# Presentation: 571373

## CD4 Testing Using the BD FACSCountTM System and Good Laboratory Practices

**Burt Houtz, CLS**
**Technical Adviser, Flow Cytometry Applications**
**BD Biosciences**

## Purpose

- To enable laboratories worldwide to perform CD4 testing using a simple, accurate, and reliable instrument system
- To provide clinical standard operating procedures (SOPs) using best practices from authoritative sources

## Objectives

- Describe and discuss flow cytometry and CD4 immunophenotyping
- Understand how CD4 Testing can be performed on the FACSCount
- Demonstrate safe laboratory practices
- Demonstrate proper specimen processing and identification techniques
- Describe and perform immunofluorescent staining, instrument operation, quality control, and data acquisition

## Objectives

- Demonstrate the proper maintenance procedure for the FACSCount instrument
- Describe and discuss Quality Assurance for CD4 Enumeration
- Describe and discuss Data Reporting
- Discuss Reference Ranges
- Describe Proficiency Testing

## Modules

**Introduction to Flow Cytometry**
**Overview of CD4 Testing and the BD FACSCount**
**Laboratory Safety**
**Specimen Processing and Identification**
**Immunofluorescent Labeling with FACSCount Reagents**
**FACSCount QC and Analysis of Patient Specimens**
**FACSCount Maintenance**
**Lymphocyte Subset and CD4 Enumeration Quality Assurance**
**Data Reporting of CD4 Test Results**
**Reference Range Studies**

## Schedule

## Introduction to Blood Cell Biology and Immunophenotyping

## Learning Objectives

- After this presentation you will be able to:
- Identify relevant cellular components of the immune system
- Describe how monoclonal antibodies can be used as reagents
- Discuss applications for immunophenotyping

## Slide 9

**Notes:**
Human Blood Smear
The most common thing to run through a flow cytometer is human blood. This slide shows a human blood smear that has been stained so that cellular differences can be visualized. Notice the differences between cells, sizes, internal components, and colors due to a reaction with the stain.  Here are some noticeable characteristics of different cell types: 
 erythrocytes - very small 
 platelets - appear like fragments 
 lymphocytes- small and contain a single large nucleus  
 monocytes - kidney-shaped nucleus 
 granulocytes - granules inside and segmented nucleus

Note: The blood smear is stained with Wright's Stain, which makes basic granules appear blue and acidic granules appear red.

## Basic Cellular Immunology

**Lymphocytes**

**Granulocytes**

**Monocytes**

**(Macrophages)**

**Erythrocytes**

**T **
**Helper**

**T**
**Cytotoxic**

**Eosinophils**

**Neutrophils**

**Notes:**
Basic Cellular Immunology 
All blood cells originate from hematopoietic stem cells.  Stem cells are precursor cells that can give rise to the different types of blood cells.  Hematopoietic stem cells are normally located in the bone marrow, but in some conditions they can be present in circulation. ProCOUNT reagents and software allow us to detect and count hematopoietic stem cells. 
This is a diagram of the blood cells.  Blood cells can be divided into different subtypes.  Each subtype has its particular function and serves a unique purpose. 
· Erythrocytes (red blood cells) carry oxygen.
· Platelets clot blood.
There are 3 types of granulocytes:
· Neutrophils  are present in pus, serve as a first line of defense, digest invaders.
· Basophils are active during allergies to produce histamine, which causes a runny nose.
· Eosinophils fight off parasitic infections, such as worms.
· Monocytes are garbage collectors or scavengers.  They engulf foreign particles and destroy them.
Lymphocytes are cells that control the immune response. They react to certain specific molecules or markers on the surface of foreign cells. There are three types: T, B, and Natural Killer.
· T cells - mature in the Thymus (therefore are called T cells).  There are two subpopulations:
- Cytotoxic T cells kill virus infected cells.  In other words, they are toxic to infected cells. Cytotoxic T cells are also responsible for rejecting grafts.
- Helper T cells activate other immune cells.  Therefore they help our immune system to escalate a response against infections. 
· NK (Natural Killer) cells kill tumor cells but not normal cells.
· B cells mature in the bone marrow and therefore are called B cells.  B cells produce special molecules, called antibodies, against foreign invaders. Antibodies serve a key role in our immune system by directing other immune cells to recognize and destroy foreign invaders. We will discuss antibodies in more detail later. 
Most of the analysis of human blood focuses on the lymphocyte population.  

## Slide 11

**Notes:**
Targets of the Immune System

 Viruses, fungi, bacteria, transplanted organs, or cancer cells can all serve as a target for the immune system.  Antibodies produced by B cells will usually recognize those foreign cells or substances and bind to them. Antibody production and binding to a foreign substance or a cell is critical as a means of signaling other cells to engulf, kill or remove that substance from the body. 

## Foreign 

**Foreign **
**Invader**

**Notes:**
Basic Cellular Immunology (Antigen/Antibody)
This slide shows a representation of foreign invader cells.  You can see that there are molecules located on a cell surface.  Those molecules are called antigens.  Our bodies produce specific antibodies that recognize those antigens.  For simplicity, different shapes are used to demonstrate different antigen molecules. In reality, the antigens are more complex in their structure. 

Antibodies bind only specific antigens.  For example, as shown here, only the antibody with the rectangular binding site will bind the antigen with a rectangular surface. Only the antibody with the triangular binding site will bind the antigen with the triangular surface, and so on. A lock and key analogy is often used to describe the antibody-antigen interaction. 

## Monoclonal Antibody Production

**T cell**

- Monoclonal Antibody Production

**Notes:**
Monoclonal Antibody Production - Continued
This slide shows the cloned B-cell hybrids producing antibodies.  Since the specific antibodies are all exactly the same and they come from a single clone, they are called monoclonal antibodies.  
Clones of interest are propagated in large bioreactors and the antibodies produced by those cells are harvested.  
After harvesting, the antibodies are purified and a fluorescent dye is attached to each one.  
The process of attaching fluorescent dyes to an antibody is called conjugation.  Another term for a fluorescent dye is a fluorochrome.  
You'll read more about fluorochromes in the Introduction to Flow Cytometry.  

## Monoclonal Antibodies

- _CD#_		_SPECIFICITY_
- 3		T-Lymphocytes
- 4		T-Helper Lymphocytes
- 8		T-Cytotoxic Lymphocytes

**Notes:**
Monoclonals Commonly Used
So far in this presentation different antigens have been represented as triangles and squares.  However, cells are not referred to as square or triangle positive. A periodic worldwide leukocyte workshop identifies antibodies that bind the same antigens. An antigen is referred to as a cluster of differentiation (CD). The first antigen identified was called CD1, etc.   There are now over 100 CD numbers!  
This nomenclature avoids problems encountered earlier when each manufacturer gave antibodies their own brand names, making it difficult to remember the binding specificities of all the antibodies.  Therefore, CD numbers, rather than trade names given by different manufacturers, now refer to antibodies. This method makes it easier for the users to identify which antibodies to purchase for their applications.   For example, we know that if someone refers to a CD3 antibody, they are referring to an antibody that is specific to all T lymphocytes. 

Familiarize yourself with those common CD numbers and their binding specificities to different types of cells.

## T-Cell Subsets

- T

- T

- C

- H

**CD3**

**CD3**

**CD3**

**CD8**

**CD4**

**T-Cell Subsets**

**Notes:**
Using Antibodies to Subset T cells 	
We can further subset T cells into two groups: T helper and T cytotoxic cells shown here.  If you remember from the last slide, CD3 antibodies bind to all T cells.  CD3 antibodies in this slide have green dye on them.  So, all T cells will appear green.  However, we can further subdivide T cells into helper and cytotoxic T cells.  CD8 antibody is specific to T cytotoxic cells and it recognizes the antigens on their cell-surface.  A red fluorochrome is attached to CD8 antibody.  Therefore, T cytotoxic cells should have both green and red dyes on their surface at the same time.    

CD4 antibody is specific for T helper cells, and it binds to antigens on the surface of those cells.   In this case, CD4 antibody has an orange fluorochrome attached to it.  T helper cells should exhibit both green and orange colors.  
In this example, we are making use of 3 fluorochromes: green, orange, and red.  This is a demonstration of how you can use those colors to identify 2 subsets of T lymphocytes.    

## Two-Color Direct Staining

- Analyze

**Two-Color Direct Staining**

**Notes:**
Two-Color Direct Staining
This slide demonstrates a simplified view of how the cells are prepared for analysis by flow cytometry.  The monoclonal antibodies conjugated to fluorochromes, depicted as orange and green in this example, are added to the blood cells.  The antibodies will bind to the cells if there are antigens on the cell surface that these antibodies recognize.  After a washing step the cells will be analyzed by flow cytometery.  Based on their fluorescent staining, a researcher will be able to determine which cell types are represented in the sample.  

Note: The use of BD MultiTest reagents simplifies the staining procedure by eliminating the wash step.   

## Slide 17

**Notes:**
Photograph of stained cells
This is a photograph of cells that have been prepared as described in the previous slide with orange- and green- labeled antibodies. 
Notice the different colors.  Previously, researchers used fluorescent microscopes to look at and hand-count cells that are green and orange.  They could also look at the size and shape of those cells.  It would be an extremely difficult task if one were to view 10,000 cells and determine what percentage was green or what percentage was both green and orange. 
Good thing we have flow cytometers, because that's the task they perform for us today! You can think of a flow cytometer as taking over for your eyes. The instrument will view the cells and make all the calculations: is the cell green? how green? how big?
One of the most useful calculations is the percentage, for example, what percent is green?  In this example, we've said that the green antibody binds T lymphocytes, therefore the percentage of green cells equals the percentage of T lymphocytes in the sample.  Of those T lymphocytes, we can also tell what percent are T helper cells or T cytotoxic cells.
Knowing the percentage of different types of cells is important.  This topic will be discussed next. 

## Reasons For Studying Blood Cell Populations

- Evaluate Immunodeficiency States
- Classify Leukemias/Lymphomas
- Monitor Graft Recipients

**Notes:**
Reasons for Studying Blood Cell Populations
Next few slides will discuss why researchers and clinicians need to determine percentages of cell populations in the immune system.  
There are many reasons to study blood cell populations, but this presentation will focus on three important ones. 
The first application includes evaluation of patients with immunodeficiency diseases such as AIDS and others.  
Another application is the study of leukemia/lymphoma. 
The third reason for studying blood cells is immune monitoring of patients with the goal of transplantation.

## Immunodeficiency - AIDS

**HIV Particle**

**T Helper Cell**

- Immunodeficiency - AIDS

**Notes:**
Immunodeficiency - AIDS
One therapeutic area where it is useful to evaluate immune cell populations of individuals is in immunodeficiency diseases (IDD). People with immunodeficiency diseases are subject to numerous infections because their immune systems are not functioning properly.   
The immune system may not be functioning properly because certain cell populations are decreased. Flow cytometry can help us determine which cell populations are decreased or missing.  
AIDS is the most commonly known type of  IDD.  This slide demonstrates the HIV virus particle targeting T helper lymphocytes.  T helper cells are infected and killed in this disease.  Therefore, as the disease progresses, the percentage of T helper cells decreases. It is useful to monitor this percentage in the blood of patients in order to determine how far the disease has progressed. As a matter of fact, the absolute number of T helper cells is used by physicians to determine when treatment should begin. One of BD products: the FACSCount, is a flow cytometer that is dedicated to providing T helper cell percentage and absolute count information for physicians.
To emphasize the important role that T helper cells play in this disease, this slide also shows that those cells are involved in the transmission of the HIV virus.  Any bodily fluid that contains infected T helper cells is able to transmit the HIV virus.  This is due to the fact that infected T helper cells actually contain and propagate the HIV particles in the course of the disease. 
Note:  A cell-free virus in body fluids also transmits HIV infection.   

## Introduction to Flow Cytometry

**Notes:**
In this presentation, we will discuss basic flow cytometry principles and how data is generated and cells are sorted on BD cytometers.

## Learning Objectives

- After this presentation, you will be able to:
- Define basic flow cytometry terms
- Describe the functions of the fluidics, optics, and electronics systems in flow cytometers

**Notes:**
After completing this module, you will be able to define basic flow cytometry terms, and describe the functions of the fluidics, optics, and electronics systems in BD cytometers. You will also be able to explain how cells are sorted on BD cytometers.

Test your understanding along the way by answering quiz questions provided.

## What Is Flow Cytometry?

- Cyto = cell     Metry = measurement
- Simultaneous measurements of multiple characteristics of a single cell or particle
- Measurements are made on a per-cell basis at routine rates of 500 to 4000 cells per second

**Notes:**
What is flow cytometry?
Cyto means cell and metry means measurement.

Hence, flow cytometry is a technology that measures multiple characteristics of cells or particles as they pass one at a time by the interrogation point.

Measurements are made as cells or particles pass by at a rate of 500 to 4000 cells per second. 

## BD FACSCount System

## 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

**Notes:**
What can a flow cytometer tell us about a cell?
It can tell us the relative size or forward scatter, abbreviated FSC,
the relative complexity or side scatter, abbreviated SSC, and
the relative fluorescence intensity.
Depending on the cytometer, up to 13 fluorescent colors can be measured at the same time.

## 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

- 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**

- Properties of FSC and SSC

**Notes:**
First, let’s discuss forward and side scatter, otherwise known as FSC and SSC.

Forward scatter signal is the light that is diffracted or bends around the cell. It is related to the cell surface area, therefore larger cells generally exhibit a high forward scatter signal intensity. This scattered light is detected at the same angle as the laser light or in the forward direction.

Side scatter is refracted or reflected light, that is light that bounces off objects such as internal components inside the cell. Cells with a lot of granules or other internal structures generally exhibit high side scatter signal intensity. These scattered light are detected at a ninety degree angle to the laser light.

## Slide 26

**Notes:**
Let’s take a look at a blood smear as seen by microscopy. The picture shows red and white blood cells.

(slide with animations: callouts appear on mouse click)
Lymphocytes are small and non-complex in comparison to neutrophils, which are larger and very complex or granular. Monocytes are larger and more complex compared to lymphocytes, but less complex compared to neutrophils.

Now that we have seen what these cells look like by microscopy, let’s see what they look like by flow cytometry.

## Lysed Whole Blood

- Side Scatter

- Forward Light Scatter

**Notes:**
By scatter alone, a flow cytometer can distinguish different populations. This is a dot plot of forward versus side scatter data of whole blood that has been treated with BD FACS Lysing solution to remove red blood cells.

Each dot represents the data from one cell. The bigger the cell, the larger the forward scatter signal and the farther to the right the dot will appear on the x-axis. The more complex or granular the cell, the larger the side scatter signal and the higher it will appear on the y-axis.  It is possible to discern lymphocytes, monocytes, and neutrophils in this plot.

(slide with animation: circles with callouts appear on mouse click)
Of the neutrophils, monocytes, and lymphocytes, the neutrophils are the largest and most complex because they are highest in forward and side scatter. The lymphocytes are the smallest and least complex because they are lowest in forward and side scatter. This is consistent with what we saw in the blood smear previously shown.

## Fluorescence

- FITC

- FITC

- FITC

- FITC

- FITC

- FITC

- FITC

- FITC

- FITC

- Emitted Fluorescence Intensity ****Binding Sites

- FITC

- Fluorescent Intensity

- Number of Events

**Notes:**
This graphic shows the fluorescence intensity emitted from the cell is proportional to the quantity of binding sites. The more binding sites, the more fluorescence emitted, and the higher on the fluorescence intensity scale the data appears.

## Emission Spectra

- Wavelength (nm)

- 400

- 450

- 500

- 550

- 600

- 650

- 700

- 1000

- 800

- 600

- 400

- 200

- 0

- RPE

- PE-Cy5

- 750

**Notes:**
As discussed previously, after a fluorescent-labeled cell encounters the laser light, the attached flurochromes absorb energy and then releases that energy as emitted light.

This graph shows some commonly used fluorochromes in flow cytometry. Note that each fluorochrome shows a spectrum of emitted wavelengths. For example, FITC emits at a range from approximately 500 nm to past 550 nm when excited by the blue laser.

With the use of optical filters we can get the majority of the signal we want to its respective detector, which we’ll discuss in more details in a few moments.

## Slide 30

**Notes:**
This is a photograph of cells that have either green or orange fluorochromes bound to them. Some cells have both fluorochromes bound to them. Notice the different colors. Looking at this slide we are being fluorescent microscopists: counting cells that are green and orange. What if I asked you to look at 10,000 cells and tell me what percentage was green or what percentage was both green and orange?  That would be a lot of work.

## Two-Color Cell Analysis

**Notes:**
(slide with animations: callouts appear on mouse click)

Good thing we have flow cytometers. The instrument will view the cells and make all the calculations.

By flow cytometry, data can be displayed in different plot types using software such as BD CellQuest Pro or BD FACSDiva. This is a dot plot of FITC versus PE. Again, each dot represents measurements from one cell.

Notice that the plot has been segmented into four quadrants. 
The lower-left quadrant signifies cells that are negative for both FITC and PE fluorescence, or double negative.
The lower-right quadrant signifies cells that are positive only for FITC.
The upper-left quadrant signifies cells that are positive only for PE.
And the upper-right quadrant signifies cells that are positive for both FITC and, or double positive.

Results, such as percent FITC positive, can be determined by looking at quadrant statistics. For example, in this case, if 40% of the dots fell in the lower-right quadrant, we could say 40% of the cells analyzed stained with the FITC reagent.

## Review

- Flow cytometry is a technology that simultaneously measures and analyzes mulitple characteristics of single cells or particles.
- True or False?

**Notes:**
We just discussed what a flow cytometer measures, now let’s talk about how a flow cytometer works.

There are three main systems in the flow cytometer:
(slide with animations: each system appears on mouse click)
The fluidics system provides a means of delivering the sample to the viewing area.

The optics system consists of a light source, focusing optics, and a means of collecting different wavelengths of light.

The electronics system processes the optical signals, converts the signals to proportional electronic signals that are then quantified and sent to the computer workstation for further analysis.

## Review

- What cell or particle characteristics can be measured by a flow cytometer?

**Notes:**
We just discussed what a flow cytometer measures, now let’s talk about how a flow cytometer works.

There are three main systems in the flow cytometer:
(slide with animations: each system appears on mouse click)
The fluidics system provides a means of delivering the sample to the viewing area.

The optics system consists of a light source, focusing optics, and a means of collecting different wavelengths of light.

The electronics system processes the optical signals, converts the signals to proportional electronic signals that are then quantified and sent to the computer workstation for further analysis.

## A Cytometer Needs a Combined System of:

**Optics**
- To generate and collect the light signals

**Electronics**
- To convert the optical signals to proportional electronic signals and digitize them for further analysis on the computer

**Fluidics**
- To introduce and focus the cells for interrogation

**Notes:**
We just discussed what a flow cytometer measures, now let’s talk about how a flow cytometer works.

There are three main systems in the flow cytometer:
(slide with animations: each system appears on mouse click)
The fluidics system provides a means of delivering the sample to the viewing area.

The optics system consists of a light source, focusing optics, and a means of collecting different wavelengths of light.

The electronics system processes the optical signals, converts the signals to proportional electronic signals that are then quantified and sent to the computer workstation for further analysis.

## Fluidics–Hydrodynamic Focusing

- Excitation Optics (laser)

**Notes:**
These diagrams show what occurs within the flow cell in a BD FACSCalibur system. What’s important to note here is that there are two fluids, the sheath and sample fluids, meeting up in the flow cell. Typically, the sheath fluid used in cytometers is a saline or salt solution.

The sample stream is focused into the center of the sheath stream, allowing cells to pass through the laser beams one at a time. This process is known as hydrodynamic focusing. This phenomena also takes place in other BD cytometers.

Also note that as the sample pressure is changed, the width of the sample stream is affected. A low sample pressure results in a small sample stream. A higher sample pressure results in a wider sample stream, allowing more cells to pass through.

## Collection Optics– BD FACSCount Flow Cytometer

- CD4, CD8
- CD3

_**Fluorochrome**_
- PE
- PE-Cy5

_**Color**_
- Yellow/Orange
- Dark Red

- Collection Optics– BD FACSCount Flow Cytometer

**Notes:**
This is a chart of which detectors receive which fluorescence signal on a FACSCalibur cytometer.  The detectors are labeled FL and then a number, so FL1 is fluorescence 1, FL2 is fluorescence 2, and so on.

Each detector has a filter in front of it that will only allow light into the detector if it is a certain wavelength.  That wavelength of light corresponds to a certain color.  The color corresponds to the emission spectra or emitted fluorescence energy of one of the dyes shown in the previous slide.

On a FACSCalibur the filters in front of the detectors are fixed, on a BD FACSVantage, BD FACSAria, or BD BD LSR II, the user has the ability to change the filters, making it more flexible in terms of which fluorochromes can be used.

## Electronics

- Quantifies voltage pulses
- Converts analog signals to proportional digital signals
- Interfaces with the computer for data transfer

**Notes:**
The electronics system performs quantification of voltage pulses, converts analog signals to digital values, and transfers data to the computer system for further analysis.

## Summary

- Flow cytometry is a technology that rapidly measures multiple characteristics of a cell or particle.
- The fluidics, optics, and electronics systems work together to provide information about relative cell size, internal complexity, and fluorescence.
- Data is further analyzed on the computer and cells can be sorted for further study.

**Notes:**
(slide with animation: bullets appear one at a time on mouse click)

In summary, flow cytometry is a technology that measures multiple characteristics of a cell or particle as they pass one at a time through the interrogation point.

The three systems in a flow cytometer work together to provide information about the cell

Last, data can be analyzed further on the computer and cells can be sorted for further study.

## Review

- The fluidics, optics, and electronics systems serve specific functions in a cytometer. Match the system with its function.

- Quiz #2

- Correct!
- Click anywhere to continue.

- Incorrect
- Click anywhere to continue.

| ____Fluidics |
| --- |
| ____ Optics |
| ____ Electronics |

| A. Provides the means for converting the optical signals to proportionate electronic signals and digitizing them for further  analysis on the computer.
 |
| --- |
| B. Provides a means of delivering the sample to the viewing area
 |
| C. Provides the means for generating and    collecting light signals. |

**Notes:**
Give participants 5 minutes to work in pairs to answer questions.

Go over answers with participants.