Hematological and biochemical profile of person with macrocytic anemia in a tertiary health care centre of Nepal

Anemia; Folic acid; Macrocytic; Red blood cell indices; Vitamin B12; Background: Anemia is a nutritional problem worldwide with an increased risk of morbidity and mortality in all age groups. Macrocytic anemia often originates from abnormalities that impair the erythroid precursor maturation in the bone marrow. Since the clinical manifestations of different types of anemias are similar, hematological parameters including hemoglobin, Red blood cell indices, and Peripheral Blood Smear examination are useful in the diagnosis of anemia.


INTRODUCTION
Anemia is a nutritional problem worldwide affecting almost two billion people with an increased risk of morbidity and mortality in young children, adolescent girls, and pregnant women. 1,2 It is broadly defined as a condition associated with a decreased total amount of Red Blood Cells (RBC) or Hemoglobin (Hb) concentration in blood impairing oxygen circulation which in turn has detrimental effects on maternal and birth outcomes, optimal child growth, impaired learning, reduced work productivity and earning during childhood. 3,4 Socioeconomic status, traditional habits of eating, irregular eating, and physiological conditions like pregnancy are the risk factors of developing anemia in adolescents. 5,6 According to World Health Organization (WHO), the Hb concentration less than 13 gm/dl in men, 12 gm/dl in nonpregnant women, 11 gm/dl in pregnant women, 12 gm/ dl in children aged 12 to 14 years, 11.5 gm/dl in children aged 5-11 years and 11 gm/dl in children (less than 5 years) are the diagnostic criteria of anemia.7 One of the basis of anemia classification is based on underlying mechanisms like impaired production, increased destruction, and huge blood loss. The second approach classifies anemia according to the change in red cell morphology and often correlates with the cause of red cell deficiency which can be morphologically divided into normocytic, microcytic, or macrocytic. 8 Depending upon the size, RBCs are called enlarged erythrocytes when Mean Cell Volume (MCV) is >100 fl which is the characteristic feature of macrocytic anemia which often originates from abnormalities that impair the erythroid precursor maturation in the bone marrow. 4,9 Megaloblastic anemia is the most common cause of macrocytic anemia due to the low availability of vitamin B12 and folic acid for coenzymes required for thymidylate and purine synthesis which results in impaired DNA synthesis, ineffective erythropoiesis, and intramedullary hemolysis. 10,11 Since the clinical manifestations of different types of anemias are similar, the differential diagnosis of macrocytic anemia can be done with the physical examination, hematological parameters including Hb, RBC indices like MCV, Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC) along with Peripheral Blood Smear (PBS) examination. The main objective of this study was to assess the hematological and biochemical parameters in a person with macrocytic anemia..

MATERIALS AND METHODS
This cross-sectional study was done in the Department of Pathology at Nepal Medical College Teaching Hospital from November 2019 to April 2020. Ethical approval was taken from the Institutional Review Committee, Nepal Medical College and Teaching hospital, and informed consent from the patients was taken before sample handling. A total of 42 patients between 14 to 62 years with low Hb concentration (gm/dl) for anemia diagnosis according to WHO criteria and MCV level >100 fl were selected. The blood samples were collected in vials containing Ethylene Diamine Tetra Acetic acid (EDTA) anticoagulant agent and were immediately analyzed in Sysmex5 parts XS-500i automated hematology analyzer in the Clinical Pathology laboratory. The evaluated parameters included Hb, MCV, MCH, MCHC, reticulocytes count, and PBS.
Hemoglobin concentration and RBC indices range for the diagnosis of anemia according to WHO 7 The reference interval for RBC indices were: When MCV was greater than 100 fl, the anemia was reported as Macrocytic anemia and undertaken for the study.

Peripheral Blood Examination
For the PBS examination, smears were made using EDTA blood and allowed to air dry. Then, 0.25 % of the Wright stain was poured on the entire slide and left for 2-3 minutes. An equal volume of distilled water was added. After 15 minutes slides were thoroughly washed, dried, and examined under a light microscope. PBS findings like macrocytic or normocytic cells, hypersegmented neutrophils, polychromatic cells, basophilic stippling, teardrop cells, etc were noted.

Reticulocyte Count
Equal volumes of the patient's EDTA blood and new methylene blue reagent were mixed in a test tube and allowed to incubate at 37°C for 10 minutes. Then the smears were made and examined under oil immersion.

Biochemical analysis
For serum Vitamin B12 and Folic acid analysis, venous blood samples were collected in gel vials, centrifuged at 3500 RPM for 10 minutes, and then estimation was done in Fully automated VITROS ECi Q, Johnson & Johnson, USA Machine.

Data analysis
Age, gender, Hb concentration, MCV, MCH, MCHC, Hematological and biochemical profile in macrocytic anemia

Pregnant women < 11
Men (15 years) < 13 reticulocyte percentage, and other parameters were determined. Serum Vit B12 and folic acid were also analyzed. The data obtained were managed in MS Excel 2016 and later analyzed using IBM SPSS version 20.

RESULTS
Our study comprised a total of 42 patients with macrocytic anemia and the age ranged from 14 to 62 years with a mean age of 31.85±12.49 years. There were 14 males (33.33%) and 28 females (66.67%) with a male: female ratio of 1:2. Figure 1 demonstrates the age-wise distribution of male and female macrocytic anemia patients with a higher incidence of 16 cases (7 males, 9 females) in the age group 21-30 years and the least 2 cases (1 male, 1 female) in >60 years of age group.
Age-wise distribution of Hb and RBC indices was compared and Hb level was found to be more than 8      Among 42 patients, only 20 had serum Vitamin B12 and Folic acid reports available out of which 15 cases had decreased levels of both biochemical markers while 5 cases had the normal level. The overall serum levels of Vitamin B12 and folic acid levels were found to be decreased in macrocytic anemia patients. Also, serum concentrations of these markers were found significantly different among male and female anemia patients (p<0.05) ( Table 4).

DISCUSSION
Primarily Vitamin B12 and folic acid deficiencies are the most probable etiology of macrocytosis (MCV>100 fl) leading to macrocytic anemia but in some cases, etiology remains unclear. However, anemia with normal MCV may indicate a chronic or mixed type of anemia. In this study, we used hematological and PBS examination to identify the macrocytic anemia and correlated the findings with their gender and age groups.
In our study, the maximum incidence of macrocytic anemia was seen in females (66.67%) than in males (33.33%) with the peak age group 21 Kannan A et al reported that out of 100 cases of macrocytosis, 62% had non-megaloblastic macrocytosis whereas 38% had megaloblastic anemia and bone marrow disorders (46%) is the most common cause of macrocytosis including acute and chronic leukemia, aplastic anemia, multiple myeloma, and myelofibrosis. A significant difference in MCV was observed between megaloblastic and non-megaloblastic macrocytosis. 17 In this study, out of 20 available reports we observed 15 cases (75%) had decreased level of both Vitamin B12 and folic acid whereas 5 cases (25%) had a normal level of those markers comprising overall decreased serum level of both Vitamin B12 and Folic acid (202.7±42.85pg/ml and 3.43±1.86ng/ml respectively) and the difference between males and females were found to be statistically significant (p<0.05). In contrast to our findings, Iqbal SP et al found out of 220 megaloblastic anemia patients, 71% of folic acid deficient patients had Vitamin B12 deficiency as well but there was no any significant difference between male and female patients. 14 Similarly, Agrawal L et al observed that out of 100 macrocytic anemia patients, 55% of patients were diagnosed with Vitamin B12 deficiency and 8% with folate deficiency. 19  In this study, the gender-wise distribution of Hb, RBC indices, PBS, and reticulocytes showed no significant differences between male and female patients. In contrast, serum Vitamin B12 and folic acid levels were found to be statistically different (p<0.05) in male and female anemic patients. A study by Sukla et al shows a similar finding which is comparable with our study. 24

CONCLUSIONS
In this study, we found the maximum incidence of macrocytic anemia in females of young age group. RBC indices like MCV, MCH, Hb, reticulocytes percent and PBS are the important factors for the diagnosis of anemia. Serum Vitamin B12 and Folic acid test further aid in the diagnosis of megaloblastic anemia. Thus the clinical, hematological, and biochemical parameters are the important tools for diagnosing macrocytic anemia and further help in differentiating megaloblastic anemia from non-megaloblastic anemia.