor 4 (PF-4) and the interventional effects of ulinastatin in rats with high-voltage electrical burns.@*Methods@#A total of 240 Sprague-Dawley rats were divided into sham injury (SI) group, simple electrical burn (SEB) group, normal saline (NS) group, and ulinastatin (UTI) group according to the random number table, with 60 rats in each group. The electrical current was applied to the outside proximal part of left forelimb of rats and exited from the outside proximal part of right hind limb of rats. Rats in groups SEB, NS, and UTI were inflicted with high-voltage electrical burn wounds of 1 cm×1 cm at current entrances and exits, with the voltage regulator and experimental transformer. Rats in group SI were sham injured through connecting the same equipments without electricity. At 2 min post injury, rats in group NS were intraperitoneally injected with 2 mL/kg NS, and rats in group UTI were intraperitoneally injected with 2×104 U/kg UTI of 10 g/L. At 15 min before injury and 5 min, 1 h, 2 h, 4 h, 8 h post injury, 10 rats in each group were selected to collect 5-7 mL blood of heart respectively. Blood of 0.05 mL were collected to make fresh blood smear for observing the number of platelet aggregation, and serum were separated from the remaining blood to determine content of β-TG and PF-4 with enzyme-linked immunosorbent assay. Data were processed with analysis of factorial design of variance, student-Newman-Keuls test, Kruskal-Wallis H test, Wilcoxon rank sum test, and Bonferroni correction.@*Results@#(1) At 15 min before injury, the numbers of platelet aggregation of rats were close among groups SI, SEB, NS and UTI (5.9±1.2, 5.8±1.2, 5.9±1.3, 5.9±1.1, respectively, with P values above 0.05). At 5 min, 1 h, 2 h, 4 h, 8 h post injury, the numbers of platelet aggregation of rats in group SEB were 57.2±16.3, 59.1±16.9, 60.8±20.6, 83.6±24.9, and 83.4±30.3, respectively, obviously more than those in group SI (6.0±1.3, 6.0±1.4, 5.9±1.4, 5.7±1.1, and 5.8±1.3, respectively, with P values below 0.001); the numbers of platelet aggregation of rats in group UTI were 29.6±7.4, 31.9±10.1, 35.0±14.2, 43.0±13.6, and 35.2±11.1, respectively, obviously more than those in group NS (58.3±16.1, 63.9±18.0, 60.8±17.7, 74.2±23.0, and 82.3±21.9, respectively, with P values below 0.001). There was no significantly statistical difference in the number of platelet aggregation of rats in group SI between each two time points within the same group (with P values above 0.05), but the number of platelet aggregation of rats in the other 3 groups at each time point post injury was significantly more than that of the same group at 15 min before injury (with P values below 0.001). (2) At 2, 4, and 8 h post injury, β-TG content of serum of rats in group SEB was significantly higher than that in group SI (with Z values from -3.780 to -3.477, P values below 0.05). At 5 min and 4 h post injury, β-TG content of serum of rats in group UTI was significantly lower than that in group NS (with Z values respectively -3.477 and -3.780, P values below 0.05). There was no significantly statistical difference in β-TG content of serum of rats in group SI at all time points of the same group (χ2=0.130, P >0.05). At 2, 4, and 8 h post injury, β-TG content of serum of rats in group SEB was significantly higher than that of the same group at 15 min before injury (with Z values from -3.780 to -3.553, P values below 0.05). At 5 min, 1 h, and 4 h post injury, β-TG content of serum of rats in group NS was significantly higher than that of the same group at 15 min before injury (with Z values from -3.780 to -3.477, P values below 0.05). At 1 and 4 h post injury, β-TG content of serum of rats in group UTI was significantly higher than that of the same group at 15 min before injury (with Z values respectively -3.250 and -3.780, P values below 0.05). (3) At 2 and 8 h post injury, PF-4 content of serum of rats in group SEB was significantly higher than that in group SI (with P values below 0.05). At 2 h post injury, PF-4 content of serum of rats in group UTI was significantly higher than that in group NS (P<0.05), and at 4 and 8 h post injury, PF-4 content of serum of rats in group UTI was significantly lower than that in group NS (with P values below 0.05). At all time points, PF-4 content of serum of rats in group SI was close (with P values above 0.05). At 2 and 8 h post injury, PF-4 content of serum of rats in group SEB was significantly higher than that of the same group at 15 min before injury (with P values below 0.05). At 1, 4, and 8 h post injury, PF-4 content of serum of rats in group NS was significantly higher than that of the same group at 15 min before injury (with P values below 0.05). There were significantly statistical differences in PF-4 content of serum of rats between all time points except for 5 min post injury and 15 min before injury (with P values below 0.05).@*Conclusions@#Increasing number of platelet aggregation and abnormal secretion of β-TG and PF-4 of rats with high-voltage electrical burns can lead to microcirculation disturbance. UTI can alleviate microcirculation disturbance caused by high-voltage electrical burns by reducing the number of platelet aggregation and inhibiting secretion of β-TG and PF-4.

Platelet Activation Markers (beta-thromboglobulin and platelet factor 4) and Clopidogrel Drug Response in Patients with Myocardiac Infarction

BACKGROUND: Dual antiplatelet therapy (aspirin and clopidogrel) is used to prevent adverse cardiac events in patients undergoing percutaneous coronary intervention (PCI). Some patients do not respond adequately to clopidogrel. Beta-thromboglobulin (beta-TG) and platelet factor 4 (PF-4) can act as markers to detect platelet activation. We investigated the relationship between clopidogrel response and the dynamics of beta-TG and PF4 concentrations. METHODS: This study included 36 myocardial infarction (MI) patients, who underwent PCI and was indicated for dual antiplatelet therapy. Platelet reactivity, using the VerifyNow P2Y12 assay, was measured on the 3rd day of PCI. At the time of admission, and on the 3rd and 10th day of PCI, the plasma beta-TG and PF4 concentrations were quantified. RESULTS: Ten patients (27.8%) were clopidogrel non-responders displaying >208 P2Y12 reaction units. At the time of admission, levels of beta-TG in patients were elevated than that in the healthy controls (P<0.001). A similar trend was observed on the 3rd and 10th day of PCI (P<0.001). The beta-TG levels on the 10th day were reduced than those at the time of admission and on the 3rd day of PCI. PF4 levels were not different between patients and controls, and were not significantly reduced after PCI. Higher beta-TG levels were observed in clopidogrel non-responders on the 10th day, but not significant. CONCLUSIONS: Clopidogrel therapy in MI reduce beta-TG concentration, but the beta-TG and PF4 levels before and after therapy are not associated with the response to clopidogrel. Platelet-derived markers may not be suitable for distinguishing clopidogrel non-responders.

The Role of Platelets in Malarial Acute Lung Injury and Acute Respiratory Distress Syndrome: A World of Possibilities.

In recent decades our understanding of platelets’ role in immune response has increased. Traditionally platelets were considered as bleeding-stopping and thrombosis-causing cells. In recent years the platelets’ role in malarial innate and adaptive immune responses is being recognized. Platelets play critical role in pathogenesis of malaria infection leading to variety of outcomes. It is being realized that platelets play dual role in case of malaria (i) by preventing early stage exponential growth of parasitemia (ii) promoting exaggerated immune responses later. Platelets role in pathogenesis of severe and cerebral malaria has been widely studied. However their role in malaria related acute lung injury and respiratory distress has gained less attention. Recently the presence of active megakaryocytes and proplatelets have been explained in human lungs. Simultaneously, the platelets role in pathogenesis of acute lung injury and respiratory distress (ALI/ARDS) was also recognized. This gives a hint that there is a possible association of platelets with malaria related respiratory diseases as well. ALI/ARDS are characterized by lung edema due to increased permeability of the alveolar-capillary barrier and subsequent impairment of arterial oxygenation. In this review we have attempted to establish the importance of role of platelets in malaria related acute lungs injury and malaria acute respiratory distress syndrome and try to explain the underlying mechanism of this process. In ALI/ARDS, including those caused by malaria, platelets participate sequestration to the vascular bundle facilitating the recruitment of immune cells viz. neutrophils. Additionally, they secrete or induce the secretion of chemokines that result into vascular damage.

Application of ELISA in Detecting Heparin-Induced Thrombocytopenia Antibodies / 现代检验医学杂志

Objective To develop an ELISA method for determination of heparin-induced thrombocytopenia (HIT)antibody. Methods The compound formed between human platelet factor 4 (PF4)and heparin was used as the coating antigen,incu-bating the patients plasma with the coating antigen in the well,after washing,the second antibody labeled HRP was added in the well to incubate and washing again,the chromogenic substrates was added in the well to incubate,when the stop reaction was finished,the absorbance A450/A630 was detected,and the test results were judged according to standard,this method was compared with IBL method and was optimized and evaluated the performance.Results An indirect ELISA method was de-velop with the purified human PF4,the optimal dilution of sample and second antibody were 1∶100 and 1∶1 500 which de-tected by the orthogonal test,the intra-and inter-assay average coefficients of variation were 7.66% and 7.76%(<10%) respectively that detected by repeated measurement the three positive standard plasma.Through measureing the 100 healthy human plasm with no history of using heparin,the positive and negative predictive reference values were 0.304 and 0.456. IBL and this method detected 100 hemodialysis patients samples at the same time,and the result of statistical analysis was that,the sensitivity,speciality and accuracy of this method were 90%,97.78% and 90%,respectively.The negative and posi-tive predictive value were 81.8% and 98.88% respectively,and the difference was statistically significant [K=0.84(0.81~1)and Pexac=0.012<0.05].The difference was statistically significant,consistency was optimal,95% confidence interval was 92.59%~92.59%.Conclusion Comparing with the IBL,the method reported by this article had the similar perform-ance and good consistency,and it could satisfy the clinical detection and diagnosis of HIT patients.

The incidence of heparin-induced thrombocytopenia and positivity of antibody in patients treated with heparin preparations / 中华内科杂志

Objectives To investigate the incidence of the heparin-induced thrombocytopenia (HIT) and positivity of anti-heparin/platelet factor 4 complex antibody (HIT-antibody) in inpatients treated with heparin preparations.Methods A total of 197 consecutive patients,including 120 men and 77 women,who were treated with unfractioned heparin (UFH) or low molecular weight heparin (LMWH),were enrolled in this study.HIT-antibody was detected by enzyme-linked immunosorbent assay (ELISA).All patients were classified into subgroups based on 4Ts (Pretest Clinical Scoring System) and types of underlying disorders.The incidence of HIT and positivity of HIT-antibody among different groups were analyzed.Results The overall incidence of HIT was 3.0％ (6/197),and the positivity of HIT antibody was 12.2％ (24/197).The positivity of HIT-antibody was 10.1％ (18/178),7.7％ (1/13) and 83.3％(5/6) in low,moderate and high HIT probability group respectively.There were significant differences of HIT positivity and mean level of HIT-antibody between the high HIT probability group and the low or moderate HIT probability group (P =0.000).Both the incidence of HIT and positivity of HIT-antibody were higher in surgical patients than in medical patients (5.8％ vs 0.9％,P =0.047 and 19.8％ vs 6.3％,P =0.004).Conclusions The incidence of HIT and positivity of HIT-antibody were 3.0％ and 12.2％ respectively,both of which were related with the types of disorders.Detection of HIT-antibody has a better applicable value in patients with high HIT probability.

CXCL4 and its receptor in colorectal cancer / 国际肿瘤学杂志

CXC chemokine ligand 4 (CXCL4) is a platelet-derived chemokine,and it belongs to CXC subfamily.Its receptor is CXC chemokine receptor 3 (CXCR3),which belongs to the seven transmebrane G-protein-coupled receptor family,and its coreceptor is glycosaminoglycan (GAG).CXCL4 and its receptors take part in the regulation of many biological processes,such as immune regulation,inflammatory response,endothelial cell proliferation and migration and angiogenesis.Recent studies show that CXCL4 and its receptor have significant influence on the cell growth,angiogenesis,invasion and metastasis of colorectal cancer,which offers a new perspective to explore the mechanism of colorectal cancer and its targeted therapy.

Los niveles de anticuerpos anti factor plaquetario 4-heparina y el índice 4T para trombocitopenia inducida por heparina / Levels of antiplatelet factor 4-heparin antibodies and 4T score for heparin induced thrombocitopenia

La trombocitopenia inducida por heparina (HIT) es un efecto adverso del tratamiento con heparina, mediada por anticuerpos anti complejo factor plaquetario 4 (PF4)-heparina (HPIA). La HIT es frecuentemente moderada pero pueden desarrollarse complicaciones trombóticas. El diagnóstico precoz es importante. La detección de HPIA por ELISA tiene alta sensibilidad pero baja especificidad (títulos bajos sin significación clínica). El índice de las 4T (índice 4T) puede detectar pacientes con alto riesgo de HIT. El propósito del estudio fue correlacionar los niveles de HPIA y el índice 4T de un grupo de pacientes derivados a nuestro centro. Evaluamos 84 pacientes, 34 de ellos desarrollaron trombosis. Cada médico completó un cuestionario clínico que fue remitido con la muestra a nuestro centro. Los cuestionarios fueron analizados por un investigador externo y el índice 4T se calculó previamente al ensayo. Los HPIA se determinaron por un ELISA (Asserachrom HPIA) que detecta los 3 isotipos, IgG, IgM e IgA, único reactivo disponible en Argentina. Los resultados se expresaron como porcentaje de absorbancia (%ABS). La correlación del índice 4T con los HPIA fue 0.472 (rho spearman, p < 0.001). Los pacientes con índice 4T ≥ 6 presentaban %ABS mayores que los ≤ 5 (67 vs. 39, p < 0.001). Aquéllos con trombosis presentaron títulos mayores que los que no la desarrollaron (%ABS 59 vs. 39, p = 0.017). En conclusión: Los títulos altos de HPIA medidos por ELISA, que detecta los 3 isotipos, correlacionaron claramente con el índice 4T ≥ 6 y fueron más frecuentes en los pacientes con trombosis, coincidiendo con lo ya descripto para ensayos de ELISA específicos para isotipo IgG.

Heparin induced thrombocytopenia (HIT) is an immune-mediated disorder due to antibodies anti platelet factor 4-heparin (HPIA). Thrombocytopenia is often moderate but certain patients can develop morbid thrombotic complications. HPIA detection by ELISA has high sensitivity but low specificity, and low titers (without clinical significance) are frequent. A pretest clinical score (4T´s) was developed in order to recognize patients that are at high risk of HIT. The aim of this study was to correlate HPIA levels and the 4T´s score of consecutive patients derived to our center. We evaluated 84 patients (35 of them developed thrombosis); the clinical questionnaire was sent along with the sample and was analyzed by an investigator who did not know the patients´ characteristics, and 4T´s scores were calculated before performing the laboratory tests. HPIA were measured by ELISA (Asserachrom HPIA) that detects IgG, IgM and IgA isotypes, (the only reagent available in our country). 4T´s score correlated with HPIA levels (rho spearman 0.472, p < 0.001). Patients with 4T´s ≥ 6 had higher absorbance percentages than those with ≤ 5 (67 vs. 39%, p < 0.001), and patients with thrombosis also presented higher titers (59 vs. 39%, p = 0.017) than those who did not develop this complication. In conclusion, high titers of HPIA measured by EIA which detects the 3 isotypes, clearly correlate with 4T´s score ≥ 6 and are more frequent in patients who develop thrombosis, just as reported when an IgG specific ELISA is used.

Usefulness of anti-PF4/heparin antibody test for intensive care unit patients with thrombocytopenia / 대한혈액학회지

BACKGROUND: It is critical to differentiate heparin-induced thrombocytopenia (HIT) from disseminated intravascular coagulation (DIC) in heparinized intensive care unit (ICU) patients with thrombocytopenia because the therapeutic approach differs based on the cause. We investigated the usefulness of PF4/heparin antibody tests in these patients. METHODS: A total of 127 heparinized ICU patients whose platelet counts were 50% after 5-10 days of heparin therapy were enrolled. PF4/heparin antibodies were measured using 2 immunoassays. We assessed the probability of HIT by using Warkentin's 4T's scoring system for antibody positive patients and compared routinely performed coagulation test results between patients with and without antibodies to evaluate the ability of these tests to discriminate between HIT and DIC. RESULTS: Positive results were obtained for 14 (11.0%) and 11 (8.7%) patients in the 2 assays. The analysis performed using the 4T's scoring system revealed that 11 of 20 (15.7%) patients with antibodies in at least 1 assay had intermediate or greater probability of HIT. Patients without antibodies had significantly higher levels of D-dimer than those with antibodies. However, there were no intergroup differences in platelet counts, PT, aPTT, fibrinogen, DIC score, and rate of overt DIC. CONCLUSION: Seropositivity for PF4/heparin antibody was 8.7-11.0% in the patients with thrombocytopenia, and more than a half of them had an increased probability of HIT. Among the routine coagulation tests, only D-dimer was informative for differentiating HIT from DIC. PF4/heparin antibody test is useful to ensure appropriate treatment for thrombocytopenic heparinized ICU patients.

The differences of platelet PAC-1,CD62P positive rates and serum platelet factor 4 levels in patients with large artery atherosclerotic stroke and cerebral small vessel disease / 国际脑血管病杂志

Objective To investigate the differences of platelet membrane glycoprotein PAC-1 and CD62P expression in patients with cerebral small vessel disease and its subtype and large artery atherosclerotic stroke and to compare the traditional platelet activation markers and inflammatory chemokine platelet factor 4(PF4).Methods Peripheral blood platelet PAC-1,CD62P positive rates and serum PF4 concentration in 30 patients with large artery atherosclerotic stroke,45 patients with cerebral small vessel disease and 30 controls were detected using flow cytometry and enzyme-linked immunosorbent assay.The differences between the groups were compared.Results The PAC-1,CD62P positive rates and serum PF4 concentration in the large artery atherosclerotic stroke group were 63.21% ± 9.78%,55.91% ± 8.17%,and 30.55 ± 15.56 ng/ml,respectively.They were significantly higher than 40.65% ± 17.42%,36.49% ± 14.60%,and 12.59 ±5.57 ng/mlin the cerebral small vessel disease group(all P=0.000),and the latter was still higher than 13.55% ± 3.14%,9.00% ± 2.32%,and 4.95 ±2.82 ng/ml in the control group(all P = 0.000).There were significant differences in all the subtypes groups of cerebral small vessel disease between the PAC-1,CD62P positive rates and serum PF4 concentration.The leukoaraiosis with lacunar infarction group(n = 15;47.72% ±15.52%,43.75% ± 12.54%,and 13.96 ± 5.23 ng/ml)and the simple lacunar infarction group (n = 15;49.87% ± 14.65%,43.98% ± 10.55%,and 14.41 ± 5.53 ng/ml)was significantly higher than the simple lacmar infarction group(n = 15;24.44% ± 8.45%,21.74% ± 7.19%,and 9.40 ±4.99 ng/ml)(P =0.000,0.000,and 0.013,respectively).There was no significant difference between the forrner 2 groups(P = 0.658,0.952,and 0.858,respectively).The peripheral blood platelet PAC-1 positive rate had significant correlation with CD62P positive rate in patients with ischemic cerebral disease(r= 0.964,P= 0.000),and the serum PF4 concentration showed linier correlation with the PAC-1(r =0.846,P =0.000)and CD62P(r =0.857,P =0.000)positive rates.Conelusions The platelet membrane glycoprotein PAC-1 and CD62P expression showed linear correlation,and they were consistent with the changes of PF4 concentration.This sugested that platelet activation and its mediated inflammatory mechanisms played an important pathophysiological role in the processes of atherosclerosis and thrombosis.This mechanism had significant difference between the different lesion types.

Utility of ELISA Optical Density Values and Clinical Scores for the Diagnosis of and Thrombosis Prediction in Heparin-induced Thrombocytopenia / 대한진단검사의학회지

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction caused by antibodies to the heparin/platelet factor 4 (PF4) complex, resulting in thrombocytopenia and prothrombotic state. HIT diagnosis is challenging and depends on clinical presentation and laboratory tests. We investigated the usefulness of clinical scores and heparin/PF4 ELISA optical density (OD) as a diagnostic marker and thrombosis predictor in HIT. METHODS: We analyzed 92 patients with suspected HIT. The heparin/PF4 antibody was measured using a commercial ELISA kit (GTI, USA). For each patient, the 4 T's score and Chong's score were calculated. RESULTS: Of the 92 patients, 28 were anti-heparin/PF4-seropositive. The 4 T's score and Chong's score showed good correlation (r=0.874). The 4 T's score and OD values showed good performance for diagnosis of the definite and unlikely HIT groups; however, OD levels showed better sensitivity (93.8%) than the 4 T's score used alone (62.5%). Of the 92 patients, 26 developed thrombosis. The OD values were significantly higher in patients with thrombosis than in those without thrombosis (0.52 vs. 0.22, P0.4) had an increased risk of thrombosis (adjusted odds ratio 9.44 [3.35-26.6], P<0.001) and a shorter 250-day thrombosis-free survival (32.1% vs. 54.7%, P=0.012). CONCLUSIONS: ELISA OD values in combination with clinical scoring can improve the diagnosis of and thrombosis prediction in HIT. More attention should be paid to the use of clinical scores and OD values as thrombosis predictors in HIT.

Expression of human platelet factor 4 mRNA and its relation with angiogenesis and lymph node metastasis in papillary thyroid carcinoma / 肿瘤研究与临床

Objective To study the expression of human platelet factor 4 (hPF4)mRNA and its relationship with angiogenesis and lymph node metastasis in papillary throid carcinoma(PTC). Methods RT-PCR was used to detect the expression of hPF4 mRNA in 40 cases of PTC and 10 cases of normal tissue surrounding carcinoma. Immunohistochemical stain was performed to detect microvessel density(MVD) whose endothelia were marked by antibody CD105. Results The positive-expression rate of hPF4 mRNA was 80 % respectively in normal tissue surrounding carcinoma. The positive-expression rate of hPF4 was 47.5 % in PTC cases. The MVD value of PTC cases with hPF4 mRNA positive expression was significantly lower than that of cases without hPF4 mRNA expression (P

Two Cases of Heparin-Induced Thrombocytopenia Diagnosed by Particle Gel Immunoassay / 대한진단검사의학회지

Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin therapy. It represents initially as thrombocytopenia and is associated with venous or arterial thrombosis. It has been reported that platelet factor 4/heparin complex antibody plays an important role in the pathogenesis of HIT. Patients on hemodialysis have a high risk of developing HIT because heparin is administrated in hemodialysis as anticoagulant. Thrombocytopenia usually occurs 5 to 10 days after the onset of administration, but occasionally, it may occur rapidly in patients who have preformed antibodies from recent heparin use. We report here 2 cases of HIT with platelet factor 4-heparin reactive antibody in hemodialysis patients.

A Convenient and Effective Approach for Gene Synthesis of Human Platelet Factor-4 / 中国药科大学学报

AIM:To synthesize the human platelet factor-4(hPF4) gene with a convenient and effective approach, and high express the hPF4 gene in E. coli BL21 (DE3). METHODS: According to the primary structure of hPF4, the nucleotide sequence was synthesized using touch-down PCR method. The resultant gene fragment containing EcoR Ⅰ and Xho Ⅰ overhangs at 5' and 3' ends was cloned into the expression vector pGEX-4T-1 to construct the recombinant plasmid pGEX-4T-1-hPF4,which was then transformed into the E. coli strain BL21 (DE3). RESULTS: hPF4 gene was successfully synthesized by touchdown PCR method. A fusion protein composed of glutathione S-transferase (GST) and the recombinant hPF4 was expressed in BL21(DE3) by IPTG induction. The expression level of the fusion protein in E. coli was about 30% of the total cellular protein. CONCLUSION: Touch-down PCR may provide a convenient and effective approach to obtain other target genes. The expressed fusion protein forms the inclusion bodies, providing sufficient material for further purification and biological activities process.

Anti-Heparin-Platelet Factor 4 Antibody is a Risk Factor for Vascular Access Obstruction in Patients Undergoing Hemodialysis

Since heparin is an anticoagulant commonly used in hemodialysis and the patients on hemodialysis are repeatedly exposed to heparin, heparin may be the cause of the development of heparin-dependent antibodies and thrombotic complications in patients on hemodialysis. The purpose of this study was to determine the prevalence and the clinical significance of the antibodies against heparin-platelet factor 4 complexes as determined by enzyme immunoassay in patients on maintenance hemodialysis. The prevalence of anti-heparin-platelet factor 4 antibodies was higher in hemodialysis patients than in normal subjects (8.8 vs 0.0%, p<0.05). The number of past episodes of vascular access obstruction per year was significantly higher in the anti-heparin-platelet factor 4 antibody positive group than antibody negative group. Anti-heparin-platelet factor 4 antibody positive patients experienced more frequent vascular access obstructions than control subjects. In conclusion, anti-heparin-platelet factor 4 antibody might be a risk factor for vascular access obstructions in patients with end-stage renal disease on maintenance hemodialysis.