Introduction

The bone marrow is one of the body’s largest organs. It constitutes 4.5% of the total body weight and weights 3375 grams in a 75 kg individual¹. The hematopoietic bone marrow is organized around the vasculature of the bone cavity. The main function of the bone marrow is to supply mature hematopoietic cells for circulating blood in a steady-state as well as to respond to increased physiologic or pathologic demands.

Recent advances in the treatment of hematologic malignancies have been paralleled by renewed interest on the part of pathologists and hematologists in methods of obtaining and preparing bone marrow for diagnostic studies.

Bone marrow aspirate is a cytologic preparation of bone marrow cells obtained by aspiration of marrow and a smear of the cells. Bone marrow aspirate smears are primarily used for the assessment of differentia count, maturational status, morphologic details, myeloid-to-erythroid (M:E) ratio. Bone marrow aspirated cell blocks provide a better and more detailed estimate of bone marrow cellularity. They are particularly useful for patients with aplastic / hypoplastic marrow. Aspiration of the marrow has been utilized from cytologic assessment, with analysis directed toward morphology and obtainment of differential cell count.

“Romanowsky Stain”is a general term which includes several specific stains or stain combinations. Romanowsky (Giemsa & Leishman) stains are routinely used in hematology²

In H&E stained sections of bone marrow, the more mature stages of the erythroid and myeloid cells, adipocytes and megakaryocytes can be identified. However lymphoid cells as well as immature progenitor cells cannot be reliably identified. The use of H&E and Romanowsky group of stains in the bone marrow analysis are very important and allow for studies of marrow’s overall cellularity, detection of focal lesions and extent of infiltration by various pathological entities.

Bone marrow studies aid in the diagnosis, staging and monitoring of several diseases Thus the present study was being conducted to compare efficacy & to explore further utility of Geimsa and Leishman stains of Romanowsky group in bone marrow aspiration smears.

Objectives

The present study was undertaken with the objectives –

Materials And Methods

The present study was done in Hamidia Hospital, Bhopal. Department of pathology from May 2008 to Sep. 2009. A consecutive marrow samples from 83 patients attending hematology OPD or ward of Hamidia Hospital, Bhopal. In the present study included 83 cases in which complete peripheral smears, bone marrow and cell blocks was available. The selection of cases was based on the clinical examination and peripheral blood smear of patients in which a hematological disorder was suspected.

Inclusion criteria were unexplained anemia, splenomegaly, hepatomegaly and lymphadenopathy, Pancytopenia, leucopenia & leucocytosis, unexplained thrombocytopenia and thrombocytosis, Leukaemia, Myeloproliferative disorders, Lymphoproiferative disorders, Plasma cell dyscrasias.

Exclusion criteria were the cases which were bleeding severely, like haemophilic patient and patient having platelet count less than 10,000.

History and clinical examination was done as per proforma. Investigations like Hemoglobin, Total WBC count, Platelet count, Reticulocyte count, Peripheral blood smear examination, Blood glucose, Blood urea, Serum creatinine, AST, ALT, Bone marrow aspiration were performed

Bone Marrow Aspiration was done through safe and preferred sites. The preffered site for young and old patients is Sternum at the level of the 2 ^nd or 3 ^rd interspace, just to one side of the midline, with the patient in a supine position. Tibia³ for use is children younger that 1 year old the flat triangular area at the proximal end of the media surface of the tibia, just below the tibial tubercle was chosen.

Other sites for Bone Marrow Aspiration⁴ are Posterior superior iliac spine, Posterior iliac crest, Anterior iliac crest, Spinous process of lumber vertebra, Upper end of tibia. The sternum remains the most commonly employed aspiration site.

Evaluation of Bone Marrow Aspiration Smears⁵: Detailed clinical history with clinical features like pallor, icterus, hepato or spleenomegaly and lymphadenopathy should be known. Peripheral blood films should be stained and evaluated. Stain 2 bone marrow smears with Leishman and Giemsa stain. Examined under scanner and low power, to asses cellularity, megakaryocyte, metastatic carcinoma cells which usually are present in the tail of the film. Select the area where cells are very well spread out. Bone marrow smears are ready for microscopic examination.

Cell blocks preparation

The clot section is prepared from the blood left over after aspirate smears have been made. The blood containing admixed marrow particles is transferred to a container with 1ml of 10% formal saline and 2ml methanol for fixation. Future processing is carried out as for routine histopathological staining.

The slides were viewed and reported. The reporting was done in conventional way as well as a scoring system created indigenously

Figure 1

The maximum score for a single case, taking into account all the four parameters was 12. Thus the maximum possible score in the study was calculated by multiplying the number of cases by 12 for each of 2 stains. A “quality index” was obtained by finding out the ratio of actual score obtained to the maximum score possible.

Quality index = Actual score obtained / Maximum score possible.

The quality index for each of two stains was compared. As the smears of all the cases were stained with Leishman stain and Geimsa stain, Quality Index of all slides were calculated and compared.

Observations

The present study was conducted for comparison between bone marrow aspirate smears stained by Romanowsky group of stains (Leishman & Giemsa) and paraffin embedded cell block section stained by Hematoxylin & Eosin stain. Total 83 cases were selected in which complete peripheral smears, bone marrow aspirate and cell blocks were available. The findings obtained are as follows-

Figure 2

Table No.1: Various Hematological disorders

Figure 3

Table No.2: Distribution of Heamatological Disorders according to age

Figure 4

Table No.3: Distribution of Hematological Disorders according to sex

Figure 5

Table No.4: Result of scoring and Quality Index of Leishman and Giemsa stains

Figure 6

Table No.5: Comparison of Quality Index in Leishman & Giemsa stained slides

Figure 7

Table No.6: Various components seen in H & E stain blocks and Romanwsky stained bone marrow aspirate smears.

Results

By Romanowsky group of stains and Hematoxylin & Eosin stained cellblocks, diagnosis of various hematological disorders was made. Out of 83 cases studied there were 60 cases (72.2%) of anaemia, 9 (10.8%) platelet disorders, 6 (7.2%) Acute Leukaemias, 3(3.6%) shift to left, 2 (2.4%) Lymph proliferative disorders, 1 (1.2%) MDS, 1(1.2%) Leishmaniasis and 1 (1.2%) hyperspleenism. Maximum cases 40 were in age group of 11-50 years of age in megaloblastic anaemia. 32 (69.5%) cases in megaloblastic anaemia showing male preponderance.

In Leishman and Giemsa stained smears parameters studied as per background of smears, overall staining, cell morphology and nuclear details, showed that except nuclear details all parameters are alike. In nuclear details the Quality was superior in Leishman than Giemsa. Quality Index in Leishman stain was 0.20 and in Giemsa stain was 0.12.

The cellularities analyzed on bone marrow aspiration showed maximum 84.3% were hypercellular marrow. Cell blocks help in better appreciation of hypocellular / acellular marrow. Megakaryocytic & Eosinophilic precursors are better identifiable in H &E stained block.

Figure 8

Photo – 1: Leishman stain (X400) Smudgy nuclear details

Figure 9

Photo – 2: Giemsa stain (X400) Smudgy nuclear details

Figure 10

Photo – 3: Leishman stain (X1000) Very clear nuclear details and prominent nucleoli

Figure 11

Photo – 4: Giemsa stain (X1000) Very clear nuclear details and prominent nucleoli

Figure 12

Photo – 5: H&E stain cell block clear morphology of Megakaryocytes

Figure 13

Photo – 6: H&E stain cell block clear morphology of Eosinophilic Precursors

Discussion

The purpose of this work is to compare efficacy of Romanowsky group of stains (Geimsa and Leishman ) in bone marrow aspiration smears & to assess the role of paraffin embedded cell block of bone marrow aspirates in bone marrow examination. In nuclear details the Quality was superior in Leishman than Giemsa. Quality Index in Leishman stain was 0.20 and in Giemsa stain was 0.12.

The cases diagnosed as megaloblastic anemia showed that male female ratio was 2:1 and the commonest age group was 21 to 30 years. This is in variance with studies of Kuperan and Rajashekhar Swamy ⁶ where the commonest age group was 31 – 40 years with male female ratio of 1.3:1.

Pizzuto J Ambriz ⁷found in their study that between 40 – 45 years Male to female ratio was 1:2.3.In the current study all the patients of Idiopathic Thrombocytopenic Purpura were in age group of 10 – 30 years & Male to female ration of 2:1 showing male preponderance

Jaishree Sharma and Shobha Mohindroo ⁸ found that acute leukemia showed male preponderance 20 (55.5%) which is in consonance with the current study with male preponderance 04 (66.66%)

Sitalakshmi et al ⁹ found that acute leukaemias were diagnostic on aspiration alone, trephine biopsy provided additional useful information. In current study we found that acute leukaemias were diagnostic on aspiration alone, clot section should be used as an adjunction to bone marrow aspiration to increase the diagnostic yield.

Rozman C et al ¹⁰ studied 329 patients, 208 (63.22%) cases were males and 121(36.78%) females. The mean age was 64.7 years. Bone marrow was infiltrated by 50% or more lymphocytes.In current study of one male patient aged 60 years peripheral smear showed absolute lymphocytosis. Bone marrow was infiltrated by 80% lymphocytes.

Conclusion

The overall staining quality is superior in Leishman stain than in Giemsa stain.Hematoxylin & Eosin stained cell blocks are particularly helpful in hypo cellular marrow aspirate. Megakaryocytes & eosinophillic precursors can be better appreciated in cell block sections. Cellblocks can be stored and used for specialized procedure like immunohistochemisty in future.