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Evaluation of vitamin B12 levels and Hypersegmented Neutrophils in pregnant women attending Rivers State University Teaching Hospital, Port Harcourt

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Ibiere Allwell Pepple, Catherine Omo Osin and Serekara Gideon Christian*

Department of Medical Laboratory Science, Rivers State University, Nkpolu-Oroworukwo, Port Harcourt, Nigeria All Correspondences to: serekara.christian1@ust.edu.ng

ABSTRACT

This study was aimed at evaluating levels of vitamin B12 and the presence of hypersegmented neutrophils in pregnant women attending Rivers State University Teaching Hospital. It is a comparative and case control study designed to evaluate the levels of vitamin B12 and presence of hypersegmented neutrophils in pregnant women. The study comprises of apparently healthy women in three groups of twenty-five females each (pregnant women who have not had miscarriage, pregnant women with previous miscarriage, and control subjects who have never been pregnant), aged between 30 to 35 years. The study was carried out from July through August 2019. Vitamin B12 levels were determined using ELISA method. Thin films were made and stained using Leishman stain to identify presence of hypersegmented neutrophils. Data was analyzed using Graph Pad prism 8.0.2 statistical package; p<0.05 was considered statistically significant. The results showed significant increase of vitamin B12 in women with miscarriages (193.78±110.63μg/day), when compared to that of women without miscarriages (174.80±53.14 μg/day), and that of non- pregnant women (98.03±9.50 μg/day). Hypersegmented neutrophils were found to be high among women with miscarriages (16%) when compared to those without miscarriage (4%), and control (0%). The miscarriages recorded among the women could be as a result of the presence of hypersegmented neutrophil which is indicative of the tendencies towards miscarriages. However, the high significant level of vitamin B12 in pregnant women with miscarriages may be due to their sea food diet despite the presence of hypersegmented neu trophils; which requ ires fu rtherinvestig a tion.

Keywords: Vitamin B12, Hypersegmented Neutrophils, Pregnancy, Miscarriage
INTRODUCTION

Pregnancy is the period from conception to birth. Pregnancy begins with the fertilization of an ovum (egg) and its implantation. The egg develops into the placenta and the embryo grows to form the foetus. Most eggs implant into the uterus upon fertilization by sperm cell. A normal pregnancy last around 40 weeks from the first day of the woman’s last menstrual period. Normal pregnancy consists of three trimesters of 3 months each (1). Miscarriage (biochemical pregnancy loss) is the pregnancy loss, which occurs after a positive urinary or serum human chorionic gonadotropin (hCG), but before ultrasound or histological detection of pregnancy (<6 weeks) (2). It can also be said to be the loss of the fetus before the 24th week of pregnancy or viability (the ability of the fetus to survive outside the uterus without artificial support). Majority of miscarriages occurs in the first trimester and may be mistaken sometimes for a late menstrual flow (1).

Vitamin B12 (cobalamin or cyanocobalamin) is a water- soluble vitamin that plays a vital role in the activities of several enzymes in the body. It is important in the production of red blood cells in the bone marrow and in the utilization of folic acid and carbohydrate in the diet and the functioning of the nervous system (1). Vitamin B12 sources for humans is food of animal origin. The highest amounts are found in liver and kidney (up to 100μg per 100 g), but it is also present in shellfish, organ and muscle meats, fish, chicken and dairy products (eggs, cheese and milk) in small amounts (6μg/L). Vegetables, fruits and all other foods of non-animal origin are free from cobalamin unless they are contaminated by bacteria. Cooking does not usually destroy cobalamin(3). Vitamin B12 maintains normal folate metabolism which is essential for cell multiplication during pregnancy.

Neutrophil hypersegmentation can be defined as the presence of neutrophils whose nuclei have six or more lobes or the presence of more than 3% of neutrophils with at least five nuclear lobes( 5 ). The presence of hypersegmented neutrophils is suggestive of cobalamin or folate deficiency( 6 ) . So pregnant women with hypersegmented neutrophils correlates with the low amount of vitamin B12 which is detrimental to red blood cell formation and a likely predisposition to neural tube defect and also pregnancy loss (miscarriage). The presence of hypersegmented neutrophils in pregnant women is also an indication of megaloblastic anaemia (5), which puts the pregnancy at risk of a miscarriage. According to Tavasoli et al., the presence of hypersegmented neutrophils indicates low levels of vitamin B12 which results in pregnancy loss due hypercoagulable states and bleeding (7).

In Sub-Saharan Africa, Nigeria inclusive, iron and folate deficiencies were reported to be the most common causes of anaemia in pregnant women (8) Lack and/or insufficient levels of iron supplementation was also reported to be among the most significant risk factors for anaemia to occur during pregnancy(9,10). An anaemic pregnant woman by implication of her condition lacks enough red blood cells for normal metabolic activities and that of the foetus. The resultant effect is that the foetus is deprived of adequate supply of the necessary nutrients required for foetal development.

There is paucity of scientific research information on the levels of vitamin B12 and presence of hypersegmented neutrophils in pregnant women in Rivers State, Nigeria, even though so much effort has been made to improve on the amount of vitamin B12 taken by pregnant women during their routine ante-natal clinic visits. This study was therefore undertaken to ascertain if levels of vitamin B12 and hypersegmented neutrophils could cause pregnancy loss or miscarriage in pregnant women. Hence the need to give this claim a scientific backing.

The aim of the study was to estimate the levels of vitamin B12 and the presence of hypersegmented neutrophils in pregnant women attending River State University Teaching Hospital. The specific objectives of the study are: To estimate the levels of vitamin B12 among pregnant women attending River State University Teaching Hospital; To estimate the number of hypersegmented neutrophils in women attending River State University Teaching Hospital; To ascertain the relationship in values of vitamin B12 estimation with the presence of hypersegmented neutrophils in pregnant women and non- pregnant women.

MATERIALS AND METHODS

Study Design

This is a comparative and case control study which is designed to assess and evaluate the levels of vitamin B12 and the presence of hypersegmented neutrophils in pregnant women attending River State University Teaching Hospital. This study was carried out from July through August 2019.

Study Area

This study was carried out in Rivers State University Teaching Hospital. The hospital is located in Port Harcourt, Rivers State, Nigeria. Port Harcourt is located on GPS coordinates of 4° 49′ 27.0012” N and 7° 2′ 0.9996” E. Rivers State University Teaching Hospital formally Braithwaite Memorial Specialist Hospital (BMSH) is a government-owned hospital, which was named after Eldred Curwen Braithwaite, a British doctor and a pioneer of surgery. It is located in Old GRA, Rivers State. It was established in March 1925.

Study Population

The subjects in this study comprised of apparently healthy pregnant women. Blood samples were drawn from seventy-five (75) women into ethylene diamine tetra acetic acid (EDTA) containers. Their age bracket was 30 to 35 years. They comprised of three (3) groups of 25 subjects each: pregnant women who have not had miscarriage(s), pregnant women that have had previous miscarriage(s) and control subjects who are the non – pregnant women. Women who were less than 30 years or more than 35years were excluded from the study. Convenient sampling method was adopted in recruiting participants.

Informed Consent/Ethical Approval

Informed consent was obtained from the pregnant women before their samples were collected upon clearance from the Department of Medical Laboratory Science, Rivers State University.

Eligibility Criteria

Only apparently healthy pregnant women attending Rivers State University Teaching Hospital and apparently healthy non pregnant women were recruited for this study.

Sample Collection and Storage

A total of 3ml of venous blood was collected by venipuncture with the use of vacutainer needle from each subject and added into individualized vacutainer tube containing 0.5ml of 1.2mg/ml dipotassium ethylene tetra- acetic acid (EDTA). The blood samples in EDTA containers were centrifuged to obtain plasma. The plasma obtained was used to analyse for vitamin B12 using an ELISA reader capable of reading absorbance at 450nm. Thin films were also made immediately before the plasma was separated.

Sample Analysis

The parameters that were analysed were vitamin B12 and presence of hypersegmented neutrophils in thin films.

      1. Determination of Vitamin B12 Using Human Vitamin B12 ELISA Kit, CALBIOTECH, Inc., El Cajon,

U.S.A. Lot No VBE5774; Expiry Date: 2020/05

Principle: It makes use of solid phase ELISA methodology based on the principle of delayed competitive binding. Streptavidin coated wells are incubated with extracted vitamin B12 standards, controls, samples and intrinsic Factor-Biotin conjugate at room temperature for 45 minutes. During the incubation, the biotin-labelled intrinsic factor binds to vitamin B12 in the sample, standard or quality control plasma, after the 45-minutes incubation, vitamin B12 enzyme conjugate is added which competes with the vitamin B12 in the sample, standard, or quality control plasma for the remaining sites on the intrinsic factor for an additional 30 minutes. All unbounded conjugates are then removed and the wells are washed, Next, a solution of tetramethylbenzidine (TMB) reagent is added and incubated at room temperature for 15 minutes, resulting in the development of blue colour. The colour development is stopped with the addition of stop solution, and the absorbance is measured spectrophotometrically at 450 nm. The colour intensity is inversely proportional to the amount of vitamin B12 in the sample. The total procedure run time is 1.5 hours.

Procedure: The EDTA blood samples were centrifuged to obtain plasma. All reagents and specimens were allowed to come to room temperature before use. Desired number of coated strips was placed into the holder. 50µl of extracted Vitamin B12 standards, controls and samples was dispensed into appropriate wells. 50 µl of biotinylated intrinsic factor reagent was dispensed into each well. The microplate was shaken gently for 30 seconds to mix. It was incubated for 45 minutes, at room temperature (250oC). 50 µl of enzyme conjugate was added into all the wells. The microplate was gently shaken for 30 seconds to mix. It was incubated for 30 minutes at room temperature (250oC). The contents were briskly shaken out of the wells. The wells were rinsed 3 times with wash buffer. The wells were stroked sharply on absorbent paper to remove residual water droplets. 100 µl of tetramethylbenzidine (TMB) substrate was dispensed into each well which resulted in the development of a blue coloured solution. The absorbance was read spectrophotometrically at 450nm.

Procedure for Thin Blood Film Preparation

One micro litre of blood was dropped near the end of a slide. The edge of the spreader was placed in front of the blood at an angle of 45o. the spreader was drawn back until it touched the drop of blood and the drop spread along the line of contact between the spreader and the slide on which the film was made. The spreader was moved along the slide with a smooth movement. The film was allowed to air dry. The subject identification was written directly on the frosted end using a lead pencil.

Film Staining using Leishman Staining Technique

Principle: Leishman stain is a mixture of eosin and methylene blue. The acidic dye, eosin variably stains the basic components of the cell which is the cytoplasm and the

basic stain, methylene blue stains the acidic components, especially the nucleus.

Procedure

The slide was placed on the staining rack. The film was flooded with Leishman stain and the stain was allowed to stain for 2 minutes. The stain was later diluted with equal volume of buffered water (pH 6.8) and was allowed to stand for 8 minutes. After 8 minutes, the stain and buffered water was washed off and slide allowed to drain. The back of the slide was then cleaned with cotton wool soaked with 70% alcohol. The film was dried on a rack in a vertical position. The stained f i lmed was examined microscopically using oil immersion objective (100x), ( Olympus microscope) for the presence of hypersegmented neutrophils.

Statistical Analysis

The data generated from this study was analysed and calculated to determine the mean, standard deviation, p- value, f-value using analysis of variance; Tukey’s multiple comparison test was done to check for significance in between groups. Graph pad prism 8.0.2 statistical package was used for the analysis.

RESULTS
    1. Demographic Details of Participants

A total of seventy-five (75) females were recruited for this study. Fifty (50) of the females were pregnant women and were grouped into two (25 were pregnant women that had previous miscarriage(s) and the other 25 were pregnant women that have not had miscarriage(s). Twenty-five (25) women served as control subjects and were non-pregnant. Their age range was between 30-35 years and they were all residents of Port Harcourt, Rivers State. Details are shown in Table 1.

Table 1: Demographic Details of Participants

Age (Years)

No. of Pregnant Women with previous Miscarriage

No. of Pregnant Women without Previous Miscarriage

Control Subjects (Non-pregnant without history of Miscarriage)

30-35 25 25 25

Analysis of Variance of Vitamin B12 in Study Population

Table 2 showed the comparison of vitamin B12 level in pregnant women with previous miscarriage(s), pregnant women without previous miscarriage(s) and non-pregnant women (control)-without history of miscarriage.

Comparatively, the analysis showed that there was statistically significant difference (p<0.05) in the values of vitamin B12 of women with miscarriages (193.78±110.63 μg/day), women without miscarriages (174.80±53.14 μg/day) and the control group (98.03±9.50 μg/day).

Table 2: Comparison of vitamin B12 level using analysis of variance in the study population

Parameter PW+M (A) PW-M (B) Control (C) p-value F-value Inference Tukey’s Multiple Mean±SD Mean±SD Mean±SD Comparison Test

Vitamin 193.78±110.63 174.80±53.14 98.03±9.50 <0.0001 11.95 HS A vs B0.4231 B12 A vs C 0.0001

(µmol/day) B vs C 0.0001

Key: PW+M = Pregnant women with previous miscarriage(s); PW-M = Pregnant women without previous miscarriage(s); HS = Highly significant; SD = Standard deviation. (Applicable to all Tables).

    1. Percentage Distribution of Hypersegmented Neutrophils in Percentage Rate in the Study Population Table 3. shows the percentage distribution of hyper- segmented neutrophils in women with miscarriages, without miscarriages and non-pregnant women. The result showed that the percentage rate of hyper-segmented

neutrophils in women with miscarriages was 4(16%) while the percentage rate of hyper-segmented neutrophils in women without miscarriages was 1(4%). No hyper- segmented neutrophils were found in non-pregnant women.

Table 3: Percentage distribution of hyper-segmented neutrophils in women with miscarriages, without miscarriages and Non-pregnant women

Parameter No. of Women with hyper-segmented Neutrophils Percentage (%)

PW+M (N=25) 4 16
PW-M (N=25) 1 4
Control (N=25) 0 0
4.0 DISCUSSION

From this study, it was observed that there was a significant increase (p<0.05) in the values of vitamin B12 in women with previous miscarriages than those without a history of miscarriage and control participants. Non-pregnant (control) in this study recorded low level of vitamin B12 as a result of them not being on drug supplements and also not having huge appetite for food when compared to pregnant women. The level of vitamin B12 in our study subjects were within the range as reported by VanderJagt et al., (11).

Though it has been reported that maternal vitamin B12 deficiency and increased presence of hypersegmented neutrophils have been associated with increased risk of common pregnancy complications, including spontaneous abortion (miscarriage), low birth weight, intrauterine

among the women could be as a result of the presence of hypersegmented neutrophil which is indicative of the tendencies towards miscarriages. However, the high significant level of vitamin B12 in pregnant women with miscarriages may be due to their sea food diet despite the presence of hypersegmented neutrophils; which requires further investigation.

REFERENCES
  1. British Medical Association. Illustrated Medical Dictionary, 2nd edition, London: Dorling Kindersley Ltd, 2008.
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findings of the study indicated that pregnant women with history of miscarriages and those without history of miscarriage had high level of vitamin B12; and so, our findings are at variance with the above reports, probably as a result of the management of the conditions that aforetime may have caused the miscarriages.

The presence of hypersegmented neutrophils is an important diagnostic feature of megaloblastic anaemia; and deficiency of vitamin B12 have been associated with megaloblastic anaemia. In regards to the percentage rate of hypersegmented neutrophils in women with history of miscarriage(s), without history of miscarriage and non- pregnant women, the result showed 4(16%) 1(4%) and 0% in the same order. The hypersegmented neutrophils were found to be high amongst women with history of miscarriage(s) (16%), and this indicates that hypersegmented neutrophils could be associated with miscarriages in some cases in women. The presence of hypersegmented neutrophils in pregnant women with history of miscarriage(s) may probably be only a reflection of their previous miscarriage(s) and tendency towards a miscarriage.

5.0 CONCLUSION

The study revealed that women with miscarriages recorded

2 0 0 5 ; 2 0 ( 1 1 ) : 3 0 0 8 – 1 1 . d o i : 1 0 . 1 0 9 3 /

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