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ORIGINAL ARTICLE  
Year : 2013  |  Volume : 56  |  Issue : 1  |  Page : 16-19
Intensive method of assessment and classification of the bone marrow iron status: A study of 80 patients


1 Consultant Pathologist, Hi-tech laboratory, KLES Dr. Prabhakar Kore Hospital and Medical Research Centre, Belgaum, India
2 Department of Pathology, KLE University's Jawaharlal Nehru Medical College, Belgaum, India
3 Department of Pathology, KLE University's Jawaharlal Nehru Medical College, Belgaum, Karnataka, India

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Date of Web Publication6-Aug-2013
 

   Abstract 

Background: The increasing prevalence of multiple co-morbidities among anemic patients with chronic diseases have made the use of serum ferritin (which is also an acute phase reactant) and transferrin saturation more challenging in diagnosing iron deficiency. Microscopic examination of bone marrow aspirate is the gold standard" for assessing marrow iron store. However, conventional Gale's method assesses iron in marrow fragments alone which provides little valuable information about functional iron deficiency seen in many chronic diseases. Aim: To perform an intensive bone marrow iron grading by assessing iron in fragments, in macrophages around fragments and in erythroblasts and to correlate the marrow iron store results with serum ferritin. Materials and Methods: A descriptive study of Perl's Prussian blue stained bone marrow aspirate smears of 80 adult patients with moderate to severe anemia. Bone marrow iron was assessed by both the Gale's method and the intensive method and correlated with serum ferritin. Results: The intensive grading system revealed normal iron stores in 37.5% cases, depleted iron stores in 16.25% patients while 23.75% and 22.5% patients had functional iron deficiency and combined deficiency, respectively. Mean log ferritin concentration was significantly lower in patients with depleted iron stores (0.91 μg/l) in comparison to those with normal iron stores (2.13 μg/l; P = 0.001), functional iron deficiency (2.65 μg/l; P = 0.000), or combined deficiency (2.04 μg/l; P = 0.002). Conclusion: Intense marrow iron examination provides a useful iron status classification which is of particular importance in cases of chronic diseases and inflammation.

Keywords: Bone marrow iron, Gale′s method, intensive method

How to cite this article:
Bableshwar RS, Roy M, Bali A, Patil PV, Inumella S. Intensive method of assessment and classification of the bone marrow iron status: A study of 80 patients. Indian J Pathol Microbiol 2013;56:16-9

How to cite this URL:
Bableshwar RS, Roy M, Bali A, Patil PV, Inumella S. Intensive method of assessment and classification of the bone marrow iron status: A study of 80 patients. Indian J Pathol Microbiol [serial online] 2013 [cited 2020 Feb 27];56:16-9. Available from: http://www.ijpmonline.org/text.asp?2013/56/1/16/116142



   Introduction Top


Nutritional anemia, particularly iron deficiency, continues to be a major public health problem worldwide, particularly in the developing nations. [1]

A combination of surrogate markers, namely serum ferritin, serum iron, total iron binding capacity, and percentage saturation of transferrin are routinely employed to assess the iron status of an individual. [2],[3],[4],[5] Serum ferritin reflects the total body iron store and a low level is indicative of a hypoferremic state. [2],[3] However, ferritin is also an acute phase response protein, whereby concentrations increase during infection, inflammation, liver disease, malignancy, hemodialysis, and thereby rendering the interpretation of normal or high serum ferritin values difficult in such conditions. [3] The serum iron markers, therefore, may not discriminate between depleted iron stores and conditions associated with defective reticuloendothelial release of iron (functional iron deficiency). [4],[6]

The microscopic examination of stainable iron in the bone marrow aspirate smear is generally considered the reference standard for determining the body iron stores. [2],[6] However, the conventional Gale's method [7] of assessing iron in marrow fragments alone provides little valuable information about the functional iron deficient state. [6]

We undertook this study with a dual purpose; firstly, to perform an intensive bone marrow iron grading [6] by assessing iron in fragments, macrophages and erythroblasts, facilitating distinction of iron store deficiency from functional iron deficiency and secondly, to correlate the marrow iron store results with serum ferritin.


   Materials and Methods Top


We conducted the study on 80 adult patients admitted with moderate to severe anemia and no history of transfusion in preceding 4 weeks, in whom a diagnostic bone marrow examination was requested by the clinician, at a tertiary care hospital from January to October 2011. The study was approved by the institution ethics committee.

Bone marrow aspirate was obtained after written informed consent from the posterior superior iliac spine observing 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 in each batch of slides. Marrow smears with at least seven fragments were subjected for microscopic examination. [2] Peripheral venous blood was collected at the same setting for hemoglobin and serum ferritin level estimation.

The Gale's grading method [7] was first employed to assess iron in the marrow fragment [Table 1]. Grades 0 and 1 corresponding to none and very slight marrow iron, respectively, were considered indicative of iron store deficiency.
Table 1: Gale's method of bone marrow iron grading

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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. [6] The marrow fragment iron was assessed as per the Gale's method. Under oil immersion, 20 fields around the fragments were examined for the presence of macrophage iron and 100 erythroblasts were examined and the percentage of sideroblasts (erythroblasts containing iron granules in their cytoplasm) noted [Figure 1]. Erythroblast iron deficiency was considered when <30% of erythroblasts had iron granules.
Figure 1: (a) Bone marrow fragment showing iron deposit (Perl's Prussian blue stain ×400); (b) Iron granules in erythroblast cytoplasm (arrow depicti ng a ring sideroblast; Perl's Prussian blue stain ×1000)

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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 [6] [Table 2].
Table 2: Bone marrow iron status classifi cati on by the intensive grading method

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Bone marrow iron results were compared with serum ferritin level determined by micro particle enzyme immunoassay (Abbott Axsym R system). Hemoglobin was estimated using the automated cell counter (Beckman Coulter LH 500).

The statistical analysis was performed using SPSS version 17.


   Results Top


Of the 80 adult patients recruited in the study, 60% (48/80) were male and 40% (32/80) were female. The minimum age was 16 years and maximum 86 years (average 41.6 years). Forty-three (53.75%) patients presented with severe anemia (hemoglobin <7 g/dl), whereas 37 (46.25%) patients had moderate anemia (hemoglobin 7-10 g/dl). The minimum hemoglobin noted was 1.9 g/dl and maximum 9.2 g/dl (mean 6.1g/dl).

The iron status category of the 80 patients assessed by both the Gale's method and intensive method is shown in [Table 3]. The Gale's grading method revealed hypoferremic state in 38.75% cases and normal iron stores in 61.25% cases. The intensive grading system demonstrated normal marrow iron store in 37.5% cases, 16.25% patients exhibited depleted iron stores, 23.75% had functional iron deficiency, and combined deficiency was detected in 22.5% patients.
Table 3: Bone marrow iron status category results (n=80)

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Mean log ferritin concentration was significantly lower in patients with depleted iron stores (0.91 μg/l) in comparison to those with normal iron stores (2.13 μg/l; P = 0.001), functional iron deficiency (2.65 μg/l; P = 0.000), or combined deficiency (2.04 μg/l; P = 0.002). However, the interpretation of disproportionately high ferritin level observed in patients with chronic diseases and functional iron deficiency proved challenging in assessing the body iron store ( P = 0.164). Also, the serum ferritin level was ineffective in distinguishing the functional iron deficiency from combined deficiency (P = 0.098) [Table 4] [Figure 2].
Figure 2: Error bar graphs for mean log ferriti n for the diff erent iron status categories using the intensive grading method

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Table 4: Serum ferriti n level in diff erent iron status categories

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   Discussion Top


The third National Family Health Survey (NFHS-3) conducted in 2005-06 estimates an alarmingly high prevalence of anemia in India, with 79% children, 70% pregnant women, and 24% adult men falling prey to the disease. [8],[9]

The clinical distinction between anemia attributable to iron store deficiency and functional iron deficient state, hallmark of anemia of chronic disease evident in patients with various inflammatory, autoimmune, and malignant disorders, is important to avoid unnecessary iron supplementation in the latter. [10]

Weiss and Goodnough [11] described the mechanism of imbalance in iron homeostasis noted in patients with chronic diseases. An increased iron uptake within the reticuloendothelial system cells consequent to up-regulation of protein divalent metal transporter 1 (DMT1) and transferring receptor expression is caused by the pro-inflammatory cytokines. This coupled with increased hepatic expression of the acute-phase protein hepcidin resulting in macrophage iron retention by internalization, degradation of ferroportin, a transmembrane protein exporter of iron, and inhibition of duodenal iron absorption explains the iron-deficient erythropoiesis despite normal or increased bone marrow iron content in anemia of chronic disease. [11]

The body iron reserve is traditionally assessed by the biochemical markers of iron metabolism, namely serum ferritin, serum iron, total iron binding capacity, and percentage saturation of transferrin. [2],[4],[5],[12]

Ferritin is the principle iron storage compound in the body. Serum ferritin concentration results from the leakage of tissue ferritin which is an intracellular hollow protein shell with a molecular weight of ~ 450 kDa and composed of 24 subunits surrounding an iron core. [3] Serum ferritin reliably reflects the storage iron in the absence of inflammation and a reduced level serves as a sensitive early indicator of depletion of body iron store. However, the fact that ferritin is also an acute phase response protein undermines its predictive ability in the setting of anemia co-existing with infection and inflammation. [2],[3],[10]

More recently, serum transferrin receptor and zinc protoporphyrin have been used as more accurate indicators of iron status. However, their use in developing nations is restricted by the limited availability of assay facility and higher cost. [13],[14]

Microscopic examination of a Perl's Prussian blue stained bone marrow aspirate smears is widely regarded as the "gold standard" for the assessment of the marrow iron store. [6],[15] The commonly practiced Gale's [7] histologic grading method evaluates storage iron in marrow fragment alone. Phiri et al.[6] employed an intensive method of assessing marrow iron in 303 children (aged 6 to 60 months) assessing iron in marrow fragments, in macrophages around fragments, and in erythroblasts. Fragment and macrophage iron reflect iron stores while iron in the erythroblast is indicative of utilizable iron which is diminished in functional iron deficiency. [16] The intensive method of marrow iron grading scores over the Gale's method and serum iron markers in facilitating distinction between functional and quantitative iron deficiency, which is particularly valuable in developing nations with high prevalence of infection and inflammation. [6]

In the present study, the intensive marrow iron assessment in adult patients with moderate to severe anemia allowed a precise iron status classification into four categories; normal, iron store deficiency, functional iron deficiency and combined store, and functional deficiency. Furthermore, we attempted correlation of the marrow iron results with the serum ferritin level.


   Conclusion Top


Intensive marrow iron examination provides a clinically useful iron status classification which is of particular importance in cases of anemia of chronic diseases characterized by functional iron deficiency as opposed to iron store depletion seen in iron deficiency anemia.

 
   References Top

1.Looker AC, Dallman PR, Carroll MD, Gunter EW, Johnson CL. Prevalence of iron deficiency in the United States. JAMA 1997;277:973-6.  Back to cited text no. 1
    
2.Hughes DA, Stuart-Smith SE, Bain BJ. How should stainable iron in bone marrow films be assessed? J Clin Pathol 2004;57:1038-40.  Back to cited text no. 2
    
3.WHO. Serum ferritin concentrations for the assessment of iron status and iron deficiency in populations. Vitamin and Mineral Nutrition Information System. Geneva: World Health Organization; 2011.  Back to cited text no. 3
    
4.Krause JR, Stolc V. Serum ferritin and bone marrow iron stores. I. Correlation with absence of iron in biopsy specimens. Am J Clin Pathol 1979;72:817-20.  Back to cited text no. 4
    
5.Baynes RD. Assessment of iron status. Clin Biochem 1996;29:209-15.  Back to cited text no. 5
    
6.Phiri KS, Calis JC, Kachala D, Borgstein E, Waluza J, Bates I, et al. Improved method for assessing iron stores in the bone marrow. J Clin Pathol 2009;62:685-9.  Back to cited text no. 6
    
7.Gale E, Torrance J, Bothwell T. The quantitative estimation of total iron stores in human bone marrow. J Clin Invest 1963;42:1076-82.  Back to cited text no. 7
    
8.NFHS India 2005-06- National Family Health Survey-3 NFHS-3-Anemia among women and children. Mumbai: International Institute for Population, Sciences; 2006.  Back to cited text no. 8
    
9.Indian Council of Medical Research, National Workshop on Micronutrients, Background document. In: Toteja GS, Singh P, editors. New Delhi: Micro-Nutrient Profile of Indian Population; 2003.  Back to cited text no. 9
    
10.Wish JB. Assessing iron status: Beyond serum ferritin and transferring saturation. Clin J Am Soc Nephrol 2006;1:S4-8.  Back to cited text no. 10
    
11.Weiss G, Goodnough LT. Anemia of chronic disease. N Engl J Med 2005;352:1011-23.  Back to cited text no. 11
    
12.Thomas C, Thomas L. Biochemical markers and hematologic indices in the diagnosis of functional iron deficiency. Clin Chem 2002;48:1066-76.  Back to cited text no. 12
    
13.Hanif E, Ayyub M, Anwar M, Ali W, Bashir M. Evaluation of serum transferrin receptor concentration in diagnosing and differentiating iron deficiency anemia from anemia of chronic disorders. J Pak Med Assoc 2005;55:13-6.  Back to cited text no. 13
    
14.Zimmermann MB, Molinari L, Staubli-Asobayire F, Hess SY, Chaouki N, Adou P, et al. Serum transferrin receptor and zinc protoporphyrin as indicators of iron status in African children. Am J Clin Nutr 2005;81:615-23.  Back to cited text no. 14
    
15.Stuart-Smith SE, Hughes DA, Bain BJ. Are routine iron stains on bone marrow trephine biopsy specimens necessary? J Clin Pathol 2005;58:269-72.  Back to cited text no. 15
    
16.Baumgartner-Staubli R, Beck EA. Sideroblast score: A sensitive indicator of iron deficiency and hypoproliferative anemia. Acta Haematol 1977;57:24-31.  Back to cited text no. 16
    

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Correspondence Address:
Maitrayee Roy
Asha Nursing Home, Chiriamore, Barrackpore, 24 PGS (N), Kolkata, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.116142

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