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Abstract

Background

Pregnancy induces physiological changes which affect biochemical and haematological parameters. As the significance of laboratory test results change throughout pregnancy, the reference interval (RI) or key result interpretive guide should be specific to pregnancy. This study sought to establish trimester-specific-RIs for routine biochemical and haematological tests in healthy white European women with singleton pregnancies with comparison to RIs for non-pregnant European adults.

Methods

A retrospective analysis of a prospective longitudinal single-centre study of healthy pregnant women conducted between November 2018 and December 2020 in a tertiary academic hospital with approximately 3000 births annually. Inclusion criteria: signed informed consent, age ≥18 years, white European, body mass index (BMI) <25 kg/m2, blood pressure <140/90mmHg, non-smoker, no previous pathology or gestational diabetes. Trimester defined as T1: up to 13 weeks + 6 days, T2: 14–27 weeks + 6 days and T3: ≥28–41 weeks + 6 days. Baseline demographics, anthropometric and laboratory measurements were recorded. In total, 31 biochemical and 10 haematological ISO15189:2012 accredited tests were measured using Roche Cobas® and Sysmex XN-9100™ analysers, respectively. RIs were established according to the International Federation of Clinical Chemistry (IFCC) recommended method.

Results

Apparently healthy pregnant women (n = 124) with bio-banked serum samples in each trimester were recruited. At the booking visit, 49.2% (n = 61) of participants were nulliparous, with median age of 34.4 (IQR: 31.3–37.3) years, gestational age of 89 (IQR: 84–93) days, BMI of 22.5 (IQR: 21.0–23.7) kg/m² and systolic and diastolic blood pressure of 116 (110–125) mmHg and 67 (61–75) mmHg, respectively.

Conclusions

Normative trimester-specific biological intervals for routinely requested biochemical and haematological medical laboratory tests were established. These RIs will be invaluable to result interpretation and the management of pregnant women.

Keywords

Reference interval trimester-specific pregnancy clinical biochemistry tests full blood count

Introduction

Reference intervals (RIs) are a range of analyte concentrations usually encompassing the 95% central distribution of values observed in healthy individuals and are designed to aid result interpretation and clinical decision-making.¹ Knowledge of physiological changes is fundamental and essential to both understanding and developing reference intervals.² Healthy physiological changes occur throughout pregnancy to accommodate the needs of both mother and fetus. These pregnancy-induced physiological adaptations impact most organ systems and many biological markers.^3,4 In laboratory medicine, this is of great importance because many biochemical and haematological test results have different significance depending on the gestational age of the woman at the time of testing.^5,6 However, despite this, many laboratories do not provide trimester-specific-RIs for healthy pregnant women and often quote RIs from non-pregnant women or adults.^4,7 The use of inappropriate RIs leads to an increased risk of misinterpreting normal pregnancy-induced changes in biomarkers as pathological or overlooking a truly abnormal result.^8,9 The clinical risk of such practice is not insignificant with the potential to lead to patient misdiagnosis and/or mismanagement; therefore, ideally laboratories should establish/provide pregnancy-specific RIs for biochemical and haematological parameters across all three trimesters.¹⁰

Establishing RIs per se is challenging¹¹ but endeavouring to do so for the different stages of pregnancy is even more demanding.² This is because to develop a RI a priori (direct) requires a minimum of 120 healthy people.¹¹ Moreover, the 9 months (40 weeks’ gestation) of human pregnancy have traditionally been divided into three 14-week periods, but often the partitioning of gestation is arbitrary.¹² Consequently, indirect or data mining methods are considered by many to be the practical way forward and perhaps the best approach to verify RIs.^13,14 Notwithstanding these issues, development of trimester-specific-RIs necessitates careful consideration of the degree of change in analyte concentration throughout pregnancy, appropriateness of timing of partitioning of data, comparison of the magnitude of the change observed to that of the non-pregnant state and, finally, deciding whether the change identified is clinically significant.⁵

There are few sources in the literature that provide RIs for white European pregnant women and those published rarely measure analytes longitudinally or tend to focus on selected analytes, particularly thyroid function tests.⁴ In addition, many define the trimesters of pregnancy differently.

To address this unmet clinical need, this study sought to establish trimester-specific-RIs for routinely requested biochemical and haematological ISO15189:2012¹⁵ accredited tests in healthy pregnant white European women.

Materials and methods

Ethical approval

Ethical approval for the study was granted by the research ethics committee at Galway University Hospitals (GUH) (Ref GUH: CA 2026). This study was conducted in accordance with the ethical principles as set out by the World Medical Association Declaration of Helsinki. Prior to study initiation, written/signed informed consent was obtained from each participant.

Study design

This was a retrospective sub-analysis of a larger prospective longitudinal study which aimed to evaluate novel biochemical tests for the diagnosis and follow-up of gestational diabetes.¹⁶ This single-centre study of healthy pregnant women was conducted between November 2018 and December 2020 in a tertiary academic hospital with approximately 3000 births annually. Participants were recruited consecutively at the first antenatal visit at Galway University Hospitals (GUH). Each participant had an early dating scan for an agreed estimated due date (EDD). This sonographically determined EDD was subsequently used to determine the gestational age of the women at each study sampling timepoint. The gestational period was divided into three 14-week periods or trimesters (T) as follows: T1: up to 13 weeks + 6 days (97 days), T2: 14–27 weeks + 6 days (98–195 days) and T3: ≥28–41 weeks + 6 days (196–293 days).¹²

Reference population

Healthy women, aged 18–47 years with singleton pregnancy, were recruited to this study. The inclusion criteria were: signed informed consent, age ≥18 years, white European, body mass index (BMI) <25 kg/m², blood pressure (BP) <140/90mmHg, non-smoker and no previous pathology or gestational diabetes.

Data collection

Following informed written consent, at the booking visit, baseline clinical data including age, ethnicity, BMI, BP, gravida, parity, anthropometric and HbA1c measurements were recorded. Weight was measured in kilogrammes using the Seca® scale and height in metres using a Seca® wall-mounted stadiometer, according to departmental standard operating procedures. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured in mmHg using an automated oscillometric device (Omron®), after participants had been seated quietly for 5 minutes in accordance with the 2013 European Society of Hypertension/Cardiology guidelines.¹⁷

Sample collection

Eligible participants were required to undergo phlebotomy at the first antenatal visit and again in trimesters 2 and 3 of pregnancy. The participants were not required to fast or make any special preparation before having blood collected at each study timepoint. Venous whole blood was collected into appropriate specimen tubes, one 7 mL (Greiner Vacuette®) plain plastic for biochemical tests and one 3 mL (Greiner Vacuette®) potassium ethylenediaminetetraacetic acid (K3EDTA) tube for measurement of haematological parameters. Samples for biochemical tests were maintained at room temperature (RT) for 30 min to allow clot formation, and all specimens were transported to the laboratory within 1 h of blood draw. On receipt, samples were centrifuged at 2000g for 15 min at RT, serum separated, aliquoted and stored at −80°C pending batch analyses. EDTA blood samples were analysed for haematological (full blood count (FBC)) parameters within 3h of phlebotomy.

Parameters evaluated and analytical methods

All biochemical and haematological parameters were measured by standard laboratory methods and in accordance with the respective manufacturer’s instructions for use.

Biochemical testing included: renal profile (sodium (Na+), potassium (K+), chloride (Cl-), urea and creatinine); liver/pancreas profile (total protein (T. Prot), albumin, bilirubin (T. Bili), aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), sex hormone binding globulin (SHBG), bile acids (BA) and amylase; bone profile (total calcium (T.Ca²⁺), adjusted (Adj) Ca²⁺ (Adj. Ca²⁺ (mmol/L) = total Ca²⁺ (mmol/L) – (0.0144 * albumin (g/L)) + 0.64 (formula derived/validated locally in a non-pregnant adult population and accredited to ISO 15189:2012), magnesium (Mg²⁺), inorganic phosphate (iPO4) and intact parathyroid hormone (iPTH)); cardiac profile (C-reactive protein (CRP), creatine kinase (CK), high sensitivity troponin T (hsTnT) and uric acid (UA); iron profile (iron and transferrin); thyroid function profile (free thyroxine (FT4) and thyroid-stimulating hormone (TSH)); and lipid profile (total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and non-HDL cholesterol (non-HDL). LDL-C was derived using the Friedewald equation¹⁸ and non-HDL was determined by subtracting HDL-C from TC. Prior to biochemical analyses, samples were removed from the −80°C freezer and allowed to thaw. Once thawed, the samples were thoroughly mixed, centrifuged at 3000g for 10 min at RT and analysed using the Roche Cobas® 8000 modular analyser series (Roche Diagnostics Limited, West Sussex, UK). Serum indices that monitor the instrument’s response to detect and flag pre-analytical interferences due to haemolysis, lipaemia and icterus (HIL indices) were performed on all samples.

The haematology full blood count (FBC) profile which includes: white cell count (WCC), red cell count (RBC), haemoglobin (Hb), haematocrit (Hct), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin content (MCHC), platelet, neutrophils, lymphocytes, monocytes, eosinophils and basophils counts were measured using Sysmex XN-9100™ platforms.

Statistical analyses

Statistical analyses were carried out using MedCalc® Statistical Software (Version 18.2.1) and Analyse-it® (Version 17). Continuous parametric data were represented as mean (±standard deviation (SD)) and non-Gaussian data, as median (interquartile range (IQR)). Categorical data are summarised with frequencies (in percentage). For non-parametric data, the Mann–Whitney U test and the Kruskal–Wallis multiple comparison test with Dunn’s post hoc multiple comparison test were used. A p-value of < 0.05 was deemed statistically significant.

The RIs were defined according to the Clinical Laboratory Standards Institute (CLSI)/International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) non-parametric method (EP28-A3c).¹¹ This requires a minimum of 120 individual reference values to establish the central 95% distribution of results and the 90% confidence interval (CI) of the lower reference limit (LRL) and upper reference limit (URL), respectively. Where reference values were less in number than those required by the non-parametric method, the Robust method¹⁹ was employed.

Reference values within each assays’ reportable range were used to establish the RIs. If results were below an assay’s limit of detection (LoD), for statistical purposes, the LoD was assigned. Firstly, data was illustrated (histogram and box and whiskers plot) together with the Shapiro–Wilk normality test to assess normality using Analyse-it® (Version 17). The frequency distribution of the individual analytes in each trimester of pregnancy was examined visually for results that were out-with the majority of reference values, that is, potential outliers. All apparent outliers were then statistically evaluated based on the Dixon²⁰ and Reed,²¹ criteria, an approach supported by the IFCC working group.¹⁰ Data was subdivided according to trimester and the appropriateness of partitioning evaluated using the Harris and Boyd’s standard deviate test.²²

Results

In total, 1522 pregnant women with singleton pregnancies were recruited to the original study, 117 withdrew or failed to complete the oral glucose tolerance test (OGTT). Of the remaining 1405 eligible participants, 279 comprised the reference population, 124 of whom had bio-banked serum samples in each trimester (Figure 1). At the booking visit, 49.2% (n = 61) of participants were nulliparous, with a median age of 34.4 (IQR: 31.3–37.3) years, gestational age of 89 (IQR: 84–93) days, BMI of 22.5 (IQR: 21.0–23.7) kg/m² and SBP and DBP of 116 (110–125) mmHg and 67 (61–75) mmHg, respectively (Table 1).

Figure 1.

Recruitment schematic.

Table 1.

Baseline characteristics of the reference population.

Variable	Reference population (n = 124)
†Age (years)	34.4 (31.3–37.3)
†GA (days)	89 (84–93)
†Gravida	2 (1–3)
†Parity	1 (0–1)
Nulliparous (%)	49.2
†Weeks of gestation at delivery	40 (39–40.8)
†BMI (kg/m²)	22.5 (21.0–23.7)
†Height (m)	1.66 (1.63–1.71)
†Weight (kg)	61.2 (56.7–65.8)
†SBP (mmHg)	116 (110–125)
†DBP (mmHg)	67 (61–75)
†HbA1c (mmol/mol)	32 (30–34)

BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure.

kg: kilogramme; m; metre; data presented as †median (interquartile range) as appropriate.

In total, 31 biochemical and 10 haematological ISO15189:2012 accredited routinely requested tests were analysed using the Roche Cobas® and Sysmex XN-9100™ analysers, respectively. Reference intervals estimated at the 2.5^th and 97.5^th percentiles were established using the IFCC non-parametric approved method (Table 2). Included in this table and for comparison purposes are RIs for healthy females previously established by this group.²³ Assay traceability and respective parameter analytical performance characteristics assessed over the period of the study are detailed in the supplemental data, Table 1.

Table 2.

Established trimester-specific-reference intervals for routinely requested biochemical and haematological parameters and comparison to those of the non-pregnant adult.

Established reference intervals (CLSI C28 A3) IFCC non-parametric method: lower (2.5th percentile) and upper (97.5th percentile) limits and (90% confidence intervals)
	Trimester 1		Trimester 2		Trimester 3		All trimesters	Non-pregnant adult		All cohorts p-value
Biochemical analyte serum (units)	Lower limit	Upper limit	Lower limit	Upper limit	Lower limit	Upper limit	p-value	Lower limit	Upper limit	p-value
Renal profile
Sodium (mmol/L)	134 (131–135)	140 (139–140)	135 (134.5–135.2)	114 (142.8–144.7)	133 (132–133.8)	143 (141.6–143.3)	<0.0001	137 (134–138)	143 (143–145)	<0.0001
Potassium (mmol/L)	3.7 (3.7–3.9)	5.2 (5.1–5.4)	4.0 (4.0–4.1)	5.4 (5.3–5.6)	3.8 (3.6–3.9)	5.2 (5.0–5.4)	<0.0001	3.7 (3.6–3.8)	5.0 (4.8–5.0)	<0.0001
Chloride (mmol/L)	98 (97–99)	105 (104–105)	100 (100–101)	109 (109–110)	99 (98–100)	107 (106–109)	<0.0001	97 (94–97)	105 (104–105)	<0.0001
Urea (mmol/L)	2.0 (1.8–2.2)	5.1 (4.9–5.5)	1.9 (1.8–2.0)	4.4 (4.4–4.7)	1.7 (1.4–1.9)	4.6 (4.2–5.1)	0.0303	2.8 (2.5–3.1)	8.1 (7.2–.5)	<0.0001
Creatinine (μmol/L)	34 (32–37)	64 (60–74)	39 (29–41)	65 (63–80)	35 (34–39)	70 (64–81)	0.0021	45 (41–49)	100 (95–100)	<0.0001
Liver/pancreas profile
T.Protein (q/L)	60 (58–63)	77 (75–78)	57 (53–58)	71 (69–74)	56 (53–57)	68 (68–69)	<0.0001	64 (61–66)		<0.0001
Albumin (q/L)	37 (32–40)	49 (48–50)	36 (31–36)	44 (43–47)	33 (33–35)	42 (41–43)	<0.0001	42 (38–43)	80 (78–87)	<0.0001
T.Bili (μmol/L)	<3 (<3–3)	16 (13–19)	<3 (<3–<3)	11 (6–13)	<3 (<3–<3)	10 (7–13)	<0.0001	3.3 (3.0–4.0)	20 (15.0–23.0)	<0.0001
AST (U/L)	5 (5–6)	19 (17–26)	4 (3–5)	19 (13–22)	6 (3–6)	23 (22–29)	<0.0001	0 (−4–0)	34 (30–35)	<0.0001
ALT (U/L)	6 (6–7)	25 (22–35)	1 (1–3)	16 (13–22)	1 (0.5–2.0)	15 (13–18)	<0.0001	0 (−5–3.5)	35 (30–35)	<0.0001
YGT (U/L)	4 (3–5)	31 (18–43)	4 (2–4)	17 (15–21)	3 (2–3)	22 (14–34)	<0.0001	0 (−11–0)	36 (30–42)	<0.0001
ALP (U/L)	30 (22–34)	89 (70–98)	29 (24–35)	102 (72–104)	55 (47–60)	286 (231–306)	<0.0001	28 (23–33)	88 (82–94)	<0.0001
LDH (U/L)	43 (38–50)	210 (189–229)	30 (10–43)	168 (159–183)	41 (9–44)	183 (175–192)	<0.0001	135	214	<0.0001
SHBG (nmol/L)	187 (142–200)	499 (474–569)	278 (188–309)	696 (634–726)	344 (307–379)	766 (670–857)	<0.0001	32	128	<0.0001
Bile acids (μmol/L)	0.7 (0.6–1.0)	8 (7–10)	0.3 (0.2–0.8)	6.0 (5.9)	2 (1–9)	14 (11–170	<0.0001	0	10	ND
Amylase (U/L)	28 (20–38)	120 (101–150)	3.4 (0.2–0.8)	127 (104–136)	32 (19–39)	133 (102–165)	0.1889	28	100	0.3257
Bone profile
T.Calcium (mmol/L)	2.18 (2.12–2.22)	2.53 (2.48–2.58)	2.03 (1.91–2.06)	2.39 (2.35–2.46)	2.08 (2.06–2.11)	2.44 (2.39–2.54)	<0.0001			<0.0001
Adj.Calcium (mmol/L)		2.49 (2.45–2.54)	2.11 (2.09–2.15)	2.43 (2.38–2.48)	2.19 (2.18–2.11)	2.52 (2.47–2.57)	<0.0001	2.16 (2.16–2.22.55)		<0.0001
Magnesium (mmol/L)		0.94 (0.92–0.97)	0.69 (0.68–0.71)	0.90 (0.87–0.93)	0.69 (0.60–0.71)	0.89 (0.86–0.92)	<0.0001	0.66	1.07	<0.0001
Phosphate (mmol/L)	0.94 (0.76)	1.56 (1.49–1.58)	0.83 (0.81–0.95)	1.43 (1.37–1.52)	0.88 (0.71–0.95)	1.45 (1.40–1.58)	<0.0001	0.8 (0.7–0.8)	1.4 (1.4–1.5)	<0.0001
Parathyroid hormone (ng/L)	6 (6–8)	28 (24–35)	5 (3–8)	35 (29–43)	4 (1–7)	33 (29–36)	0.0451	12 (8.1–16.0)	62 (49.0–64.6)	<0.0001
Cardiac profile
C-reactive protein (mg/L)	0.2 (0.2–0.3)	21 (11–43)	0.3 (0.1–0.5)	12 (8–19)	0.3 (0.2–0.4)	11 (7–26)	0.2089	0.6 (0.6–0.6)	7.2 (4.3–8.9)	<0.0001
Creatine kinase (U/L)	11 (6–15)	96 (76–110)	7 (4–11)	87 (74–155)	12 (0–16)	155 (112–204)	0.0001	20	180	<0.0001
Hs troponin T (ng/L)	3 (3–3)	2 (3–6)	3 (3–3)	4 (3–4)	3 (3–3)	5 (4–6)	<0.0001	3 (3–3)	8 (8–10)	<0.0001
Uric acid (mmol/L)	82 (75–990)	227 (211–249)	113 (73–142)	284 (246–43)	135 (106–158)	344 (309–357)	<0.0001	150 (1.5–2.1)	3.7 (3.2–3.9)	<0.0001
Iron profile
Iron (μmol/L)	9 (7–10)	33 (30 = 36)	7 (7–8)	36 (26–41)	6 (5–7)	45 (37–65)	<0.0001	8 (6–10)	28 (26–30)	<0.0001
Transferrin (mg/L)	2.1 (12.0–2.2)	3.9 (3.5–4.1)	2.5 (2.4–2.6)	4.6 (4.1–4.7)	3.0 (2.8–3.1)	5.1 (4.8–5.7)	<0.0001	2.0 (1.5–2.1)	3.7 (3.2–3.9)	<0.0001
Thyroid function tests
FT4 (pmol/L)	11.7 (10.6-)	20.0 (19.1–24.1)	10.3 (9.1–10.9)	16.8 (16.4–17.2)	9.4 (9.0–9.9)	16.8 (15.4–18.0)	<0.0001	12.4 (11.3–1322.9)	(20.8–24.0)	<0.0001
TSH (mIU/L)	0.16 (0.11-	3.38 (2.59–3.85)	0.42 (0.23–3.85)	3.94 (3.62–4.59)	0.46 (0.25–0.69)	3.28 (2.91–3.89)	<0.0001	0.60 (0.28–0.74)	68 (4.19–5.73)	<0.0001
Lipid profile
T.C (mmol/L)	3.0 (2.4–3.1)	6.0 (5.8–6.0)	3.8 (3.3–4.6)	8.1 (7.8–9.6)	4.7 (3.8–5.2)	9.6–10.5	<0.0001	3.0 (2.9–3.3)	6.0 (5.8–6.3)	<0.0001
TG (mmol/L)	0.5 (0.5–0.6)	2.6 (1.8–2.9)	0.9 (0.8–0.9)	2.9 (2.7–3.1)	1.3 (1.4–1.5)	4.6 (3.9–6.1)	<0.0001	0.4 (0.4–0.5)	2.8 (2.3–4.7)	<0.0001
HDL-C (mmol/L)	1.06 (0.93-)	2.36 (2.16–2.61)	1.07 (1.3–2.2)	2.70 (2.45–3.18)	1.04 (0.76–1.20)	2.81 (2.52–2.90)	0.0023	1.0 (0.73–1.10)	2.8 (2.3–3.1)	0.0024
LDL-C (mmol/L)*	1.2 (0.9–1.4)	3.7 (3.5–4.1)	1.7 (1.3–2.2)	5.5 (5.1–6.8)	2.1 (1.7–2.6)	6.3 (5.9–7.2)	<0.0001	1.2 (1.1–1.14)	3.8 (3.5–4.0)	<0.0001
Non-HDL (mmol/L)*	1.5 (1.1–1.7)	4.4 (4.1–4.8)	2.0 (1.7–2.8)	6.5 (6.1–7.8)	3.1 (2.4–3.8)	8.4 (7.3–8.6)	<0.0001	1.5 (1.4–1.6)	4.4 (4.3–4.7)	<0.0001

Haematological parameters	Trimester 1		Trimester 2		Trimester 3		All trimesters	Non-pregnant adult		All cohorts
Plasma (units)	Lower limit	Upper limit	Lower limit	Upper limit	Lower limit	Upper limit	*p-Value	Lower limit	Upper limit	*p-Value
Full blood count profile
WCC (10*9/L)	4.9 (4.2–5.9)	12.3 (11.6–14.7)	6.3 (6.2–6.7)	1.3 (12.5–13.9)	5.9 (5.5–6.3)	14.5 (13.0–15.4)	0.0240	3 (2–3)	10 (9–11)	<0.0001
RBC (10*12/L)	3.6 (3.4–3.8)	4.8 (4.7–4.9)	3.2 (3.0–3.5)	4.4 (4.2–4.4)	3.3 (3.1–3.5)	4.5 (4.3–4.7)	<0.0001	3.8 (3.7–4.0)	5.1 (5.0–5.3)	<0.0001
Haemoglobin (g/dL)	11.0 (10.7–11.6)	14.6 (14.3–14.9)	10.2 (9.9–10.6)	13.0 (12.8–13.1)	10.1 (10.0–10.6)	13.2 (13.1–13.3)	<0.0001		15.5 (15.1–15.9)	<0.0001
Hct (L/L)	0.33 (0.30-	0.42 (0.41–0.43)	0.29 (0.28–0.32)	0.42 (0.40–0.42)	0.31 (0.30–0.32)	0.42 (0.40–0.44)	<0.0001	0.35 (0.34)	0.46 (0.45–0.47)	<0.0001
MCVV (fL)*	82 (78–84)	95 (94–95)	81 (80–86)	98 (98–99)	82 (81–83)	99 (98–100)	<0.0001	81 (79–83)	98 (97–100)	<0.0001
MCH (pg)*	28 (26–29)	33 (32–33)	27 (26–28)	34 (33–34)	27 (27–27)	34 (33–340	0.1238	27 (27–28)	33 (33–34)	0.1136
MCHC (q/dl)*	32 (32–33)	36 (36–37)	30 (29–28)	36 (35–36)	30 (29–30)	36 (35–36)	<0.0001	31.7 (31.4)	35.5 (35.1–35.9)	<0.0001
Platelet count (10*9/L)	177 (159–188)	370 (360–547)	166 (163–176)	386 (324–400)	150 (110–169)	352 (309–412)	0.0018	151 (135-)	358 (338–375)	0.0048
Neutrophils (10*9/L)	3 (2–4)	9 (9–11)	4 (4–5)	11 (10–12)	4 (4–4)	12 (10–13)	0.0027	0.9 (0.5–1.4)	3.0 (2.8–3.3)	<0.0001
Lymphocytes (10*9/L)	1.0 (0.9–1.2)	2.9 (2.6–3.4)	0.8 (0.7–1.1)	2.5 (2.2–2.6)	0.9 (0.8–1.0)	3.2 (2.9–3.6)	0.0764	0.6 (0.4–0.9)	3.0 (2.8–3.3)	0.0271
Monocytes (10*9/L)	0.3 (0.3–0.3)	0.9 (0.8–0.9)	0.3 (0.3–0.4)	0.9 (0.9–1.0)	0.4 (0.3–0.4)	1.3 (1.0–1.4)	<0.0001	0.1 (0.1–0.2)	0.6 (0.6–0.6)	<0.0001
Eosinophil (10*9/L)	0.0 (0.01–0.02)	0.5 (0.4–0.7)	0.0 (0.02–0.04)	0.3 (0.27–0.35)	0.0 (0.0–0.0)	0.5 (0.3–0.7)	0.5905	0.0 (0.0–0.1)	0.4 (0.1–0.5)	0.0017
Basophils (10*9/L)	0.0 (0.0–0.01)	0.1 (0.07–0.11)	0.0 (0.01–0.01)	0.1 (0.06–0.07)	0.0 (0.0–0.0)	0.1 (0.0–0.1)	0.1318	0.0 (0.0–0.0)	0.0 (0.0–0.0)	<0.0001

Of the biochemical parameters assessed, only the comparison of the observed reference intervals for amylase and C-reactive protein between trimesters was non-significant. Comparison with the non-pregnant female RIs found no significant difference between respective trimester-specific-RIs for amylase but the difference observed for C-reactive protein was both statistically (Table 3) and clinically significant (Table 2). A significant decrease was seen in sodium, creatinine, albumin, total protein and PTH values (p < 0.05) across all trimesters, while ALP and SHBG increased in comparison to the non-pregnant RIs. Differences observed for the transaminases, ALT and AST, although statistically significant both across trimesters and in comparison to the non-pregnant RIs, were not clinically significant. TC, TG, LDL-C and non-HDL values remained within the non-pregnant RI in the first trimester of pregnancy (p > 0.05) and increased significantly in the second and third trimesters (p < 0.05). HDL-C trimester-specific-RIs were significantly different (p = 0.0023), with values increasing as pregnancy progressed but remained within the non-pregnant RI. Both FT4 and TSH were decreased in comparison to the non-pregnant RI with statistically significant differences observed in all trimesters for FT4 and in the first trimester for TSH.

Table 3.

Comparison of established trimester-specific-reference intervals to those of the non-pregnant adult.

Apparently healthy European pregnant and non-pregnant adult compared
Biochemical analyte serum (units)	Trimester (T) all	T1 Vs T2	T1 vs T3	T2 Vs T3	Cohorts all	Non-preg vs T1	Non preg vs T2	Non-preg vs T3
Biochemical analyte serum (units)	p-value	^p-value	^p-value	^p-value	^p-value	^p-value	^p-value	^p-value
Renal profile
Sodium (mmol/L)	<0.0001	<0.0001	0.1227	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
Potassium (mmol/L)	<0.0001	<0.0001	0.8786	<0.0001	<0.0001	0.0280	<0.0001	0.0172
Chloride (mmol/L)	<0.0001	<0.0001	<0.0001	0.0001	<0.0001	0.7091	<0.0001	<0.0001
Urea (mmol/L)	0.0303	0.2786	0.2786	0.1043	<0.0001	<0.0001	<0.0001	<0.0001
Creatinine (μmol/L)	0.0021	0.0012	0.0054	0.7234	<0.0001	<0.0001	<0.0001	<0.0001
Liver/pancreas profile
T.Protein (g/L)	<0.0001	<0.0001	<0.0001	0.7004	<0.0001	<0.0001	<0.0001	<0.0001
Albumin (g/L)	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
T.Billi (μmol/L)	<0.0001	<0.0001	<0.0001	0.5999	<0.0001	<0.0001	<0.0001	<0.0001
AST (U/L)	<0.0001	0.0399	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
ALT (U/L)	<0.0001	<0.0001	<0.0001	0.8160	<0.0001	0.0033	<0.0001	<0.0001
YGT (U/L)	<0.0001	<0.0001	<0.0001	0.4978	<0.0001	<0.0001	<0.0001	<0.0001
ALP (U/L)	<0.0001	0.8046	<0.0001	<0.0001	<0.0001	0.0017	0.0058	<0.0001
LDH (U/L)	<0.0001	0.0002	<0.0001	0.6349	<0.0001	<0.0001	<0.0001	<0.0001
SHBG (nmol/L)	<0.0001	<0.0001	<0.0001	0.0003	<0.0001	<0.0001	<0.0001	<0.0001
Bile acids (μmol/L)	<0.0001	0.0001	<0.0001	<0.0001	ND	ND	ND	ND
Amylase (U/L)	0.1889	0.3275	0.0696	0.3935	0.3257	0.3376	0.6951	0.8933
Bone profile
T.Calcium (mmol/L)	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	0.0156	<0.0001	<0.0001
Adj.Calcium (mmol/L)	<0.0001	<0.0001	0.2839	<0.0001	<0.0001	0.0017	<0.0001	0.0586
Magnesium (mmol/L)	<0.0001	<0.0001	<0.0001	0.5491	<0.0001	0.0429	<0.0001	<0.0001
Phosphate (mmol/L)	<0.0001	<0.0001	<0.0001	0.1115	<0.0001	<0.0001	0.3402	0.8092
Parathyroid hormone (ng/L)	0.0451	0.0740	0.0189	0.4552	<0.0001	<0.0001	<0.0001	<0.0001
Cardiac profile
C-reactive protein (mg/L)	0.2089	0.1514	0.9718	0.1031	<0.0001	<0.0001	<0.0001	<0.0001
Creatine kinase (U/L)	0.0001	0.0084	0.0580	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
Hs troponin T (ng/L)	<0.0001	0.1462	0.0001	0.6956	<0.0001	<0.0001	<0.0001	<0.0001
Uric acid (mmol/L)	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
Iron profile
Iron (μmol/L)	<0.0001	0.0002	<0.0001	0.073	<0.0001	0.0608	0.3467	<0.0001
Transferrin (mg/L)	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	0.0003	<0.0001	<0.0001
Thyroid function tests
FT4 (pmol/L)	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	0.0003	<0.0001	<0.0001
TSH (mlU/L)	<0.0001	<0.0001	<0.0001	0.0151	<0.0001	<0.0001	0.4992	0.1106
Lipid profile
T.c. (mmol/L)	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
TIRIG (mmol/L)	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
HDL-c (mmol/L)	0.0023	0.0011	0.0063	0.6956	0.0024	0.6116	0.0111	<0.0001
LDL-C (mmol/L)*	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
Non-HDL (mmol/L)	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
Apparently healthy European pregnant and non-pregnant adult compared
Haematological parameters plasma	Trimester (T) all	T1 Vs T2	T1 vs T3	T2 Vs T3	Cohorts all	Non-preg vs T1	Non preg vs T2	Non-preg vs T3
Haematological parameters plasma	p-value	^p-value	^p-value	^p-value	^p-value	^p-value	^p-value	^p-value
Full blood profile
WCC (10*9/L)	0.02400	0.0100	0.0280	0.3550	<0.0001	<0.0001	<0.0001	<0.0001
RBC (10*12/L)	<0.0001	<0.0001	<0.0001	0.8968	<0.0001	0.0029	<0.0001	<0.0001
Haemoglobin (g/dL)	<0.0001	<0.0001	<0.0001	0.3185	<0.0001	0.0107	<0.0001	<0.0001
Hct (L/L)	<0.0001	<0.0001	<0.0001	0.7806	<0.0001	<0.0001	<0.0001	<0.0001
MCV (fL)*	<0.0001	<0.0001	<0.0001	0.978	<0.0001	0.0308	0.1311	0.1473
MCH (Pg)*	0.1238	0.2456	0.5050	0.5182	<0.0001	0.1136	0.1805	0.0503
MCHC (g/dl)*	<0.0001	<0.0001	<0.0001	0.3938	<0.0001	<0.0001	0.3897	0.8889
Platelet count (10*9/L)	0.0018	0.1805	0.0003	0.1437	0.0271	0.0857	0.6916	0.2399
Neutrophils (10*9/L)	0.0027	0.001	0.0280	0.2020	<0.0001	<0.0001	<0.0001	<0.0001
Lymphocytes (10*9/L)	0.0764	0.0585	0.0675	0.8662	0.0271	0.3382	0.0169	0.0168
Monocytes (10*9/L)	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
Eosinophil (10*9/L)	0.5905	0.6616	0.5026	0.2399	0.0017	0.0005	0.0061	0.0006
Basophils (10*9/L)	0.1318	0.1048	0.0923	0.8379	<0.0001	<0.0001	<0.0001	<0.0001

Of the FBC parameters evaluated, no differences were observed between trimester-specific-RIs for MCH, lymphocytes, eosinophils and basophils. Comparison with those for non-pregnant women determined that there was no statistical difference in MCH RIs for any trimester. While the differences observed for eosinophils and basophils were statistically different and for lymphocytes for trimesters 2 and 3 only, all were clinically insignificant. For all remaining trimester-specific biochemical and haematological RIs, the observed differences were statistically different, as was the overall comparison with the respective RIs established in the non-pregnant cohort. Levels of RBC, Hb and Hct were lower, and levels of WCC, platelets, neutrophils and monocytes were higher across trimesters and in comparison to non-pregnant healthy women. These differences were both clinically and statistically significant.

Discussion

Main findings

Normative trimester-specific-RIs for 41 routine biochemical and haematological tests in white European pregnant women with singleton pregnancies were established.

Strengths and limitations

This study comprised a comprehensive retrospective analysis of a longitudinal study establishing trimester-specific-RIs for routinely requested biochemical and haematological tests in healthy white European women with singleton pregnancies. This study design permitted the identification of pregnant women in good health (the reference population),²⁴ reducing the potential for confounders to influence the established trimester-specific-RIs.

We acknowledge that an increased sample size would likely increase the reliability of results. Restricting the study to white European women may mean the findings are not generalizable to other ethnic groups. We accept that the study cohort of healthy pregnant women precludes comments on the performance of any of the analytes evaluated to detect disease.

The increased use of supplements (B12, folate, iron, etc.) in pregnant women can cause certain analytes to fluctuate greatly. Moreover, high doses of biotin supplements (e.g. ≥5 mg) lead to excess biotin concentrations in the blood, and biotin-streptavidin–based immunoassay tests may be subject to immunoassay interference as a result. We acknowledge that participants were not asked or advised about any supplementation prior to providing blood samples and that this is a potential limitation of the study design. However, no unexpected results were observed in the immunoassays performed in the study cohort and the assumption was made that levels of biotin in samples were within the tolerance of the assays. Furthermore, as normal physiological changes occur throughout pregnancy impacting routine biochemical and haematological tests, it is critical for appropriate result interpretation to acknowledge that the established RI are exclusive to the gestational week of blood sampling (Table 4). For example, the RI defined for trimester 1 is specific for the end of trimester 1 as all participants gave their trimester 1 sample after 8 weeks’ gestation with a median gestational week of 12.7 (Table 4).

Table 4.

Trimester reference intervals for clinical biochemistry and haematology tests during normal pregnancy.

Trimester-specific-reference intervals for clinical biochemistry and haematology tests during normal pregnancy (CLSI C28-A3) IFCC non-parametric method percentiles: 2.5% (median), 97.5% (upper limit)
gestational age (weeks): median (range: minimum–maximum)	Trimester 1 12.7 (8.8–13.8)			Trimester 2 24.2 (15.7–27)			Trimester 3 32.4 (28.2–39.7)
Biochemical analytepercentiles plasma (units)	2.5% lower limit	50% median	97.5% upper limit	2.5% lower limit	50% median	97.5% upper limit	2.5% lower limit	50% median	97.5% upper limit
Renal profile
Sodium (mmol/L)	134	137	140	135	138	144	133	137	143
Potassium (mmol/L)	3.7	4.4	5.2	4.0	4.7	5.4	3.8	4.4	5.2
Chloride (mmol/L)	98	101	105	100	104	109	99	103	107
Urea (mmol/L)	2	3.1	5.1	1.9	3.0	4.4	1.7	2.9	4.6
Creatinine (μmol/L)	34	48	64	39	51	65	35	50	70
Liver/pancreas profile
T.Protein (q/L)	60	69	77	57	63	71	56	63	68
Albumin (g/L)	37	44	49	36	39	44	33	38	42
T.Bili (μmol/L)	<3	5	16	<3	3	11	<3	3	10
AST (U/L)	5	9	19	4	8	19	6	11	23
ALT (U/L)	6	12	25	1	5	16	1	6	15
¥GT (U/L)	4	9	31	4	7	17	3	7	22
ALP (U/L)	30	46	89	29	48	102	55	89	286
LDH (U/L)	43	118	210	30	101	168	41	94	183
SHBG (nmol/L)	187	322	499	278	463	696	344	583	766
Bile acids (μmol/L)	0.7	3	8	0.3	2	6.0	2	4	14
Amylase (U/L)	26	59	120	34	62	127	32	64	133
Bone profile
T.Calcium (mmol/L)	2.18	2.32	2.53	2.03	2.19	2.39	2.06	2.34	2.44
Adj.Calcium (mmol/L)*	2.22	2.35	2.49	2.11	2.27	2.43	2.19	2.33	2.52
Magnesium (mmol/L)	0.70	0.84	0.94	0.69	0.78	0.90	0.69	0.76	0.89
Phosphate (mmol/L)	0.94	1.25	1.56	0.83	1.17	1.43	0.88	1.13	1.45
Parathyroid Hormone (ng/L)	6	15	28	5	16	35	4	17	33
Cardiac profile
C-reactive protein (ng/L)	0.2	2.1	21	0.3	2.8	12	0.3	2.3	211
Creatine kinase (U/L)	11	37	96	7	28	87	12	41	155
Hs troponin T (ng/l)	3	3	3	3	3	4	3	3	5
Uric acid (mmol/L)	82	159	227	113	189	264	135	209	344
Iron profile
Iron (μmol/L)	9	19	33	7	15	36	6	14	45
Transferrin (mg/L)	2.1	2.7	3.9	2.5	3.4	4.6	3.0	3.9	5.7
Thyroid function tests
FT4 (pmol/L)	11.7	16.2	20.0	10.3	13.7	16.8	9.4	12.4	16.8
TSH (mlU/L)	0.16	1.36	3.38	0.42	1.95	3.94	0.46	1.71	3.28
Lipid profile
T.C. (mmol/L)	3.0	4.6	6.0	3.6	6.0	8.1	4.7	6.8	9.8
TG. (mmol/L)	0.5	1.1	2.6	0.9	1.6	2.9	1.3	2.4	4.6
HDL-C (mmol/L)	1.1	1.6	2.4	1.1	1.8	2.7	1.0	1.74	2.81
LDL-C (mmol/L)*	1.2	2.4	3.7	1.7	3.5	5.5	2.1	3.8	6.3
Non-HDL (mmol/L)*	1.5	3.0	4.4	2.0	4.3	6.5	3.1	5.0	8.4
Full blood count profile
WCC (10*9/L)	4.9	8.6	12.3	6.3	9.9	13.6	5.9	9.1	14.5
RBC (10*12/L)	3.6	4.3	4.8	3.2	3.8	4.4	3.3	3.8	4.5
gestational age (weeks): median (range: minimum–maximum)	Trimester 1 12.7 (8.8–13.8)			Trimester 2 24.2 (15.7–27)			Trimester 3 32.4 (28.2–39.7)
Haematological parameterspercentiles plasma (units)	2.5% lower limit	50% median	97.5% upper limit	2.5% lower limit	50% median	97.5% upper limit	2.5% lower limit	50% median	97.5% upper limit
Haemoglobin (g/dL)	11.0	13.1	14.6	10.2	11.7	13	10.1	11.4	13.2
Hct (L/L)	0.33	0.38	0.42	0.29	0.35	0.42	0.31	0.33	0.42
MCV (fL)*	82	89	95	81	91	98	82	91	99
MCH (pg)*	28	30	33	27	31	34	27	31	34
MCHC (g/dL)*	32	34	36	30	33	36	30	34	36
Platelet count (10*9/L)	177	255	370	166	255	386	150	231	352
Neutrophils (10*9/L)	3	6	9	4	7	11	4	7	12
Lymphocytes (10*9/L)	1.0	1.7	2.9	0.8	1.6	2.5	0.9	1.6	3.2
Monocytes (10*9/L)	0.3	0.5	0.9	0.3	0.6	0.9	0.4	0.6	1.3
Eosinophil (10*9/l)	0	0.1	0.5	0	0.1	0.3	0	0.1	0.5
Basophils (10*9/L)	0	0	0.1	0	0	0.1	0	0	0.1

Interpretation

The quality of the RI is critically dependent on the selection of the reference population.¹ Stringent inclusion criteria ensured that the health of the reference population was well-characterized, representative, and closely resembles the population where the tests will be used;¹ while the numbers failing to meet the inclusion criteria are significant, a similar study recruited 801 healthy Caucasian pregnant women amongst 2147 attending first trimester screening with 391 participants comprising the reference population.⁴

Few biochemical analytes measured remained unchanged during pregnancy (Table 2). The reference values for sodium were lower than those for non-pregnant adults’ findings supported by others^4,8,10 and consequent to the increased plasma volume (30%–50%), extracellular volume and cardiac output.²⁵ Potassium values were slightly increased, with 97.5^th percentile values in each trimester > 5.0 mmol/L and in agreement with Larsson et al.⁸ In contrast, Klajnbard et al. reported lower potassium levels in all trimesters, while Friis Petersen et al. reported lower 2.5^th percentile values in the first trimester than in non-pregnant adults.^4,10 This may be related to pre-analytical issues, for example, the use of lithium heparin as compared to serum in the current study.

The observed lower RIs for creatinine concur with the well-recognized increase in glomerular filtration rate (GFR), approximately 50% in early pregnancy.²⁶ It is readily apparent that the use of non-pregnant RIs could potentially result in a missed diagnosis of pregnancy associated acute kidney injury and suboptimal patient care and mismanagement.

The haemodilution effect which occurs because of increased extracellular volume results in a decrease in protein concentrations in each trimester, findings that are consistent with others.^4,8 ALT and AST values remained stable across all trimesters and correlate with previous studies.^4,27,28 However, others observed lower levels of ALT and AST during pregnancy⁸ which may relate to assays that do not include pyridoxal phosphate.⁴ Other liver profile analytes (Table 2) increased with each trimester.

Intrahepatic cholestasis of pregnancy (ICP) and haemolysis, elevated liver enzymes and low platelets (HELLP) syndrome are fatal life-threatening conditions that can occur during pregnancy.⁶ ICP, characterized by pruritus and elevated BA, is associated with an increased risk of adverse pregnancy outcomes. In our study, total BA levels ranged from 0.3 to 14 μmol/L and rose minimally in the third trimester. A similar study performed in a predominantly white European population (94%) gave comparable results.²⁹

Preeclampsia and eclampsia are hypertensive disorders of pregnancy which cause significant amounts of cellular damage. While pregnancy itself does not affect LDH levels, an increase in LDH reflects the occurrence of pre-eclamptic complications and the severity of pre-eclamptic disease.^6,30 Our study shows lower levels of LDH in pregnant women than in non-pregnant women. Friis Petersen et al. reported decreasing levels of LDH with increasing weeks’ gestation in the first trimester, illustrating how gestational changes can already be seen in early pregnancy.¹⁰

Calcium homeostasis is altered during pregnancy to ensure the growing fetus receives sufficient minerals for bone mineralization.³¹ Calcium in serum is either present in an ionised (or free) state or bound to proteins, principally albumin. Commonly, calcium is measured as total calcium (i.e. the sum of protein-bound, complexed and free (ionised) calcium) in plasma or serum. Consequently, variations in albumin concentration will alter total calcium levels independently of the physiologically active ionised (free) calcium concentration. In patients with hypoalbuminaemia, formulae are used to adjust patients’ total calcium result to that expected had the albumin concentration been normal. Albumin levels are lower in healthy pregnant women and the use of these formulae is essential to prevent result misinterpretation. Our results show a slight decrease in calcium and magnesium, while phosphate levels decreased after the first trimester; these findings are in accord with Larsson et al.⁸

PTH RIs were significantly lower in each trimester compared to the non-pregnant adult interval. A lack of awareness of these changes in pregnancy may mask primary hyperparathyroidism or incorrectly diagnosis hypoparathyroidism. While previous studies have reported lower PTH levels^31–33 during pregnancy which is consistent with our findings, others have reported unchanged levels in comparison to the non-pregnant state.³⁴

Sepsis has been reported as the third most common cause of maternal mortality worldwide.³⁵ Pregnancies complicated by sepsis are associated with increased risk of adverse outcomes.³⁶ CRP is widely used as a marker of inflammation in the diagnosis of sepsis.³⁷ In our study, CRP concentrations increased throughout pregnancy which is broadly in accord with a recent study reporting a range of 0.5–34 mg/L in a healthy pregnant cohort.³⁸

Cardiac troponin tests are essential for diagnosing myocardial injury; however few studies have investigated higher-sensitivity assays in pregnancy. In this study, hs-TnT values remained unchanged across the trimesters of pregnancy and were not clinically different from the non-pregnant RI established in healthy women.³⁹ hs-TnT has rarely been compared in pregnant and non-pregnant women. However, Adamcova et al. reported normal hs-TnT levels during the third trimester of pregnancy although the demographics of the reference population and 99th percentile URL are unknown.⁴⁰

RIs for TC, TG, LDL-C and non-HDL were significantly and clinically different from the non-pregnant RIs. The changes observed are dramatic; TC, TG and LDL-C concentrations increase throughout normal pregnancy due to reduced activity of lipoprotein lipase, insulin resistance and oestrogen stimulation.^6,41 Lipid results in pregnancy are routinely assessed using RIs specific to normal non-pregnant adults. During pregnancy, normal lipid results may be considered as highly abnormal if interpretated using these inappropriate RIs.

As a result, the potential risk for misdiagnosis (e.g. familial hypercholesterolaemia) or mismanagement is increased. Moreso, evolving evidence suggests that lipids can be used for identifying maternal and foetal complications, as well as individuals at risk of developing GDM, highlighting the importance of providing RI specific to pregnancy.^42–45 Women with underlying hypertriglyceridaemia are at risk of a severe rise in triglycerides during pregnancy and potentially pancreatitis.⁴⁶

Thyroid disease is common in pregnancy and when untreated can lead to a plethora of adverse outcomes. In 2014, the European Thyroid Association recommended the use of trimester-specific-RIs to avoid misclassification of thyroid dysfunction in pregnant women.⁴⁷ Our results show a decrease in both FT4 and TSH across all three trimesters in comparison to the non-pregnant RI, findings which are in accord with numerous studies reported on this topic.⁴⁸

The haematological trimester-specific-RIs for RBC, WCC, Hb, Hct, platelet count and neutrophils were strongly in agreement with those reported by Markus et al.⁴⁹ The decrease in Hb which is often referred to as ‘physiologic anaemia’ of pregnancy and decrease in Hct are common phenomena observed in pregnant women and have been previously reported.^4,50 The elevation of WCC and neutrophils in all trimesters is consistent with Dockree et al.⁵¹ The WCC is frequently measured when there is suspicion of infection; thus, it is imperative that WCC results are interpreted in the context of normal pregnancy. Furthermore, it is critical to appreciate the influence of ethnicity on haematological parameters. Compared with whites, Africans show significantly lower levels of Hb, Hct, WBC, neutrophils and platelet counts.^52,53 This highlights the importance of knowing the characteristics of the reference population from which the RIs are established to ensure accurate diagnosis and patient management.

Conclusion

Education of clinicians and healthcare professionals to the physiological changes that occur in normal pregnancy together with the provision of appropriate trimester-specific-RIs on laboratory reports is essential to mitigate the clinical risk to pregnant women of missed diagnoses, misdiagnoses or mismanagement. Normative biological intervals defined in a healthy pregnant white European population for routinely requested laboratory parameters will be invaluable to result interpretation and management.

Supplemental Material

Supplemental Material - Reference intervals for clinical biochemistry and haematology tests during normal pregnancy

Supplemental Material for Reference intervals for clinical biochemistry and haematology tests during normal pregnancy by Wendy Groenendijk, Delia Bogdanet, Louise Dervan, Oisin Finn, Md Nahidul Islam, Helen Doheny, Tomas P Griffin, Liam Blake, Mark Lyons, Mary Kilcooley, Janusz Krawczyk, Ruth Gilmore, Damian G Griffin, Geraldine Gaffney, Fidelma P Dunne and Paula M O’Shea in Annals of Clinical Biochemistry

Footnotes

Author Contributions

PMOS: study concept and design, responsibility for laboratory analysis, data integrity, statistical analysis and interpretation. PMOS/DB/DGG/RG/JK/GC/FD: study conduct and protocol development. WG/DB/LD: patient recruitment, clinical data acquisition and collation. WG/LD/OF/MNI/HD/TPG/LB: laboratory analysis. HD/ML/MK: laboratory analysis, laboratory data integrity, acquisition and collation. WG and PMOS: first draft of this manuscript. All authors critically reviewed, edited and approved the final version of the manuscript.

Acknowledgements

We wish to express our gratitude to all volunteers who made this study possible. Special thanks to the scientific, nursing and medical staff at the Centre for Endocrinology, Diabetes and Metabolism and the Departments of Obstetrics and Gynaecology, Clinical Biochemistry and Haematology at Saolta University Health Care Group (SUHCG), University Hospital Galway, Ireland.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical approval

Ethical approval for the study was granted by the research ethics committee at Galway University Hospitals (GUH) (Ref: GUH: CA 2026 on 10.10. 2018).

Guarantor

PMOS.

Contributorship

ORCID iDs

Wendy Groenendijk

Delia Bogdanet

Tomás P. Griffin

Fidelma P. Dunne

Paula M. O’Shea

Supplemental material

Supplemental material for this article is available online.

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