Establishment of Reference Intervals of Complete Blood Count and Coagulation Tests in Pregnant Women at Hung Vuong Hospital
Conditions: Pregnancy Related
Sponsor: Hung Vuong Hospital

Study Protocol ID: CS/HV/23/13 
Page 1 of 4 
 
Cover page for Clinical Trials Document 
Study title 
Establishment of Reference Intervals of Complete Blood Count and 
Coagulation Tests in Pregnant Women at Hung Vuong Hospital 
NCT Number 
NCT05929326 
Document date 
August 27, 2023 
 
 
 
 

Study Protocol ID: CS/HV/23/13 
Page 2 of 4 
 
I. 
STUDY PROTOCOL 
Reference intervals are defined as the 2.5th percentile to 97.5th 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, 3], but none was conducted in Vietnam. Therefore, 
we conduct this study to determine trimester specific reference intervals for healthy pregnant 
women. 
Patient selection 
From June 2023 to August 2023, pregnant women visiting obstetrics clinics at Hung 
Vuong Hospital are selected if eligible after history taking and clinical examination. Inclusion 
criteria was singleton pregnancy. Exclusion criteria were hypertension, diabetes, pre-
eclampsia, gestational diabetes, hemoglobinopathy, current infection, positive screening for 
treponema pallidum, hepatitis B virus, HIV, usage of anticoagulant drug. Each patient will 
have their blood drawn 4 millimeters (mL), 2 mL into citrate anticoagulated tube to test for 
APTT, PT, fibrinogen, and 2 mL into Ethylenediaminetetraacetic acid (EDTA) tube to test for 
complete blood count. 
To establish reference intervals using non-parametric method, the minimum sample size 
needed is 120, according to CLSI EP28-A3c [4], because of the 90% confidence interval 
reported for the 2.5th percentile. However, Sample Sizes for Clinical, Laboratory and 
Epidemiology Studies [5] suggested a calculation for larger minimum required sample size 
𝑁𝑅𝑎𝑛𝑘= 𝜂√3 × [
𝑍1−𝛾/2
𝜌𝑝𝑙𝑎𝑛×𝑍1−𝛾/2]
2
where 𝜂=
√(𝛾/2)[1−(𝛾/2)]
𝜙1−𝛾/2
 = 2.11  (𝜙1−𝛾/2 = 
1
√2𝜋𝑒
𝑍1−𝛾/2
2
2
) 
with α = 0.05, γ = 0.1.  ρplan is margin of error, which is the estimate of the percentage that the 
width of the confidence interval of the reference limits is of the width of the reference 
interval. The recommended ρplan is 10%. From the formula above, the calculated sample size 
required is 258 patients. Because we want to establish trimester specific reference intervals, 
the total combined number of each trimester is at least 774 patients.  
Ethical approval 
The study was approved by the Ethical committee of Hung Vuong Hospital, Vietnam. 
Written consent was obtained before study participation. 
Samples collection and instrument 
Venous blood samples were collected in trisodium citrate 3.2% and K2EDTA plastic 
whole blood tube and then immediately analyzed.  
Instrument and analysis 
Coagulation assays, which comprises of Activated partial thromboplastin time 
(APTT), prothrombin time (PT), fibrinogen (FIB), was performed on ACL TOP 550 
automated coagulation analyzer (Werfen, Barcelona, Spain) and complete blood count, 
including White blood cell (WBC) counts and percentage and absolute count of different 

Study Protocol ID: CS/HV/23/13 
Page 3 of 4 
 
leukocytes (neutrophils, lymphocytes, monocytes, eosinophils and basophils), red blood cells 
(RBC), hemoglobin (Hgb), hematocrit (HCT), mean corpuscular volume (MCV), mean 
corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), RBC 
distribution width (RDW), platelet counts (PLT), mean platelet volume (MPV), was 
performed on DxH900 automated hematology analyzer (Beckman Coulter, CA, USA).  
All instruments were maintained and calibrated according to the manufacturer’s 
instruction. Two levels of controls were done on ACL TOP 550 analyzer every eight-hour 
shift and three levels of controls were run DxH900 once a day. 
II. 
STATISTICAL ANALYSIS PLAN 
 
Patients are divided into three groups, based on their gestational age. The three groups 
are the first, second and third trimester. The non-parametric method, recommended to 
establish reference intervals by CLSI EP28-A3c, is used to determine the lower reference 
limit 2.5th percentile and upper reference limit 97.5th percentile for each group. Each 
reference limit was reported along with its 90th confidence interval. Groups are then 
compared using Mann-Whitney U and Kruskal-Wallis tests. A two-tailed P-value < 0.05 was 
considered significant. 
 
 
 

Study Protocol ID: CS/HV/23/13 
Page 4 of 4 
 
REFERENCES 
1. 
Torgersen CKL and Curran CA. A systematic approach to the physiologic 
adaptations of pregnancy. Critical care nursing quarterly 2006;29:2-19. 
2. 
Cui C, Yang S, Zhang J, Wang G, Huang S, Li A, et al. Trimester-specific 
coagulation and anticoagulation reference intervals for healthy pregnancy. 
Thrombosis research 2017;156:82-6. 
3. 
Gong JM, Shen Y, He YX. Reference intervals of routine coagulation assays 
during the pregnancy and puerperium period. Journal of clinical laboratory 
analysis 2016;30:912-7. 
4. 
CLSI. Defining, Establishing, and Verifying Reference Intervals in the Clinical 
Laboratory; Approved Guideline—Third Edition. CLSI document EP28-A3c. 
In: Wayne, PA: Clinical and Laboratory Standards Institute, 2008. 
5. 
Reference Intervals and Receiver Operating Curves. In: Sample Sizes for 
Clinical, Laboratory and Epidemiology Studies, 2018:339-59