Evaluation of Macrocytosis in Routine Hemograms

Abstract

Macrocytosis, a condition in which erythrocytes are larger than normal manifests as an increase in mean corpuscular volume (MCV) more than 100 fl. The aim of this study was to identify the underlying causes of macrocytosis, detected in routine hemograms and to evaluate the hematological features in different etiologies. This study included 178 adult patients whose detailed medical history was recorded, and Vitamin B12 assay, folate assay, thyroid function tests, liver function tests, complete blood counts and peripheral smear evaluation was performed. Alcoholism was identified as the etiological factor in 65 cases (36.5%), Vitamin B12 deficiency in 43 cases (24.1%) and drug related in 23 cases (12.9%). These three conditions accounted for 73.6% of macrocytosis. Other causes identified were folate deficiency, liver disease, Myelodysplastic syndrome, chronic renal failure and Aplastic anemia. In 41 cases, the cause of macrocytosis could not be explained. Anemia was observed in 95 cases (53.3%) being most common in Vitamin B12 deficiency. 9 cases (20.9%) of Vitamin B12 deficiency presented with isolated macrocytosis without anemia. It was observed that mean hemoglobin was lower and red cell distribution width (RDW) higher in megaloblastic conditions. Peripheral smear revealed hypersegmented neutrophils in 86% and macro-ovalocytes in 72% of the megaloblastic cases. Complete medical history, red cell parameters and peripheral blood smear are simple, inexpensive tools which assist in identifying the underlying cause of macrocytosis, particularly in resource limited settings. Macrocytosis needs to be evaluated even in the absence of anemia, as it may be the first clue to an underlying pathology.

Introduction

The term macrocytosis refers to a condition in which erythrocytes are larger than normal. This manifests in routine hemograms as an increase in mean corpuscular volume (MCV). The normal MCV in adults ranges from 80 to 100 femtolitres [1, 2]. An MCV greater than 100 fl is generally regarded as macrocytosis [3–5]. This cut off value is lower in children, as normal MCV is lower in children than in adults. Macrocytosis can be an incidental finding in asymptomatic patients or it may be associated with symptoms of underlying disease. The prevalence of macrocytosis in general population is reported to range between 1.7 and 3.6% [1, 2].

Physiological macrocytosis is seen in pregnancy, newborns and infants. Macrocytosis without anemia may sometimes be a normal variant found in members of the same family, suggesting a genetic predisposition. Cold agglutinins, hyperglycemia, leukocytosis, reticulocytosis and delayed sample processing can result in false elevation of MCV [1, 3].

The mechanisms underlying the formation of macrocytes are diverse and complex. In Vitamin B12/folate deficiencies, there is a defect involving nuclear maturation, which affects cell division. Hemoglobin synthesis proceeds normally, while nuclear division lags behind, thereby resulting in larger than normal red cells. Macrocytosis in alcoholism is related to the direct toxic effect of alcohol on the red cell membrane [3]. In myelodysplastic syndrome, macrocytosis results from the dysplastic changes in the erythroid precursors [6]. In hepatobiliary disease and myxoedema, there is deposition of excess lipids on the red cell membrane, which results in increased MCV [7, 8]. Reverse transcriptase inhibitors interfere with DNA production, thereby resulting in megaloblastic changes and macrocytosis [1, 3].

The aim of this study was to identify the causes of macrocytosis detected in routine hemograms and to evaluate the hematological features in macrocytosis of different etiologies.

Materials and Methods

This study was conducted in Department of Pathology of our Institute, from July 2009 to December 2010. The study included 178 adult patients with an MCV >100 fl. Newborns, pregnant women, reticulocytosis and spurious macrocytosis were excluded from the study.

In all these cases, a complete medical history including diet, medication and alcohol intake was recorded. Sustained and regular excessive drinking, alcohol tolerance and withdrawal symptoms were considered as ‘Alcoholism’ [9, 10]. All cases were subjected to serum Vitamin B12 (Cobalamin) assay, folic acid assay and thyroid function tests using fasting samples on Beckman Coulter AccessR 2 immunoassay system and AccessR reagents and calibrators. Liver function tests were done using Cobas Integra 400 plus, Roche diagnostics. A detailed evaluation of red cell parameters and peripheral smear was done in all cases.

The concentrations considered for defining deficiencies were serum Vitamin B12 <150 pmol/l (203 pg/ml) and serum folate <10 nmol/l (4 ng/ml) [11]. Cases with Hb <11.6 g/dl (females) and Hb <13.2 g/dl (males) were classified as anemic macrocytosis [12]. Bone marrow was done in 28 cases, where it was clinically indicated. Based on the etiological factors identified, cases were segregated into megaloblastic and nonmegaloblastic categories. Red cell parameters and peripheral smear findings were compared between these two categories.

Results

Of the 178 cases evaluated, underlying etiological factor/factors could be identified in 137 cases (76.9%) as shown in Table 1. Alcoholism was the most common cause of macrocytosis, identified in 65 cases (36.5%), followed by Vitamin B12 deficiency found in 43 cases (24.1%) and drug related macrocytosis in 23 cases (12.9%). These three conditions accounted for 73.6% of macrocytosis. Of the 43 cases diagnosed as Vitamin B12 deficiency, 31 had serum levels between 100 and 199 pg/ml and 12 cases had <100 pg/ml. Of these 12 cases, 9 had normal folate levels and 3 had reduced folate levels. As the clinicians opted to start combined Vitamin B12 and folate treatment, and there was no facility to perform serum homocysteine and methyl melanoic acid, we could not decide if the low cobalamin levels reflected actual Vitamin B12 deficiency or resulted from concomitant folate deficiency. These 3 cases were therefore considered as combined deficiency [13].

Table 1.

Etiological factors identified in macrocytosis

Etiological factor	No of cases in which the condition was present (%)
Alcoholism	65 (36.5)
Vitamin B12 deficiency	43 (24.1)
Folate deficiency	8 (4.4)
Drug induced	23 (12.9)
Hypothyroidism	3 (1.7)
Liver disease	8 (4.4)
Hemodialysis	4 (2.2)
Myelodysplastic syndrome	3 (1.6)
Aplastic anemia	2 (1.1)
Unexplained	41 (23)

Drug (medication) induced macrocytosis was the third most common cause of macrocytosis identified in 23 cases (12.9%). Reverse transcriptase inhibitors, anticonvulsants and chemotherapeutic agents were the most common drugs encountered. The duration of treatment varied in different drugs, ranging from 2 months to 7 years as indicated in Table 2. Liver disease, hypothyroidism, chronic renal failure (on hemodialysis), myelodysplastic syndrome and aplastic anemia were the other causes identified. In 22 cases (12.3%) more than one etiological factor was detected (Table 3). In 41 cases (23%), the cause of macrocytosis could not be explained.

Table 2.

Drugs causing macrocytosis

Name of the drug	Duration of treatment	No of cases
Reverse transcriptase inhibitors	6 months to 3 years	7
Chemotherapy	2–6 months	5
Phenytoin	3–7 years	6
Carbamazepine	1–2 years	3
Sulfasalazine	5–7 months	2
Total		23

Table 3.

Cases with combined etiological factors

Combined etiological factors identified	No of cases
Alcoholism with Vitamin B12 deficiency	7
Alcoholism and liver disease	4
Vitamin B12 deficiency and medication	4
Alcoholism and medication induced	1
Hypothyroidism and Vitamin B12 deficiency	1
Liver disease and Vitamin B12 deficiency	1
Combined deficiency of Vitamin B12 and folic acid	4
Total no of cases with more than one contributing factor	22

Anemia was present in 95 cases (53.3%) of macrocytosis and the remaining 83 cases had normal hemoglobin levels. The relative incidence of anemia varied in different etiologies (Fig. 1). In Vitamin B12/folate deficiencies anemia was present in 80% (41 cases), while in alcoholism only 24.6% (16 cases) were anemic.

Fig. 1.

Prevalence of anemia in various conditions causing macrocytosis

Bone marrow evaluation was done only when clinically indicated. Of the 28 cases, 17 cases revealed megaloblastic marrow, 9 cases appeared normoblastic and 3 cases showed dysplastic changes. Marrow iron stores were increased in two cases, of which one was MDS and the other was alcoholic liver disease. The 38 cases resulting from Vitamin B12/folate deficiencies, with no additional etiological factors were categorized as megaloblastic macrocytosis. 53 cases resulting from alcoholism (without secondary vitamin deficiency or other contributing factors) were categorized as nonmegaloblastic (normoblastic) macrocytosis.

Hematological parameters and blood smear findings observed in both these cataegories are compared in Table 4. The mean hemoglobin (Hb) was 8.8 g/dl in Vitamin B12/folate deficiency, as compared to 12.7 g/dl in alcoholism. The mean MCV was higher in megaloblastic cases. The maximum MCV observed in Vitamin B12 deficiency was 146.8 fl, while in alcoholism it was 114 fl. The mean RDW, an indicator of anisocytosis was significantly higher in the megaloblastic group. Megaloblastic anemia showed lower red cell counts, which was proportional to the degree of anemia. Peripheral smear in megaloblastic cases revealed hypersegmented neutrophils in 86% (32 cases) and macro-ovalocytes in 72% (27cases). In alcoholism, these features were found in 9.5 and 17.4% respectively. 82.6% cases of alcoholism showed uniform, round macrocytes with normal RDW.

Table 4.

Hematological profile in megaloblastic and nonmegaloblastic macrocytosis

Parameter	Megaloblastic category (38 cases)	Nonmegaloblastic (alcoholic) category (53 cases)
Hb (g/dl)	8.8 (2.0–14.5)	12.69 (8.7–16.2)
MCV (fl)	111.8 (100.5–146.8)	107.80 (100.3–114)
MCH (pg)	35.1 (28.8–45.2)	34.8 (31.7–40)
Red cell count × 1012/l	2.34 × 1012/l (0.49–4.41)	3.8 × 1012/l (3.2–5.36)
RDW-CV %	21.6% (12.7–46.4)	13.7% (12.3–28.4)
Total leukocyte count × 10−9/l	5.13 (1.0–9.4)	7.28 (2.8–16.5)
Platelet count × 10−9/l	192.4 (15–754)	246.17 (80–680)
Hypersegmented neutrophils	86.3%	9.5%
Macro-ovalocytes	72.2%	17.4%

Mean values are presented along with the range in brackets

Discussion

The causes of macrocytosis reported varies in different populations studied. Savage et al. [14] in their study of 300 hospitalized patients in a New York City teaching hospital have reported drug therapy and alcohol abuse as the most frequent causes, and Vitamin B12/folate deficiency as uncommon (6%). Davidson et al. [7] have also reported medications as the most frequent cause. Keenan et al. [15] have observed that alcoholism followed by hematological disorders as the most frequent causes. However, their study has excluded drug induced causes. Mahmoud et al. [16] have studied 124 patients in the age group of ≥75 years and found a higher incidence of hematological diseases and malignancy associated with macrocytosis. Breedveld et al. [17] and Seppa et al. [5] have reported Vitamin deficiency and alcoholism as the most frequent etiological factors, which is similar to the findings in the present study.

Drugs reported to cause macrocytosis include reverse transcriptase inhibitors (Zidovudine, lamivudine, stavudine), anticonvulsants (valproic acid, phenytoin), folate antagonists (methotrexate), chemotherapeutics (alkylating agents, pyrimidine and purine inhibitors), sulfasalazine (anti inflammatory), pyrimethamine, trimethoprim, sulfamethoxazole, metformin and nitrous oxide. Most patients with HIV, being treated with reverse transcriptase inhibitors will display macrocytosis without anemia. In these patients, macrocytosis is considered a surrogate marker indicating the patients compliance in taking medications [1, 3].

Colon-Otero et al. [18] have reported that 60% patients with macrocytosis present without anemia. In the present study, 83 cases (46.7%) presented without anemia and the remaining 95 cases (53.3%) had associated anemia. The relative incidence of anemia in our study varied among different etiologies, being most frequent in Vitamin B12/folate deficiency (Fig. 1). Alcoholism was commonly associated with nonanemic macrocytosis. Another important observation in this study was that 9 cases (20.9%) of Vitamin B12 deficiency manifested isolated macrocytosis, without anemia. This re-emphasises the fact that macrocytosis can be an early diagnostic harbinger of Vitamin B12 deficiency, preceding anemia by months. It must be noted that MCV is an average value, and can be masked by a coexisting microcytic anemia like iron deficiency anemia, anemia of chronic illness and thalassemia trait [19].

In the present study, the maximum MCV observed in Vitamin B12/folate deficiency was 146.8 fl, whereas in alcoholism it was 114 fl. In alcoholism, the MCV is usually reported to range between 100 and 110 fl [6]. Ethanol appears to have a direct toxic effect on the marrow erythroid precursors. As the life-span of erythrocytes is 120 days, it may take 3–4 months for MCV to return to normal following abstinence from alcohol [10]. The mean RDW (21.6%) in megaloblastic cases was significantly higher compared to nonmegaloblastic conditions (13.7%).

Gupta et al. [20] have compared the red cell parameters in macrocytosis of aplastic anemia and megaloblastic anemia and observed that RDW in megaloblastic anemia was significantly higher than the RDW in aplastic anemia. An increased RDW has been reported in megaloblastic anemia, myelodysplastic syndromes and chemotherapy [19, 21]. In liver disease and aplastic anemia, RDW is normal or mildly increased [20, 22]. Seppa et al. [23] have compared the red cell parameters in macrocytosis of pernicious anemia and alcohol abuse. They found that a combination of low red cell count (<4 × 10¹²/l), high RDW (>15%), with a normal thrombocyte count and a normal thrombocyte mean cellular volume was highly sensitive in detecting Vitamin B12/folate deficiency. Bessmann et al. [24] have developed an improved classification of anemias based on MCV and RDW.

Megaloblastic processes are characterized by the presence of oval macrocytes (macro-ovalocytes) and hypersegmented neutrophils. However, hypersegmented neutrophils can sometimes be absent in megaloblastic conditions, if associated with marked left shift or severe neutropenia [25]. Macro-ovalocytes are reported to be more sensitive but somewhat less specific than neutrophil hypersegmentation in predicting marrow megaloblastic changes [6]. It is reported that an MCV >115 fl, increased RDW, macro-ovalocytes, and hypersegmented neutrophils suggest a megaloblastic disorder [1]. Nonmegaloblastic conditions display uniformly round macrocytes and normal RDW [3, 19]. Round macrocytes are commonly seen in alcoholism and round, target-appearing macrocytes are characteristic of liver disease such as hepatitis and obstructive jaundice [1]. In the present study, hypersegmented neutrophils and macro-ovalocytes were more frequently observed in megaloblastic macrocytosis. However, these features were also observed in a small percentage of alcoholism cases, who predominantly exhibited round, uniform macrocytes.

Though not specific for one condition, an elevated MCV of >100 fl, is a useful parameter directing further investigations which can ultimately lead to a definitive diagnosis in 75% of cases [16]. It may be the only indicator of vitamin deficiency and alcoholism [17]. Mahmoud et al. [16] believe that unexplained macrocytosis in the elderly (≥75 years) could be an early sign of Myelodysplastic syndrome. Joseph et al. [26] have observed that refractory, unexplained macrocytosis may be an early sign of smoldering leukemia. Therefore regardless of the hemoglobin levels, close attention should be paid to MCV [27, 28].

Macrocytosis needs to be evaluated even in the absence of anemia, as it may be the first clue to an underlying pathology. Complete medical history including alcohol and drug intake, proper evaluation of red cell parameters and peripheral smear assist in arriving at a provisional diagnosis, thereby directing further management. This approach helps in determining the cause of macrocytosis, particularly in resource limited settings.

Conclusion

Macrocytosis is most commonly caused by alcoholism, Vitamin B12 deficiency and medications. Even in the absence of anemia, an increased MCV needs to be evaluated, as it may be the only clue to an underlying pathological condition. Complete history including medication and alcohol intake, proper evaluation of the red cell parameters and peripheral blood smear help in arriving at a provisional diagnosis in majority of cases. Red cell parameters and peripheral smears are obtained routinely and serve as simple, inexpensive and valuable tools in identifying the cause of macrocytosis, particularly in resource limited areas.