Participants and blood samples
This study was carried on 80 cases of Rh (D) positive pregnant women who were examined in Huangdao district people's Hospital in Qingdao. All of them were negative in irregular antibody screening, and their blood types were different from their husbands. There were 36 pregnant women with A-O blood group, 41 with B-O blood group and 3 with AB-O blood group, aged from 20 to 45 (30.44 ± 5.30) years.
The studied pregnant women were divided into two groups:Controls(n = 38), the healthy controls;ABO-HDFN group(n = 42), whose baby were positive in release test were identified as hemolytic disease, regardless of the results of direct anti-hemolysis test and free-blood test, the pregnant women were assigned to the ABO-HDFN group.
Exclusion criteria:(1) neonatal hemolysis caused by other blood group systems and other causes (thalassemia, hereditary spherocytosis, deficiency of glucose-6-phosphate dehydrogenase, etc.) ; (2) Co-infection: clinically diagnosed as infection (including sepsis, pneumonia, etc.) or CRP > 10mg/L; (3) neonatal hypoxia: fetal distress in utero, perinatal asphyxia; (4) large head hematoma or severe contusion; (5) maternal smoking or chronic hypertension, diabetes, systemic lupus erythematosus or other autoimmune diseases.
Peripheral venous blood or umbilical cord blood were collected into ethylene diamine tetra-acetic acid (EDTA) (1.2 mg/mL) for haematological profiling.For the irregular antibody screening, direct anti-human globulin test, free antibody test, and antibody release test,chemical analysis and C-reactive protein assays (CRP), clotted samples were obtained and serum was separated by centrifugation for 15 minutes at 1,000×g.
100µL of maternal plasma was mixed with the same amount of β -mercaptoethanol and incubated at 37℃ for 10min. After dilution with normal saline, Red blood cells with ABO reagent of the same blood group as her husband were added. After incubation at 37℃ for 30 min, IgG antibody titer of corresponding blood group was determined .
The maternal plasma obtained by centrifugation of EDTA anticoagulant specimens was used for enzyme-linked immunosorbent assay, and the OD value was measured at 450nm wavelength with a microplate reader. All operations were performed in accordance with the reagent operating instructions.
This study was reviewed and approved by the ethics Committee of our hospital. All subjects obtained the consent of pregnant women and their families and voluntarily signed informed consent.
The measurement data were tested for normality by KS (D) test, and the indicators of normal distribution were expressed as mean± standard deviation (Mean±SD). Inter-group comparisons were analyzed by independent sample T test, and inter-group comparisons were analyzed by One Way ANOVA method. Indicators with non-normal distribution were represented by median and quartile spacing (IQR), mann-Whitney U test was used for inter-group comparison, and Kruskal-Wallis H test was used for inter-group comparison. Statistical data were expressed as percentage (%), and comparisons between groups were performed by Chi-square test or Fisher's exact probability method. Spearman correlation analysis was used to analyze the correlation between the two factors. Diagnostic efficiency was evaluated by ROC curve. P (probability) <0.05 was accepted as a significant difference in the analyses.