The Bone Marrow Plasma Cell Labeling Indexby Flow Cytometry

Belinda Pope,* Ross Brown, John Gibson, and Doug JoshuaInstitute of Haematology, Royal Prince Alfred Hospital, Sydney, New South Wales, Australia

The bone marrow plasma cell labeling index is the most important prognostic indicator for patients withmultiple myeloma. Traditionally, this test has been performed as a two color immunofluorescent microscopetechnique which is time consuming and requires a degree of subjectivity in its interpretation. We haveassessed various adaptations of this method to flow cytometry. A bromodeoxyuridine method has beencompared with a propidium iodide DNA method to detect cells in S phase and CD38-FITC has been comparedwith CD38-FITC 1 CD138-FITC and CD38-biotin 1 streptavidin FITC to identify plasma cells. The meanchannel fluorescent intensity of the plasma cell peaks for each of these markers was 12.7, 17.4 and 35.3respectively demonstrating the superiority of CD38-biotin 1 streptavidin FITC. Analysis after propidiumiodide staining provided a good correlation with the slide technique (r 5 0.71; P F 0.0001) but thebromodeoxyuridine method did not correlate with the slide method (r 5 0.09; P 5 NS). The labeling indexvalues obtained from either of the flow methods were greater than the microscopic method. Thus a labelingindex of G4% will replace the traditional G1% threshold for identifying patients with a significantlyincreased labeling index. The advantages of the new method are that it takes less time to perform, is moreobjective and provides additional data on ploidy and cell cycle status. Cytometry (Comm. Clin. Cytometry)38:286–292, 1999. r 1999 Wiley-Liss, Inc.

Key terms: myeloma; DNA ploidy; flow cytometry; prognosis

Prognostic factors for patients with multiple myelomaare based on a measurement of either the tumor bulk, thepresence of a genetic abnormality, the host response orthe proliferative activity of the tumor (10). Of the latter,the plasma cell labeling index is acknowledged as the mostimportant parameter and has been extensively used inboth research and diagnostic laboratories either to deter-mine prognosis (1,5,6), to detect escape from plateaudisease into progressive disease (1,3–6,9,24) or to helpdifferentiate between smoldering myeloma and monoclo-nal gammopathy of undetermined significance (4,15). In amultivariate analysis, the combination of plasma cell label-ing index and serum b2-microglobulin provided the mostprognostic significance while other parameters lost anysignificance evident in univariate analysis (5).

The plasma cell labeling index has been traditionallydetermined using a microscope immunofluorescent assay.This technique is labor-intensive and like other micro-scope enumeration assays suffers from a lack of sensitivityespecially when there are a limited number of cellspresent. Several groups, including our own, have at-tempted to adapt the microscope bromodeoxyuridineassay to the flow cytometer (11,19). Although thesemethods have proven to be technically feasible, themethodology is complex and the results have failed toprovide a correlation with the traditional microscope

method. An alternative approach has been to label the Sphase cells with propidium iodide and perform a DNA cellcycle analysis on the plasma cell population (17,18).

We have studied several possible modifications of theplasma cell labeling index method in an attempt todevelop the most suitable flow cytometric assay for aroutine diagnostic laboratory. In particular we have com-pared the use of CD38, CD138 and cytoplasmic immuno-globulin (cIg) with various conjugates and amplificationsteps to identify plasma cells and then compared DNAlabeling using propidium iodide (PI) with the incorpora-tion of bromodeoxyuridine to determine the percentage ofS phase cells. This report presents the results of theseinvestigations and outlines the methodology used in ourroutine diagnostic laboratory.

MATERIALS AND METHODSPatient Samples and Preparation

Bone marrow samples were obtained after informedconsent from patients with multiple myeloma who pre-

*Correspondence to: Belinda Pope, Institute of Haematology, RoyalPrince Alfred Hospital, Missenden Rd., Camperdown, NSW 2050,Australia.

Received 19 January 1999; Accepted 26 July 1999

Cytometry (Communications in Clinical Cytometry) 38:286–292 (1999)

r 1999 Wiley-Liss, Inc.

sented for routine assessment of their disease. Bonemarrow samples were washed with Hanks Balanced SaltSolution (HBSS; Trace Biosciences, Castle Hill, Australia)and resuspended in RPMI-1640 media (CSL, Melbourne,Australia) containing 10% FCS (ICN Biomedicals, Costa),aspirated through a 26G needle to obtain a single cellsuspension and mononuclear cells were isolated using aFicoll-paque gradient (Pharmacia Fine Chemicals, Uppsala,Sweden) and then washed in HBSS.

The Bromodeoxyuridine Labeling Index Microscope Assay

Bone marrow mononuclear cells were incubated inRPMI-1640 and 10µM 58-bromo-28deoxyuridine (BrdUrd,Boehringer Mannheim, Penzburg, Germany) at 37°C for1h. Cytospin preparations were prepared and fixed in 95%ethanol for 10 min and then incubated with 50ul of 1/10diluted mouse anti BrdUrd (Boehringer Mannheim) at37°C for 1h. The slides were washed in phosphate-buffered saline (PBS) and incubated a further 30 min with50ul 1/50 RITC-labeled sheep anti-mouse immunoglobulinIg (Silenus, Hawthorn, Australia) and either 50ul 1/50FITC-labeled sheep anti-human Ig (Silenus) or 50ul 1/100FITC-labeled goat anti-human kappa or lambda (Coulter,Hialeah, FI). Using a fluorescent microscope, positive cellshave a red nucleus and green cytoplasm. Where possible500 plasma cells per slide were counted but in all reportedcases a minimum of 200 plasma cells were evaluated.

The Bromodeoxyuridine Labeling IndexFlow Cytometric Assay

BrdUrd treated cells were permeabilized and fixed withperiodate lysine paraformaldehyde solution at 210°C for15 min (2), washed twice in PBS and then 13106 cells wasadded to 50ul of murine anti BrdUrd diluted 1/10 (Boeh-ringer Mannheim) at 37°C for 30 min. After washing withPBS, 50ul of a 1/50 dilution of RED 613 conjugated goatanti-mouse Ig (GibcoBRL, Gaithersburg, MD) was addedfor 30 min at 4°C. The cells were washed with PBS,incubated with normal mouse serum for 5 min and thenlabeled with 20ul anti CD38-PE (Becton Dickinson, Moun-tain View, CA) and 10ul anti CD45-FITC (Dako, Glostrup,Denmark). Cells were analysed on a Coulter EPICS flowcytometer (Coulter). A relevant control tube with isotypecontrol matched antibodies was also assayed.

The DNA Flow Cytometric Assay

The flow cytometric assay involved 3 tubes per patient.Tube 1 contained 10ul IgG1 FITC (Becton Dickinson) and10ul CD38 PE (Becton Dickinson). Tube 2 contained 10ulCD138 FITC (B-B4) (Immuno Quality Products, Gro-ningen, The Netherlands) and 10ul CD38 PE (BectonDickinson). The 3rd tube contained 5ul CD38 Biotin(Caltag Laboratories, Burlingame, CA). Each tube received1–3 3106 mononuclear cells in 100ul, spun briefly to mixcells and antibodies and then vortex to resuspend. Tubeswere incubated at room temperature for 10 min. Tubes 1and 2 were lysed with 1ml of lysing buffer (0.16Mammonium chloride, 0.01M sodium bicarbonate and

0.0001M EDTA at pH 7.3) before washing with 1ml PBSand resuspended in 0.5ml PBS before storage in the darktill ready to read. Tube 3 was washed once with PBS,before adding 50ul of 1/50 streptavidin FITC. It wasincubated at room temperature for 10 min and washedonce in PBS. While gently vortexing, 100ul of DNA-Preplysing and permeabilizing reagent (Coulter) and then 2mlDNA-Prep Stain (Coulter) was added and the sample wasallowed to incubate for 15 min at room temperature. Cellswere analysed on a Coulter EPICS XL flow cytometer. Toimprove the accuracy of the results every attempt wasmade to collect as many plasma cell counts as possible.Multicycle for Windows was used to analyse the DNAhistogram.

Reproducibility Testing

The reproducibility of the slide based BrdUrd methodand the flow cytometric DNA assay was tested by repeatingboth methods 6 times on three patient samples represent-ing a low, intermediate and high assay value. Five thousandto 10,000 events were analysed on the flow cytometer.Data was reanalysed on System II (v2.0) software (Coulter)with stops being placed at various cell number intervals totest reproducibility at different cell numbers. One thou-sand cells were counted manually on a fluorescent micro-scope with results being recorded at regular 100 cellintervals. The results were analysed for standard deviation(SD), mean and coefficient of variation (CV).

RESULTSIdentification of Plasma Cells by Flow Cytometry

Plasma cells were identified by high intensity CD38(CD3811) as previously described (8,13,15,16,23). While

FIG. 1. Identification of plasma cells in bone marrow of a patient withmultiple myeloma using plasma cell markers CD38 and CD138. Plasmacells are in quadrant 2. These cells are high intensity CD38 and CD138positive.

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CD38 is also an activated T cell and B cell marker, there is aclear peak of cells with high intensity CD38 expression,which are plasma cells. Cytoplasmic immunoglobulin(cIg), cytoplasmic kappa or cytoplasmic lambda, andCD138 have been used to confirm that these cells areplasma cells (16). In another study (13) CD3811 cellswere sorted and stained for a panel of antibodies. It wasfound that 9664% were plasma cells and were negative forCD2, CD3, CD10 and CD16 antigens. In this study aseparate tube containing CD38 and CD138 (syndecan-1, aspecific plasma cell marker) without propidium iodidewas used to estimate the percentage of plasma cells forevery patient in the study (Fig. 1).

The Bromodeoxyuridine Method by Flow Cytometry

In preliminary studies we simply adapted the traditionalslide method to flow cytometry. Details of these studieshave been published previously (10). Figure 2b shows anexample of a dot plot of bromodeoxyuridine against CD38.Figure 2a shows the appropriate negative control for thissample. This technique enabled more plasma cells to becounted and an objective cut off to be established for eachsample based on the fluorescence intensity of controls.The mean labeling index of bone marrow plasma cells bythe bromodeoxyuridine flow method was significantlyhigher (mean 5 2.7%) than by the slide method (mean 50.6%) (t55.2; P,0.01; n565) and there was no significantcorrelation between the slide and bromodeoxyuridineflow method (Fig. 3; r50.09; P5NS; n565).

Optimization of Staining for Plasma Cellsin the Presence of PI

We evaluated modifications of the propidium iodidemethod in an attempt to develop a flow cytometric assay,which had a better correlation with the traditional slide-based method. A series of studies were undertaken toidentify the best method to identify plasma cells, whichare simultaneously stained with propidium iodide. In

FIG. 3. The correlation between labeling index determined by thetraditional slide technique and a bromodeoxyuridine flow method (r50.09; P5NS).

FIG. 2. Bromodeoxyuridine labeling index. Negative control (a) and Dot plot of CD38 vs. bromodeoxyuridine (b). The plasma cell labeling index is thepercentage of plasma cells that are bromodeoxyuridine positive.

288 POPE ET AL.

particular, staining with CD38 FITC, CD38 FITC plusCD138 FITC and CD38 biotin plus streptavidin FITC werecompared. The mean channel fluorescent intensities ofthese reagents were 12.7, 17.4 and 35.3 respectivelyindicating that CD38 biotin plus streptavidin FITC pro-vided the clearest discrimination of the plasma cell popula-tion and the highest mean channel fluorescence intensity.Figure 4a,b,c illustrates the CD38 histograms from onepatient. Permeabilization and staining of cells with prop-idium iodide decreased the mean channel fluorescence ofplasma cells stained with CD38 biotin 1 streptavidin from91 to 35.3. Despite variations in staining intensity, thepercentage of plasma cells was not significantly differentfor each of the three methods. Figure 5 shows a dot plot ofCD38 vs. PI with high intensity CD38 gated for separate

DNA analysis. Cytoplasmic immunoglobulin was not con-sidered as a plasma cell marker for this study as otherstudies (13) have shown that CD38 gave a much betterseparation between the plasma cell peak and other cells.

Comparison of the DNA Method With the TraditionalSlide Method

Reportable results were obtained for 44 out 54 samplesby flow cytometry and 42 out of 54 samples by the slidetechnique. The primary reason for failing to completeeither assay was insufficient plasma cells. In the slide basedtechnique, although 1000 cells were counted if possible, aminimum of 200 plasma cells with a mean of 741 cellswere counted. The minimum number of cells required forDNA flow cytometry analysis varied and for each samplewas determined by the Multicycle DNA cell cycle analysisprogram. The program used a complex algorithm toanalyse wether the data was acceptable for S phaseestimation. Only samples, which passed the softwarecriteria, were used in this study. The numbers of plasmacells counted ranged from 522 to 19795 with a mean of4218. The CVs of the non-plasma cell diploid peak rangedfrom 2.5–7.4 (mean 4.7) indicating acceptable CVs accord-ing to the clinical DNA guidelines (20). The quality ofsamples was excellent with the mean background aggre-gates and debris (BAD) equalling 0.3% (range 0–2.1). Thusfor 81% of the samples studied, DNA analysis using theMulticycle for Windows program allowed an objectivedetermination of the number of CD3811 cells in S phase(Fig. 6a). Determination of the number of both aneuploidand diploid plasma cells in S phase was often difficult asthe 2 graphs overlaid each other (Fig. 6b). However formost patients an average S phase result for both diploidand aneuploid plasma cells was able to be calculated usingMulticycle for Windows. Figure 7 illustrates the correla-tion between the DNA flow cytometric assay and thetraditional slide technique (r50.71; P,0.0001; n5 39).Although the flow method provided values which werehigher than the slide method, there was an excellent

FIG. 5. Dot plot of CD38 vs. PI. Region A is gated to obtain the cellcycle analysis for plasma cells.

FIG. 4. Identification of plasma cells from the same patient using CD38FITC (a), CD38FITC 1 CD138FITC (b) and CD38 biotin 1 streptavidin FITC (c).

289PLASMA CELL LABELING INDEX

agreement in the interpretation of the data (x25 27.5; P,0.0001). If a cut off of 4% is used for the flow method and1% for the manual slide method then 37 out of 39 sampleswere either both high or normal. Two samples gave a highflow result and a normal slide result. One sample had adramatically big difference between the two methods witha DNA flow cytometric labeling index of 10.3% and slidelabeling index of 0.1%. For this patient 70%, of the cellswere plasma cells and over 11,000 plasma cells were

counted for S phase analysis. The patient was in progres-sive disease at diagnosis according to other myelomastaging tests. This suggests that the slide based labelingindex suffered technical difficulties.

Reproducibility of Labeling Index Assay

Table 1 shows the means and CVs of the flow cytometricand the slide based plasma cell labeling index. While insome instances it is possible to obtain a result for the flowcytometric labeling index with as low as 500 plasma cells,in these three examples a minimum cell count of 1,500cells was required to get an adequate cell cycle fit by theMulticycle for Windows program. Results for the averagenumber of cells and maximum number of cells analysedfor both tests are also included. There was a definiteimprovement of CVs obtained for the flow cytometricplasma cell labeling index compared to the slide basedplasma cell labeling index even at a low cell number. CVsat 5,000 cells were excellent with values ranging from2.67% to 12.70% in contrast to the CV of the slide basedtechnique which was never less than 50%. Patient 3 had ahigh flow cytometric labeling index but a lower slideplasma cell labeling index than expected. Slides from thispatient were difficult to interpret because of a high level ofsubjectivity.

Detection of DNA Aneuploidy

To detect DNA aneuploidy, non-plasma cells (CD38-)were used as an internal diploid control. A DNA aneuploidpeak was defined as a plasma cell peak with a meanchannel fluorescence intensity at least 10% higher than thenon-plasma cell control and two distinct peaks werepresent (20). Of 52 samples, 44% had a DNA aneuploidplasma cell population of greater DNA content. None of

FIG. 7. The correlation between labeling index determined by thetraditional slide method and the propidium iodide flow method (r50.71;P,0.0001).

FIG. 6. Examples of diploid (a) and aneuploid (b) DNA histograms of CD3811 cells from 2 different patients.

290 POPE ET AL.

the 52 samples tested had a DNA aneuploid plasma cellpopulation with less than normal DNA content.

DISCUSSION

The bone marrow plasma cell labeling index assay hastraditionally been performed as a manual enumerationmicroscopic assay. This study reports a series of differentmodifications, which were evaluated during an attempt toadapt this assay to flow cytometry. A number of QualityAssurance programs have demonstrated that microscopicenumeration assays are prone to significant errors whichcan be avoided when automated counting instruments areused. For example the accuracy of reticulocyte counting isgreatly improved when performed on a modern auto-mated cell counter (12). This study demonstrates that thebone marrow labeling index is another example of how anassay can be improved by automating the counting proce-dure. After many different attempts to determine theoptimal conditions to perform this assay, it was found thata combination of DNA analysis after propidium iodidestaining and the use of biotinylated CD38 staining plusstreptavidin FITC proved to be the best. This method takesadvantage of an amplification of the signal with streptavidin-FITC. The reproducibility of the test has improved fromthe BrdUrd slide method and has the added advantage ofbeing faster, a larger number of plasma cells can becounted and the test is more objective. The BrdUrd assayon the flow did not correlate with the slide BrdUrdlabeling index which could be due to the poor sensitivityof the slide based technique or increased non specificbinding of the flow BrdUrd assay.

Previous studies from our group and others have clearlyidentified the use of CD38hi as a marker for plasma cells inthe bone marrow of patients with myeloma (2,10,21).These cells exhibit clonal light chain expression and areCD32, CD142, CD332 (10,13). It has also been previ-ously been demonstrated that any rise in the bone marrowlabeling index is due to a rise in the labeling index of theprimitive plasma cell (CD38hi, CD45hi) population (10).Thus a further refinement of the bone marrow plasma cell

labeling index assay would be to analyse the number ofcells in S phase in the CD38hi CD45hi population.

Whether or not the additional data obtained from thedetermination of plasma cell DNA ploidy has any clinicalsignificance is not clear. One report has suggested thatpatients with hyperploidy (7) have a poor prognosis whilein other studies either hypoploidy (21) was associatedwith a poor prognosis or hyperploid patients had a goodprognosis (18,21,22). These apparent discrepancies wouldsuggest that the identification of ploidy per se has limita-tions. More precise data using either cytogenetic or FISHtechniques have subsequently been shown to provideexcellent prognostic information (14).

This modification of the bone marrow plasma celllabeling index is well suited to the work flow of a busydiagnostic laboratory which performs flow cytometry. Theoptimum method as described here is easier to performthan the manual slide method and correlates well with thetraditional method. Analysis of cell cycle status afterpropidium iodide staining has proved to be a far bettermodification for flow cytometry than an adaptation of thebromodeoxyuridine uptake method, which lacks correla-tion with the traditional method.

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Table 1Flow Cytometric Plasma Cell Labeling Index*

Numberof cells

Patient 1 Patient 2 Patient 3Mean CV Mean CV Mean CV

1,500 2.23 38.1 3.95 16.3 9.50 3.54,217 2.05 17.4 3.33 7.0 8.83 5.25,000 2.02 12.7 3.22 5.4 9.07 2.7

10,000 1.80 14.1 n.d. n.d. n.d. n.d.

*n.d., not done.Slide Plasma Cell Labeling Index

Numberof cells

Patient 1 Patient 2 Patient 3Mean CV Mean CV Mean CV

200 0.20 137.0 0.92 41.1 0.83 82.0741 0.22 94.8 0.92 37.5 0.94 65.2

1,000 0.26 58.3 0.95 23.8 0.82 55.0

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