Flow Cytometry in Haematology

APPLICATIONS

• Diagnosis, subtyping of lymphoproliferative disorders & leukaemias

• Detection of residual disease in above

• CD34 assays

• Lymphocyte subsets

• PNH studies

• Foeto-maternal hemorrhage

• DNA Ploidy, Apoptosis

• Platelet aggregation

PNH• Although Ham’s test and Sucrose lysis tests are

sensitive & specific, their accuracy is strongly operator dependent and are cumbersome (HT).

• Principle of FCM: Absent or markedly diminished expression of glycosyl phosphatidylinositol-anchored protein (GPI-AP) on red cells and/or white cells in the appropriate clinical setting.

• GPI-AP - CD59 (MIRL) & CD55 (DAF)• Use two GPI-AP for confirmation

PNH

• Can detect small clones in multiple lineages but red cells usually adequate; granulocyte analysis ? technically more challenging & need to be tested immediately.

• Quantify clone size (down to 0.1%)• Granulocytes not influenced by haemolysis or

transfusion but the red cell testing is reliable even with significant transfusion.

• Obviously the % of abnormal red cells would be affected by both above factors

PNH

• A more sensitive assay (0.004%) using a bacterial toxin-aerolysin which spares PNH cells

• Red cells show clearest delineation between phenotypes

• Assay both erythrocytes and granulocytes• Small proportion (esp PNH/AA) may only

be detected on granulocyte clones

PNH

• Summary: Analysis of GPI-AP – highly specific test for PNH.

• No other disease in which the erythrocytes are a mosaic of both GPI-AP+ & GPI-AP-

cells• Patients with isolated deficiency of either

CD59 or CD55 are extremely rare & in those cases 100% of cells are abnormal & expression of only one GPI-AP is deficient

CD34 assays

• CD34 – surrogate marker of hemopoietic progenitor cells

• 0.1% of PB mononuclear cells & 1-3% of human bone marrow cells

• Various antibodies available

• Prefer PE conjugated ab because of the rarity of these cells

CD34 assay

• Method (ISHAGE- ISCT) - Sequential gating to define CD45+cells (low to intermediate), CD34+ & with side scatter similar to blast cells

• + viability using 7 AAD• RMH - ISHAGE CD34 Coulter Stem Kit

Enumeration Method• 3 RCPA- QAP surveys each year

CD34 assay

• CD34 cell count in an autograft correlates well with the rate of hemopoietic recovery.

• Values of 2-5x106 cells/kg predicting rapid & durable engraftment following PBSCT.

• It has been demonstrated in one study that a PB CD34+ cell count of > 5.0x106/l predicts a satisfactory harvest.

• Before apheresis WBC > 5x109/l and blood CD34 conc. > 1x 104/ml

ANZSBT Guidelines for Laboratory Assessment of Fetomaternal Haemorrhage

1st edition - 2002

• Summary & Recommendation • Page 6 Section 3.

– “Flow cytometry is accepted as the most accurate quantitative test for FMH…The Scientific Sub-Committee believes this is the method of choice for quantitation if readily available”

Example of calculation of FMH volume using flow method

• Foetal cells assumed to be 22% larger than maternal cells

• Assumed average maternal red cell volume of 1800 ml– E.g. for flow result of 0.5% foetal red cells– Uncorrected foetal RBC vol: 1800 x 5/1000

= 9– Corrected for foetal RBC vol: 9 + (9 x

22/100) = 10.98 ml

Revised Guidelines on Immunophenotyping in Acute

Leukaemias and Chronic Lymphoproliferative Disorders.

BSH: Clinical & Laboratory Haematology 2002;24:1-13.

Background

• Major technical advances– Improved instrumentation– New monoclonal antibodies– Up to 6 colour staining– Permeabilizing agents– CD45 gating strategies

Pre-analytical factors

• Blood and bone marrow samples– Anticoagulant (EDTA or Heparin)– Specimen to be analysed within 24 hours– Storage at 2-200C– No need to isolate mononuclear cells– Care with lysing procedure

 General Recommendations

• Immunophenotyping should be assessed in conjunction with clinical features and cell morphology.

• Need for Quality System Essentials to be adhered to.

Selection of FluorochromesRuiz-Arguelles et al Clinical Cytometry, 70B,

39-44, 2005

• Abs whose normal expression is rather dim (eg CD7, CD10, CD11b, CD13, CD2, CD34, CD64, CD117 or TdT) should be labelled with the most bright fluorochromes: phycoerythrin and, if instrumentation allows, allophycocyanin.

LEUKEMIA IMMUNOPHENOTYPING STRATEGY

• Identify blasts/abnormal cells

• Determine lineage (B, T-lymphoid or myeloid)

• Determine immunological subtype (EGIL)• Search for leukemia aberrant phenotypes• Customise follow up panel for MRD

Reporting FCM results

• Should include:– Light scatter properties– Presence or absence of each of tested ags– Intensity, modality or coexpression of ags– Description of the normal cell population– For MRD, the estimated number of malignant

cells

The value of autoflourescence as a diagnostic feature of APML

• Hayden et al, Dublin, Haematologica 2006;91:417-418

Acute leukemia – first presentation/initial screen FITC PE ECD PC5

G1 Control G1 Control G1 Control CD45 CD19 CD10 CD34 CD45

HLA-DR CD33 CD34 CD45 CD7 CD2 CD34 CD45 CD65 CD13 CD34 CD45

AML – secondary panel FITC PE ECD PC5

G1 Control G1 Control G1 Control CD45 CD11b CD56 CD34 CD45

HLA-DR CD117 CD34 CD45 CD15 CD14 CD34 CD45

As indicated Glycophorin A CD33 CD34 CD45

CD61 CD33 CD34 CD45 Plus Cytoplasmic MPO and TdT

B ALL – secondary panel

FITC PE PC5

G1 Control G2 Control CD45

CD20 CD22 CD45

Kappa Lambda CD45 Plus Cytoplasmic Tdt, CD22, IgM heavy chains and CD79a

T ALL – secondary panel FITC PE PC5

G1 Control G2 Control CD45 CD8 CD4 CD45

Plus Cytoplasmic TdT and CD3

AML – follow upFITC PE ECD PC5

G1 Control G2 Control G2 Control CD45CD7 CD13 CD34 CD45

CD33 CD56 CD34 CD45

B ALL – follow upFITC PE ECD PC5

G1 Control G2 Control G2 Control CD45CD19 CD10 CD34 CD45CD33 CD13 CD34 CD45

T ALL – follow upFITC PE ECD PC5

G1 Control G2 Control G2 Control CD45CD19 CD10 CD34 CD45CD8 CD4 CD34 CD45CD1a CD2 CD34 CD45

Multiple myelomaFITC PE PC5

G1 Control G2 Control CD38CD19 CD56 CD38CD45 CD86 CD38CD45 CD138 CD38

                                            

          

Chronic Lymphoproliferative Disorders

Lymphoma/CLL

FITC PE PC5

G1 Control G2 Control CD45

CD19 CD5 CD45

CD20 CD10 CD45

CD23 CD79b CD45

CD16 CD56 CD45

CD3 CD4 CD45

CD3 CD8 CD45

FMC7 CD2 CD45

Kappa CD19 CD45

Lambda CD19 CD45 Plus CD38FITC/CD20PE in cases of CLL

New disease identification/classification

• T-CD4+ (CD56+, CD57+, TCR+) : a new clonal T-LPD (San Miguel 2004)

• 2% of LPD

• no cytopenias

• no AID

• frequently associated with neoplasia

• CD4+ CD56+ lin- pDC leukemia

– a new entity, 1st reported by GEIL in 2002• Elderly, Cutaneous lesions, CNS disease common

• Initial CR common but then aggressive relapse

• Only 25% 2 year survival

Monoclonal B lymphocytes with the characteristic of

“indolent” CLL are present in 3.5% of adults with normal

blood counts

• Rawstron A et al. Leeds, UK

• Blood 15 July 2002

The Natural History of “Early CLL”

Rawstron et al Blood ASH 2003 #656

• Progression to clinically relevant disease with ~ 1% annum requiring treatment

• Identifying an absolute cut-off for diagnosis is less clinically relevant than identifying the specific cell phenotype & genotype

Monoclonal CD5+ and CD5- B-lymphocyte expansions are

frequent in the peripheral blood of the elderly

• Paolo Ghia et al Multicentre, Italy– Blood March 15, 2004

• The question we are facing is whether the presence of monoclonal B cells in the PB of otherwise healthy subjects may have a clinical bearing and if so, to what extent. The results of the present study call for increased caution in interpreting FCM results in a clinical setting. The widespread use of the evaluation of the / ratio, during common diagnostic procedures, suggests that clinically silent circulating B-cell clones may be rather easily reported during routine controls, bringing along the

difficulty of the interpretation in terms of clinical prognosis. Prospective studies are definitely needed in order to define the features, if any, that can discriminate between "benign B-cell clones" and "progressive B-cell clones" as well as to identify those individuals who would benefit from clinical follow up. The experience with MGUS suggests that this may be a clinical result quite difficult to reach.

Immunophenotyping of Leukaemias Using a Cluster of Differentiation Ab Microarray:

Belov et al Cancer Research June 2001

• Suspension of cells is applied to an array of >50 abs on a glass slide

• Enables concurrent determination of > 50 antigens

Medsaic Immunomicroarray

NanoarrayGlass slide with

immobilised capture molecules specific for each discriminatory

marker

ScannerProprietary slide

reader capture image and transfers this file

to attached PC

SoftwareEmbedded algorithm compares captured

image with proprietary database of consensus

binding patterns

•Full proteomic analysis•Detailed diagnostic report

Diagnostic kitNanoarray plus other

consumables

Exp 4/2005

Discriminatory Patterns for Leukaemia

CLLAML

Immunomicroarray (contd)

• Advantages:– Test for many more antigens.– Simple technique– No flow cytometer required

• Disadvantages:– Multiparameter studies not possible– Various cell populations can’t be separated– Antigen co-expression

B-ALL subtypes

PrecursorB-ALL

CommonALL

Pre-B-ALL

Mature-B-ALL

HLA-DRcCD22CD79aCD19

Positive

TdT Positive NegativeCD10 Negative Positive NegativecIgM Negative Positive NegativesIg Negative Positive

B lymphoid development

CD34

CD22

T-ALL

Pro-T-ALL

Pre-T-ALL

Cortical-T-ALL

Mature-T-ALL

TdT Positive NegativecCD3 PositiveCD7 PositiveCD2 Negative PositiveCD5 Negative PositiveCD4 NegativeCD8 Negative

Positive forCD4 and

CD8

Positive forCD4 or

CD8CD1a Negative Positive NegativesCD3 Negative Positive

T lymphocyte development

CD34

CD3

Normal myeloid antigen expression

AML

• Myelomonocytic

– MPO+,CD13+,CD33+,CDw65+,CD117+

• Erythroid (M6)

– Glycophorin A

• Megakaryocytic (M7)

• Poorly differentiated (M0 AML)

• TdT+ AML

• AML with lymphoid markers

• Biphenotypic acute leukemiasEGIL, Leukemia 1995, 9, 1783-1786

Leukemic aberrant phenotypes

• Cross-lineage infidelity

• Asynchronous antigen expression

• Antigen overexpression

• Ectopic phenotypes

• Abnormal light scatter

High frequency of immunophenotype changes in acute myeloid leukemia at relapse: implications for residual disease detection (CALGB Study 8361)

Maria R. Baer

Blood. 2001;97:3574-3580

• 136 AML patients• Immunophenotyped at diagnosis and relapse

Detecting residual leukemic disease

• Morphology of peripheral blood/marrow

• Immunohistology of paraffin sections

• Cytogenetics

• FISH

• Molecular PCR– fusion genes– IgH and TCR

• Immunophenotyping

Benefits of using flow vs PCR for MRD detection

• Greater applicability– Not dependent on specific fusion gene

• Less problems with contamination

• Rapid availability of results

• Viability of cells can be determined

• More widely available technology

• Multiple “clones” can be identified

MRD in AML

% Studiable Cases

Relative Sensitivity

Karyotype >75% 10-1

FISH >40% 10-1- 10-2

FCM >90% 10-2- 10-4

PCR, RT-PCR >50% 10-4- 10-6

Determination and quantitation of MRD

•250 000 cells for MRD (at least 100 events of interest)

•Aberrant phenotype diagnosed when expressed on >20% blast cells

•Patients without AML should have <0.004% aberrant cells

MRD analysis• AML and ALL

– High detection rate for aberrant populations (>90%)

– Prognostic value after induction chemo

• Myeloma– >90% have aberrant plasma cells

– predicts earlier relapse after autograft

• CLL– sensitive detection of clonality

Quantifying MRD

Normal marrow

ALL at diagnosis MRD

Dario Campana, Cytometry (Communications in Clinical Cytometry) 38:139–152 (1999)

Monitoring kinetics of leukemia with flow

May 2003

Early immunophenotypical evaluation of minimal residual disease in acute myeloid leukemia identifies different patient risk groups and may contribute to postinduction treatment stratification

Jesu´s F. San Miguel

BLOOD, 15 SEPTEMBER 2001 VOLUME 98, NUMBER 6

IMMUNOLOGICAL RELAPSE PRECEDES MORPHOLOGICAL

RELAPSE BY

• AML 3-9 months

• ALL 10+/-9 months

Asynchronous coexpression of CD34 and CD15 at diagnosis (A); low-level MRD at follow-up (B). Overexpression of CD34 and CD13 at diagnosis (C); high-level MRD at follow-up (D).

Applications of MRD testing in acute leukemia

• Quantitative MRD

• Functional prognostic indicator post- induction

• Effectiveness of chemotherapy and marker of tumor resistance

– resistant subclones

– clonal switching

Applications of MRD testing in acute leukemia

• Allows intervention before florid relapse– DLI

– Further chemo pre-autograft collection and assessment of autograft contamination

• Prediction of relapse in “favourable cytogenetic” group

• Assessment of extramedullary relapse eg CNS

ZAP-70 Compared with Immunoglobulin Heavy-chain Gene

Mutation Status as a Predictor of Disease Progression in CLL

Rassenti et al, CLL Research Consortium N E J M Aug 26, 2004

• ZAP-70 is a stronger predictor of the need for treatment in B-CLL than the presence of unmutated IgVHgene.

ZAP 70

ZA

P70

Diagnosis of Acute Leukaemia: Can flow cytometry replace

molecular techniques

• JJM van Dongen – ISLH Amsterdam 2006

Diagnosis and classification of leukemias

informativity per leukemia*Techniques Speed Estimated Costs**

ALL AML CLL CML €Cytomorphology <1 day ++ ++ + ++ 50Immunophenotyping <1 day +++ ++ + + 250Cytogenetics 2-3 wks ++ ++ + + 500FISH 2-3 days ++ ++ + ++ 200PCR fusion genes 2-3 days ++ ++ - ++ 200Clonality testing 1 week + - + - 250 via Ig/TCR genesCGH arrays 2-3 days + + + - 250Gene expression 1 week + + + - 1000 profiling

*+++/++ very informative; + fairly information; + limited informativity;-no informativity; ** includes reagents, depreciation of equipment,-laboratory infrastructure, personnel, and overhead

Techniques for MRD monitoring in leukemia patients

informativity per leukemia*Techniques Speed Sensitivity

ALL AML CLL CML

Cytomorphology <1 day 10-1 to 10-2 - - - - Flow cytometric 1-2 days 10-3 to 10-4 + + + - immunophenotypingCytogenetics 2-3 wks 10-1 to 10-2 - - - +FISH 2-3 days 10-1 to 10-2 - - - +RQ-PCR fusion 2-3 days 10-4 to 10-6 + + - ++ transcriptsRQ-PCR of Ig/TCR 2-4 days** 10-4 to 10-5 ++ - ++ - genes

*informativity: ++ highly informative; + moderate informativity; + limited informativity; - no informativity; ** junctional region sequencesOf the involved Ig/TCR targets should have been identified at an early stage

Comparison between molecular techniques and flow cytometry in hematological malignancies*

Speed 2-3days (up to 1wk) 1-2hrs

Target DNA or RNA (RNA is an unstable target)

protein/cells (end-product)

Applicability depends on disease (chromosome aberration)

broad

Multiplexing technically demanding relatively easy (even 25 to 100 test/tube)

Accurancy (semi-) quantitative Quantitative

Focus all cells in sample, unless prior purification

any subpopulation

Facilities special lab needed (pre-PCR lab, PCR lab, etc)

only standard lab needed (+flow cytometry)

Molecular techniques Flow cytometry

PARAFFIN SECTION IMMUNOTYPING IN

HAEMATOLOGY

PSI - Pros

• may be the only modality

• can be superior to FCM

• potential to provide more accurate quantitative information about neoplastic cells & residual hemopoiesis

• potential to detect molecular abnormalities e.g. ALK in ALCL

Immunohistology - Cons

• Inability to detect multiple ags on the same cell

• ? Turn around time

Technical considerations Works on tissues fixed in any of the usual fixatives -

formalin, Bouin’s or B5.• Labelled polymer method with DAB as a chromogen.• DAKO Autostainer & Vision Bio Systems• Antigen retrieval:

– Dako- microwave pressure cooker in 10mM Na citrate pH 6.0 for 2 mins or trypsin digestion or

EDTA pH 8 (bcl-6)– Vision Bio systems -Heat method

• BM control preferable

Antibody selection in immunohistochemical detection of cyclin D1 in MCL – Torlakovic et

al AM J Clin Path, Nov. 2005

• Sensitivity of 4 different abs varied from 53%-100%

PSI vs FCM• No definite data

• In a limited comparison, PSI was less sensitive than FCM in the detection of HLA-DR, CD34, CD41, CD61.

• A number of abs applicable on FCM are not available for PSI e.g. CD13, CD33, CD19 and vice versa

• Leukaemia Diagnosis

• Clinical features

• Morphology including cytochemistry

• Flow cytometry

• Cytogenetics

• Molecular studies

• Immunohistology

• Immunohistologic staining of bone marrow trephine bxs or clot sections.

• Extramedullary myeloid tumours.

Acute Leukaemia• Bone marrow aspirate dry tap .• Difficulty in establishing lineage by FCM.• Detection of small number of residual leukaemic

blasts post-chemotherapy not detectable by morphology or by FCM.

• Establish focal blastic change in the marrow in CML or in MDS.

• Blast cells not localized clearly on scatter plot on FCM.

• Extramedullary myeloid tumours

ALL• B-lineage

• CD10, CD79a, CD20, pax-5

• T-lineage

• CD3, F1, CD1a, CD4, CD8 & 2TL 242

• TdT & CD7 are not lymphoid lineage specific.

Collaborators

• Lynne Trutte

• David Westerman

• Deon Venter

• John Seymour

• Miles Prince

AML

• Anti-MPO, lysozyme, CD68, WGM1.

• CD13 and CD33 not applicable on PSI.

• M0 - Unclassifiable on routine morphology & cytochemistry may be positive with MPO on tissue sections.

• Pax-5 in t(8;21)

• M3 - Negativity with CD34 & HLA-DR & ? aberrant expression of CD79a.

• M4/M5 - CD68 & lysozyme can be helpful in establishing monocytic differentiation but are not specific.

• M6 - Leukaemic erythroblasts or residual erythroblasts within a leukaemic infiltrate -glycophorin A (or C) or spectrin.

• M7 - CD41 & CD61.

BAL

• Usually over-diagnosed due to failure to exclude non-leukaemic cells from the analysis, over-interpretation of weak or non-specific binding & failure to recognize the lineage specificity of many abs.

• Abs most useful are CD79a,CD10,CD20 CD3, MPO, lysozyme, TdT.

Other Types of Leukaemias

• Plasma Cell Leukaemia: Abs to Ig heavy & light chains and VS38c ab.

• Mast Cell Leukaemia- Mast cell tryptase ab

• NK Cell Leukaemia: CD56 & CD57.

• Cytotoxic T cell lymphocytosis/leukaemia-CD8.

MRD in Acute Leukaemias

• Residual myeloblasts post-chemotherapy distinguished from erythroblasts.

• TdT + lymphoblasts

• CD20+ lymphoblasts*

CD20

Chronic Leukaemias

• HCL

• dry tap at diagnosis

• Semi-quantitative assessment of residual disease post-chemotherapy.

• Follow-up in cases with sub-clinical minimal residual disease.

• Abs used are DBA.44, CD20, anti-TRAP ab, Annexin A1

Simple diagnostic assay for hairy cell leukaemia by

immunocytochemical detection of annexin A1 (ANXA1)

• Brunangelo Falini et al, The Lancet June 6, 2004

CLL

• Minimal disease post-chemotherapy not demonstrable by flow cytometry.

• Richter’s transformation.

Panel for Immunotyping of Acute Leukaemias at diagnosis

• TdT, CD3, CD10, CD20, CD79a, MPO, Glycophorin (A or C), CD61 or CD41, CD34, Lysozyme & CD68.

Detection of Minimal Disease

• Individualize depending of the subtype.

• AML: MPO, CD68 & lysozyme.

• ALL: CD3, CD79a, CD10, CD20, TdT.

LYMPHOMAS

• Lymphoma vs reactive vs non-lymphoid

• Lineage (T, B or NK)

• CD5 coexpresion

• Loss of pan-T cell antigens in a T-cell infiltrate

• CD30, ALK, Ki-67

• Hodgkin lymphoma

• Prognostic markers (bcl-6, bcl-2)

Confirmation of molecular classification of DLCL by

IHC using tissue microarray

• Hans et al, Multinational , Blood Jan 1, 2004

• Sections stained with abs to CD10, bcl-6, MUM1, FOXP1, cyclin D2 and bcl-2

GCB

Non-GCB

http://www.bloodjournal.org/content/vol103/issue1/images/large/h80145429003.jpeg

Limitations of Routine Morphology in Myeloma

• Variable aspirate quality

• Patchy disease

• Difficulties in plasma cell recognition

• Small aggregates often missed on H & E

Potential Uses of Immunohistology in Myeloma

• Establishing clonality

reactive vs neoplastic

• Differential Diagnosis

eg MGUS vs MM & Plasmacytoma vs MM

• Monitoring of disease – semi-quantitaive

• Detection of minimal disease

Antibody Panel

• CD138

• Bcl-2

• VS38c

• CD79a

• CD20

• Light chains

• EMA

Angiogenesis in Myeloma

• Microvessel density (MVD)

• Detected in trephine biopsies by staining with CD31, CD34 & VWF

• MVD correlates with prognosis

• Main action of some new agents eg thalidomide is anti-angiogenesis

Summary• PSI has definite role in the diagnosis,

characterization & monitoring residual disease in many cases of haematological malignancies.

• Scope likely to increase as the the ab panel expands and the technique is automated & more widely available.

C linical features

M orpho logy(cytochem is try)

F lo w c yto m etry Im m unohis to logy C ytogene tics M o lecu la r s tud ies

B iopsy materia l (B M / P B / L N )

P atient w ith haem ato logic al m alignanc y

Hemispheres

Rightx 4 Objective

Leftx 100 Objective

“Lost the plot” area of WF

Wernike’s GOBSAT Area

FRONTAL LOBE

CI A/AFI P truth centre

Tunnel Vision

H&EColour Vision

MGGColour Vision

VISION

FAB CharismaComplex

Rappaport’s nucleus

Dyslexogenesis Areaof Lukes

Circumlocutory SpeechArea of Collins

Postulated I ntegrativearea of REAL/WHO

James Isbister 2000