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Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
Flow Cytometry in Haematology
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Flow Cytometry in Haematology

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  • This revised guideline will focus on new techniques and reagents and define new panels of McAb recommended for lineage assignment in acute leukaemias and for the characterization of chronic lymphoproliferative disorders
  • 1994 same group published guidelines
    Major technical advances since last guidelines published
    New monoclonal antibodies- improved diagnosis of AL
    allows the identification of leucocytes and the exclusion of platelets and debris. Thereafter, another gating is performed which includes the cells positive with the McAb under study. (Borowitz et al., 1993). A minimum number of CD45-positive events must be collected in order to obtain reliable results, particularly when estimating the numbers of low frequency cells such as CD34-positive cells (Barnett et al., 1999). It should also be noted that there is a marked variation between laboratories as to the number of CD45-positive events collected - ranging from 10   103 to 10   106
  • Specimen to be analysed within 24 hours In this situation results should be assessed with caution as antigen loss, particularly of markers which are weakly expressed, may occur. Suspected cases of Burkitt's lymphoma/leukaemia and cerebrospinal fluid samples require rapid transport and processing of the sample.
    Flow cytometry can be applied to blood or bone marrow samples without the need to isolate mononuclear cells, thus simplifying laboratory procedures and making immunophenotyping of high risk, e.g. HIV-positive, samples safer. The samples should be treated with a hypotonic erythrocyte lysing solution with NH4Cl-based reagents which causes minimal selective loss of cell populations
  • Immunophenotyping should not be considered in isolation particularly in specimens containing a mixture of normal and neoplastic cells
    Immunophenotyping be assessed in conjunction with clinical features and cell morphology,
    whole composite phenotype should be taken into account as most immunological markers are not strictly lineage specific; for instance cluster designation (CD)7 is a T-cell marker but it is also expressed in a proportion of cases of acute myeloid leukaemia (AML).
    Need for Quality System Essentials
    Controls. Positive and negative controls for each McAb should be carried out only if a small number of tests are performed infrequently or if a specific antigen is used infrequently, to monitor the validity of the immunostaining and the lysing procedures.
  • FIGURE 1 here
    immunophenotyping is indicated if it is not possible to establish a definitive diagnosis of AML on the basis of a Romanowsky stained film and standard cytochemistry.
  • Here is the BCSH AML Panel
    based on the recommendations by the EGIL group
    two step process with the first panel of markers being applicable to all cases of acute leukaemia unless a clear myeloid committment can be demonstrated by morphology and cytochemistry.
    A second panel is selected, when necessary, to deal with specific diagnostic problems
    Highlight CD79a and CD117 as new
    Philosophy-The first line panel permits the diagnosis of the majority of cases of acute leukaemias and their classification into the three major subtypes: AML, B and T-lineage ALL whilst the second line panel is aimed at the identification of uncommon types of AML such as those with megakaryocytic or erythroid differentiation and at the exclusion or confirmation of a diagnosis of a non- haemopoietic malignancy.
  • Natural killer (NK) associated markers, e.g. CD16, CD56, CD57 and CD11b may be investigated in cases with a presumptive diagnosis of large granular lymphocyte (LGL) leukaemia, whether or not cells express T-cell specific markers such as CD3 or T-cell receptor (TCR). Use of these McAb is strongly recommended in those cases in which cells are CD2 positive but lack expression of specific T and B cell markers.
     TIA-1, a McAb that recognizes an intragranular protein in cytotoxic T lymphocytes and NK cells may be useful in cases in which an expansion of CD8-positive cells is documented as it may help to distinguish CD8-positive LGL-leukaemia from the minority of cases of other T-cell leukaemias that are CD8-positive, e.g. T-cell prolymphocytic leukaemia and Sezary syndrome which, unlike LGL leukaemia, are TIA-1 negative (Matutes et al., 1996). In addition, this marker is characteristically expressed in a well defined subtype of T-cell lymphoma, the hepatosplenic / T-cell lymphoma, which arises from cells bearing the TCR / .
    Investigation of expression of the nuclear enzyme TdT is optional but is recommended in those cases shown to have a T-cell phenotype and immature or blastic morphology in order to exclude or confirm a diagnosis of T-lymphoblastic lymphoma/T-ALL.
    Markers associated with activated T-cells such as CD25 may be assessed in T-cell leukaemias and lymphomas in which the cells lack expression of most T-cell and NK associated markers and/or when a diagnosis of HTLV-I positive adult-T-cell leukaemia lymphoma is entertained. McAb anti-TCR / may be used if a diagnosis of hepatosplenic T-cell lymphoma is suspected.
  • This panel is essentially the same as the one previously recommended (General Haematology Task Force of the BCSH, 1994b) with minor modifications. As for the diagnosis of acute leukaemias, it comprises a first line panel of markers applied to all cases and a second line to be selectively applied if indicated by the results with the first line panel.
    FIRST LINE
    Pan-T cell marker: CD2 (CD2 is recommended instead of CD3 because a proportion of T-cell diseases, whether positive or not with natural killer-associated markers, are CD3 negative).
    (we do not adopt the same first/ second line approach-both CD2 and 3 are included in the lymphoma panel.
    The first line panel will permit distinction of B-cell from T-cell neoplasms and, within the B-cell disorders, will demonstrate whether or not the immunophenotype typical of CLL is present (Elaborate on the flow chart on how CLL scores 4-5 Table 6)
  • Transcript

    • 1. Flow Cytometry in Haematology
    • 2. 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
    • 3. 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
    • 4. 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
    • 5. 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
    • 6. 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
    • 7. 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
    • 8. 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
    • 9. 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 /lpredicts a satisfactory harvest. • Before apheresis WBC > 5x109 /l and blood CD34 conc. > 1x 104 /ml
    • 10. 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”
    • 11. 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
    • 12. Revised Guidelines on Immunophenotyping in Acute Leukaemias and Chronic Lymphoproliferative Disorders. BSH: Clinical & Laboratory Haematology 2002;24:1-13.
    • 13. Background • Major technical advances – Improved instrumentation – New monoclonal antibodies – Up to 6 colour staining – Permeabilizing agents – CD45 gating strategies
    • 14. Pre-analytical factors • Blood and bone marrow samples – Anticoagulant (EDTA or Heparin) – Specimen to be analysed within 24 hours – Storage at 2-200 C – No need to isolate mononuclear cells – Care with lysing procedure
    • 15. General Recommendations • Immunophenotyping should be assessed in conjunction with clinical features and cell morphology. • Need for Quality System Essentials to be adhered to.
    • 16. Selection of Fluorochromes Ruiz-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.
    • 17. 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
    • 18. 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
    • 19. The value of autoflourescence as a diagnostic feature of APML • Hayden et al, Dublin, Haematologica 2006;91:417-418
    • 20. 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
    • 21. 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
    • 22. 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
    • 23. T ALL – secondary panel FITC PE PC5 G1 Control G2 Control CD45 CD8 CD4 CD45 Plus Cytoplasmic TdT and CD3
    • 24. AML – follow up FITC PE ECD PC5 G1 Control G2 Control G2 Control CD45 CD7 CD13 CD34 CD45 CD33 CD56 CD34 CD45
    • 25. B ALL – follow up FITC PE ECD PC5 G1 Control G2 Control G2 Control CD45 CD19 CD10 CD34 CD45 CD33 CD13 CD34 CD45
    • 26. T ALL – follow up FITC PE ECD PC5 G1 Control G2 Control G2 Control CD45 CD19 CD10 CD34 CD45 CD8 CD4 CD34 CD45 CD1a CD2 CD34 CD45
    • 27. Multiple myeloma FITC PE PC5 G1 Control G2 Control CD38 CD19 CD56 CD38 CD45 CD86 CD38 CD45 CD138 CD38
    • 28. Chronic Lymphoproliferative Disorders
    • 29. 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
    • 30. New disease identification/classification • T-CD4+ (CD56+, CD57+, TCRαβ+) : 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
    • 31. 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
    • 32. 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
    • 33. 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
    • 34. • The question we are facing is whether the presence of monoclonalB cells in the PB of otherwise healthy subjects may have a clinicalbearing and if so, to what extent. The results of the presentstudy call for increased caution in interpreting FCMresults in a clinical setting. The widespread use of the evaluationof the / ratio, during common diagnostic procedures, suggeststhat clinically silent circulating B-cell clones may be rathereasily reported during routine controls, bringing along the difficulty of the interpretation in terms of clinical prognosis.Prospective studies are definitely needed in order to definethe features, if any, that can discriminate between "benignB-cell clones" and "progressive B-cell clones" as well as toidentify those individuals who would benefit from clinical followup. The experience with MGUS suggests that this may be a clinicalresult quite difficult to reach.
    • 35. 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
    • 36. Medsaic Immunomicroarray Nanoarray Glass slide with immobilised capture molecules specific for each discriminatory marker Scanner Proprietary slide reader capture image and transfers this file to attached PC Software Embedded algorithm compares captured image with proprietary database of consensus binding patterns •Full proteomic analysis •Detailed diagnostic report Diagnostic kit Nanoarray plus other consumables Exp 4/2005
    • 37. Discriminatory Patterns for Leukaemia CLLAML
    • 38. 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
    • 39. B-ALL subtypes Precursor B-ALL Common ALL Pre-B- ALL Mature-B- ALL HLA-DR cCD22 CD79a CD19 Positive TdT Positive Negative CD10 Negative Positive Negative cIgM Negative Positive Negative sIg Negative Positive
    • 40. B lymphoid development CD34 CD22
    • 41. T-ALL Pro-T- ALL Pre-T- ALL Cortical-T- ALL Mature-T- ALL TdT Positive Negative cCD3 Positive CD7 Positive CD2 Negative Positive CD5 Negative Positive CD4 Negative CD8 Negative Positive for CD4 and CD8 Positive for CD4 or CD8 CD1a Negative Positive Negative sCD3 Negative Positive
    • 42. T lymphocyte development CD34 CD3
    • 43. Normal myeloid antigen expression
    • 44. 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 leukemias EGIL, Leukemia 1995, 9, 1783-1786
    • 45. Leukemic aberrant phenotypes • Cross-lineage infidelity • Asynchronous antigen expression • Antigen overexpression • Ectopic phenotypes • Abnormal light scatter
    • 46. 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
    • 47. Detecting residual leukemic disease • Morphology of peripheral blood/marrow • Immunohistology of paraffin sections • Cytogenetics • FISH • Molecular PCR – fusion genes – IgH and TCR • Immunophenotyping
    • 48. 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
    • 49. 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
    • 50. 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
    • 51. 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
    • 52. Quantifying MRD Normal marrow ALL at diagnosis MRD Dario Campana, Cytometry (Communications in Clinical Cytometry) 38:139–152 (1999)
    • 53. Monitoring kinetics of leukemia with flow
    • 54. May 2003
    • 55. 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
    • 56. IMMUNOLOGICAL RELAPSE PRECEDES MORPHOLOGICAL RELAPSE BY • AML 3-9 months • ALL 10+/-9 months
    • 57. 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).
    • 58. 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
    • 59. 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
    • 60. 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.
    • 61. ZAP 70 ZAP70
    • 62. Diagnosis of Acute Leukaemia: Can flow cytometry replace molecular techniques • JJM van Dongen – ISLH Amsterdam 2006
    • 63. Diagnosis and classification of leukemias informativity per leukemia* Techniques Speed Estimated Costs** ALL AML CLL CML € Cytomorphology <1 day ++ ++ + ++ 50 Immunophenotyping <1 day +++ ++ + + 250 Cytogenetics 2-3 wks ++ ++ + + 500 FISH 2-3 days ++ ++ + ++ 200 PCR fusion genes 2-3 days ++ ++ - ++ 200 Clonality testing 1 week + - + - 250 via Ig/TCR genes CGH arrays 2-3 days + + + - 250 Gene expression 1 week + + + - 1000 profiling *+++/++ very informative; + fairly information; + limited informativity; -no informativity; ** includes reagents, depreciation of equipment, -laboratory infrastructure, personnel, and overhead
    • 64. 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 + + + - immunophenotyping Cytogenetics 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 + + - ++ transcripts RQ-PCR of Ig/TCR 2-4 days** 10-4 to 10-5 ++ - ++ - genes *informativity: ++ highly informative; + moderate informativity; + limited informativity; - no informativity; ** junctional region sequences Of the involved Ig/TCR targets should have been identified at an early stage
    • 65. 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
    • 66. PARAFFIN SECTION IMMUNOTYPING IN HAEMATOLOGY
    • 67. 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
    • 68. Immunohistology - Cons • Inability to detect multiple ags on the same cell • ? Turn around time
    • 69. 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
    • 70. 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%
    • 71. 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
    • 72. • Leukaemia Diagnosis • Clinical features • Morphology including cytochemistry • Flow cytometry • Cytogenetics • Molecular studies • Immunohistology
    • 73. • Immunohistologic staining of bone marrow trephine bxs or clot sections. • Extramedullary myeloid tumours.
    • 74. 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
    • 75. ALL • B-lineage • CD10, CD79a, CD20, pax-5 • T-lineage • CD3, β F1, CD1a, CD4, CD8 & 2TL 242 • TdT & CD7 are not lymphoid lineage specific.
    • 76. Collaborators • Lynne Trutte • David Westerman • Deon Venter • John Seymour • Miles Prince
    • 77. 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.
    • 78. • 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.
    • 79. 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.
    • 80. 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.
    • 81. MRD in Acute Leukaemias • Residual myeloblasts post-chemotherapy distinguished from erythroblasts. • TdT + lymphoblasts • CD20+ lymphoblasts*
    • 82. CD20
    • 83. 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
    • 84. Simple diagnostic assay for hairy cell leukaemia by immunocytochemical detection of annexin A1 (ANXA1) • Brunangelo Falini et al, The Lancet June 6, 2004
    • 85. CLL • Minimal disease post-chemotherapy not demonstrable by flow cytometry. • Richter’s transformation.
    • 86. Panel for Immunotyping of Acute Leukaemias at diagnosis • TdT, CD3, CD10, CD20, CD79a, MPO, Glycophorin (A or C), CD61 or CD41, CD34, Lysozyme & CD68.
    • 87. Detection of Minimal Disease • Individualize depending of the subtype. • AML: MPO, CD68 & lysozyme. • ALL: CD3, CD79a, CD10, CD20, TdT.
    • 88. 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)
    • 89. Confirmation of molecular classification of DLCL by IHC using tissue microarray • Hans et al, Multinational , Blood Jan 1, 2004
    • 90. • Sections stained with abs to CD10, bcl-6, MUM1, FOXP1, cyclin D2 and bcl-2
    • 91. GCB Non- GCB
    • 92. Limitations of Routine Morphology in Myeloma • Variable aspirate quality • Patchy disease • Difficulties in plasma cell recognition • Small aggregates often missed on H & E
    • 93. 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
    • 94. Antibody Panel • CD138 • Bcl-2 • VS38c • CD79a • CD20 • Light chains • EMA
    • 95. 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
    • 96. 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.
    • 97. C lin ic a l fe a t u r e s M o r p h o lo g y ( c y t o c h e m is t r y ) F lo w c y t o m e t r y I m m u n o h is t o lo g y C y t o g e n e t ic s M o le c u la r s t u d ie s B io p s y m a te r ia l ( B M / P B / L N ) P a t ie n t w it h h a e m a t o lo g ic a l m a lig n a n c y
    • 98. Hemispheres Right x 4 Objective Left x 100 Objective “Lost the plot” area of WF Wernike’s GOBSAT Area FRONTAL LOBE CIA/AFIP truth centre Tunnel Vision H&E Colour Vision MGG Colour Vision VISION FAB Charisma Complex Rappaport’s nucleus Dyslexogenesis Area of Lukes Circumlocutory Speech Area of Collins Postulated Integrative area of REAL/WHO James Isbister 2000

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