Introduction to Flow Cytometry Dr. Fatma Al-Qahtani

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

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 4000 cells per second

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)

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

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

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

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

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

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

Two-Color Direct Staining Analyze Incubate Wash

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

Three-Color Direct Staining Analyze Wash Incubate

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

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

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)

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

Flowcytometery 1979

1995 1986 2004

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.

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.

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.

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.

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.

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.

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

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

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.

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

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.

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.

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)

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.

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.

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).

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.

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.

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.

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

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