Bone Marrow Morphology: A Key Diagnostic Tool in Various Hematological and Non Hematological Disorders

Bone marrow morphology means microscopic examination of bone marrow cells and tissue samples obtained by aspirate and trephine biopsy. An invaluable tool for diagnosis of many hematological and non-hematological disorders, various types of cancer staging and metastases detection. Different kinds of anemia and leukemia can be accurately diagnosed and monitored. Objective: To check the spectrum of diseases which could be identi�ed and diagnosed with the help of bone marrow morphology examination thus demonstrating its diagnostic utility and to check diagnostic concordance between aspirate and core biopsy. Methods: Cross sectional study with consecutive sampling technique conducted over period of one year. Samples of both Bone Marrow Aspiration (BMA) and trephine biopsy (BMB) were collected from all patients included in study after consent using standard protocols. Air dried smears were prepared from bone marrow aspiration samples while trephine biopsy tissues were processed by histopathological techniques. Routine staining such as Wright Giemsa stain, H and E stain and special cytochemistry were used to visualize the cellular architecture of bone marrow. Results: Out of 471 samples, males were predominant with the frequency of 63.9% and maximum patients (31.8%) were from the age group of 35-40 years. Most common clinical indication to conduct bone marrow examination was unexplained Cytopenia which accounted for 35.2% cases, followed by suspicion of Leukemia (28.5%). Malignant hematological disorders were more common as compared to benign disorders (64% vs 17.5%). Acute leukemia was the most commonly identi�ed cancer with frequency of 27.6%. Conclusions: Bone marrow morphology till date remains low risk, economical, crucial diagnostic tool in especially in under resource country like Pakistan. It can guide physicians to plan proper and timely management of patients.

Pakistan only major medical institutions such as those in Karachi, Lahore, and Rawalpindi, likely have been among the early adopters of this procedure.Bone marrow examination must be preceded by complete clinical assessment of the patient including clinical history, baseline hematological examination including complete blood count and peripheral smear examination followed by targeted radiological, microbiological and biochemical investigations if required [4].Bone Marrow Aspirate (BMA) is a diagnostic procedure that involves extracting a small amount of liquid bone marrow for examination under light microscope after air dried smears are stained with routine Wright Giemsa stain.Information obtained from Bone Marrow Aspiration includes overall bone marrow cellularity including exact percentage of hematopoietic / abnormal cell counts, which can help in differentiating between proliferative and hypoplastic bone marrow disorders like Myelodyplasia and Aplastic anemia respectively [5].It can also give signi cant information about morphological/ cytological detail about size, shape, cellular outline, cytoplasm and nuclei of different cells under investigation such as characteristic appearance of Lymphoblast, Myeloblast and other malignant Plasma cells, thus enabling Pathologists (Hematologists) to correctly diagnose different types of acute lymphoid leukemia, acute myeloid leukemia and plasma cell myeloma respectively [6].Bone marrow aspirate smears can also be used for cytochemical staining such as Sudan Black B (SBB), Myeloperoxidase (MPO), Periodic Acid Schiff (PAS), Speci c and Nonspeci c Estrases (NSE) etc., which can help in classifying different types of Leukemia.Bone marrow aspirate can also be used for diagnosis of different infectious diseases by doing bone marrow aspirate cultures and sensitivity.Bone marrow morphology examination after staining aspirate smears with another special stain i.e., Perl's stain is still gold standard for diagnosis of Iron De ciency anemia [7].Bone Marrow Trephine Biopsy (BMB) is a diagnostic test where a small core tissue of bone marrow is removed for examination under microscope.It complements the information obtained from a bone marrow aspiration.Its examination under light microscopy gives detailed panoramic view of bone marrow cellularity and provides c o m p re h e n s i ve ove r v i ew of i n a m m a to r y c e l l s percentage, presence of any extra medullary cells, dysplasia, and cellular atypia, patterns of in ltration by malignant lymphoma, metastatic disease, marrow packing with blasts and any degree of brosis.Presence of Histiocytosis, and Storage disorders are clearly diagnosed on trephine biopsy [8].Granulomatous disease is another di cult diagnosis detected by trephine biopsy.Bone marrow trephine biopsy has another advantage that its can be used for another important diagnostic modality that is

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immunohistochemistry to study con rmation of diagnosis, extent of bone marrow in ltration and prognosis of many malignant neoplasms.Two pillars of bone marrow morphology exam such as Bone marrow aspirate and trephine biopsy are both complementary to each other however sometimes aspirated specimen is of very low volume and sometimes it is diluted with sinusoidal blood, in both these conditions bone marrow aspirate is devoid of cellular components and suboptimal for reporting [9].Adequate core length trephine biopsy makes it ideal technique for providing information regarding marrow cellular and architectural changes occurring in marrow cavity due to various underlying malignant and nonmalignant processes.Therefore, aim of present study was to check the spectrum of diseases which could be identi ed with the bone marrow e x a m i n a t i o n a t i n i t i a l s t a g e s .F r e q u e n c y a n d categorization of benign and neoplastic lesions were also checked to compare diagnostic concordance between BMA and BMB.
The present study was conducted at the Pathology department (Hematology section) of King Edward Medical University/Mayo Hospital Lahore over the study period of one year from March 2023 to March 2024.Ethical approval was taken from the institute Ethical Review Board (Reference Number: 329/RC/KEMU).It was cross sectional study and samples were collected through consecutive sampling technique.Sample size was calculated by using win-pepi ver: 11.15 software with con dence level of 95%, acceptable difference = 0.05 [2].A Total of 471 cases which ful lled the inclusion criteria were selected in the study period.Inclusion criteria included cases from the both gender of age groups (1-60 years) in the study.All the patients that were referred for examination of bone marrow and also for staging of hematological malignancies were included in the current study.Exclusion criteria de ned as diluted bone marrow aspirate and samples from the patients less than 1 year of age were excluded from the study.Demographic data like age and gender were recorded from all the included cases.Clinical indications were determined by taking into account, patients thorough clinical history and on the basis of complete blood count CBC and peripheral smear results.Both BMA and BMB were taken from each patient according to standard ICSH (International council for standardization in Hematology) protocol of bone marrow sample collection [5].Written informed consent was taken from each patient before the procedure.Bone marrow aspirate was done from posterior iliac crest using aseptic techniques.Anterior superior iliac spine was used for the bone marrow sample collection from obese patients.Selected area from where the sample was taken was locally anesthetized rst and then commercial 127 bone marrow needle (with removable stylet) of appropriate size was used to take small amount (0.5ml) of bone marrow aspiration followed by collection of bone marrow trephine biopsy sample placed in Bouin's solution.Air dried smears were made from the bone marrow aspirate while for trephine biopsy, (>1cm) biopsy on gross examination was considered adequate.For the processing of bone marrow aspirate, a mono-layered smear was made from the concentrated marrow cells on the slide.After air methanol used to x the smears.Slides were stained with Wright-Giemsa stain which contain methylene blue and eosin dissolved in methanol.After staining, slides were air dried and were observed under light microscope Olympus CX43 ve head Microscope using 10x, 20x, 40x and 100x magni cation by counting 400 cells to visualize the cytological detail and in ltration pattern of the bone marrow.For processing of bone marrow trephine biopsy, samples were initially decalci ed.After this, samples were processed according to standard histopathological techniques including tissue processing and embedding in para n wax.Special stains such as Sudan Black B, Non-Speci c Esterase (NSE), Acid Phosphatase, Periodic Acid Schiff (PAS), Reticulin stain was used according to the patient's clinical symptoms and probable disease identi cation.Photomicrographs were taken by digital camera attached to Olympus CX43 ve head light microscope EP50 with 2592x1944 pixel resolution.Normal morphology of bone marrow was distinguished from pathological bone marrow by the presence of abnormal cells in ltrate in latter.Acute Leukemia was diagnosed and differentiated into Acute Lymphoblastic (ALL)and Acute Myeloblastic Leukemia (AML) according to WHO 2016 diagnostic criteria stating presence of 20% Bone marrow aspirate Blasts and classi ed according to FAB morphological classi cation by using special staining methods (SBB, MPO, PAS, AP, NSE) and subtyped by using immunohistochemistry on trephine biopsy.Similarly, remission assessment was done morphologically according to percentage of bone marrow blast (<5%).Chronic leukemia such as Chronic Myeloid Leukemia (CML) was diagnosed morphologically according to WHO 2022 criteria de ned by Granulocytic Hyperplasia with variable number of blasts according to different stages of disease.Lymphomas were differentiated on the basis of morphologically different atypical lymphoid population and subtyped by immunohistochemistry. Multiple Myeloma was differentiated due to presence of >10% abnormal plasma cells in bone marrow morphology as part of WHO 2016 diagnostic criteria.MPN especially Myelo brosis was one of the disorders that can only be diagnosed on bone marrow e x a m i n a t i o n w i t h R e t i c u l i n s p e c i a l s t a i n.N o n hematological metastatic disease was differentiated from hematological malignancies using speci c morphology of malignant cells and use of immunohistochemistry [9].Amongst Benign Hematological disorders, Dimorphic anemia was differentiated from Megaloblastic anemia by presence of two different erythroid maturation pattern with special stain (Perl's stain) used to diagnose iron de ciency anemia.Hypoplastic bone marrow disorders showed less than 20% cellularity in BMB exam.Malaria infection showed ring forms and gametocytes in bone marrow, Leishmania infection exhibited LD bodies in bone marrow while tuberculous granulomatous disease was differentiated on marrow morphology showing giant epitheloid cells and in ammatory cell in ltrate.While bone marrow aspirate culture isolated salmonella typhi.Storage disorders showed bone marrow in ltration by large histiocytic gaucher cells and foam cells.Data were collected and statistically analyzed by using SPSS software 27.0.Numerical data were expressed as mean.Frequencies and percentages were calculated for the univariate variables.Diagnostic concordance was the rate of agreement between BMA and BMB was expressed as percentage.Chi square test was used to evaluate any statistically signi cant difference between BMA and BMB.

R E S U L T S
A total of 471 samples of bone marrow aspiration and trephine biopsy for bone marrow morphology examination was processed during the study period.Out of which 63.9% (n=301) samples were collected from the male patients while 36.1% (n=170) samples were collected from the female patients.Age distribution showed that maximum samples were collected from the age group of 30-45years n=150, followed by age group of 45-60yrs and 15-30yrs (n=100) each table 1.

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Most frequent complications associated with the sampling of bone marrow aspirate and bone marrow trephine biopsy were pain, anxiety, bleeding and dizziness.About 90% in individuals feel pain during the procedure in spite of the local anesthesia.While ratio of dizziness and bleeding was too low as compared to pain which was 1.09% and 1.82% respectively.Total 471 samples of bone marrow examination were processed for the identi cation of hematological and non-hematological diseases by using Giemsa and special stains.72 samples showed normal bone marrow cellular architecture.Most common clinical condition observed was Leukemia (both acute and chronic leukemia/Lymphomas) which was 48.8% (n=130), followed by Remission of malignant tumors 11.8% (n=56).Detailed disease pattern observed in bone marrow examination was summarized in table 3.In this disorder bone marrow trephine morphology was mandatory for staging of disease, without which treatment can't be started (Figure 1).Photomicrographs were taken by digital camera attached to Olympus CX43 ve head light microscope EP50 with 2592x1944 pixel resolution.In present study, diagnostic concordance rate between BMA and BMB for diagnosis of various disorders was 88% while only 27 cases were missed on BMA alone and was diagnosed on BMB making diagnostic disagreement between both tools of bone marrow morphology almost negligible as depicted in table 4. Comparison of the cases diagnosed by bone marrow aspirate and bone marrow trephine biopsy was statistically done by using Chi-square test.Diagnosed cases and missed diagnosis in both modalities were compared which showed statistically signi cant value of 0.012.This table showed there was a statistically signi cant difference between BMA and BMB diagnosis (p < 0.05).This means that the proportion of diagnoses being done or missed were statistically different between BMA and BMB, which shows the complementary nature of both modalities in bone marrow morphology interpretation (Table 5).
disorder (MPN-ET).This patient p r e s e n t e d w i t h p o r t a l v e i n t h r o m b o s i s a n d thrombocytosis.Without bone marrow morphology, diagnosis of this disease was not possible without timely bone marrow exam showing characteristic large megakaryocytes with abundant cytoplasm and lobulated, stag horn nuclei.Photomicrograph D shows Bone marrow metastatic disease.

Table 1 :
Age Distribution of Patients

Table 2 :
Clinical shows a rare nding of presence of bone marrow Calcium Oxalate crystals in a patient who presented with cytopenias (anemia, platelet dysfunction) and bone pains.It's crucial to start early and individualized treatment.Photomicrograph

Table 4 :
Diagnostic Concordance between BMA and BMB Among non-malignant hematological conditions, hypocellular marrow was observed in 7% (n=33) of cases.Another study also reported similar results with the percentage of 5.3%[13].It's important to mention here that aplastic anemia and other hypocellular bone marrow syndromes can only be PJHS VOL. 5 Issue.8 Aug 2024 Copyright 2024.Published by Crosslinks International Publishers 129