The document outlines the development and validation of a 10-color, 14-antibody flow cytometry screening tube designed to identify lymphoproliferative disorders and myelodysplasia in bone marrow samples. It covers phases of optimization in sample preparation, antibody titration, and instrument setup, as well as results indicating high concordance in detecting cell populations and aberrant expressions. Future developments aim to enhance accuracy and expand the panel for improved immunophenotyping capabilities.

1. Ten-Color, 14 Antibody Flow cytometry
(FCM) Screening Tube for
Lymphoproliferative Disorders and
Myelodysplasia (MDS) – Related Changes in
Bone Marrow Samples
EMAIL: Amr.Rajab@uhn.ca
Amr Rajab, BSc, MLT, QCYM (ASCP)
Clinical Flow Cytometry Laboratory
Laboratory Medicine Program
Toronto General Hospital/University Health Network
Ontario, Canada

2. TOPICS
• Development
• Validation for LL/AL screening (Phase 1)
 Optimization of sample preparation
 Titration of antibodies
 Verification of cocktail stability
 Instrument setup and compensation
 Comparison study
 Analysis
 Cases
• Validation for MDS Screening (Phase 2)
• Current & Future Developments

3. GOAL
 Develop a 10 Color, 14 antibody Flow
cytometry (FCM) screening tube that
correctly identifies lymphoproliferative
disorders and myelodysplasia (MDS) –
related changes in bone marrow samples

4. FLOW CYTOMETRY LAB - UHN
Section A Section B
Tests performed AL & LL CD34, CD4/CD8 & PNH
Number of samples- 2014
Referred in samples
8143
40%
7174
40%
Instruments Two 3L 10C Navios Two 1L 5C FC500
Number of Technologists 7 1
Total number of samples processed during 2014= 15317

5. Why the new screening tube (ST)? (1)
1. To be used for immunophenotyping of
hypocellular samples, dry taps, FNAs, and
body fluids (e.g. CSF).
2. To be used for “referred in” samples (mainly
bone marrow aspirates and peripheral blood
samples) not accompanied by sufficient clinical
information, provisional diagnosis, or prepared
smears (50% of referred samples)

6. Why the new ST? (2)

7. Why the new ST? (3)
• These samples were usually screened using the
UHN lymphoma protocol (2 tubes, 20 antibodies).
• The outcome determined how much further
investigation was needed.
• 50% do not show any pathological population.
Tube FITC PE ECD PC5.5 PC7 APC APC-
AF700
APC-
AF750
PB KO
B-cell kappa lambda CD19 CD38 CD20 CD34 CD23 CD10 CD5 CD45
T-cell CD57 CD11c CD8 CD3 CD2 CD56 CD7 CD4 CD5 CD45

8. Similar approach has been applied by other groups in 4-
color, 6-color, 8-color and 10-color settings –
Focusing on lymphoid population
• Quijano S, …etc. Spanish Group for the Study of CNS Disease in NHL.Identification
of leptomeningeal disease in aggressive B-cell non-Hodgkin's lymphoma: improved
sensitivity of flow cytometry. J Clin Oncol. 2009 Mar 20;27(9):1462-9.
• Preijers FW…etc. B. OMIP-010: a new 10-color monoclonal antibody panel for
polychromatic immunophenotyping of small hematopoietic cell samples. Cytometry A.
2012 Jun;81(6):453-5.
• Costa ES, …etc. A new automated flow cytometry data analysis approach for the
diagnostic screening of neoplastic B-cell disorders in peripheral blood samples with
absolute lymphocytosis. Leukemia. 2006 Jul;20(7):1221-30
• van Dongen JJ,…etc. EuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708).
EuroFlow antibody panels for standardized n-dimensional flow cytometric
immunophenotyping of normal, reactive and malignant leukocytes. Leukemia. 2012
Sep;26(9):1908-75.
• Hedley BD, Keeney M, Popma J, Chin-Yee I. Novel lymphocyte screening tube
using dried monoclonal antibody reagents. Cytometry B Clin Cytom. 2015 May 4

9. SCREENING TUBE
10 COLORS, 14 ANTIBODIES
• Enumerate Major Populations:
• Blasts
• B- lymphocytes, T- lymphocytes,
• NK cells
• Monocytes
• Neutrophils
• Enumerate Sub-populations:
• T-Helper cells, T- Suppressor cells
• CD34+/CD19+ B- cell progenitors
• CD34+/CD33+ myeloid progenitors
• CD33+/CD10+ mature granulocytes
• B-cell clonality status: Kappa Lambda
• B-cell maturation status CD20/CD10
expression.

10. VALIDATION FOR
LYMPHOMA/LEUKEMIA (PHASE 1)
• Optimization of sample preparation
• Titration of antibodies
• Verification of cocktail stability
• Instrument setup and compensation
• Comparison study

11. VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1)
(Selection of lysing solution)

12. VALIDATION FOR
LYMPHOMA/LEUKEMIA (PHASE 1)
1. Sample preparation
• Wash cells x 2 with warm PBS
• Resuspend cells in 1% BSA in PBS
• Adjust cells to 5 x109 /L
• Add Ab 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)

13. VALIDATION FOR
LYMPHOMA/LEUKEMIA (PHASE 1)
(Titration of antibodies)
All antibodies titrated to determine the optimal concentrations
Ab PN Titer (uL)
FITC
CD4 IM0448U 10
Kappa A64828 10
PE
CD8 IM0452U 10
Lambda A64827 10
ECD
CD3 IM2705U 5
CD14 IM2707U 3
PC5.5 CD33 A70198 3
PC7
CD20 IM3629U 5
CD56 A51078 10
APC CD34 IM2472U 5
A700 CD19 A78837 3
A750 CD10 A89310 5
PB CD5 A82790 5
KO CD45 A96416 5
 Optimal signal-to-noise separation
 Minimize background fluorescence

14. VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1)
(Verification of cocktail stability)
 Antibodies cross reactivity
 Stability over two weeks

15. VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1)
(Instrument setup)
• FSC/SSC settings were optimized using a whole blood
sample lysed with VersaLyse
• FL1-FL9 were optimized using blood samples stained
with CD4
• FL10 was optimized using blood samples stained with
CD45 KO

16. VALIDATION FOR LYMPHOMA/LEUKEMIA
(PHASE 1)
(Optimizing Instrument Compensation)
• The compensation matrix was calculated using Kaluza software (BC) and list-
mode files (LMD) from blood samples stained with single CD45 antibodies for
each fluorescence channel .
• Compensation for the screening tube was adjusted using normal BMA stained
with the full screening panel.

17. • FlowCheck Pro (Beckman Coulter)
• FlowSet Pro (Beckman Coulter)
• Compensation verifier
VALIDATION FOR LYMPHOMA/LEUKEMIA
(PHASE 1)
(Instrument QC)

18. VALIDATION FOR LYMPHOMA/LEUKEMIA
(PHASE 1)
(Comparison study)
Study Overview
The validation was done using 32 patient samples
The samples included 17 BMA, 11 blood samples (PB), 3 lymph node
cell suspensions (LNB) and one body fluid (BF, pleural fluid).
16 female and 16 male patients, age 25-93(mean age 57)
Diagnosis included 7 normal/reactive samples (3 BMA, 1 BF and 3
PB), 12 samples with B or T lymphoproliferative disorders (LPD), 1
Multiple myeloma (MM), 6 acute leukemias (AML/ALL), 5 MDS and one
paroxysmal nocturnal hemoglobinuria (PNH).

19. KALUZA ANALYSIS TEMPLATE

20. NORMAL BM SAMPLE
Living Cells = NOT Debris

21. NORMAL BM SAMPLE
(BLAST ANALYSIS)

22. Dim CD45+CD45-ve
NORMAL BM SAMPLE
(CD45-/dim ANALYSIS)

23. NORMAL BM SAMPLE
(B-CELL ANALYSIS)

24. NK = Lymphs AND (NOT CD19+)
NORMAL BM SAMPLE
(T-CELL/ NK ANALYSIS)

25. NORMAL BM SAMPLE
(Grans & Monos Aanalysis)

26. KALUZA SELECTED STATISTICS

27. EXPORT KALUZA STATISTICS

28. SCREENING TUBE REPORT

29. FLOW CYTOMETRY LAB - UHN
Paperless reporting
All staff members have access to analysis
software and share drive
Shared drive storage/reporting

30. Lymphoproliferative neoplasm
B- CLL
FCL
T-LPD

31. Plasma Cell Neoplasms
ST
MM

32. Paucicellular Cytologic Specimens (1)
CSF
WBC= 5 x106/L
1,315 events collected
Reactive lymphoid T-cell population
Rajab A, Boerner S, da Cunha Santos G, Geddie W, Ko HM, Porwit A. Ten-Color 14
antibody flow cytometry panel for immunophenotyping of paucicellular cytologic
specimens. Cytometry Part B: Clinical Cytometry, 2013, Oct 25, 84, (6): 419
(abstract).

33. CSF
WBC= 2 x106/L
3,262 events collected
B-cell LPD
Paucicellular Cytologic Specimens (2)

34. CSF
WBC= 22 x106/L
6,560 events collected
ATLL
Paucicellular Cytologic Specimens (3)

35. Acute Leukemia
AML
B-ALL
T-ALL

36. PNH -POSITIVE
CD14
NEGATIVE
PNH-
MONOCYTE
CLONE

37. VALIDATION FOR LYMPHOMA/LEUKEMIA
(PHASE 1- RESULTS)
(The comparison study showed 100% concordance)

38. VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1)
SUMMARY (1)
• During August 2012 – December 2013 we have analyzed 1025
samples using ST. In 94% of the cases we could render a final FCM
report and 6% of cases required further immunophenotyping.
• The Screening Tube is capable of enumerating major blood and
bone marrow cell populations.
• It can detect aberrant antigen expression on B-cells (CD5, CD10,
CD20, Kappa/Lambda)
• Establish B-cell clonality status
• Establish B-cell maturation status (CD10/CD20)
• Detecting aberrant expression on T-cells (CD3, CD5, CD4, CD8, &
CD10)

39. VALIDATION FOR
LYMPHOMA/LEUKEMIA (PHASE 1)
SUMMARY (2)
• The Screening Tube can detect abnormal
Myeloid populations
• The Screening Tube can reduce TAT
• The Screening Tube reduces the need for Hem-
Path consultation
• The Screening Tube can reduce costs by up to
30%
• Unable to detect minor clonal plasma cell
population

40. Flow cytometry has recently been recommended as a
significant parameter in the integrated diagnostics of
patients with cytopenia and suspected MDS
• Malcovati L, Hellstrom-Lindberg E, Bowen D, Ades L, Cermak J, del CC, et al. Diagnosis and
treatment of primary myelodysplastic syndromes in adults: recommendations from the European
LeukemiaNet. Blood 2013 Oct 24;122(17):2943-64.
• Porwit A. Role of flow cytometry in diagnostics of myelodysplastic syndromes--beyond the WHO
2008 classification. Semin Diagn Pathol 2011 Nov;28(4):273-82.
• Porwit A, van de Loosdrecht AA, Bettelheim P, Brodersen LE, Burbury K, Cremers E, et al.
Revisiting guidelines for integration of flow cytometry results in the WHO classification of
myelodysplastic syndromes-proposal from the International/European LeukemiaNet Working
Group for Flow Cytometry in MDS. Leukemia 2014 Jun 12.
• Porwit A. Is There a Role for Flow Cytometry in the Evaluation of Patients
With Myelodysplastic Syndromes? Curr Hematol Malig Rep. 2015 Sep;10(3):309-17
VALIDATION FOR MDS SCREENING
(PHASE 2 background)

41. VALIDATION FOR MDS SCREENING
(PHASE 2 background)
Study enrolled patients from 6 institutions (Europe, USA,
Japan)
417 low grade MDS (with <5% blasts)
380 pathological controls with non-clonal cytopenias
FCM analysis was performed with various antibody
combinations and various flow cytometers
Haematologica 2012; 97(8)

42. VALIDATION (PHASE 2 background)
FCM score > 2 was significantly associated with MDS diagnosis
High values were associated with multilineage dysplasia,
transfusion dependency and high-risk cytogenetics
Della Porta et al. 2012

43. FCM- score for MDS using our screening tube
(NORMAL BM)
Gated Myeloblasts in all nucleated cells: 1.6% (If ≥2%, FCM score= 1)
Gated B-cell progenitor in CD34+ cells: 5.3% (If ≤5%, FCM score=1)
Lymphocyte to myeloblast CD45 MFI ratio= 6.4 (If ≤4 or ≥7.5, FCM score=1)
Granulocyte to lymphocyte SSC mode ratio: 8 (If ≤6, FCM score=1)
Total FCM score= 0

44. FCM- score for MDS (MDS case)
Gated Myeloblasts in all nucleated cells: 3.3 (If ≥2, FCM score= 1)
Gated B-cell progenitor in CD34+ cells: 0.4 (If ≤5, FCM score=1)
Lymphocyte to myeloblast CD45 MFI ratio: 12.5 (If ≤4 or ≥7.5, FCM score=1)
Granulocyte to lymphocyte SSC mode ratio: 4.7 (If ≤6, FCM score=1)
Total FCM score= 4

45. VALIDATION (PHASE 2)
• 741 BM samples were analyzed within 2013
• Results of MDS score were correlated with morphological and
cytogenetic evaluation in a selected group of 440 bone marrow samples
from patients with full clinical information:
 8 normal BM samples
 207 BM samples hospital controls*
 18 B-cell or plasma cell neoplasm
 33 post treatment for other malignancy
 21 post BM transplant
 47 MPN
 106 samples from patients with MDS or MDS/MPN diagnosis
(based on morphological, cytogenetic and clinical findings)
* Patients with cytopenia(s) with no evidence of underlying malignancy, BM samples with reactive
inflammatory changes or lymphoma staging specimens without lymphoma involvement.

46. RESULTS- VALIDATION (PHASE 2)
Rajab A, Porwit A. Screening bone marrow samples for abnormal lymphoid populations and myelodysplasia-related features
with one 10-color 14-antibody screening tube. Cytometry B Clin Cytom. 2015 Jul-Aug;88(4):253-60
PPN= 96%
NPV= 82%
PPN= 75%
NPV= 86%

47. RESULTS- VALIDATION (PHASE 2)
Rajab A, Porwit A. Screening bone marrow samples for abnormal lymphoid populations and myelodysplasia-related features
with one 10-color 14-antibody screening tube. Cytometry B Clin Cytom. 2015 Jul-Aug;88(4):253-60

48. 10 color acute leukemia panel
at University Health Network, Toronto
AML 1 AML 2 AML 3 B-ALL T-ALL cytopl
FITC CD65 CD36 CD71 CD58 CD7 nTdt
PE CD13 CD64 CD11c CD22 CD1a cMPO
ECD CD14 CD56 CD4 CD38 CD8 CD14
PC5.5 CD33 CD33 CD33 CD33 CD3 CD33
PC7 CD34 CD34 CD34 CD34 CD34 CD34
APC CD117 CD123
CD2 CD123 CD2 cCD79a
APC_AlexaF700 CD7 CD19
CD10 CD10 CD10 cCD22
APC_AlexaF750 CD11b CD38
CD235a CD19 CD4 CD19
Pacific_BLUE CD16 HLA-DR CD15 CD20 CD5 cCD3
Krome Orange CD45 CD45 CD45 CD45 CD45 CD45

50. CONCLUSION
• Developed 14 MAb 10-color FCM tube can be applied
for a quick screening for aberrant lymphoid populations
and myelodysplasia-related features.
• MDS score 3 or 4 is highly indicative of MDS/MDS-MPN
but can also be seen in MPN.
• Score 2 in patients with differential diagnosis of MDS or
MDS/MPN should prompt further investigation for
myelodysplastic syndrome using comprehensive FCM
panel.
• MDS diagnosis should always be based on integrated
diagnostics (morphology, FCM, cytogenetics)

51. CURRENT & FUTURE DEVELOPMENTS
A. Improve the accuracy of MDS score
B. Develop Ten-Color 15 Antibody Flow
Cytometry Panel for Immunophenotyping of
Lymphocyte population

52. Improve the accuracy of MDS score
Porwit A, et al. Revisiting guidelines for
integration of flow cytometry results in the WHO
classification of myelodysplastic syndromes-
proposal from the International/European
LeukemiaNet Working Group for Flow Cytometry
in MDS. Leukemia 2014 Jun 12.

53. Ten-Color 15 Antibody Flow Cytometry Panel
for Immunophenotyping of Lymphocyte
population
• 80% of LPD cases we diagnosed in 2014 in blood are
CLL/MBL.

54. Ten-Color 15 Antibody Flow Cytometry Panel for
Immunophenotyping of Lymphocyte population
Ab
FITC CD4 + Kappa
PE CD8 + Lambda
ECD CD3 + CD14
PC5.5 CD5
PC7 CD20 + CD56
APC CD10
APC-A700 CD19
APC-A750 CD200
PB CD57 +CD23
KO CD45
ICCS 2015, Abstract #88, Poster Session 2 on Monday, October 12, 2015
Ten-Color 15 Antibody Flow Cytometry Panel for Immunophenotyping of Lymphocyte population
Amr Rajab, Graeme Quest, Jennifer Leung, Anna Porwit
Department of Laboratory Hematology, University Health Network Toronto, ON, Canada

55. CLL

56. MCL

57. T-cell LGL

58. AITL

59. ACKNOWLEDGEMENTS
• Anna Porwit
• Jennifer Leung
• Jessie Leung
• Jordan Ngo
• Josello Mandawi
• Liz Valenzuela
• May Ly
• Sajid Dawji
• Reza Jafari

60. Thank you for your attention