李啟誠, 朱崧肇, 蔡喜修, 許淑敏, 謝馨慧 Michael R. Loken Practical Application of Multi-dimensional Flow Cytometry in Hematological Disorders 李啟誠, 朱崧肇, 蔡喜修, 許淑敏, 謝馨慧 Michael R. Loken

Introduction Immunophenotyping has become a powerful tool in the characterization of different hematological disorders The key is to realize normal cellular differentiation pathways on the flow cytometric histographs and detect any “deviation” from the normal patterns

Different in strategy of flow cytometric development rather than to design novel markers and apply more and more fluorescences, which are high-priced, time consuming, and only suitable for study of MRD but not the other hematological disorder such as MDS. For any suspicious cell population found on flow, the best way is to sort them and send for molecular confirmation.

Types of antigeneic abnormalities Lineage infidelity Maturational asynchrony Antigeneic absence Quantational abnormality Wells DA, Benesch M, Loken MR et al, Blood, 2003, 102: 394-403

Sensitivity of MRD by flow cytometry Two variables: degree of phenotypic difference between target cells and remaining cells; number of cells can be analyzed 1 in 107 or more similar to PCR Maximal sensitivity of 1 in 105 cells during analysis in clinical sample Consistent sensitivity: 1 in 104 Campana D, Acta haematologica, 2004, 112:8-15

Principle of flow cytometry

Structure of flow cytometry

Normal bone marrow with orderly myeloid maturation Neu Mye/Meta Neu Promye Meta Promye Mye

Normal B lymphoid maturation

Normal B lymphoid maturation III, IV II I T cell I IV III II II III I IV

Normal B lymphoid maturation

Antibodies panels used for myeloid assessment Tube FITC PE PerCP # 1 isotope isotope CD45 # 2 HLA-DR CD11b CD45 # 3 CD5 CD19 CD45 # 4 CD56 CD38 CD45 # 5 CD16 CD13 CD45 # 6 CD15 CD34 CD45 # 7 CD14 CD33 CD45 # 8 CD7 CD56 CD45 # 9 HLA-DR CD34 CD45

Antibodies panels used for B-lymphoid assessment Tube FITC PE PerCP # 1 isotope isotope CD45 # 2 HLA-DR CD11b CD45 # 3 CD5 CD19 CD45 # 4 CD56 CD38 CD45 # 5 CD20 CD10 CD45 # 6 CD22 CD34 CD45 # 7 kappa CD19 CD45 # 8 lambda CD19 CD45 # 9 FMC7 CD19 CD45

Examples of flow cytometric application in hematological disorders Minimal residual detection for AML Minimal residual detection for ALL Flow cytometric diagnosis of MDS Flow cytometric diagnosis of Lymphoma Flow cytometric diagnosis of multiple myeloma Flow cytometric analysis of tissue sample Flow cytometric diagnosis of non-malignant disorder Application of cell sorting for MRD confirmation

Minimal Residual Detection for AML

Over-expression of CD34 & HLA-DR Diagnostic sample MRD 4.0%

Aberrant expression of CD56 & CD7 Diagnostic sample MRD 0.1%

Aberrant expression of CD19 Diagnostic sample MRD 0.6%

Minimal Residual Detection for ALL

Precursor B-ALL, D15 s/p induction C/T Diagnostic sample MRD 0.03%

CALLA (-) precursor B-ALL

Relapse s/p BMT, MRD 0.3%

Flow Cytometric Diagnosis of MDS

56 y/o female, pancytopenia, blast 5 56 y/o female, pancytopenia, blast 5.0%, CD13+, CD33+, aberrant CD15+, aberrant CD34-, asynchronous myeloid maturation on CD13 vs CD16 panel Convex shape CD15+ CD34-

53 y/o female, MDS, CD56+ on blast, monocyte & neutrophil

Flow Cytometric Diagnosis of Lymphoma

66 y/o man, Maltoma, lacrimal gland, for staging HE staining of BM

7.0% clonal marginal zone lymphoma, lower CD45 as compared to lymphocyte, CD19+, CD20+, FMC7+, kappa+, lambda-

L26 staining

Flow Cytometric Diagnosis of Multiple myeloma

68 y/o female, multiple myeloma with cytoplasmic lambda restriction, (30.0%)

AML, M4eo, in remission; MGUS, 0.2% cyto-kappa+

Flow Cytometric Analysis of Tissue Sample

55 y/o man, left axillary LN: CD10+, CD20+, CD19/lambda+, diagnostic of follicular center B-cell lymphoma

Flow Cytometric Diagnosis of Non-malignant Disorder

27 y/o female, macrocytic anemia, Acid-Ham test (-) PB flow: PNH, heterozygous clone Neutrophil: dim CD16 Monocyte: lose CD14

BM flow

Application of Cell Sorting for MRD Confirmation

53 y/o man, follicular center B-cell lymphoma

Cell sorting for follicular B-cell lymphoma Unsorted cells Sorted cell: CD10-/CD45+ Sorted cell: CD10+/CD45+ Monoclonal Peaks 345.7 bp for FR1 and 280.7 bp for FR2 in unsorted and sorted cell fraction (CD10+/CD45+) not in sorted control cell fraction (CD10-/CD45+) [blue=FR1; black=FR2; green=FR3; red=size standard]

19 y/o female, precursor B-ALL s/p BMT, 0.3% relapse

Cell sorting for precursor B-ALL

Conclusion Hierarchical approach to diagnostics Integration of multiple techniques Establish diagnosis and prognosis with minimum of tests Develop strategy for monitoring treatment Requires close interaction between hematologist/pathologist/hematological core Lab

Diagnostic Techniques Morphology; IHC Cytogenetics FISH Flow Cytometry Molecular Analysis; DNA/RNA

Strategy of development at SCTC

Standard processing & instant report BM or PB or tissue sample come in Immediate morphology & flow screening (within 24 hours) Molecular study (sorted or unsorted) if needed (within 48 hours) Cytogenetics or FISH (sorted or unsorted) if needed (within 72 hours)

Member Recruitment & Training Technician for morphology & flow cytometry Technician for molecular analysis (include all hematological malignancies) Flow & sorting training: Hematologics, Seattle Honor Dr. Michael Loken by: Inviting Hematologics affiliate to SCTC Honor Dr. Loken to be distinguished professor at SCTC

Compassionate support to hematological society in Taiwan Cooperate with non-medical centers that can not perform standard hematological study Free charge for half a year since 2009 Establish potential strategical alliances in the long run, including publication and patients referral Charge as government controlled insurance fee after solid relationship created

Thank You!