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Comparative evaluation of bone marrow aspirate particle smears, imprints and biopsy sections.
Correspondence Address:
Comparative evaluation of bone marrow aspirate particle smears, imprints and biopsy sections was done on 30 haematological problems. Core needle biopsy of the bone marrow is a safe and useful procedure. It is a valuable diagnostic aid for measurement of marrow cellularity, metastatic tumours and fibrosis. It should not be taken as a substitute for examination of the marrow by aspiration smear but is a complementary procedure which affords several advantages. Bone marrow biopsy was of maximum utility in myelofibrosis which was diagnosed on biopsy alone. There were three additional cases with normal bone marrow aspiration in which specific diagnosis could only be made from bone marrow biopsy sections. New methodologies i.e. plastic embedding and semi thin sections of undecalcified bone marrow, can be expected to improve the cytological details of tissue obtained by biopsy. Imprint preparations obtained from biopsy can be useful in patients of malignancy but we have found them to be of limited value except in cases of dry tap.
Marrow biopsy by surgical trephine is an older procedure than needle aspiration. It is only since the late 1950s that core needle biopsy of the bone marrow has been widely used. Since that time, it has had a considerable effect on diagnostic haematology, pathology and oncology. Wide acceptance is associated with the introduction of a simple procedure using the Jamshidi needle to improve the procedure, as well as the quality and size of specimens. The uses and advantages of needle biopsies are numerous. Metastatic deposits, degree of cellularity, fibrosis and assessment of dry taps can readily be determined. There are virtually no contraindications to needle biopsy of the bone marrow. The standard procedure using the Jamshidi needle is technically simply, safe and relatively easily learned. Since the initial description[7], a broad experience has been gained and many articles have been written[1],[5],[6],[9]. The present study is conducted to evaluate the procedure and its results to consider if it has reached its expectations.
This study has been conducted at the Department of Pathology, Dayanand Medical College and Hospital, Ludhiana. Patients included in the study were from the associated hospital and its oncology clinic. As and when bone marrow aspiration was performed, bone marrow biopsy was undertaken at the same time. The standard technique was employed in obtaining the samples from posterior iliac crest using a Jamshidi needle. For preparing the aspirate particle smears, about 0.25 to 0.5 ml of aspirate was obtained into a syringe and delivered onto clean glass slides and smears prepared. The biopsy imprints were made by gently touching the core on slides. The cores were then fixed in Zenkers formalin, decalcified, embedded in paraffin and 2 um thin sections made. The particle smears and biopsy touch preparations were stained by the Wright-Giemsa and the biopsy sections were stained by the Wright-Giemsa and haematoxylin and eosin methods. In addition Gomori's reticulin stain and prussian blue stain for iron were also performed. Evaluation of slides: The cellularity, megakaryocyte density and differential counts were done on all three preparations of bone marrow samples and recorded in a proforma. They were assessed subjectively. The cellularity was graded as hypocellular (+), normocellular (+ +), or hypercellular (+ + +). Multiple areas of each slide were screened and an estimate made. The bone marrow aspirate smears and core biopsies were evaluated for the presence of iron. They were graded as absent (0), decreased (+), normal (+ +), and increased (+ + +). Samples were evaluated for tumour staging and assessed for the presence of tumour cells in the three different preparations. Reticulin grading was done according to Bauermeister et al[4] and normal bone marrow reticulin was graded as 0 to 1 +.
The present study was conducted on patients coming to haematology section of DMC and Hospital, over a period of 2 years i.e. from Jan. 1987 to Dec. 1988. A comparative evaluation of bone marrow aspirate particle smears, imprints and biopsy section was done on 30 cases. Based on their haematological findings and other relevant investigations, the cases were broadly classified into 8 groups [Table - 1]. Aplastic anaemia: According to the criteria used by SM Lewis[8] aplastic anaemia was diagnosed in two cases. Both showed a markedly hypocellular marrow with increase in the fat cells and decreased haematopoietic tissue and sinusoids (See [Figure - 1]. Lymphocytes were found to be increased along with few plasma cells, isolated clusters of normoblasts, few myeloid cells and megakaryocytes. Bone marrow aspiration was tried more than once from different sites in both the cases and dry tap was obtained. Idiopathic thrombocytopaenic purpura (ITP): There was only one case of ITP included in the study. As compared to smears and imprints it was seen that megakaryocytes were increased and diffusely distributed in the biopsy section, and were better estimated from biopsy than from the aspirate or imprint smear. Chronic myeloid leukemia (CML): Four cases of CML were studied and marrow biopsy revealed a packed marrow with little remaining fat spaces. All classes of neutrophils and neutrophil precursors were increased, as also increased numbers of megakaryocytes, which were readily apparent in sections of biopsy specimens. Increased myeloid; erythroid ratio with fairly orderly neutrophil maturation was found. Fibrosis was not seen in any of the cases studied. However marrow examination was of little diagnostic help in the cases of chronic myeloid leukaemia. Myelofibrosis: Seven cases of myelofibrosis were present in our study and diagnosis was made from the biopsy sections alone in all the cases. Bone marrow histology in myelofibrosis was classified according to the criteria by Bard R et a1[2]. Reticulin was graded from 1 + to 4 + as per the grading system proposed by Bauermeister [4]. Five of the seven cases were found to be in the intermediate stage of myelofibrosis i.e. grade II, characterised by hyperplastic areas of marrow mixed with fibrotic areas consisting of both reticulin and collagenous fibres, with reduction in the erythrocytic and myelocytic elements (See [Figure - 2] & [Figure - 3]). Megakaryocytes were however present. One case was found to be in the cellular phase of myelofibrosis characterised by panhyperplasia of all the three elements with a slight increase in reticulin (See [Figure - 4]). One case was diagnosed as acute myelofibrosis as per the histological criteria proposed by Bartl et al[1]. Myelodysplastic states (MDS): Three cases of MDS were diagnosed in this series on aspiration. It was seen that features of abnormal erythropoiesis and myelopoiesis were easily detected on the aspirate smears. However certain advantages of bone marrow biopsy over bone marrow smears were found. Firstly the biopsy in MDS gives an exact assessment of the cellularity; secondly the presence of reticulin fibres, which may be increased can be evaluated; thirdly dysmegakaryopoiesis is more easily detected in biopsy; finally abnormal behaviour of myeloblasts, clustering centrally in the bone marrow is frequently observed. Multiple Myeloma: In the case diagnosed as multiple myeloma the aspirate smears were inadequate and marrow imprints were infiltrated by sheets of plasma cells both mature as well as immature. Biopsy section showed masses of plasma cells with no stroma. The cells were plasmacytic type-1 i.e. mature Marschalko type with eccentric cartwheel nuclei and basophilic cytoplasm. Metastatic deposits: Three cases of metastatic deposits in the bone marrow biopsy showed squamous cell carcinomatous deposits in two cases and adenocarcinomatous deposits in one case (See [Figure - 5]). Imprints showing clumps of malignant cells were positive in all three cases but aspirate was positive in only one case (33%). Biopsy in all three cases showed an increase in fibrosis as evident by reticulin stain, due to tumour associated demoplastic reaction. Though the number of cases is less, biopsy was positive in all cases and aspirate was positive in only one case. Acute myeloid lukemia (AML): In all cases of AML, cytological details of the blast cells were evident more clearly on the contact imprints; biopsy further demonstrated the associated fibrosis in two of the cases. In the case diagnosed as AML-M6, abnormal and bizarre erythroblasts were seen quite well in the biopsy sections. Lymphomas: The features, which were helpful in the diagnosis of lymphoma involving the bone marrow were extensive paratrabecular lymphocytic infiltrate with disruption of normal marrow architecture, or a diffuse interstitial growth pattern. Cytological atypia and immaturity of cells with clefting and indentations were seen in the case diagnosed as poorly differentiated lymphocytic lymphoma. No nodules or lymphoid aggregates were seen. Lymphoplasmacytoid lymphoma was diagnosed in one case as per the criteria proposed by Bartl et a1[3]. The infiltration mainly consisted of small lymphocytes with variable numbers of plasma cells, lymphoplasmacytoid cells in a hypo or normocellular marrow with an interstitial growth pattern.
Questions concerning the value of, and indications for the biopsy as compared to the aspirate have been considered at least as long as needle biopsy has been used. The answer, although somewhat complicated, remains essentially the same. Aspiration and biopsy, using standard fixation, embedding and cutting techniques, generally complement each other with the aspirated smears being primarily useful for cytologic diagnosis and biopsy sections mainly helpful for histologic diagnosis as cellularity, fibrosis, metastatic deposits and architectural patterns. Imprint preparations obtained from biopsies can be useful in patients with malignancy but we have found them to be of limited value except in cases of dry taps. This is in close agreement with the observations made by other workers (Westerman MP)[9]. The overall cellularity of the samples was comparable in the three preparations. However the biopsy touch preparation and aspirate particle smears under estimated the cellularity in about a third of the cases. Not enough samples with low cellularity were evaluated to draw any conclusions regarding their accuracy. Megakaryocytes could be readily identified in biopsy sections, imprints and aspirate particle smears but their quantity could be more accurately assessed in the biopsy section. The diagnosis of aplastic anaemia in two cases could only be made bone marrow biopsy section. Biopsy imprints and sections were positive in all three cases of metastatic carcinoma (100%) whereas aspirate smear was positive in only one case (33%). The poor sensitivity (33%) of our aspirate smears in the diagnosis of metastatic carcinoma compares with the data reported in the literature (30-50%)[6]. Biopsy imprint is of high sensitivity in the diagnosis of non-haematological metastatic tumours. Many studies have confirmed the higher incidence of positive biopsies in lymphoproliferative disorder over aspiration as reported in the literature, and this demonstrated the value of bone marrow biopsy over aspiration [1],[3]. Our study on the two cases reflects a 100% positive, for both aspirate particle smears and biopsy sections. Biopsy is of discriminating value in differentiating myelomatous from non-myelomatous plasmacytosis, since compact masses of plasma cells with no stroma is a crucial histological feature for such differentiation, thereby emphasising the role of bone marrow biopsy. In our study also the diagnosis of the single case of myeloma was clinched by the bone marrow biopsy. The diagnosis of myelofibrosis could only be made from biopsy sections. Two of the cases of myelofibrosis diagnosed on biopsy section, showed the aspirate smears to be normal. One case which was diagnosed as acute myeloid leukaemia, later proved to be acute myelofibrosis after evaluating the biopsy section as per the criteria proposed by Bard et al [1]. The association of fibrosis with acute leukaemia in three cases as evident on biopsy section further emphasises the role of bone marrow biopsy since assessment of reticulin, fibrosis and collagen can only be done on biopsy sections. We were not able to recognise histologically the five types of MDS. However certain features, as, exact assessment of cellularity, increased reticulin, dysmegakaryopoiesis and clustering of myeloblasts were better evaluated on biopsy sections. There were three cases (10%) in which specific diagnosis could only be made from bone marrow biopsy sections. They correlates well with the incidence observed by various other authors as by Ellis L. D. et a1[5] who presented a report with a large series of needle biopsies (1,445) and concluded that bone marrow biopsy sections was of specific diagnostic value in 11% of cases whose diagnosis were not apparent from bone marrow aspirate. The accurate cytological identification on decalcified, thin, paraffin-embedded sections poses many problems, and the differentiation of cell morphology remains difficult. Hence cellular morphology and detail can only be done on the aspirate particle smears and biopsy imprints. Assessment of reticulin, collagen, and fibrosis can only be done on the biopsy sections. In the present study, significantly different amounts of stainable iron were observed in needle biopsy sections when compared to aspirated smears. The needle biopsy sections in all cases assessed for iron showed lesser amount of iron, and most showed no marrow haemosiderin, in contrast to definite deposits in simultaneously obtained aspirate smears. The discrepancy may be related to difference in sample preparations and processing. The processing of biopsy section may be associated with loss of iron due to its solubility in acid during decalcification.
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