Histology and immunohistology of bone marrow biopsy in multiple myeloma

Eur J Haematol Suppl 51, Vol 43, 52-59

Key words: plasmacytoma - multiple myeloma - microscopy - immunohistochemistry - monoclonal antibodies

Histology and immunohistology of bone marrow biopsy in multiple myeloma

S. Pileri'., S. Poggi", P. Baglioni", M. Montanari", E. Sabattini", P. Galieni', P. L. Tazzari', M. Gobbi', M. Cavo', B. Falini', H. Stein3, and S. Tura'.

'Institute of Haematology "L. & A. Seragnoli", Bologna University, 'Haernolymphopathology Section, 'Department of Internal Medicine, Perugia University, Italy, and 31nstitute of Pathology, Free University, Berlin, Fed. Rep. Germany

B5-fixed/paraffin-embedded Jamshidi needle biopsies from 125 multiple myeloma patients were reviewed according to both morphological and immunohistological criteria. At microscopic examination, the following parameters were evaluated: i) grade of malignancy (low = 56; intermediate = 50; high = 19); ii) growth pattern (interstitial + /- sheets/nodules = 90; nodular = 13; packed marrow = 18; sarcomatous = 4); 111) histological stage (I = 64; I1 = 35; 111 = 26). Comparison of the findings in trephine biopsies and aspirates showed that in 30% of the cases the latter led to an understimation of the tumor burden. Immunohistochemical determination of Ig easily allowed: i) differential diagnosis from exuberant reactive plasmacytosis; ii) recognition and counting of neoplastic plasma cells; iii) detection of minimal residual disease after treatment. Immunohistochemistry also confirmed phenotypic aberration of neoplastic plasma cells, showing positivity for CD45, EMA, and cytokeratins in 14%, 59'70, and 25 % of the cases, respectively. Furthermore, it displayed expression of the P-glycoprotein in 4/8 resistant cases. These findings underline that routinely processed Jamshidi needle biopsies can be of great value in the study of patients with multiple myeloma.

It is well-known that bone marrow examination represents the cornerstone in establishing the diagnosis of multiple myeloma (MM) (1-15). In particular, it allows distinction from other closely related disorders and gives information concerning elements - such as the tumor burden and cytological details - which are of great prognostic relevance (1-15).

Although, in patients with MM, bone marrow examination is more often carried out in aspirates, it is evident from Bartl's studies that Jam-

shidi needle biopsy is mandatory, as it permits evaluation of both the histological stage and grade of malignancy of the tumor (1-5). These data supplement any clinical staging system at present in use and can direct decisions on treatment modalities (1-5). Furthermore, sequential bone marrow biopsies can usefully be employed in monitoring the effects of therapy during the course of the disease (1-5, 7).

While immunophenotyping of MM has often been performed in cytological preparations and/

TREPHINE BIOPSY AND MM 53

or frozen sections showing great heterogeneity in antigen expression (16-23), very little is known as to the impact of immunohistochemistry in the study of routinely processed tissue samples (7, 24-28). This might partially be due to the diffu- sion of plastic-embedding techniques which are not compatible with the use of highly sensitive immunocytochemical methods (29, 30).

Herein, we report on 125 plasmacytoma cases which have extensively been studied in routine sections both on morphological and immunohistochemical grounds.

Material and methods All the biopsies were fixed in B5, decalcified in Decal, processed for routine histology, and embedded in Para- plast at 57’C (29, 30). 3-pm thick sections were stained with H. & E., Giemsa, P.A.S., and Gomori silver impregnation for reticulin fibers (29, 30). Histological evaluation was carried out according to the criteria of Bartl et a1 (1-5).

Routine sections were also used for immunohistochemical analysis which was performed by applying a large panel of monoclonal antibodies and specific antisera (Table I ) , which were revealed with the APAAP (31) and the PAP technique (32), respectively.

In cases in which one part of the Jamshidi needle biopsy had been frozen in liquid nitrogen and stored at

-80°C (30), cryostat sections were cut and tested with the antisera, anti-Ig light chains and the monoclonal antibody Ki-67, which is directed at a proliferation- associated nuclear antigen (33). In particular, the Ki-67 and specific antisera were revealed with a double-staining technique, as previously described (30).

Results and discussion At microscopic examination, cytological details were well preserved in all instances, despite decalcification and paraffin-embedding. According to Bartl et a1 (1-5), out of 125 cases, 46 were of the Marschalko’ type, 10 of the small cell type, 4 of the cleaved type, 25 were polymorphous, 21 asynchronous, and 19 blastic. Therefore, 56, 50, and 19 cases belonged to the categories with low-grade, intermediate-grade, and high-grade of malignancy, respectively.

In comparison with the data previously reported by Bartl et a1 (1-5), our series showed a higher incidence of cases with intermediate- and high- grade histology. In particular, it is worthy of note that the blastic type accounted for 15.2% in our series versus 2% in Bartl’s series. This might be due to a fortuitous selection. Our figures, however, are similar to those shown by others: Greipp et a1 (13) reported 15% plasmablastic tumors in their series.

TABLE 1 Panel of specific antisera and monoclonal antibodies applied to paraffin sections

Specific antisera Source Reactivity

Anti-I@. Anti-IgD Anti-IgG Anti-IgM Anti-Kappa Anti-Lambda

DAKO DAKO DAKO DAKO DAKO DAKO

Ig a-heavy chain Ig &heavy chain Ig y-heavy chain Ig p-heavy chain Ig K-light chain Ig Might chain

Monoclonal Ab.s Source Reactivity

Anti-Lc Anti-EMA Anti-Elastase Anti-GpIIIa Anti-HLA-DR MTl LNl KL 1 Ber-H2 2F8 C219 MRK16

DAKO DAKO DAKO DAKO DAKO

CLONAB CLONAB

IMMUNOTECH Prof. Stein Dr. Tavari

Dr. Ling Dr. Tsuruo

Leuc. common Ag./CD45 Epithel. membrane Ag.

Elastase GpIIIa (CD61)

HLA-DR CD43

B-Lymphocytes Cytokeratins

CD30 Multidrug resistance

54 PILERI ET AL

4

Concerning the growth of MM in the bone marrow, we observed the following patterns: i) interstitial with loosely dispersed plasma cells in 64 cases; ii) interstitial with sheets of plasma cells in 15 cases; iii) interstitial with nodules of neoplastic elements in 11 cases; iv) nodular in 13 cases; v) packed marrow in 18 cases; vi) sarcomatous in 4 cases. These data differ from those of Bart1 et a1 (1-5) only in the higher incidence of nodular forms. It is noteworthy that in the cases with the interstitial growth pattern, neoplastic plasma cells were sometimes located around cap- illaries. This finding questions whether pericapil- lary location of plasma cells of the Marschalko’ type can always be regarded as diagnostic of reactive plasmacytosis (1-5).

According to histopathology, 64 patients were in stage I, 35 in stage 11, and 26 in stage 111.

When correlating the above-mentioned parameters, we found that the patients with low-grade histology usually had an interstitial growth pattern (with or without sheets of neoplastic elements) and were in stage I (p < 0.005). On the other hand, those with a high-grade tumor most often showed a packed marrow or a sarcomatous pattern (68.8%), and stage 111 disease (p < 0.0001).

On morphological grounds, it has to be further underlined that in patients who underwent repeat- ed biopsies the relapse phase was associated with an increase of the tumor burden and frequent conversion to a more aggressive histologic subtype. This is in kepping with the data previously reported by one of us (F. B.) who described emergence of a B-immunoblastic lymphoma in 10 patients with multiple myeloma and interpreted this as expression of the transition from a low-turn- over neoplasm to a kinetically more aggressive tumor, with a correspondingly poor prognosis


(34). This concept has further been confirmed by our group through immunohistochemical evaluation of the growth fraction in plasmacytoma cases with marked extramedullary spread occurring sev- eral years after the onset of the disease (35).

It is worthy of note that in 4 cases of the present series the development of a second neoplasm could be ascertained: Hodgkin’s disease and acute leukemia, in 2 instances each.

In 45 out of 125 patients, adequate follow-up data were available (Table 2). As expected, these showed that the disease ran a different clinical course, according to the grade of malignancy, pattern of growth, and histological stage. It has to be underlined that, conversely to Bartl’s series (1-5), we did not observe a clear-cut difference in survival between the low-grade and intermediate- grade categories: however, this might be due to the lower number of cases and shorter follow-up peri- od in our series.

Finally, we tried to correlate the histological stage with the clinical one, as well as the findings in Jamshidi needle biopsy with those in aspirates. This analysis revealed only a rough correspondence between histological and clinical stages; furthermore, it showed that in 30% of the cases aspirates led to an understimation of the tumor burden with respect to the more accurate Jamshidi needle biopsy technique. This finding is of great practical value, as in aspirates a plasma cell content higher than 20-30% is generally regarded as an unfavorable prognostic factor (9, 15, 18, 19). However, especially in cases with large nodules or sheets of neoplastic cells, it can happen that aspiration fails to show the real tumor burden.

Although plasmacytomas were amongst the first type of lymphoid tumors to be categorized using immunohistological techniques in routine

Figure 1. Neoplastic plasma cells of the Marschalkb type, with interstitial distribution, show monotypic restriction for k light chain. (Immunoalkaline phosphatase; nuclear counterstaining with Gill’s hematoxylin; x 500). Figure 2. Tumoral elements express the leucocyte common antigen (CD45). (Immunoalkaline phosphatase; nuclear counterstaining with Gill’s hematoxylin; x 500). Figure 3. Strong cytoplasmic staining for cytokeratins in a plasmacytoma of the polymorphic type. (Immunoalkaline phosphatase; nuclear counterstaining with Gill’s hematoxylin; x 500). Figure 4. Neoplastic plasma cells display a clear-cut positivity with the monoclonal antibody 2F8 (anti-MDR). (Immunoalkaline phosphatase; nuclear counterstaining with Gill’s hematoxylin; x 500).

56 PILERI ET AL

TABLE 2 Clinicopathological correlations in 45 cases with follow-up available

MM: SURVIVAL/GRADE OF MALIGNANCY R

0 20 40 m m Months

a-Lw b-mmu. C-HIM

MM: SURVIVAL/GROWTH PATTERN px

O ~ O ~ M ~ H ) B O ~ O M I Months

- 1 = INT + INT/SHE + INT/NDD 2 = NOD + PAC + SW -

MM:SURVIVAL/HISTOLOGlCAL STAGE Pz

01 I o i o m w o o 5 o m o o

Months a-1 b- II c - 111

sections (25), studies were restricted to the use of anti-Ig specific antisera until recently, when fixa-

tive-resistant monoclonal antibodies became available (36-43).

To the best of our knowledge, our study represents the first attempt to characterize a series of multiple myeloma cases by applying a large panel of specific antisera and monoclonal antibodies to paraffin sections.

In particular, specific antisera against Ig heavy and light chains (Figure 1) appeared to give useful information in cases in stage I and with interstitial infiltration, as well as after chemotherapy and/or bone marrow transplantation. In fact, they allowed: i) clear-cut differential diagnosis from exuberant reactive plasmacytosis, ii) easy recognition and counting of neoplastic plasma cells, iii) detection of minimal residual disease after treatment. The latter finding may be of great practical value for therapeutic decisions, especially in cases in which a monoclonal paraprotein is not present (nonsecretory myelomas) or difficult to quanti- tate. It is noteworthy that in one of the nonsecretory cases in our series, Ig storage was respon- sible for a marked pleomorphism of neoplastic elements, which appeared multinucleated, fusi- form, and spindle-shaped. In a previous biopsy performed at another hospital, this had led to a misdiagnosis of bone-warrow metastatic involve- ment by rhabdomyosarcoma. Finally, concerning the light chain ratio in multiple myeloma and M-GUS, our data do not support the hypothesis of Buss et a1 (7) that a value less than 16 indicates a nonmalignant process.

At immunohistochemical analysis with monoclonal antibodies, 14% of the cases showed expression of the leucocyte common antigen CD45 (Figure 2): conversely to previous observations (19), this finding was not correlated with a more aggressive clinical course of the disease. Further- more, it was encountered in any histological subtype: this does not support the view (24) that CD45 antigen expression occurs in borderline cases between plasmacytoma and non-Hodgkin lymphoma either of the immunocytic or B-immunoblastic type.

Either membrane-bound or dot-like cytoplasmic staining for the epithelial membrane antigen was observed in 59% of the cases and was un-


related to both the clinical behavior and histological pattern. This finding further supports the concept that EMA expression characteristically shown by normal plasma cells is retained in most plasmacytoma cases (36).

The monoclonal antibody KL1 raised to cytokeratins of low/intermediate molecular weight, gave a clear-cut cytoplasmic staining in 25% of our cases (Figure 3). This confirms the anti-cytokeratin reactivity by plasma cell tumors previously reported by Sewell et a1 (24) and Wotherspoon et a1 (28) in 1 and 4 cases, respectively. It is noteworthy that in our series the staining with KLI was un- related to both immunoglobulin phenotype and histology, while it occurred in 50% of the resistant cases. Although the true nature of the antikeratin- reactive moiety is still not clear and supportive biochemical work is needed, our findings allow some interesting speculations:

1. they confirm the possibility that on rare occa- sions cytokeratin positivity may occur also in non-epithelial tumors, including those of the immune system (24, 28, 44);

2. they suggest that in plasmacytomas this phe- nomenon is more widespread than could be explained by isolated malignant aberrant gene expression (24);

3. they underline that in anaplastic plasmacytomas the use of a limited panel of monoclonal antibodies consisting of reagents anti-cytokeratins, EMA, and CD45 could lead to an errone- ous diagnosis of undifferentiated carcinoma.

As to the results obtained with the other monoclonal antibodies, it is worthy of note that we could observe positivity with the MTl in roughly 50% of the cases, independently from the type of secreted immunoglobulin. This monoclonal is raised to the CD43 molecule, which is expressed by T cells, immature B cells, histiocytes and myeloid cells (41). Our findings differ from those of Woth- erspoon et a1 (28) who reported positivity with MTI in 2/14 cases and association with the IgD phenotype. On the other hand, while confirming possible expression of the LN1 antigen by plasma cell tumors as observed by others (37), our results

showed constant negativity with the monoclonal antibody Ber-H2 (45), as well as with the ones directed at the elastase and GpIIIa (CD61) (46, 47).

Taken together, our data underline that plasmacytomas display aberrant phenotype also in paraffin sections. In fact, conversely to normal plasma cells, they do not react with the monoclonal antibody Ber-H2, while they present inconstant EMA staining, occasional CD45 expression, and possible cytokeratin positivity. Besides reactivity with MT1, no staining was appreciated in our series with the monoclonals directed at myeloid/megakaryocytic markers (19, 20). This might be due either to the limited panel of antibodies which can be applied to routine sections, or the loss of antigens during fixation and/or decalcification procedures.

Interestingly, the anti-MDR monoclonal antibody 2F8 (48) gave a strong cytoplasmic staining in 48 resistant cases (Figure 4). No staining was seen in normal and neoplastic plasma cells in 20 Jamshidi needle-biopsies, which were employed as controls. On the other hand, no reactivity was found either in resistant or in control cases with the antibodies MRK16 (49) and C219 (50). This finding is not unexpected, as the MRK16 and C219 detect, respectively, a surface and a cytoplasmic epitope of the gp-170, which does not resist B5 fixation (51). Our results suggest the possibility of assessing the expression of P-glycoprotein also in paraffin sections by adequate monoclonal antibodies: this proce- dure may be of practical value for therapeutic decisions (20, 52).

Finally, in cases with frozen material, available evaluation of the growth fraction with the monoclonal antibody Ki-67 showed that the content of positive cells as determined with a double-staining technique (30) varied signifi- cantly in M-GUS, multiple myeloma, and relapse of multiple myeloma (35). It is noteworthy that the Ki-67 marking on cryostat sections displays some advantages over the bro- modeoxyuridine technique in aspirates (53), as it does not require incorporation of the marker, avoids DNA denaturation procedures, and al-

58 PILERI ET AL

lows easy cell kinetics evaluation, also in cases where prominent stromal reaction gives rise to dry tap at aspiration.

Conclusions According to the above-mentioned data, we think that our study:

1 . gives further confirmation of the role of Jam- shidi needle biopsy in the management of patients with multiple myeloma, owing to its prognostic relevance and accuracy in detecting the tumor burden and monitoring the disease;

2. suggests the possibility of avoiding resin-embedding procedures which do not allow immunophenotyping in routine sections;

3. underlines the usefulness of immunohistochemical techniques in the study of multiple myeloma.

Acknowledgments This paper was partially supported by grants from Ministry of Public Education (Rome), AIRC (Milan), and Labometrics (Milan).

References 1. Bartl R, Frisch B, Burkhardt R, et al. Bone marrow

histology in myeloma: its importance in diagnosis, prognosis, classification and staging. Br J Haematol 1982;51:361- 75.

2. Bartl R, Frisch B, Burkhardt R, Jager K, Pappenberger R, Hoffmann-Fezer G . Lymphoproliferations in the bone marrow: identification and evolution, classification and staging. J Clin Pathol 1984;37:233-54.

3. Bartl R, Frish B, Burkhardt R. Bone marrow biopsies revisited. A new dimension for haematologic malignancies. Basel: Karger, 1985.

4. Bartl R, Frisch B, Fateh-Moghadam A, Kettner G , Jager K, Sommerfeld W. Histologic classification and staging of multiple myeloma. A retrospective and prospective study of 674 cases. Am J Clin Pathol 1987;87:342-55.

5 . Bartl R. Histologic classification and staging of multiple myeloma. Hematol Oncol 1988;6:107-13.

6. Bataille R, Durie BGM, Grenier J, Sany J. Prognostic factors and staging in multiple myeloma: A reappraisal. J Clin Oncol 1986;4:80-7.

7. Buss DH, Prichard RW, Cooper MR. Plasma cell dys- crasias. Hematol/Oncol Clin North Am 1988;2:603-15.

8. Canale DD, Collins RD. Use of bone marrow particle sections in the diagnosis of multiple myeloma. Am J Clin Pathol 1974;61:383-92.

9 . Cavo M, Baccarani M, Gobbi M, Lipizer A, Tura S. Bone marrow plasma cell infiltration in multiple myeloma. Br J Haemat 1984;57:352-3.

10. Durie BGM, Salmon SE. A clinical staging system for multiple myeloma. Correlations of measured myeloma cell mass with presenting clinical features, response to treatment and survival. Cancer 1975;36:842-54.

1 1 . Frish B, Lewis SM, Burkhardt R, Bartl R. Biopsy pathology of bone and bone marrow. London: Chapman and Hall, 1985.

12. Fritz E, Ludwig H, Kundi M. Prognostic relevance of cellular morphology in multiple myeloma. Blood 1984;63: 1072-9.

13. Greipp PR, Raimond NM, Kyle RA, O’Fallon WM. Mul- tiple myeloma: significance of plasmablastic subtype in morphological classification. Blood 1985;65:305-10.

14. Isobe T, Ikeda Y, Ohta H. Comparison of sizes and shapes of tumor cells in plasma cell leukemia and plasma cell myeloma. Blood 1979;53: 1028-30.

15. Kyle RA. Prognostic factors in multiple myeloma. Hema- to1 Oncol 1988;6:125-30.

16. Barlogie R, Epstein J, Alexanian R. Genotypic and phenotypic characteristics of multiple myeloma. Hematol Oncol 1988; 6: 99- 102.

17. Caligaris-Cappio F, Janossy G , Bergi L, et al. Identifica- tion of malignant plasma cell precursors in the bone marrow multiple myeloma. J Clin Invest 1985;76:1243-51.

18. Durie BMG, Grogen TM. CALLA-positive multiple myeloma: an aggressive subtype with poor survival. Blood 1986;66:229-32.

19. Durie BMG. The biology of multiple myeloma. Hematol Oncol 1988;6:77-81.

20. Barlogie B, Epstein J, Selvanayagam P, Alexanian R. Plasma cell myeloma - New Biological insights and advances in therapy. Blood 1989;73:863-79.

21, Falini B, Mason DY, Stein H, et al. The immunohistological analysis of undecalcified human bone trephine biopsies. Haematol 1984;69:346-76.

22. Foon KA, Schroff RW, Eale RP. Surface markers in leukaemia, lymphoma cells: Recent advances. Blood 1982;60: 1-19.

23. Tazzari PL, Gobbi M, Dinota A, et al. Normal and neoplastic plasma cell membrane phenotype: studies with new monoclonal antibodies. Clin Exp Immunol 1987;70:192-200.

24. Sewell HF, Thompson WD, King DJ. IgD Myeloma/ immunoblastic lymphoma cells expressing cytokeratin. Br J Cancer 1986;53:695-6.

25. Taylor CR, Russell R, Chandor S. An immunohistologic study of multiple myeloma and related conditions, using an immunoperoxidase technique. Am J Clin Pathol 1978;70612-22.

26. Taylor CR. Immunomicroscopy: A diagnostic tool for the surgical pathologist. Philadelphia: Saunders WB, 1986:116-34.

27. Thiele J, Arenz B, Klein H, Vierbuchen M, Zanovich R, Fischer R. Differentiation of plasma cell infiltrates in the bone marrow. A clinicopathologic study on 80 patients


including immunohistochemistry and morphometry. Vir- chows Arch [Pathol Anat] 1988;412:553-62.

28. Wotherspoon AC, Norton AJ, Isaacson PG. Immunoreac- tive cytokeratins in plasmacytomas. Histopathol 1989; 14:141-50.

29. Pileri S. Linfomi maligni non-Hodgkin. Dalla tecnica alla diagnostica. Bologna: Esculapio Editrice, 1985.

30. Falini B, Pileri S. Histological and immunohistological analysis of human lymphomas. Boca Raton: CRC Press Inc, 1989 (in press).

31. Cordell JL, Falini B, Erber WN, et al. Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 1984;32:219-29.

32. Sternberger LA, Hardy PH, Cuculis JJ, Meyer HG. The unlabeled antibody-enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in the identification of spirochetes. J Histochem Cytochem 1970;18:315-33.

33. Gerdes J , Schwab U, Lemke H, Stein H. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer l983;3 1:13-20.

34. Falini B, De Solas I, Levine AM, Parker JW, Lukes RJ, Taylor CR. Emergence of B-Immunoblastic sarcoma in patients with multiple myeloma: A clinicopathologic study of 10 cases. Blood 1982;59:923-33.

35. Falini B, Canino S, Sacchi S, et al. Immunocytochemical evaluation of the percentage of proliferating cells in patho- logical bone marrow and peripheral blood samples with the Ki-67 and anti-bromo-deoxyuridine monoclonal antibodies. Br J Haematol 1988;69:311-20.

36. Delsol G, Gatter KC, Stein H, Erber WN, Pulford KAF, Zinne K, Mason DY. Human lymphoid cells express epithelial membrane antigen. Lancet 1984;ii:1124-9.

37. Epstein AL, Marder RJ, Winter JN, Fox RI. Two monoclonal antibodies (LN1, LN2) reactive in B5 formalin- fixed, paraffin embedded tissues with follicular center and mantle zone human B lymphocytes and derived tumours. J Immunol 1984;133:1028-36.

38. Makin CA, Borrow LG, Bodmer WF. Monoclonal antibody to cytokeratin for use in routine histopathology. J Clin Pathol 1984;37:975-85.

39. Norton AJ, Ramsey AD, Smith SH, Beverley PCL, Isaac- son PG. Monoclonal antibody (UCHLl) that recognises normal and neoplastic T cells in routine fixed tissues. J Clin Pathol 1986;39:399-405.

40. Norton AJ, Isaacson PG. Detailed phenotypic analysis of B-cell lymphoma using a panel of antibodies reactive in

routinely fixed wax-embedded tissue. Am J Pathol 1987;128:225-39.

41. Poppema S, Hollema H, Visser L, Vos H. Monoclonal antibodies (MTl, MT2, MB1, MB3) reactive with leucocyte subsets in paraffin-embedded tissue sections. Am J Pathol 1987; 127:418-29.

42. Warnke RA, Gatter KC, Falini B, et al. Diagnosis of human lymphoma with monoclonal anti-leucocyte antibodies. N Engl J Med 1983;309:1275-81.

43. West KP, Warford A, Fray L, Allen M, Campbell AC, Lauder I. The demonstration of B-cell, T-cell and myeloid antigens in paraffin sections. J Pathol 1986;150:89-101.

44. Zauli D, Gobbi M, Crespi C, et al. Vimentin and keratin intermediate filamants expression by K562 leukemic cell line. Leuk Res 1986;10:29-33.

45. Schwarting R, Gerdes J, Falini B, Pileri S, Stein H. Ber- H2: a new high avidity monoclonal antibody to formalin resistant epitope of the Ki-1 (CD30) antigen. Immunohisto- chemical and immunobiochemical characterization. Blood 1989 (in press).

46. Pulford KAF, Erber WN, Crick JA, Olsson I, Micklem KJ, Gatter KC, Mason DY. Monoclonal antibody against human neutrophil elastase for the study of normal and leukaemic myeloid cells. J Clin Pathol 1988;41:853-60.

47. Erber WN, Mynheer LC, Mason DY. APAAP labelling of blood and bone marrow samples for phenotyping leukaemia. Lancet 1986;i:761-6.

48. Tazzari PL. A new monoclonal antibody directed at the multidrug resistance (MDR) agent. Manuscript in preparation.

49. Fitgerald DJ, Willingham MC, Cardarelli CO, et al. A monoclonal antibody-Pseudornonas toxin conjugate that specifically kills multidrug-resistant cells. Proc Natl Acad Sci USA 1987;84:4288-92.

50. Kartner N, Evernden-Porelle D, Brandley G, Ling V. De- tection of P-glycoprotein in multidrug-resistant cell lines by monoclonal antibodies. Nature 1985;316:820-3.

51. Pileri S. Unpublished observation. 52. Fojo AT, Ueda K, Slamon DJ, Ppolack DG, Gottesman

MM, Pastan I. Expression of a multidrug-resistance gene in human tumors. Proc Natl Acad Sci USA 1987;84:265-9.

53. Pileri S, Gerdes J, Rivano MT, et al. Monoclonal antibodies as markers of the growth fraction: proper choice and application. Bas Appl Histochem 1987;31:155.

Correspondence to: Prof. Stefan0 Pileri Istologia Emolinfopatologica Istituto di Ematologia “Sertignoli” Via Massarenti 9 40138 Bologna Italy

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Histology and immunohistology of bone marrow biopsy in multiple myeloma