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Effect of megaloblastic anemia on hemoglobin A2 and diagnosis of β-thalassemia trait


1 Department of Pathology, Kalinga Institute of Medical Sciences (KIMS), KIIT University, Bhubaneswar, Odisha, India
2 Department of Pediatrics, Kalinga Institute of Medical Sciences (KIMS), KIIT University, Bhubaneswar, Odisha, India

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Date of Submission02-Mar-2021
Date of Decision24-Apr-2021
Date of Acceptance01-May-2021
Date of Web Publication24-May-2022
 

   Abstract 


Context: β-thalassemia trait is usually diagnosed by raised hemoglobin A2 (HbA2). The presence of megaloblastic anemia can cause an increase in HbA2 and create a diagnostic dilemma. Here, we have analyzed the effect of vitamin B12 and folic acid supplementation on HbA2 and diagnosis of β-thalassemia trait in cases of megaloblastic anemia with raised HbA2. Methods and Material: Cases of megaloblastic anemia with raised HbA2 on high-performance liquid chromatography (HPLC) were supplemented with vitamin B12 and folic acid. Post-treatment evaluation was done after 2 months. Cases showing adequate hematological response were subjected to statistical analysis. Based on post-treatment HbA2 value, the cases were diagnosed as normal, borderline raised HbA2, or β-thalassemia trait. Pre- and post-treatment values of red cell parameters and HbA2 were analyzed. Results: There was a significant decrease in HbA2 value after vitamin B12 and folic acid supplementation. The diagnosis was changed in 70.97% of the cases after treatment. The chance of inconclusive diagnosis was decreased from more than 50% to less than 10%. Pre-treatment mean corpuscular volume (MCV) and HbA2% showed a significant difference between the thalassemic and normal groups. Conclusions: Megaloblastic anemia can lead to false-positive diagnosis of β-thalassemia trait on HPLC. Repeat HPLC should be done after adequate supplementation of vitamin B12 and folic acid in cases of megaloblastic anemia with raised HbA2. Red cell parameters are not helpful to suspect β-thalassemia trait in presence of megaloblastic anemia. However, HbA2% on HPLC can be a useful parameter to suspect or exclude β-thalassemia trait in cases of megaloblastic anemia.

Keywords: β-thalassemia trait, hemoglobin A2, high-performance liquid chromatography, megaloblastic anemia


How to cite this URL:
Sahoo S, Sahu N, Das P, Senapati U. Effect of megaloblastic anemia on hemoglobin A2 and diagnosis of β-thalassemia trait. Indian J Pathol Microbiol [Epub ahead of print] [cited 2022 Oct 5]. Available from: https://www.ijpmonline.org/preprintarticle.asp?id=345862





   Introduction Top


Hemoglobinopathies are the most common inherited red cell disorders affecting approximately 5% of the world population.[1],[2],[3] β-thalassemia is one of the most common hemoglobinopathy worldwide.[4],[5],[6] Though β-thalassemia trait remains mostly asymptomatic, it can result in severe forms like homozygous thalassemia or different compound heterozygous forms in the next generation. Hemoglobinopathies are generally not curable. But the incidence of more severe forms can be decreased by population screening, prenatal diagnosis, and genetic counseling.[7]

The best and most accurate method of diagnosing hemoglobinopathies is molecular study. But most of the laboratories do not have this facility. Hence, hemoglobinopathies are usually diagnosed by correlating the clinical findings, peripheral blood examination, hemoglobin analysis, ancillary studies like sickling test and Hemoglobin H (HbH) inclusion test, and family study.[8],[9]

β-thalassemia trait is diagnosed by raised hemoglobin A2 (HbA2). Nowadays, high-performance liquid chromatography (HPLC) is the method of choice for HbA2 measurement. HbA2 value of >4% is suggestive of β-thalassemia trait.[2],[5],[10] But the presence of some non-hemoglobinopathy diseases can alter the HbA2 value making the diagnosis of β-thalassemia trait difficult. Megaloblastic anemia, presence of HbS, hyperthyroidism, and antiretroviral therapy can cause an increase in HbA2 while iron deficiency anemia can cause a decrease in HbA2.[11],[12],[13]

Effect of iron deficiency on HbA2 and diagnosis of β-thalassemia trait is studied extensively.[13],[14],[15],[16],[17] But studies related to the effect of megaloblastic anemia on HbA2 are quite few and have variable opinions on the significance of the effect.[11],[12] Here, we have analyzed the effect of vitamin B12 and folic acid supplementation on HbA2 and diagnosis of β-thalassemia trait in cases of megaloblastic anemia with raised HbA2.


   Subjects and Methods Top


The present study was a prospective study conducted after getting approval from the Institutional Ethical Committee. Among all the cases received for hemoglobin analysis by HPLC from May 2018 to April 2020, cases having megaloblastic anemia were identified. Megaloblastic anemia was diagnosed by correlating the findings of complete blood count (CBC), peripheral blood smear examination, reticulocyte count and serum vitamin B12 and serum folate levels. Hemoglobin concentration of <13 gm/dL for male and <12 gm/dL for female were taken as cut-off point to detect anemia.[18] All the cases had a deficiency of vitamin B12 and/or folic acid. Serum vitamin B12 level of <200 pg/mL and serum folate level of <2 ng/mL were taken as the cut-off point for this. Patients with concomitant iron deficiency (serum ferritin <30 ng/mL) were excluded because iron deficiency is known to cause a decrease in HbA2. The presence of other common causes of increased HbA2 like sickle cell disorder, hyperthyroidism, and antiretroviral therapy was ruled out. Hemoglobin analysis was done by HPLC using the instrument BIO-RAD D-10TM dual-mode, manufactured by the Bio-Rad laboratories. The cases were divided into two groups: one with normal HbA2 (≤3.5%) and the other with raised HbA2 (≥3.6%). Vitamin B12 and folic acid supplementation were given to all. Cases with raised HbA2 were followed up and assessed for treatment response after 2 months. Patients with a history of blood transfusion within the last 4 months of analysis were excluded because of its interference in the HPLC findings.

Vitamin B12 supplementation was started at 1,000 μg oral every day for a week, followed by every alternate day for 2 weeks, then 2 days a week for 2 weeks, and then, once a week. Folic acid supplementation was given at a dose of 0.5–1.0 mg tablets daily for 3–4 weeks, then a maintenance dose of 0.2 mg daily for 3 months. Cases with severe anemia were given packed red blood cells (RBCs) as per requirement. Post-treatment evaluation in these cases was done only after 4 months of blood transfusion if possible.

The cases were evaluated again at the end of the second month of treatment by doing CBC, peripheral blood smear examination, and HPLC. Reticulocyte count, serum vitamin B12, and folic acid estimations were done in cases where hemoglobin level was low. Hemoglobin levels of 12 gm/dL in males and 11 gm/dL in females (representing >90% response to therapy) were taken as the cut-off to assess adequate hematological response to treatment except in a few cases which had microcytic hypochromic blood picture with target cells and HbA2 value >4% suggesting an associated β-thalassemia trait component. The cases which were lost to follow-up or did not show adequate hematological response at the end of the second month of treatment were excluded from the analysis. Based on post-treatment HbA2%, the cases were diagnosed as normal (≤3.5%), borderline raised HbA2 (3.6–4%), or β-thalassemia trait (≥4.1%). The pre- and post-treatment values of different red cell parameters like hemoglobin (Hb), total RBC count (TRBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and red cell distribution width (RDW), HbA2% and diagnosis based on HbA2% were analyzed.

Statistical analysis: All the statistical analyses were done using STATA 15.1 software. Comparison between pre- and post-treatment red cell parameters and HbA2 was done by paired t-test and P value calculated. Pre-and post-treatment diagnoses based on HbA2% were compared and the percentage of change in diagnoses was calculated. Comparison of pre-treatment red cell parameters and HbA2% was done between the two groups thalassemia and normal by unpaired t-test.


   Results Top


We had a total of 133 cases of megaloblastic anemia undergoing HPLC to detect any hemoglobinopathy. Among these, 61 (45.86%) cases had raised HbA2. After 2 months of treatment, 23 cases were lost to follow-up. Out of the remaining 38 cases, adequate hematological response was seen in 31 cases while 7 cases showed inadequate response to treatment.

The median age of presentation was 15 years (range: 10–57 years); 29 cases (93.54%) were between 10–23 years of age. Females outnumbered males with a male:female ratio of 1:4.1.

Comparison of red cell parameters and HbA2 % before and after treatment

We found a significant increase in hemoglobin concentration and TRBC after treatment [Table 1]. However, three cases (all of them diagnosed with β-thalassemia trait after treatment) had post-treatment lower hemoglobin (<12 gm/dL for males and <11 gm/dL for females) concentration. There was a significant decrease in MCV, MCH, MCHC, and RDW values after treatment. The pre-treatment MCV was <80 fL in 14 (45.16%) cases and >100 fL only in 7 (22.6%) cases. It was decreased in 26 (83.88%) cases, increased in 4 (12.90%), and remained constant in 1 case after treatment.
Table 1: Comparison of different red cell parameters and HbA2 value before and after treatment

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HbA2% was decreased in all with a statistically significant decrease in mean HbA2 value (P = 0.0001). The decrease in HbA2 varied from 0.1 to 1.8% with a mean decrease of 0.75%.

Interpretation of HbA2 in relation to diagnosis of β-thalassemia trait

Before treatment, 16 (51.6%) cases had borderline raised HbA2 while the rest 15 (48.4%) had HbA2 in the thalassemic range. Out of the 16 cases in the borderline group, 15 (93.75%) cases became normal after treatment while 1 remained as borderline raised HbA2 [Table 2]. Out of the 15 cases with pre-treatment HbA2 in thalassemic range, 5 (33.33%) were diagnosed as normal and 2 (13.33%) as borderline raised HbA2 while the other 8 (53.34%) remained as β-thalassemia trait after treatment. So, after treatment, the diagnosis remained the same in 9 (29.03%) cases while the other 22 (70.97%) cases showed a change in the diagnosis. The number of cases with borderline raised HbA2 decreased from 51.6 to 9.68% after treatment. The group with normal post-treatment HbA2 (n = 20, 64.51%) was named as normal and the group with HbA2 in thalassemic range (n = 8, 25.81%) as thalassemia. The diagnoses of the three cases with borderline HbA2 after treatment were not confirmed.
Table 2: Diagnosis based on HbA2 before and after treatment

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Comparison of different parameters at the time of presentation

We have compared the pre treatment values of different red cell parameters and HbA2 between the two groups: (1) Thalassemia and (2) Normal [Table 3]. MCV value was significantly lower and HbA2 value was significantly higher in the group with thalassemia as compared to the normal group. There was no significant difference in the other parameters.
Table 3: Comparison of pre-treatment parameters between the two groups (Thalassemia and Normal)

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   Discussion Top


Our review of literature on the diagnosis of hemoglobinopathies found that HbA2 value either remains normal or increased in cases of megaloblastic anemia.[8],[11],[19],[20] So, we have not considered the diagnosis of β-thalassemia trait in cases of megaloblastic anemia with normal HbA2 (≤3.5%) and excluded them from our analysis.

The pre-treatment mean hemoglobin level (5.77 gm/dL) was lower than that found by Srikanth et al.[21] (6.195 gm/dL) and Pandya et al. (7.22 gm/dL).[22] This was probably due to the associated β-thalassemia trait component in some of the cases and also because only the cases of raised HbA2 (maybe having more a severe deficiency of vitamin B12 and folic acid) were included in the study. There was a significant increase in hemoglobin concentration and TRBC after vitamin B12 and folate supplementation. The three cases with post-treatment low Hb value had associated β-thalassemia trait. In the present study, the mean MCV (85.28 fL) before treatment was lower compared to the results found by Srikanth and Pandya et al.[21],[22] All cases with low MCV had normal serum iron and ferritin levels which rules out associated iron deficiency. The low MCV value in these cases was probably due to the presence of schistocytes and associated thalassemia component in some cases. The presence of schistocytes, sometimes leading to low mean corpuscular volume is a known hematological manifestation of megaloblastic anemia particularly in cases with severe vitamin deficiency.[23],[24],[25] Four (12.90%) cases showed a post-treatment increase in MCV value. The lower pre-treatment MCV value in these cases was probably due to the presence of schistocytes.

The significant decrease in post-treatment HbA2 may be attributed to the correction of anemia by vitamin B12 and folic acid supplementation. A few previous studies have mentioned the increase in HbA2 in megaloblastic anemia but they have not mentioned the significance of this in the diagnosis of hemoglobinopathies.[8],[13],[19],[20] However, Seema Rao et al.[11] have done a one-time study and compared the HbA2 levels between two groups of population with normal HPLC pattern: one group with megaloblastic anemia and another group without megaloblastic anemia. They found a significantly higher level of HbA2 among megaloblastic anemia cases.

In the present study, 45.86% cases of megaloblastic anemia had raised HbA2. Studies related to this type of incidence are few. While in one study the incidence was 55.55% among pernicious anemia cases[26] in another study it was only 2.83% among cases of megaloblastic anemia.[12] Josephson et al. (1958)[26] found raised HbA2 level in five out of nine (55.55%) cases of pernicious anemia. The increase in HbA2 was mild in four of them while in one case it was in the thalassemic range. But they have not assessed the effect of vitamin B12 therapy on HbA2 in these cases.

Henshaw et al.[12] studied the effect of therapy on HbA2 in cases of megaloblastic anemia with raised HbA2. They had 106 cases (50 cases with vitamin B12 deficiency, 50 with folate deficiency, and six having deficiency of both) of megaloblastic anemia. Only three (2.83%) cases (all belonging to vitamin B12 deficiency group) had raised HbA2. Also, the increase was mild and not up to the level of β-thalassemia trait to cause any difficulty in diagnosis. In these three cases, HbA2 level became normal after vitamin B12 supplementation. There was no increase in HbA2 among folate deficiency and combined deficiency groups.

But the findings in our study were quite significant. After treatment 20 (64.51%) cases became normal, 8 (25.81%) were diagnosed with β-thalassemia trait while 3 (9.68%) remained in the borderline group. A change in diagnosis was observed in 70.96% of the cases (22 out of 31) after treatment [Table 2]. However, the diagnoses in the three cases with borderline raised HbA2 were not confirmed because the molecular study was not done. The percentage of inconclusive diagnosis (i.e., borderline raised HbA2) was decreased from more than 50% to less than 10% after treatment. So, in a case of megaloblastic anemia with raised HbA2 repeat HPLC should be done after adequate vitamin B12 and folic acid supplementation particularly if the molecular analysis is not available to solve the dilemma in the diagnosis of β-thalassemia trait.

The pre-treatment values of different RBC parameters and HbA2% were compared between the two groups: thalassemia (n = 8) and normal (n = 20) to find out the significant difference in any parameter that can be helpful in suspecting an associated β-thalassemia component [Table 3]. Except for MCV no other RBC parameters showed significant difference. MCV value was significantly lower in the thalassemia group. Similar findings were documented earlier by Castellanos-Sinco et al.[27] and Devalia et al.[28] It was most probably due to the associated thalassemia component but because of marked overlap in MCV values among the two groups, it was very difficult to decide a cut-off point of MCV to suspect an associated β-thalassemia component at the time of initial presentation.

The HbA2% was significantly higher in the thalassemia group. The mean HbA2% of the the thalassemia group was 6.50% while it was 3.84% in the normal group. After treatment, the highest decrease in HbA2 observed among the thalassemic group was 1.2% and among the normal group was 1.8%. There was no overlap in the pre-treatment HbA2 value between the two groups. The lowest pre-treatment HbA2 value observed among the thalassemic group (n = 8) was 5.3%. The highest pre-treatment HbA2% observed among the normal group (n = 20) and the borderline group (n = 3) were 4.6% and 4.3%, respectively. So all cases with pre-treatment HbA2 value >5% were diagnosed as β-thalassemia trait after treatment. Among the cases with pre-treatment HbA2 value <5%, a majority (n = 20; 86.96%) of them became normal after treatment except a few (n = 3; 13.04%) cases which remained in the borderline range. But none of the cases with pre-treatment HbA2 value <5% were diagnosed as thalassemia after treatment. Hence, at the time of presentation, HbA2 value of 5% can be taken as a cut-off point to exclude or suggest the diagnosis of β-thalassemia trait in presence of megaloblastic anemia. However, repeat HPLC should be done after adequate supplementation of vitamin B12 and folic acid.

Study limitations

The present study is limited by less number of cases and a lack of molecular study for confirmation of post-treatment borderline raised HbA2 cases. Also, the difference in HbA2 value in relation to the severity of anemia and type of nutritional deficiency (deficiency of vitamin B12 or folic acid or both) was not established.


   Conclusion Top


Megaloblastic anemia can cause an increase in HbA2 and lead to false-positive diagnosis of β-thalassemia trait. Adequate vitamin B12 and folic acid supplementation lead to a significant decrease in HbA2% in cases of megaloblastic anemia with raised HbA2. The overall diagnosis was changed in more than 70% of the cases after treatment. So, one should rule out the presence of megaloblastic anemia in cases of raised HbA2, and repeat HPLC should be done after supplementation of vitamin B12 and folic acid in these cases particularly when the molecular study is not feasible. None of the red blood cell parameters appear helpful in suspecting or excluding associated β-thalassemia trait component in the presence of megaloblastic anemia. But pre-treatment HbA2 value of 5% on HPLC can be taken as a cut-off point to suspect or exclude the presence of associated β-thalassemia trait component in cases of megaloblastic anemia. However, studies with larger series of cases are required to support this fact.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Nageswar Sahu,
Department of Pathology, Kalinga Institute of Medical Sciences, Bhubaneswar, Odisha - 751 024
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijpm.ijpm_233_21




 
 
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