Year : 2010 | Volume
: 53 | Issue : 1 | Page : 96--100
Morphological findings in bone marrow biopsy and aspirate smears of visceral kala azar: A review
Kajal Kiran Dhingra, Parul Gupta, Vijay Saroha, Namrata Setia, Nita Khurana, Tejinder Singh
Department of Pathology, Maulana Azad Medical College and Lok Nayak Hospital, Bahadur Shah Zafar Marg, New Delhi - 110 002, India
Kajal Kiran Dhingra
Room No. 62, Department of Pathology, Maulana Azad Medical College, New Delhi
Context: Visceral leishmaniasis (VL) is endemic in India and may simulate and cause many hematological disorders like pancytopenia, myelofibrosis, myelodysplasia and hemophagocytosis. Aims: The study aims to investigate the hematological manifestation of Visceral Leishmaniasis and associated changes that may be observed in bone marrow aspirate smears and biopsy which may warn a pathologist of possible infections. Settings and Design: This is a retrospective study of 18 VL cases on B (b) one marrow aspirate and biopsy in the department of Pathology in a tertiary care teaching hospital in New Delhi. Methods and Material: Giemsa stained slides of bone marrow aspirates and hematoxylin and Eosin stained biopsy slides were reviewed in detail by two competent pathologists. All the findings were tabulated and discussed and comparisons made with the previous similar studies. Results: Hyper cellular marrow, increased lymphocytes and plasma cells, marrow granulomas, hemophagocytosis, myelofibrosis, myelodysplasia and gelatinous transformation of the marrow were notable features the presence of which together or individually should caution a pathologist to search for Leishman Donovan (LD) bodies in patients especially in a non-endemic zone in a tropical country.
|How to cite this article:|
Dhingra KK, Gupta P, Saroha V, Setia N, Khurana N, Singh T. Morphological findings in bone marrow biopsy and aspirate smears of visceral kala azar: A review.Indian J Pathol Microbiol 2010;53:96-100
|How to cite this URL:|
Dhingra KK, Gupta P, Saroha V, Setia N, Khurana N, Singh T. Morphological findings in bone marrow biopsy and aspirate smears of visceral kala azar: A review. Indian J Pathol Microbiol [serial online] 2010 [cited 2021 Jan 18 ];53:96-100
Available from: https://www.ijpmonline.org/text.asp?2010/53/1/96/59193
Visceral leishmaniasis (VL), also known as kala azar, is an endemic disease in tropical and subtropical regions of India. It may mimic and lead to a variety of hematological disorders like pancytopenia, myelofibrosis, myelodysplasia and hemophagocytosis. Although the diagnostic utility of bone marrow aspirate smears is well proven no study describes the associated histomorphological features of leishmania in the biopsy sections. The cytological and histological features associated with VL have been described individually in literature. However, there is no such report from the Indian subcontinent that describes all these morphological features together. We aim to retrospectively study in detail the various findings in 18 cases of kala azar reported in our institute for which both bone marrow aspirate and biopsy were performed.
Material and Methods
This retrospective analysis was done from the available records of patients diagnosed as VL/ kala azar on bone marrow aspirate followed by biopsy in the department of Pathology in a tertiary care teaching hospital in New Delhi. Medical records were retrieved and details including clinical presentation and examination findings collected for each of the 18 patients. Various hematological and biochemical investigations were recorded from the patient files. Giemsa-stained slides of bone marrow aspirates and hematoxylin and eosin stained biopsy slides were taken out from the records and reviewed in detail by two competent pathologists. All the findings were tabulated as shown below and have been discussed in detail with special reference to the aspirate and the biopsy features. These were compared with the observations made previously by various observers.
A total of 18 patients of VL diagnosed either on bone marrow aspiration or biopsy from the year 2000 to 2006 were reviewed. There were 14 male and four female patients. The age ranged from 14 to 42 years (median 18 years) in males and eight to 32 years (median 21 years) in females. Fever was present in 13 out of 18 cases (mean duration 4.2 months). Abdominal pain and swelling was seen in eight cases (44.44%). Splenomegaly as seen in all cases (100%), moderate splenomegaly in 22.22% of cases and marked splenic enlargement was present in the remaining 77.77% of cases. Hepatomegaly was present in 8 of the 18 cases (44.44%). Lymphadenopathy was present in 4 cases (22.22%). Pallor and fatigue were the main presenting symptoms in 13 cases (72.22%). Weight loss and loss of appetite was present in 14 cases (77.77%). Bleeding manifestations including purpuric spots and epistaxis were seen in 4/18 cases (22.22%). The observations in our study are tabulated below. Anemia was present in 17 cases with a mean hemoglobin concentration of 7.5 g. Similarly, leucopenia was present in 13 cases with a mean total leukocyte count of 4400/cumm; thrombocytopenia was present in 11 out of 18 cases with a mean platelet count of 84000/cumm. Pancytopenia in the form of anemia, leucopenia and thrombocytopenia was therefore most common hematological finding. Relative lymphocytosis was present in five of our patients. Macrocytosis was also seen in six cases (mean MCV 88.73 fl).
Leishmania donovani (LD) bodies were presenting, both intracellular and extracellular, in 12 cases [image I [Figure 1]A and B]; six cases had only intracellular LD body. Intracellular organisms were found within the macrophages and rare ones in the neutrophil. A nuclear body along with a distinct kinetoplast was appreciated in each of these cases. Increased cellular correspondence to age assessed on bone marrow biopsy was seen in 14 of our cases. Erythroid hyperplasia with Myeloid: Erythroid ratio ranging from 2:1 to1:4 were seen in four cases; three of them also had megaloblastic erythroid reaction with open nuclear chromatin and asynchronous nucleocytoplasmic maturation. Increased fibrosis was seen in three cases (image I [Figure 2]A). Prominent marrow eosinophils were present in three cases. Increased marrow lymphocytes and plasma cells were seen in 13 cases, with formation of lymphoid follicles in four of these cases. Increased macrophages were seen in nine cases and four of these also revealed non caseating epithelioid cell granulomas [image I [Figure 2]B]. Gelatinous transformation (acellular amorphous eosinophilic material) was seen in one of our cases. [image II [Figure 3]A and B]. Hempohagocytosis was observed in two cases. [Image II [Figure 4]A] Feature of myelodysplasia which included dyserythropoeisis in the form of karryorhexis, bi and multinuclearity in the erythroid series, giant and hypolobated myeloid precursors and hypolobated and micromegakaryocytes were seen in four cases.[image II [Figure 4]B] Marrow iron stores assessed on Perls (Perls') stain revealed increased stores (grade 4-5) in six cases.
VL is caused by the protozoan parasite Leishmania Donovani and transmitted by the bite of infected sandfly Phlebotomus argentipes, dog is the most common reservoir. Also known as kala azar (Indian name meaning black fever), VL is endemic in tropical and subtropical regions. It is a chronic illness characterized by irregular fever, hepatosplenomegaly and pancytopenia, progressive weakness and emaciation which can result in death if left untreated. Globally, the incidence and prevalence of kala azar cases per year are 0.5 and 2.5 million, respectively.  More than 90 per cent of the world's VL cases are from the Indian subcontinent. Ninety per cent of all these cases are reported from the States of Bihar, West Bengal and Uttar Pradesh alone.  Emergence of AIDS epidemic has further caused resurgence in incidence of VL in India, which has now become an important opportunistic infection. 
Confirmation of diagnosis is made by demonstration of the parasite by microscopic examination of material obtained by bone marrow aspiration biopsy or rarely splenic aspirates. The parasites are found intracellular in the reticuloendothelial system as the amastigote form which is aflagellate, round and two to four µm in diameter, known as the Leishman-Donovan (LD) body. Various cytological and histological features associated with VL have been described individually in literature. However, no study in the Indian subcontinent describes all morphological features together. Since the disease burden in our country is enormous a study to evaluate bone marrow biopsy and aspirate findings would guide the pathologist to a probable diagnosis of VL when the suspicion is otherwise low.
We received a total of 288 VL cases diagnosed on bone marrow aspirate smears in the 2000 to 2006 period. The cases (18) presented here are very few in number compared to the VL cases diagnosed in our hospital. This is because some cases were accompanied by a biopsy for suspected unrelated diagnosis; we excluded all such cases without a biopsy. Fourteen cases in our study had hyper cellular bone marrow aspirate smears confirmed on biopsy. The other four cases were normocellular for age. Erythroid hyperplasia was prominent in four of these cases. According to reviewed reports hypercellularity has been reported almost universally in the bone marrow aspirate smears. ,, The mechanism suggested postulates that LD infection of marrow stromal macrophages selectively enhances myelopoiesis by GM-CSF and TNF-alpha overproduction.  Patients with hypercellular marrow have been reported to respond well to therapy.  Increased eosinophilic precursors were seen in three of the 18 bone marrow aspirates. No other cause of secondary eosinophilia could be demonstrated. It is not understood if this was unrelated to VL or as a result of the parasitism by LD. We did not find any report of similar observations in the searched medical literature. The presence of abundant lymphocytes and plasma cells was seen in 13 of our cases.
Most of the lymphocytes were interstitial in location with admixed maturing plasma cells. Two of our cases also had benign lymphoid nodules with secondary germinal centre formation. Plasma cells were particularly observed as perivascular aggregates. This perivascular location and admixture with interstitial lymphocytes helps differentiate the suspicious cases from a probably plasma cell dyscrasia, especially in older age group. Kumar et al. have reported similar lymphoid nodules with many LD bodies.
Dabiri et al.  reported hypolymphpoiesis in VL.  Patients with benign lymphoid nodules have been reported to respond well to therapy. Increased histiocytes in the aspirate as well as biopsy were seen in 10 cases. Most of them demonstrated intracellular LD bodies. Increased histiocytes which may or may not be associated with hemophagocytosis have been reported by other authors.  Ill defined granulomas were seen in four of our cases. Granuloma formation is rarely seen in VL. Migration of histiocytes at the time of the initial infection in the bone marrow results in the formation of noncaseating granuloma. This may also be confused with tubercular granuloma due to the presence of fewer LD bodies; especially in countries like India where both diseases are endemic. 
Various other authors have described granuloma formation and reported paucity of LD bodies. ,, This paucity of LD bodies in the granuloma may lead to an under diagnosis of VL. Correlation with response to treatment has been attempted and Kumar et al.  observed, in 2007, that patients with granulomas did not respond to glucantime therapy but responded to amphotericin B.
One of our 18 cases revealed Gelatinous Marrow Transformation (GMT) characterized by fat cell atrophy, focal loss of hematopoietic cells, and deposition of extra cellular gelatinous substances rich in hyaluronic acid. Alcian-B(b)lue staining on the marrow aspirate and smears is useful in confirmation of these cases. Gelatinous transformation associated with leishmaniasis has been reported in VL only once. Although it is reported in patients of anorexia nervosa, acute febrile states, and AIDS, it is yet to be investigated whether it is a direct consequence of parasitization or a manifestation of gelatinous transformation occurring in febrile states. 
Three of the 18 cases in our series had marrow fibrosis. Myelofibrosis suspected on routine H and E sections was confirmed on reticulin staining. Increased reticulin (grade 3-4) was observed in these cases. The association of myelofibrosis with VL has been reported in literature only on rare occasions. Therefore, the three cases described here are significant. To the best of our knowledge, this is the fourth report describing secondary bone marrow fibrosis associated with kala azar. Kumar et al.  have reported diffuse fibrosis with rare LD bodies in the biopsy. The bone marrow fibrosis is transient and these patients are expected to show regression after treatment.  Therefore, we suggest a routine screening for LD body in biopsies received for suspected myelofibrosis. Also, a reticulin staining should be performed regularly to look for bone marrow fibrosis and to grade it, in known cases of kala azar. ,,,
On reviewing the peripheral smears of these cases a substantial number of tear drop cells and nucleated red cells were observed. Kumar et al.  reported that patients with marrow fibrosis and marrow necrosis were resistant to any type of therapy and therefore may predict a poorer prognosis. Hempohagocytosis in the form of engulfed erythroid and myeloid precursors within the marrow was seen in two of our cases. The marrow and other reticuloendothelial organs are infiltrated by activated lymphocytes and macrophages engulfing hematopoeitic cells. This can result in in pancytopenia, fever, organ enlargement, neurological dysfunction and disseminated intravascular coagulation constituting the hemophagocytic syndrome.  It has been reported as a complication of VL in various other reports, especially as a life threatening complication in children. ,, Also it has been reported to delay the diagnosis by misleading the pathologist away from the primary diagnosis. Differentiation between primary and secondary hemophagocytic syndrome is very important because hemophagocytosis associated with infectious diseases like VL resolves with treatment of the underlying infections, while cytotoxic drugs are used for the treatment of primary cases. 
Case reports of VL masquerading as myelodysplasia have been described in literature. ,, This prompted us to retrospectively investigate the available peripheral blood and bone marrow aspirate smears diagnosed as VL. We observed bilineage and trilineage myelodysplasia in four cases. Dyserythropoiesis alone has been reported by authors  along with megaloblastic features  Elevated levels of tumor necrosis factor-alpha (TNF-alpha) may cause nonclonal trilineage myelodysplasia in the patients with VL.  This myelodysplasia is one of the factors responsible for pancytopenia. This is a cause of misdiagnosis if LD bodies if the organism is not carefully screened for. Also, it is a potentially reversible cause of myelodysplasia. Therefore, timely and adequate therapy can lead to normal hematological profile.
Increased iron stores have been observed in six cases of VL. Both increased and decreased stores have variably been reported in VL. Increased stores could result from cytokine production especially tumor necrosis factor which also leads to anemia of chronic disease.  Also, variable degrees of dyserythropoiesis lead to intramedullary destruction of erythroid precursors causing increased marrow stores. One study reported low iron stores in the marrow.  This is explained by the poor nutritional status of patients belonging to low socioeconomic strata.
To conclude, a few of our findings in the bone marrow biopsy and aspirate smears have also been observed by other authors but no analysis had been done noting all the associated findings together. A note of such aforementioned observations should warn a pathologist to search for a probable Leishmania infection. Since correlation of various bone marrow findings has also been attempted with response to therapy, a mention of these findings on the report may guide the clinician to the line of therapy to be followed.
We thank clinicians from the medicine and pediatric departments for providing us with valuable bone marrow aspirate smears and biopsy. We would also like to thank technical staff of the department of hematology for help rendered.[Table 1],[Table 2],[Table 3]
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