Clinical Significance Of Detection Of IgG-H/PF4 Antibody In Patients Undergoing Hemodialysis

Background:Heparin-induced thrombocytopenia(HIT) is one of the most important immunologic drug reactions that can lead to thromboembolism, cardiovascular events,and death.HIT can be divided into two types.TypeⅠis considered to be more common and mainly occurs in 1 to 3 days after the initial use for common unfractionated heparin.It belongs to non-immune mediated response. TypeⅡalways presents with significant thrombocytopenia which lasts for a longer time and even results in occlusive vascular disease of extremities or life-threatening arterial and venous thromboembolism,which usually occurs in 5 to 10 days after the heparin treatment.It is caused by platelet-activating antibodies directed against complexes of heparin with a special platelet protein,platelet factor 4(PF4).HIT is mainly caused by immunoglobulin G(IgG) class among anti-heparin/platelet factor 4 antibodies that bind to epitopes on platelet factor 4(PF4) released from activated platelets that developed when it form s complexes with heparin.Platelet aggregation and hype rcoagulation status result from this process.Besides,the reactions between antibodies and vascular endothelial cells and monocytes are involved in HIT. Laboratory detection of anti-heparin/platelet factor 4 antibodies after heparin adminis tration may help diagnose HIT early.Tests for de tecting antibodies to the heparin/ PF4 complex can be classified into functional platelet assays(which rely on the dem-on stration of platelet activation) and immunoassays(which detect the presence of an antibody without regard for its functional ability).But there is no simple and effective test available currently.When heparin-induced thrombocytopenia is suspected,testing is indicated for heparin-dependent antibodies with the use of serologic or functional assays,or both.Serologic assays are available at most clinical laboratories,and they detect circulating IgG,IgA,and IgM antibodies.Although immunoassays have high sensitivity(greater than 97 percent),their specificity(74 to 86 percent) is limited by the fact that they also detect H-PF4 antibodies in patients who do not have heparin-induced thrombocytopenia.Thus,the positive predictive value of the immunoassay can be low(range,10 to 93 percent,depending on the population) but the negative predictive value is high(greater than 95 percent).The specificity of serologic testing for clinical disease can be improved if only IgG antibodies are measured,but IgG-specific assays are not commercially available.Functional assays measure platelet activation and detect heparin-dependent antibodies capable of binding to and activating the Fc receptors on platelets.The sensitivity of platelet-aggregation testing is greater than 90 percent at experienced laboratories.Its specificity ranges from 77 to 100 percent,depending on the clinical context of the heparin exposure.An assay measuring the 14C-serotonin release from activated platelets has high sensitivity(88 to 100 percent) and specificity(89 to 100 percent) but is not widely available.Because of the variability in responsiveness among platelet donors to PF4-heparin antibodies,the positive predictive value of functional assays tends to be higher(89 to 100percent) than the negative predictive value(81 percent).These antibodies are associated with the development of both venous and arterial thrombosis.These antibodies are present in nearly all patients who receive a clinical diagnosis of the disorder and cause disease in animals.However, they are also present in many patients who have been exposed to heparin in various clinical settings but in whom clinical manifestations do not develop.It is uncertain why complications occur in some patients but not in others.The time to the onset of thrombocytopenia after the initiation of heparin varies according to the history of exposure.A delay of 5 to 10 days is typical in patients who have had no exposure or who have a remote(more than 100 days) history of exposure,whereas precipitous declines in platelet counts(within hours) occur in patients with a history of recent exposure to heparin and detectable levels of circulating PF4-heparin antibodies. Platelet counts seldom drop below 10,000 percubic millimeter,are rarely associated with bleeding,and typically recover within 4 to 14 days after heparin is discontinued, although recovery may take longer in some patients.Previous studies indicated that the antibody would be prevalent in 8%of patients using the heparin,of which 1%to 5%patients would develop heparin-induced thrombocytopenia and thrombosis (HITT).In patients with heparin-induced thrombocytopenia,the thrombotic risk is more than 30 times that in control populations.The risk of thrombosis remains high for days to weeks after discontinuation of heparin,even after the platelet count normalizes.A typical manifestations include heparin-induced skin necrosis,venous gangrene of the limbs,and anaphylactic-type reactions after receipt of an intravenous bolus of heparin.Thrombotic complications may affect any vascular bed and frequently occur at sites of vascular injury.Venous thromboses predominate in medical and orthopedic patients,whereas arterial and venous thromboses occur at a similar frequency in patients who have undergone cardiac or vascular surgery.Limb ischemia may result in amputation in 5 to 10 percent of patients with heparin-induced thrombocytopenia.Rarely,thromboses occur at unusual sites,such as the adrenal veins or cerebral venous sinuses.The mortality rate is high(8 to 20 percent), regardless of therapy Heparin is the most often used anticoagulant during hemodialysis,which patients with end-stage renal disease on maintenance hemodialysis are repeatedly exposed to.Thus,this group of patients is at risk for developing heparin-platelet factor 4(H-PF4) antibodies and for developing HIT. Zwicker et al.suggested that the prevalence of anti-H/PF4 antibodies was correlated with thrombosis-related complications in patients using heparin for long-term maintenance hemodialysis.Furthermore,thrombotic complications were easy to occur with the elevation of serum level of IgG-H/PF4 antibodies for their effect on vascular endothelial dysfunction.Embolism in hemodialysis patients usually occurred in arteriovenous fistula,internal jugular vein indwelling pipe,etc which might be found as deep vein thrombosis,pulmonary embolism,myocardial infarction,cerebral infarction,limb artery embolism,and so on.Treatment of heparin-induced thrombocytopenia requires anticoagulation with one of two classes of anticoagulant agents,directthrombin inhibitors or heparinoids.Three direct thrombin inhibitors are currently available for patients with heparin-induced thrombocytopenia:lepirudin, argatroban,and bivalirudin.These agents directly bind and inactivate thrombin and,unlike heparin,do not require antithrombin.Direct thrombin inhibitors have short half-lives and show no cross-reactivity to heparin.Therapeutic dosing is recommended for patients who have isolated thrombocytopenia or heparin-induced thrombocytopenia with thrombosis.A recent study showed that IgG-H/PF4 antibodies could significantly be associated with increased mortality in patients with maintenance hemodialysis,it was also indicated that there might be some difference in the prevalence of serum IgG-H/PF4 antibodies in various races.Lee et al.tested anti-PF4-H antibodies in 91 cases of maintenance hemodialysis patients and the results showed that the positive rate of anti PF4-H antibody was 34%and was 10 times higher than that in normal control in Korea.However,they did not test IgG specific H/PF4 antibodies instead of all kinds of anti-PF4-H antibodies which result in higher prevalence but less specific.In this study,we investigate the prevalence of IgG-H/PF4 antibodies in Chinese patients with maintenance hemodialysis.We firstly detected IgG-H/PF4 antibodies in a large cohort of Chinese patients with maintenance hemodialysis.Then we further analyzed the associations between the levels of IgG-H/PF4 antibodies and vascular thrombosis.Objective:The aim of this study was to determine the prevalence of IgG-anti-heparin-platelet factor 4(IgG-H/PF4) antibodies and its association with vascular thrombosis in patients with maintenance hemodialysis using unfractionated heparin or low-molecular-weight heparin as anticoagulant.Methods:The experiment was carried out in March,September 2008 by Peking University First Hospital Institute of Nephrology.2008-01/2008-10 in the Peking University First Hospital and Peking University People's Hospital blood purification center dialysis patients with chronic renal failure.Inclusion criteria:1.Patients with serum creatinine.etc indicators meet the diagnosis standard of end-stage renal failure; 2.there are no serious complications 3.Patients received a bolus of 1000-2000 units of unfractionated heparin(UFH) or low molecular weight heparin at the start of each dialysis treatment;4.The patients are willing to accept this experiment and signed informed consent.A total of 147 patients with end-stage renal disease on maintenance hemodialysis from Peking University First Hospital and Peking University People's Hospital were enrolled in this study,65 were males and 82 females,former incidence of the disease:69 cases of chronic glomerulonephritis,chronic interstitial nephritis 25 cases,7 cases of adult polycystic kidney disease,hypertension 16 cases,15 cases of diabetic nephropathy,for unknown reasons 14 cases.Dialysis method:All patients were given dialysis,hemodialysis bicarbonate,and dialysis calcium level 1.5mmol/L, two or three times per week.4 hours each time,vascular access autogenous arteriovenous fistula used for dialysis or polysulfone(F6),Selection Blood samples were obtained prior to the hemodialysis procedure and were stored at -20℃until use. All serum samples were tested for IgG-H/PF4 antibodies using a commercial ELISA kit(GTI PF4 Enhanced,GTI Diagnostics,Waukesha,WI,USA).The assay was performed according to the procedures provided by the manufacture.Briefly,the microtiter plate was coated by heparin-platelet factor 4 complexes.Sera were diluted to 1:50 in phosphate-buffered saline.The volumes of this step and subsequent steps were 50μl and were carried out at 37℃for 35 minutes.The plates were washed three times with 300μl diluted Wash Solution between stages.Dilute the anti-human IgG antibodies 1 to 100 in phosphate-buffered saline.The volumes of this step and subsequent steps were 50μl and were carried out at 37℃for 35 minutes.The plates were washed three times with 300μl diluted Wash Solution between stages.Bound antibodies were detected by anti-human IgG antibodies.The reaction was revealed using PNPP Substrate in dark and was stopped with 100μl Stop Solution.The color intensity was read at 405nm in an ELISA reader.The IgG-H/PF4 ELISA assay was considered positive if the sample had an OD above 0.40.For statistical analysis, statistical software SPSS 13.0 was employed.Quantitative data were expressed as mean±SD.For comparison between patients with positive and negative antibodies, the Student's t-test and Chi-square test were used.Statistical significance was considered as P＜0.05.Results:.The number of episodes of vascular access thrombotic was significant higher in the IgG-H/PF4 antibody positive group than that in the negative group(P＜0.05).The levels of serum albumin were significantly lower in the IgG-H/PF4 antibody positive group compared with that in the negative group(P＜0.05).There were no other significant differences in age,sex,hemoglobin,Kt/V,or the type of anticoagulant(UFH or low molecular weight heparin) between IgG-H/PF4 antibody positive and negative groups(P＞0.05).Conclusion:The number of past episodes of vascular access thrombosis was significantly higher in the patients with positive IgG-H/PF4 antibodies than those without IgG-H/PF4 antibodies,which suggests that the IgG-H/PF4 antibodies might be a risk factor for vascular access thrombosis in patients with end-stage renal disease on maintenance hemodialysis.