1. Introduction to Flow Cytometry
Dr. Fatma Al-Qahtani

2. What is Flow Cytometry? Flow refers to a fluid stream
Cyto refers to a cell
metry refers to measurement.

3. What Is Flow Cytometry?
Simultaneous measurements of multiple characteristics of a single cell
Measurements are made on a per-cell basis at routine rates of 500 to cells per second

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 (FL1, FL2, FL3, FL4, and FL5)

5. Properties of FSC and SSC
Right Angle Light Detector  Cell Complexity
Forward Light Detector  Cell Surface Area
Incident Light Source
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

7. Lysed Whole Blood Side Scatter Forward Light Scatter 200 400 600 800
200
400
600
800
1000
Neutrophils
Side Scatter
Monocytes
Lymphocytes
200
400
600
800
1000
Forward Light Scatter

8. What is Fluorescent Light?HO O
 = 488 nm
  530 nm C
Incident Light Energy
CO2H
Emitted Fluorescent Light Energy
Fluorescein Molecule
Antibody
The fluorochrome absorbs energy from the laser
The fluorochrome releases the absorbed energy by:
Vibration and heat dissipation
Emission of photons of a longer wavelength

9. Absorption and Emission Spectra of a Fluorochrome
FITC
1000
800
600
400
200
400
450
500
550
600
650
700
Wavelength (nm)

10. A Cytometer Needs a Combined System of:
Fluidics
To introduce and focus the cells for interrogation
Optics
To generate and collect the light signals
Electronics
To convert the optical signals to proportional electronic signals and digitize them for computer analysis

11. Optics–FACSCalibur . FL1 530/30 SSC 488/10 FL2 585/42
90/10 Beam Splitter
FL4
661/16
DM 560SP
DM 640LP
Half Mirror
670LP
Fluorescence Collection Lens
FL3
Beam Combiner .
Flow Cell
488 nm Blue Laser
Red Diode Laser ~635 nm
FSC Diode
488/10
Focusing Lens

12. Two-Color Direct Staining
Analyze
Incubate
Wash

14. Two-Color Cell Analysis
CD19 PE
CD3 FITC
10 0
10 1
10 2
10 3
10 4

15. Three-Color Direct Staining
Analyze
Wash
Incubate

17. Three-Color Cell Analysis
10 4
10 4
10 4
10 3
10 3
10 3
CD4 FITC
10 2
CD8 PE
10 2
CD8 PE
10 2
10 1
10 1
10 1
10 0
10 0
10 0
10 0
10 1
10 2
10 3
10 4
10 0
10 1
10 2
10 3
10 4
10 0
10 1
10 2
10 3
10 4
CD3 APC
CD4 FITC
CD3 APC

18. DNA Histogram Counts FL2-Area 250 200 150 100 50 200 400 600 800 1000
G0/G1
200
150
Counts
100 50 S
G2/M
200
400
600
800
1000
FL2-Area

19. Flow Cytometric Applications in Clinical Laboratories
HIV immunophenotyping (CD4/CD8 ratio)
Leukocytes Subsets
Leukemia and lymphoma immunophenotyping
Cell cycle and ploidy analysis of tumors
Reticulocyte enumeration
Flow crossmatching (organ transplantation)
Stem cell enumeration
Residual white blood cell detection (QC platelet, red blood cell packs)

20. Flow Cytometric Applications (cont.)
TdT Assay
Diagnosis of PNH
Assessment of Fetal Maternal Hemorrhage
Detection of Platelet associated Antibodies
DNA Index
Detection of acrosomal reaction in sperms

21. Flowcytometery 1979

23. 1995
1986
2004

24. Case 1
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.

26. 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.

27. 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.

28. Here we are looking at unusual pattern of staining for the CD10/CD19/CD20. We can first see that the normal lymphocytes (R1) are staining negative for CD10 and positive for CD19/CD20; and this is what we expect of normal B-cells, at the same time this pattern of staining makes a good internal control for the reactivity of CD10. On the other hand we can see that the Blasts (R2) not only staining for CD10/CD19 but they are also expressing CD20. This finding is important because in normal B-cell development as the cell start to express surface CD20, they will start to lose the expression of CD10. Any time that we find an asynchrony in the expression of surface markers usually this will be a clear indication of malignancy such as the case.

29. 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.

30. 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.

31. Antigen profile: positive for CD10, CD19, CD20, CD22, CD9 and surface Kappa, HLA-DR and CD45. FISH: using IGH/MYC t(8;14) DNA probe signal was positive in 92% of the cells
Diagnosis: Findings consistent with Burkitt Lymphoma

32. Case 2
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 to role out M3

34. 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.

35. 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

36. Here we are looking at more characteristics phenotype of APL
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.

37. Again we are looking at additional markers , and we find that the blasts are clearly positive for CD9, CD64 and slightly partially for CD15 and negative for CD117, CD11b and CD2.

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

40. Case 3
Seven (7) year old, male patient, referred with Clinical history of Mediastinal Mass.
Peripheral Blood Examination showed high Leukocyte Count with 26% Blasts.

42. 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.

43. 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).

44. Looking at this slide we learn more about the nature of these cells
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.

45. The abnormal cells are positive for CD2 and CD11b and negative for HLA-DR. Usually in most cases of the T-cells they tend to be negative for HLA-DR. The positivity of CD11b is also atypical of this kind of malignancy.

46. 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.

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

48. Antigen profile: Positive for CD4, CD8, CD7, CD2, CD5, CD56, partially CD1a, cCD3 and negative for HLA-DR. Molecular: Positive for TCR gamma gene rearrangement by PCR
Diagnosis: Acute T-cell Leukemia