Abstract
Bone marrow examination entails study of aspirate smears, touch imprints and trephine biopsy. Bone marrow aspirate smears can be prepared by the squash (crush) or wedge method. Both techniques have their own advantages and disadvantages. There is paucity of studies that have compared these smear types. This study was conducted over a period of one year. Two hundred and five bone marrow aspirates were included. Both squash and wedge smears were made. Blinded slide review was done. Bone marrow cellularity, megakaryocyte number, myeloid to erythroid (M:E) ratio, morphology and final diagnosis on each smear type was compared. Chi square test, t-test and Kappa were applied to study the agreement between the wedge and squash smears. Among the 205 patients studied, squash smears showed significant over estimation of cellularity and megakaryocyte number (p < 0.05). There was no significant difference (p > 0.05) in the M:E ratio and morphological scores. Most patients (188/205 [91.7%]) showed complete diagnostic concordance while 17 (8.3%) patients had discrepancy in diagnosis between the squash and wedge smears. In 8 (3.9%) of these, major discrepancies were seen while 9 (4.4%) patients had minor discrepancies. Bone marrow cellularity and megakaryocyte numbers were underestimated in wedge smears with no differences in M:E ratio or morphology. Acceptable agreement for diagnosis was seen for patients with most disorders. Major diagnostic discrepancies were seen in patients with lesions known to have focal distribution—lymphoma, myeloma and tuberculosis (granulomas). Both squash and wedge smears should be studied for bone marrow examination.
Keywords: Aspiration, Bone marrow, Crush, Smears, Squash, Wedge
Introduction
Bone marrow examination is an important diagnostic procedure in clinical practice. It is useful in the evaluation and diagnosis of various benign and malignant hematological disorders [1–3]. Bone marrow examination entails study of aspirate smears, trephine biopsy, touch imprints and clot section. These modalities complement each other and a combined evaluation is recommended [4–11]. Bone marrow aspiration is the most consistently carried out procedure while the rest are not done in all patients in many centers.
Bone marrow aspirate smears can be prepared by the squash (crush) and the wedge method. International Council for Standardization in Hematology (ICSH) guidelines recommend that a minimum of six wedge smears and two particle squash (crush) slides should be studied in patients who undergo bone marrow examination [8]. However, most of the laboratories use only one of these two techniques depending upon their personal preference, familiarity and technical expertise.
Both techniques have their own advantages and disadvantages with differences in estimation of marrow cellularity and megakaryocyte number. Hemodilution, variable preservation of cell morphology and under or over estimation of cell populations has also been reported [12,13].
Previous studies have shown differences in opinion regarding the advantages, disadvantages and usage of squash and wedge techniques [12,14,15]. There is paucity of studies till date that have compared squash and wedge aspirate smears. The present study addresses this question by attempting to find out the differences in bone marrow assessment between the two techniques.
Materials and Methods
This observational prospective study was carried out in a tertiary care hospital in North India over a one year period. Two hundred and five bone marrow aspirates done in adult patients and having adequate particles (at least 3 particles) per slide, were included. Cases with inadequate marrow particles for making both types of smears, those with anticoagulated bone marrow specimen and unsatisfactory aspirate smears or trephine biopsy were excluded. Bone marrow procedure was performed by clinical hematologists, clinicians and certified physician assistants of the clinical hematology department. Single needle different track technique was used. Disposable Jamshidi needles (Suretech Medical Inc.) were used to perform bone marrow examination.
Preparation of Bone Marrow Smears
Bone marrow aspiration was done from the posterior superior iliac spine in all cases. About 0.5–1.0 ml of aspirated bone marrow from the first attempt was placed on a clean watch glass disc. The fragments were isolated using a surgical blade and carefully transferred to a clean glass slide. The smears were made only by two specially designated technicians within 1–2 min of the procedure. Two wedge smears were prepared first followed by two squash smears. (Fig. 1).
Fig. 1.
Shows unstained and stained (May-Grunwald-Giemsa stain) squash and wedge bone marrow aspirate smears. In the squash smears, marrow particles are present in the center of the slide while in wedge smears, they are dragged to the tail end of the smear
Wedge Smears
A small drop of marrow with particles was placed 1 cm from the end of the slide. Following this a spreader was placed in front of the drop of aspirate at an angle of 30 degrees and pulled back to make contact with the drop which spread along the line of contact with the slide. A smear of 3–5 cm length was made by dragging the spreader forward.
Squash (Crush) Smears
Marrow particles were placed close to the middle of a glass slide. A second slide (held with its axis perpendicular to the first slide) was gently placed on top of the first and both slides were dragged away from each other in the direction of the long axis of the first slide. Aspirate slides were stained by May-Grunwald-Giemsa (MGG) stain.
Assessment of Bone Marrow Aspirate Smears
Blinding: The observers (NK and KSK) were blinded to the findings of the corresponding smear type. Blinding was done by an individual other than the observers. For each case, four selected slides (2 wedge and 2 squash smears), were assigned coded numbers and randomly allocated to the observers for assessment. Both smear types were examined according to ICSH guidelines [8].
Bone marrow cellularity and megakaryocyte number was assessed under × 100 and × 400 magnification while a 400-cell differential cell count for each smear type was done using × 1000 magnification. Erythropoiesis and granulopoiesis along with any other specific feature was noted.
The following characteristics were compared between the wedge and squash smears:
i) Bone marrow cellularity ii) megakaryocyte number iii) myeloid to erythroid (M:E) ratio [M:E ratio- 2–4:1 was considered as normal] iv) nuclear and cytoplasmic morphology for all hematopoietic and other cell lines using a scoring system, (Table 1, Fig. 2) and v) final diagnosis, which was categorized as: a) normal marrow findings b) benign disorders c) malignant disorders and d) indeterminate (non-specific) findings. The scoring criteria for morphological scoring were designed by the authors since there were no clearly defined criteria available in the literature. The morphological scores were used for analysis of trilineage hematopoiesis as well as for the abnormal cell population (blasts, plasma cells, metastatic clusters, lymphoid population, etc.) including benign disorders.
Table 1.
Scoring system for cell morphology
| Morphological scoring | Score | Morphological description |
|---|---|---|
| Excellent | 2.0 | Nuclear and cytoplasmic details very clear, margins sharp and well outlined. No overlapping |
| Good | 1.5 | Nuclear and cytoplasmic details clear, cell outline well preserved, cytoplasmic granules clearly visible, minimal overlap |
| Fair | 1.0 | Quality intermediate between good and suboptimal |
| Suboptimal | 0.5 | Poor nuclear and cytoplasmic details, ill-defined cell outlines, marked cellular overlapping |
Fig. 2.
Squash smears with different morphological scores in four patients: a Acute myeloid leukemia with 95% myeloblasts (score-0.5) [MGG × 400], b Acute promyelocytic leukemia, hypergranular type with faggot cells [arrow] (score-1.0) [MGG × 1000], c Megaloblastic anemia with giant metamyelocytes (score-1.5) [MGG × 1000], d Mild plasmacytosis (score-2.0) [MGG × 400]
Findings of bone marrow aspiration by squash and wedge technique were correlated with the trephine biopsy. This was used as a guide for i) diagnostic concordance ii) verification of final marrow cellularity and iii) megakaryocyte number. Clot section and touch imprints were also studied.
Once the findings of individual observers were recorded for both squash and wedge smears, the cases were unblinded. In case of significant discrepant findings between the observers, a combined slide review was done and a consensus diagnosis was arrived at. Between the smear types, major discrepancies were defined as cases in which one of the smears types did not show the important diagnostic element or a related obvious finding. Minor discrepancies were defined as cases in which both smears types showed the same primary diagnosis but with variation in the degree of diagnostic features.
Statistical Analysis
Categorical variables were analysed by Chi square test. The mean morphological scores between the two smears were compared using t-test. Test of agreement (Kappa) was applied to study the agreement between the wedge and squash smears.
Results
Of the 205 patients included in the study, the age ranged from 5 to 83 years. The mean age was 47.1 ± 19.5 years. There were 121 males and 84 females (M:F = 1.4:1).
Indications of Bone Marrow Examination
Bone marrow was done for hematological indications in 179 (87.4%) patients and for non-hematological indications in 26 (12.6%) patients. The common indications are shown in Fig. 3a. Other indications included: Cytopenia/s under evaluation (12.2% cases), myeloproliferative disorders (6.3%), nutritional anemia (5.8%), immune thrombocytopenic purpura (ITP), suspected hematological malignancy (3.9% each, myelodysplastic syndrome (MDS), aplastic anemia (2.4% each), evaluation of hepatomegaly or splenomegaly (0.9%) and auto-immune hemolytic anemia (0.4%). The final diagnosis made on each type of smear was categorized as benign, malignant, non-specific findings (erythroid or myeloid hyperplasia, increased megakaryocytes-singly or in combination) and normal bone marrow (Fig. 3b).
Fig. 3.
a Clinical indications for which bone marrow was done (n = 205). Leukemia, lymphoma and multiple myeloma were the common indications. Non-hematological indications included pyrexia of unknown origin, infective/reactive conditions, hypersplenism and metastasis from solid tumours. b Major diagnostic groups after bone marrow examination
Squash smears showed more accurate estimation of bone marrow aspirate cellularity compared to wedge smears (p = 0.047). This correlated well with cellularity seen in trephine biopsy. When subgroup comparison was done within hypercellular (Fig. 4), normocellular and hypocellular aspirates, normocellular and hypocellular marrows showed good concordance between the two smear types (p = 0.078 and p = 0.495 respectively) (Table 2).
Fig. 4.
Note the smear hypercellularity in squash technique compared to the wedge technique. The patient was a 69 years old lady with chronic myeloid leukemia (MGG × 100)
Table 2.
Comparison of bone marrow aspirate cellularity, megakaryocyte number, Myeloid:Erythroid (M:E) ratio and morphological scores between squash and wedge smears
| Parameter | Squash smear | Wedge smear | p-value |
|---|---|---|---|
| Number | Number | ||
| Cellularity | |||
| Hypercellular | 123 (60) | 103 (50.2) | 0.047 |
| Normocellular | 63 (30.8) | 80 (39.1) | 0.078 |
| Hypocellular | 17 (8.3) | 21 (10.2) | 0.495 |
| Variablea | 02 (0.9) | 01 (0.5) | – |
| Megakaryocyte number | |||
| Increased | 142 (69.2) | 85 (41.5) | 0.0001 |
| Normal | 41 (20) | 94 (45.8) | 0.0001 |
| Decreased | 22 (10.8) | 26 (12.7) | 0.538 |
| Myeloid: Erythroid ratio | |||
| Erythroid hyperplasia | 85 (51.5%) | 87 (52%) | 0.915 |
| Normal | 66 (40%) | 59 (35.4%) | 0.379 |
| Myeloid prominence | 14 (8.5%) | 21 (12.6%) | 0.225 |
| Morphology scores | |||
| Erythroid series | 1.65 ± 0.16 | 1.64 ± 0.24 | 0.484 |
| Myeloid series | 1.65 ± 0.18 | 1.64 ± 0.24 | 0.474 |
| Megakaryocytic series | 1.63 ± 0.17 | 1.63 ± 0.21 | > 0.999 |
| Malignant disordersb | 1.61 ± 0.20 | 1.61 ± 0.26 | > 0.999 |
aNumber not sufficient for statistical analysis
bAnalysis of 61 patients with malignancy
Squash smears also overestimated megakaryocyte numbers (p = 0.0001). On subgroup analysis, there was good concordance (p = 0.538) between the squash and wedge smears in patients with reduced megakaryocytes. There was no significant difference when M:E ratio was compared between smears with erythroid hyperplasia (p = 0.915), normal smears (p = 0.379) and those with myeloid prominence (p = 0.225). Student t-test was used for comparing mean morphological scores between the squash and wedge smears including those for benign pathological findings and malignant disorders. Scores for erythroid series (p = 0.484), myeloid series (p = 0.474), megakaryocytes (p > 0.999) and malignant disorders (p > 0.999) showed no significant difference. (Table 2).
Almost perfect agreement was seen for most hematological conditions (Kappa: 0.81–1.0) while moderate agreement (Kappa-0.445) was seen in patients with tuberculosis which often shows focal distribution of granulomas. (Table 3).
Table 3.
Agreement for final diagnosis between squash and wedge smears
| Diagnosis | Kappa |
|---|---|
| Multiple myeloma | 0.855 |
| Lymphoma | 0.831 |
| Acute leukemia | 1.0 |
| Chronic leukemia | 1.0 |
| Tuberculosis | 0.455 |
| Aplastic anemia | 1.0 |
| Myeloproliferative disorders | 1.0 |
| Bone marrow for remission status | 1.0 |
| Suspected malignancy/ metastasis | 1.0 |
Cohen’s Kappa interpretation: ≤ 0-No agreement, 0.01–0.20-None to slight agreement, 0.21–0.40-Fair agreement
0.41–0.60-Moderate agreement, 0.61–0.80-Substantial agreement, 0.81–1.00-Almost perfect agreement
Discrepant Diagnosis Between Squash and Wedge Aspirate Smears
Of the 205 patients studied, 188 (91.7%) showed complete diagnostic concordance while 17 (8.3%) patients had discrepant diagnosis when squash and wedge smears were compared. In 8 (3.9%) patients, major discrepancies were seen. Of these 4 patients showed focal lymphocytosis with 2 of these diagnosed to have NHL infiltration while the other 2 had benign lymphoid nodules. The latter were not pursued further by immunohistochemistry as the clinical background and morphology was not indicative of NHL. In two patients, granulomas were seen in the squash smear only. In another patient, focal presence of increased plasma cells was seen only in squash smears. One patient showed erythrophagocytosis in the squash smears. (Table 4) Nine (4.4%) patients showed minor discrepancies. Four of these patients showed numerical differences in the diagnostic cell population (atypical lymphoid cells, plasma cells, histiocytes and neutrophils). In another 3 patients, there was a subjective observation difference (normal erythropoiesis vs. megaloblastoid). In the remaining 2 patients, the discrepancy related to a qualitative difference (unilineage vs. bilineage dysplasia and prominent vs. rare hemophagocytosis). (Table 5) (Figs. 5, 6, 7, 8).
Table 4.
Cases with major discrepancies on comparison between squash and wedge aspirate smears (n = 8)
| Patient | History/clinical profile | Squash smear findings | Wedge smear findings | Trephine biopsy finding | Final diagnosis |
|---|---|---|---|---|---|
| 1 | 62 years/male with gastric MALT lymphoma, bone marrow done for staging | Marrow lymphocytosis (42%). Mature small lymphoid cells | No increase in lymphocytes | Marrow lymphocytosis (focal) | Suspected NHL infiltration |
| 2 | 49 years/male, diagnosed case of multiple myeloma. Bone marrow done to rule out progressive disease | Focal plasmacytosis (90%) seen | No increase in plasma cells (3%) | Focal plasma cell collections | Progressive disease (multiple myeloma) |
| 3 | 35 years/female with anemia. No background of sepsis or malignancy | Hemophagocytosis of erythroblasts seen | No hemophagocytosis | No hemophagocytosis | Hemophagocytosis |
| 4 | 74 years/male with suspected acute leukemia | 58% blasts with marrow lymphocytosis (87%) | 53% blasts, no lymphocytosis seen | No lymphoid collection seen | AML with ?Benign lymphoid nodulea |
| 5 | 60 years/female with pancytopenia | Hypercellular marrow with marrow lymphocytosis (45%) | No lymphocytosis seen | Single discreet lymphoid nodule | ?Benign lymphoid nodulea |
| 6 | 27 years/female with miliary tuberculosis | Epithelioid cell granulomas | No granulomas seen | Many caseating granulomata seen | Tuberculosis |
| 7 | 53 years/female with disseminated tuberculosis | Epithelioid cell granulomas | No granulomas seen | Non-caseating granulomata | Tuberculosis |
| 8 | 60 years/female with anemia and massive (22 cm below left costal margin) splenomegaly | Marrow lymphocytosis (80%)-mostly mature appearing | No lymphocytosis seen | Marrow lymphocytosis (focal) | NHL infiltration |
AML Acute myeloid leukemia, MALT Mucosa associated lymphoid tissue, NHL non-hodgkin lymphoma
aIn both patients with ?benign lymphoid nodule, immunohistochemistry was not done and the suspicion of benign nodule was considered in view of morphology, distribution pattern, age of the patients and the clinical setting
Table 5.
Cases with minor discrepancies on comparison between squash and wedge aspirate smears (n = 9)
| Case | Age/Sex | Squash smear findings | Wedge smear findings |
|---|---|---|---|
| 1 | 22/F | Megaloblastoid erythropoiesis | Normoblastic erythropoiesis |
| 2 | 35/M | Megaloblastoid erythropoiesis | Normoblastic erythropoiesis |
| 3 | 30/F | Normoblastic erythropoiesis | Megaloblastoid erythropoiesis |
| 4 | 76/M | Normal granulopoiesis | Granulocytic maturation arrest beyond metamyelocyte stage |
| 5 | 60/M | MDS-MLD with bilineage dysplasia (dysgranulopoiesis and dysmegakaryopoiesis) | MDS with unilineage dysplasia (dysmegakaryopoiesis) |
| 6 | 25/F | Disseminated Tb, many histiocytes with AFB | Disseminated Tb, few histiocytes with AFB |
| 7 | 55/M | Prominent hemophagocytosis | Rare histiocyte showing hemophagocytosis |
| 8 | 24/M | Atypical lymphoid cells-40% (NHL) | Atypical lymphoid cells-24% (NHL) |
| 9 | 70/M | Mild increase in plasma cells (average-12%) | No significant increase in plasma cells (average- 4%) |
AFB Acid fast bacilli, CML Chronic myeloid leukemia, MDS Myelodysplastic syndrome, MLD Multi lineage dysplasia, NHL non-Hodgkin Lymphoma, Tb Tuberculosis
Fig. 5.
a, b Atypical lymphoid cells (arrows) in a patient with NHL infiltration. The cells were pleomorphic with fine chromatin, occasional nucleoli and moderate light basophilic cytoplasm (MGG × 1000) c, d Increase in histiocytes with intracytoplasmic negative linear shadows (MGG × 400) which showed numerous acid fast bacilli. The patient was a 25 years old lady with disseminated tuberculosis d [Inset] on Ziehl Neelsen stain
Fig. 6.
a Focal dense cellular clumps (arrows) in the squash smears (MGG × 100) which on higher magnification showed sheets of plasma cells (90%) with syncytial pattern and indistinct cytoplasmic outlines (Inset). The wedge smear b did not show increase in plasma cells. The patient, a 49 years old male was a diagnosed case of multiple myeloma in whom bone marrow had been done to rule out progressive disease. (MGG × 100) c Shows a distinct granuloma comprised of epithelioid cells. (MGG × 400) This finding was not seen in the wedge smear. The patient was a 27 years old lady with disseminated tuberculosis d. (MGG × 200)
Fig. 7.
a Squash smear shows erythrophagocytosis which was not seen in the wedge smear. The erythroblast shows nuclear budding b (MGG × 1000). c, d Leishman Donovan (LD) bodies. (MGG × 1000) The squash smear shows a histiocyte with numerous intracytoplasmic LD bodies (amastigote forms). More LD bodies were seen in the squash smear compared to the wedge smear
Fig. 8.
a, b Myelodysplastic syndrome with multi-lineage dysplasia (MDS-MLD) showing dysmegakaryopoiesis with disjointed megakaryocytic nuclei. The squash smear also shows a dwarf megakaryocyte (MGG × 1000) c Gastric MALT lymphoma with focal increase in with small to intermediate sized lymphoid cells (42%). d No increase in lymphoid cells was seen in the wedge smears. (MGG × 400)
Discussion
Our comparative study has shown good overall agreement between squash and wedge smears. Although bone marrow cellularity and megakaryocytes number were overestimated in the squash smears, no difference in M:E ratio or morphology was seen. Acceptable agreement for diagnosis was seen for patients with most malignant and benign disorders. In eight patients with major diagnostic discrepancy, the diagnostic element was seen in the squash smears only. In these patients, the diagnosis could have been missed if only wedge smears were studied.
In our study, squash smears offered the advantage of accurate estimation of cellularity that roughly correlated with the trephine biopsy cellularity. The marrow particles are useful for assessing cellularity whereas the peripheral portion is good for cytomorphology.
Similar to our observations, Lewandowski et al. in their study of 105 normal subjects reported higher cellularity in squash smears (p = 0.005) [14]. In addition, the squash smears in our study showed a significant increase (p < 0.0001) in megakaryocyte number as compared to wedge smears and trephine biopsy. Similar to our study, Lewandowski et al. also reported megakaryocyte number to be significantly higher (p < 0.00001) in squash smears as compared to wedge smears [14].
Squash smears tend to overestimate bone marrow cellularity and megakaryocyte number due to the nature of the technique in which marrow fragments (particles) are brought together in the center of the slide during the smearing process causing overlapping at places. In wedge smears, the marrow particles are dragged away during smearing and the cellular trails give a better actual representation of the cells associated with that particular fragment. In squash smears, although the marrow particles are closely placed with overlapping at places, areas at the periphery of the particles show good cell morphology and avoid the effect of hemodilution that is seen more often with wedge smears [12,14]. As the wedge smears in our study were made immediately after aspiration, the effect of hemodilution was minimized.
As the disease spectrum in our patients was very wide, we did not compare proportion of individual hemopoietic cell types between the squash and wedge smears but estimated the overall myeloid to erythroid ratio. The comparison of M:E ratio did not show a significant difference (p > 0.05) between squash and wedge smears. However, in contrast to our findings, Lewandowski et al. reported a higher median M:E ratio of 2.7:1 in wedge smears as compared to 1.6:1 for squash smears. The authors attributed this difference to possible hemodilution in wedge smears leading to increase in the neutrophil number [14].
The mean morphological scores for erythroid, myeloid and megakaryocytic series in our study were comparable (p > 0.05). Lewandowski et al. reported good morphology with squash smears in areas with well smeared cells [14]. However unlike the present study, they did not use objective scoring criteria. In contrast, Prashant et al. opined that in their experience, wedge smears showed better cell morphology. They attributed this to the artefacts and thicker nature of squash smears with poor stain penetration [12]. However, in our study we found that cellular areas towards the margins of the fragments in squash smears show good morphology and hemodilution was not apparent in these areas. The technique of smear preparation and quality of stain used could also be a confounding factor in different studies.
In our study, almost perfect agreement (Kappa: 0.81–1.0) for both smear types was seen for patients for whom bone marrow was done for multiple myeloma, lymphoma, acute and chronic leukemia, myeloproliferative disorder and metastatic malignancy. In the study by Lewandowski et al. acute leukaemia was confirmed in 21 patients with slides prepared using squash technique and in 16 patients using wedge technique. The authors attributed this difference to possible hemodilution in the wedge smear. They also reported 13 patients with NHL who showed bone marrow infiltration in the squash smears but not in the wedge smears [15].
In the present study, except for one case in which multiple myeloma was identified in squash smear and not on the wedge smear, minor clinically inconsequential differences were seen in plasma cell counts between the two smear types. Higher plasma cell counts were seen in squash smears. In 24 patients with multiple myeloma in their study, Lewandowski et al. also reported higher plasma cell count in the squash smears [15]. In multiple myeloma and systemic mastocytosis in which plasma cells and mast cells respectively often remain within fragments, these cells may be identified better in the squash than in the wedge smears [13].
Between the two smear types, we found diagnostic discrepancies in 17 (8.3%) cases. Of these, eight cases had major discrepancies while nine cases had minor discrepancies.
Disorders known to have a focal bone marrow involvement like lymphoma, multiple myeloma, granulomatous pathology or metastatic disease often show discrepancies in findings between the aspirate smears and trephine biopsy [13,16–18].
The major discrepancies in our study were seen in diseases known to have focal bone marrow involvement. In patients with minor diagnostic discrepancy, the differences observed were related to the percentage of cell counts, morphological variation and the quantity of diagnostic material. These differences could be due to possible subjective variation in interpretation and were clinically inconsequential.
In our opinion, the difference in diagnostic ability of squash and wedge smears for focal bone marrow lesions seen in the present study may reflect a chance occurrence rather than an indication of the superiority of the squash smear over the wedge smears. A study with larger sample size for diseases known to have focal distribution in the bone marrow may be able to address this issue better.
Both squash and wedge smears have their own advantages and disadvantages. Squash smears offer the advantage of lack of dilution with blood resulting in more accurate cell counts. In patients with focal lesions, the cells of interest may be trapped within the marrow particles. It is also useful in assessment of fibrotic marrows and detection of abnormal cells of low incidence. Its limitations are presence of artefacts like bare nuclei, closely packed nuclei and disrupted cells, difficulty in distinguishing mature erythroblasts from lymphocytes and plasma cells, poor stain penetration in the thicker parts of the aspirate and variable staining of cytoplasmic granules.
The advantage of wedge smears is that it can avoid overestimation of marrow cellularity and megakaryocytes with good preservation of cellular morphology. However cellular loss from the fragment trails resulting in paucicellular trails may lead to underestimation of cellularity especially in anti-coagulated marrow specimens. Also, if the smears are not made rapidly after aspiration, considerable hemodilution may occur [8,12,16].
In accordance with the ICSH guidelines [8] and the findings of our study, centers presently making only one type of smear may consider examination of both types of smears in the future. A possible limitation of our study is that we did not have sufficiently large numbers in each category of the malignant diagnosis. A larger study only on malignant disorders may provide more robust data regarding the diagnostic differences between the two smear types.
Conclusion
Our study has shown overestimation of marrow cellularity and megakaryocytes in the squash smears as compared to wedge smears with no difference in M:E ratio or morphology. Acceptable agreement for diagnosis was seen for most malignant and benign disorders. Major diagnostic discrepancies between the smear types were seen in patients with lesions known to have focal distribution. Squash and wedge smears are complementary techniques and both smear types should be studied for bone marrow examination.
Acknowledgement
The study was conducted at Christian Medical College & Hospital, Ludhiana. The authors acknowledge the contribution of Mr. Prasan Das, laboratory technician, for retrieval of the bone marrow aspirate slides.
Funding
No funding was required for the study.
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no conflict of interest.
Availability of Data and Material
Yes.
Consent for Publication
Yes.
Consent to Participate
Consent was taken from all patients.
Ethical Approval
This study was a thesis for the post-graduation in pathology by the first author. Ethical and research committee approval was obtained prior to the study.
Human and Animal Rights
No animals were used in this study. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed Consent
Informed consent from the patients was taken.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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