ten-color, 14 antibody flow cytometry (fcm) screening tube for lymphoproliferative disorders and...

Ten-Color, 14 Antibody Flow cytometry

(FCM) Screening Tube for

Lymphoproliferative Disorders and

Myelodysplasia (MDS) – Related Changes in

Bone Marrow Samples

EMAIL: [email protected]

Amr Rajab, BSc, MLT, QCYM (ASCP)

Clinical Flow Cytometry Laboratory

Laboratory Medicine Program

Toronto General Hospital/University Health Network

Ontario, Canada

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

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

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

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)

Why the new ST? (2)

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

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

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.

VALIDATION FOR

LYMPHOMA/LEUKEMIA (PHASE 1)

• Optimization of sample preparation

• Titration of antibodies

• Verification of cocktail stability

• Instrument setup and compensation

• Comparison study

VALIDATION FOR LYMPHOMA/LEUKEMIA (PHASE 1)

(Selection of lysing solution)

VALIDATION FOR


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)

VALIDATION FOR


(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


(Verification of cocktail stability)

Antibodies cross reactivity

Stability over two weeks


(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

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.

• FlowCheck Pro (Beckman Coulter)

• FlowSet Pro (Beckman Coulter)

• Compensation verifier


(PHASE 1) (Instrument QC)


(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).

KALUZA ANALYSIS TEMPLATE

NORMAL BM SAMPLE

Living Cells = NOT Debris

NORMAL BM SAMPLE

(BLAST ANALYSIS)

Dim CD45+ CD45-ve

NORMAL BM SAMPLE

(CD45-/dim ANALYSIS)

NORMAL BM SAMPLE

(B-CELL ANALYSIS)

NK = Lymphs AND (NOT CD19+)

NORMAL BM SAMPLE (T-CELL/ NK ANALYSIS)

NORMAL BM SAMPLE (Grans & Monos Aanalysis)

KALUZA SELECTED STATISTICS

EXPORT KALUZA STATISTICS

SCREENING TUBE REPORT

FLOW CYTOMETRY LAB - UHN

Paperless reporting

All staff members have access to analysis

software and share drive

Shared drive storage/reporting

Lymphoproliferative neoplasm

B- CLL

FCL

T-LPD

Plasma Cell Neoplasms

ST

MM

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).

CSF

WBC= 2 x106/L


B-cell LPD


CSF

WBC= 22 x106/L


ATLL


Acute Leukemia

AML

B-ALL

T-ALL

PNH -POSITIVE

CD14

NEGATIVE

PNH-

MONOCYTE

CLONE


(PHASE 1- RESULTS)

(The comparison study showed 100% concordance)


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)

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

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)

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)

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

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

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

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.

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%

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

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

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)

CURRENT & FUTURE DEVELOPMENTS

A. Improve the accuracy of MDS score

B. Develop Ten-Color 15 Antibody Flow

Cytometry Panel for Immunophenotyping of

Lymphocyte population

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.

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.

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

T-cell LGL

ACKNOWLEDGEMENTS

• Anna Porwit

• Jennifer Leung

• Jessie Leung

• Jordan Ngo

• Josello Mandawi

• Liz Valenzuela

• May Ly

• Sajid Dawji

• Reza Jafari

Thank you for your attention

ten-color, 14 antibody flow cytometry (fcm) screening tube for lymphoproliferative disorders and...

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