Assessment of bone marrow iron stores using Gale's grading and its correlation with iron deficiency anemia

Ishani Gupta; Bhavneet Kour; Roopali Jandial; Subhash Bhardwaj

doi:10.4103/jmedsci.jmedsci_420_20

ORIGINAL ARTICLE

Year : 2022 | Volume : 42 | Issue : 4 | Page : 160-165

Assessment of bone marrow iron stores using Gale's grading and its correlation with iron deficiency anemia

Ishani Gupta, Bhavneet Kour, Roopali Jandial, Subhash Bhardwaj
Department of Pathology, Government Medical College, Jammu, Jammu and Kashmir, India

Date of Submission	16-Dec-2020
Date of Decision	28-Apr-2021
Date of Acceptance	22-May-2021
Date of Web Publication	25-Nov-2021

Correspondence Address:
Dr. Bhavneet Kour
Department of Pathology, Government Medical College, Jammu, Jammu and Kashmir
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jmedsci.jmedsci_420_20

Abstract

Background: Iron deficiency anemia (IDA) is a very common condition worldwide, especially in low- and middle-income countries. The most accurate method to evaluate iron status is the measurement of bone marrow iron content by potassium ferrocyanide (Prussian blue)-stained aspirate. Microscopic examination of Prussian blue-stained bone marrow aspirate has been considered the “gold standard” for determining iron-depleted states. Aim: The aim of this study was to assess and classify the iron status in the bone marrow by both Gale's and intensive method, thereby differentiating iron store deficiency from functional deficiency. Methods: The present study was carried out in the Department of Pathology, Government Medical College and Hospital, Jammu, over a period of 1 year. A total of 135 cases of anemia with hemoglobin <10 g/dl were included in the study. Bone marrow aspiration was done; iron assessment was done by both Gale's and intensive method. Iron status assessed by the intensive method was categorized as normal, functional iron deficiency, iron stores deficiency, or combined functional iron and iron stores deficiency. Results: A total of 135 cases were included in the study, out of which 47% (64/135) were male and 53% (71/135) were female. Gale's study revealed hypoferrimic state in only 40 cases out of 135 (29.6%). According to the intensive method, out of these 40 cases, only nine cases were truly deficient cases that too had combined deficiency, two cases had iron store deficiency, 25 cases had functional iron deficiency, and four cases had normal iron stores. In the present study, according to the intensive method, maximum number of cases had functional iron deficiency (54%, 73/135). There were 1.4% (2/135) pure iron-deficient cases and 11.1% (15/135) revealed combined deficiency. Conclusion: Differentiation between IDA and functional iron deficiency is important for accurate diagnosis and treatment of the patient. Although intensive method of grading iron requires more expertise, it has proved to be superior and has more precision in diagnosing iron-deficient cases.

Keywords: Anemia, Gale's method, intensive method, iron deficiency anemia

How to cite this article:
Gupta I, Kour B, Jandial R, Bhardwaj S. Assessment of bone marrow iron stores using Gale's grading and its correlation with iron deficiency anemia. J Med Sci 2022;42:160-5

How to cite this URL:
Gupta I, Kour B, Jandial R, Bhardwaj S. Assessment of bone marrow iron stores using Gale's grading and its correlation with iron deficiency anemia. J Med Sci [serial online] 2022 [cited 2023 May 31];42:160-5. Available from: https://www.jmedscindmc.com/text.asp?2022/42/4/160/353048

Introduction

Iron deficiency anemia (IDA) is the most common condition worldwide, especially in countries having population falling under low- and middle-income groups.^[1] Majority of cases of anemia are due to IDA. It is one of the major causes of maternal and child mortality, low physical performance, and referrals to healthcare professionals.^[2] According to the 2003 report by National Nutrition Monitoring Bureau, India, the incidence of IDA is more among young children, adolescents, and pregnant women.^[3] Lactating mothers (78%), pregnant women (75%), adolescent girls (70%), and preschool children (67%) are the major population groups, which are at risk of IDA.^[1]

The most conventional method of assessing iron stores is through bone marrow fragments, which represent the presence of iron stores in the form of insoluble iron storage compound hemosiderin.^[4],[5] The most accurate method for evaluation of iron status is by measuring the bone marrow iron content by potassium ferrocyanide (Prussian blue)-stained aspirate.^[6],[7] Bone marrow aspiration is an invasive procedure; therefore, assessment of serum ferritin (S. ferritin) and transferrin saturation is used frequently in daily clinical practice for assessing iron status as they are noninvasive.^[8] Nowadays, S. transferrin receptor and zinc protoporphyrin have been put to use mainly in inflammatory conditions. However, due to limited availability of assay facilities and higher cost, their use is restricted in developing countries.^[9],[10] The iron stores in the bone marrow start to decrease long before its reflections are seen in the levels of hemoglobin and in the appearance of clinical manifestations of anemia. S. ferritin is mainly found in the cytoplasm of cells of reticuloendothelial system.^[11] Measurement of these levels helps in identification of the bone marrow iron store levels in iron-deficient patients, but a minimum optimum level below has yet not been defined in the literature. This is mainly because S. ferritin is also a positive acute-phase reactant.^[12] Therefore, the use of serum iron (S. iron) markers may not differentiate between depleted iron stores and conditions associated with defective reticuloendothelial release of iron (functional iron deficiency).^[5] Although mass spectrometry is being recently used to give a definitive determination of iron in tissue, microscopic examination of Prussian blue-stained bone marrow aspirate smears has been considered as the “gold standard” for determination of iron depletion of stores.^[13]

According to the World Health Organization, IDA is diagnosed with S. ferritin cutoff levels of <34 pmol/L (15 ng/mL) among adults, <27 pmol/L (12 ng/mL) among children aged <5 years, and 67 pmol/L (30 ng/mL) where inflammation is coexistent in adults.^[14] The normal reference range is 29–508 pmol/L (13–226 ng/mL).^[15] The reference range and cutoff for iron deficiency based on the marrow iron in anemic patients might be different compared to those without anemia. In most studies, absent iron was taken as reference, but it may not be appropriate when Gale's grading is used as grades 0 and 1 are deficient. However, the conventional Gale's method^[4] of assessing iron in the marrow fragments alone provides little information about the functional iron-deficient state.^[5]

In view of importance of assessing the iron status, we performed this study with a dual purpose; first, to perform an intensive bone marrow iron grading^[5] by assessing iron in fragments, macrophages, and erythroblasts, thereby differentiating iron store deficiency from functional iron deficiency; and second, to assess and classify the iron status in bone marrow by both Gale's and intensive method.^[4],[5]

Materials and Methods

This retrospective study was carried out in the Department of Pathology, Government Medical College and Hospital, Jammu, over a period of 1 year (March 2019 to February 2020). A total of 135 cases of anemia with hemoglobin <10 g/dl were included in the study. All the subjects with hemoglobin <10 g/dl, on iron and folic acid therapy, or with recent history of blood transfusion were excluded from the study. Peripheral venous blood was collected for the assessment of hemoglobin and other hematological parameters. The study was approved by the institutional ethical committee (IEC/GMC/Cat C/2020/160).

After written informed consent, bone marrow aspirate was obtained from the posterior superior iliac spine under strict asepsis, spread on to a slide, air-dried, fixed with methanol, and stained with Perl's Prussian blue stain. A positive control was included with each batch of slides. Bone marrow smears with minimum of seven fragments were subjected for microscopic examination.^[2] Peripheral venous blood of the subjects was collected at the same setting for hemoglobin and S. ferritin level estimation.

The Gale's grading method^[4] was first used to assess iron in the marrow fragment [Table 1]. Grades 0 and 1 corresponding to none and very slight marrow iron, respectively, indicate iron store deficiency [Table 1].

Table 1: Grading for bone marrow iron status according to Gale et al. [Figure 2]

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Next, a more intensive method of assessing marrow iron in three sites, notably in the fragments, macrophages around the fragments, and erythroblasts, was performed^[5] [Table 2]. The marrow fragment iron was assessed according to the Gale's method. Under oil immersion, 20 fields around the fragments were examined for the presence of iron in macrophage, and 100 erythroblasts were examined and the percentage of sideroblasts was (erythroblasts containing iron granules in their cytoplasm) noted.

Table 2: Classification of iron status using the intensive grading method [Figure 3]

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Iron status assessed by the intensive method was categorized as normal, functional iron deficiency, iron stores deficiency, or combined functional iron and iron stores deficiency.^[5]

The statistical analysis was performed using SPSS statistics software version 17.

Results

A total of 135 cases were included in the study, out of which 47% (64/135) were male and 53% (71/135) were female. The minimum age of the study subjects was 1 year and the maximum age was 79 years. 36.2% (49/135) of cases presented with mild anemia (8–10 g/dl), and 33.3% (45/135) and 30.3% (41/135) of cases presented with moderate anemia (6–8 g/dl) and severe anemia (<6 g/dl), respectively. The minimum hemoglobin noted was 4 g/dl, and the maximum was 10 g/dl. In the present study, Gale's grading was done, and it revealed hypoferrimic state (Gale's Grade 0–1+) in only 40 cases out of 135 (29.6%) [Table 3]. According to the intensive method, out of these 40 cases, only nine cases were truly deficient cases that too had combined deficiency, 2 cases had iron store deficiency, 25 cases had functional iron deficiency, and 4 cases had normal iron stores [Table 4]. In the present study, according to the intensive method, the maximum number of cases had functional iron deficiency (54%, 73/135). There were 1.4% (2/135) pure iron-deficient cases and 11.1% (15/135) revealed combined deficiency.

Table 3: Distribution of iron status of study subjects according to gale's grading

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Table 4: Comparison of grades of both Gale's and intensive method of all the cases included in the study

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Mean log ferritin concentration was significantly lower in patients with depleted iron stores (0.96 μg/l), in comparison to those with normal iron stores (2.32 μg/l; P = 0.001), or functional iron deficiency (2.75 μg/l; P = 0.0024), or combined iron deficiency (2.09 μg/l; P = 0.0023) [Table 5].

Table 5: Serum ferritin level in different iron status categories

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Discussion

It is a well-known fact that IDA is an important cause of nutritional anemia. Functional iron-deficient cases constitute a large percentage of the clinically diagnosed iron-deficient cases, and they need to be segregated from the pure iron-deficient cases. Functional iron deficiency develops when our normal physiological system for transporting iron is not able to deliver iron to the target tissues, despite the presence of satisfactory iron stores.^[14] This occurs because of release of cytokines as an acute-phase response to infection, which leads to impaired erythropoiesis and anemia which is also known as “anemia of inflammation.”^[15]

The clinical distinction between anemia attributable to iron store deficiency and functional iron deficient state is important so as to avoid unnecessary iron supplementation in the latter.^[16] In the present study, all the study subjects were anemic and were prescribed iron on an empirical basis without proper assessment of their iron status. After performing the study, it was found that the actual number of patients requiring iron supplementation was very low. It is very important to diagnose the iron-deficient cases, before starting iron therapy as unnecessary supplementation of iron, and its overtreatment can lead to toxicities and diversion from the correct approach to diagnosis.

The literature shows that various studies in the past have included serum ferritin (S. ferritin) and other markers as an important component for assessing body iron reserve as it reflects the total body iron stores and its low level indicates hypoferrimic state.^[3],[5] On the contrary, it is a well-known fact that low S. ferritin can help in diagnosing iron deficiency, but high S. ferritin does not necessarily exclude out iron deficiency as it is a false-positive indicator for iron deficiency in cases of chronic inflammation, which constitutes a major percentage of cases of anemia.

Microscopic examination of a Perl's Prussian blue-stained bone marrow aspirate smears is widely considered as the “gold standard” for the assessment of the marrow iron store.^[5] The commonly used Gale's histologic grading method evaluates storage iron in the marrow fragment alone, whereas intensive method of assessing marrow iron assesses iron in marrow fragments, in macrophages around fragments, and in erythroblasts.^[4] Fragment and macrophage iron in the bone marrow reflects iron stores [Figure 1], [Figure 2], [Figure 3], [Figure 4] while iron in the erythroblast indicates utilizable iron which is decreased in functional iron deficiency.^[17] The intensive method of marrow iron grading scores over the Gale's method and S. iron markers in differentiating between functional and quantitative iron deficiency, which is particularly valuable in developing nations with high prevalence of infection and inflammation.^[5]

Figure 1: Bone marrow smear showing iron in macrophage (Perl's Prussian blue stain, ×100)

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Figure 2: Bone marrow smear showing iron in erythroblast (Perl's Prussian blue stain, ×100)

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Figure 3: Bone marrow smear showing dense large clumps of iron in throughout marrow fragment-Gale's Grading-5 (Perl's Prussian blue stain, ×40)

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Figure 4: Bone marrow smear showing very large deposits of iron particles both in intra- and extra-cellular marrow fragment-Gale's Grading-6 (Perl's Prussian blue stain, ×40)

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It has been proved in the present study that functional iron-deficient cases comprise maximum percentage of anemic cases, which account to 54% (73/135) by the intensive method of iron assessment. This was also seen in study by Kaushik et al., in which 57.26% (67/117) of cases were functional deficient.^[13] Another study by Phiri et al. proved that the functional iron deficiency constituted the most common iron status category comprising 39.6% (74/187) cases.^[5]

This study demonstrated iron deficiency in 29.6% of cases and normal iron status in 69.4% of cases, which correlates with the results of another study by Bableshwar et al.^[18] Bableshwar et al. found iron deficiency in 38.75% of cases and normal iron stores in 61.25% of cases. These 29.6% of cases (40/135) were further narrowed down to 6.66% (9/135) of cases by the intensive method. Hence, the results of the present study prove that the intensive method for assessing iron is superior over Gale's method.

The intensive method is proved to be better for diagnosis and specificity in IDA. It segregates the iron-deficient cases from functional iron-deficient cases which is a drawback of Gale's method. Intensive method can accurately diagnose the iron-deficient cases and helps in correct treatment. The importance of assessment of bone marrow iron stores cannot and should not be ignored especially in the gray zone conditions where the use of S. iron markers gives borderline and fallacious results. Cases having raised S. ferritin should be segregated, especially if the history suggests the association of any infective/inflammatory condition. These cases should be subjected to iron assessment by bone marrow studies for getting precise and accurate results.

Limitation

The limitation of our study is that the comparison of IDA could not be done with various biochemical parameters such as S. ferritin, S. iron, and total iron-binding capacity as these tests were not available in our department. In future, a more extensive study which includes all these parameters is recommended.

Conclusion

Differentiation between IDA and functional iron deficiency is important for accurate diagnosis and treatment of the patient. S. iron parameters should be used for the diagnosis of IDA, but our observation lays emphasis on the use of bone marrow iron staining as a valuable test for excluding IDA in patients with indeterminate biochemical indices. Although intensive method of grading iron requires more expertise, it has proved to be superior and has more precision in diagnosing iron-deficient cases. Moreover, this method provides a clinically useful iron status classification which is of utmost importance in cases of anemia of chronic diseases characterized by functional iron deficiency, contrary to iron store depletion seen in IDA.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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