3.1. Clinic characteristics of twin pregnancy and definitions of exclusion criteria
The incidence of complications was analyzed in 1153 cases of twin pregnancy(Table 1). Twin pregnancy combined with diabetes, premature rupture of membranes, placental abruption, preeclampsia, anemia, hepatitis B carrying, and thyroid dysfunction were of higher incidence comparing to a singleton pregnancy, indicating that twin pregnancy was one of the high-risk factors in pregnancy. Diabetes mellitus was the most prevalence complication, accounting for 21.3%, while a group of premature rupture of membrane and anemia followed at 16.0%. The third rank was scarred uterus (10.8%).
All these women with twin pregnancy were delivered by planned cesarean. The outcomes were then grouped into the adverse ones and the better ones (the remaining). According to single-factor analysis, it was found that TTTs, hypertension, preeclampsia, selective fetal growth restriction, premature rupture of membranes and placental abruption, scar uterus, chorioamnionitis, and pelvic inflammation are the high-risk factors for the adverse outcome of twin pregnancy (Table 1). Chorioamnionitis shows the most significant positive correlation to adverse outcomes, whose OR value was 5.40. Premature rupture of membrane and pelvic inflammatory disease were also observed high positive correlation to adverse outcomes with OR values of 3.31 and 3.81, respectively. However, the OR value of anemia was 0.79, indicating that the diagnosis of anemia based on the existing reference intervals appeared to be a protective factor.
After screening the clinical figure of these pregnant women, 253 cases without following exclusion criteria were then selected as the reference cohort (Fig. 1).
(1) with Twin-to-twin transfusion syndrome (TTTs), preeclampsia, diabetes, hypertension, G6PD deficiency, hyperthyroidism, hepatitis B carriers, hyper-viscosity, anemia, intrahepatic cholestasis,
(2) fetal malformation, infection, proteinuria, uterine tumor/cancer, selective fetal growth restriction, chorioamnionitis, umbilical cord around the neck, premature rupture of fetal membrane, scar uterus, and amniotic fluid,
(3) time of delivery is under 36 weeks of gestation,
(4) the weight of either neonate is below 2000 grams,
(5) neonate with major congenital anomalies.
However, when looking up the complete blood count records of these 253 cases in LIS informatic system, only 158 records of them had that of 30-35w (late-term) and 98 records of 20-25w (mid-term) of gestation.
3.2. Clinical laboratory analyses of complete blood count of the reference cohort
The CBC data of reference cohort within mid-term and late-term pregnancy were retrieved from the Sysmex XN9000 analysis platform. It is found that only 98 and 158 cases could be tracked, respectively. The mean pregnancy duration of this group was 36.8weeks, and the mean dates of the CBC data from specimens were 23 0/7w and 32 4/7w on average. The above parameters are all normal distribution through the analysis of the Kolmogorov–Smirnov test, and the mean values ± SD were shown in Table 2.
As the table indicated, both the mid-term and late-term pregnant women of twin pregnancy have the lower level of RBC, HGB, HCT and PLT than those in healthy nulligravida and the difference was statistically significant (P<0.05), and RBC, HGB and HCT increased as the gestation developing from mid-term to late-term. The count of white blood cells, neutrophils, and NEU% in the mid-term and late-term pregnancy increased than those in the healthy nulligravida group, and statistical significance (P<0.05) showed in the mid-term. The absolute count of WBC and NEU were 11.36±4.92 and 9.76±5.00 in mid-term, and drop to 8.54±4.17 and 7.17±4.17 when entered into late-term gestation. During the pregnancy, we found that the absolute count of LY was stable, but the LY% of reference decreased compared to that of the healthy nulligravida both in mid-term and late-term, and the difference of LY%in the mid-term was statistically significant (P<0.05). In conclusion, according to Chinese healthy unpregnant females, the reference intervals of RBC and PLT in twin pregnancy decreased; on the contrary, the WBC and neutrophils increased during pregnancy.
The requirements of Clinical and Laboratory Standards Institution (CLSI) suggest that any laboratory has its own reference intervals if no more than two results were outside the proposed reference interval when 20 samples are adopted to evaluate a new reference interval. Thus, a small group of 20 samples chosen at random, as described in methods, was tested for the reference interval of the late-term pregnancy (Fig. 2). The HGB index and HCT index of different two samples exceeded the upper and lower limits of the reference range of the twin pregnancy individually, while the RBC index of one sample was lower than the lower limit of that. Besides, all the other values fell within the established reference range. The verification passed, and the reference interval can be applied directly.
However, when using the Chinese normal female population reference intervals, 12 (60.0%) of the group would have out of range values of RBC count,13 (65.0%) women would have out-of-range hemoglobin and hematocrit value. All of these out-of-range values were below the lower limit of the reference range. From the perspective of WBC, 10 (50.0%) of the group would have out of range WBC count,12 (60.0%) and 11 (55.0%) women would have out-of-range count and percentage of neutrophils, 12 (60.0%) women would have out-of-range lymphocyte using the Chinese normal female population reference intervals (Fig.2).
3.3. Indexes of CBC might be potential indicators for adverse outcome of twin pregnancy
After establishing the reference intervals, we then further assessed its application potential in clinical diagnosis. Eighty-nine cases of twin pregnancy had a PTB during the period of 30-35 weeks of gestation, and 16 cases who suffered PPH were analyzed, while 3 of them were overlapped (Fig. 3). We excluded the three overlapped cases in order to see a single characteristic indicator relating to the particular outcome. The mean dates of first CBC records as they admitted to our center were 32 5/7w and 35 0/7 w of gestation, respectively of PTB and PPH groups, and the mean dates of delivery were 33 4/7w and 37 4/7w. Based on the statistical analysis of CBC among group REF, PTB, and PPH (Table 3), we found that the level of HGB and HCT significantly drop in the PTB group (p<0.05), while absolute WBC and NEU increased in PTB group. In the PPH group, it showed a significant decrease in HGB (p<0.05), but no statistical differences in other indexes (Fig. 4).
Using the established reference intervals for twin pregnant women in this study, 10.47% (9/86) of PTB population had beyond-upper limit WBC values during late pregnancy, while 12.79% (11/86) had beyond-upper limit NEU values. Conversely, 17.44% (15/86) of PTB population had the out-of-range value of HGB and/or HCT, 14 of which were below of lower limit and should be considered to have any grade of anemia (Fig. 5A). Otherwise, of the PPH population, 30.77% (4/13) were below the lower limit of established reference intervals of HGB value (Fig. 5B).