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CLINICAL PATHOLOGY

FLOW CYTOMETRY
DAPHNE ANG, MD
2ND SHIFT, OCTOBER 19, 2017

TOPIC OUTLINE 2. PLATELET FUNCTION ANALYSIS


I. Introduction to Flow Cytometry Platelet receptor quantitation
A. Clinical Applications o Diagnosis of congenital platelet disorder
1. Lymphocyte Subset Enumeration o Detection of surface markers
2. Platelet Function Analysis o Bernard Soulier: GP1b
B. Principle o Glanzmanns thrombasthenia: GP IIb/IIIa
C. Interpreting Data o Platelet function abnormalities: in patients with bleeding,
normal CBC and platelet count, normal PT and aPTT
II. Panel
o Run peripheral blood in flow cytometry, incubate with
A. Lymphoid Malignancies
antibodies against the specific glycoprotein, if absent
B. Myeloid Malignancies
expression of glycoproteins diagnosis of the specific
III. Two-Parameter Histogram platelet function abnormality
IV. Review
B. PRINCIPLE
I. INTRODUCTION TO FLOW CYTOMETRY
Sample can be lymph node, bone marrow aspirate, or
Method of measuring multiple physical and chemical peripheral blood (last two are the most common)
characteristics of particles by optical means
Incubate first the sample with fluorescently-labeled
Involves the analysis of the fluorescence and light scatter antibodies of interest before injecting into the flow sheath
properties of a single particle/cell during their passage
Laser will activate fluorescently-labeled probes
through narrow, precisely defined liquid stream
Fluorescence emitted translated into histogram where
individual dots represent one cell
A. CLINICAL APPLICATIONS
Machine can show in the histogram the relative percentage
Immunophenotyping: used in diagnosing hematological
and count of cells with the specific marker
diseases; faster diagnosis than immunohistochemistry
Fluorescent dyes commonly used in Flow Cytometry
o Acute leukemias
o Fluorescein isothiocyanate (FITC)
o Chronic lymphoproliferative
o Texas red
o Disorders malignant lymphomas
o Phycoerythrin (PE)
Monitor progress of patients after chemo and bone marrow
transplantation
Detection of residual disease
o Monitoring of minimal residual disease
o Mainly used in patients with leukemia after the patient
receives chemotherapy
o Send bone marrow aspirates to be checked
microscopically and also perform flow cytometry
o Residual leukemic blasts in the patient not seen
morphologically in the slides but can be detected with
flow cytometry because of its sensitivity (can go down up
to 0.1% blast)

1. LYMPHOCYTE SUBSET ENUMERATION


Monitoring CD 4 T cells in HIV
o Decreased CD 4 T helper lymphocytes in HIV patients
o Only know the percent lymphocytes with CBC and
differential count but cannot detect if CD 4 or CD 8
o Count the CD 4 cells and determine the CD4:CD8 ratio C. INTERPRETING DATA
with flow cytometry Single parameters can be displayed as a histogram
Stem cell transplantation
o For patients with worst prognosis who have molecular
markers that indicate that the patient will have recurrence
soon after remission
Immunodeficiency
o T cell: CD 3, CD 4, CD 8
o B cell: CD 19 (CD 20)
o NK: CD 16, CD 56

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TAYAG, P.I.L.
[CLINICAL PATHOLOGY] FLOW CYTOMETRY
DAPHNE ANG, MD

Double parameter data can be displayed in two dimensions


using dot, density or contour plots

II. PANEL
CD 45: differentiates hematologic malignancies from other
B. MYELOID MALIGNANCIES
neoplasms
Myeloid all other cells except lymphocytes
o Also known as common leukocyte antigen: seen in all
AML
WBCs/leukocytes
o CD 13, CD 33, CD 117
CD 34 and HLA DR Ags: markers of hematopoietic stem
Monocytic
cells; quality assurance in bone marrow transplant
o CD 4, CD 11b, CD 11c, CD 14, CD 36, CD 64 or CD 68
o CD 34: marker currently used to identify stem cells
Stem cell transplant: stem cell from umbilical cord or Megakaryocytic
o CD 41 and CD 61
donors; count CD 34 cells in peripheral blood from
donor to know if it reaches the cut-off Erythro Leukemia
o HLA DR antigens: seen positive in a lot of blasts but o CD 235 (glycophorin)
generally not used for stem cell counting Myeloperoxidase: most specific marker of myeloid lineage;
Pan B cell: CD 19, CD 20, CD 22 M0-M7 all (+) MPO
Pan T cell: CD 2, CD 3, CD 4 and/or CD 8, CD 7 (CD 5 also
included as a marker in the lecture but not indicated in ppt) FAB Classification of AML (review WBC disorder trans)
o TTN: Pan meaning all, B for Big numbers (19, 20, 22), T for o M0: AML Minimally Differentiated
Tiny numbers (2, 3, 4, 5, 8, 7) 90% blasts, <3% MPO (+)
CD 13, 33, 117
A. LYMPHOID MALIGNANCIES o M1: AML without Maturation
Lymphoid lymphocytes (CD 4 T helper cells, CD 8 T 90% blasts, >3% MPO (+)
cytotoxic cells, B cells, NK cells) CD 13, 33, 117
B cell neoplasm o M2: AML with Maturation
o Precursor B malignancy is 75-85% of ALL <90% blasts
o Use CD 10 and 19, CD 34, Tdt CD 13, 33, 117
CD 19, 20, 22 o M3: Acute Promyelocytic Leukemia
CD 10: non-specific Associated with DIC
CD 34: present in all blasts, doesnt help in Chromosome 15 and 17 translocation
differentiation CD 13, 33, 117
Tdt: marks immature lymphoblasts; mostly negative in o M4: Acute Myelomonocytic Leukemia
myeloblasts 20% myeloblast, >20% but <80% monoblast
T cell neoplasm Use monocytic markers CD 4, 14, 64, 11b, 11c (CD
o 15-25% of ALL 64 and 68 are seldomly used) to identify monocytic
o Use CD 7, CD 3, CD 34, Tdt differentiation
CD 4: T helper cells o M5: Acute Monoblastic Leukemia/Schilling Leukemia
CD 8: T cytotoxic cells >80% monoblasts
Cytoplasmic CD 3: most specific for diagnosis of T o M6: Acute Erythroid Leukemia/Di Guglielmo
cell neoplasm Syndrome
o Older males with high blast count and mediastinal mass Blasts should present with erythroid lineage marker
FAB VS WHO CD 235 (glycophorin)
o FAB o M7: Acute Megakaryoblastic Leukemia
Morphology, cytochemical stains CD 41 and CD 61
ALL = 3
AML = 7
o WHO
Morphology, cytochemical stains, flow cytometry,
karyotype and molecular data

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TAYAG, P.I.L.
[CLINICAL PATHOLOGY] FLOW CYTOMETRY
DAPHNE ANG, MD

III. TWO-PARAMETER HISTOGRAM

X-axis: CD 45 (common leukocyte antigen)


Y-axis: Side scatter (granularity)
Orange population: dim CD 45, low side scatter blasts

Order of CD 45 expression (highest/brightest to lowest)


Lymphocytes>Monocytes>Neutrophils
*Blasts: usually have dim CD 45 expression; usually sits right
under lymphoid cells

IV. REVIEW
Forward scatter: size Step 1: Diagnosis of acute leukemia
Side scatter: granularity
o TTN: Fansign in Singapore/FS in SG (FS: Forward, size; SG:
Side, granularity)

X-axis: CD 45 (common leukocyte antigen)


Y-axis: Side scatter (granularity)
Upper black population: high side scatter, (+) CD 45
X-axis: Forward scatter (size) expression neutrophils
Y-axis: Side scatter (granularity) Lower black population: low side scatter, bright CD 45
In assessing the size and granularity, look at each axis expression lymphocytes
individually Red encircled population: dim CD 45 expression blasts
Red population: high forward scatter, high side scatter Patient has acute leukemia: abnormal number of blasts
neutrophils (large size, high granularity)
Purple population: low forward scatter, low side scatter
lymphocytes (small size, low granularity)

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TAYAG, P.I.L.
[CLINICAL PATHOLOGY] FLOW CYTOMETRY
DAPHNE ANG, MD

Step 2: Identify the type of leukemia (rule out) X-axis: CD 34 (marker to identify stem cells; present in all
blasts)
Y-axis: CD 14 (monocytic marker)
Red population: (+) CD 34, (-) CD 14 proves that they are
blasts, verify AML diagnosis

X-axis: CD 10 (marker for B cell neoplasm)


Y-axis: CD 19 (marker for B cell neoplasm)
Red population: (-) CD 10, (-) CD 19 rule out B cell
lymphoid malignancy
Immunophenotyping by Flow Cytometry (edited, see original
Step 3: Identify type of leukemia (rule out) diagram at WBC disorder trans page 6)

OTHER EXAMPLES
Example 1: blast (+) CD 34, (+) Tdt, (+) CD 5, (+) CD 3 T
cell Acute Lymphoblastic Leukemia (ALL)
o CD 34: stem cell marker; present in all blasts
o Tdt: marks immature B and T lymphoblasts
o cCD 3: most specific T cell marker
Example 2: blast (+) MPO, (+) CD 235, (+) CD 34 M6
Acute Erythroid Leukemia
o MPO: most specific marker of myeloid lineage
o CD 235: glycophorin; erythroid lineage marker
o CD 34: stem cell marker; present in all blasts
Example 3

X-axis: CD 33 (AML marker)


Y-axis: CD 7 (T cell marker)
Red population: (+) CD 33, (-) CD 7 AML

Step 4: Use other markers to verify diagnosis

X-axis: CD 8 (T cytotoxic cell marker)


Y-axis: CD 4 (T helper cell marker)
(+) CD 4, (-) CD 8: T helper cells
(-) CD 4, (+) CD 8: T cytotoxic cells
(-) CD 4, (-) CD 8: possibly B cells and NK cells
(+) CD 4, (+) CD 8: double-positive thymocytes

-END-

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TAYAG, P.I.L.

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