| Objective:Anaphylactic shock is caused by the drugs,food,etc of the triggers of systemic hypersensitivity,mainly happened via type I hypersensitivity.In the practice of forensic medicine,cases death of anaphylactic shock are common,but due to the lack of characteristic pathomorphological changes,it is often a difficult problem in forensic pathology diagnosis.A large number of experiments have shown that postmortem biochemistry is to objectively determine the pathophysiological state before death by using various biochemical indexes in body fluids such as postmortem serum,pericardial fluid,and vitreous fluid.In particular,the detection of postmortem serum total Immunoglobulin E(IgE)can help the diagnosis of anaphylactic shock and has been applied.However,due to the cryopreservation of corpses in China,in the practice of forensic medicine,the serum cannot be separated from the blood because of the serious hemolysis after the freezing and thawing.In the clinical test,the hemolysis sample is refused to do the test and redraw the blood to separate the serum for detection.This makes it almost impossible to use normal clinical tests on postmortem samples,further complicating the detection of total IgE after death.Moreover,in the actual work,samples need to be temporarily stored due to such factors as untimely sample submission,transfer or detection,which is generally frozen or refrigerated and sometimes requires long-term cryopreservation for detection.The extent to which these two practical issues affect total IgE testing is unclear.Although in the light of the clinical research of clinical samples and postmortem samples in forensic science research has been of some indicators(such as the NT-proBNP)and different preservation conditions on the stability of frozen,in view of the samples after the death of total IgE under the condition of hemolysis and frozen,and blood serum total IgE freeze-thaw stability,the stability of the different conditions in vitro,and test results of the research has not yet been reported.Therefore,this study focused on the stability of total IgE in hemolysis and freezing,and explored how to obtain the total Ig E detection value in hemolyzed samples which can reflect the serum level.Methods:This study is divided into two parts.Part I: Autopsy samples were collected from 40 non-frozen cadavers within 48 hours after death,including 21 plasma samples and 19 whole blood samples.The samples were stored at-20?,4?,25? and-80?,respectively.The concentration of IgE in the samples was detected by electrochemical luminescence on days 0,3,7,14,21 and 28,and the stability of IgE in plasma and hemolytic samples after death was investigated by using samples that were freeze-thaw treated for 5 times.Total IgE in plasma and hemolytic samples before and after treatment was detected by electrochemical luminescence.Part II: 33 autopsy samples were collected from non-frozen cases within 72 hours after death.In each case,plasma was separated and total hemolytic samples were prepared by repeated freezing-thawing.Total hemolytic samples were ultrafiltrated to obtain ultrafiltrate.The hemoglobin concentrations in plasma,total hemolytic samples and ultrafiltrate were determined by the method of Vanzi's liquid-cyanidation methemoglobin.Total IgE in plasma and ultrafiltrate was detected by electrochemical luminescence and ELISA.SPSS 20.0 software(IBM)was used for statistical analysis.Linear regression was used to analyze the ultrafiltrate of ELISA and ECL and the corresponding plasma IgE values,and the correlation coefficients were calculated.Wilcoxon symbol rank sum test was used for comparative analysis.Graph with GraphPad Prism 5.0 Results: 1.The degradation rate of plasma IgE increased with the extension of the preservation time under the three storage conditions of-20?,4? and 25?,and the descending rate of the three storage conditions was close to each other.The degradation rate was around 15% after 28 days.IgE degradation rate in hemolytic samples was faster than that in plasma,especially>40% at 28 days under 25?.2.There was no statistically significant difference between the detected values of plasma samples after freezing at-80? for one year and those before freezing,while the hemolytic samples were degraded after freezing at-80? for one year,and the difference was statistically significant compared with that before freezing.3.The results of plasma and hemolytic samples after 5 freezing-thawing cycles after death were not significantly different from those before freezing-thawing cycles.4.The hemoglobin concentration in ultrafiltrate was significantly lower than that in the whole hemolytic sample(P<0.05).The ultrafiltrate was correlated with plasma by ELISA and electrochemical luminescence(r=0.984),which was better than ELISA(r=0.635).The ultrafiltrate measured by the two methods was corrected by their respective equations(ELISA: y=0.503 6x + 285.45;Electrochemical luminescence: y=3.059 3x + 2.017 3.There was no significant difference between the adjusted values and the plasma(P>0.05).Conclusion: 1.Under three storage conditions of-20?,4? and 25?,the stability of plasma IgE is better than that of hemolytic samples.So the blood sample should separate plasma or serum as early as possible for better preserve after death.2.IgE in plasma and hemolytic samples after death has good freeze-thaw stability and can withstand 5 freeze-thaw cycles.3.IgE remained relatively stable in plasma after death and could be stored at-80? for one year;The stability of IgE in hemolytic samples was poor,and the degradation was significant even after one-year preservation at-80?.Therefore,for samples requiring long-term storage at-80?,it is recommended to store them in the form of plasma or serum.4.Ultrafiltration technology has a good treatment effect on total hemolytic samples.The correction values of ultrafiltrate detected by electrochemical luminescence and ELISA after correction by correction equation are close to the plasma level before hemolysis.Ultrafiltration combined with electrochemical luminescence had the best results and could be used for total IgE detection of frozen hemolytic samples. |
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