Cutaneous Hyperpigmentation in Megaloblastic Anemia: a Five Year Retrospective Review

Somanath Padhi¹, RajLaxmi Sarangi², Anita Ramdas¹, Kandasamy Ravichandran³, Renu G’Boy Varghese¹, Thomas Alexander⁴, George Kurian⁴ and Sudhagar Mookkappan⁵

Departments of ¹Pathology, ²Biochemistry, ³Biostatistics, ⁴Gastroenterology, and ⁵General Medicine, Pondicherry Institute of Medical Sciences, Puducherry, India.

Published: April 10, 2016
Received: January 31, 2016
Accepted: March 21, 2016
Mediterr J Hematol Infect Dis 2016, 8(1): e2016021, DOI 10.4084/MJHID.2016.021
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Introduction

Megaloblastic anemia (MA) is a heterogeneous group of reversible bone marrow failure syndromes characterized by a variable degree of peripheral blood cytopenia (s) in the presence of a normo or hypercellular bone marrow. The hallmark pathophysiologic mechanism of MA is an impairment of DNA synthesis in all nucleated cells secondary to vitamin B₁₂ (B₁₂) and/or folate deficiency, resulting in nuclear-cytoplasmic asynchrony; distinctive megaloblastic changes, increased apoptosis, and ineffective hematopoiesis in the bone marrow.[1] The manifestations of MA are diverse and may range from nonspecific signs and symptoms of anemia to gastrointestinal disturbances and potentially fatal neuropsychiatric and cardiovascular disorders.[2]
Megaloblastic anemia is not uncommon in the Indian subcontinent as well as other parts of Asia with females and vegetarians being more susceptible to B₁₂ deficiency. Various studies in the past have shown that occult B₁₂ deficiency may be rather prevalent among Indian urban and rural population.[3-5]
Cutaneous manifestations associated with B₁₂ deficiency include characteristic mucocutaneous hyperpigmentation (most common), vitiligo, angular cheilitis, and hair-nail changes, which are often missed or overlooked in early, asymptomatic phases of the disease.[6] In this manuscript, we describe the association of cutaneous hyperpigmentation (CP) with bone marrow changes in a series of 25 cases from a tertiary center in South India with a correlation of hematological and biochemical parameters, and also present a concise review of relevant literature. Furthermore, the association of pyrexia in MA is also briefly highlighted.

Materials and methods

In this retrospective study the bone marrow records of all adult cases (> 16 years), who underwent bone marrow aspiration (BMA) and trephine biopsy (Bx) over the last five years (October 2010 to December 2015) in the Department of Pathology of our Institute, were reviewed for the presence of CP. Informed written consent had been obtained from each case prior to the BM procedure; and the study was approved by the Institutional Ethics Committee.
As a part of the protocol for BM procedure, CP was prospectively documented in the bone marrow record by one of the authors (SP), prior to performing the procedure. Two types of pigmentation were documented: 1) dominant brownish-black pigmentation over dorsal aspect of hands and/or feet with accentuation over interphalangeal joints and periungual areas [knuckle pad (KP) group], and 2) diffuse and/or patchy, macular, dusky brownish-black discoloration/pigmentation over palms and/or soles (diffuse pigmentation (DP) group). Nature of BMA and gross appearance of Bx core were also recorded in each case. Data pertaining to routine hematological parameters such as hemoglobin (Hb), mean corpuscular volume (MCV), total leukocyte count (TLC), total platelet count (Plt), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and peripheral blood smear (PBS) findings were collected from laboratory records in each case at the time of BM procedure.
BMA smears and Bx sections of all the above cases were retrieved from the departmental archives. Two pathologists (AR, RGV) who were blinded to the presence and nature of the pigmentation, reviewed the slides for the presence of definite megaloblasts. In each case, two MGG (May Grunewald Giemsa) and one Prussian blue (Perl stain) stained BMA smears were available for morphological study and assessment of the iron stores, respectively. A definitive diagnosis of MA was rendered with a constellation of PBS and BM findings such as erythroid hyperplasia, altered myeloid to erythroid ratio (M:E), presence of definite megaloblasts with sieve-like chromatin, nuclear-cytoplasmic asynchrony, giant abnormal shaped metamyelocytes and/or band forms, and/or abnormal (multinucleated) megakaryocytes with adequate (1-3+) or increased (≥ 4+) iron stores. A diagnosis of dimorphic marrow picture was rendered when there was admixture of both megaloblast and micronormoblast (with reduced hemoglobinization) and reduced (1+) or absent (0) iron store on Perl stain.[7] When definite megaloblasts were not seen or very sparse, but dyspoietic changes were present, a possibility of macronormoblastic erythroid maturation with dyspoiesis was reported. Furthermore, those with dyspoietic changes and increased iron stores (≥ 4+) were also screened for the presence of ringed sideroblasts for a possible diagnosis of myelodysplastic syndrome (MDS) in the appropriate clinical setting.
Bone marrow changes were then correlated with the presence or absence and nature of hyperpigmentation, and serum biochemical parameters such as serum B₁₂, folate, ferritin, and iron levels (wherever available). Serum B₁₂ (ref.: 190 to 800 pg/ml), folate (ref.: 4 to 20 ng/ml), and ferritin levels [ref.: 30 to 400 ng/ml (males), 15 to 150 ng/ml (females)] were measured by electrochemiluminescence immunoassay (ECLIA) technique in Cobas e411 automated analyzer (Roche, Germany). Serum iron [ref.: 50 to 168 μg/dl (males), 35 to 145 μg/dl (females)] was measured in a semi-auto analyzer (Microlab 300, Merck) by using commercially available kits. Cases with B₁₂ level <190 pg/ml were considered as B₁₂deficient and those with levels < 100 pg/ml were considered as severe B₁₂ deficiency.[2] Similarly, cases with folate levels < 4ng/ml were considered as folate deficient. Cases with serum ferritin < 30 ng/ml were considered as depleted iron stores; and those with < 15 ng/ml were considered as a severe iron deficiency.
Demographic profile, place of origin, dietary habits, history of prior illness and medications (if any), provisional clinical diagnoses, relevant clinical, microbiological and/or serological data were collected from the case records. All cases where a diagnosis of MA was made received parenteral (intramuscular) cyanocobalamin (1000 μg/day) for 7 days, followed by a weekly dose of the same for a minimum of 10 to 12 weeks. Those with dimorphic anemia were prescribed both parenteral and/or oral cobalamin in addition to hematinics and multivitamins.
Statistical analysis: Continuous variables were described by mean (± SD) and Box plot were used to highlight the distribution of variables between two groups. Comparison between two groups was made using Fisher’s exact method for categorical variables, student t-test for normally distributed continuous variables, and Mann-Whitney test for not normally distributed continuous variables. All the tests were 2 sided; and a P value less than 0.05 was considered as statistically significant. SPSS software (version 20.0) was used for analyzing the data.

Results

During the study period, a total of 304 patients underwent a BM examination, out of which 198 were personally performed by one of the authors (SP). All of these cases had been assessed for the presence or absence of CP at the time of the BM examination and hence were included in the analysis. CP was documented in 25 of these 198 patients (12.6%).
The demographic and clinico-laboratory characteristics of all 25 cases are presented in Tables 1 and 2. There were 16 males (64%) and 9 females (36%) with a mean age of 41.2 ± 16.7 years. Among the cases, 21 (84%) were dark skinned South Indians (in and around Puducherry). Seventeen of 25 (68%) were taking a mixed diet while only 8 (32%) were vegetarians. Seven of 16 (43.7%) males and none of the females had a history of alcohol abuse. Fatigue was the most common clinical presentation noted in 15/25 (60%) cases; 3/25 (12.0%) were diagnosed cases of autoimmune hepatitis on periodic follow-up with maintenance drugs such as azathioprine and prednisolone, 3/25 (12%) had evidence of gastric atrophy (anti-intrinsic factor antibody positive in one), 5 (20%) presented with fever, 2 (8%) had history of diarrhoea, 1 (4%) was a case of non-alcoholic steatohepatitis (NASH) secondary to uncontrolled type 2 diabetes mellitus, and 1 (4%) was a case of paranoid schizophrenia on olanzapine.

Table 1. Base line characteristics of cases with cutaneous hyperpigmentation which was documented during bone marrow procedure (October 2010-December 2015).

Table 2. Cutaneous hyperpigmentation: clinicolaboratory profile of 25 cases (October 2010-December 2015).

The association of MA with CP was highly significant (P<0.001) with 21 (out of 25) patients having MA [20 had pure MA and 1 had mixed MA and iron deficiency anemia (IDA)] as compared to only 12 patients with MA (5 had pure MA and 7 had mixed MA and IDA) among 173 patients without pigmentation (Table 3). Prominent knuckle pad hyperpigmentation (KP) was documented in 16 of 25 (64%) cases whereas 9/25 (36%) cases had patchy or diffuse, dusky, brownish-black pigmentation over palms and/or soles with accentuation over palmar creases (DP) (Figures: 1A, 1B, 1C, 1D). Eighteen of 25 (72%) had pancytopenia (13 with KP, 5 with DP) and 7 (28%) had bicytopenia (3 with KP, 4 with DP). The mean Hb, MCV, TLC, and Plt were 75.4 ± 28.4 g/L, 103.7 ± 11.4 fL, 3.2 ± 1.2 x 10⁹/L, and 57.4 ± 32.5x10⁹/L, respectively.

Table 3. Association of megaloblastic anemia and presence of cutaneous hyperpigmentation.

Peculiar cutaneous hyperpigmentation from cases with megaloblastic anemia: typical diffuse, brownish-black discoloration of the palms (1A), knuckle pad hyperpigmentation in the dorsum of hands (1B) in a case of megaloblastic anemia (prior to therapy) in a 52 year old female who had gastric atrophy, proven on endoscopic biopsy (case no;18, Table 2). This patient had pancytopenia, macrocytosis (MCV; 116fL), and severe B12 deficiency (55pg/ml). Note the reversal of pigmentation (4A, 4B) in the same patient, 12 weeks after initiation of parenteral cyanocobalamin therapy. Diffuse brownish-black pigmentation over dorsal aspect of feet (1C) and dusky, brownish-black discoloration of palms with accentuation of palmar creases (1D) in a 52 year old vegetarian male with fever, jaundice, pancytopenia, macrocytosis (MCV; 115fL), and florid megaloblastic anemia proven on bone marrow examination (B12 and folate assay not done) (case no; 23, Table 2).

The marrow findings were consistent with MA in 20/25 (80%) cases [severe in 12, moderate in 5, and mild/focal in 3); dimorphic anemia with both megaloblasts and micronormoblasts in 1 case (4%); and dyspoietic changes with ringed sideroblast (3% of erythroid nuclei) suggestive of MDS was noted in one case (4%) (Figures: 2A, 2B, 2C). Nonspecific marrow findings and dyspoietic erythroid changes in the form of either macronormoblastic erythroid maturation, binucleation, nuclear budding, internuclear bridging were described in remainder 3 (12%) cases. There was no evidence of any granulomas, necrosis, organisms, hemoparasites, or any malignancy noted in any of the cases on the bone marrow. Detailed systemic, radiological, and endocrinological examinations were unremarkable in all cases and blood and BMA culture did not reveal any growth in cases with pyrexia. 

Figure 2. Bone marrow aspirate in megaloblastic anemia with cutaneous hyperpigmentation. Note the richly particulate bone marrow aspirate (2A) obtained during bone marrow procedure in cases with cutaneous hyperpigmentation and cytopenia (s). Bone marrow aspirate smears demonstrating erythroid hyperplasia and megaloblasts with sieve-like nuclear chromatin (2B, thick arrow) and giant, abnormal shaped stab forms (2C, thin arrow). These findings were consistent with a diagnosis of megaloblastic anemia (May Grunewald Giemsa, X400).

Serum B₁₂ and folate levels were available in 17/25 cases. It was found that 11/17 (64.7%) were B₁₂ deficient (<190 pg/ml) among which 8 had severe B₁₂ deficiency (<100 pg/ml); 4/17 (23.5%) were folate deficient (< 4 ng/ml); and 2/17 (11.8%) had combined B₁₂ and folate deficiency.
KP group had lower Hb (69.6 ± 24.2 vs. 86.3 ± 33.9 g/L, respectively, P=0.19), higher MCV (106.1 ± 12.6 vs. 99.2 ± 7.6 fL, respectively, P=0.18), lower Plt (50.9 ± 29.3 vs. 69.6 ± 36.5x10⁹/L, respectively, P=0.15) than the DP group (Table 4). Similarly, the median (50^th quartile) and interquartile (25^th to 75^th quartile) range for Hb and Plt were lower in the former group than the latter [64.0 (52.0 - 80.0) vs 81.0 (60.3 - 116.0) g/L; 41.0 (30.0 - 56.0) vs. 58.0 (36.8 - 100.0) x 10⁹/L, respectively]. However, median and interquartile range for MCV were higher between two groups [108.0 (99.0 - 114.0) vs. 97.0 (94.3-102.8) fL, respectively, P>0.05]. Median and interquartile range of serum B₁₂ values in the KP group was lesser [100.0 (30 – 822.0) pg/ml] compared to the later group [316.0 (142.0 –1617.3) pg/ml] (P = 0.17); whereas median serum folate levels were similar between two groups [6.0 (3.0 – 18.0) vs. 5.0 (4.3 – 17.0) ng/ml, respectively, P=0.79] (Table 4, Figures: 3A-3E).

Table 4. Comparative characteristics between two groups of cases with cutaneous hyperpigmentation.

Figure 3. Box plot diagram depicting the comparison of median (50th quartile, black horizontal line) and interquartile (25th to 75th) range of mean corpuscular volume (MCV) (A), Hb (B), Platelets (C), B12 (D), and folate (E) levels among two groups of pigmentation [knuckle pad (KP) vs diffuse type (DP)]. Note that the median and interquartile range of Hb, Platelets, and serum B12 were lower in the KP group than in the DP group; whereas the median and interquartile range of MCV were higher in KP group than the DP group. Also note that the group with DP has a wider B12 value compared to the KP group (D). The median value of serum folate was similar among two groups.

Six of 25 cases where follow-up data were available, showed dramatic improvement (> 85%) in their hyperpigmentation following 12 weeks B₁₂ therapy (Figures: 4A and 4B), whereas the rest were lost to follow-up. In all the five cases who presented with pyrexia, their fever subsided following day 3 to 4 of starting parenteral cyanocobalamin therapy.

Figure 4. Reversal of hyperpigmentation in the patient of figure 1A and 1B, 12 weeks after initiation of parenteral cyanocobalamin therapy.

Table 5. Review of literature related to hyperpigmentation in vitamin B12 and/or folate deficiency.

Figure 5. The postulated biochemical mechanism of hyperpigmentation in megaloblastic anemia.[6,16] The 4 most accepted mechanisms involved are: 1) low methylcobalamin level in melanocytes leads to reduced level of reduced glutathione (GSSH); which in turn activates Tyrosinase enzyme in melanin synthesis pathway, 2) defective DNA synthesis activates Microphthalmia-associated transcription factor (MITF); which causes activation of both Tyrosinase and Tyrosinase related protein 1 and 2 (TRP 1 and 2),[16] 3) hyperhomocysteinemia leads to accumulation of cysteine leading to increased melanin synthesis, 4) defective melanin transfer from the melanocytes to adjacent megaloblastic keratinocytes. Increased angiogenesis secondary to upregulation of dermal vascular endothelial growth factor (VEGF) may also lead to increased pigmentation.[18] Both histopathologic and ultrastructural studies have postulated that hyperpigmentation is due to increased number of basal melanocytes as well as increased melanosomes.[19]