Flowcytometry

1. Application of Flow
Cytometry in the
Diagnosis of
Hematological Disorders

2. What Is Flow Cytometry?
 Flow cytometry (FC) plays an indispensable role in a multimethodology approach to
diagnosis of hematologic diseases , by providing data on the immunophenotype
 Flow ~ motion
 Cyto ~ cell
 Metry ~ measure
 It is a laser-based biophysical method capable of providing both qualitative and quantitative
measurements of multiple characteristics of a single cell or any other particle by measuring a
particle’s optical and fluorescence characteristics.
 Flow cytometry can provide information not only on cell size but also on cytoplasmic
complexity, DNA or RNA content, and a wide range of membrane-bound and intracellular
proteins.

3. Principles of Flow Cytometry
Flow cytometry relies on the application of fluorescent conjugates or
fluorochromes attached to a monoclonal antibody (McAb) that has
specific affinity for an antigen on the cell surface, in the cytoplasm or in
the nucleus. Each fluorochrome has a characteristic peak excitation and
emission wavelength, and the emission spectra often overlap, requiring
compensation. Consequently, the combination of fluorescent labels that
can be used depends on the wavelength of the laser used to excite the
fluorochromes and on the detectors available.

4. What Can a Flow Cytometer
Tell Us About a Cell?
 Its relative size (Forward Scatter—FSC)
 Its relative granularity or internal complexity
(Side Scatter—SSC)
 Its relative fluorescence intensity

5. Properties of FSC and SSC
When cells pass through the laser intercept, they
scatter laser light
Forward Scatter—diffracted light
 Related to cell surface area
 Detected along axis of incident light in the forward
direction
 Side Scatter—reflected and refracted light
 Related to cell granularity and complexity
 Detected at 90° to the laser beam
Right Angle Light Detector
 Cell Complexity
Forward Light Detector
 Cell Surface Area
Incident
Light
Source

8. Lysed Whole Blood
Neutrophils
Monocytes
Lymphocytes
SideScatter
Forward Light Scatter
0 200 400 600 800 1000
02004006008001000

9. Flowcytometery 1979

10. 1986
1995
2016

11. Flow Cytometric Applications
in Clinical Laboratories
 Diagnosis of Hematological Malignancies
 Detection of Minimal Residual Disease
 Lymphocyte Subset Enumeration for Immunodeficiency Disease
 Analysis of DNA Ploidy, the Cell Cycle, and Cell Death
 Measurement of the Efficacy of Cancer Chemotherapy
 Reticulocyte Enumeration
 Platelet Function Analysis
 Stem cell enumeration
 Diagnosis of PNH
 Assessment of Fetal Maternal Hemorrhage

12. Diagnosis of hematological malignancies

13. Diagnosis of acute leukemia
Immunophenotyping by flow cytometry
(FC) plays an important role in the
diagnosis, subclassification, and monitoring
of patients with acute leukemias

14.  The classification of acute leukemia is based on
morphologic,
immunophenotypic, cytogenetic, molecular, and clinical data
Generally, acute leukemias are divided into acute
lymphoblastic
leukemia (ALL) and acute myeloid leukemia (AML),
with a subset of leukemias displaying mixed phenotypic
features
(acute biphenotypic or bilineal leukemia).

15. WHO 2008 criteria for lineage
assignation in acute leukaemia.
Lineage Relevant antigen
Myeloid :MPO positive
Monocytic antigens (at least two of CD11c,
CD14 or CD64)
T-lineage :Cytoplasmic CD3 or surface
CD3 (rare in mixed
phenotype AL)
B-lineage :Strong CD19 with at least one
of: CD79a,
cCD22, CD10 strongly expressed
Weak CD19 with at least two of:
CD79a, cCD22, CD10 strongly expressed

16. Panel Markers in Acute
Leukaemia
First-line screening: CD34, TdT, HLA-DR,
MPO,
CD19, CD79a, CD22, CD3
Second-line B ALL :CD10, cμ, CD20, CD58,
CD38
Second-line T ALL :CD1a, CD2, CD3, CD4,
CD5,
CD7, CD8, TCR
Second-line myeloid :CD13, CD33, CD117,
CD16,
CD11b
Second-line monocytic :CD14, CD64, CD36,

17. CASE 1
A 16-year old male was referred as a new leukemia. Peripheral blood had a high
WBC with 53% promyelocytes. Sample was sent for flow cytometry

19. The CD45/SSC display looks different than your typical bone marrow and peripheral blood patterns. You
can see that there are two clusters of cells one is the normal lymphocytes (red) and the other (green) are the
abnormal cells. For the observer the later population may look like neutrophils, but if you look closely you
will see that they are not. Usually the normal PMNs in peripheral blood start from approximately the
channel-600 on the SSC and spread higher (see arrow). In this particular case the distribution of the
abnormal cells start from the area where “blasts” tend to be present and gradually move higher overlapping
with the area where PMNs tend to be located. So definitely we are not dealing with PMNs but with cells
that are large and very granular.

21. This display is self explanatory in term of the CD3/CD4/CD8 staining on the normal lymphocytes (red). I would
like you now to pay attention to pattern of staining of the abnormal cells (green) with these monoclonal antibodies.
You can see that there is a considerable increase in the autofluoresence of the blasts (see brackets). This increase in
autofluoresence is characteristic of APL due to the high content of protein in the granules. Care should be taken
when placing the markers so there will be no false results

22. Here we are looking at more characteristics phenotype of APL. The blasts are homogenous positive for CD33,
heterogeneous positive for CD13, negative for CD34, HLA-DR and CD7. It is the lack of HLA-DR and CD34 that
kindly differentiate APL from other types of AML.

23. Again we are looking at additional markers , and we find that the blasts are clearly positive for , CD64 and slightly
partially for CD15 and negative for CD117, CD11b and CD2.

24. Antigen profile: Positive for CD45, CD33, CD13,
CD9 and cMPO.
Negative for HLA-DR, CD117 and CD34.
 Diagnosis: Acute Myeloid Leukemia, FAB(M3)

25.  Seven year old, male patient, referred with
Clinical history of Mediastinal Mass.
 Peripheral Blood Examination showed high
Leukocyte Count with 26% Blasts.
Case 2

27. Looking at this case display you can tell that there is an abnormal population (green) that is v.bright CD45
“brighter than normal lymphocytes” and has a high side scatter. There is almost no normal lymphocyte in
this bone marrow specimen.

28. This is a very informative slide and probably gave you the diagnosis already. Looking at the CD3/CD4/CD8
patterns we can see that the majority of the gated cells are dual positive for CD4/CD8 and negative for surface CD3.
This pattern is very uncommon in bone marrow and indicate the immature T-cell nature of the blasts. Among the
abnormal cells you can recognize the normal lymphocytes pattern (see red arrow).

29. Looking at this slide we learn more about the nature of these cells. We find that they are negative for
CD10,CD19,CD34,CD33,CD13 and positive for CD7. The later antibody “CD7” confirm the T cell lineage of the
abnormal cells.

30. Again the abnormal cells continue to confirm its T-cell lineage; they are positive for CD56, CD5, partially CD1a
and negative for CD117 and CD64. Usually the expression of CD56 in leukemias is associated with worse
prognosis.

31. The cytoplasmic staining of TdT and CD3 confirm the immature nature of the blasts.

32. Antigen profile: Positive for CD4, CD8, CD7, CD2,
CD5, CD56, partially CD1a, cCD3 .
Negative for HLA-DR. CD10,CD19,CD34,CD33,CD13
 Diagnosis: Acute T-cell Leukemia

33. flow cytometry in mixed
phenotype acute leukaemia
show simultaneousco-expressionof antigens specific
for two different lineages:
myeloid and lymphoid The myeloid component in
MPAL is defined exclusively by MPO positivity and/or
by clear evidence of a monocytic component (CD64,
CD14). The T-lymphoid component is defined by the
expression
of CD3, cytoplasmic or surface. Since there is no
marker specific for the B-lymphoid lineage, evidence
0f B-lymphoid differentiation should be based on the
expression of strong CD19 plus another B-cell marker
(CD10, CD22 or CD79a) or weak CD19 and strong
expression of two of the other specified B-cell
markers

34. Immunological markers in chronic
lymphoproliferative disorders

35. Immunological markers in chronic
lymphoproliferative disorders
A list of potential indications is listed
below:
Significant lymphocytosis (>6  109/L)
Lymphadenopathy and
lymphocytosis
Abnormal lymphoid morphology
Splenomegaly +/cytopenias where an
LPD is possible
Unexplained cytopenias where an
LPD is possible
Persistent unexplained erythroderma
Splenomegaly and lymphadenopathy
where a node

36. Diagnosis of B-cell disorders

38. Case 3
CLINICAL PRESENTATION :
•72 year-old white male who presented with hepato-splenomegaly and a 20- pound
weight loss in the past 8 weeks.
•CBC: markedly elevated WBC count with a lymphocytosis and 55% 'blasts' on the
peripheral smear.
•A bone marrow biopsy& aspirate were performed.
•BMA sample was sent for flow-cytometry.
•C.T. of the thorax and abdomen showed bilateral pleural effusions, small lymph
nodes in the axillae, marked splenomegaly with multiple low-density lesions&
extensive retroperitoneal lymphadenopathy.
•Other tests: cytogenetic and molecular genetic studies .

45. Antigen profile: Positive for CD19, CD 5, CD20 and FMC7, Kappa
restriction.
Negative for : CD3, CD 10, CD 23.
Diagnosis: Mantal cell lymphoma

46. Case 4
 A 2-year old female presented with fascial palsy
and bilateral nephromegaly, renal failure and high
LDH and uric acid with low hemoglobin.
 The aspirate was heavily infiltrated by immature
cells, with 100% blasts. The blasts were medium
to large in size with relatively high N/C ratio,
basophilic cytoplasm and vacuolations.
Myelopoiesis and erythropoiesis were suppressed.
Megakaryocytes were absent.

48. The first thing that you notice when you look at these two plots that there is a well defined cluster of cells
that have a dimmer CD45 and a slightly higher SSC signal when compared to the normal lymphocytes (R1).
The increase in the SSC signal usually indicate either the presence of granules or vacuoles in the cytoplasm
of the cells. The location of the blasts (R2) on the CD45 vs SSC plot fall in the same place that normally we
find Myeloblasts; but when we look at their location in term of FSC vs SC we find that they tend to be
slightly larger than lymphocytes (red) and smaller than monocytes . So we can not jump to conclusion when
we see these two patterns that we are dealing with myeloblasts.

49. Again we see here that the blasts (R2) are completely negative for CD3/CD4/CD8 staining; while the normal
lymphocytes(R1) express a normal pattern of staining for the same antibodies. You may see few events that are
positive for CD4 only (arrow). These cells most likely are normal Monocytes as the blasts area overlaps with the
normal monocytes; and normal monocytes tend to express CD4.

50. We see here that the blasts (R2) have additional marker that lead us in the direction that we are dealing with more
mature cells and that is the absence of CD34. Another indication of malignancy is the the bright expression of HLA-
DR compared to its expression on the surface of the normal lymphocytes (R1). Again one of the indications that
you are dealing with a malignant clone is the over or under expression of different antigen levels on the surface of
cell membrane.

51. In this slide we are looking at a very important combination of antibodies and that is Kappa/Lambda/CD19. If you
look closely we can see that there are clearly two different patterns of staining for these antibodies combination.
The first is the normal lymphocytes (red arrows) pattern of staining which is clearly show that the normal B- cells
are polyclonal in their expression for the kappa/lambda. What we mean with polyclonal is that approximately half
of the B-cells express kappa and the other half express lambda (arrows). The blasts (R2) on the other hand show a
definite clonal expression for the kappa light chain; meaning that all of the blasts express a single kind of light chain
(kappa) while they completely lack the expression of the lambda light chain.

52. In this slide we are looking at a very important combination of antibodies and that is Kappa/Lambda/CD19. If you
look closely we can see that there are clearly two different patterns of staining for these antibodies combination.
The first is the normal lymphocytes (red arrows) pattern of staining which is clearly show that the normal B- cells
are polyclonal in their expression for the kappa/lambda. What we mean with polyclonal is that approximately half
of the B-cells express kappa and the other half express lambda (arrows). The blasts (R2) on the other hand show a
definite clonal expression for the kappa light chain; meaning that all of the blasts express a single kind of light chain
(kappa) while they completely lack the expression of the lambda light chain.

53. Antigen profile: positive for CD10, CD19, CD20, CD22, CD9 and
surface Kappa, HLA-DR and CD45.
Diagnosis: Findings consistent with Burkitt Lymphoma

54. Diagnosis of T cell disorder

55. Flow cytometry in
myelodysplastic
syndromes

56. Flow cytometry in myelodysplastic
syndromes
The flow cytometric abnormalities typically detected in MDS include:
- abnormal co-expression of antigens that are normally present at different stages
of differentiation
- abnormal intensity of antigen expression
- presence of aberrant lymphoid lineage antigens expressed on myeloid
precursors, and abnormally decreased side scatter properties due to hypo
granularity of granulocytes.

57. Blasts in MDS can show increased
fluorescence intensity for CD117, CD13
or CD33, decreased CD45 , or aberrant
expression of lymphoid antigens such as
CD2, CD5, CD7, and CD56, and
paradoxical expression of mature
myeloid antigens, such as CD65, CD15,
CD10, CD11b.

58. Detection of Minimal Residual Disease
 Flow cytometry is used as a simple, rapid method for
detection of minimal residual disease (MRD), the
persistence of malignant cells in the bone marrow or other
tissues of patients with hematologic malignancies after
remission at levels below the limit of detection by
conventional morphologic assessment.

59. IMPORTANCE OF FLOWCYTOMETRY
Having the ability to rapidly analyze thousands of cells per second
also allows for quick answers.
We can have the results of a flow cytomtery test back to a
clinician within hours, allowing for prompt treatment and ultimately
supporting better outcomes, particularly for acute leukemia, a
disease that gets worse rapidly if not promptly treated.
In addition to clinical applications, flow cytometry has diverse
applications in basic science and cancer research, making it an
invaluable tool for clinical and research teams alike.