Bone Marrow Transplantation, (1997) 20, 205–210 1997 Stockton Press All rights reserved 0268–3369/97 $12.00 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 (CD3811) 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- CD341Lin2Thy1 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 CD3811/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 CD341 on a magnetic column fol- patients were immature (CD451/CD4511) rather than lowed by flow activated cell sorting, no malignant cells mature (CD452). 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 (CD452) were analysed but only in 9% of harvests cells. Furthermore, these methods are not able to quantitate when immature (CD451/CD4511) 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- CD191 cells. There was concordance between the ratio metric assay to analyse light chain restriction in the plasma of malignant (CD192/CD561) to normal (CD191/ cell population in PBSC harvests to determine whether light CD562) 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 mg/kg/day) or GM-CSF (5 mg/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 3 106) med as part of the routine investigations. Informed consent were placed in tubes, primary antibodies IgG1 (50 ml) and was obtained from all patients for all procedures. CD45 (10 ml) were added to the cells and incubated for 30 min at 4°C. Cells were washed in PBS and the second- ary antibody, 50 ml (1/50 dilution in PBS) of GaM 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 3 109/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 ml), or anti-lambda FITC USA). Where possible studies were performed on each m m 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 ml), then incubated for 15 min at room a small aliquot of cells was quickly thawed at 37 C and m 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 3 106 CD341 Coulter Epics XL with XL software. At least 80 000 events cells/kg. Yields of CD341 cells/kg ranged from 0.03 to 6.6 were collected in listmode data. All cells except debris and 3 106 and CD341 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 CD341 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, (CD3811) as previously described.10,13,14 Cytoplasmic USA); anti-CD38 FITC (Caltag, San Francisco, CA, USA); immunoglobulin (cIg), cytoplasmic kappa, cytoplasmic 9 a 9 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); GaM 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 3 106) were stained with permeabilisation kit. We compared relative fluorescence anti-CD38 PE (20 ml) and B-B4 FITC (10 ml) for 30 min intensity (RFI) on bone marrow cells from a patient with on ice. Cells were then washed twice and resuspended in IgGk myeloma and obtained the following results for the 200 ml of PBS, ready for analysis on flow cytometer. Cells RFI of the kappa1ve cells: PLP = 53.4, PermeaFix = 258.5, were also stained with anti-ICB-61 (50 ml) and incubated Caltag Fix and Perm = 937.1, STF = 160.5 and Harlan on ice for 30 min.