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Principle of flowcytometry

&

diagnostic applications

Principle of flow cytometer

Diagnostic applications

Introduction of BriCyte E6

Principle of flow cytometer

Principle of sheath flow

Principle of fluorescence

Principle of optical filter

Principle of signal detection

Reaction of antigen and antibody

Principle of fluorescence staining

Data analysis

Leakage and compensation of fluorescence

Principle of sheath flow

Laminar flow

Turbulent flow

Fluid motion

Laminar flow

Turbulent flow

A fluid flows in parallel layers, with no disruption between the layers

The fluid flows are no longer keep laying and the layers are confused

Sheath flow

Sample flow

Flow cell

Principle of fluorescence

the emission of light by a substance that has absorbed light or other electromagnetic radiation

Fluorescence

The substance can emit fluorescence after excitationFluorescein

Generation process of

fluorescence

Excited state

Ground state

Laser Fluorescence

wavelength

Intensity

Principle of optical filter

480 535 580 480 535 580

530LP 530LP 530/30LP

480535 580

Optical filter

Long pass filter（LP）

Short pass filter（SP）

Band pass filter（BP）

Principle of signal detection

Forward scatter（FSC）

Side scatter（SSC）

fluorescence（FL）

FSC detector

FSC detector

SSC detector SSC detector

t

v

Height （H）

Width （W）

Area （A）

Signal detection

Principle of fluorescence staining

Fluorescent probe

Role of fluorescein

Fluorescence label

Fluorescein

fluorescein antibody Cell with antigen

Reaction of antigen and antibody

Specificity

Proportionality

reversibility

characteristics

Principle of measuring

FL4

FL6

FL5(PE-cy7)

FL3(PerCP)

FL2(PE)

FL1(FITC)

SS

488/10

530/30

FS

488/10

585/40

670LP

785/60

491/LP

550LP

645LP660/20

785/60740LP

488/10

反光镜

t

v

FL6(APC-cy7)

FL4(APC)

反光镜

t

vt

v

t

v

t

v

t

v

t

v

t

v

FSC

SSC

FL1

FL2

FL3

FL5

Lasers and fluorescein

488nm：– FITC

– PE

– PerCP

– PerCP-Cy5.5

– PE-Cy7

– PE-Cy5

– PE-Texas Red

638nm：– APC

– APC-Cy7

Data analysis—Graphs

Histogram

Data Presentation

Dot plot

Contour plot

Density plot

3-D plot

Data analysis—Gate

Polygonal gate & Quadrant gate

Interval gate & Bifurcated gate

Data analysis—statistics

Percentage 、absolute number

mean、SD、CV……

Fluorescent leakage and compensation

FITC530/30

PE585/40

+ += =

Fluorescent leakage：1. Each fluorescein has its own spectra of emission light;

2. Spectrum of different fluorescence may overlap each other；

Signals in FITC channel

signals of interest

Signals in PE channel

signals of interest

Fluorescent leakage and compensation

FL1

FL2

Q1 Q2Q3 Q4

FL1

FL2

FL1

FL2

FL1

FL2

Eliminate the signals in FL2 from FL1

Eliminate the signals in FL1 from FL2

Y-medianQ3=Q4

X-medianQ1=Q3

Q1 Q2Q3 Q4

Q1 Q2Q3 Q4

Q1 Q2Q3 Q4

Diagnostic applications

Leukaemia diagnosis

Immune deficiency diagnosis

Predicting response to mono clonal

antibody therapy

Rare event detection

Screening for genetic disease

Case studyA four-year-old female

Lethargic for three weeks without clear cause.

petechiae on her arms and legs

low platelet count

Examination of a blood film immature cell (blast).

A bone marrow slide A large population of small blasts with scanty cytoplasm

samples of anticoagulated bone marrow.

Flow cytometryCD2 (pan T-cell) and CD7 (pan T-cell)

CD10 (precursor B-cells) and CD19 (pan B-cells)

CD33 (myeloid cells) and CD45 (pan leukocyte)

Cytoplasmic CD3 (pan T-cells), cytoplasmic CD79a (pan B-cells) and

cytoplasmic myeloperoxidase (pan myeloid cells)

precursor B-cell acute lymphoblastic leukaemia (ALL)

Leukaemia diagnosis

Interpretation

The cells were positive (>20% expression) for CD19, CD10 and cytoplasmic CD79a

Interpretation of leukemia immunophenotyping data: expression of non-lineage markers(CD13 in a case of B precursor ALL = aberrant expression

The specificity of different markers in the assignment of lineage is recognized in a scoring system which allows distinction between leukemia cases with aberrant expression of a marker from the far rarer cases where two distinct lineages are involved in the leukemic process (mixed phenotype acute leukemia)

Immune deficiency diagnosis

Case study A four-year-old male with recurrent ear infections

a full blood count and immunoglobulin are requested.

The immunoglobulin results are as follows:

IgG = 0.97 g/L (reference range 5.9–15.5 g/L);

IgA = 0.06 g/L (reference range 0.4–2.0 g/L);

IgM = 0.08 g/L (reference range 0.5–2.1 g/L).

Flow cytometry is requested to enumerate T, B and NK cells.

Flow cytometry

Total lymphocyte count = 3.1 × 109/L (normal)

CD3 (total T-cells) = 89%, absolute count 2.76 × 109/L (reference range 1.8–3.0 × 109/L)

CD4 (T helper cells) = 65%, absolute count 2.02 × 109/L (reference range 1.0–1.8 × 109/L)

CD8 (T suppressor/cytotoxic cells) = 24%, absolute count 0.75 × 109/L (reference range

0.8–1.5 × 109/L)

CD19 (B-cells) = <1%, absolute count <0.01 × 109/L (reference range 0.7–1.3 × 109/L)

CD16+CD56+ (NK cells) = 11%, absolute count 0.34 × 109/L (reference range 0.2–0.6 ×

109/L)

Interpretation

Hypogammaglobulinaemia and absent B-cells suggestive of a primary immune deficiency associated with defects in Bruton's tyrosine kinase (Btk).

This can be confirmed by Western blotting for Btk protein and genetic analysis.

Other causes of absent B-cells with hypogammaglobulinaemia include thymoma, common variable immune deficiency, related to therapeutic drugs or other rare congenital defects in the B-cell development pathway.

Predicting response to monoclonal

antibody therapy

Humanized monoclonal antibodies therapeutic

use

Rituximab :anti-CD20 present on B-cells

Gemtuzumab :anti-CD33 present on myeloid

cells

Alemtuzumab :anti-CD52 present on lymphoid

cells

Case study A 48 -year-old female with a diagnosis of CLL has had a poor response to

first-line chemotherapy.

the addition of immunotherapy with Alemtuzumab.

Prior to commencing treatment, assess the expression of CD52 on the patient's tumor cells.

Flow cytometry

The following antibody combination is used:

CD52 (binding site for Alemtuzumab), CD19 (pan B-cells), CD5 (pan T-cells and B CLL cells) and CD3 (pan T-cells).

The expression of CD52 is assessed on these cells. The expression of normal T-cells within the sample (CD3-positive) acts as a control confirming the activity of the CD52 antibody. (All normal T-cells highly express CD52).

Interpretation

The malignant cells express CD52. This confirms that it would be reasonable to use Alemtuzumab in this patient.

Rare event detection

High-speed analysis coupled with multicolour staining essential for rare events

Detect residual leukaemic cells after the initiation of therapy (MRD)

Rh(D)-positive fetal red cells transferred to an Rh(D)-negative mother across the placenta at birth

estimating the progenitor cell content in bone marrow transplantation

enumeration of contaminating white cells in filtered blood

detection of abnormal red and white blood cells in patients with bone marrow failure disorders (subclinical PNH clone detection)

Case study

A 56-year-old male who is being treated for B-cell non-Hodgkin's lymphoma

peripheral blood stem cell (PBSC) collection following his fourth course of chemotherapy

The stem cells will be stored to allow an autologous PBSC transplant as part of his future therapy.

To allow the collection to be timed to ensure the efficient collection of PBSCs

peripheral blood progenitor cell count is measured from the first day of neutrophil recovery.

Flow cytometry

Peripheral blood CD34 count (progenitor cells) enumeration

A graphical representation of absolute CD34 count in cells per microlitre is shown for

consecutive days

Peripheral blood CD34+ cell counts measured on consecutive days following

chemotherapy in a patient with non-Hodgkin's lymphoma

Reliable counting of rare events needs large numbers of events

To achieve an intra-assay coefficient of variation of 10%, 100 positive events need

to be collected.

In practice, this means that between 500,000 and 1 million cells need to be

acquired.

Screening for genetic disease

Some genetic diseases result in the failure to

express a protein on the surface of cells

This methodology is employed in the

investigation of platelet disorders

(Glanzmann's thrombasthaenia and Bernard–

Soulier syndrome)

leukocyte adhesion deficiency

X-linked hyper IgM syndrome

Case study

A nine-month-old male with respiratory distress.

There is no history of HIV in the family and the child tests negative for HIV.

PCP is confirmed by culture

The lymphocyte count and T- and B-cell subsets are normal.

Immunoglobulins show undetectable IgG and IgA with a normal IgM.

X-linked hyper IgM syndrome is suspected and samples are sent to check for normal expression of CD40 ligand

Flow cytometry

Whole blood from the patient and control are incubated for six hours at 37°C with phorbolmyristate acetate and calcium ionophore to activate the cells. Aliquots of activated whole blood are stained with the following combinations:

CD3 (pan T-cells) and CD154 (CD40 ligand)

CD3 (pan T-cells) and CD69 (activation marker).

Introduction of BriCyte E6

Characteristics

Characteristics

Carrousel mode& Single tube mode

Compatible for more tubes

Convenient voltage adjusting

Automatic compensating

More suitable for clinical application

Support LIS

Configuration

Loading mode

Manual loading

Auto loading

Optical system

2-laser,4-color

2-laser,5-color

2-laser,6-color

Characteristics

Flow Rate

Low rate：~ 10μL/min

Mid rate：~ 50μL/min

High rate：~ 100μL/min

Data Format : FCS3.0/export built-in data

format

Dynamic Range :20 Bit

Workstation Resolution:2 ^ 20

Parameters

Types: FSC, SSS and FL1-FL6, each channel includes peak value (Height), pulse integration(area), pulse width, time and self-defined parameters (including ratio etc.), with the enumeration of CD3/4/8 absolute number and percentage

Quantity: 8×4+N (8 is the number of channels: FSC, SSS, FL1-FL6; 4 is the parameter quantity of each channel: H、A、W and T; N represents the number of self-defined parameters)

Characteristics

• Minimum Sample Volume :100 μL

• Max Acquisition Rate :12,000 events/sec

Thank you