QBEND10 for the diagnosis of myelodysplastic syndromes in routinely processed bone marrow biopsy specimens.

AIM--The assessment of the value of the antibody QBEND10, which is directed against the haemopoietic stem cell related antigen CD34, in the immunohistochemical diagnosis of myelodysplastic syndrome in routinely processed bone marrow biopsy specimens. METHODS--581 formalin fixed, paraffin embedded trephine biopsy specimens of the iliac crest were immunostained with QBEND10 (avidin-biotin complex/ABC method). The number of CD34+ haemopoietic stem cells/blast cells (referred to hereafter as CD34+ cells) was determined in each case. The Wilcoxon test was used for statistical analysis. RESULTS--The following diagnostic categories were defined: (1) normal or reactive bone marrow (n = 356), (2) lymphoproliferative disorders, usually non-Hodgkin's lymphoma of low grade malignancy or multiple myeloma (n = 118), (3) myelodysplastic syndrome (n = 22), (4) acute leukaemia (n = 44), and (5) myeloproliferative diseases (n = 41). The average number of CD34+ cells was very low (0.2/HPF) in normal and reactive bone marrow, in lymphoproliferative disorders and in the myelodysplastic syndrome subtypes RA and RARS. Myeloproliferative diseases showed an average of three CD34+ cells/HPF. However, the average number of CD34+ cells was significantly higher (p < 0.05) in the myelodysplastic syndrome subtypes RAEB and RAEB-T (8.7/HPF) and in acute leukaemia (including both myeloid and lymphoblastic leukaemia; 111.7/HPF). CONCLUSIONS--QBEND10 is of value for the identification of RAEB and RAEB-T in routinely processed bone marrow biopsy specimens because it enables the detection of even small increases in the number of CD34+ cells.     

Chronic myelomonocytic leukemia: The role of bone marrow biopsy immunohistology.

The World Health Organization criteria for diagnosing chronic myelomonocytic leukemia (CMML) are largely based on findings observed in the peripheral blood and bone marrow aspirate. A specific diagnostic role for the bone marrow biopsy has not been adequately explored. We examined whether bone marrow biopsy supplemented by immunohistochemistry may be helpful in distinguishing CMML from cases of chronic myelogenous leukemia and atypical chronic myeloid leukemia (aCML). We immunostained 25 cases of CMML with paraffin reactive antibodies which included CD68 (KP1), CD68R (PG-M1), and CD163, and compared the results with those observed in six cases of chronic myelogenous leukemia and in three cases of atypical CML. In addition, we examined whether CD34 immunohistochemistry could be useful in separating cases of CMML with less than 10% blasts (type-1) from cases of CMML with blasts accounting for 10-19% (type-2), and cases of CMML in acute transformation to acute myeloid leukemia (blasts > or = 20%). The presence of nodules of plasmacytoid monocytes was investigated by CD123 staining. CD42b was used to highlight abnormal megakaryocytes. Our results demonstrate significant differences between the groups. CD34 analysis allowed separating CMML type-1 from type-2 and the former from CMML in acute transformation. CD123-positive plasmacytoid monocyte nodules were found only in CMML and not in the other two disease groups. Overlap between CMML and the other two groups were observed with CD68 immunostaining. CD68R was more restricted to bone marrow macrophages and monocytes than CD68, but the differences between CMML and chronic myelogenous leukemia or atypical CML were still not significant. Although CD42b immunostaining facilitated the detection of dwarf megakaryocytes often present in CMML, the distinction between those and the small forms seen in chronic myelogenous leukemia was still problematic.     

Discordant immunophenotypic profiles of adhesion molecules and cytokines in acute myeloid leukemia involving bone marrow and skin

We investigated the role of adhesion molecules in skin involvement by acute myeloid leukemia (AML) using immunohistochemical analysis. Ten paired cases of skin and bone marrow biopsy specimens from patients with myeloid leukemia cutis (MLC) and 15 bone marrow biopsy specimens from patients without MLC were studied with antibodies directed against CD29, CD34, CD54, CD62-L, CD183, and cutaneous lymphocyte antigen (CLA). CLA was expressed in all cases of leukemia whereas CD54 was negative within blasts. CD62-L was expressed in 4 of 10 specimens of marrow infiltrates with MLC and 6 of 10 specimens of matching skin infiltrates; in marrows without MLC, only 2 of 15 were positive. CD29 was expressed in 1 of 10 marrow infiltrate specimens with MLC and 4 of 10 matching skin infiltrate specimens; in marrows without MLC, only 1 of 15 were positive. CD183 was expressed in 1 of 10 marrow infiltrate specimens with MLC and 4 of 10 matching skin infiltrate specimens; in marrows without MLC, CD183 was negative. The gain of CD62-L, CD29, and CD183 expression in bone marrow and skin infiltrates in leukemia cutis, relative to bone marrow infiltrates of cases without MLC, suggests a role for these markers in AML homing to skin.     

Immunophenotypic profile of myeloid cells in granulocytic sarcoma by immunohistochemistry. Correlation with blast differentiation in bone marrow

The present study was designed to evaluate the lineage differentiation (particularly monocytic differentiation) of immature myeloid cells in granulocytic sarcoma (GS) by immunohistochemistry and correlate the results with lineage differentiation of blasts in the bone marrow and to determine the degree of maturation of the infiltrating myeloid cells in GS by immunohistochemistry using CD34 and HLA-DR. Immunohistochemical stains were performed on paraffin-embedded tissue from 17 GS lesions with lineage-associated markers: myeloperoxidase, CD68 (KP1), CD68 (PG-Ml), glycophorin A, factor VIII, and CD56; and with markers for blasts and immature myeloid cells: CD34 and HLA-DR. Our results show that positive staining with PG-M1, but not KP1, suggests monocytic differentiation of myeloid cells in GS and correlates with the monocytic differentiation of blasts in the bone marrow. Expression of CD56 is frequent in GS, especially when the marrow blasts have monocytic differentiation, and should not be interpreted as a primary natural-killer cell process. The immature myeloid cells in GS are frequently HLA-DR positive. However, CD34 positivity of the immature myeloid cells is relatively uncommon, except in cases with underlying myelodysplastic syndrome or chronic myelogenous leukemia.     

Evaluation of bone marrow specimens with acute myelogenous leukemia for CD34, CD15, CD117, and myeloperoxidase

CONTEXT: Immunophenotyping of bone marrow (BM) specimens with acute myelogenous leukemia (AML) may be performed by flow cytometric (FC) or immunohistochemical (IH) techniques. Some markers (CD34, CD15, and CD117) are available for both techniques. Myeloperoxidase (MPO) analysis may be performed by enzyme cytochemical (EC) or IH techniques. OBJECTIVE: To determine the reliability of these markers and MPO by these techniques, we designed a study to compare the results of analyses of these markers and MPO by FC (CD34, CD15, and CD117), EC (MPO), and IH (CD34, CD15, CD117, and MPO) techniques. MATERIALS AND METHODS: Twenty-nine AMLs formed the basis of the study. These AMLs all had been immunophenotyped previously by FC analysis; 27 also had had EC analysis performed. Of the AMLs, 29 had BM core biopsies and 26 had BM clots that could be evaluated. The paraffin blocks of the 29 BM core biopsies and 26 BM clots were stained for CD34, CD117, MPO, and CD15. These results were compared with results by FC analysis (CD34, CD15, and CD117) and EC analysis (MPO). RESULTS: Immunodetection of CD34 expression in AML had a similar sensitivity by FC and IH techniques. Immunodetection of CD15 and CD117 had a higher sensitivity by FC analysis than by IH analysis. Detection of MPO by IH analysis was more sensitive than by EC analysis. There was no correlation of French-American-British (FAB) subtype of AML with CD34 or CD117 expression. Expression of CD15 was associated with AMLs with a monocytic component. Myeloperoxidase reactivity by IH analysis was observed in AMLs originally FAB subtyped as M0. CONCLUSIONS: CD34 can be equally detected by FC and IH techniques. CD15 and CD117 are better detected by FC analysis and MPO is better detected by IH analysis.     

PG-M1: A New Monoclonal Antibody Directed against a Fixative-Resistant Epitope on the Macrophage-Restricted Form of the CD68 Molecule

A new anti-macrophage monoclonal antibody (PG-M1) was produced by immunizing BALB/c mice with fresh spleen cells from a patient with Gaucher's disease. PG-M1 reacts strongly with a fixative-resistant epitope of an intracytoplasmic molecule, selectively expressed by virtually all macrophages of the human body. Although attempts to immunoprecipitate the molecule recognized by PG-M1 have failed so far, the reactivity of the antibody with COS-1 and WOP cells transfected with a human complementary DNA clone encoding for the CD68 antigen suggests that PG-M1 is a new member of the CD68 cluster. However, unlike other CD68 antibodies (KP1, EBM11, etc.), which react with both macrophages and myeloid cells, PG-M1 detects a fixative-resistant epitope on the macrophage-restricted form of the CD68 antigen. In 957 routinely fixed, paraffin-embedded samples, PG-M1 showed a more restricted reactivity with elements of the monocyte/macrophage lineage than the previously described monoclonal antibodies MAC-387 (anti-calgranulins), KP1 (CD68) and Ki-M1P. Among hematological malignancies, PG-M1 only labels acute leukemias of M4 and M5 type and rare examples of malignant histiocytosis/true histiocytic sarcoma. In contrast, acute leukemias of the M1, M2, M3, M6, M7, and L1-L3 types, non-Hodgkin's lymphomas, and Hodgkin and Reed-Sternberg cells of Hodgkin's disease are consistently PG-M1-negative. In the daily diagnostic practice, PG-M1 seems to be particularly valuable for the diagnosis of myelomonocytic or monocytic leukemia and neoplasms of true histiocytic origin in routine paraffin sections.     

Immunocytochemical detection of megakaryocytes by endothelial markers: a comparative study.

By endothelial markers such as FVIII, UEA-I, CD31 and QBEND/10, the Authors have investigated the immunohistochemical pattern of bone marrow megakaryocytes in myelodysplastic (8 cases) and myeloproliferative (35 cases) disorders as well as in control biopsies (8 cases). An evident cytoplasmic positivity with FVIII and UEA-I was encountered in all normal and pathological specimens, whereas the immunolocalization of CD31 was limited to megakaryocytes present in normal bone marrow biopsies or in cases of myelofibrosis. No immunostaining with QBEND/10 was observed in any case, although the most selective staining of endothelial cells of bone marrow vessels was noted with this antibody. The usefulness to utilize endothelial markers in order to identify atypical and immature forms of megakaryocytes as well as micromegakaryocytes, especially in myelofibrosis by the aid of CD31, was also discussed.     

Immunoarchitecture of normal human bone marrow: a study of frozen and fixed tissue sections.

To provide baseline information on the immunoarchitecture of normal bone marrow, we studied cryostat-cut, frozen, and paraffin-embedded, fixed tissue sections prepared from 21 core biopsies of normal bone marrow obtained during bone marrow harvests for transplantation. A large panel of antibodies was applied that included, for frozen tissue, Leu-6 (CD1), T11 (CD2), Leu-3a (CD4), Leu-1 (CD5), Leu-2a (CD8), J5 (CD10), My7 (CD13), Leu-11 (CD16), B4 (CD19), B1 (CD20), B2 (CD21), Tac (CD25), My9 (CD33), T200 (CD45), NKH-1 (CD56), kappa and lambda chains, beta F1, Ki-67, HLA-DR, TQ1, and keratin, and for fixed tissue, leukocyte common antigen (CD45), L26 (CD20), LN1 (CDw75), LN2 (CD74), LN3, LN4, LN5, MB1 (CD45R), MB2, MT1 (CD43), MT2 (CD45R), UCHL1 (CD45R0), BM1, Ki-1 (CD30), Leu-M1 (CD15), lysozyme, KP1 (CD68), actin, S100, neuron-specific enolase, vimentin, and keratin. On fresh-frozen sections CD19 and CD2 were the most reliable and sensitive markers for B and T cells, staining 5% and 9% of marrow cells, respectively. Immunoglobulins generally showed heavy background staining, which frequently precluded an accurate assessment. The CD4 to CD8 ratio in the bone marrow was reversed from that of peripheral blood. On fixed tissues, leukocyte common antigen was found in 14% of the marrow cells, corresponding roughly to the lymphocyte population. L26, a pan-B-cell marker, stained 3% of the marrow cells. Among the other B-cell markers, LN1 and MB2 stained a large number of cells (40% to 70%), indicating reactivity with cells of the myeloid or erythroid series in addition to lymphocytes. Among the T-cell markers, UCHL1 and MT1 stained 66% and 50% of the cells, respectively, which could be explained by their cross-reactivity with myeloid cells. Nonspecific myelomonocytic markers (Leu-M1, KP1, and lysozyme) also showed reactivity in a high percentage of cells. No particular architectural distribution patterns of B or T lymphocytes were noted in either frozen or fixed bone marrow specimens. The results of this study provide normal baseline data for the immunohistologic application of hematopoietic and lymphoid markers on frozen or fixed bone marrow biopsy specimens.     

Analysis of immunohistochemical markers in bone marrow sections to evaluate for myelodysplastic syndromes and acute myeloid leukemias.

BACKGROUND: Accurate bone marrow (BM) blast counts (BCs) are essential for diagnosis (dx) of myelodysplasia (MDS), MDS/myeloproliferative (MDS/MPD) disease, or acute myeloid leukemia (AML), and may be difficult in hemodiluted bone marrow aspirates (BMAs). Erythroid precursors (EPs) may be indistinguishable from myeloblasts in BM sections (aspirate clots/cores). We compare the usefulness of immunohistochemistry (IHC) [ie, CD34, CD117, myeloperoxidase (MPO), Hemoglobin A1 (HbA1), and terminal deoxynucleotidyl transferase (TdT)] of BM sections (IHC-BM) with BMA, bone marrow touch preparation (BMTP), and flow cytometry (FC) BCs. DESIGN: The initial BC (48), percentage (%) of Eps (38) (both based on initial 100 to 600-cell counts), and FC expressions of CD34, CD117, Glycophorin A(GLY A), and TdT (44) were tabulated from 50 BMs (MDS, MDS/MPD, or AML). BMAs (48) and BMTPs (25) subsequently received 500-cell counts. IHC-BM was performed (45:formalin, 5:B5-fixed) [CD34 (46), CD117 (45), HbA1 (45), TdT (42), and MPO (45)]. RESULTS: Retrospective BMA BCs revealed a 31% (15/48) discrepant rate between the original/retrospective BMA BCs; 80% revealed an underestimated initial BC. There was a 28% discordance rate between the retrospective BMA and BMTP reviews; 77% showed a higher BMTP BC. IHC showed significantly higher BCs in 19% (9/47), resulting in a different dx (5). However, CD34 and CD117 IHCS revealed lower BCs in 38% and 48%, respectively. The CD34 IHC results were primarily due to CD34-negative blasts by FC. The CD117 IHC results were largely unexplained. EPs were CD34 and CD117-negative. CONCLUSIONS: (1) Evaluation for MDS/AML requires 500-cell counts of BMAs and/or BMTPs. (2) CD34 and/or CD117 blasts by FC indicate IHC-BM may increase BC accuracy. (3) CD34 is more reliable than CD117 by IHC; however, in combination, they are most reliable and should be performed on BM clots/cores due to variable reactivity.     

Anti-CD34 immunoperoxidase staining in paraffin sections of acute leukemia: comparison with flow cytometric immunophenotyping.

Anti-CD34 is a monoclonal antibody that reacts with bone marrow progenitor cells and leukemic blasts, and is expressed on 30% to 50% of all acute leukemias. Detection of CD34 has previously been restricted to flow cytometric studies. To expand the utility of CD34, we immunostained 46 paraffin-embedded bone marrow specimens with acute leukemia; results were compared with flow cytometric studies. CD34 reactivity was also evaluated in nine chronic leukemia cases, 27 malignant lymphoma cases (Hodgkin's disease and non-Hodgkin's lymphoma), six normal bone marrow specimens, and three benign, hyperplastic lymph node specimens. All cases that were CD34 positive by flow cytometry (11 of 19 B-cell precursor acute lymphoblastic leukemia cases, one of six T-cell acute lymphoblastic leukemia cases, and seven of 21 acute myeloblastic leukemia cases) were also CD34 positive in paraffin sections. Both cell membrane and cytoplasmic staining was seen. The positivity percentage and fluorescence intensity by flow cytometry correlated with the estimated number of stained cells and the intensity of immunoperoxidase staining in 18 of 19 CD34-positive cases. The remaining bone marrow and lymph node cases studied were CD34 negative; prominent endothelial cell staining, however, was noted. This is the first report of anti-CD34 staining of acute leukemia in paraffin-embedded sections. In contrast to other monoclonal antibodies reactive in bone marrow paraffin sections with leukemia, anti-CD34 immunoperoxidase staining is limited to leukemic blasts and may provide useful diagnostic information when flow cytometric studies are not available.     

Different immunoreactivity of endothelial markers in well and poorly differentiated areas of angiosarcomas

This study evaluated the immunohistochemical staining of four endothelial cell markers in well differentiated and poorly differentiated areas of angiosarcomas. Formaldehyde-fixed, paraffin-embedded sections from eight angiosarcomas were studied using the antibodies anti-factor VIII-related antigen (FVIII-RA), Ulex europaeus I agglutinin, anti-CD34 (QBEND/10) and anti-CD31 (JC70). The immunostaining of the angiomatous (well differentiated) and solid (poorly differentiated) areas was separately analysed and specificity was evaluated in 20 non-vascular tumours. The antibody anti-CD31 and Ulex europaeus were the most sensitive markers staining well differentiated vasoformative structures and poorly differentiated solid areas. Anti-FVIII-RA and anti-CD34 did not stain undifferentiated malignant endothelial cells from solid areas. Ulex europaeus and anti-CD34 showed very low specificity; in contrast, none of the non-vascular tumours expressed CD31 or FVIII-RA. JC70 (anti-CD31) appears to be the most useful marker in elucidating the vascular nature of angiosarcomas. Is important to emphasize the lack of specificity of Ulex europaeus and the low sensitivity of anti-CD34 and anti-FVIII-RA for poorly differentiated lesions.     

Immunostaining for CD31 and CD34 in Kaposi sarcoma.

AIMS--To evaluate antibodies directed against CD31 (JC70/A) and CD34 (QBEND/10 and anti-HPCA-1) more extensively in Kaposi sarcoma; to assess their value in routine diagnosis; and to compare them with the traditional endothelial cell markers Ulex europaeus agglutinin 1 (UEA-1) and factor VIII related antigen. METHODS--Twenty four cases of Kaposi sarcoma were studied retrospectively. All specimens had been fixed in formalin and embedded in paraffin wax. The antibodies were applied using the Streptavidin biotin technique in all cases except for UEA-1, for which an indirect two stage method was used involving peroxidase conjugated anti-ulex as the secondary antibody. RESULTS--Tumours were classified into those showing angiomatoid or lymphangiomatoid elements and spindle cell lesions. Universal labelling of all lesions and virtually all elements within lesions was seen with the anti-CD34 antibodies QBEND/10 and HPCA-1. Labelling of spindle cells was less consistent with JC70/A but both markers were superior to the traditional endothelial cell markers UEA-1 and factor VIII related antigen. CONCLUSIONS--These data confirm that Kaposi sarcoma is a tumour of endothelial cell origin. They shed further light on the histogenesis of this complex tumour and demonstrate that immunostaining for CD34 and CD31 can be used as an aid to diagnosis in routinely processed tissue.     

Acute lymphoblastic leukaemia: correlation between morphological/immunohistochemical and molecular biological findings in bone marrow biopsy specimens.

BACKGROUND: Although numerous antibodies suitable for use on paraffin wax embedded sections are available for the subtyping of acute leukaemia (acute myelogenous leukaemia (AML) and acute lymphoblastic leukaemia (ALL)) in bone marrow biopsy sections, unequivocal identification of the cell line involved is sometimes impossible. METHODS: Forty eight formalin fixed, paraffin wax embedded bone marrow biopsy specimens that had been decalcified in EDTA were investigated, including 42 thought to exhibit ALL on the basis of bone marrow smears. Five specimens exhibited AML and one biphenotypic leukaemia, as diagnosed immunohistochemically in bone marrow biopsies. Immunostaining was performed with antibodies against relatively specific B and T cell antigens. The blasts were investigated for rearrangements of the immunoglobulin heavy chain (IgH) and the T cell antigen receptor (TCR) genes. RESULTS: Amplifiable DNA was obtained from all 48 specimens. An IgH gene rearrangement was detected in 20 of 23 c-ALL specimens. Four of seven T cell ALL (T-ALL) specimens had a TCR-gamma gene rearrangement, and the one B cell ALL (B-ALL) specimen exhibited a clonal IgH gene. Three of four cases of unclassifiable ALL could be assigned to the B cell lineage on the basis of gene rearrangement analysis. Seven cases originally diagnosed in smears as ALL were rediagnosed as AML (n = 5) or biphenotypic leukaemia (n = 2) because of immunohistochemical reactivity for myeloperoxidase or lysozyme. Two of these AML cases and two of three cases of biphenotypic leukaemia exhibited a monoclonal IgH gene rearrangement. CONCLUSIONS: Acute leukaemia can be subtyped in bone marrow sections with a limited panel of antibodies suitable for use on paraffin wax embedded sections (against CD3, CD10, CD20, CD79a, myeloperoxidase, and lysozyme). In patients with ALL and a diagnostically equivocal immunophenotype, gene rearrangement analysis might indicate whether the B or T cell lineage is involved.     

The detection of CD14 and CD16 in paraffin-embedded bone marrow biopsies is useful for the diagnosis of chronic myelomonocytic leukemia

Histopathological study of bone marrow biopsy (BMB) in chronic myelomonocytic leukemia (CMML) is often difficult and might benefit from an immunohistochemical approach. We immunostained 15 cases of CMML, focusing at two new antibodies staining for CD14 and CD16 on paraffin-embedded tissues. CD68 (KP1), CD68 (PG-M1), and CD163 were not differentially expressed between CMML and chronic myelogenous leukemia (CML). In CMML BMB, we found a significant increase in the number of CD14⁺ monocytes. This increase was made of dispersed cells in the interstitium, often exhibiting bilobated nuclei, and being difficult to differentiate from neutrophils. There was no expansion of CD16⁺ monocyte-like cells. However, we found a significant decrease in the number of granulocytes expressing CD16, MPO, and CD15 in CMML compared to CML and control BMB, probably related to dysgranulopoiesis. Indeed, BMB immunohistochemistry can be helpful in CMML by identifying both the monocyte expansion with CD14 and the dysgranulopoiesis with CD16.     

Immunohistochemical analysis of decalcified paraffin-embedded human bone marrow biopsies with emphasis on MHC class I and CD34 expression

 

Aims:

To identify the stromal structures and haematopoietic cell lineages in normal bone marrow. The optimal conditions were studied for the reactivity of a panel of antibodies, applicable to paraffin sections of decalcified trephine biopsies using antigen retrieval methods.  

Methods and results:

Two methods of antigen retrieval (pepsin and acid citrate buffer) were tested. For the demonstration of most antigens and for reduction of background staining, heating in acid citrate buffer was preferred. In the case of elastase and von Willebrand Factor (factor VIIIrAg) pepsin pre-treatment was optimal, whereas Ulex europaeus agglutinin (UEA-1) and α-smooth muscle actin (α-SMA) required no pre-treatment. Staining patterns obtained after 48 h EDTA decalcification and short electrolytic decalcification were identical. Both methods allowed recognition of HLA-A and HLA-B antigens, isolated CD34+ cells, mono-histiocytic cells (CD68+), myeloid cells (elastase and myeloperoxidase), erythroid cells (glycophorin C) and of megakaryocytic cells (Factor VIIIrAg). A relative simple lymphocyte-subset analysis was possible in decalcified paraffin sections allowing recognition of B-cells (CD20+) and T-cells (CD3+ and CD45RO+) in frequencies comparable to frozen sections. Suitable stromal cell staining was achieved by vimentin and desmin antibodies, whereas the bone marrow capillary network was visualized by CD34, factor VIIIrAg and UEA-1.  

Conclusions:

This immunohistochemical study indicates that all cellular components of the haematopoietic microenvironment of the bone marrow can be identified in decalcified paraffin sections using antigen retrieval methods and that the time of decalcification can be reduced to 1–1.5 h.     

CD34/QBEND10 immunostaining in the bone marrow trephine biopsy: a study of CD34-positive mononuclear cells and megakaryocytes

CONTEXT: The immunohistochemical detection of CD34 protein using QBEND10 antibody in bone marrow trephine biopsies was shown recently to be a precise method for quantitation of blasts and a possibly useful approach in diagnosis and classification of myelodysplastic syndrome. OBJECTIVES: To evaluate CD34+ cells in bone marrow biopsies with various diagnoses and to assess how counts obtained using this method correlate with blast counts obtained by traditional morphologic evaluation of bone marrow smears. DESIGN: Bone marrow trephine biopsies from 108 adult patients were evaluated by immunohistochemistry using anti-CD34 antibody (QBEND10). CD34+ mononuclear cells were counted and compared with the blast counts in the bone marrow aspirate smears or imprints. CD34+ mononuclear cell clusters and CD34+ megakaryocytes were also recorded. The type of positivity (membranous vs cytoplasmic) and the percentage of CD34+ megakaryocytes were evaluated because the presence of CD34+ megakaryocytes was recently suggested to be present in myelodysplastic syndrome, but not in myeloproliferative disease or nonneoplastic bone marrow. RESULTS: Six of 24 biopsies with partial involvement by non-Hodgkin lymphoma and 5 of 60 biopsies with reactive changes had 5% to 10% CD34+ mononuclear cells and were associated with lymphocytosis and increased hematogones. The CD34+ mononuclear cell clusters were found only in myelodysplastic syndrome and myeloproliferative disease. The CD34+ megakaryocytes were present in all diagnostic groups. CONCLUSION: The number of CD34+ mononuclear cells was often slightly higher than the number of myeloid blasts in the bone marrow smears, probably due to increased hematogones. The presence and the number of CD34+ megakaryocytes do not appear to have diagnostic value, but this finding should be further investigated in relation to clinical parameters.     

Incidence, sensitivity, and specificity of leukemia-associated phenotypes in acute myeloid leukemia using specific five-color multiparameter flow cytometry

We assessed the usefulness of 5-color multiparameter flow cytometry to detect leukemia-associated phenotypes (LAPs) in the bone marrow of patients with newly diagnosed acute myeloid leukemia (AML) and determined its usefulness for detection of minimal residual disease (MRD). Overall, 94% of patients (51/54) with AML had LAPs at diagnosis. The frequency of leukemic bone marrow/median frequency of LAPs in normal or regenerating bone marrow samples using maximum log difference statistics revealed that CD2, CD56, CD11b, CD7, and CD19 expression on AML blasts represented the most sensitive and reliable markers for detection of MRD. Serial dilutional experiments showed that the sensitivity level of immunophenotyping was between 10-4 and 10-5 and that the approach was highly reproducible.Immunophenotypic analysis using a CD45 gating strategy, 5-color staining, and an extensive panel of monoclonal antibodies allowed the identification of LAPs in 94% of AML cases, and these immunophenotypes can be used for MRD monitoring with a sensitivity limit of 10-4 to 10-5.     

The tryptase positive compact round cell infiltrate of the bone marrow (TROCI-BM): a novel histopathological finding requiring the application of lineage specific markers

AIMS: Compact tryptase-positive round cell infiltrates of the bone marrow (TROCI-BM) are very rare histopathological findings and may pose challenging problems with regard to the cell type involved (either mast cells or basophilic granulocytes) and the exact diagnosis. METHODS: A selected panel of immunohistochemical markers against mast cell and basophil related antigens, including CD25, CD34, CD117/Kit, and the 2D7 antigen (which is found only in basophilic granulocytes) on a total of 410 routinely processed bone marrow biopsy specimens (including 88 cases of systemic mastocytosis (SM), 20 cases of chronic myeloid leukaemia (CML), 92 cases of myeloid neoplasms other than CML, and 210 controls with normal/reactive bone marrows). RESULTS: In total, 17 cases with TROCI-BM could be identified: 11 SM (including two cases of well-differentiated SM and two mast cell leukaemias; MCL), 2 myelomastocytic leukaemia (MML), 2 CML with excess of basophils (secondary basophilic leukaemia (CMLba)), and 2 tryptase positive acute myeloid leukaemia (AML). Regarding the cell types involved, TROCI-BM cells were found to express CD117/Kit in all cases of SM and MCL. In MML and tryptase postitive AML, TROCI-BM cells were found to coexpress CD34 and Kit. The basophil specific antigen 2D7 was only detected in CD34/Kit negative TROCI-BM cells in two patients with CMLba. The activating point mutation D816V was detected in 8/11 patients with SM but not in any of the other haematological malignancies. CONCLUSIONS: In summary, a total of six rare myeloid neoplasms may present with a novel immunohistochemical phenomenon tentatively termed TROCI-BM.     

脾脏窦岸细胞血管瘤临床病理观察

目的研究脾脏窦岸细胞血管瘤（LCA）的临床病理及免疫表型特征,探讨脾脏窦岸细胞血管瘤的本质。方法对17例脾脏LCA进行了回顾性研究,包括临床表现、影像学和病理形态学等,并进行了多种抗原标记的免疫表型检测,以正常脾脏、淤血性脾肿大、脾海绵状血管瘤病例作为对照。结果（1）17例脾脏LCA有相似的临床及影像学表现,即脾脏肿大伴单发或多发占位性病变。5例伴肝囊肿,1例伴卵巢浆液性囊腺瘤。（2）大体病理改变为脾脏体积不同程度增大,切面见单个或多个暗褐色结节,结节的直径为0．2～6．0cm不等。（3）镜下改变主要为病变由相互吻合的窦状腔隙组成,腔隙常形成乳头状突起或呈囊性扩张,其中可见两类细胞,一类是衬在腔隙内表面的体积较小的细胞,形似窦岸细胞;另一类是脱落于窦状腔隙内的大细胞,两类细胞均无明显异型性。（4）免疫表型检测：17例标本中的小细胞均呈CD31、多克隆第八因子相关抗原阳性,CD34阴性,1例呈CD8阳性,1例呈CD21阳性,均不表达组织细胞分化抗原;大细胞均表达各种组织细胞抗原,包括CD68（KP1和PG-M1）、CD163和溶菌酶等,各例中均有少数大细胞表达CD31抗原,但均不表达CD34,且相关抗原的表达模式与对照组的三类脾脏良性病变有所不同;大、小细胞均不表达S-100蛋白。结论脾脏窦岸细胞血管瘤是一种良性病变,可能是由于某种原因导致局部血流动力学的改变,致脾脏窦岸细胞增生,脾血窦扩张并相互吻合而形成的局部血管瘤样病变伴组织细胞反应。特征性的形态学改变以及CD31和组织细胞相关抗原的检测有助于该疾病的病理诊断。     

Immunophenotyping of acute lymphoblastic leukaemia in routinely processed bone marrow biopsy specimens

AIMS: To assess the value of immunophenotyping of acute lymphoblastic leukaemia (ALL) in routinely processed bone marrow trephine biopsy specimens and to establish a minimum panel of antibodies to assess lymphoid lineage and enable differentiation from acute myeloid leukaemia. METHODS: 45 routinely processed bone marrow biopsy specimens (formalin fixed, paraffin embedded and mildly decalcified in EDTA) reported to contain leukaemic infiltrates on the basis of cytomorphological and enzyme-cytochemical analysis of bone marrow smears (22 c-ALL, 11 T-ALL, 2 B-ALL, 10 u-ALL (unclassified)) were immunostained by the ABC method with a broad panel of 26 antibodies against various haemopoietic antigens. RESULTS: Staining with antibodies directed against myeloperoxidase and lysozyme showed that seven cases were either biphenotypic or mixed leukaemias (2), or of myelogenous origin (acute myeloid leukaemia (AML)-M1 (2); AML-M4 (2); AML-M5a (1)). Five of these seven cases had been diagnosed initially as u-ALL. Three further cases with no compact leukaemic infiltrates were excluded. ALL was confirmed in the remaining 35 cases. Because of revised diagnoses, the total numbers of ALL subtypes changed (23 c-ALL, 8 T-ALL, 2 B-ALL, 2 u-ALL). Immunostaining of more than 10% of blast cells in at least one case was found with 19 of the 26 antibodies. The most sensitive lineage specific antibodies for diagnosis were found to be anti-CD10 for c-ALL (22/23) and beta F1 for T-ALL (6/8). Expression of aberrant antigens was fairly common--for example, 7/23 cases of c-ALL stained with antibodies against T cell associated antigens. CONCLUSIONS: Immunohistochemical investigation of routinely processed bone marrow biopsy specimens enables reliable detection of ALL subtypes c-ALL and T-ALL. A minimum panel of antibodies, against TdT, CD34, myeloperoxidase, lysozyme, CD10, CD79a, and CD20, and the antibody beta F1, is proposed for the immunophenotyping of acute leukaemia.