1. ICCS e-Newsletter- CSI Case Spring 2015
Amr Rajab and Anna Porwit
Flow Cytometry Laboratory, Department of Laboratory Medicine, University Health Network, Toronto General Hospital, Toronto, ON, Canada

2. Clinical History and Specimens Submitted
79 year old male diagnosed with splenic marginal cell lymphoma 2010.
Bone marrow aspirate and biopsy were performed due to suspect progression (increasing splenomegaly and some lymphadenopathy).

3. Peripheral Blood Cell Counts (CBC)
Hb: g/L
MCV: 92
WBC: x10e9/L
Plt: x10e9/L
Neutrophils: 2.1 x10e9/L
Eosinophils: 0.2 x10e9/L
Lymphocytes: 3.2 x10e9/L
Monocytes: 0.6 x10e9/L
Basophils: 0 x10e9/L
Abnormal cytoplasm-rich lymphocytes are present in blood, some have villous cytoplasm, no increase of plasma cells in blood

4. Bone Marrow Aspirate Sample Quality: Adequate Fragments: Present
Cellularity: Moderate
Erythropoiesis: Increased Left shifted
Erythroid Morphology: Normal
Granulopoiesis: Adequate for age
Granulocyte Morphology: Normal
Megakaryopoiesis: Present
Megakaryocyte Morphology: Normal
Lymphocytes: Increased
Plasma Cells: Increased, some binucleated forms seen
Iron Status: Normal
Ringed Sideroblasts: Absent
Clot Section: Confirms findings in smears: variable cellularity, clusters of lymphocytes and plasma cells noted

5. Bone Marrow Differential
Number of cells counted: 200
Blasts: %
Promyelocytes: %
Myelocytes: 7 %
Metamyelocytes: 10 %
Neutrophils: %
Eosinophils: 1 %
Basophils: 0 %
Monocytes: 2 %
Lymphocytes: 26 %
Plasma cells: 13 %
Erythroblasts: 26 %
Plasma cells in BM smears

6. Flow Cytometric Analysis
Flow cytometric analysis was performed on bone marrow aspirate.
Data acquisition was performed on a 10-Color Navios Flow cytometer from Beckman Coulter.
Flow cytometric data was analyzed using Kaluza software from Beckman Coulter.
B Tube
Multiple Myeloma
KAPPA FITC
cyt. KAPPA FITC
LAMBDA PE
cyt. LAMBDA PE
CD19 ECD
CD56 ECD
CD38 PC5.5
CD138 PC5.5
CD20 PC7
CD34 PC7
CD34 APC
CD117 APC
CD23 APC-A700
CD19 APC-A700
CD10 APC-A750
CD38 APC-A750
CD5 PB
CD20 PB
CD45 KO

7. Flow Cytometric Analysis
Surface staining
Wash cells x 2 with warm PBS
Resuspend cells in 1% BSA in PBS
Adjust cells to 5 x109 /L
Add Abs cocktail to 100ul (5 x105 cells) of sample
Incubate for 15 minutes in dark at RT.
Lyse with 1mL VersaLyse (BC ref. IM3648) plus 25 uL IOTest 3 Fixative (BC ref. IM3515)
Wash and suspend in 1 mL PBS and 12.5 uL IOTest 3 Fixative (BC ref. IM3515)

8. Intracellular staining
Flow Cytometric Analysis
Intracellular staining
Wash cells x 2 with warm PBS
Resuspend cells in 1% BSA in PBS
Adjust cells to 5 x109 /L
Add surface Abs cocktail to 50ul of sample
Incubate for 15 minutes in dark at RT.
Add 100 mL of IntraPrep Reagent ‘1’ (BC ref. A07803) to each tube
Vigorously vortex tubes.
Incubate at room temperature for 15 minutes, in the dark.
Wash and suspend in 50uL PBS
Add 100 mL IntraPrep Reagent ‘2’ (BC ref. A07803) to each tube and GENTLY mix.
Incubate at room temperature, in the dark, for 5 minutes.
Add cytoplasmic antibodies.
Gently vortex tubes
Incubate at room temperature, in the dark, for 15 minutes
Wash and Re-suspend cells in ~500 uL of PBS

9. Flow Cytometric Data Analysis – B Tube
Plasma cells
Non-viable cells are gated using forward (FSC) vs. side scatter (SSC) gating (gate Debris, plot A). All consequent dot plots are created of Boolean gate (Living cells) excluding Debris.
Various cell subpopulations are mapped on CD45/Side scatter (SSC) plot (B)
Gray: Granulocytes
Yellow: Monocytes
Cyan: Blasts
Blue: Plasma cells
Red & Purple: Lymphocytes

10. Flow Cytometric Data Analysis – B Tube
B-cells are enumerated using CD19+/SSC dot plot (A)
Kappa vs. Lambda plot shows Lambda light chain restriction (Red) (B).

11. Flow Cytometric Data Analysis – B Tube
The B-cell population (Red dots, 93% of B-cells) is negative for CD10, CD5, CD23, and CD34 (not shown) but brightly positive CD20 and CD38 (compared to background normal B cells – Green dots, 3% of CD19+). A small population of CD10+ B-cell precursors was noted (Cyan dots, 4% of CD19+)

12. Flow Cytometric Data Analysis – B Tube
T-cells are enumerated using CD5 (A&B).
CD34+ cells are enumerated out of the total living cells (C).
Plasma cells are enumerated using CD38++/SSC dot plot (D). Increased plasma cells prompted adding of the multiple myeloma panel. D

13. Flow Cytometric Data Analysis – MM Tube
Debris and doublets were removed using sequential gating (A &B).
PCs were gated on CD38/CD138 plot (C) and viable PCs on FSC/SSC plot (D). Note many PC in the area for apoptotic cells (Myeloma panel performed 24 hours after aspiration).

14. Flow Cytometric Data Analysis – MM Tube
PCs are positive for cytoplasmic Kappa, CD56 and CD117, but negative with CD19 and CD20 (A, B & C)
Eight-parameter Radar plot (D): Kappa and lambda are displayed horizontally from East to West. Antigens expressed on normal PCs are displayed vertically towards the North and those expressed aberrantly on clonal PCs vertically towards the South. D

15. Immunophenotypic Findings
BMA:
9% of B-cells positive for lambda, CD19, CD20, CD38, negative for CD5, CD10, CD23, and CD11c (not shown, evaluated in T-cell tube)
4% kappa+ monotypic plasma cells population positive for CD138, CD38++, CD56+, CD117+ and negative for CD19, CD20 and CD45.
PB (received separately): 21% B-cells positive for lambda, CD19, CD20, CD38, negative for CD5, CD10, CD23, and CD11c.

16. Morphology Findings
Bone marrow smears showed up to 13% plasma cells near fragments, less in other more diluted areas
Bone marrow biopsy confirmed presence of CD20+ B-cell infiltrates and an increase of intra sinusoidal B-cells.
Clusters of plasma cells with positivity for kappa, CD56 and CD117 were noted.

17. CD20+ infiltrates in BM biopsy

18. Clusters of CD138+ plasma cells

19. Diagnosis: Bone marrow involvement by splenic marginal zone lymphoma (lambda+) and plasma cell neoplasm (kappa+).

20. Discussion
Approximately 70 patients with concomitant involvement of the bone marrow by plasma cell myeloma and B-cell lymphoma (mainly of CLL type) have been described in the literature (1,2). In most cases B-cell lymphoma preceded plasma cell myeloma.
Two processes may arise independently from the same stem cell (3) or arise from different B cells coincidentally (4).
Splenic marginal cell lymphoma may show marked plasmacytic differentiation and molecular study may be necessary to identify the 2 monotypic populations if the light-chain restriction is the same for both monotypic processes (5). In our case this scenario is unlikely since the plasma cell neoplasm showed kappa light chain restriction. Previous studies have shown that lambda loci in kappa-producing neoplastic B-cells are largely unrearranged while kappa loci in lambda producers are often rearranged and inactivated by rearrangements of the kappa-deleting element (6,7). Thus a possibility of kappa+ plasma cell neoplasm developing as progression of lambda+ B-cell lymphoma is not likely.

21. References
Alley CL, Wang E, Dunphy CH, Gong JZ, Lu CM, Boswell EL, Burchette J, Lagoo AS. Diagnostic and clinical considerations in concomitant bone marrow involvement by plasma cell myeloma and chronic lymphocytic leukemia/monoclonal B-cell lymphocytosis: a series of 15 cases and review of literature. Arch Pathol Lab Med Apr;137(4):
Hussein S, Gill K, Baer LN, Hoehn D, Mansukhani M, Jobanputra V, Bhagat G, Alobeid B. Practical diagnostic approaches to composite plasma cell neoplasm and low grade B-cell lymphoma/clonal infiltrates in the bone marrow. Hematol Oncol Jan3 (E-Pub)
Saltman DL, Ross JA, Banks RE, Ross FM, Ford AM, Mackie MJ. Molecular evidence for a single clonal origin in biphenotypic concomitant chronic lymphocytic leukemia and multiple myeloma. Blood. 1989;74(6):2062–2065.
Pereira Wde O, Bacal NS, Correia RP, Kanayama RH, Veloso ED, Borri D, Hamerschlak N, Campregher PV. Development of plasma cell myeloma in a B-cell chronic lymphocytic leukemia patient with chromosome 12 trisomy. BMC Res Notes Oct 29;6:433.
Molina TJ, Lin P, Swerdlow SH, Cook JR. Marginal zone lymphomas with plasmacytic differentiation and related disorders. Am J Clin Pathol ; 136(2): 211–225.
Korsmeyer SJ, Hieter PA, Sharrow SO, Goldman CK, Leder P, Waldmann TA. Normal human B cells display ordered light chain gene rearrangements and deletions. J Exp Med Oct 1;156(4):
Bräuninger A, Goossens T, Rajewsky K, Küppers R. Regulation of immunoglobulin light chain gene rearrangements during early B cell development in the human. Eur J Immunol Dec;31(12):3631-7