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ORIGINAL ARTICLE
Year : 2014  |  Volume : 3  |  Issue : 4  |  Page : 221-224

A study of bone marrow iron storage in hematological disorder


Department of Pathology, P.D.U. Medical College, Rajkot, Gujarat, India

Date of Web Publication16-Oct-2014

Correspondence Address:
Krupal M Pujara
Matru-Ashish, 3, Navjyot Park, Kalavad Road, 150 F.T. Ring Road, Rajkot - 360 005, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2278-344X.143052

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  Abstract 

Background: Study of marrow in iron deficiency anemia is very much essential, and it helps to decide whether iron therapy would be of any use or not. Where sophisticated instruments and facilities are not available for measuring iron in the blood by chemical method, this simple technique, is efficient, cost-effective, and result oriented. Aim: The aim was to study the marrow finding in various blood disorders. To establish the fact "old is gold" as "bone marrow iron store by Prussian blue stain" still has its stand in hematology in era of genetic engineering markers study and ferrokinetic study. Materials and Methods: Bone marrow aspiration was performed in 73 patients. Marrow films were stained with hematoxylin and eosin stain, observed and then submitted for Prussian blue stain. Results: In a study of iron deficiency anemia 92.7% of cases received iron store grade in marrow in range of 0-1 (P = 0.001). In megaloblastic anemia, 70% of cases received in the range of 3-4 (P = 0.164), whereas all cases of dimorphic anemia received in the range of 2-3 (P = 0.000) when compared to the normal range of 1-2. Conclusion: Prussian blue stain is simple and helpful technique to see and measure body iron store semi-quantitatively. Furthermore, it helps to decide whether iron therapy would be useful or not.

Keywords: Bone marrow iron stores, iron deficiency anemia, Prussian blue staining


How to cite this article:
Pujara KM, Bhalara RV, Dhruva GA. A study of bone marrow iron storage in hematological disorder. Int J Health Allied Sci 2014;3:221-4

How to cite this URL:
Pujara KM, Bhalara RV, Dhruva GA. A study of bone marrow iron storage in hematological disorder. Int J Health Allied Sci [serial online] 2014 [cited 2019 Sep 20];3:221-4. Available from: http://www.ijhas.in/text.asp?2014/3/4/221/143052


  Introduction Top


Grohe (1861), Quinken (1868), Lavullory (1935), Mallory (1938), Highman (1942), Lillie (1954) used various methods for demonstration of iron. Perl's (1867) Prussian blue method for demonstration of iron is a standard method for iron store estimation in bone marrow till now. Principle given by Perl's was: Ferric iron deposits in tissue (present mostly as ferric iron within the storage protein ferritin) then react with the soluble ferrocyanide in the stain, to form insoluble Prussian blue dye (a complex hydrated ferric ferrocyanide substance) in situ (i.e. in place). They are then visualizable microscopically as blue or purple deposits, within cells. Nutritional anemia, particularly iron deficiency, continues to be a major public health problem worldwide, particularly in the developing nations. A combination of surrogate markers, namely serum ferritin, serum iron, total iron binding capacity (TIBC), and percentage saturation of transferrin are routinely employed to assess the iron status of an individual. To check iron status of an individual, most commonly performed tests are serum ferritin, serum iron, TIBC, percentage saturation of transferrin, etc., Also, anemic patients suffer from multiple co-morbidities, so their serum ferritin and transferrin saturation are altered in many conditions other than iron deficiency. Hence, these two parameters cannot be made the gold standard/absolute criteria for diagnosing iron deficiency anemia. Diseases associated with defective reticuloendothelial release of iron may be difficult to distinguish from the iron-deficient state since serum iron parameters (serum iron, percentage saturation of transferrin < 15%) may overlap. Assessment of the bone marrow biopsy iron stores was often necessary to resolve the diagnosis. Microscopic examination of bone marrow aspirate is the gold standard for assessing marrow iron store. [1] However, the conventional Gale's method of assessing iron in marrow fragments alone provides little valuable information about the functional iron-deficient state. [2]

Grading for iron storage

  • 0: No stainable iron [Figure 1]
  • 1+: Small iron particles just visible in reticulum cell in oil immersion objective [Figure 2]
  • 2+: Small, sparse iron particle in low power field
  • 3+: Numerous small granules in all marrow particles [Figure 3]
  • 4+: Large granules in small clumps [Figure 4]
  • 5+: Dense large clumps of granules
  • 6+: Very large deposits obscuring marrow details.
Figure 1: Absent iron store

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Figure 2: Normal iron store, Grade 1

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Figure 3: Increased iron store, Grade 3

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Figure 4: Increased iron store, Grade 4

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Minimum seven particles are required to give the absence of iron content (Grade 0).

  • 2+: Normal
  • 3+: Slightly increased
  • 4+: Moderately increased
  • 5+, 6+: Markedly increased.


Scope and limitation

Scope

  • Semi quantitation of iron stores in anemia and various blood disorders can be measured
  • In iron deficiency anemia, macrocytic anemia and dimorphic anemia, it is very much helpful
  • In various form of sideroblastic anemia
  • Useful prognostic marker in case of idiopathic sideroblastic anemia undergoing leukemic transformation
  • The presence or absence of iron in marrow may be regarded as an index for iron therapy in anemia as well as for diagnosis of refractory anemia where response to iron therapy can be observed.


Limitation

As it is a semi-quantitative method, it gives rough estimation of iron store. Subjective variation is likely to occur from person to person, but with experience it can be minimized. The body iron reserve is traditionally assessed by the biochemical markers of iron metabolism, namely serum ferritin, serum iron, TIBC, and percentage saturation of transferrin. [3] 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. [4] 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. [5]


  Materials and methods Top


We conducted the study on 73 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 2013. The study was approved by the Institution Ethical Committee. Bone marrow aspirate was obtained after written informed consent from the posterior superior iliac spine observing strict asepsis, spread onto a slide, air-dried, fixed with methanol, and stained with hematoxylin and eosin (H and E), observed microscopically and also simultaneously stained with Prussian blue stain as under. Equal volume of 2% of potassium ferrocynide and 2% hydrochloric acid solution are mixed in staining jar and slides are immersed in the solution for 15-20 min. Then removed and rinsed with distilled water. Counterstaining with saffranin for 30 s. Then allowed to dry and then examined. The marrow was rejected when it was found to be diluted with blood or poor material or inadequate material on H and E stain. This study comprise of 73 patients, 3 are rejected due to this criterion. In all cases patient's age, sex, clinical history and other laboratory investigations were recorded. Peripheral venous blood was collected at the same setting for hemoglobin and serum iron and serum TIBC estimation. Bone marrow iron results were compared with serum ferritin level determined by micro particle enzyme immunoassay (Abbott Axsym system). Hemoglobin was estimated using the automated cell counter (Sysmex KX 21). The statistical analysis was performed using SPSS version 17 by IBM.


  Results Top


In the present study majority of cases (30.0%) were affected in 21-30 years age group, next group was (27.1%) in 31-40 years, third was in 11-20 years age group (21.4%). Male was more affected during first and second decade of life while female in third and fourth decade of life and in fifth decade and above both are equally affected. In a study of iron deficiency anemia 92.7% of cases received iron store grade in marrow in range of 0-1 (P = 0.001), in megaloblastic anemia, 70% of cases received in the range of 3-4 (P = 0.164), all cases of dimorphic anemia received in the range of 2-3 (P = 0.000) as compared to the normal range of 1-2. In aplastic anemia, 66.6% of cases received in the range of 0-1. All cases of hemolytic anemia received normal iron store grade in marrow. Patient on multiple transfusion received Grade 3. Other conditions unrelated to anemia, were leukemoid reaction, idiopathic thrombocytopenic purpura, chronic myeloid leukemia in which iron grade were 3, 2, and 1 respectively [Table 1] and [Table 2]. The serum iron [Table 3] and TIBC [Table 4] have a direct correlation with the bone marrow iron stores in the present study (P = 0.127 and P = 0.153) respectively. A good correlation between low serum ferritin levels and absence of iron stores in biopsy specimens was found (P = 0.146) [Table 5].
Table 1: Incidences of various hematological conditions in the study

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Table 2: Predominant marrow findings and marrow iron storage in various hematological conditions

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Table 3: Relationship of serum iron with bone marrow iron stores

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Table 4: Relationship of TIBC with bone marrow iron stores

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Table 5: Relationship of serum ferritin with bone marrow iron stores

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  Discussion Top


Various workers in the diagnostic field have emphasized the importance of iron storage by Prussian blue method. Being very old technique many old studies are there, but now days it has gained a firm stand, being a simple technique with temptation and enthusiasm to know and see what our ancients have founded. An attempt of the short study has been put forth to establish its importance in clinical ground and to assess its result with a wish to continue as a routine practice in hematology. Prussian blue positive iron stained granules were seen in most of cases in macrophage and as free particles. There is also the presence of siderotic granules in erythroblasts in variable numbers in few cases of megaloblastic anemia and also in patients who had received multiple transfusions. The study correlates the findings obtained by Bableshwar et al. [1] in study of 80 patients and Krause and Stolc [6] in a study on 104 patients. The microscopic examination of stainable iron in the bone marrow aspirate smear is generally considered the reference standard for determining the body iron stores. [7] 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. [8] Thus, Prussian blue stain is simple and helpful technique to see and measure body iron store semi-quantitatively. Study of marrow in iron deficiency anemia is very much essential, and it helps to decide whether iron therapy would be of any use or not. Where sophisticated instruments and facilities are not available for measuring iron in the blood by chemical method, this simple technique, is efficient, cost-effective and result oriented, can be used as a routine hematological procedure.

 
  References Top

1.
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.  Back to cited text no. 1
[PUBMED]  Medknow Journal  
2.
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. 2
[PUBMED]    
3.
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. 3
    
4.
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. 4
    
5.
Hanif E, Ayyub M, Anwar M, Ali W, Bashir M. Evaluation of serum transferrin receptor concentration in diagnosing and differentiating iron deficiency anaemia from anaemia of chronic disorders. J Pak Med Assoc 2005;55:13-6.  Back to cited text no. 5
    
6.
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. 6
    
7.
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. 7
    
8.
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. 8
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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