Influence Of Hemolyzed Sample Nitrogen-containing Compound And Amino-terminal Pro-brain Natriuretic Peptide In Postmortem Biochemical Detections

Objective:Postmortem biochemistry is an applied discipline that uses the biochemical indicators of post-mortem serum,pericardial fluid,vitreous humor and other body fluids to objectively determine the pathophysiological state before death and assist in forensic pathology.Serum urea(Urea),creatinine(Crea),and uric acid(UA)are relatively stable in the early post-mortem period and have important auxiliary effects in the diagnosis of death due to renal failure and hyperosmolar dehydration.The level of Amino-terminal pro-brain natriuretic peptide(NT-proBNP)in the post-mortem pericardial fluid is of great help in objectively determining the state of cardiac function.However,due to post-mortem changes and corpse freezing often lead to blood hemolysis,pericardial fluid is also contaminated by hemolysis.Conventional biochemical detection are very strict on the hemolysis of samples.The samples taken at autopsy,especially frozen corpses,often have a very severe degree of hemolysis.Therefore,this study aimed to investigate the severe hemolysis of serum(and whole blood)samples of urea.Therefore,the purpose of this study was to investigate the interferences of severe hemolysis on the biochemical determination of hemolytic pericardial fluid NT-proBNP and urea,creatinine,uric acid in serum(and whole blood)samples.Methods:This study is divided into two parts.Part I: Collect 32 unhemolytic blood samples(25 of which were from unfrozen bodies,autopsies within 48 hours after death;and 7 from peripheral venous blood from patients with chronic renal failure).Each blood sample was divided into two parts,one was immediately centrifuged to separate the serum for testing,and the other was used to prepare a hemolyzed sample in which the degree of hemolysis was sequentially increased in the three groups A,B,and C.The prepared hemolyzed sample was ultrafiltered(Groups A and B using Centrifree? 30 KD ultrafiltration tube;Group C using hollow fiber ultrafiltration module and Amicon? Ultra-0.5 30 KD ultrafiltration tube),the obtained ultrafiltrate and serum,Untreated hemolyzed samples were assayed for urea,creatinine,and uric acid concentrations using an automated biochemical analyzer.Part II: The second part: collected 1 case of left heart blood(no hemolysis)and 10 cases of pericardial fluid(no macroscopic hemolysis,electrochemiluminescence(ECL)and dry immunofluorescence method to determine the NT-proBNP concentration is greater than the lower limit of detection).After sampling,the blood sample was immediately prepared to concentrate the hemolyzed blood.Each of the pericardial fluids was prepared into a total of five concentration gradient pericardial fluid samples containing hemoglobin(Hb)from 0 g/L to 60 g/L.The concentration of NT-proBNP in a total of 50 gradient hemolytic pericardial fluids was determined by dry immunofluorescence(n=25)and electrochemiluminescence method(n=25).A hemolysis pericardial solution with a Hb concentration of 60 g/L was selected and divided into 3 groups(6 parts per component),which were stored at-20 ℃,4 ℃,and room temperature(controlled between 24 ℃ and 26 ℃).The concentration of NT-proBNP was detected by electrochemiluminescence method on days 1,3,9,15,24,and 40,respectively.The two parts used Bias% and Mean Absolute Percent Error(MAPE)as indicators to measure the degree of interference.Statistical analyses were performed with SPSS 24.0 and GraphPad Prism 5.0.Results: 1.The Bias% of uric acid in the three groups A,B,and C(range:-16.76% to-1152.65%)are all negative,that is,the detection value is decreased.No consistent direction of bias was observed in urea(range:-839.85% ~ 380.12%)and creatinine(range:-50.92% ~ 589.06%).At the same time,it was observed that as the degree of hemolysis increased,the MAPE of urea,creatinine and uric acid concentrations gradually increased.2.The hemolyzed sample is a colorless transparent solution after ultrafiltration treatment.The correlation analysis between the ultrafiltrate and the corresponding serum showed that urea and creatinine were strongly correlated(r2>0.95),and uric acid was relatively dispersed(r2=0.683).3.After the ultrafiltrate is corrected,it can better reflect the serum urea,creatinine and uric acid concentrations.4.Electrochemiluminescence method(|Bias%|<5.5%)is superior to immunofluorescence method(|Bias%|<20%).5.-20℃ storage conditions,degradation of 26.4% in 40 days;4℃ storage conditions,degradation of 40.5% in 40 days;room temperature storage conditions,degradation of 90.0% in 40 days.Conclusion: 1.Hemolyzed samples seriously interfere with the biochemical determination of urea nitrogen,creatinine and uric acid.After ultrafiltration treatment,the concentration of urea nitrogen,creatinine and uric acid in the ultrafiltrate can be relatively accurately reduced to the level before the hemolysis.2.Both detection methods have good anti-interference ability for hemolysis interference,and the interference direction is negative.As the degree of hemolysis increases,the interference is gradually increased.The anti-interference ability of electrochemiluminescence is better than immunofluorescence.3.The degradation of NT-proBNP is accelerated under severe hemolysis.