|Year : 2016 | Volume
| Issue : 2 | Page : 181-185
Study of sideroblasts and iron stores in bone marrow aspirates using Perls' stain
Arpana Dharwadkar, Shruti Vimal, Narayanan Krishnakutty Panicker, Shirish S Chandanwale, Vidya Viswanathan, Harsh Kumar
Department of Pathology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India
|Date of Web Publication||1-Mar-2016|
Department of Pathology, Pad Dr. D. Y. Patil Medical College, Pune, Maharashtra
Source of Support: None, Conflict of Interest: None
Introduction: A bone marrow examination is a critical part of the evaluation of patients with a variety of hematopoietic and nonhematopoietic diseases. A Perls' or Prussian blue stain demonstrates hemosiderin in bone marrow macrophages and iron within sideroblasts. Examination of iron stain allows detection not only of an increased or decreased proportion of sideroblasts but also of abnormal sideroblasts. We undertook this study to evaluate the iron stores in bone marrow as judged by Perls' stain. Materials and Methods: A Prospective analysis of 55 bone marrow aspirates in a tertiary care center. Results: Sideroblasts were present in all the cases. They were decreased in iron deficiency anemia (IDA), and few other chronic diseases whereas normal to increase in megaloblastic anemia. According to Gale's method, cases of IDA had absent or decreased iron stores while those of megaloblastic anemia or normoblastic marrows showed normal to increased stores. Grading by the recent intensive method showed combined functional and iron stores deficiency. Conclusion: Perls' stain on bone marrow aspirates is an important tool not only for assessing the iron stores, increased or decreased but also to study abnormal sideroblasts.
Keywords: Bone marrow aspirates, iron store, Perls′ stain, sideroblasts
|How to cite this article:|
Dharwadkar A, Vimal S, Panicker NK, Chandanwale SS, Viswanathan V, Kumar H. Study of sideroblasts and iron stores in bone marrow aspirates using Perls' stain. Med J DY Patil Univ 2016;9:181-5
|How to cite this URL:|
Dharwadkar A, Vimal S, Panicker NK, Chandanwale SS, Viswanathan V, Kumar H. Study of sideroblasts and iron stores in bone marrow aspirates using Perls' stain. Med J DY Patil Univ [serial online] 2016 [cited 2020 Apr 9];9:181-5. Available from: http://www.mjdrdypu.org/text.asp?2016/9/2/181/177655
| Introduction|| |
The marrow produces cellular elements of blood, including platelets, red blood cells and white blood cells. While much information can be gained by testing the blood itself, it is sometimes necessary to examine the source of the blood cells in the marrow.  Bone marrow aspirates are suitable for many diagnostic investigations in addition to routine microscopy of Romanowsky-stained bone marrow film. Other diagnostic procedures that may be of use in individual cases include cytochemistry, immunophenotyping, cytogenetic and molecular genetic analysis and ultrastructural examination. Some of these techniques are used routinely, like the Perls' stain to demonstrate hemosiderin in a bone marrow aspirate as it is very useful. However, the other methods are applied selectively. A Perls' or Prussian blue stain demonstrates hemosiderin in bone marrow macrophages and iron within the erythroblasts.
A proportion of normal erythroblasts have few (1-5) iron-containing granules randomly distributed in the cytoplasm. Such erythroblasts are designated as sideroblasts. Examination of iron stain allows detection not only of an increased or decreased proportion of sideroblasts but also of abnormal sideroblasts. Assessment of the storage iron may be done as normal, decreased or increased or may be graded from 0 (absent) to 6+. Consequently, it allows the assessment of both the amount of iron in reticulo-endothelial stores and availability of iron to developing erythroblasts. 
We undertook this study to evaluate the iron stores in bone marrow as judged by Perls' stain.
| Materials and Methods|| |
This study was undertaken in the Department of Pathology of a tertiary care hospital over a period of 1 year. A total of 55 bone marrow aspirates of patients with various hematological, nonhematological disorders or when otherwise indicated were examined. A detailed clinical history was obtained from all the patients followed by a thorough clinical examination. Routine laboratory investigations were done. A written and informed consent was taken from the patients for bone marrow aspiration.
Bone marrow aspiration was done according to the standard procedure using Salah's bone marrow aspiration needle. Of the five smears prepared, two were fixed in absolute alcohol for Perls' staining and others were stained by Leishman's stain. A peripheral smear of patients was also done at the same time.
Procedure of Perls' Staining: This method is considered by many to be the first classical histochemical reaction.  Ferrocyanide solution is prepared freshly using 2% potassium ferrocyanide (25 ml) and 2% hydrochloric acid (25 ml). Control and test smears were treated with freshly prepared acid ferrocyanide solution for about 30 min. Washed well in distilled water. Treated with 0.5% aqueous neutral red for 30 s. Washed rapidly in distilled water. Dehydrated, cleared and mounted. Results: Iron-blue; nucleus-red; cytoplasm-pink (control-bone marrow aspirates of normal healthy individuals with normal hemoglobin levels).
Leishman's stained slides were routinely assessed for cellularity, M/E ratio, erythroid series, myeloid and lymphoid series, megakaryocytes, plasma cells, parasites and any other abnormal cells. The smears stained with Perls' stain were assessed for the presence of iron in sideroblasts, macrophages and extracellular matrix. The percentage of sideroblasts in the smear was assessed.
Perls' positivity for storage iron was graded as per the system proposed by Gale et al.  and also by the recent intensive method proposed by Phiri et al. 
| Results|| |
In this study, 55 bone marrow aspirations were examined. The following observations were made. Age ranged from 16 years to 78 years (mean-47 years). Thirty-three (60%) males and 22 (40%) females were included in the study showing a male preponderance.
Forty-one aspirates (74.54%) showed hypercellularity, 12 (21.81%) were normocellular and only 2 cases (3.63%) were hypocellular. Of the 41 cases which were hypercellular, 17 showed micronormoblastic maturation, 11 showed megaloblastic maturation, 10 cases showed dual maturation and only 3 cases showed normoblastic maturation.
The presence of sideroblasts was noted in all the cases. Sideroblasts were <5% of nucleated red cells in 21 (38.8%) cases. More than 20% sideroblasts were seen in only 11 (20%) marrow samples.
Sideroblasts in cases reported as iron deficiency anemia (IDA) ranged from 2 to 12% (mean 7%). Cases of megaloblastic anemia had a higher range of sideroblasts varying from 12% to 38% (mean 25%). Dimorphic anemia and normoblastic marrows showed moderate numbers of sideroblasts with mean of 16.5% and 19%, respectively. One case each of leukemia, multiple myeloma, refractory anemia and chronic granulomatous lesion were noted which showed 8%, 10%, 10%, and 8% sideroblasts, respectively. Two cases of megaloblastic anemia showed ringed sideroblasts [Figure 1]. The siderotic granules, around 8-10 in number were arranged in a ring around the nucleus. The prevalence of sideroblasts was also on a higher range of normal being ≥35%. These cases showed abundant storage iron of grade 3.
|Figure 1: Ring sideroblast showing blue colored granules around the nucleus (Perls' stain, oil immersion)|
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Gale's grading method
All the smears were graded using this method. [Table 1] shows the distribution of cases using Gale's method. Grade 0 was present in 8 cases (14.54%) indicating the absence of iron [Figure 2]. Thirty-five cases (63.63%) showed grade 1+, 8 cases (14.54%) showed grade 2+ [Figure 3] and 4 cases (7.27%) showed grade 3+. None of the cases showed grades above 3+. Of the 23 cases of IDA, 7 cases had grade 0, 15 cases had grade 1+, and only 1 case showed grade 2+ iron score. None of the cases of megaloblastic anemia had grade 0. Six cases were graded as 1+, 4 as grade 2+ and 3 cases as grade 3+ [Figure 4]. Normoblastic marrows showed 2 cases of grade 1+ and 1 case of grade 2+. Cases of leukemia, multiple myeloma and granulomatous lesion showed grade 1+ iron score.
|Figure 2: Bone marrow aspirate showing grade 0 iron store (Perls' stain, high power)|
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|Figure 3: Bone marrow aspirate showing grade 2+ iron store (Perls' stain, low power)|
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Grading iron according to intensive method
All the cases were graded using the intensive method [Table 2]. Normal iron status was noted in 5 cases, functional iron deficiency in 7 cases and combined functional and iron stores deficiency in 43 cases. Isolated iron stores deficiency was not seen in any of the cases.
| Discussion|| |
Bone marrow iron staining has been the "gold standard" method of assessing the iron stores. However, the commonly used method of grading iron stores remains highly subjective. 
Most (75%) of the aspirates that we received in our study were hypercellular for the age and sex of the patient. Forty-one percentage of these showed micronormoblastic maturation suggestive of IDA. Eleven cases showed megaloblastic and 10 cases dual maturation. It has been documented that the marrow aspirates of untreated IDA and nutritional anemia are hyperplastic showing predominantly erythroid hyperplasia.  Hypocellular smears were noted in only 2 cases (4%).
Sideroblasts in bone marrow
Normally 20%-50% of late erythroblasts contain 1-2 Prussian-blue granules.  In cases of IDA Hill et al. described that fewer than 10% of the marrow normoblasts are sideroblasts.  In our study, the range of sideroblasts in cases of IDA was between 2% and 12%. This correlates well with the above study and also with another study conducted by Kaplan where he assessed 150 cases of microcytic hypochromic anemia and found that there was a consistent reduction in the range of sideroblasts varying from 0.5% to 15%, vast majority falling below 1%.  The iron granules in the sideroblasts indicate the presence of stainable nonhemoglobin iron. Reduction in the sideroblasts indicates that iron is not available to the normoblasts, thus highlighting iron deficiency. When iron is administered parenterally to subjects with iron deficiency, there would be a prompt rise in sideroblasts. , This indicates that the iron granules represent iron taken up from the available stores.
The range of sideroblasts in megaloblastic and dimorphic anemia was 12-38% and 3-30% respectively in our study. Dacei describes that the percentage of sideroblasts is greatly increased in megaloblastic anemia and hemolytic anemia. Kaplan also studied cases of megaloblastic anemia in infants where the incidence of sideroblasts was above 90%. , These studies do not correlate well with our study in which the range of sideroblasts was in the normal range.
A single case of refractory anemia in our study showed 10% sideroblasts. It is known that refractory anemia shows increased number of sideroblasts with abnormal ringed sideroblasts. Such features were not seen in our case as the aspirate was hypocellular with a very poor cell yield.
Two cases of megaloblastic anemia showed ringed sideroblasts. Warren, in his study of abnormal ringed sideroblasts commented that few ringed sideroblasts (1-4%) are also seen in cases of megaloblastic anemia.  This is related to the ineffective erythropoiesis seen in erythroblasts of such cases.
Grading of storage iron
According to Gale's method Majority of IDA cases had either grade 0 or grade 1 iron score, indicating a reduction or absence of iron stores. This is in accordance with a study conducted by Rath and Finch where they described that there is a complete absence of iron in iron deficiency. 
None of the cases of megaloblastic anemia showed a complete absence of iron. Majority of the cases showed grade 1, while others showed either grade 2+ or grade 3+. It has been documented that the marrow iron stain usually shows large amounts of iron in the fragments and the reticulum cells in cases of megaloblastic anemia. 
According to the intensive method by Phiri et al. This new method assesses the iron in three different sites in the fragments (as in Gale's method), macrophages and erythroblasts. Iron in the fragments and macrophages represented iron stores while iron in the erythroblast represented utilizable iron. In addition, 20 fields around the fragments were examined at high power and all macrophages in these fields were studied. Results of iron assessment were interpreted as normal status; functional iron deficiency; iron stores deficiency; and combined functional iron and iron stores deficiency. The ability to distinguish the state of decreased cellular iron delivery to erythroblasts in the presence of adequate iron stores (functional iron deficiency) compared to a state with limited availability due to lack of available iron stores is of particular importance. This is of importance in chronic diseases characterized by functional iron deficiency as opposed to iron store depletion seen in iron-deficiency anemia.  In our study, combined functional and iron stores deficiency was most commonly seen.
| Conclusion|| |
Perls' stain on bone marrow aspirates is an important tool to study the sideroblasts and for assessing the iron stores. The most commonly practiced Gale's method of grading helps to assess iron stores in the marrow while the recent intensive method distinguishes functional iron deficiency seen in chronic diseases from iron store depletion seen in iron deficiency anemia.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]