1. 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

2. 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

3. 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.

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

5. 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

6. Principle of flow cytometry

7. Structure of flow cytometry

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

9. Normal B lymphoid maturation

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

11. Normal B lymphoid maturation

12. Antibodies panels used for myeloid assessment
Tube FITC PE PerCP
# isotope isotope CD45
# HLA-DR CD11b CD45
# CD CD CD45
# CD CD CD45
# CD CD CD45
# CD CD CD45
# CD CD CD45
# CD CD CD45
# HLA-DR CD CD45

13. Antibodies panels used for B-lymphoid assessment
Tube FITC PE PerCP
# isotope isotope CD45
# HLA-DR CD11b CD45
# CD CD CD45
# CD CD CD45
# CD CD CD45
# CD CD CD45
# kappa CD CD45
# lambda CD CD45
# FMC CD CD45

14. 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

15. Minimal Residual Detection for AML

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

17. Aberrant expression of CD56 & CD7
Diagnostic sample
MRD 0.1%

18. Aberrant expression of CD19
Diagnostic sample
MRD 0.6%

19. Minimal Residual Detection for ALL

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

21. CALLA (-) precursor B-ALL

22. Relapse s/p BMT, MRD 0.3%

23. Flow Cytometric Diagnosis of MDS

24. 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-

25. 53 y/o female, MDS, CD56+ on blast, monocyte & neutrophil

26. Flow Cytometric Diagnosis of Lymphoma

27. 66 y/o man, Maltoma, lacrimal gland, for staging
HE staining of BM

28. 7.0% clonal marginal zone lymphoma, lower CD45 as compared to lymphocyte, CD19+, CD20+, FMC7+, kappa+, lambda-

29. L26 staining

30. Flow Cytometric Diagnosis of Multiple myeloma

31. 68 y/o female, multiple myeloma with cytoplasmic lambda restriction, (30.0%)

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

33. Flow Cytometric Analysis of Tissue Sample

34. 55 y/o man, left axillary LN: CD10+, CD20+, CD19/lambda+, diagnostic of follicular center B-cell lymphoma

35. Flow Cytometric Diagnosis of Non-malignant Disorder

36. 27 y/o female, macrocytic anemia, Acid-Ham test (-) PB flow: PNH, heterozygous clone
Neutrophil: dim CD16
Monocyte: lose CD14

37. BM flow

38. Application of Cell Sorting for MRD Confirmation

39. 53 y/o man, follicular center B-cell lymphoma

40. Cell sorting for follicular B-cell lymphoma
Unsorted cells
Sorted cell: CD10-/CD45+
Sorted cell: CD10+/CD45+
Monoclonal Peaks bp for FR1 and 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]

41. 19 y/o female, precursor B-ALL s/p BMT, 0.3% relapse

42. Cell sorting for precursor B-ALL

43. 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

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

45. Strategy of development at SCTC

46. 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)

47. 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

48. 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

49. Thank You!