Contribution of flow cytometric immunophenotyping and bone marrow trephine biopsy in the detection of lymphoid bone marrow infiltration in non-Hodgkin's lymphomas

The bone marrow (BM) is a frequent site of involvement in non-Hodgkińs lymphomas (NHL) and evidence of an infiltrated BM may implicate different therapeutical regimens. Flow cytometric immunophenotyping of bone marrow aspirates now is included in the assessment of patients with NHL and used as an adjunct to morphologic evaluation in the staging of lymphoma. The aim of the study was to compare flow cytometric immunophenotyping of BM and paraffin section staining of BM biopsies in the marrow involvement of NHL. Cytometric immunophenotyping of bone marrow and immunohistochemical paraffin section staining of bone marrow biopsies in 53 B- and T-cell lymphoma patients were performed. We used the following fluorochrom conjugated monoclonal antibodies specific for: CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD23, CD79B, FMC7 and Ig kappagamma light chain. Unilateral BM trephine biopsies were obtained in all cases, fixed, decalcified and paraffin-embedded. Morphologic marrow involvement by lymphoma was found in 24 cases; flow immunophenotyping identified 26 cases with NHL: morphology-positive/flow-positive (n=21), morphology positive/flow-negative (n=3), morphology-negative/flow-positive (n=4), and morphology-negative/flow-negative (n=23). The concurrence rate of BM trephine biopsy and flow cytometric immunophenotyping in evaluation of NHL bone marrow infiltration was 88.7%. Immunophenotyping of the bone marrow of NHL patients by flow cytometry is helpful for assessment of bone marrow infiltration, especially in B-cell disorders. Both trephine biopsies and flow cytometry are better than single investigation for detection of infiltration in NHL.     

Audit of bone marrow aspirates and trephine biopsies in multiple myeloma--a single centre study

We compared 79 simultaneous marrow aspirates and trephine biopsies from multiple myeloma patients for sensitivity, concordance, quality and clinical relevance. A total of 60 examinations had been performed for initial diagnosis, i.e. in cases of suspected myeloma and 19 at follow-up. Of which, 45 (57%) of trephine biopsies were less than 1.6 cm before processing and 33 (42%) were crushed and/or fragmented. Overall, only 19/79 (24%) of trephine biopsy specimens were of at least 1.6 cm length prior to processing and not disrupted. On the other hand, 75% of aspirates were particulate and satisfactory. Mean time between receipt of a trephine biopsy specimen and issuance of a histopathological report was 9 days. Although 40% of trephine biopsies yielded information that could not be reliably obtained from a bone marrow aspirate such information was in all cases clinically irrelevant or obtainable by non-invasive means. In all cases where myeloma was detected in a trephine biopsy it was also detected in a simultaneous bone marrow aspirate, if particulate. However, there were four (5%) cases in which myeloma was detected in such aspirates but not in simultaneously taken trephine biopsies. In cases (n=19) where repeat aspirates/trephine biopsies were taken for surveillance, concordance was found between reported changes in plasma cell ratio. Our data failed to demonstrate any added benefit from routinely performing trephine biopsies after a particulate specimen had been aspirated for the diagnosis or surveillance of myeloma. Furthermore, they suggest that particulate aspirates may be at least as sensitive as trephine biopsies for detecting myeloma.     

Localisation and distribution of hyaluronan in normal bone marrow matrix: a novel method to evaluate impending fibrosis?

Bone marrow trephine biopsies from 30 healthy volunteers, 10 men and 20 women aged 18-60 yr were obtained for identification and localisation of hyaluronan (HYA). Fixation, decalcification and embedding were performed by two different methods, with identical results in both. For comparison bone marrow trephine biopsies from three patients with different haematological diseases and known fibrosis were studied. All bone marrow specimens were also stained for reticulin grading. HYA was found in the bone marrow specimens from healthy individuals in a pattern that was concordant with the reticulin staining, the common way of visualising bone marrow fibrosis. In bone marrow from the patients with known fibrosis the HYA and reticulin staining were both more intense and abundant. Interestingly, HYA was also found intracellularly in eosinophilic cells in normal bone marrow. HYA is a polysaccharide unique both in structural and biological properties, and in excess it may predict bone marrow fibrosis.     

Audit of bone marrow aspirates and trephine biopsies in multiple myeloma – a single centre study

We compared 79 simultaneous marrow aspirates and trephine biopsies from multiple myeloma patients for sensitivity, concordance, quality and clinical relevance. A total of 60 examinations had been performed for initial diagnosis, i.e. in cases of suspected myeloma and 19 at follow‐up. Of which, 45 (57%) of trephine biopsies were less than 1.6 cm before processing and 33 (42%) were crushed and/or fragmented. Overall, only 19/79 (24%) of trephine biopsy specimens were of at least 1.6 cm length prior to processing and not disrupted. On the other hand, 75% of aspirates were particulate and satisfactory. Mean time between receipt of a trephine biopsy specimen and issuance of a histopathological report was 9 days. Although 40% of trephine biopsies yielded information that could not be reliably obtained from a bone marrow aspirate such information was in all cases clinically irrelevant or obtainable by non‐invasive means. In all cases where myeloma was detected in a trephine biopsy it was also detected in a simultaneous bone marrow aspirate, if particulate. However, there were four (5%) cases in which myeloma was detected in such aspirates but not in simultaneously taken trephine biopsies. In cases (n = 19) where repeat aspirates/trephine biopsies were taken for surveillance, concordance was found between reported changes in plasma cell ratio. Our data failed to demonstrate any added benefit from routinely performing trephine biopsies after a particulate specimen had been aspirated for the diagnosis or surveillance of myeloma. Furthermore, they suggest that particulate aspirates may be at least as sensitive as trephine biopsies for detecting myeloma.     

Deposition of autofluorescent eosinophil granules in pathologic bone marrow biopsies

Eosinophil granules are intensely autofluorescent when excited by green light. To determine if eosinophils degranulate in the bone marrows of patients with a variety of diseases, we used green light epifluorescence microscopy to examine deparaffinized and dezenkerized sections of 49 bone marrow core biopsies. In 14 of the biopsies, there was striking extracellular deposition of intensely autofluorescent eosinophil granules in addition to numerous intact eosinophils. Among the 14 specimens with extracellular autofluorescence were seven cases of leukemia, four cases of non-Hodgkin's lymphoma, two cases of myelofibrosis, and one case of pancytopenia with eosinophilia. In the remaining 35 specimens, only intact eosinophils were identifiable. There was no extracellular autofluorescence in three normal marrows, four marrows from AIDS patients, or three biopsies from patients with idiopathic thrombocytopenic purpura (ITP). We conclude that green light epifluorescence microscopy identifies extracellular deposits of eosinophil granules in bone marrow biopsies of some neoplastic disorders and in diseases associated with reticulin fibrosis.     

Aberrant positivity for CD79a in erythroid lineage cells--a finding observed in a subset of re-staging bone marrow trephine biopsies after treatment cell pain

Aberrant expression of CD79a has been reported in neoplastic cells in peripheral T cell lymphoma, T-cell acute lymphoblastic leukemia and acute myeloid leukemia (especially those with t(8;21)). In this report, we document the first report of CD79a positivity in erythroid precursor cells in bone marrow. In all, we document this finding in five of 18 re-staging bone marrow trephine samples in patients of lymphoma treated with chemotherapy (one index case and 17 additional validation cases). It is important to appreciate this finding especially in rituximab treated patients where one tends to rely on CD79a to identify minimal marrow disease.     

Bone Marrow Involvement in Neuroblastoma: A Study of Hemato-morphological Features

Bone marrow involvement in neuroblastoma indicates advanced stage of disease. The recent use of autologous bone marrow “rescue”, has provided an additional important reason for accurate assessment of bone marrow status in newly diagnosed patients. In this study, we analyzed 44 cases of neuroblastoma for bone marrow infiltration status and their hematological parameters. Eighty-eight bone marrow aspirate and trephine touch imprint smears and 44 trephine biopsy sections were examined in these 44 patients. Of these, 24 cases (54.5 %) showed marrow infiltration. Leucopenia and bicytopenia were significantly (p < 0.05) associated with marrow infiltration. Both bone marrow aspirate and biopsy were positive for infiltration in 16 out of 24 positive cases. Only aspirate smears were positive in 4 and only trephine biopsy in another 4 cases. The pattern of infiltration consisted of rosette formation in 40.7 % cases on aspirate smears and 22.2 % cases in trephine biopsies. Remaining cases showed diffuse and interstitial presence of tumor cells and cases positive only on trephine biopsy, showed marked stromal reaction. Bilateral trephine biopsies combined with aspirate smears picked up all positive cases compared to when they were assessed alone.     

Random skin biopsy and bone marrow biopsy for diagnosis of intravascular large B cell lymphoma

Intravascular lymphoma (IVL) is a rare type of extranodal lymphoma in which the lymphoma cells proliferate exclusively in the lumina of small vessels. The diagnosis of IVL requires histological confirmation. Although random skin biopsy from healthy-appearing skin in patients with suspected IVL appeared to be useful, the sensitivity of this method for the diagnosis of IVL remains unknown. We performed a random skin biopsy from 12 consecutive cases of IVL diagnosed at our institution over the past 4 years and evaluate its relevance of clinical and laboratory characteristics, presence or absence of skin lesions, and bone marrow involvement. All 12 patients were diagnosed antemortem by either random skin biopsy or bone marrow biopsy and treated with rituximab-containing chemotherapy. Random skin biopsy was performed in all 12 patients, and the results were positive in ten patients (83.3%). Erythematous skin lesions were seen in 3 of 12 patients, but biopsy was positive for lymphoma lesion in two patients. Bone marrow invasion was seen in 11 of the 12 patients (91.6%) by bone marrow smear and/or flow cytometric analysis, but was detected in only half of the patients by trephine biopsy. We concluded that random skin biopsy from normal-appearing skin is highly sensitive in the diagnosis of IVL comparable to bone marrow trephine biopsy. It should be performed irrespective of the presence or absence of skin lesions in patients who were suspicious of IVL.     

Anaplastic large cell Ki-1 lymphoma involving bone marrow: marrow findings and association with reactive hemophagocytosis.

This report describes the bone marrow findings in four patients whose marrow was involved by anaplastic large-cell Ki-1 lymphoma, an uncommon event in this type of lymphoma. In the marrow aspirate smears, the involvement was subtle, and was in the form of isolated large cells with irregular nuclear configuration, coarse chromatin, prominent nucleoli, and basophilic cytoplasm which might be vacuolated. One case showed paradoxically massive involvement in the trephine biopsy taken from the same site as the marrow aspirate. Reactive histiocytic proliferation with hemophagocytosis was also present. Since marrow aspirate or biopsy may be the first pathologic specimen examined in patients having anaplastic large-cell Ki-1 lymphoma, it is important to be able to recognize the small population of neoplastic cells, which should lead to prompt treatment or further investigations as deemed necessary.     

Detection of lymphomatous bone marrow involvement with magnetic resonance imaging.

We reviewed magnetic resonance (MR) staging examinations of 98 patients with malignant lymphoma who failed other therapy and were under evaluation for bone marrow transplantation. MR scan results were compared with blind posterior iliac crest aspirations and biopsies. Images of vertebral, pelvic, and femoral marrow were obtained using a standard T1-weighted, short repetition time (TR), short time to echo (TE) (TR700/TE22), spin-echo (T1-SE) method in 92 patients and short TI inversion recovery (STIR) technique (TR1,500/TE36/TI100) in all. On standard T1-SE sequence, normal marrow is bright due to the predominance of marrow fat, and tumor is dark. With STIR images, water containing tumor has a very high signal intensity in a dark (fat suppressed) background. Thirteen patients had positive MR scans and marrow biopsies, whereas 49 had negative MR scans and biopsies. Of 36 discordant MR/histology results, 10 had positive biopsies and negative MR exams; eight of these had microscopic infiltration (less than or equal to 5%) with tumor. MR detected marrow tumor either in the crests or elsewhere in 25 of 75 (33%) patients with negative study biopsies. We could confirm marrow involvement in 15 of these 25 (60%) by clinical methods. Therefore, up to one third of the patients evaluated with routine biopsies may have occult marrow tumor detectable by MR exam. In patients with negative marrow biopsies, especially those with Hodgkin's disease or intermediate to high-grade non-Hodgkin's lymphomas, MR scans found focal lesions distant from the crests. Biopsy better detected lower grade microscopic involvement. We conclude that optimal marrow staging of lymphoma patients incorporates both biopsy and MR imaging.     

Trephine needle bone marrow biopsy in the initial staging of Hodgkin disease: sensitivity and specificity of the Ann Arbor staging procedure criteria

The purpose of this study was to test the value of the Ann Arbor staging procedures committee criteria in defining a group of newly diagnosed patients with Hodgkin disease who do not have involvement of the bone marrow and do not need this procedure performed. One hundred sixty-six bilateral and 16 unilateral trephine bone marrow biopsies were performed in a consecutive series of 182 patients undergoing initial staging for Hodgkin disease. Bone marrow involvement was found in 13 patients. Advanced stage, defined as stage III or IV, occurring in 92%, anemia occurring in 100%, and "B" symptoms present in 100% were found to be the most sensitive indicators for bone marrow involvement. Osseous disease (99%), platelet count less than 150,000/mm3 (98%), and WBC less than 4,800/mm3 (94%) were the most specific parameters. The known association of bone marrow involvement with older patients, lymphocyte depleted histology, lower blood cell counts, anemia, advanced stage, and poorer survival is verified. The Ann Arbor staging procedures committee criteria for performing a bone marrow biopsy were shown to be 100% sensitive and 40% specific. Use of these criteria would have found all 13 patients with bone marrow involvement. Of the 73 patients who did not meet the criteria, no patient had bone marrow involvement. Use of the Ann Arbor staging procedures committee criteria is recommended.     

Hematological profile in pyrexia of unknown origin: role of bone marrow trephine biopsy vis-à-vis aspiration

Abstract

Background: Bone marrow examination, by aspiration and/or trephine biopsy, is an important procedure in arriving at a diagnosis for long-duration febrile illness. The role of trephine biopsy in immunocompromised host, especially HIV-positive patients, has been well studied in the literature. However, its utility in immunocompetent patients is still shrouded by controversy. Thus, the authors attempted to evaluate the utility of marrow aspirate vis-à-vis trephine biopsy in establishing a diagnosis in cases of pyrexia of unknown origin in immunocompetent individuals, along with an analysis of haematological alterations in these patients.

Materials and methods: Over a period of 8 years, 121 patients with pyrexia of unknown origin underwent both bone marrow aspiration and trephine biopsy as a part of diagnostic work-up. These cases were reviewed for their clinical data and hematological findings, including detailed morphological features in aspiration smears and trephine biopsies. Bone marrow aspiration and biopsy were compared for their diagnostic efficacy in these patients.

Results: A wide age range (2–65 years) was noted with a slight male predominance (2 : 1). Anemia was the most common feature in peripheral blood findings, seen in 97·5% of patients. Bone marrow aspiration was diagnostic in only 16·5% of cases, which revealed leishmaniasis or pure red cell aplasia. Granulomas were infrequent in marrow aspiration smears, as only two cases (1·6%) showed ill defined epithelioid cell collections. Compared to this, trephine biopsy offered a diagnosis in 76% of the cases. Granulomas were a frequent finding in the trephine biopsy, being present in 70% of the cases included. Additional cases diagnosed on biopsy (over those diagnosed with aspiration smears) included lymphoma, tuberculosis, fungal infection, sarcoidosis and hypocellular marrow.

Conclusion: Bone marrow trephine biopsy is an important adjunct to aspiration in arriving at an aetiological diagnosis of patient with long-duration febrile illness, and should be routinely performed in such cases. The presence of granulomas in trephine biopsy increases the likelihood of an etiologic diagnosis in these patients.     

Immunohistochemical distinction of haematogones from B lymphoblastic leukaemia/lymphoma or B-cell acute lymphoblastic leukaemia (B-ALL) on bone marrow trephine biopsies: a study on 62 patients

Haematogones are normal, maturing B-cell precursors. They can be confused with neoplastic immature lymphoid cells of B lymphoblastic leukaemia/lymphoma or B-cell acute lymphoblastic leukaemia (B-ALL). Though multi-colour flow-cytometry strategies for distinguishing haematogones from cells of B-ALL are well-described, similar strategies have not been determined for bone marrow trephine biopsies (BMTB). We revisited the morphological and immunohistochemical features (CD20, CD34, TdT and PAX5 expression) in 69 BMTB from 62 patients - 27 with excess haematogones; seven with residual B-ALL after therapy; 18 with no reported excess of haematogones or residual acute leukaemia on BMTB; and 17 diagnostic samples of B-ALL. The distinctive immunophenotypic pattern of BMTB with excess haematogones was of CD34, TdT, CD20 and PAX5 accounting for increasing proportions of cells in the order mentioned, whereas among B-ALL, the immunohistochemical pattern was of CD20, PAX5 and TdT accounting for an equal proportion of cells. Furthermore, among haematogones, the intensity of CD20 expression was extremely heterogeneous as compared to the neoplastic cells in CD20-positive B-ALL. The TdT-positive haematogones were generally small and uniform, while a certain degree of heterogeneity was noticed among neoplastic B-ALL cells. This study provides a practical strategy to distinguish haematogones from B-ALL cells in BMTB.     

Fragment filtration: a method for the accurate determination of flow cytometric kinetic data from bone marrow aspirates

The extent to which bone marrow obtained by conventional aspiration is contaminated by peripheral blood has been confirmed and quantitated. In marrow aspirates from normal subjects the median percentage of nucleated cells that had originated from the peripheral blood was 32% (range 2.5%-64%), in patients with acute leukemia 23% (range 0.5%-96.5%), in patients with chronic leukemia 59% (range 17%-76%), and in patients with lymphoma 31% (range 0.5%-74%). Flow cytometric (FCM) DNA analysis of conventional marrow aspirates from a range of subjects significantly underestimated the proportions of S-phase cells present, when compared with results from trephines obtained at the same time. Having shown, using 51Cr-labeled red cells in mice, that circulating red cells do not reenter the marrow parenchyma, a mathematical correction for contaminating blood similar to that described by Holdrinet et al. was devised. This correction improved the S-phase cell estimate from aspirated marrows, and the corrected values were not significantly different from values from paired trephine samples. A previously described technique for collecting fragments by filtration of aspirated marrow has been adapted for FCM analysis as a more direct way of overcoming problems due to blood contamination. This method was shown to yield estimates of S-phase cells not significantly different from those in paired marrow trephines and offers an alternative to routine trephine biopsies for FCM analysis of marrow cell kinetics.     

Cell cycle status in AML blast cells from peripheral blood,bone marrow aspirates and trephines and implications for biological studies and treatment

Using immunohistochemistry and flow cytometry to define phases of the cell cycle, this study shows that a high proportion of acute myeloid leukaemia (AML) blasts obtained from trephine biopsies are cycling, whereas >95% of peripheral blood‐derived blasts are arrested in G₁. Results obtained from bone marrow aspirates are more similar to those from blood rather than from trephine biopsies. These differences were confirmed by gene expression profiling in a patient with high count AML. This has implications for cell cycle and other biological studies using aspirates rather than trephine biopsies and for the use of cell mobilising agents before chemotherapy.     

Results of bone marrow examination in 275 patients with histological features that suggest an indolent type of cutaneous B-cell lymphoma

Whether or not bone marrow biopsies should be performed routinely in patients with skin lesions that show histological features consistent with an indolent B-cell lymphoma [marginal zone lymphoma (MZL) or follicle centre lymphoma (FCL)] has been debated. As no studies have addressed this question for this group of lymphomas, we evaluated the results of bone marrow biopsy examination in 275 patients with histological features consistent with MZL ( n  = 82) or FCL ( n  = 193) first presenting in the skin. In the MZL group, two of 82 patients (2%) showed bone marrow involvement, which was the only extracutaneous localization in one of these patients. In the FCL group, 22 of 193 patients (11%) had bone marrow involvement and was the only extracutaneous localization in nine of them. FCL patients with skin lesions and a positive bone marrow had a significantly worse prognosis when compared with patients with only skin lesions (5-year disease-specific survival 63% vs. 95%; P  = 0·001). These results indicate that bone marrow investigation is essential for staging patients with a FCL first presenting in the skin. Bone marrow examination appears to have limited value in patients with MZL presenting in the skin.     

Trephine biopsy of the bone marrow in disseminated histoplasmosis.

Twenty-two patients had bone marrow aspirates for culture and marrow trephine biopsies for histologic examination during the evaluation of an illness eventually proved to be disseminated histoplasmosis. These procedures provided evidence of involvement of the bone marrow with Histoplasma capsulatum in 19 cases. Fungi were demonstrated in trephine biopsy sections in 15 of the patients, permitting a rapid specific diagnosis. Three patterns of bone marrow morphology were observed; each reflected a variation of macrophage proliferation. In two of the patterns, diffuse macrophage proliferation and loose aggregates of macrophages, typical 2 to 4 microns intracellular hisoplasma organisms were numerous and obvious in hematoxylin and eosin-stained sections, were infrequent and were often relatively large (6 to 12 microns). There were four patients from whom H. capsulatum was cultured when organisms could not be demonstrated in either hematoxylin and eosin- or silver-stained sections. The patients with diffuse proliferation of macrophages in the marrow comprised a distinct clinical group associated with immunosuppression, a fulminant course and a fatal outcome despite therapy. Patients in the other two morphologic groups responded well to therapy even though the immune responses on many were also suppressed. Bone marrow examination is an excellent diagnostic procedure in disseminated histoplasmosis. Trephine biopsies permit a rapid diagnosis in most cases and may be of prognostic significance.     

Intrasinusoidal bone marrow infiltration: a common growth pattern for different lymphoma subtypes

We report a retrospective immunohistochemical study on bone marrow biopsies of 43 patients with different types of lymphomas showing unusual intrasinusoidal infiltration. Most of these patients presented with splenomegaly (74.4%) and peripheral lymphocytosis (83%). In 20/43 patients, lymphoid infiltrates were not detectable on haematoxylin-eosin sections. After immunohistochemistry on bone marrow biopsies and blood and bone marrow smear examinations, the following diagnoses were made: splenic marginal zone lymphoma with villous lymphocytes (SLVL) in 24 patients, large granular lymphocyte (LGL) leukaemia in 14 patients, hepatosplenic T-cell lymphoma in two patients, anaplastic large cell lymphoma in two patients and intravascular large B-cell lymphoma in one patient. In the presence of intrasinusoidal infiltrates of small lymphocytes, a B-cell phenotype (CD20+, CD76/DBA44+/-) was associated with splenic marginal zone lymphoma whereas intrasinusoidal CD3/CD45RA-positive T-cell infiltrates were strongly suggestive of LGL leukaemia. Intrasinusoidal bone marrow infiltration appears to be a common feature of distinct lymphoma subtypes. Immunohistochemical analysis is essential to detect intrasinusoidal medullary infiltrates (which may be minimal) and should be systematically performed in patients with splenomegaly and peripheral lymphocytosis.     

Is bone marrow trephine biopsy always mandatory in staging Hodgkin's disease?

We reviewed data from 690 adult patients with Hodgkin's disease (HD) to determine whether bone marrow trephine biopsy (BMTB) is mandatory for all patients. The data suggest that it is not necessary in clinical stage I-IIA. However, bilateral BMTB is recommended in the presence of B symptoms also in patients with localized stage disease.     

Bone marrow origin of a B-cell lymphoma

To search for precursors of the neoplastic B cells in a patient with a nodular lymphoma, we produced a monoclonal antibody to a variable region idiotope on the lymphoma IgM heavy chain. Clonal ancestors of the lymphoma cells were identified by this marker among bone marrow pre- B cells (5% to 26%). A second antiidiotype (anti-Id) antibody specific for the complete lymphoma IgM kappa recognized 10% of B cells in bone marrow and blood and greater than 95% of B cells in lymphomatous lymph nodes, including one obtained after tumor conversion to a diffuse large cell lymphoma. Immunoglobulin gene analysis surprisingly revealed expansion of multiple clones of early B lineage cells in bone marrow, including members of the neoplastic clone. The data suggest that this lymphoma arose through a progression of transformational events beginning in bone marrow: first, creation of an oligoclonal pre- neoplastic pool of pre-B cells, subsequent conversion of a single subclone into low grade neoplastic B cells that homed to the lymph node follicles, and later progression to a more invasive form of the B-cell lymphoma.