Reference intervals of complete blood count and coagulation tests in Vietnamese pregnant women

Reference interval is defined as the interval between and including two numbers, an upper and lower reference limit, which are usually the 2.5^th percentile to 97.5^th percentile of a normal population. Therefore, it is common to interpret results as abnormality based on normal reference intervals in pregnant women due to their biophysiological changes [1]. As a result, it is crucial to define normal reference intervals for pregnant women. Complete blood count and coagulation tests such as activated partial thromboplastin time (APTT), prothrombin time (PT), and fibrinogen are of a routine hematology panel. There have been many studies conducted on Asian pregnant women [2‐4], but none was conducted in Vietnam. Therefore, we conduct this study to determine trimester specific reference intervals for healthy pregnant women.

A total of 879 patients were enrolled but 12 patients’ samples failed to run, leaving 867 patients. Out of 867 patients, due to unqualified quality, 837 samples were eligible for complete blood count test and 836 samples for coagulation tests. The characteristics of the women measured before being enrolled were described in Table 1. Overall, most women are in the age of 25 to 33. In the first trimester, one outlier was detected in APTT parameter, one outlier in monocyte parameter and one outlier in basophil parameter. In the second trimester, two outliers were detected in RDW-SD parameter. In the third trimester, only one outlier was detected in eosinophil parameter. Tables 2 and 3 illustrate the reference intervals, for complete blood count test and coagulation tests, respectively, along with 90^th confidence intervals for upper and lower limits. Table 4 describes verification study for transference of reference interval from two studies on Asian pregnant women.

During pregnancy, the mother has an increase in blood volume, up to 50% increase. This contributes to slightly decreased red blood cell indices, also known as dilutional anemia. However, red blood cell production also increases by 25–30% until term gestation [1]. In our study, RBC, HCT, and HGB (Fig. 1) started to decline in the second trimester but then rise again in the third trimester, which is explained by the physiology adaptation in pregnancy. The 2.5^th percentiles of HGB (g/dL) in our study are 10.33, 9.71, 9.80 in first, second and third trimester, respectively. Nevertheless, according to the definition of anemia recommended by Centers for Disease Control and Prevention (CDC) [8], HGB levels below 11 g/dL, 10.5 g/dL, 11 g/dL in first, second and third trimester, respectively, are considered to be anemic. 11% of patients were classified as anemia if we use CDC criteria despite taking taking iron supplements. Several studies including Aiwei Li et al. and Yi Jin et al. [4, 7] also found that the 2.5^th percentiles were below the diagnostic cutoff for anemia. An explanation for this phenomenon may be due to the use of 5^th percentile of data aggregated from four European studies of healthy iron-supplemented pregnant women to establish the cutoff [9]. Therefore, CDC conventional diagnostic criteria for anemia may not be approriate for Vietnamese pregnant women and we suggest the use of 2.5^th percentile as a cutoff for anemia diagnosis.

In addition, Torgersen CKL and Curran CA [1] reported white blood cell count increases in the beginning of the first trimester and then remains stable in the second and third trimester. In white blood cell count, neutrophils contributes the most to the rise, followed by eosinophils. In our study, white blood cell count from all trimesters ranges from nearly 6 × 10⁹/L to roughly 15 × 10⁹/L, with the number of white blood cell count increases slightly in second trimester, compared to the first trimester, but decreases in the third trimester (Fig. 2). Thus, the difference between the third trimester and first trimester shows no statistical significance.

Because of dilutional effect in pregnancy, platelet count also suffers from the same decrease as red blood cell count. Figure 3 shows the platelet count are roughly equaled. Only 9 (1%) women in our study suffered from thrombocytopenia but they did not show any signs or symtomps. Nonetheless, the 2.5^th percentile of platelet count in each trimester is well above the 150 × 10⁹/L cutoff for thrombocytopenia [10]. Hence, platelet count in pregnant women is almost the same as non-pregnant women.

PT, APTT, Fibrinogen reference intervals are presented in Table 3. Figure 4 also shows that PT and APTT get shorter in every trimester while fibrinogen level shows an ascending trend as gestational age increase. This can be explained by the hypercoagulable state of pregnant women. Our results coincide with the results of C. Cui et al. [2], which also used the ACL TOP coagulation analyzer. Moreover, the first trimester medians of PT (s), APTT (s) in our study are 11.35 and 30.1, respectively, which are close to non-pregnancy medians in C. Cui’s study (11.0, 32.8). A meta-analysis study by Mina Abbassi-Ghanavati et al. [11] also found a similar hypercoagulation trend. The difference between each trimester reference interval was statistically significant.

Apart from establishing new reference intervals, we performed a verification study which is described in Table 4. Before verifying the transferability, we randomly selected 20 persons from our study and perform a Tukey outlier test until there were no outliers for all tests from the complete blood count panel or coagulation tests. After we had successfully chosen 20 persons with no outliers in the data, we tested if there were more than two results that fell outside of the targeted reference interval. If less than or equal to two results fall outside, the reference interval is verified. This procedure is recommended by CLSI EP28-A3c. Overall, in complete blood count verification, we used data from Aiwei Li’s study [7], which used Sysmex XE-2100 hematology analyzer. Most of the parameters were verified except for platelet parameter in third trimester and MPV parameter in first trimester and monocyte in first and third trimesters. In coagulation test verification, we used data from C. Cui’s study [2], which used ACP TOP coagulation analyzer. All coagulation parameters fell inside the limits, proving that the verification process can be successfully achieved if the chosen study had the same equipment as the laboratory’s. Another point to make is that the patient selection step for verification can be cumbersome because we must find another patient to replace the one that has outliers in her test results.

Reference intervals are important tools to aid clinicians in deciding their next step in treatment. Thus, it is important that laboratories use appropriate reference intervals via verifying or establishing new ones. In our study, the trimester-specific reference intervals were derived from random and independent women. In order to determine the real change in women during pregnancy, data throughout three trimesters should be taken from one participant. Therefore, our study’s results may be affected by the biologic variations between subjects. Despite that, by obtaining data from a large sample, we hope to minimize that effect and bring clinicians a useful tool to evaluate and other laboratories our own target population reference intervals.