Laboratory tests are often requested during pregnancy to exclude pathological complications that may affect maternal or fetal health [23, 24]. Diagnostic accuracy is based on the evaluation of results in relation to accurate reference values . Although changes in normal laboratory values induced by pregnancy are well known, few studies have been conducted to establish reference intervals for pregnant women, especially those with twin pregnancies [7, 10,11,12,13]. In this study, we report x ± 1.96 s as reference intervals for CBC parameters in twin pregnancy. We found that the RBC, HGB, HCT, PLT levels and the LY% in mid- and late-term pregnant women with twin pregnancies were lower than those in healthy nulligravida women, while the WBC, NEU counts and NEU% in mid- and late-term twin pregnancy were higher [7, 10, 13]. We used these results to create reference intervals and tested our reference intervals using blood samples from 20 women with twin pregnancies. After confirming the correctness of the reference intervals, we found that the levels of RBCs, HGB, HCT, and PLTs decreased in women with twin pregnancies, and WBCs and NEUs increased, which suggests that these factors may be prognostic indicators of this adverse outcome. The establishment of suitable reference intervals for women with twin pregnancies has the potential to improve diagnostic quality, which could lead to increased survival, reducing unnecessary treatment and cost savings.
We Initially found that 16.0% of twin-pregnant women were diagnosed with anemia, the OR value of which for adverse outcomes was 0.79 (Table 1). These values strongly suggested that the use of the HBG level of healthy Chinese nulligravid individuals may result in incorrect interpretations in the clinic. The development of an accurate set of CBC reference intervals for twin pregnancies is urgently needed. Based on the reference intervals established in this study, we found that RBC, HGB, HCT, and PLT levels decreased during normal twin pregnancy during the mid- and late-term, which is consistent with findings from studies of singleton pregnancy [10, 11]. The MCV was unaffected by twin pregnancy and remained the same as the levels in healthy nulligravida women, which indicates that RBC, HGB, and HCT levels were affected by hemodilution rather than nutritional deficiencies . Consistent with other studies of singleton pregnancy, we inferred that this result occurred due to the expansion of plasma volume during pregnancy with a concomitant lower expansion in red-cell volume . RBC, HGB and HCT showed the lowest values in the mid-term, and these values increased slightly in the late-term. This result may be attributed to the fact that as a pregnancy develops from mid- to late-term, the body activates the metabolic system to compensate for the physiological change, and the blood dilution stabilizes [18, 26].
In contrast, HCT and RBC counts decreased with increasing gestational age in our data, but the absolute WBC count reference interval was elevated during pregnancy, which was due to the significant increase in NEU count during a normal twin pregnancy. This result may be related to a stress response, the redistribution of WBCs between the marginal and circulating pools or pain, nausea, vomiting, and anxiety in the absence of infection [19, 27]. WBC and NEU counts are expected to increase during pregnancy. Therefore, the reference intervals for these parameters should especially be considered with caution in the clinic. The use of these parameters should be limited to markers for infection during pregnancy in case proper reference intervals are lacking [20, 28]. We found that the increase in WBC count resulted primarily from an increase in NEU counts, which peaked in the mid-term and decreased in the late-term and was slightly different from the results of other studies. The absolute WBC and NEU counts decreased from 11.36 ± 4.92/8.54 ± 4.17 to 9.76 ± 5.00/7.17 ± 4.17 when entering the late-term of gestation, and the NEU% was stable. We found that the absolute count of LY was stable during twin pregnancy, and LY% was lower than that of healthy nulligravida women. Significantly lower values were detected in the mid-term (P < 0.05), which is consistent with the literature that absolute NEU counts peaked at the mid-term [14, 20, 28].
Investigation of potential risk markers of adverse outcomes has been performed at the molecular, biochemical, and metabolic levels [29,30,31]. Multiple pregnancies are associated with an increased risk of adverse maternal and perinatal outcomes, primarily due to obstetric complications, preterm birth, and birth weight discordance . Cervical length, fetal fibronectin, and uterine activity monitoring, which used to be potential risk factors for interest, are not recommended because no new evidence has been identified to confirm their accuracy . Using ultrasound screening, crown-rump length and placental location were measured to assess their association with adverse pregnancy outcomes [34,35,36]. The current study identified risk factors using more routine laboratory tests and determined that absolute NEU in CBC values may be a predictor of PTB. According to the established reference intervals for a twin pregnancy, NEU increased to above the upper limit (> 11.31 × 109/L) in 12.79% of the PTB population. The association between NEU count and PTB needs further confirmation using evidence from a larger sample.
While the strength of this study is the large screened population (1153 cases) in one institute, the limitations hould not be overlooked. First, the sample size for reference interval establishment was small due to the high incidence of adverse outcomes in twin pregnancy, although it met the requirement of CLSI guidelines. Second, CBC data of candidates were obtained primarily from examinations in the second and third trimesters due to the retrospective nature of this study. Therefore, we may have missed the earlier changes in maternal hematology that corresponded to twin pregnancies. However, compared with the use of standard whole blood reference intervals from nonpregnant women, which may result in an increase in out-of-range values among twin pregnant women, the limitations of the current study may not have significantly affected the accuracy of the reference intervals for this population.
Twin pregnancy clinics were established in our center to provide better medical services for such a high-risk population. Currently, the reference interval we established is in a further stage of clinical verification and cannot be presented as a formal reference value inroutine blood reports, but rather as a comment. However, according to feedback from obstetricians of the twin pregnancy clinics, the reference interval allows them to better assess the status of the twin pregnant women, which enables them to eliminate an “infection” deduced by an increasing WBC count and arrange an amniocentesis, as well as to eliminate “anemia” deduced on the basis of a decreasing HGB value and avoid excessive iron supplementation. Compared with the current officially adopted reference interval, the interval we established, particularly for women with twin pregnancies, can aid obstetricians in precise clinical decision-making and reduce artificial interference. After long-term and large-scale clinical sample verification, we will upgrade the LIS to identify the application department of test and diagnosis keywords (twin, gestational age), then retrieve the reference interval of the corresponding stage and present it in the report automatically.
In summary, CBC indexes varied to different degrees with the development of twin pregnancy. The present study established reference intervals for several hematological variables in healthy women with twin pregnancies that may be used for clinical management in our center. These values should be considered for the development of region-specific reference intervals for women with twin pregnancies in China. Further validation and practice in the clinic of these established reference intervals are warranted. We hope that the presentation of these reference values will assist clinicians in distinguishing between physiological changes and pathological states during twin pregnancy, and help clinicians make effective judgments and administer treatment in a timely manner.