Plasma Cells in Peripheral Blood Stem Cell Harvests from Patients with Multiple Myeloma Are Predominantly Polyclonal

Plasma cells in peripheral blood stem cell harvests from patients with multiple myeloma are predominantly polyclonal

B Pope, R Brown, J Gibson and D Joshua

Institute of Haematology, Royal Prince Alfred Hospital, Sydney, NSW, Australia

Summary: Recent studies have reported conflicting levels of tumour cell contamination in PBSC harvests. Radioactive finger- A flow cytometric technique has been developed to printing PCR techniques for the immunoglobulin heavy detect individual plasma cells in PBSC harvests and to chain genes have found tumour cell contamination in 36%5 establish light chain restriction as a surrogate marker and 44%6 of harvests. However when patient-specific oli- of their clonality. Plasma cells were identified by high gonucleotide primers were used, 100% of patient samples intensity CD38 (CD38؉؉) and cytoplasmic immunoglob- were PCR positive for tumour cells even after a double ulin (cIg) expression. The ratio of cytoplasmic kappa to transplant.7 Gazitt et al8 sorted PBSC harvests for lambda expression was used to detect light chain restric- CD34ϩLinϪThyϩ cells and demonstrated by PCR a 2.7 to tion. All 25 PBSC harvests studied contained 7.3 log reduction of tumour cells was possible.8 In this -CD38؉؉/cIg plasma cells (mean 0.7%, range 0.03–2%). study there were no plasma cells detected by flow cytome Harvests from non-myeloma patients also contained try based on the presence of CD38bright cells in the sorted plasma cells (mean 0.4%, range 0.01–1.5%). Most of the population. In contrast, another study found that after the plasma cells detected in the harvests from myeloma cells were sorted for CD34ϩ on a magnetic column fol- patients were immature (CD45؉/CD45؉؉) rather than lowed by flow activated cell sorting, no malignant cells mature (CD45؊). When the total plasma cell population could be detected by patient-specific oligonucleotide PCR.9 was studied, definite isotype restriction could be While these PCR methods are extremely sensitive for detected in only 16% of harvests. Light chain restriction detecting specific tumour cell populations they have limited was found in 53% of harvests when the mature plasma ability to quantitate cell numbers and analyse individual cells (CD45؊) were analysed but only in 9% of harvests cells. Furthermore, these methods are not able to quantitate when immature (CD45؉/CD45؉؉) plasma cells were the number of normal plasma cells present in the PBSC analysed. Five percent of patients with myeloma had harvest. detectable light chain restriction in peripheral blood The aim of this study was to use a sensitive flow cyto- CD19؉ cells. There was concordance between the ratio metric assay to analyse light chain restriction in the plasma of malignant (CD19؊/CD56؉) to normal (CD19؉/ cell population in PBSC harvests to determine whether light -CD56؊) plasma cells and light chain expression in 86% chain restriction can be used as a surrogate marker of clon of patients studied. This study has demonstrated that ality. We also wished to correlate light chain restriction and the majority of plasma cells in PBSC harvests from CD19/CD56 expression which has been reported to be a patients with myeloma are not only immature but are marker of malignant plasma cells.10,11 also predominantly polyclonal and that monoclonality is best detected in mature plasma cells. Keywords: multiple myeloma; PBSC; plasma cells; light Materials and methods chain restriction; flow cytometry Patient selection Autologous PBSC were collected from 25 patients with High-dose therapy supported by autologous peripheral myeloma between March 1994 and May 1996. Harvests blood stem cell (PBSC) transplantation is frequently used in were performed after mobilisation with chemotherapy the treatment of patients with myeloma. PBSC collections (cyclophosphamide 4 g/m2) and either G-CSF (5– obtained following chemotherapy and cytokine mobili- 10 ␮g/kg/day) or GM-CSF (5 ␮g/kg/day). Nine patients sation contribute to faster haemopoetic recovery and are were IgA, 14 were IgG and two patients had light chain 1,2 easier to obtain than autologous marrow. It has been sug- only disease. Seventeen patients were kappa and 8 lambda. gested that PBSC may be contaminated by fewer plasma Eighteen patients were in progressive disease and seven in cells than autologous bone marrow3 but there are concerns stable disease at the time of harvest. 4 that G-CSF may mobilise tumour cells into the harvest. PBSC harvests from nine non-myeloma patients were also studied (one AML, five non-Hodgkin’s lymphoma, two Correspondence: B Pope, Institute of Haematology, Royal Prince Alfred germ cell cancers and one rhabdomyosarcoma). These Hospital, Missenden Rd, Camperdown, NSW, 2050, Australia patients had also undergone chemotherapy and G- or GM- Received 14 February 1997; accepted 20 April 1997 CSF mobilisation. Multiple myeloma PBSC contain polyclonal plasma cells B Pope et al 206 Bone marrow cells were obtained from a biopsy perfor- Cytoplasmic kappa/lambda/Ig staining: Cells (1 ϫ 106) med as part of the routine investigations. Informed consent were placed in tubes, primary antibodies IgG1 (50 ␮l) and was obtained from all patients for all procedures. CD45 (10 ␮l) were added to the cells and incubated for 30 min at 4°C. Cells were washed in PBS and the second- ary antibody, 50 ␮l (1/50 dilution in PBS) of G␣M RED Collection and cell preparation of PBSC harvest 613, was added to each tube, incubated for 30 min at 4°C PBSC were collected by apheresis using a Baxter CS3000 and then washed. One hundred microlitres of Solution A Plus (Deerfield, IL, USA). Cells were frozen in 10% from the cell permeabilisation kit were added for 15 min DMSO at a concentration of 1–10 ϫ 109/l in a Control Rate at room temperature and then washed. Fifty microlitres of Freezer (Kyro 10/16 Series II; Planer Biomed, Middlesex, 1/10 dilution of normal mouse serum were added for UK) and then stored in vapour phase in a liquid nitrogen 10 min for blocking purposes. One hundred microlitres of storage facility (XLC1110; MVE, Bloomington, MN, Solution B from the cell permeabilisation kit was added along with anti-kappa FITC (10 ␮l), or anti-lambda FITC USA). Where possible studies were performed on each ␮ ␮ patient from day 2 of apheresis harvest. Prior to analysis (10 l) or anti-IgG FITC (50 l of a 1/50 dilution) and ° anti-CD38PE (20 ␮l), then incubated for 15 min at room a small aliquot of cells was quickly thawed at 37 C and ␮ anticoagulant citrate dextrose solution formula A (ACD-A; temperature. Cells were washed and resuspended in 200 l Baxter, Old Toongabbie, NSW, Australia) was added (1/5) of PBS. to avoid clotting. The number of harvests ranged between one and nine Flow cytometry: Flow cytometric data was acquired on a (mean = 4), aiming for a minimum yield of 2 ϫ 106 CD34ϩ Coulter Epics XL with XL software. At least 80 000 events cells/kg. Yields of CD34ϩ cells/kg ranged from 0.03 to 6.6 were collected in listmode data. All cells except debris and ϫ 106 and CD34ϩ cells were analysed according to the aggregates were gated. Forward scatter, side scatter and Sutherland method.12 Five patients failed to provide suf- three fluorescent signals (to detect FITC, PE and RED613 ficient CD34ϩ cells and were not eligible for transplan- or PERCP) were collected for each sample. The data was tation. Currently, 19 patients have been transplanted. analysed on a remote workstation using Coulter Elite V.4 software.

Monoclonal antibodies and reagents Results The following antibodies were used in this study. Anti- CD56 FITC, anti-CD38 PE, anti-IgG1 PERCP, anti-CD45 PERCP (Becton Dickson Immunocytometry Systems, San Identification of light chain expression of plasma cells in Jose, CA, USA); anti-IgG1 FITC, anti-CD19 PE, poly- bone marrow and PBSC from patients with multiple clonal anti-kappa FITC, polyclonal anti-lambda FITC, anti- myeloma CD38 and anti-CD45 (DAKO Corporation, Carpinteria, Plasma cells were identified by high intensity CD38 CA, USA); anti-IgG1 (Coulter Corporation, Miami, FL, (CD38ϩϩ) as previously described.10,13,14 Cytoplasmic USA); anti-CD38 FITC (Caltag, San Francisco, CA, USA); immunoglobulin (cIg), cytoplasmic kappa, cytoplasmic Ј ␣ Ј 15 anti-IgG (H&L) F(ab )2 FITC, S M Ig F(ab )2 FITC lambda, ICB-61 and B-B4 were used to confirm that these (Silenus, Hawthorn, Victoria, Australia); G␣M RED 613 cells were plasma cells. (Gibco BRL, Gaithersburg, MD, USA); anti-B-B4 FITC To determine the optimum method to detect cytoplasmic (Immuno Quality Products, Groningen, The Netherlands); kappa and lambda expression we evaluated the following ICB-61 was a gift from Dr D Mason (Oxford). The cell cell permeabilisation reagents: periodate-lysine-paraformal- permeabilisation kit was obtained from Harlan Sera-Lab dehyde (PLP), PermeaFix (Ortho Diagnostic Systems, Rari- (Crawley Down, Sussex, UK). tan, NJ, USA), Caltag Fix and Perm (Caltag), STF (Streck Laboratories, Omaha, NE, USA) and Harlan Sera-Lab cell Cell surface staining: Cells (1 ϫ 106) were stained with permeabilisation kit. We compared relative fluorescence anti-CD38 PE (20 ␮l) and B-B4 FITC (10 ␮l) for 30 min intensity (RFI) on bone marrow cells from a patient with on ice. Cells were then washed twice and resuspended in IgG␬ myeloma and obtained the following results for the 200 ␮l of PBS, ready for analysis on flow cytometer. Cells RFI of the kappaϩve cells: PLP = 53.4, PermeaFix = 258.5, were also stained with anti-ICB-61 (50 ␮l) and incubated Caltag Fix and Perm = 937.1, STF = 160.5 and Harlan on ice for 30 min. Cells were washed in PBS and the sec- Sera-lab cell permeabilisation kit 943.8. Of all these ondary antibody S␣M IgG FITC (50 ␮l, 1/50 dilution in reagents Harlan Sera-Lab cell permeabilisation kit gave the PBS), was added to each tube and incubated for 30 min at clearest distinction between kappa and lambda plasma cells. 4°C, then washed. Fifty microlitres 1/10 dilution of normal Figure 1 shows typical cytoplasmic kappa, lambda and mouse serum were added for 10 min for blocking purposes, Ig staining of bone marrow cells from a patient with kappa anti-CD38PE (20 ␮l) was then added and incubated for myeloma. These histograms clearly show a predominant 30 min on ice. The cells were washed in PBS and resus- kappa expression with a ␬/␭ ratio = 61. Of the CD38ϩϩ pended in 200 ␮l of PBS. A similar procedure was used to cells, 98% were cIg positive. These cells were also positive indirectly label anti-CD38 with G␣M RED 613 (50 ␮l if a for B-B4 and ICB-61. This technique for detecting cyto- 1/50 dilution in PBS) followed by direct labelling with anti- plasmic kappa and lambda expression was reproducible for CD19 FITC and anti-CD56PE. all multiple myeloma bone marrows studied (n = 13) with Multiple myeloma PBSC contain polyclonal plasma cells B Pope et al 207 abc 1000 1000 1000 100 100 100 10 10 10 CD38 PE CD38 PE CD38 PE 1 1 1

0.10.1 1 10 100 1000 0.1 1 10 100 1000 0.10.1 1 10 100 1000 Kappa FITC Lambda FITC Ig FITC Figure 1 Histograms of CD38 against cytoplasmic kappa (a), cytoplasmic lambda (b) and cytoplasmic Ig (c) from a bone marrow of a patient with kappa myeloma.

abc 1000 1000 1000 100 100 100 10 10 10 CD38 PE CD38 PE CD38 PE 1 1 1 0.1 0.1 0.11 10 100 1000 0.11 10 100 1000 0.10.1 1 10 100 1000 Kappa FITC Lambda FITC Ig FITC

Figure 2 Histograms of CD38 against cytoplasmic kappa (a), cytoplasmic lambda (b) and cytoplasmic Ig (c) from a PBSC harvest of a patient with kappa myeloma. an average CD38ϩϩ/cIg expression of 98.3% (range 95– number of CD38ϩϩ cells for both myeloma and non-mye- 99.7%). The same technique was applied to PBSC harvests loma samples. The non-myeloma patients tended to have from patients with myeloma. Figure 2 shows histograms fewer CD38ϩϩ cells in their PBSC harvests (mean = 0.4%, from a PBSC harvest in which the CD38ϩϩ cells were also range 0.01–1.5%) but this was not statistically significant cytoplasmic Ig positive. However in this specimen signifi- (t = Ϫ0.7, P = 0.5). We also evaluated the absolute numbers cant numbers of both kappa and lambda cells were present of plasma cells found in PBSC harvests from myeloma (␬/␭ ratio = 2.0). patients, the mean was 2.5 ϫ 107 CD38ϩϩ/l, range 3.1 ϫ 106–9.3 ϫ 107 CD38ϩϩ. In non-myeloma harvests Frequency of plasma cells in PBSC harvests the mean absolute number of plasma cells was 3.4 ϫ 107 ϩϩ ϫ 5 ϫ 8 ϩϩ CD38 /l, range 7.8 10 –1.3 10 . The difference in Plasma cells (CD38 ) were found in all 25 stem cell col- absolute numbers was also not significantly different lections studied with a mean of 0.7% and a range of 0.03– (t =−0.7, P = 0.5). 2%. CD38ϩϩ cells were also easily demonstrated in the PBSC harvests of non-myeloma patients. Figure 3 plots the

Maturity of plasma cells in PBSC harvests

1 CD45 was used as a marker of maturity of plasma cells. As plasma cells mature they lose CD45 expression.16,17 Three clear peaks of CD45 expression in plasma cells were analysed as previously described.17 These populations were cells ϩϩ Ϫ

++ mature plasma cells (CD38 , CD45 ), immature plasma ϩϩ ϩ 0.1 cells (CD38 , CD45 ) and primitive plasma cells (CD38ϩϩ, CD45ϩϩ). Most of the plasma cells found in % CD38 PBSC harvests from myeloma patients were immature (CD45ϩ) or primitive (CD45ϩϩ). This is demonstrated in Figure 4. The mean percentage of immature and primitive 0 .01 cells in the harvests was 83%, range 37–100% and the Myeloma Non-Myeloma mean percentage of mature plasma cells was 15%, range Figure 3 Dot plot showing the number of CD38ϩϩ/cIgϩ cells in mye- 0–62%. loma and non-myeloma PBSC harvests. Multiple myeloma PBSC contain polyclonal plasma cells B Pope et al 208 CD38++ CD38++ CD38++ CD45– CD45+ CD45++ 1000

10 100

5 10 CD38 PE ratio λ / κ 1

0.5 0.1 1 10 100 1000

CD45 RED 613

Figure 4 Histogram of CD38 against CD45 for a PBSC harvest from a patient with myeloma. In this sample 88% of the plasma cells were immature (CD45ϩ/CD45ϩϩ).

0.1

Cytoplasmic Ig relative fluorescent intensity Figure 5 Dot plot of ␬/␭ ratios from 25 PBSC harvests. Monoclonality ϩϩ was defined as a ␬/␭ ratio of у5.0 or р0.5. The grey area shows the CD38 cells were positive for cIg in all CD45 subpopula- ␬ ␭ tions. Overall 98% CD38ϩϩ cells were also cIg positive. range for normal / ratios. In the CD45Ϫ subpopulation, CD38ϩϩ cells were on average 99% cIg positive and in the CD45ϩ/ϩϩ subpopulations, the CD38ϩϩ cells were on average 97% cIg positive. The contrast the immature plasma cell population demonstrated RFI of cIg in the mature (CD45Ϫ) cells was significantly restriction in only 9% of harvests. In another series of 44 different from the immature and primitive (CD45ϩ/ϩϩ) patients with myeloma, light chain restriction was demon- regions (t = 4.3, P Ͻ 0.0003). The CD45Ϫ region had a strated on CD19ϩ cells in the peripheral blood of two mean RFI of 626 (range 30–1008) compared to the patients (5%). Thus cytoplasmic light chain restriction is CD45ϩ/ϩϩ region, mean 383 (range 74–712). The reduction even more rare in B cells than in primitive plasma cells. in intensity of cIg staining in CD45ϩ/ϩϩ cells compared The percentage of CD38ϩϩ cells in the PBSC harvest did with CD45Ϫ cells correlates with the degree of maturity as not correlate with the presence or absence of light chain defined by CD45 expression. restriction.

Light chain restriction in plasma cell populations Clonal plasma cells identified by CD19ϪCD56ϩ expression Light chain restriction was used as a surrogate marker for clonality. A kappa clone was defined as a ratio of ␬/␭ у 5.0 Several groups have suggested that malignant plasma cells and a lambda clone was defined as a ratio of ␬/␭ р 0.5.18 have a CD19Ϫ/CD56ϩ phenotype while normal plasma Of the 25 patients studied (Figure 5), four patients (16%) cells are CD19ϩ/CD56Ϫ.10,11 We have evaluated this cri- displayed definite plasma cell light chain restriction. These terion to discriminate between normal and clonal cells in patients also had a relatively high number of CD45Ϫ PBSC and compared this phenotype with light chain (mature) plasma cells in their PBSC collections (mean 59% expression in our series of patients. The ratio of CD38ϩϩ of the total plasma cells) and all had progressive disease at CD19Ϫ/CD56ϩ plasma cells (malignant) to CD38ϩϩ the time of PBSC harvest. In contrast, 18 out of 25 patients CD19ϩ/CD56Ϫ plasma cells (normal) was used to deter- (72%) had no detectable light chain restriction and had few mine a ratio of malignant/non-malignant plasma cells. CD45Ϫ plasma cells (mean 5%). Seventy-six percent of Table 1 shows that most of the harvests (n = 14) had low these patients had progressive disease at the time of harvest. ratios of malignant to non-malignant CD19/CD56 Three patients (12%) had a reversed ␬/␭ ratio, one of whom expression. There was concordance between the ratio of had progressive disease at time of harvest. malignant to non-malignant plasma cells as defined by When the analysis was confined to mature plasma cells, CD19/CD56 expression and light chain expression in 86% light chain restriction was observed in 53% of harvests. In of patients studied. Multiple myeloma PBSC contain polyclonal plasma cells B Pope et al

Ϫ ϩ 209 Table 1 Comparison of the presence of malignant cells by cytoplas- plasma cells have a CD19 /CD56 phenotype and that nor- mic light chain or CD19/CD56 expression mal plasma cells express CD19ϩ/CD56Ϫ. In this study there was concordance between the ratio of malignant to non- Patient Light chain CD19/CD56 Concordance malignant plasma cells as defined by CD19/CD56 expression expression ␬/␭ ratio malignant/ expression and light chain expression in 86% of patients non-malignanta studied. The remaining patients all had a malignant CD19/CD56 phenotype but light chain restriction was not 1 0.2b 1.4c demonstrated. This could infer that CD19/CD56 expression 2 0.1b 3.5c is a more sensitive means of detecting malignant plasma 3 54.7b 1.5c cells, however Harada et al10 have demonstrated that while 4 2.1 Ͻ0.1 Ϫ ϩ Ͻ malignant plasma cells are usually CD19 /CD56 they 5 0.6 0.1 Ϫ Ϫ ϩ ϩ 6 1.8 Ͻ0.1 may also be CD19 /CD56 or CD19 /CD56 . 7 2.0 Ͻ0.1 Previous flow cytometric studies of light chain 8 1.4 0.1 expression have either determined surface expression or 9 1.2 2.1c — have not optimised the technique of detecting cytoplasmic 10 1.2 0.2 19 11 1.3 5.5c — light chain expression. Prior to this study we optimised 12 0.8 0.4 the assay with bone marrow samples and tested five differ- 13 1.5 0.3 ent permeabilisation and fixation techniques in order to 14 1.4 0.1 determine the most suitable for clearly differentiating light chain expression. The cell permeabilisation kit from Harlan a ϩϩ Ϫ ϩ Malignant plasma cells CD38 /CD19 /CD56 ; non-malignant plasma Sera-Lab was used in these studies as it provided the clear- cells CD38ϩϩ/CD19ϩ/CD56Ϫ. bOutside normal range 0.5 р ␬/␭ у 5.0. est separation of cytoplasmic kappa and lambda staining. cMalignant to non-malignant plasma cell ratio Ͼ1. The findings reported here and by other workers4 suggest that the process of PBSC mobilisation with chemotherapy and G- or GM-CSF leads to the release of both clonally Discussion restricted and polyclonal plasma cells. The cause of this phenomenon is not known. Plasma cells are present in the PBSC harvests of both mye- A large number of reports have now shown that PBSC loma and non-myeloma patients in sufficient numbers to be from patients with myeloma contain malignant cells and studied by flow cytometry. The present study has demon- many studies have used PCR-based techniques to demonstrated that there is considerable heterogeneity in the strate the presence of clonally restricted (malignant) cells.5–8 plasma cell population present in PBSC harvests obtained Attempts to quantitate PCR are technically difficult, prone from patients with multiple myeloma. Using a flow cyto- to error and do not estimate the residual non-malignant metric analysis of cytoplasmic kappa and lambda plasma cells. Accurate quantitation of plasma cell numbers expression after cell permeabilisation we have demon- in PBSC has been performed by flow cytometry using high strated that for most patients with myeloma, the majority expression of CD38 as a marker for plasma cells.10,13,14 The of plasma cells in PBSC harvests are polyclonal. Only a present study is important because it demonstrates that the small percentage (16%) of collections clearly demonstrated majority of these CD38ϩϩ cells in PBSC from myeloma light chain restriction consistent with the presence of a pre- patients are normal plasma cells. Thus studies which infer dominantly clonal population. Studies of CD45 coex- that CD38 expression in PBSC is related to the number of pression have shown that PBSC plasma cells are predomi- malignant cells must be treated with caution. nantly immature (CD45ϩ/ϩϩ) rather than mature (CD45Ϫ). An analysis of light chain expression in these plasma cell subpopulations demonstrated that a clonal population was Acknowledgements clearly detectable in 53% of harvests when the mature plasma cells were studied but only in 9% when primitive This work was supported in part by grants from the Leo and Jenny plasma cells were studied. This apparent discrepancy can Leukemia and Cancer Foundation and the National Health and be explained by the observation that the majority of Medical Research Council. PBSC harvests contain predominantly immature (CD45ϩ/CD45ϩϩ) plasma cells which are mostly polyclonal. Thus the large number of polyclonal immature cells References mask the light chain restriction of the mature (CD45Ϫ) population when the total plasma cell population is evalu- 1 Gianni AM, Siena S, Bregni M et al. Granulocyte–macro- ated. The four patients with definite light chain restriction phage colony-stimulating factor to harvest circulating haemo- were the only four patients with mostly mature (CD45Ϫ) poietic stem cells for autotransplantation. Lancet 1989; ii: plasma cells in their PBSC and hence no masking effect 580–584. 2 Boiron J-M, Marit G, Faberes C et al. Collection of peripheral from polyclonal immature plasma cells. When peripheral blood stem cells in multiple myeloma following single high- blood was studied light chain restriction could be detected dose cyclophosphamide with and without recombinant human in even fewer patients (5%). Thus monoclonality is best granulocyte–macrophage colony-stimulating factor (rhGM- detected by analysing mature plasma cells. CSF). Bone Marrow Transplant 1993; 12: 49–55. It has been suggested by other workers10,11 that malignant 3 Vescio RA, Han EJ, Schiller GJ et al. Quantitative comparison Multiple myeloma PBSC contain polyclonal plasma cells B Pope et al 210 of multiple myeloma tumor contamination in bone marrow of two subpopulations of plasma cell in MGUS patients. Util- harvest and leukapheresis autografts. Bone Marrow Trans- ity in the differential diagnosis with multiple myeloma. Blood plant 1996; 18: 103–110. 1996; 88: (Suppl. 1): 640a. 4 Vora AJ, Toh CH, Peel J, Greaves M. Use of granulocyte 12 Sutherland RD, Keating A, Nayar R et al. Sensitive detection colony-stimulating factor (G-CSF) for mobilising peripheral and enumeration of CD34ϩ cells in peripheral and cord blood blood stem cells: risk of mobilizing clonal myeloma cells in by flow cytometry. Exp Hematol 1994; 22: 1003–1010. patients with bone marrow infiltration. Br J Haematol 1994; 13 Brown RD, Pope B, Luo X-F et al. The oncoprotein pheno- 86: 180–182. type of plasma cells from patients with multiple myeloma. 5 Mariette X, Fermand J-P, Brouet J-C. Myeloma cell contami- Leuk Lymphoma 1994; 16: 147–156. nation of peripheral blood stem cell grafts in patients with 14 Terstappen LWMM, Johnsen S, Segers-Nolten IMJ, Loken multiple myeloma treated by high-dose therapy. Bone Marrow MR. Identification and characterization of plasma cells in nor- Transplant 1994; 14: 47–50. mal human bone marrow by high-resolution flow cytometry. 6 Dreyfus F, Ribrag V, Leblond V et al. Detection of malignant Blood 1990; 76: 1739–1747. B cells in peripheral blood stem cell collections after chemo- 15 Wijdenes J, Vooijs WC, Clement C et al. A plasmocyte selec- therapy in patients with multiple myeloma. Bone Marrow tive monoclonal antibody (B-B4) recognizes syndecan-1. Br J Transplant 1995; 15: 707–711. Haematol 1996; 94: 318–323. 7 Corridini P, Astolfi M, Ladetto M et al. High-dose (HDS) 16 Hata H, Xiao H, Petrucci MT et al. Interleukin-6 expression chemotherapy in multiple myeloma. Residual tumor cells are in multiple myeloma: a characteristic of immature tumor cells. detectable in PBPC and BM cell harvests and after single or Blood 1993; 81: 3357–3364. double autografting. Blood 1995; 86: (Suppl. 1): 206a. 17 Joshua D, Petersen A, Brown R et al. The labelling index 8 Gazitt Y, Reading CC, Hoffman R et al. Purified CD34ϩLin- of primitive plasma cells determines the clinical behaviour of ϪThyϩ stem cells do not contain clonal myeloma cells. Blood patients with myelomatosis. Br J Haematol 1996; 94: 76–81. 1995; 86: 381–389. 18 Billadeau D, Van Ness B, Kimlinger T et al. Clonal circulat- 9 Willems P, Croockewit A, Raymakers R et al. CD34 selection ing cells are common in plasma cell proliferative disorders: a from myeloma peripheral blood cell autografts contain comparison of monoclonal gammopathy of undetermined sig- residual tumour cells due to impurity, not to CD34ϩ myeloma nificance, smouldering multiple myeloma and active mye- cells. Br J Haematol 1996; 93: 613–622. loma. Blood 1996; 88: 289–296. 10 Harada H, Kawano MM, Huang N et al. Phenotypic difference 19 King MA, Radicchi MA. Monitoring circulating B cells in of normal plasma cells from mature myeloma cells. Blood patients with multiple myeloma at diagnosis or in plateau 1993; 81: 2658–2663. phase: how prevalent is light chain isotype suppression? Br J 11 San Miguel JF, Almeida J, Ocqueteau M, Orfao A. Detection Haematol 1992; 81: 218–222.