| Objective:To establish a simple,stable method for measuring total complement activity in plasma which on the basis of the CH50 hemolysis method.In this method,human own red blood cells(RBCs)were used to replace sheep red blood cells(SRBCs),the multi-tube method was changed to a single tube method.Methods:1.First of all,the hemolysis experiment which using human own RBCs replace SRBCs,rabbit-anti-human RBC antibody replace rabbit-anti-SRBC antibody(hemolysin)attempted to prove the the efficiency of hemoglobin release after RBC destruction in different RBC and plasma concentrations.The CH50 muti-tube hemolysis experiment attempted to identify the optimum concentration of various reagent and establish the single-tube hemolysis colorimetric method.At the same time,the linear research and repeated experiment were used to prove the accuracy and stability of new method.2.The CH50 standard method was used to measure the total complement activity in plasma and serum.The new method was used to measure the complement activity of same plasma which using different human RBCs as the hemolysis indicator system,further optimized the single-tube hemolysis colorimetric method,verified the feasibility of the new method.3.The CH50 method and single-tube hemolysis colorimetric method were used to measure the complement activity of the same plasma,the experiment attempted to prove the correlation of two methods.4.The single-tube hemolysis colorimetric method was used to measure the complement activity of intensive care unit(ICU)patients,Coronary heart disease(CHD)patients,healthy humans,the experiment attempted to prove the new method has a certain clinical application value.At the same time,polymerase chain reaction(PCR)detection technology was used to measure the condition of MICA*008 gene-carry,analyze the relationship of CHD patients complement activity and MICA * 008 gene.Results:1.The human RBCs and rabbit-anti-human RBC antibody were used to replace SRBC and hemolysin to do the hemolysis experiment.The result showed that the hemolysis activity influence of different RBC concentration was smaller than different plasma concentration.At the same time,when the plasma concentration was 20%,the influence of different RBC concentrations in the hemolysis experiment was smaller.The proportion of each component(20% RBC suspension and 20% plasma concentration tube)was calculated as the reference for each component in the single-tube hemolysis colorimetric method.The single-tube hemolysis colorimetric method was used to measure the total complement activity of different plasma concentration and the same plasma for five consecutive days,the result showed that the total complement activity of different plasma concentration has a linear distribution,the coefficient variation(CV)of the complement activity for five days was 11.3%,it concluded that the single-tube hemolysis colorimetric method has a certain accuracy and stability.2.The CH50 standard method was used to measure the complement activity of plasma and serum,the result showed that the complement activity of plasma and serum were similar completely,therefore the plasma could replace serum to measure the complement activity,the complement detection technology optimization was obtained.At the same time.the complement activity of the same plasma which using different human RBCs as hemolysis indicator system were similar roughly(p>0.05).It concluded that different RBCs could not affect the complement activity in plasma,the single-tube hemolysis colorimetric method has a certain feasibility.3.The OD values of single-tube hemolysis colorimetric method and the complement activity value of CH50 method has a linear relationship,it showed that single-tube hemolysis colorimetric method and CH50 method has significant correlation,single-tube hemolysis colorimetric method can replace CH50 hemolysis method to measure the total complement activity.4.The complement activity of ICU patients and CHD patients were significantly decreased than healthy humans,single-tube hemolysis colorimetric method has a certain clinical application value.The MICA*008 gene distribution of CHD patients and healthy persom was similar roughly.At the same time,the complement activity of CHD patients may not have a relationship with MICA*008 gene.Conclusion:1.Single-tube hemolysis colorimetric method to measure complement activity has good accuracy,stability and feasibility.The new method has significant correlation with CH50 method and to optimize the CH50 hemolysis method,it has a certain clinical value.2.The total complement activity of ICU patients and CHD patients were lower,it showed that ICU patients has an immune dysfunction,CHD patients may be in the stage of the inflammatory response.3.The complement activity of CHD patients may not have a significant relationship with MICA*008 gene. |
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