Download - Flow Cytometry in Haematology
![Page 1: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/1.jpg)
Flow Cytometry in Haematology
![Page 2: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/2.jpg)
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
![Page 3: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/3.jpg)
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
![Page 4: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/4.jpg)
![Page 5: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/5.jpg)
![Page 6: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/6.jpg)
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
![Page 7: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/7.jpg)
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
![Page 8: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/8.jpg)
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
![Page 9: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/9.jpg)
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
![Page 10: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/10.jpg)
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
![Page 11: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/11.jpg)
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
![Page 12: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/12.jpg)
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”
![Page 13: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/13.jpg)
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
![Page 14: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/14.jpg)
![Page 15: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/15.jpg)
![Page 16: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/16.jpg)
Revised Guidelines on Immunophenotyping in Acute
Leukaemias and Chronic Lymphoproliferative Disorders.
BSH: Clinical & Laboratory Haematology 2002;24:1-13.
![Page 17: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/17.jpg)
Background
• Major technical advances– Improved instrumentation– New monoclonal antibodies– Up to 6 colour staining– Permeabilizing agents– CD45 gating strategies
![Page 18: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/18.jpg)
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
![Page 19: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/19.jpg)
General Recommendations
• Immunophenotyping should be assessed in conjunction with clinical features and cell morphology.
• Need for Quality System Essentials to be adhered to.
![Page 20: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/20.jpg)
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.
![Page 21: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/21.jpg)
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
![Page 22: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/22.jpg)
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
![Page 23: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/23.jpg)
The value of autoflourescence as a diagnostic feature of APML
• Hayden et al, Dublin, Haematologica 2006;91:417-418
![Page 24: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/24.jpg)
![Page 25: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/25.jpg)
![Page 26: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/26.jpg)
![Page 27: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/27.jpg)
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
![Page 28: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/28.jpg)
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
![Page 29: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/29.jpg)
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
![Page 30: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/30.jpg)
T ALL – secondary panel FITC PE PC5
G1 Control G2 Control CD45 CD8 CD4 CD45
Plus Cytoplasmic TdT and CD3
![Page 31: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/31.jpg)
AML – follow upFITC PE ECD PC5
G1 Control G2 Control G2 Control CD45CD7 CD13 CD34 CD45
CD33 CD56 CD34 CD45
![Page 32: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/32.jpg)
B ALL – follow upFITC PE ECD PC5
G1 Control G2 Control G2 Control CD45CD19 CD10 CD34 CD45CD33 CD13 CD34 CD45
![Page 33: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/33.jpg)
T ALL – follow upFITC PE ECD PC5
G1 Control G2 Control G2 Control CD45CD19 CD10 CD34 CD45CD8 CD4 CD34 CD45CD1a CD2 CD34 CD45
![Page 34: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/34.jpg)
Multiple myelomaFITC PE PC5
G1 Control G2 Control CD38CD19 CD56 CD38CD45 CD86 CD38CD45 CD138 CD38
![Page 35: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/35.jpg)
![Page 36: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/36.jpg)
Chronic Lymphoproliferative Disorders
![Page 37: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/37.jpg)
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
![Page 38: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/38.jpg)
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
![Page 39: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/39.jpg)
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
![Page 40: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/40.jpg)
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
![Page 41: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/41.jpg)
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
![Page 42: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/42.jpg)
• 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.
![Page 43: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/43.jpg)
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
![Page 44: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/44.jpg)
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
![Page 45: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/45.jpg)
Discriminatory Patterns for Leukaemia
CLLAML
![Page 46: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/46.jpg)
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
![Page 47: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/47.jpg)
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
![Page 48: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/48.jpg)
B lymphoid development
CD34
CD22
![Page 49: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/49.jpg)
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
![Page 50: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/50.jpg)
T lymphocyte development
CD34
CD3
![Page 51: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/51.jpg)
Normal myeloid antigen expression
![Page 52: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/52.jpg)
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
![Page 53: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/53.jpg)
Leukemic aberrant phenotypes
• Cross-lineage infidelity
• Asynchronous antigen expression
• Antigen overexpression
• Ectopic phenotypes
• Abnormal light scatter
![Page 54: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/54.jpg)
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
![Page 55: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/55.jpg)
Detecting residual leukemic disease
• Morphology of peripheral blood/marrow
• Immunohistology of paraffin sections
• Cytogenetics
• FISH
• Molecular PCR– fusion genes– IgH and TCR
• Immunophenotyping
![Page 56: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/56.jpg)
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
![Page 57: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/57.jpg)
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
![Page 58: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/58.jpg)
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
![Page 59: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/59.jpg)
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
![Page 60: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/60.jpg)
Quantifying MRD
Normal marrow
ALL at diagnosis MRD
Dario Campana, Cytometry (Communications in Clinical Cytometry) 38:139–152 (1999)
![Page 61: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/61.jpg)
Monitoring kinetics of leukemia with flow
![Page 62: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/62.jpg)
May 2003
![Page 63: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/63.jpg)
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
![Page 64: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/64.jpg)
IMMUNOLOGICAL RELAPSE PRECEDES MORPHOLOGICAL
RELAPSE BY
• AML 3-9 months
• ALL 10+/-9 months
![Page 65: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/65.jpg)
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).
![Page 66: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/66.jpg)
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
![Page 67: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/67.jpg)
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
![Page 68: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/68.jpg)
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.
![Page 69: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/69.jpg)
ZAP 70
ZA
P70
![Page 70: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/70.jpg)
Diagnosis of Acute Leukaemia: Can flow cytometry replace
molecular techniques
• JJM van Dongen – ISLH Amsterdam 2006
![Page 71: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/71.jpg)
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
![Page 72: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/72.jpg)
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
![Page 73: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/73.jpg)
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
![Page 74: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/74.jpg)
PARAFFIN SECTION IMMUNOTYPING IN
HAEMATOLOGY
![Page 75: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/75.jpg)
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
![Page 76: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/76.jpg)
Immunohistology - Cons
• Inability to detect multiple ags on the same cell
• ? Turn around time
![Page 77: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/77.jpg)
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
![Page 78: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/78.jpg)
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%
![Page 79: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/79.jpg)
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
![Page 80: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/80.jpg)
• Leukaemia Diagnosis
• Clinical features
• Morphology including cytochemistry
• Flow cytometry
• Cytogenetics
• Molecular studies
• Immunohistology
![Page 81: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/81.jpg)
• Immunohistologic staining of bone marrow trephine bxs or clot sections.
• Extramedullary myeloid tumours.
![Page 82: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/82.jpg)
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
![Page 83: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/83.jpg)
ALL• B-lineage
• CD10, CD79a, CD20, pax-5
• T-lineage
• CD3, F1, CD1a, CD4, CD8 & 2TL 242
• TdT & CD7 are not lymphoid lineage specific.
![Page 84: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/84.jpg)
![Page 85: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/85.jpg)
Collaborators
• Lynne Trutte
• David Westerman
• Deon Venter
• John Seymour
• Miles Prince
![Page 86: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/86.jpg)
![Page 87: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/87.jpg)
![Page 88: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/88.jpg)
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.
![Page 89: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/89.jpg)
• 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.
![Page 90: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/90.jpg)
![Page 91: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/91.jpg)
![Page 92: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/92.jpg)
![Page 93: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/93.jpg)
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.
![Page 94: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/94.jpg)
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.
![Page 95: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/95.jpg)
MRD in Acute Leukaemias
• Residual myeloblasts post-chemotherapy distinguished from erythroblasts.
• TdT + lymphoblasts
• CD20+ lymphoblasts*
![Page 96: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/96.jpg)
CD20
![Page 97: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/97.jpg)
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
![Page 98: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/98.jpg)
![Page 99: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/99.jpg)
Simple diagnostic assay for hairy cell leukaemia by
immunocytochemical detection of annexin A1 (ANXA1)
• Brunangelo Falini et al, The Lancet June 6, 2004
![Page 100: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/100.jpg)
![Page 101: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/101.jpg)
CLL
• Minimal disease post-chemotherapy not demonstrable by flow cytometry.
• Richter’s transformation.
![Page 102: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/102.jpg)
Panel for Immunotyping of Acute Leukaemias at diagnosis
• TdT, CD3, CD10, CD20, CD79a, MPO, Glycophorin (A or C), CD61 or CD41, CD34, Lysozyme & CD68.
![Page 103: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/103.jpg)
Detection of Minimal Disease
• Individualize depending of the subtype.
• AML: MPO, CD68 & lysozyme.
• ALL: CD3, CD79a, CD10, CD20, TdT.
![Page 104: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/104.jpg)
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)
![Page 105: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/105.jpg)
Confirmation of molecular classification of DLCL by
IHC using tissue microarray
• Hans et al, Multinational , Blood Jan 1, 2004
![Page 106: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/106.jpg)
• Sections stained with abs to CD10, bcl-6, MUM1, FOXP1, cyclin D2 and bcl-2
![Page 109: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/109.jpg)
![Page 110: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/110.jpg)
![Page 111: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/111.jpg)
![Page 112: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/112.jpg)
![Page 113: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/113.jpg)
Limitations of Routine Morphology in Myeloma
• Variable aspirate quality
• Patchy disease
• Difficulties in plasma cell recognition
• Small aggregates often missed on H & E
![Page 114: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/114.jpg)
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
![Page 115: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/115.jpg)
Antibody Panel
• CD138
• Bcl-2
• VS38c
• CD79a
• CD20
• Light chains
• EMA
![Page 116: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/116.jpg)
![Page 117: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/117.jpg)
![Page 118: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/118.jpg)
![Page 119: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/119.jpg)
![Page 120: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/120.jpg)
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
![Page 121: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/121.jpg)
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.
![Page 122: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/122.jpg)
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
![Page 123: Flow Cytometry in Haematology](https://reader035.vdocument.in/reader035/viewer/2022081418/56812ea3550346895d943d54/html5/thumbnails/123.jpg)
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