CD4 Module 7: CD4 Levels as an Immunological Clinical Marker for Monitoring HIV Infection Purpose: To provide an overview of CD4

and the role CD4 counts play in monitoring and treating HIV. Pre-requisite Units: None Learning Objectives: Define CD4 and the role of CD4 cells in the immune system function. Describe the utility of CD4 cell enumeration for program planning and monitoring. Describe the effect of anti-retroviral treatment on monitoring. Determine if appropriate data exists for determining adult and pediatric reference range for your local population.

CONTENT IMMUNE SYSTEM: Introduction to the Immune System Protection of Body - Two divisions Innate Immunity: o Non-specific o First line of defense o Inflammation o Repeated exposure Adaptive Immunity: o Specific o Second line of defense o Repeated exposure augmented memory no augmentation

Innate Immunity Components: Biochemical: o Enzymes, Complement, etc. o Secretions o pH Physical: o Skin o Cilia Cells: o Phagocytes, NK

Adaptive and Innate Interactions Infectious Exposure Innate Immunity holds

NO DISEASE

Innate Immunity Fails

Adaptive Immunity / Specific memory

DISEASE

Adaptive Immune system

Recovery

Second Infectious Exposure with same organism

The Innate Immune System* Components: Biochemical: o Enzymes, Complement, etc. o Secretions o pH Physical: o Skin o Cilia Cells: o Phagocytes, NK *NOTE: The innate immune system is the first contact with an infecting organism. Innate immunity is not extremely efficient, but it is effective. For example, HIV needs to bypass the skin to infect someone.

Adaptive/Antigen Specific Immunity Humoral immunity: Mediated by antibodies (proteins), which attack foreign substances. Cell-mediated immunity:

Mediated by cells (T-lymphocytes), which attack foreign substances directly.

Lymphocytes These cells originate from progenitor cells in the bone marrow. Differentiation and maturation occurs in the bone marrow (B-cells) and thymus (T-cells). During maturation, they begin to express antigen receptors and become responsive to antigenic stimulation.

Lymphocyte Development* This chart shows the different types of blood cells: T-cells are a subset of lymphocytes that develop in the thymus. T-helper cells are a subset of T lymphocytes. *NOTE: The lymphocytes are located on the LEFT side of the figure.

B-Lymphocytes These cells are derived from stem cells and mature in the bone marrow. B-cells produce antibodies. The antibodies (immunoglobulins) may be secreted by the B-cell or remain bound to the B-cell surface.

T-Lymphocytes Precursors arise in the bone marrow, migrate to and mature in the thymus. Further divided into: Helper T-cells Cytotoxic T-cells Antigen receptor on T-lymphocytes is the T-cell receptor (TcR) which is structurally related to antibodies.

T-Helper Cells Secrete small proteins (cytokines) in response to antigenic stimulation. These cytokines signal T-cells, B-cells and macrophages to begin or sustain an immune response. This immune response is designed to fight off and eliminate an infectious organism.

T-B Cells Interaction

This image shows how a T-helper cell can interact with a B cell. The lymphokines (red dots) are secreted by the T-helper cell. The lymphokines act upon the B cell inducing the B cell to differentiate into a plasma cell. The plasma cell releases the antibodies into the circulation to fight off the invading organisms.

CD4 on T-Helper Cells T-helper cells have a protein on their surface called CD4. The gp120 protein on HIV binds to this CD4 protein and can thus infect T-helper cells. As an HIV infection worsens, the number of cells expressing CD4 will drop. Thus, knowing the number of CD4 cells in an HIV-infected individual provides an estimate of the severity of his/her disease.

Gp120 on HIV binding to CD4 and Chemokine Receptor on a T-Helper Cell

HIV T-Helper Cell

gp120

T-Cytotoxic Cells A subset of T cells. These cells can destroy a target cell which contains an infectious organism, such as a virus. T-cytotoxic cells have a protein on their surface called CD8.

CD8 T-Cell Cytotoxicity*

*NOTE: The top (pink) cell produces a virus and thus expresses a viral protein on its surface. The CD8 (orange) cell recognizes that protein as foreign. The T-cytotoxic cell attaches to the foreign protein. This binding is the first step in the CD8 cytotoxic T-cell destroying the virus infected target cell.

How to Identify Different Lymphocytes Lymphocytes have proteins on their surface which are related to the cells function. For Example: CD4 on T-helper cells and CD8 on T-cytotoxic cells.

We can identify these proteins in the laboratory and thus count the number of each type of lymphocyte. The term CD refers to a uniform nomenclature system which has been adopted to identify these proteins.

What does CD mean?* CD refers to cluster of differentiation which refers to cell surface proteins. These proteins are often associated with the cells function; cells with different functions will express different CD molecules. Over 300 CD molecules have been described.

*NOTE: You will NOT need to know all 300 CD antigens. Also, it is important to note that CD4 is the antibody and the CD4 antigen is the protein on the cell that CD4 binds to.

CD4* The CD4 protein is found on the surface of different cell types, including T-helper cells. This protein is also bound by the gp120 molecule which is expressed on the outside of HIV. This binding is the first contact between HIV and its target cell. Other molecules, known as chemokine receptors, are also required for HIV to infect a cell. *NOTE: A chemokine is a substance that will attract cells across a barrier or gradient.

CD Molecules Used for Identifying Different Lymphocyte Subclasses CD45 CD3 CD4 CD8 CD19 CD56 All White Blood Cells T-cells T-helper cells T-cytotoxic cells B-cells Natural killer (NK) cells

CD4 AND HIV INFECTION: HIV Binding to CD4 and a Chemokine Receptor

HIV infection is mediated through binding of HIV to CD4 and chemokine receptor molecules. The horizontal black line represents the cell membrane. CD4 and the chemokine receptor are part of the cell and gp41 and gp120 are part of the virus.

CD4 on T-helper Cells* T-helper cells are required for proper functioning of the adaptive (antigen specific) immune system. HIV suppresses the immune response by infecting and destroying the CD4 positive T-helper cells. So, the number of CD4 positive T-cells in the peripheral blood provides an estimate of the degree of immune suppression caused by HIV.

The CD4 Number Does Not Diagnose HIV Infection* CD4-positive T-cells may be reduced in other viral infections or immune deficiency states not associated with HIV. The number of CD4-positive T-cells may be used to stage disease or monitor immune deterioration in individuals who have HIV infection, documented by antibody, antigen, and or nucleic acid detection studies. *IMPORTANT: CD4 count is NOT a diagnostic test.

Clinical Utility of CD4 Cell Monitoring* The US Centers for Disease Control and WHO include a CD4-positive T-cell level in their staging of HIV infection.

The number of CD4-positive T-cells is also used to initiate antiretroviral therapy (ART) and treatment of HIV-related diseases. The CD4 T cell number can be used as an indicator of anti-retroviral treatment effectiveness. The absolute number of CD4-positive T-cells is reported in cells per microliter (cells/ L). Physicians will also be interested in the percent of T-lymphocytes which express CD4, especially in the HIV-infected pediatric population.

Remember* While CD4-positive T-cell numbers are a critical tool used to monitor HIV disease, diminishing CD4-positive T-cell numbers alone are not a diagnostic test for HIV. Serology, western blots, and DNA/RNA detection are the main assays for laboratory diagnosis of HIV infection. There are other primary immunodeficiency conditions that will result in lowered T-helper cell counts. In these conditions, there is no progressive decline of CD4 counts overtime as seen in HIV infection. *IMPORTANT: CD4 count is NOT a diagnostic test.

CD4 T-Cells & HIV Decrease in CD4-positive T-cells is associated with increased risk for opportunistic infections and HIV disease progression. CD4 counts of less than 200/L are associated with greater risk for pneumocystis infections. CD4 counts <100/L are associated with greater risk for Mycobacterial infections. CD4-positive T-cell count is used for clinical prognosis and for monitoring therapeutic response for inclusion criteria for antiretroviral therapy.

Time Course of CD4-Positive T-Cell Decrease HIV infection will progress to AIDS at different rates in different patients. The rate of loss of CD4-positive T-cell count may be used as an adjunct to monitoring individuals to determine if they are fast progressors, long term non-progressors, or normal nonprogressors.

Clinical Consequences of HIV Infection: Fast Progressors*

*NOTE: Recent studies have identified super fast progressors who have developed full blow AIDS within a year. Whether this is related to the virus type or the host is NOT currently clear.

Clinical Consequences of HIV Infection: Slow Progressors

CD4 REFERENCE RANGES: Variability of Lymphocytes with Age

Newborns and young children will have higher absolute lymphocyte counts, and higher absolute CD4-positive T-cell counts than adults. Generally, absolute number counts for each lymphocyte subclass will decrease with age. In contrast to absolute counts, the lymphocyte subset % and the CD4:CD8 ratio remains rather constant across age ranges. Worldwide, clinicians use CD4-positive T-cell % for pediatric patients and CD4 T-cell absolute count for adults in the management of patients.

Reference Values for CD4 T-Cell Counts CD4-positive T-cell level is the most commonly used marker for monitoring of immune status of HIV- infected individuals. CD4-positive T-cell counts in HIV seronegative individuals are influenced by age, race, geographical location, sex, time of blood draw and exercise. Both genetic and environmental factors presumably contribute to differences seen in various populations, although no definitive reasons have been identified. This variability demonstrates the need for labs to establish or verify the lymphocyte subclass reference range for the local healthy population. Laboratories performing CD4-positive T-cell testing must determine appropriate reference ranges for the population they serve. There have been published studies of reference values of lymphocyte subsets in various geographic locations.

Determining CD4 Reference Ranges* CD4-positive T-cell counts should be determined for a large number of healthy individuals, representative of the age and sex of the population to be tested. The range encompassing 95% of the values determined for the healthy individuals will be the reference range for that population. *NOTE: CD4 counts are NOT normally distributed in a population. Thus, the use of mean and standard error of the mean should be avoided. The median and 95% range should be used as a reference range.

Protocol for Determining CD4 Adult T cell Reference Ranges Study Population:

Normal healthy donors, across the adult age range (18-65 yrs), no medication for apparent illness. If your region has a blood donor program, specimens obtained from healthy blood donors may be useful for determining reference ranges.

Test Protocol: The reference range should be determined using the instrument you will use for patient testing. Determine reference ranges for all lymphocyte subclasses that you may use in your HIV monitoring program. o Some regions or physicians will also use a CD8 value to monitor their patients.

Pediatric CD4 Reference Ranges It may not be possible to establish pediatric reference ranges in a given location due to an inability to test sufficient numbers of healthy children. Published reference ranges may be used as a guide in the absence of locally determined ranges. The published ranges should be validated by testing a small number of local volunteers.

Circadian Variations in CD4 Cells* CD4-positive T-cell levels (and total lymphocyte counts) tend to increase during the hours one is awake (0800-2200). This increase may approach 60 CD4-positive T-cells/ L in HIV infected patients and hundreds of cells/ L in healthy controls. Thus, follow up specimens should be obtained at approximately the same time as the previous specimen to minimize this circadian variation. *NOTE: The CD4 cell level varies depending on activeness of an individual. This variation known as circadian.

RESEARCH STUDIES: Application #1: HIV Patient Case Study

CD4 counts in HIV patients are measured periodically to monitor patients immune capabilities. An HIV infected patient has a CD4 absolute count of 75/L on 1 April. On July 1, he has a CD4 absolute count of 110/L. The April specimen was collected at 0900. The July specimen was collected at 1630.

Question: Does this represent a significant increase in the absolute CD4 count? Why? Answer: This difference does not necessarily represent a significant increase in the absolute CD4 count. Absolute CD4 numbers will increase from 0900 until 1630. The patient should be redrawn at approximately 0900 for the comparison to be meaningful.

Case Study #1: CD4 Counts in Tanzania Lower CD4 % were found in healthy Tanzanians when compared to Swedish controls.

Lymphocytes Subsets in HIV-1 Antibody Negative Adult Tanzanian Subjects Determined Using MultiSET in Relation to Sex Males (n=107) Females (n=107) Males and Females (n=214) Parameters MEAN (95% Cl) 2445 Lymphocytes/ L (1047,6013) 67.1 CD3+ T-cells % (49.8, 82.3) 1381 CD3+ T-cells/ L (714, 2407) 37.6 CD4+ T-cells % (24.4, 55.3) 748 CD4+ T-cells/ L (383, 1321) 25.8 CD8+ T-cells % (15.0, 43.5) 526 CD8+ T-cells/ L (222, 1007) (300, 1300) (261, 1033) (15.7, 41.0) 569 (15.0, 41.0) 548 (514, 1686) 24.8 (405, 1500) 25.3 (31.0, 55.3) 940 (27.0, 55.0) 843 (881, 3016) 42.6 (769, 2798) 40.1 (56.0, 83.0) 1629 (53.7, 82.6) 1505 (1276, 4056) 70.4 (1189, 4941) 68.8 MEAN (95% Cl) 2427 MEAN (95% Cl) 2436

1.38 CD4:CD8 ratio

1.63

1.50

(0.59, 2.43) (0.74, 2.92) (0.61, 2.73) Reference: Urassa W et al. J Immunol. Methods (2003) 277 65-74

Comparison of Median Levels of Lymphocyte Subsets among Tanzanian and Swedish HIV Seronegative Adult Males Tanzanian Swedish P value CD4 % CD4/ L CD8 % CD8/ L B-cells % B-cell/ L NK % NK/ L (n=107) 38.0 725 26.0 479 12.0 238 18.0 348 (n=50) 45.0 693 28.0 453 13.0 212 11.5 186 <0.001* 0.572 0.082 0.291 0.0157* 0.003* <0.001* <0.001*

*NOTE: Statistically significant value

Comparison of lymphocyte subsets among HIV seronegative adults in other studies 12.1% of Tanzanian males compared to 28.5% of Ethiopian males had <500 CD4 cells/ L, (Kalinkovich 1998). 12.1% of Tanzanian males compared to 44.4% of Ethiopian males had a CD4:CD8 <1.0, (Kalinkovich 1998). Tanzanians had lower mean CD4-positive T-cell counts compared to Ugandan (Tugume et al 1995) or European individuals (Bofill 1992, Maini 1996). Tanzanians had similar median CD4-positive T-cells as reported from a study in Guinea Bissau, (Norrgren 1998).

SUMMARY: CD4 is a protein expressed on the surface of T-helper cells. CD4 is a receptor for HIV. HIV infects and destroys CD4 T-cells. The number of CD4-positive T-cells is used as a surrogate marker of the immune deterioration in HIV infected individuals. A low number of CD4-positive T-cells is not diagnostic of HIV infection. The reference range for CD4-positive T-cells varies with patient age, time of specimen collection and geographic location. Each laboratory performing CD4 testing must determine, or validate, the appropriate reference range for the population being tested. Current literature indicates that CD4 reference ranges in African nations are lower than the published ranges for European countries. CD4 Module 8: Basic Principles of Flow Cytometry

Purpose: To provide an introduction to Flow Cytometry and its applications in the laboratory. Pre-requisite Units: CD4 Module 7 Learning Objectives: Define flow cytometry. Describe the basic components of a flow cytometer. Describe what is measured by a flow cytometer.

Discuss the basic capabilities of a flow cytometer, including multiparameter analysis and gating. Discuss the advantages and disadvantages of flow cytometry. Describe some clinical and research applications of flow cytometry. Define immunophenotyping. Discuss the basic protocol for preparation of cells for immunophenotyping. Describe strategies that can be used to analyze immunophenotyping data.

CONTENT FLOW CYTOMETRY: Flow Cytometry is the automated analysis of cells passing in a fluid stream through a light source.

Flow Cytometers: Four basic components: Light source: usually a laser or mercury arc lamp. Sample chamber, flow cell & optical assembly. Electronic system: converts light impulses to digital signals.

Computer system: controls instrument operations, collects data and performs analytical routines. Flow Cytometers: What is measured? Forward angle light scatter (FSC): proportional to cell size. Side angle light scatter (SSC): proportional to cell granularity. Fluorescence signals: usually two to eight but can be more.

Flow Cytometers: Basic Capabilities: Multiparameter analysis:

o The capability of measuring multiple parameters (size, granularity, associated fluorescence) of each cell simultaneously Gating:

o The ability to select a specific subset of events or cell population for analysis

Advantages of Flow Cytometry Statistical strength Rapidity Objectivity Quantitative Simultaneous analysis of several parameters Sensitivity: <2,000 molecules/cell Small amount of specimen required

Disadvantages of Flow Cytometry Must have single cell suspension Tissue architecture lost Cell sorting is slow & few cells obtained Cost of equipment Trained personnel required Limitations in other fields Data management difficult

APPLICATIONS: Clinical Uses Immunophenotyping DNA/Cell Cycle Analysis Reticulocyte Enumeration

Research Uses Many, including cell sorting; measures of cell activation such as Ca++ flux, pH changes and membrane potential; oncogene expression; etc.

Immunophenotyping Based upon definition of surface membrane antigens by monoclonal antibodies Useful for: Classification of leukemias and lymphomas Monitoring CD4 level in HIV patients Monitoring organ transplant rejection Monitoring patients with autoimmune diseases or primary immunodeficiency states

Immunophenotyping Cell Preparation: Samples are usually peripheral blood, bone marrow or lymph node tissue depending on purpose of analysis. o For CD4 determinations, peripheral blood is used. Two basic methods are used:

o Lyse/wash method o Lyse/no wash method Must be used with single-platform instruments that employ microfluorospheres for absolute counts. Immunophenotyping Cell Preparation: Lyse/wash method:

o Add fluorochrome-labeled monoclonal antibody to small amount of sample. o Incubate 20 minutes, add lysing agent for 10 minutes, wash and resuspend in fixative. o Analyze. Lyse/no wash method:

o Add fluorochrome-labeled monoclonal antibody to precise amount of sample. o Incubate 20 minutes, add lyse/fixing agent for 10 minutes. o Analyze.

Immunophenotyping Information obtained is both diagnostic and prognostic: Using panels of specific monoclonal antibodies, a profile of the cell types present is obtained. In peripheral blood, absolute numbers can be obtained using CBC values or comparison to known amount of microfluorospheres.

Antibody Panels for CD4 Counting The panel of monoclonal antibodies used for CD4 counting will depend on a number of factors: The method used for CD4 determinations:

o Semi-automated o Manual The type of instrumentation available:

o Two color to six color flow cytometer o In lab (e.g. FACSCalibur), rapid (e.g. FACSCount), portable (e.g. Partec CyFlow) The intended gating strategy:

o FSC vs. SSC or CD45 vs. SSC Cost

Antibody Panels for CD4 Counting Examples of antibody panels for CD4 counting*: For manual methods, only anti-CD4 will likely be used

o Anti-CD14 also used to block CD4 on monocytes

For two color systems:

o Anti-CD3/Anti-CD4 and Anti-CD3/Anti-CD8 o Anti-CD3/Anti-CD16+56 and Anti-CD3/Anti-CD19 For four color systems:

o Anti-CD4/Anti-CD8/Anti-CD45/Anti-CD3 o Anti-CD19/Anti-CD16+56/Anti-CD45/Anti-CD3 For six color systems:

o Anti-CD3/Anti-CD16+56/Anti-CD45/Anti-CD4/Anti-CD19/Anti-CD8 *NOTE: For two and four color systems, tubes for B cell (CD19) and NK cell (CD16+56) analysis can be added for complete documentation of lymphocyte subsets and to aid in assessing accuracy of CD4 count.

GATING STRATEGIES: There are two types of gating strategies: 1. Forward Scatter vs. Side Scatter (FSC vs. SSC): Selection of the population to be analyzed is determined by the light scatter characteristics of the cell populations. o This method is often combined with separate measurement of CD45 vs. CD14 fluorescence in a back-gating procedure. o Requires tube with control antibodies to establish background autofluorescence level to be run separately. NOTE: Image illustrates FSC vs. SSC Gating Strategy

2. CD45 vs. Side Scatter (CD45 vs. SSC)*: Selection of population to be analyzed is based on combined CD45 fluorescence and 90 light scatter. o Requires presence of anti-CD45 in all analyses. o When analyzing lymphocytes, eliminates all other cell types from examination. o Subpopulations are then defined by the presence or absence of specific markers (e.g., CD3). o Isotype controls are not usually required since negative populations in a scattergram can be used to set background autofluorescence levels.

NOTE: Image illustrates CD45 vs. SSC Gating Strategy

*NOTE: When using the FSC vs. SSC gating strategy, other cells, especially basophils, which have light scatter characteristics similar to lymphocytes, can contaminate the gate. The CD45 vs. SSC gating strategy eliminates these cells from the analysis by taking advantage of the differential expression of CD45 on these cells. Therefore, a more accurate CD4 count is obtained.

SUMMARY: Flow cytometry is a semi-automated method that can be used to determine both the physical characteristics of cells and other characteristics based upon association of cells with analytes that are either themselves fluorescent or attached to fluorescent molecules. Flow cytometers can measure multiple parameters of each cell simultaneously. Immunophenotyping is a technique that uses fluorescent-labeled monoclonal antibodies to define the cell types present in a patient sample. The methods used for analyzing CD4 counts and the strategies for data analysis should be tailored to the needs and capabilities of each laboratory.

CD4 Module 9: Immunophenotyping

Purpose: To provide an introduction to Immunophenotyping and its applications in the laboratory. Pre-requisite Units: CD4 Module 8 Learning Objectives: Identify and differentiate CD-positive cell types (CD3+, CD4+ T-Helper and CD3+, CD8+ T-Cytotoxic cells). Define immunophenotyping and the method used to determine T-cell subsets (e.g. BD FACSCount).

CONTENT IDENTIFYING AND DIFFERENTIATING CD4 AND CD8 POSITIVE T-CELLS: The Goal Quantifying CD4 and CD8 positive T-cells is useful for monitoring HIV-induced immune suppression. So, our goal is to identify and determine the absolute number of CD4 and CD8 positive Tcells per microlitre of blood in HIV infected individuals.

The Problem* CD4 and CD8 T-cells are a MINOR component of total blood cells. Among lymphocytes, T-cells and their subsets CANNOT be specifically identified by morphology. How, then, can we accurately identify them?

*NOTE: As you know from hematology, the lymphocytes and, specifically, the T-cell subsets are a minor component of the total cells in the peripheral blood. They are less than 1% of all cells therefore we need a fast way to examine many, many cells. What is more important? While we can identify lymphocytes by morphology, we cannot identify the different types. For the most part, T-cells cannot be distinguished from B-cells and certainly CD4 and CD8 T-cells are morphologically identical. The problem then becomes how can we accurately identify these minor components fast?

The Solution: Immunophenotyping Immuno-: Use of immunologic markers, e.g. antibodies.

-phenotyping: Determining type of cell by appearance and predict function.

Immunophenotyping: Is a method used to analyze blood, bone marrow or other tissues in order to determine the expression quantities of certain proteins on or in the component cells. In our case we are using fluorochrome-labeled monoclonal antibodies against CD3, CD4, and CD8 to see what is on the cell surface of lymphocytes in the blood and to identify them.

The Markers* Antibodies to various cellular proteins have been designated by CD#s. Question: How many markers are required to uniquely identify T-helper and T-cytotoxic cells? Answer: T-helper cell makers: CD3+CD4+; T-cytotoxic cell markers: CD3+CD8+

*NOTE: The markers that are used are monoclonal antibodies to various cell surface proteins, which are classified by giving the antibodies to these proteins cluster of differentiation numbers or CD numbers. Over 330 CD antibodies have been produced to various surface and intracellular molecules. Often the proteins that these antibodies react to are referred to by their CD number.

For example, the CD4 molecule or CD4 antigen. This chart demonstrates the ontogeny of the lymphoid system, but, more importantly, it demonstrates that the various lymphocytes can be exactly characterized by the CD antigens on their surface. It would only take one if CD4 and CD8 were unique to these two cells. However, that is not the case. You can see here that CD8 antigen is also on NK cells and CD4 is also on monocytes (not shown). So if we add one marker, the T-cell receptor designated by CD3, then the combination of both markers is now unique to T-helper and Tcytotoxic cells.

Review: CD Antigens on T-Cells* CD3 Mature T-cells (all T-cells) CD4 Helper/inducer cells and monocytes CD8 Cytotoxic T-cells and some NK cells T-CYTOTOXIC CELL T-HELPER CELL

*NOTE: The expression of both CD4 and CD3 antigens will uniquely characterize a cell as a Thelper cell and, also, the coexpression of both CD3 and CD8 uniquely characterize a cell as a Tcytotoxic cell. Unfortunately, we cannot see these molecules on the cells without an aid. This is where immunophenotyping comes into play.

IMMUNOPHENOTYPING / STRATEGY FACSCount: Immunophenotyping CD4 T-cells* 1. Add whole blood to tube with PE-Cy5-labeled anti-CD3 and PE-labeled anti-CD4. 2. Incubate. 3. Analyze or run sample.

*NOTE: For the FACSCount, there are two tubes. The first tube has PE-labeled anti-CD4 and PE-Cy5-labeled anti-CD3, you add whole blood. You then incubate, which allows the antibodies to react to their respective antigens, in our case CD4 and CD3 antigens on T-cells and T-helper cells. Cells without these antigens will not be labeled. At this point, we still cannot visualize any differences because the fluorochromes on the antibodies are colorless. However, when we illuminate the cells with the right wavelength of light (in this case 633nm red diode laser light), the fluorochrome will emit colored light at their characteristic wavelengths. In the case of PE, the colored light is orange and, for PE-Cy5, the colored light is reddish. Now we can visually see the differences, which the analyzer can also see and then quantitate.

Immunophenotyping CD8 T-cells* 1. Add whole blood to tube with PE-Cy5-labeled anti-CD3 and PE-labeled anti-CD8. 2. Incubate. 3. Analyze or run sample. *NOTE: So the CD8 T-cells tube is similar to the previous example except that the PE-labeled antibody is now anti-CD8 but the anti-CD3 remains the same (PE/Cy5). Immunophenotyping Quantification* Using BD FACSCount Flow cytometer: Semi-automated instrument that can analyze and count thousands of cells per minute in each of the previous two tubes. Absolute numbers are derived by comparison to known numbers of reference beads in each tube. *NOTE: The quantitation of the different cells is seen and counted by the BD FACSCount. These cells are analyzed and counted relative to fluorescent reference beads.

REAGENTS: Selecting Appropriate CD4 Immunophenotyping Reagents Most clinical flow cytometers are designed with a simple protocol for determining CD4 and CD8 T-cell counts using a specified antibody combination. The BD FACSCount uses anti-CD3, anti-CD4 and anti-CD8 in proprietary tubes.

These reagents are evaluated in the specific system for which they are designed and are not interchangeable.

Storage and Handling of Reagents Conjugated antibodies will come with specific storage and handling instructions.

Normally, they require refrigeration: 2 to 8 degrees Celsius

Most have relatively long expiration dates: 3 months to 1 year

SUMMARY: Immunophenotyping is a technique that uses labeled antibodies to identify specific proteins on the surface of cells. CD4 and CD8 positive T-cells in peripheral blood can be uniquely identified by a combination of fluorochrome-labeled CD3 and CD4 antibodies in one tube and anti-CD3 and anti-CD8 antibodies in another tube. The BD FACSCount is a semi-automated instrument, which immunophenotypes lymphocytes and determines the absolute number of CD4 and CD8 positive T-cells. The antibody panel and reagents are specific to the manufacturers instrument. They are not interchangeable, (BD FACSCount and Calibur). Reagents must be properly stored and handled.

CD4 Module 10: CD4 Counting Maintenance and Calibration for Instrumentation and Pipettes

Purpose: To provide information regarding maintenance on analyzers and pipettes within the immunophenotyping laboratory. Pre-requisite Units: CD4 Module 9 Learning Objectives: Discuss essential elements of a preventative maintenance plan in an immunophenotyping laboratory. Discuss reasons for performing maintenance on immunophenotyping analyzers and pipettes. Describe examples of routine maintenance for immunophenotyping analyzers. Describe procedures for pipette calibration and maintenance.

NOTE: Maintenance of CD4 analyzers, equipment, and pipettes helps to provide quality performance in the CD4 laboratory. Establishing Standard Operating Procedures (SOPs) regarding maintenance and having technicians follow these guidelines, as well as securing service contracts is essential to quality assurance. In this module, we will discuss when and how CD4 analyzer maintenance is performed, along with when and how to check pipette precision.

CONTENT LABORATORY MAINTENANCE: Flow Cytometry Instrument Maintenance* Maintenance is an important aspect of quality control in every laboratory. Maintaining good functioning instrumentation is imperative for: Providing accurate and precise results. Reducing the risk of the breakdown of the system, minimizing instrument repair costs. Ensuring continuous patient care.

*NOTE: Good maintenance practices can really minimize instrument repair cost and limit the downtime or workflow interruptions in the long term.

Laboratory Maintenance* Laboratory maintenance can be described as: The systematic operation of cleaning and adjusting the laboratory equipment.

The periodic replacement of certain components in order to ensure laboratory equipment continues to work for a very long time.

Laboratory Maintenance* There are two maintenance types: Systematic preventive maintenance:

o Performed periodically (daily, weekly, monthly) o Is the responsibility of the laboratory personnel Occasional curative maintenance:

o Occasional repair or replacement of parts of an analyzer o Usually performed by the manufacturers service representative *NOTE: Both systematic preventative and curative maintenance is necessary and should always be documented when performed. STANDARD OPERATING PROCEDURES (SOPs):

Maintenance Procedures* Necessary procedures and documentation Establish standard operating procedures (SOPs) for equipment maintenance so that the technician has specific, clear, and detailed description of how and when to carry out maintenance. These SOPs should be done in a vocabulary appropriate for those persons who are going to be using them while clearly following the manufacturers instructions. *NOTE: SOPs for daily, weekly and monthly, as well as annually and bi-annually should be available.

Maintenance Procedures* Records that should be kept for documentation should include: An error/corrective actions log of all problems and actions taken to correct the problem.

A maintenance log(s), recording all maintenance performed (daily, weekly, bi-weekly, monthly, semi-annually, and annually). *NOTE: Corrective action documentation allows technicians to review repeating problems, and anticipate and maybe avoid future problems, which may interrupt service/workflow. Documentation helps to ensure that all required cleaning and maintenance will be carried out.

SERVICE CONTRACTS:* A service contract should be arranged and budgeted for when equipment or an analyzer is purchased or leased. This is arranged with the manufacturer; usually on an annual basis (first year is usually included in purchase as warranty). *NOTE: When negotiating for purchases and/or leasing, it is a good time to discuss service contracts and remember that service is an important factor when deciding on which analyzer/vendor to choose. Make sure the services to be provided (how often and what is to be covered under the contract in the way of analyzer parts, reagents, etc.) are clear and understandable.

Service Contracts The manufacturer sends trained representatives for both equipment problems and annual, biannual, and preventative maintenance (PM) on the analyzer. Annual PMs are necessary and generally arranged and should be scheduled by the laboratory, so general cleaning and checks may be performed.

*NOTE: Service contracts differ depending on the manufacturer. It is important to note exactly what service and parts will be covered or serviced when instruments are purchased. All settings are checked to make sure that your analyzer is performing optimally, replacement of lasers and/or other parts may be necessary.

Analyzer/Contract Documentation* Once a service contract is in place, you will be expected to provide information about the analyzer when calling for service for a problem or scheduling a PM. Keep this information readily available for all of the appropriate laboratory personnel.

*NOTE: Immediately after installation, make this information available. Make sure all technicians know where to locate this information when needed.

List of Necessary Vendor Information* Name of Laboratory Salespersons name and contact of local representative Model Number Number of maintenance contract Serial or Series Number Expiration date of the maintenance contract Location Dates of previous service and detail of service provided Manufacturer Contact Lists of replacement pieces *NOTE: Generally when an analyzer is NOT operational, the laboratory environment becomes hectic. Make sure all the following information is readily available so that service/vendor can act immediately.

MAINTENANCE PROTOCOLS FOR INSTRUMENTS: Immunophenotyping Laboratory Maintenance Essentials*

It is vital for equipment to have regular cleaning and maintenance performed to ensure accuracy and precise CD4 results. It is also necessary to perform regularly scheduled (six months) calibration checks on pipettes to ensure they are measuring accurately and with precision. The daily analyzer cleaning standard procedure takes very little time and can prevent many problems. *NOTE: If you get into a habit of proper cleaning and maintenance, your analyzer will perform better, more accurately, etc.

Daily Maintenance Protocols* It is recommended to follow the manufacturers procedure for basic daily cleaning regiments. There are rules that are general to all immunophenotyping analyzers. Certain aspects of cleaning are specific to each analyzer.

*NOTE: While there are essential cleaning procedures common to all CD4 immunophenotyping, always make certain that you follow the manufacturers guidelines for maintenance, troubleshooting, and cleaning.

General Recommended Cleaning* Three cleanings are recommended: 1. Cleaning procedure recommended after several (8) hours of operation, or a specific number of specimens run. 2. 3. Cleaning performed before shut-down of the analyzer. Monthly cleaning, which is a more extensive cleaning cycle.

*NOTE: In general, (busy) labs with large volumes of work will need to perform cleaning procedures more often. Analyzers are always cleaned before shut-down. Monthly cleaning involves longer rinse times, checking and replacing filters, and or tubing.

General Recommended Cleaning* Check the fluidics system for debris, proteins, salt crystals (sheath fluid). Identify and clean obstructions at the sample injection probe point and tubing of the fluidics system: This optimizes the functionality of the fluidics system. This also reduces the debris found during the course of analyzing.

*NOTE: While performing cleaning and analyzer start-up, always inspect the system for problems with/in tubing, sampling, etc. occurring from normal use. Even though you are diligent in following the recommended cleaning, clogs from salt crystals in sheath fluid can frequently build up and clog the analyzer filters, and tubing.

General Recommended Cleaning* Rinse the lines when debris is detected, following the general cleaning procedure or follow manufacturers procedure. Clean with a solution of bleach: 5% sodium hypochlorite (home bleach) diluted 1:10 with distilled water. (Important: Follow manufacturers recommendations for bleach strength.) *NOTE: It is important to read the manufacturers recommended procedures for cleaning and maintenance. For example, while a *1:10* dilution of 5% bleach is mentioned and used with many analyzers, the BD recommends a *1:3* dilution for their FACSCount instrument, but not with some other BD analyzers.

Daily Cleaning for the FACSCount Analyzer* Perform the cleaning procedure at the end of each day, when the screen message appears recommending that you run the cleaning cycle (Note: this message appears after at least 51 reagent pairs have been run), and when recommended to do so by the troubleshooting section. *NOTE: By reading through the cleaning procedure for the FACSCount CD4 analyzer, you can see how simple it is. Go through this reading of the cleaning procedure to demonstrate a typical cleaning cycle.

Daily Cleaning for the FACSCount Analyzer* When not using the instrument or you are turning off the power, leave a tube of distilled water on the instrument with the sample holder up. (Note: Do NOT leave bleach on the sample holder.) This protects the sample injection probe from the formation of salt deposits.

*NOTE: Read through the cleaning procedure to demonstrate a typical cleaning cycle. Most CD4 immunophenotyping analyzers have you leave the sample probe in distilled water.

Additional Maintenance Avoid spillage of saline liquids in the cytometer. Avoid interruptions or fluctuations of electric power by using a surge protector/APC of appropriate capacity. Protect the cytometer from ambient dust (cover the instrument, close windows).

Air-conditioned rooms are advisable in most COUNTRY settings. After shut-down, clean the workbench area with 0.5% concentration of bleach, as well as wiping down all other equipment used.

Recording of Maintenance For quality assurance, recording of all routine daily, weekly, monthly maintenance must be done to document and ensure that all laboratory personnel perform maintenance. Annual and bi-annual preventative maintenance (PM) service must also be documented.

Maintenance Log Example* *NOTE: This is one example of a maintenance log for daily, monthly and quarterly maintenance for BDs FACSCalibur/FACScan analyzers.

Corrective Action Log Example* CORRECTIVE ACTION LOG Date Problem Action Taken Tech

*NOTE: Corrective action logs allow you to track problems and action taken. Always document problems in this type of log.

Pipette Maintenance:

Mechanical Pipette Maintenance* The accuracy and precision should be checked the first time of use and periodically thereafter. Accuracy and precision should be checked at least every 6 months: If either fails, have another technologist confirm.

If either fails again, it is important to follow the manufacturers instructions for repair and calibration. *NOTE: The manufactures instructions show how to disassemble the pipette, clean and lubricating it and replacing some parts such as springs and rubber gaskets. Following this pipette maintenance, precision and accuracy needs to be tested again.

Mechanical Pipette Maintenance* Maintain complete records of pipette calibration function check: Include serial and other identifying numbers of each pipette

SUMMARY: Follow an internal maintenance program based on written procedures and follow the plan to the letter. All personnel should follow laboratory procedures. Annual maintenance contracts are needed for the immunophenotyping analyzer. High maintenance standards maintain a high performance level and improve the efficiency and overall quality of the laboratory. The ability of manufacturers field service representatives to provide service for your instruments is critical for the choice of your equipment you acquire. CD4 Module 11: Flow Cytometry Quality Assurance

Purpose: To provide an introduction to quality assurance and its application in flow cytometry. Pre-requisite Units: CD4 Module 10 Learning Objectives: Discuss the importance of quality assurance. Define the components of a quality assurance system including monitoring records.

Outline policies and procedures needed to be reviewed and monitored to assure quality in a flow cytometry laboratory.

Differentiate between precision and accuracy and how these terms relate to test reliability. Define mean, median, mode, standard deviation, and coefficient of variation.

CONTENT INTRODUCTION TO QUALITY ASSURANCE: Definition of Quality Assurance (QA)* Standard and systematic set of activities for the purpose of providing adequate confidence that quality requirements will be met. The purpose of quality assurance is the maintenance of the overall quality of patient results. It takes into consideration all factors that affect the test result, from the time the test is ordered to result reporting:

Pre-analytic

Analytic

Post-analytic

1) Pre-analytic Processes: (Pre-analytic means before testing) Quality of collection Specimen transport Specimen acceptability

2) Analytic Processes: (Analytic means testing (which includes QC)) Result accuracy Clerical errors Analytical errors Assay repeat rates

3) Post-analytic Processes: (Post-analytic means after testing) Timely results Timely reporting to patient file Record keeping

*NOTE: Pre-analytic refers to everything impacting on the patient specimen and result, before it is tested for the analyte. If the patient specimen isnt identified, collected or handled properly, the specimen wont be worthy of testing. You will notice that aspects of analytical included the accuracy of the result, analytical errors, clerical errors, and these all impact the patient result.

And the post-analytical aspect includes timely reporting to the patient file for the clinician and record keeping. All phases impact overall quality of reported patient results.

SPECIMEN COLLECTION, HANDLING AND STORAGE: What should be included in a quality assurance SOP covering specimen collection, handling, transport and storage?* Procedures for sample collection, optimal handling conditions for transport and storage should be properly followed. Written procedures with criteria for rejection of unacceptable specimens should be made available to all institutions. *NOTE: Documentation in Requisition logs and corrective action logs is an essential part of quality assurance.

PROFICIENCY TESTING: Participation in Proficiency Testing* Laboratories should enroll, and satisfactorily participate in, a performance evaluation/assessment program: If conventional proficiency testing is not available, the laboratory must exercise an alternative performance assessment system for determining the reliability of analytic testing (sample splitting for inter-laboratory testing). o Test samples must be run as if it were a patient sample. If the lab has more than one method-system for performing tests for an analyte, it must be checked against each other at least twice a year for correlation of patient results (bias scatter plots). *NOTE: Splitting samples with nearest laboratory is an alternative to proficiency testing. Always run samples as though if they were a patient. This is very important as there is the temptation to do extra steps (i.e., run more than once, etc.). So dont do it (run proficiency specimen more than once etc.) as doing this defeats its purpose, which is to find potential problems.

Proficiency Testing* IMPORTANT: Well-delivered external quality assessment monitoring can have profound impact on global HIV management! A number of regional EQA programmes exist for CD4 T lymphocyte testing, but there are two that provide international coverage: QASI, Bureau of HIV/AIDS, STD & TB, Ottawa, Canada operated 2-3 times per annum (approx 300 international sites) (contact Frank_Mandy@hc-sc.gc.ca).

UK NEQAS for Leukocyte Immunophenotyping, Sheffield, England operated 6 times per annum (2 samples per send out) (approx 500 international sites) (contact d.barnett@btconnect.com or visit www.ukneqasli.org).

Role of Quality Assurance Systems International (QASI)* Provide quality assessment program for CD4 T-cell immunophenotyping. Transfer the QAP management process to regional authorities. Assist with data processing, analysis and performance reports. Provide skill building workshops and technology transfer sessions wherever needed. *NOTE: The utility of QASI is to provide the Quality Assurance program (QAP) for the CD4 T-cell Immunophenotyping. It will transfer our knowledge of QAP procedures and processes to laboratories. It will also collect, process and analyse data. It will produce and distribute performance reports. And, finally it will provide workshops, lectures and training sessions in-house or on their premises, if requested.

QASI Shipments Provides external QAP for CD4 T-cell enumeration where there is none available. The external QAP shipments include challenge survey material with simulated specimens. Collect, process and analyze external QAP data. Provides rapid return of survey results to assure maximum time for remedial action.

REFERENCE RANGES: How to determine reference ranges?* Reference ranges are established using data from a minimum of 25 normal controls (better with more) drawn from normal, healthy donors. As you add/gather more control samples, recalculate your references ranges until you have about 100 samples. Set up a system for review. *NOTE: Care must be taken when establishing normal ranges, especially in highly endemic areas. Obvious outliers should be excluded or further investigated for exclusion.

What to do when controls are out of established reference ranges?* Results of the normal donor control are expected to be within the established reference range. If results exceed reference range limits, follow corrective action: Repeat test using same antibody aliquot. If the results still exceed the limits, do not automatically invalidate patient results.

o Due to the 95% confidence interval, 1 in 20 specimens from healthy individuals drawn at random can be outside reference range limits due to biological factors. May be true result - misrepresentation of healthy status (especially true in highly endemic areas). o If still exceeds limits, repeat with new antibody aliquot. o If this corrects the problem, all patient specimens must be repeated using the new antibody aliquot. Document all these steps on a corrective action log.

*NOTE: Look for trends. If more than expected are falling outside, it suggests an assay/reagent problem.

Exercise #1: Mean, SD Calculation For 20 different normal adult specimens, the following data was obtained from the BD FACSCount for CD4 absolute values: DAY RESULT DAY RESULT DAY RESULT DAY RESULT 1

750 6 850 11 1100 16 800 2 850 7 1150 12 750 17 1125 3 850 8 1300

13 950 18 1100 4 1200 9 900 14 1200 19 1150 5 850 10 950 15 1200 20

900 Question: Determine the reference range to be used by calculating the mean and standard deviation. Mean (average) can be determined by adding the results of the 20 normal controls and dividing by 20. Standard deviation can be determined by a complicated formula that is prone to errors due to multiple steps needed for calculation. o It is best done with a calculator (most inexpensive calculators have this function). 95% confidence limits = range determined from these two numbers.

o mean - 2 x standard deviation o mean + 2 x standard deviation So, the reference range for 20 normal samples =? Answer: Find the mean for 20 normal samples (Total sum of results divided by 20) = 996.25 Standard Deviation = 173.3 95% confidence limits = range determined from these two numbers

o mean 2 x standard deviation = 996.3 (173.3 x 2)= 649.7 o mean + 2 x standard deviation = 996.3 + (173.3 x 2)= 1342.9 Overall Answer: Reference range is 650 1343

TRAINING AND COMPETENCY ASSESSMENT: Competency Assessment Program should be established to include: Competency training evaluation and revalidation evaluation. When there is a change in methodology or instrumentation, the technicians performance must be reevaluated to include new test methodology or instrumentation, prior to reporting patient results: Laboratory director evaluates supervisors; all others are evaluated by supervisor. Keep evaluations in Quality Assurance Review Book.

RESULT REPORTING: Proper procedure includes:

All data entry results should be verified by a section head, supervisor, or colleague for final interpretation and release of results. In the event that a report has already been sent out and needs correction, a new report is issued with updated report written on it. The old report remains in the patient file.

Verbal result reports should be documented, listing the time of the receipt of the report.

DOCUMENTATION OF CORRECTIVE ACTION: Start an Out of Control Book. Remedial actions to be taken when values for controls do not fall within the normal range limits MUST be documented in a Out of Control Book. Equipment malfunction, reagent problems and corrective measures taken in response to unacceptable proficiency testing results must also be documented in the same book. Clerical errors on results must be corrected immediately and must also be documented in the Out of Control Book. IMPORTANT: Document, Document, Document!!

QUALITY IMPROVEMENT: Monitor all phases of pre-analytic, analytic, and post-analytic testing continuously for quality improvement: Analyze problems encountered develop solutions to resolve and prevent future issues. Monitor and study routine operations for areas of improvement develop solutions.

Quality Control: Definition and Purpose of Quality Control (QC) System used to assure that all operational techniques and activities meet the specified predetermined requirements. QC ensures valid and reproducible results. Quality Control Quality control is a vital part of quality assurance. All labs benefit from quality control in terms of confidence in and reproducibility of test results.

Recording and monitoring test variables such as temperature, reagents, controls and equipment function allows one to look objectively and retrospectively at parameters vital to the accuracy and precision of the test. Documentation allows one to foresee a potential problem before the situation requires corrective action and adversely effects patient results. Levey-Jennings type plots are useful. These documents must be reviewed daily. Any trends outside the expected range should be investigated. IMPORTANT: Do Not Ignore!!

Quality Control in Flow Cytometry Control QC: Stabilized prep of normal human blood Method controls

Reagents Instrument QC: Analyzers Pipettes Centrifuges Refrigerators, freezers Thermometers

QC CONTROLS: Positive control of stabilized normal human peripheral blood leukocytes (if available/or budget permits) or fresh healthy volunteer blood donor control: Stabilized control or fresh control is stained for all subsets using the same amount of monoclonal antibody reagent used for patients. It is run each day on each machine, results are recorded, and mean, SD, %CV calculated.

Reagents for Stabilizing Blood Samples* Guidelines for CD4+ T-lymphocyte counting state that analysis must be complete within 18 hours.

Some hematology analyzers will have difficulty producing a differential after 24 hours.

To overcome this, reagents are available that can be added to whole blood to slow down the decay process. CytoChexTM (Streck laboratories): Member of family of non-cross-linking fixatives Designed to preserve WBCs in whole blood (1:1) for up to 7 days at 40 degrees Celsius

TransFixTM (Cytomark Ltd, UK): TransFixTM that lasts > 10 days, (1:10), < 40 degrees Celsius Termed Transfix because it allows transportation of fixed samples Low dilution factor

Both CytoChexTM and TransFixTM compatible with flow technology. No data on stabilized blood and manual CD4 counts.

*References: Turpen & Collins. Amer Clin Lab 1996 15:30.; Barnett et al. Cytometry 1996 26:216.; Jani et al. J Imm Meth 2001 257:145.

Quality Control Whole blood specimen from a healthy donor or stabilized control should be run each day to determine if the procedure for preparing and processing are optimal. Record values for all that are provided and reported by your analyzer on a Levey-Jennings type plot. Possible recorded values include: CD3+, CD3+4+, CD3+8+, and CD8+ and helper/suppressor ratio in a control log.

Knowledge Check Question: If values repeatedly fall outside the new established range, what is the next action you should take? Answer: Evaluate and rule out problems with: Instrument PMT, Compensation, Linearity Operator technique skills, procedural Product deterioration

QC REAGENTS: How to store reagents* Always store according to the manufacturers recommendations. Reagents must be dated and initialed upon receipt. Lot numbers must be recorded in a reagent quality control record book. After preparation and/or when placed in use, reagents must be labeled when put in use according to the manufacturers suggested recommendations. *NOTE: Reagents are very expensive and deterioration of your reagents will result in invalid results in your laboratory. Get into a habit of maintaining your reagents in proper temperatures, documenting lot numbers and discarding reagents when their expiration dates have expired or if there is a possibility of expiration.

Reagent parallel testing* New reagent lots must be checked with old lots using a normal control before use. The variability for new lots of reagents compared to the current lot should not be greater than the variability found for triplicate samples of the current lot. Variability should be within 5%. Results of reagent checks must be recorded, dated and initialed. Document all lot-to-lot procedures with date and variability results. *NOTE: Result values obtained from a new lot number must be compared to those obtained from the current lot in use.

QC EQUIPMENT: All equipment in the CD4 laboratory Should have instruction manuals, as well as SOPs, regarding proper use and maintenance requirements. Should be monitored and recorded for quality control procedures, function checks, preventative maintenance and repairs. These should be documented and filed in separate log books.

Cytometers* Record Serial numbers for each.

Perform function verification (PMT, Linearity, Compensation, if applicable, on a daily basis). Perform preventive maintenance (daily and monthly checks, along with bi-annual Preventive Maintenance checks). Troubleshooting procedures should be available to all staff. *NOTE: Follow SOP for performing daily analyzer checks, (mentioned above), which should clearly spell out the manufacturers instructions for verifying that the analyzer is functioning appropriately. There are different degrees of preventative maintenance (i.e., longer wash times for monthly vs. daily analyzer cleaning) depending on daily, monthly bi-annual, etc.

BD FACSCalibur PMT and Linearity Daily Check* BD CaliBRITE 3 and APC Beads are tested using B/D FACSComp. Changes in PMT voltages and Compensation may indicate there is an instrument problem. PMT voltages and Fluorescence Compensation are checked for significant day to day variation. *NOTE: This print out of a daily PMT check for BDs FACSCalibur indicates that the photomultiplier tube voltages and fluorescence compensation are checked and have passed. If this test fails, follow manufacturer's suggested follow-up and be sure to document in a corrective action log.

QC Equipment Before putting new equipment or a new method into service it must be validated. This is accomplishment by correlation and/or agreement studies. The new method or equipment is validated against old method and/or equipment.

Refrigerators and freezers* Record serial numbers. Record temperatures daily. Maintaining correct temperatures is vital to maintaining the integrity of reagents and should be maintained at 2 to 8C. Freezer should be maintained at -100 to -200C. *NOTE: All laboratory equipment must be properly monitored and maintained in order to maintain quality assurance in the laboratory.

Pipettes Improperly calibrated pipettes will affect your assay and should be checked for precision and accuracy bi-annually. Pipettes not passing accuracy checks should be cleaned and checked for worn parts, but if you follow manufacturers instructions and the problem is not apparent, the pipette may be sent to and serviced by reliable vendors, and is not typically very expensive.

SUMMARY: To ensure that a laboratory practices and provides quality assurance for all phases of testing, the laboratory should: All enroll and satisfactorily participate in a performance evaluation/assessment program. Establish reference ranges for analytes being tested. Document training and competency assessment for their technicians. Provide review and verification of all results released, including verbal result reports.

Provide documentation of all problems with equipment malfunction, reagent problems and unacceptable proficiency testing results. Review, evaluate and attempt to improve laboratory processes continuously. Provide quality control for reagents, equipment, and analyzers in the laboratory.

CD4 Module 12: CD4 Laboratory Set-Up and Design

Purpose: To provide the information that is necessary to configure an efficient and safe CD4 laboratory. Pre-requisite Units: CD4 Module 11 Learning Objectives: Discuss how to optimally configure a CD4 laboratory with regard to workflow, equipment, space and staffing requirements. Describe various services a CD4 laboratory supports. Identify and describe various components essential to CD4 laboratory management.

CONTENT LABORATORY CONFIGURATION: CD4 Laboratory Configuration*

The need for CD4 counts is expected to grow in HIV treatment programs. There is always pressure for the laboratory to produce quality results, using current technology, while keeping up with increasing demand to aid clinicians and program managers. There must be strict adherence to following Standard Operating Procedures for each and every stage of the testing process. It is necessary to clearly-delineate work areas for each process stage to avoid overlap, oversights, and sample and/or result mix-ups. *NOTE: As in other clinical laboratories, it is necessary to maintain and follow SOPs in every stage of laboratory process. This module will point out that when configuring your laboratory it is important to separate each work area or process stage.

LOCATION: Considerations for location of a laboratory Accessibility: Is the laboratory strategically located to be reasonably accessible to the clinics served? Must consider specimen transport issues

Infrastructure: Will the infrastructure at the location support the scope of the planned laboratory activities? Must consider power and water requirements, space issues, type of instrumentation, waste disposal. The type of instrumentation should be commensurate with the intended use and with the level of expertise of the health care facility. Laboratories with flow cytometry instruments should be air-conditioned to maintain proper room temperature and control dust. Instrumentation should be placed in an area that is free from vibration and noise.

WORKFLOW: Strict workflow is required: Deterioration over time of samples requires real time testing and tracking of sample batch age. Occasional repeat testing requires a system for specimen tracking and temporary storage.

Standard Operating Procedures, testing guidelines, equipment specifications and testing configuration should follow international recommendations, when available (e.g., WHO, CDC, and manufacturers).

Increase staff efficiency and reduce reagent wastage.

Laboratory Configuration The key to a reliable routine CD4 testing lab is for it to be optimally configured. Configuration involves: Large and small equipment layout Workflow staging and direction Space and electrical requirements Consideration for staffing resource requirements

Work Area Arrangement* Work areas should be arranged to allow unidirectional sample flow and defined space for each test step. Defined areas are needed for: Specimen receiving and storage Specimen preparation Specimen testing-instrument Results production, validation and release Reagent and consumable storage

Avoid overlapping procedures. *NOTE: Avoiding the overlapping of procedures increases workflow efficiency and decreases the change of errors.

CD4 Laboratory Configuration #1.JPGCD4 Laboratory Configuration #1

This diagram of a CD4 laboratory illustrates work areas separated by process and flow in one direction, from sample processing and preparation to result processing and sample storage for a small volume, CD4 laboratory.

Laboratory Configuration #2 CD4 Laboratory Configuration #2.JPGIn this 2nd diagram, a high-volume CD4 laboratory is illustrated. You can see workflow is also setup in one direction, but here there are additional peripherals of components to aid in the increased volume. Notice there is an automated specimen preparation component along with a computer and printer used to run a high-volume CD4 analyzer. An automated specimen preparation instrument replaces some manual processes, allowing for some walk-away time for the technician to attend to other functions. The computer allows for data saving until results are needed to be printed or transferred.

CD4 Laboratory Configuration* Forecast, if possible. Consider and plan space for additional instrumentation based on anticipated testing/processing increases. *NOTE: As mentioned before, testing volume will probably increase and so there will be a need for high-volume analyzers to replace low volume, additional analyzers and other equipment.

STAFF REQUIREMENTS: Technical Staff* Staff categories based on function: Specimen reception and queries

o Specimen receptionist (e.g. assisting with specimen handling and biosafety training) Specimen processing and testing

o Trained technician Results validation and release

o Trained technician *NOTE: Although not always possible, try to anticipate and plan for adequate staffing for these areas listed so that no one process or area slows down workflow.

Minimum Staffing Requirements* Low-volume instrument (e.g. FACSCountTM): 1 specimen receptionist 1 technician/instrument/50 - 100 specimens per day

High-volume instrument (e.g.FACSCaliburTM) 1 specimen receptionist 2 technicians/instrument/100-150 per day 3 technicians/instrument 150-300 per day

* NOTE: For continuous, high-volume CD4 laboratories, more than one technician is optimal. A specimen receptionist for 20-24 specimens is not always applicable in resource poor countries.

Staff Requirements Low Volume Testing This diagram illustrates workstations or workflow staging for a low-volume laboratory. Notice workflow is unidirectional.

Staff Requirements High Volume Testing This diagram illustrates workstation areas and the number of staff required for each for a high volume CD4 laboratory. The CD4 lab testing workstation as well as the result validation and release workstation show that they require additional staffing to provide a continuous workflow.

SPACE REQUIREMENTS: Environmental and Space Requirements* Do not have unrelated activities in the same area. Ensure sufficient room for all testing processes. Air should be free from external dust, e.g. closed windows and doors. Control temperature: Testing should be conducted without extremes in room temperature.

*NOTE: Having unrelated activities can add to confusion and errors. If possible, allow for more than required space for all testing processes. Air-instruments pull air from the room through fans for cooling, so air should be free from dust, which keep filters on cooling fans from becoming clogged. Temperature is critical to instruments and test performance. Analyzers may turn off if temperatures are too high.

Space Requirements* Avoid placement that requires you to move instrument frequently for cleaning and maintenance. Bench strength should be adequate enough to support instrument weight for the long-term without sagging.

Bench width should accommodate the entire instrument with sufficient space behind instrument to allow cooling and access for repairs. *NOTE: Many CD4 analyzers contain delicate lasers and optical sensors. Moving theses analyzers, especially high-capacity analyzers, may require service representatives to move to guarantee parts are not damaged. Stable floor and bench top is important to support an analyzer. Vibration may loosen analyzer parts over time, and damage lasers.

Storage Requirements* Ensure adequate supply of consumables, e.g. pipette tips, gloves, paper. Ensure sufficient cold storage space for reagents. If testing volume is high, you may require a cold storage room. First-Expired, First-Out (FEFO) stock. Monitor cold storage temperature (all refrigeration). *NOTE: If you are able to plan ahead, and can afford to purchase cold room storage, it would be wise to do so. Most reagents (not pointcare) require refrigeration.

Electrical Specifications* Follow specification recommended by instrument supplier. If instrument is moved, recheck electrical supply. Flow cytometers and ancillary equipment require protection from major and minor fluctuations of electrical supply. Testing does not allow for interruptions in electrical supply, install adequate uninterrupted power supply (UPS) (if possible). *NOTE: A stable voltage supply with uninterrupted power is necessary for CD4 analyzers.

LABORATORY MANAGEMENT: Lab Support Services: Laboratory Services Fundamentals* Laboratory services are required to be: Reliable Fast Cost-Effective

Easy to Access High Quality Sustainable

Resources are often inadequate to always deliver services. *NOTE: Healthcare clinicians rely on CD4 results for optimal utilization of resources. Maintaining statistical data is critical for improved patient outcomes. There is always pressure for the laboratory to do more with fewer resources. Demand for CD4 counts will increase. That is why it is essential to take time for careful planning and configuration of your laboratory.

Demand for CD4 Counts Testing demand for CD4 counts will be high. Demand is high at the start of a treatment program (CD4 count is a screening test). Demand will increase significantly as the program expands.

Optimal utilization of current resources and planning for future demands are vital. CD4 testing is: A common criterion for treatment eligibility. The major laboratory indicator for individual therapy success.

Statistical Monitoring and Evaluation of CD4 Counts* Constant monitoring and evaluation for treatment program performance requires data on CD4 counts: Proportion of HIV-positive tested for CD4 count HIV population CD4 count average after 6 months and 1 year of treatment HIV population baseline CD4 count average Mortality with different CD4 count baselines Proportion of CD4-eligible treated

Total number and proportion of CD4 cell counts below treatment cut-off (e.g. <200 cell/mm3) *NOTE: Begin as soon as possible to standardize data collection, electronic database, etc.

CD4 Count Testing* Evaluate the testing facilities in the following areas: Physical infrastructure Equipment Human resources and training Quality management and safety Sample transport logistics Consumable delivery and storage logistics Data management

Resolve bottlenecks and inefficient steps. *NOTE: Evaluate and re-evaluate for continual quality improvement in all of these areas.

Equipment Management:* Ensure adequate testing capacity at each laboratory based on patient and testing volumes. Select instruments with capacity to cope with current and future demand. Besides the analyzer, ancillary equipment is needed for testing (e.g. pipettes, vortex mixer, blood rockers, refrigeration, tube racks, etc.). Ensure the equipment has regular preventive maintenance and access to service when needed. Strengthen systems for reporting and repairing instrument breakdowns: Specimens will need to go to another lab if your instrument is not repaired or replaced in 1-2 days. Reagents are expensive and must be stored at an appropriate temperature. *NOTE: Quality Assurance of a laboratory includes laboratory equipment management. Analyzers and equipment must be maintained, serviced and documentation of that maintenance is an essential part of quality assurance.

Maintenance Record Example This form is an example of a maintenance record. When utilized it demonstrates service and maintenance has been done, helping to ensure that your analyzer is working optimally. This also documents and allows one to follow analyzer problems, if they are reoccurring, so that you may be able to possibly anticipate how long a particular part will last so that you can plan for the next time, and not be left with workflow interruptions.

Daily Temperature Log Example Reagents are expensive and will deteriorate, if not stored properly. By maintaining a daily temperature charts for your refrigeration, you are able to track problems which could lead ultimately to result errors.

Routine Centrifuge Quality Control Record Example

Another piece of equipment which must be maintained and documented are centrifuges, usually bi-annually.

Thermometer Accuracy Quality Control Form Example Thermometers should be monitored for accuracy and documented.

Reagent and Consumable Management: Reagents and Consumables Uninterrupted reagent supply is critical to continued testing. Besides the test reagent kit, you need other consumables, (e.g. pipette tips, gloves, printing paper). Strengthen systems to prevent reagent stock-outages. Specimens have to be sent to other testing laboratories or discarded if reagent supply is interrupted for more than 1-2 days. Careful forecasting of reagents and other laboratory consumables from all levels is necessary. Reagent and consumable forecasts should be communicated early and in a standard format to planning purchasing authorities. Maintain good storeroom inventory, FEFO stock and provide security for inventory.

Reagent Record Chart Example By keeping an inventory of reagents, you will better be able to anticipate how quickly your inventory is used and when supplies are low. This chart allows for recording of lot numbers and expiration dates also.

Positive and Negative Control QC Chart Example This form documents that quality control was done with positive and negative control values.

SPECIMEN TRANSPORT LOGISTICS:* Reliable specimen transport systems from clinics to CD4 laboratories will be needed with SOPs for pre-analytical, analytical and post-analytical phases of testing: Specimen labeling and test requisitions Storage and transport Delivery and receipt Resolution of problem specimens

*NOTE: Maintaining specimen logs is critical. Auditing of each step following and documenting specimen transport should be mandatory.

DATA MANAGEMENT:* Ensure reliable and rapid delivery of results to clinic sites. Ensure clinics have systems for receiving and processing result data. Ensure the laboratory maintains records of result data for defined periods. Systems are needed to collate, aggregate CD4 count data from laboratories-need for monitoring and evaluation of program success. Use standard reporting formats. Ensure dedicated human and other resources for data management are assigned. *NOTE: Have Standard Operating Procedures (SOPs) for all phases of result reporting. It is not enough to just analyze and get a result. That result must have a reliable way to be delivered to clinical sites. Always keep a backup or copy of all results in case originals are lost.

Example of Report Form for Pediatric Patients

Example of Patient Report Form Notice necessary information needs to be recorded.

SAFETY:* The following are required: Safe blood collection devices Safe disposal of biohazardous waste Training on safety procedures and universal precautions Post-exposure prophylaxis readily available and procedures for obtaining it in place

*NOTE: See the safety information provided in the safety module. Document when laboratory staff are trained and recertified on safety procedures and universal precautions. Plan ahead and have in place all of the necessary post-exposure prophylaxis before it is ever needed with SOPs for EXPOSURE, should it be needed.

Daily Workstation Decontamination Log Example This form is one example of how to document that safety practices are performed.

QUALITY SYSTEMS: Ensure testing in all laboratories follow Standard Operating Procedures based on international guidelines. CD4 count SOPs should fall within the overall laboratory quality management system. Establish internal and external quality policies. Establish intra-and inter- laboratory monitoring and corrective action programs for testing quality. Ensure dedicated human and other resources for the above.

Corrective Action Log Example Part of providing quality in a laboratory is to document and evaluate corrective action logs on all instrument, equipment and workflow process problems. This corrective action log allows for documentation so that there is no question as to what problems occurred.

Human Resources and Training* CD4 counting is not a common test. Theory and training has not traditionally been offered in the past.

Three levels of training are required for existing staff: 1. Instrument and software operation and applications (provided by instrument manufacturer/vendor) 2. 3. CD4 laboratory lab operations and management CD4 specimen collection, transport and biosafety for CD4 clinic staff

Training should be facility-based. *NOTE: Documentation of training is an important part of quality assurance and another part of laboratory management

Personnel Training and Documentation in Flow Cytometry Example This form documents that laboratory personnel has been trained and when the training occurred.

Competency Revalidation Employee Documentation Example Personnel should demonstrate competency an annual basis.

Timetable for QA/QC Procedures & Reviews Example Overall Timetable for QA/QC procedures laboratory director is responsible for. By glancing at this chart, you can quickly view all when and what actions are necessary in order to maintain quality control and quality assurance, maintained in laboratory management.

Conclusions CD4 counting requires both reliable and competent instrument operation and laboratory management to ensure high quality results, adequate testing resources and short turn-aroundtime. CD4 laboratory management and planning are key to sustainable testing services and meeting the demands of the HIV treatment and care program.

STANDARD OPERATING PROCEDURES (SOPs): Writing a Standard Operating Procedure (SOP) All standard operation procedure/policies are written to describe current technical practices. If a change in reagent, or instrumentation or methodology is changed, the SOP will be revised.

All technical procedures will be checked for compliance with standards of practice as defined in the most recent edition of accepted clinical laboratory policies and practices. All documents generated should be clearly identified with the facility identification: Name of lab Name of Institute Address

Policies are numbered with a unique ID and define the manner in which standard operation procedures (SOPs), policies and work forms are designed, prepared, maintained, placed into service, archived and retired from service. A policy describes a definite course of action or prescribes adherences to proper procedure or ethical standards of practice. Establish a Written Procedure: Elements of a Procedure Principle of test Purpose Safety precautions Specimen collection and labeling Reagents, supplies, equipment Step-by-step procedure Calibration Quality control Interpretation Calculations Reportable result range Limitations of assay References, distributions Attachments

CD4 Module 13A: Flow Cytometry Instrumentation Troubleshooting Part A

Purpose: To provide a working knowledge of flow cytometry theory and troubleshooting. Pre-requisite Units: CD4 Module 12

Learning Objectives: Describe the basic theory of flow cytometry and the single platform analyzer, FACSCountTM. Analyze automated flow cytometer results to determine if patient results are acceptable. Suggest how to troubleshoot for when quality control results fail to meet expected results.

Recognize the significance of flagged error messages and what appropriate actions must be taken.

CONTENT INITIAL SET-UP: Out of the Box* For the service engineer: Check instrument for visual damage. Check for any loose parts or connections. Make sure all computer boards are properly seated. Check the socket to verify proper voltage outlet. Plug instrument power cord into (voltage stabilizer) electrical supply. Confirm the correct voltage on instrument. Plug in and charge the electronic pipette (min. 10hrs).**

*NOTE: Each instrument is typically installed by the manufacturer, in this case, BD Bioscience. It is a good idea to check for these items along with the service engineer so that you become familiar with these features. **ADDITIONAL NOTE: The reverse-pipette is separate from the analyzer and needs to be plugged in for a minimum of 10 hours. Ask about purchasing extra battery backup or extra pipette, if possible.

Exterior Parts of Analyzer Becton Dickinson FACSCountTM.JPG Here are various exterior parts on the front of the analyzer. The fluidics compartment is housed behind the cover shown on the front of the analyzer.

Instrument Set-Up:*

Follow manufacturers instructions to: Load the software. Prepare the fluidics system. Align and optimize the optical system.

Remove crossover signals (set the compensation in high volume analyzers, not the FACSCount). Standardize settings across instrumentation. Establish sensitivity and linearity. Establish analysis ranges.

*NOTE: Basically, the service engineer will instruct you to: load software, or insert a floppy disc into the disc drive and allow the software to load. Fill the fluidics, which is sheath fluid reservoir and prepare the waste reservoir with a dilution of bleach. Go through the proper checks of the optical, laser system. In general, most CD4 analyzers will require that you do the following for instrument set-up. Notice that fluorochrome dye compensation is a part of quality control, required with high volume, dual platform, CD4 analyzers, in order to capture only the desired amount of each different signal. However, this is will not be required with the FACSCount.

Load the Software* A magnetic floppy disk is the software with instructions for the FACSCountTM to function. Load the Software.JPG *NOTE: The software supplies instructions for the analyzer to operate. Once the disc is inserted, the process of providing instructions will begin.

Prepare the Fluidics.JPGPrepare the Fluidics* Before turning on the instrument, it is necessary to fill the fluid reservoir with sheath fluid for the days specimen run. This involves pinching the connection tubing, removing, filling and replacing the sheath reservoir and turning on the instrument. *NOTE: Fluidics system begins with the sample probe and, under pressure, once the sample is taken in goes through the fluidics system, passing through the laser intersection and into the waste receptacle. The sheath fluid reservoir is easily removed to fill by disconnecting the system fluid connector tubing.

QUALITY CONTROL (QC): Reviewing Quality Control in Flow Cytometry Purpose of QC: Assures proper functionality of instrumentation. Means of assuring accuracy of unknowns. Monitoring the integrity of the instrument set-up.

Flow Cytometer Quality Control* Optical alignment is checked daily. Most clinical flow cytometers capable of 3- and 4-color immunophenotyping have fixed optical systems (i.e., relative position of the flow cell with respect to the optical elements is fixed) and the operator cannot optimize o Regardless of whether it can be fixed or can be aligned, optical alignment must be checked to ensure that the cytometer gives acceptably bright fluorescence measurements and that homogenous peaks are produced for all parameters to be used in sample analysis. *NOTE: Coulter FC500 has user-adjustable optics.

Instrument Set-up: Instrument QC Initial Set-up: Flow cytometers are aligned by the manufacturer and technical service to established specifications. Daily Monitoring: On a daily basis, the lab verifies that the flow cytometers performance is consistent with established specifications.

Quality Control* Establish normal population reference ranges for adults, and when possible, children. The more normal specimens run the better. Calculate the mean of these normal specimen samples and the standard deviation. Your reference range will be the mean +/- 2 standard deviations.

*NOTE: It is critical that patient normal values be established for a given population; both adult and pediatric values must be established due to the big difference in normal values for adult and pediatrics. Materials for this procedure are patient specimens from patient who are considered to be in a healthy state. These specimens may come from a patient admitted for a non-critical condition such as routine surgeries or non life threatening diseases. A minimum of twenty specimens must be run. For most accurate representative results, 30 specimens should be run over a three-day period. This will permit more variance to be associated with the results; therefore, a more representative of true testing conditions. In order to maximize on specimen availability and eliminate differences, the specimen should be run by all methods at the same time that is automated and manual. When adequate numbers have been assayed for all CD4 values, a mean and SD will be calculated for each analyte for all methods. The range for the method is from a minus 2SD TO PLUS 2SD. For example, if the CD4 mean value = 1000 and the standard deviation is 100, the range would be 800-1200. These normal values should be a standard part of a patient report in order for a physician to evaluate a patient as normal or abnormal. If manual and automated methods normal value were found to differ, the physician should be alerted when an alternate method was used, and the patient report should reflect the normal values for that specific test method.

LINEARITY: Quality Control Method* FACSCountTM quality control is facilitated by: Running FACSCount Control Beads:

o Four Control Bead populations (or three when using the FACSCount CD4/CD3 Reagent) with normal human blood or process control. o Beads check count linearity. Running a process control:

o Can be run with Control Beads. o Commercially-available preserved specimen, or normal blood specimen can be tested in parallel with patient samples. Software surveillance of instrument performance.

*NOTE: It is necessary to obtain a normal blood specimen for each day you use the instrument. Control beads are stable for one month at 2-8C.

Optical Alignment: Running QC Use stable calibration material such as microfluorospheres labeled with fluorochromes that have been measurable and known forward-scatter, and fluorescence properties in each channel to be used for sample analysis.

FACSCountTM reagent uses reference beads that are used by the system software to check instrument alignment.

Quality Control* Paired Tubes Containing Beads Tube #1: CD3 PE-Cy5 and CD4 PE Tube #2: CD3 PE-Cy5 and CD8 PE Controls to Verify Count Linearity (4 concentrations of beads) Zero = 0 beads/ L Low = approx. 50 beads/ L Medium = approx. 250 beads/ L High = approx. 1000 beads/ L Uses fixative solution: formaldehyde 5% *NOTE: Control runs set up the instrument and check the linearity as well as the reagent activity.

FACSCount Quality Control* Run Control Beads each day the instrument is turned on and whenever you open a new lot of reagent. Data for each control run is stored on the protocol diskette until the next time controls are run. Normal donor blood samples should be run with the Control Beads added, prior to running patient samples. *NOTE: Run controls (beads with a normal blood specimen) each day when the instrument is turned on and whenever you open a new lot of reagent. Also, for quality control, after analyzed and passes, you must also remember to run the normal blood specimen along with your patients, using this normal specimen as a control.

Procedure for Staining the Controls: 1. Label 2 pairs of reagent tube pairs as: Pair One: Zero and Low; and Pair Two: Medium and High.

2. Vortex the reagent tube pairs upside down for 5 seconds at a midrange setting. 3. Vortex pair upright up for 5 seconds. 4. Open the reagent tube pairs with the coring station by placing reagent tube onto coring station and pulling the lever down to core the tubes. Then, place tubes in workstation to keep tubes upright, and cover them from light by placing the cover over tubes. 5. Mix the whole blood control by inverting the vacutainer tube 5 times. 6. Pipette 50 L of normal blood into each of the four reagent tubes. 7. Cap the tubes and vortex upright for 5 seconds. 8. Incubate the tubes for 60 to 120 minutes at room temperature. Close the cover to protect tubes from light. 9. Uncap the tubes and pipette *50 L* of fixative solution into each reagent tube, changing tips between tubes. 10. Recap and vortex the tubes. These samples can be held 24 hrs before adding control beads. Uncap just before step 11. 11. Remove one pair of zero/low control beads and one pair of medium/high control beads from the control kit and place them in the control area of the workstation. Add control beads to the reagent tubes just before running the tubes on the instrument. Store reagent tubes in the workstation until you are ready to add control beads. 12. Vortex the Zero/Low control bead pair and vortex the Zero bead. Pipette 50 L of the Zero control beads (yellow) into the CD4 reagent tube (green top) labeled zero. Remember: Add these beads just prior to running sample. 13. Pipette *50 L* of the Low control beads (red top) into the CD8 tube labeled Low (clear top). 14. Pipette *50 L* of Medium control beads (blue top) into the CD4 tube labeled Medium (green top). 15. Pipette *50 L* of High control beads (purple top) into the CD8 tube labeled High (clear top).

FACSCountTM Control Results* *NOTE: In addition to a message of Passed, other results need to be checked before considering that your controls have passed. Your T-sum or the sum of the CD4 tube and your CD8 tube must equal the total CD3 (averaged). All the CD3 tubes (4) must be compared to be within 10% of one another.

BD FACSCalibur Sensitivity Check BD CaliBRITE 3 and APC Beads are tested using B/D FACSComp. Changes in PMT voltages and compensation may indicate that there is an instrument problem.

PMT voltages and Fluorescence Compensation are checked for significant day to day variation.

PROBLEMS: Quality Control: Problems/Failure* What do you do when controls are not within the expected range? Do not analyze and report out-patient results yet. Verify instrument functioning. Check for shifts and trends. Check integrity of material (reagents not refrigerated or out-dated). Troubleshoot. Repeat the assay.

*NOTE: In general, for all flow cytometry instruments, you are going to go through a series of steps to figure out why your controls did not work. Check your instruments fluidics, electric, function, if you have been running the assay now for some time, check if this lot number of control has been failing previously by checking your corrective action log. By plotting bead control results, you will be able to then go back and look at this for individual lots of control. Troubleshoot by ruling out one variable at a time. Repeat the assay after looking at these variables.

What do you do when controls are outside expected range?* Ensure the control Material was mixed/vortex prepared properly.

Material was pipetted properly. Also, check pipette precision and accuracy (especially if CD3 tube variability was not within 10%). Check if controls were not stored at proper temperature. Identification information was entered correctly (numeric keyboard not entered correctly).

Setup information, assigned values, and expected ranges matches the control package insert for the current lot number being used. *NOTE: We outlined the steps for preparing our reagents on the FACSCount for the purpose of identifying the variables just in the preparation of our controls. Pipetting errors account for variability and control run failures, so be sure to practice pipetting, especially if your CD3 tube to tube variability was not within 10%.

What do you do when controls are outside expected range? If any of the problems existed, re-run the control; otherwise proceed to the next step.

Always record the problem in a correction log and re-run the control to ensure the problem was not a statistical outlier. If the control fails again, open a new control packet to rule out control sample problems.

PATIENT RESULTS: FACSCountTM Results The CD4 and CD8 absolute counts are CD3+/CD4+ and CD3+/CD8+ T-lymphocyte counts, respectively. The CD3 absolute counts are reported as the average of the total CD3 value obtained from the CD3/CD4 and CD3/CD8 tubes. CD4/CD8 is the ratio of CD3+CD4+ lymphocytes to CD3+CD8 T-lymphocytes. CD3/CD4 is the ratio of CD4+CD3+ T-lymphocytes to the average CD3+ value from both tubes. CD3/CD8 is the ratio of CD8+CD3+ lymphocytes to the average CD3+ value from both tubes. PROBLEM SOLVING Troubleshooting: When to Troubleshoot the Assay CD4 outside expected result CD3 tube to tube check not within 10% CD4 and CD8 sum is not equal to CD3 T- cell, within +10%

When Outside the Expected Result: In order to troubleshoot why your patient result is outside the expected result, it is important to take into consideration all factors that affect the test result, from the time the test is ordered:

Specimen Related Problems (Pre-Analytical) An instrument problem is differentiated from a specimen-related problem by running a control. If the control results are acceptable, check for: Clots Hemolysis

Minimum sample, <25% full vacutainer

If the CD4+ T-cell result does not correlate with the patients clinical picture or expected value, and the controls are within range, review of the specimen history is vital. Anticoagulant used:

o Verify that the proper anticoagulant was used to support cell viability for the period of time preceding flow analysis. Age of specimen:

o The time drawn and time received at the laboratory allow prediction of specimen quality. Specimen temperature:

o Discuss with pre-analytic personnel and review laboratory receipt records to verify the temperature conditions. Documentation audit:

o All paperwork should be audited for any abnormal times, temperatures, or history. Patient history:

o Medications may affect patient results, refer to history.

Potential Problems with Specimen Integrity CAUSES RESULTS Transport temperature too high Debris increase in light scatter Holding temperature too low, which can cause hemolysis & also loss of CD4 Light scattering patterns smear; decreased CD4 Specimen too old Neutrophils degranulate, increase in debris, CD4/8 count decreased

When Outside the Expected Ranges: Analytic problems, which may cause results outside of range with regard to specimen integrity:

Lymphopenia Poor viability Membrane debris

Post-Analytic Assessment Any analytic explanations for low/abnormal values? Laboratorys proficiency test performed? How many cells were analyzed? Level of laboratorys experience? What reagents were used?

When Outside the Expected Result: Analytical Investigation If the control shows similar problems to the patient results tested, it indicates a general instrument, pipetting problem, or reagent problem.

For sources of erroneous results, check: Fluidics: Clog in system, perform maintenance, system cleaning/priming, empty reservoir, check for tubing leaks. Optics: Calibration and control checks must be done.

Electronics: Check power supply, use power surge protector.

Reagents: Check for expiration dates, date of opening, storage history, and perform and document lot to lot checks.

When Outside the Expected Result: Analytic Investigation Troubleshoot your assay.

Go over your technique: Did you vortex enough and at the proper times? Did you keep prepared samples in the dark during incubation and before analyzing? Did you mix contents of two different reagent kits with different lot numbers?

Corrective Actions of Instrument Malfunction Many instruments have computerized on-board monitoring systems for detecting laser output, photomultiplier tube voltage, reagent levels, wash and sampling functions. Error flags will often print out to warn the operator of instrument malfunction and prevent running a patient test or printing out questionable patient test results. For example, the FACSCountTM prints out error numbers and messages indicating instrument malfunction: The following tables briefly explain the error codes and messages that can occur on a FACSCount, and other Flow Cytometers printout. For detailed information on causes and corrective actions, refer to the Printed Code Errors in the Troubleshooting Chapter or Section of the Users Guide or Manual provided by the manufacturer of your flow cytometer.

Error Codes for FACSCountTM CODES POSSIBLE CAUSES RECOMMENDED SOLUTIONS 01 Pipetting error made (50 L of blood not added to both tubes). Prepare sample again. See Appendix A for instructions of pipetting techniques. Blood in CD4 tube and CD8 tube not matching, for example different donors. Prepare sample again. Sample not properly mixed.

Prepare sample and vortex immediately before running on instrument. 02 CD4 and CD8 tubes run out of sequence. Run CD4 tube followed by CD8 tube. Pipetting error. Prepare sample again. See Appendix A for instructions. Ensure that the volume of blood added to reagent tube is 50 L. Blood in CD4 tube and CD8 tube not matching, for example different donors. Prepare sample again. 03 CONTACT YOUR LOCAL BD REPRESENTATIVE IF YOU SEE THIS ERROR CODE. ------

Errors/Issues for FACSCountTM OBSERVATION POSSIBLE CAUSES RECOMMENDED SOLUTIONS Instrument not powering on Unconnected power cord Check power cord connection to both the instrument and the wall outlet Blown fuse Change fuse, see page 94 A buzzing sound

Instrument equipped with internal buzzer that sounds for full waste reservoir Replace the full reservoir with the spare reservoir. Instrument-equipped internal buzzer sounding for damaged waster reservoir filter Low event rate No reagent tubes in sample holder Make sure there is no air in the flow cell. Then place sample in sample holder. If air was introduces into the flow cell, see Priming System page 44. Air in flow cell

Instrument Service: When to Call When cleaning procedures fail to resolve the problem and you have consulted the troubleshooting section of the Instrument Manual/Users Guide with no success. When controls repeatedly fail with several different normal human controls, where specimen integrity problems are ruled out. When troubleshooting error instructs you to do so. Exercise #1: Troubleshooting Case Study After control results pass, with CD3 tube to tube check just within 10%, two of 3 patient samples tested fail the CD3 tube to tube average check. Specimen integrity is checked, and all preanalytical variables are ruled out. Question: What could be the problem? Answer: Pipetting technique is crucial to accurate results. It is important to practice technique and to check pipette precision and accuracy.

Exercise #2: Control Run Case Study A technologist notices that the days control run is outside the acceptable range. The CD3 tube average is within 10%, but the tech notices that the scatter on the service report is abnormal with a large amount of debris and the pattern smearing.

Question #1: What could be the problem? Answer #1: Pre-analytical problem, possibly specimen being exposed to temperature extremes, too hot or cold, and/or hemolyzed while waiting to be used as a control.

Question #2: What should be the course of action? Answer #2: Select another blood sample.

SUMMARY: While troubleshooting your assay and results, it is important to systematically rule out problems, looking at quality control, and the pre-analytic/ analytic process. CD4 Module 13B: Flow Cytometry Instrumentation Troubleshooting Part B

Purpose: To provide a working knowledge of flow cytometry theory and troubleshooting. Pre-requisite Units: CD4 Module 13A Learning Objectives: Identify problems that can occur with the immunophenotyping systems (e.g. BD FACSCountTM). Recognize the significance of flagged error messages and what appropriate actions must be taken. Suggest how to troubleshoot for when instrument fails to meet expected performance. Perform common troubleshooting procedures.

CONTENT INSTRUMENT INFORMATION: Make and model of flow cytometer: Becton Dickinson FACSCount Serial number and/or system ID number: D12345 Manufactured Date: April 2005 Date of Installation: September 2005 Type of laser: Convection-cooled Helium-Neon Laser Computer Operating System and Software Version: FACSCount SW Version 1.5 4/05 on DOS 5.0 Peripheral equipment ID (printer, auxiliary power supply, auto sample loader, etc.): FACSCount Electronic Pipette S/N AB12345 Calibrated September 2005

Technical Support Contact Information: BD Biosciences and contact your local BD representative

PRECISION AND ACCURACY: Troubleshooting Exercise: Method to Ensure Pipetting PRECISION Use the following procedure to check that you are consistently pipetting a precise volume: Pipette 20 replicates of blood and record the weight of each. Calculate the coefficient of variation (%CV). The %CV should be <2%.

If the CV is >2%, performances of the BD FACSCount system will not meet the expected results.

Method to Ensure Pipetting PRECISION 1. 2. 3. 4. 5. Place a plastic weigh boat or similar container in an analytical balance. Tare the balance so it reads zero. Pipette 50 L of normal blood into the container. Record the weight of the blood to the nearest 1/10,000 g. Tare the balance.

6. Repeat steps 3 through 5 for a total of 20 replicates. (Change pipette tips between replicates.) 7. Calculate the %CV.

Before calculating the %CV, you must calculate the mean and the standard deviation (SD). The %CV should be <2%. Example: Replicate Weight (g) Replicate Weight (g) Replicate Weight (g)

Replicate Weight (g) 1 0.0526 6 0.0524 11 0.0528 16 0.0527 2 0.0526 7 0.0530 12 0.0518 17 0.0533 3

0.0521 8 0.0512 13 0.0530 18 0.0526 4 0.0534 9 0.0532 14 0.0530 19 0.0533 5 0.0520 10 0.0521 15

0.0526 20 0.0527 Example Answers: MEAN: Mean = sum of all replicate weights number of replicates Mean = 1.0524 20 Mean = 0.05262

STANDARD DEVIATION: SD = 0.00056

%CV: %CV = (SD mean) X 100 %CV = (0.00056 0.05262) X 100 %CV = 1.064%

Troubleshooting Exercise: Method to Ensure Pipette ACCURACY The following procedure allows you to verify that the BD FACSCount pipette is delivering an accurate volume of fluid: Pipette five replicates of distilled water and record the weight of each. Calculate the mean. The mean weight should fall within 0.0490 and 0.0510 grams.

Method to Ensure Pipette ACCURACY 1. 2. 3. Place a plastic weigh boat or similar container in an analytical balance. Tare the balance so it reads zero. Pipette 50 L of liquid into the container.

4. 5. 6. 7.

Record the weight of the water to the nearest 1/10,000 g. Tare the balance. Repeat steps 3 to 5 for a total of five replicates. (Change pipette tips between replicates.) Calculate the mean weight.

The mean should fall within 0.0490 and 0.0510 grams. See the following example: Replicate Weight (g) 1 0.0502 2 0.0501 3 0.0504 4 0.0499 5 0.0502 Example Question: Calculate the mean. Example Answer: Mean = sum of all replicate weights number of replicates Mean = 0.2508 5 Mean = 0.0502

Troubleshooting Problem: High Event Rate*

POSSIBLE CAUSES RECOMMENDED SOLUTIONS Sample was aspirated too quickly Check that saline filter is full if not open clamp to fill it up to the top Flow cell dirty Perform periodic cleaning procedure *NOTES: Dirty or clogged sample probes and flow cells contribute to errors in testing. Daily and periodic cleaning procedures are part of routine instrument maintenance, but also are primary troubleshooting steps when quality control results do not meet expected results. These cleaning procedure exercises are written for the FACSCount instrument, but are also applicable to FACSCalibur and FACScan cytometers.

Troubleshooting Problem: Low Event Rate or No Events* POSSIBLE CAUSES RECOMMENDED SOLUTIONS Loose fluid connectors Check and tighten all fluid connectors Clogged sample injection Perform cleaning procedure(s): Daily Cleaning Periodic Cleaning Also, clean injection probe Waste reservoir failure Replace the full reservoir with the spare reservoir *NOTES: Dirty or clogged sample probes and flow cells contribute to errors in testing. Daily and periodic cleaning procedures are part of routine instrument maintenance, but also are primary troubleshooting steps when quality control results do not meet expected results. These cleaning

procedure exercises are written for the FACSCount instrument, but are also applicable to FACSCalibur and FACScan cytometers.

CLEANING PROCEDURES: Daily Cleaning Clean the BD FACSCount instrument before you turn off at the end of each day. Proper cleaning ensures that your instrument maintains its reliability. Use the cleaning tubes and dispensing bottles provided with the system and follow the prompts that the software displays.

Perform the cleaning procedure at the following times: At the end of each day

When the screen message appears recommending that you run the cleaning cycle (the message appears after at least 51 reagent pairs have been run). When recommended to do so by the troubleshooting section of the users guide.

If you are not using the instrument or you are turning off the power, leave a tube of distilled water on the instrument with sample holder up. This protects the sample injection probe from the formation of salt deposits.

FACSCount Cleaning Step-by-Step 1. Fill one cleaning tube with a 1:3 dilution of bleach and a second cleaning tube with distilled water. 2. Press [UTILITY] on the FACSCount screen to display the UTILITY screen.

3. Press [CLEAN] to access the following message: Move cleaning solution tube into position. Press the RUN key. 4. Place the tube of 1:3 bleach solution in the sample holder and press [RUN].

o A message appears informing you of a cleaning boost, followed by a message that the cleaning solution will run for approximately 3 minutes. o When the cleaning cycle is complete, the sample holder lowers and the following message is displayed: Move rinsing solution tube into position. Press the RUN key. 5. Place the tube of distilled water in the sample holder and press [RUN].

o A message appears informing you of a rinsing boost, followed by a message that the rinsing solution will run for approximately 3 minutes.

o When the rinsing cycle is complete, the following message is displayed: Cleaning protocol is finished.

CHANGING POWER SUPPLY FUSE: Troubleshooting Problem: Instrument Not Powering On or No Screen Display POSSIBLE CAUSES RECOMMENDED SOLUTIONS Unconnected power cord Check power cord connection to both the instrument and the wall outlet Blown fuse Change the fuse

Changing a Fuse Change a fuse when you are instructed to do so in the troubleshooting section of the users guide or by a BD service representative. The fuse drawer is located at the back of the instrument to the left of the AC plug. CAUTION: Unplug the instrument before attempting to change fuses.

Make sure the instrument has been shut down before turning off the power. Changing a Fuse 1. 2. 3. Turn off the instrument power. Unplug the instrument from the wall outlet. Unplug the AC plug from the back of the instrument.

o This allows easy access to the fuse drawer. 4. Place a small screwdriver into the slot at the right of the fuse drawer and pry open the drawer. o The fuse drawer will pop out. 5. 6. Pull out the fuse drawer. Remove each fuse and examine it.

o If the filament is broken or you see any discoloration of the glass, discard the fuse. If the fuse appears to be in good condition, check with a continuity tester. 7. Replace any damaged fuses with new fuses from your spares kit.

o CAUTION: For protection against risk of fire, replace the fuses with fuses of the same type and rating. 8. Slide the fuse drawer into the instrument.

o The clip should be to the right. Push gently on the drawer until it snaps into place. 9. Plug the AC power cord into the instrument.

10. Plug the other end into the wall outlet.

DIFFERENT LOTS OF REAGENT IN USE: Troubleshoot Problem: Different Lots of Reagents Used for Sample and Control Preparation* The reagent used for patient samples should match the reagent lot for the control run. In this exercise, prepare and run sample using both same and different lots of reagent from the control. Compare report printouts of the samples prepared with matched and unmatched lots. *NOTES: Several FACSCount Printed Error Codes identify the condition that a different lot of reagent was used for sample and control preparation, as the possible cause of the error. Also, follow routine procedures to prepare and run both the controls and samples.

Create a Troubleshooting Worksheet What problem did you encounter? What troubleshooting steps did you take? Which troubleshooting steps led to a resolution of the problem?

REPLACING SYTSTEM FLUID FILTER: Troubleshooting Problem: Sample Acquisition Error (Code 04) POSSIBLE CAUSES RECOMMENDED SOLUTIONS Dirty system fluid

Replace system fluid with clean system fluid System fluid filter to be replaced Change system fluid filter

Changing the System Fluid Filter The system fluid filter cleans the system fluid before it enters the flow cell. Contaminated system fluid can affect sample results. The filter will be changed by your BD service representative as part of the regular preventive maintenance. However, you might be instructed by your service representative or the troubleshooting section of the users guide to change the filter.

Changing the System Fluid Filter This diagram of the system fluid assembly shows the locations of the various parts of the system fluid filter assembly:

Changing the System Fluid Filter Unclamp the plastic pinch clamp located on the air vent tubing and allow the sheath filter to fill with system fluid. You will hear the air being aspirated from the filter as it fills. If air bubbles are visible in the filter, tap the filter gently to dislodge the bubbles, forcing them into the air vent tubing.

Changing the System Fluid Filter: Step-by-Step Procedure 1. 2. 3. Turn off the BD FACSCount instrument. Open the fluidics compartment door by pressing the top center of the door. Remove the system fluid and waste reservoirs.

o This allows you to easily access the system fluid filter to remove it. 4. Disconnect the fluid filter input and output tubing from the fluidics panel by squeezing the metal clips on the connectors and pulling. 5. Unscrew the cap located on the filter port.

6. Unscrew the two thumbscrews located below the mounting bracket and set the thumbscrews aside.

7. 8. 9.

Lift the filter up and out of the mounting bracket. Lift the securing bracket over the top of the filter and set the bracket aside. Discard the old filter.

10. Slip the securing bracket over the top of the new filter. o Make sure the securing bracket studs are pointing down. 11. Install the filter in the mounting bracket. o Align the securing bracket studs with the holes in the mounting bracket. o Make sure the filter input and output tubing are pointing toward you. 12. Screw the two thumbscrews onto the studs. 13. Replace the cap by screwing it onto the filter port. 14. Connect the fluid input and output tubing connectors to the fluidics panel by pushing firmly until you hear a click. 15. Replace the system fluid and waste reservoirs. 16. Turn on the instrument. 17. Remove the air from the sheath filter. 18. When all air is removed from the filter, secure the pinch clamp. 19. Prime the system to remove any air that might be trapped in the flow cell. 20. Close the fluidics compartment door.

CLEANING THE SAMPLE INJECTION PROBE: Perform this procedure when instructed by a BD service representative. 1. 2. Press [UTILITY] on the FACSCount screen to display the UTILITY screen. Press [CLEAN].

o The following message is displayed: Move cleaning solution tube into position. Press the RUN key. 3. Place a tube of hot tap water in the sample holder and press [RUN].

o A message appears informing you of a cleaning boost, followed by a message that the cleaning solution will run for approximately 3 minutes. o When the cleaning cycle is complete, the sample holder lowers and the following message is displayed: Move rinsing solution tube into position. Press the RUN key.

4.

With the tube of hot tap water still in the sample holder, press [RUN].

o A message appears informing you of a rinsing boost, followed by a message that the rinsing solution will run for approximately 3 minutes. o When the rinsing cycle is complete, the following message is displayed: Cleaning protocol is finished. Cleaning the Sample Injection Probe Insert a stylet (a thin metal wire included in the spares kit) in the opening of the sample injection probe. While holding the end of the stylet, gently push it up into the injection probe. Remove the stylet. After unclogging the sample probe, perform a drain to flush the dirt to the waste!

PERIODIC CLEANING:* Perform the periodic cleaning procedure when recommended to do so by the troubleshooting section of the users guide or by a BD service representative. *NOTE: Allow approximately 1hr 30min to complete periodic cleaning exercise.

Flow Cell:

System Fluid and Waste Fluid Reservoir Connectors:

Periodic Cleaning: Step-by-Step Procedure 1. Open the fluidics compartment door to access the system fluid and waste reservoirs.

o Push the top center of the door; then release to open. 2. Disconnect the system fluid tubing from the fluidics panel.

o Squeeze the metal clip on the white connector and pull the tubing from the fitting.

3.

Disconnect the system fluid detection probe connector from the fluidics panel.

o Squeeze the white tabs at the sides of the connector and pull. 4. 5. Remove the system fluid reservoir from the instrument. Empty the system fluid completely from system fluid reservoir.

o Rinse the system fluid reservoir thoroughly with DI water and drain. 6. 7. 8. Fill the system fluid reservoir with 1-2 liters of freshly prepared 1:10 dilution of bleach. Recap the reservoir and replace it in the instrument. Remove the waste reservoir, empty waste fluid and replace reservoir in the instrument.

9. Disconnect the fluid filter output tubing from the fluidics panel by squeezing the metal clips on the connectors and pulling. 10. Connect the system fluid tubing directly to the output tubing connector, bypassing the filter. o CAUTION: Do not connect the system fluid tubing of the 10% bleach solution tank to the system fluid connector. This would allow the bleach solution to pass through the filter unit and damage the filter. Leave the system fluid tubing connector port unconnected during this cleaning procedure. o Bypass the saline filter to prevent damage from the bleach solution. 11. Fill one cleaning tube with a 1:10 dilution of bleach and a second cleaning tube with distilled water. 12. Press [UTILITY] on the FACSCount screen to display the UTILITY screen. 13. Press the [DRAIN] to access the following message: Put on tube of distilled water. Press DRAIN to start draining. 14. Place the cleaning tube with 10% bleach into the holder and press [DRAIN] again. o The following message is displayed: Press STOP to stop draining. 15. When all of the fluid has drained from the flow cell, press [STOP].

o The flow cell fills automatically. The following message is displayed, followed by the ULTILITY screen: Refilling flow cell. Please wait. 16. Repeat steps 12 14 two times. 17. Press [CLEAN] TO access the following message. o CLEAN Move cleaning solution tube into position. Press the RUN key. 18. Place the tube of 1:10 bleach solution in the sample holder and press [RUN]. o A message appears informing you of a cleaning boost, followed by a message that the cleaning solution will run for approximately 3 minutes. 19. Perform the cleaning procedure with only the bleach solution for a total of five times. o When the following prompt appears after the first 3 minutes of each cleaning cycle, do not remove the bleach tube and replace with DI water: Move rinsing solution tube into position. Press the RUN key. o Press [RUN] to allow the bleach to run an additional 3 minutes each cycle. This will result in a total of 30 minutes of bleach run for five cleaning cycles. 20. Replace the bleach in both the sheath tank and cleaning tube with DI water. o Rinse the sheath tank thoroughly with DI water to eliminate any remaining bleach solution. 21. Repeat steps 12 to 15 with DI water in the both the sheath tank and cleaning tube. 22. Perform the cleaning procedure with only DI water for a total of five times. This will result in a total of 30 minutes of DI water run for five cleaning cycles. 23. Replace the DI water in the sheath tank with system fluid (sheath fluid). o Rinse the sheath tank with sheath fluid to eliminate any remaining DI water. 24. Disconnect the system fluid tubing from the output tubing connector and reconnect to the system fluid tubing connector in the fluidics panel. 25. Reconnect the system fluid detection probe connector to the fluidics panel. 26. Reconnect the sheath filter output connector to its original location.

INSTRUMENT SERVICE: When to Call When cleaning procedures fail to resolve the problem and you have consulted the troubleshooting section of the Instrument Manual/Users Guide with no success. When controls repeatedly fail with several different normal human controls, where specimen integrity problems are ruled out. When troubleshooting error instructs you to do so

TEST QUESTIONS: Question #1: The control run failed and the error code identifies possible pipetting error. What steps should be taken to ensure that the pipetting is accurate and precise?

Question #2: An error code appears that identifies a dirty or possibly clogged system. What procedure(s) can be applied to resolve this problem?

Question #3: In performing the periodic/monthly cleaning procedure, the technologist does not bypass the sheath filter and allows the bleach solution to run through the filter system. What should the technologist do when he/she realizes the error? Is acceptable to use the filter if the technologist rinses the filter thoroughly with DI water?

Question #4: The instrument does not power on. What are the possible causes?

Question #5: In performing the daily workload of patient testing, the technologist finishes a box of reagents. The technologist opens another box of reagents that are a different lot from the previous set. The technologist enters the new reagent lot into the system data information and continues staining the remainder of the samples. Since the control passed with the previous lot of reagents, the technologist does not repeat the controls. Is this acceptable? Why or why not?

CASE STUDIES: Case Study #1: Troubleshooting After control results pass, with CD3 tube to tube check just within 10%, two of 3 patient samples tested fail the CD3 tube to tube average check. Specimen integrity is checked, and all preanalytical variables are ruled out. Question: What could be the problem? Answer: Pipetting technique is crucial to accurate results. It is important to practice technique and to check pipette precision and accuracy.

Case Study #2: Control Run Case Study A technologist notices that the days control run is outside the acceptable range. The CD3 tube average is within 10% but the tech notices that the scatter on the service report is abnormal with a large amount of debris and the pattern smearing. Question #1: What could be the problem?

Answer #1: Pre-analytical problem, possibly specimen being exposed to temperature extremes, too hot or cold, and/or hemolyzed while waiting to be used as a control.

Question #2: What should be the course of action? Answer #2: Select another blood sample

SUMMARY: While troubleshooting your assay and results, it is important to systematically rule out problems, looking at quality control, and the pre-analytic/ analytic process. CD4 Module 14: Recording and Reporting of Results

Purpose: To provide a working knowledge of proper record-keeping and specimen tracking. Pre-requisite Units: CD4 Module 13B Learning Objectives: Describe proper record keeping and methods for tracking specimens through the entire testing process (pre-analytical, analytical, and post-analytical stages).

CONTENT DOCUMENTATION: Documentation of Patient Results* Laboratories must examine all specimens received to ascertain that they meet the proper criteria for data entry and processing. There must be verification that the results are correct and for the intended patient. Correct data entry and processing is extremely important to assure that the results are given out in a correct and efficient manner. Proper record keeping of patient results is vital for providing optimal patient care and gaining knowledge from patient data collected. *NOTES: Throughout the entire testing process complete record keeping is essential to ensuring that results given out for a patient are correct. It does not matter how accurately an analyzer processes a sample if the wrong result is for a particular patient, or if a result never reaches a patient file. Formatting and maintaining patient results so that they may later be easily aggregated and transformed into knowledge is important to improving patient outcomes. If data

is scattered, incomplete, or illegible, it jeopardizes the patient as well as a chance of improving patient outcomes.

TESTING WORKFLOW:* Pre-analytical stage: CD4 T-cell count procedures start with blood collection. Specimens need to be transported to laboratory under correct conditions.

Analytical stage: Specimens are tested in laboratory. Post-analytical stage: Results are produced and communicated. In clinic, results are received and placed in patient file.

*NOTES: This is a brief summary of the testing process, broken down into the pre-analytical, analytical and post-analytical stage: Pre-analytical stage: It is the laboratory responsibility. A number of factors can induce a change in CD4 T-cell counts. Acceptable criteria for your analysis need to be given. List the factors: anticoagulant, filling volume, time of collection, etc. For example: If you are using Dynabeads, your blood need to be analyzed within 6 hours. The kind of tube required also needs to be given. Analytical stage: Parameters that need to be known during the analytical stage: storage space, number of technicians doing the analysis, storage of sample test requisition forms, etc. Post-analytical stage: Factors that are important during the post-analytical stage: results given out to patients, clinicians, logistical limits e.g. if patients is coming from a rural area, etc.

Overview of Testing Workflow This diagram illustrates the record keeping and written information for all 3 stages of the testing process. With each stage is a set of important documentation formatting and/or forms, which is crucial to data management. Work list sample is optional; it will depend on the number of samples received in the laboratory and the general organization of the laboratory.

PRE-ANALYTICAL: Pre-analytical Stage* The pre-analytical stage of processing results begins with phlebotomy: Phlebotomy should be conducted with adherence to safe blood collection procedures and universal precautions. After blood specimen is collected, the tube is labeled immediately with key information: Patient ID, time and date of collection, test type (e.g. CD4), and initials of phlebotomist.

*NOTES: It is important that the tube is labeled; work with the phlebotomist to define the information that should be written on the tube. Training and procedures should be given to the phlebotomist with focus on storage condition of the empty tube, filling volume, labeling the tube, etc. Also, a requisition form should accompany the tube. The tube and requisition form should be placed in a plastic bag. A check list (summary) can be included.

Test Requisition Form A test requisition form should accompany all specimens and has multiple functions: Instructs laboratory to conduct specific test(s) Provides authorization record for test Provides laboratory with patient ID Provides laboratory with specimen age information (date and time of collection) Provides information on clinic site or health care worker to send results to

IMPORTANT: A well-designed test requisition form should fulfill all of these functions.

Specimen Transport* Transported specimens should be accompanied by: Test requisition forms Delivery checklist form - specifying patient specimens included in the delivery

A delivery checklist form: Helps to identify and resolve inconsistencies in labeling and packing of specimens Allows formal agreement between clinic and laboratory on samples delivered

*NOTES: Work with the clinicians to determine the transport conditions. Communicate to the clinicians the criteria that might force you to refuse the sample.

Sample Reception When a specimen is received in the laboratory: Check integrity (age, haemolysis, clotting). Resolve delivery problems (lost specimens, requisitions, mislabeling). Specimens are logged in for testing in a specimen reception logbook or electronic log. Assign:

o Internal laboratory testing ID: unique identifier associated with test requisition data (patient ID, date, clinic) o This unique identifier is helpful in tracking the specimen through the laboratory system.

Knowledge Check Question: What are the different forms/logs we have mentioned so far? Answer: Requisition form Delivery checklist form (if transported) Specimen reception logbook/electronic logbook

Specimen Rejection If the specimen is rejected for any reason (e.g. received clotted, or on ice), information from that specimen should be noted in a corrective action log. All specimens entered in the corrective action log should be followed up with notification to the clinic/physician to re-draw the patient and record the notification in the corrective action log.

ANALYTICAL: Sample Testing* Those specimens received by the CD4 testing section are recorded onto a Work list. Use paper-based or electronic lists (electronic databases allow for easier retrieval of patient results). Run specimens and verify correct result for correct specimen. *NOTES: During the analytic phase, it is necessary to identify the sample you will be testing in the analyzer by writing identification of the sample tubes, etc. Never leave specimen tube order up to memory.

Laboratory Reporting of Results It does not matter how accurate the patients result is if it does not find its way to the patients file in the clinic. Proper processing of the result is vital to providing patient care.

Patient Report Form* Results should be entered on both a CD4 Work list and Patient Result Form. Work lists should be filed by date in the laboratory for easy result retrieval. Patient Report Form results should be entered and processed. *NOTES: By recording your patient results in both a work list and on the patients result form, the lab always maintains a record of the result.

Archiving Results* Electronic and paper-based archives: Use consistent system based on one or more variables (collection date, clinic site, patient ID) that allows easy reference and retrieval of records. Establish system for clinic queries for missing CD4 results. *NOTES: Decide how your laboratory will file patient records/results and be consistent. Make all technicians aware of these protocols so that results may be found by everyone.

Results Release* Authorized results can be released through a variety of mechanisms, e.g.: Pick-up trays Hand delivered Courier Fax E-mail

For each clinic, establish standard procedures for results release (any governmental/institutional requirements should be fulfilled). The test is not complete until the result is made available for the next patient visit (placed in the patient file). *NOTES: SOPs for releasing results must be written and followed by each individual laboratory, with your own criteria.

POST- ANALYTICAL:

Results to the Clinic A laboratory-clinic results delivery/pick-up checklist should be used, particularly for intermediate and high volume testing situations. Clinics should establish standard procedures for the receipt, handling and filing of results: Avoid results accumulating without filing. Designate staff to manage results.

Communication Good coordination and communication between clinics and the laboratory is essential. Testing is the responsibility of both the clinic and laboratory. Non-conformance to SOPs should be noted and corrected.

SUMMARY: Good record keeping is a way to audit the CD4 laboratory testing process. Successful CD4 count testing requires the establishment of standard procedures in both the clinic and the laboratory. The following forms/logs containing essential data are necessary to maintain good records and track specimens: Test requisition form Sample delivery checklist form (if transported) Specimen reception logbook/electronic logbook Corrective action log Testing work list Patient result form

CD4 Module 15: Manual Methods for CD4 Counting

Purpose: To provide a working knowledge of the principles and procedures behind the manual CD4 count procedure. Pre-requisite Units: CD4 Module 14 Learning Objectives:

Understand the principles and procedures of the manual CD4+ count. Describe the components of the Manual CD4+ Count kit (Beckman Coulter). Understand the procedures for:

o Sample collection o Sample preparation o Reagent preparation o Reagent storage Quality assurance/quality control procedures

CONTENT INTRODUCTION: Brief Background CD4 is a protein found on the surface of: T-helper cells (a sub-class of lymphocytes) Monocytes

CD4 is a receptor for binding gp120, which is a glycoprotein in the envelope of the human immunodeficiency virus (HIV). HIV infection causes a progressive decline in CD4+ cell number that correlates with the degree of immunodeficiency. Knowledge of CD4 cell number is crucial for patient care.

CYTOSPHERE METHOD: Principles of Coulter Cytosphere Method* The test employs inert latex beads coated with murine monoclonal antibodies specific for CD4. CD4-coated latex beads can also react with monocytes, but are blocked by prior exposure to MY4 (anti-CD14)-coated beads. Beads attach to CD4+ cells forming cell-Cytosphere rosettes Rosettes can be easily identified visually and quantified in a hemacytometer: T-cell and monocyte-specific rosettes are distinguished by size and coloration of the attached beads.

*NOTES: Attachment of the CD14-coated beads (the small latex spheres) to monocytes has the effect of blocking the attachment of anti-CD4 coated beads (the large latex spheres) to these cells.

Cytosphere Kit Components* Reagent A: MY4 Cytospheres Monocyte Blocking Reagent (2% suspension of spheres in PBS with 0.2% BSA) o Sphere size is 0.55 0.65 microns in diameter Reagent B: CD4 Cytospheres Reagent (1% suspension of spheres in PBS with 0.2% BSA)

o Sphere size is 1.8 2.4 microns in diameter Reagent C: Lysing Reagent (2% acetic acid in distilled water and 0.025% crystal violet stain)

*NOTES: The crystal violet in the lysing reagent is used to stain the nuclear material of the leukocytes to facilitate identification (especially of neutrophils) in a hemacytometer.

Additional Required Materials Hemacytometer with cover slips (0.1 mm or 0.2 mm depth) Microscope Tally counter Timer Absorbent tissues Pipette tips 12 x 75 mm test tubes Petri dish and filter paper (to serve as a moisture chamber) 10 100 L mechanical pipette

Reagent Storage Keep an inventory of all kits with lot numbers and expiration dates.

Rotate stock. Store Cytosphere kits at 2-8C. Avoid extreme conditions of freezing and temperatures above 37C. Always check for evidence of deterioration: Leakage Change in color Solidification, macroscopic clumping, layering

Reagent Preparation Use reagents directly from the vial: No special preparation is required

Reagents must be at room temperature prior to use: Let sit for 15 30 minutes @ 20-25C

Mix reagents thoroughly by inverting gently until smooth suspension is obtained. Normal appearance of reagents: Reagent A: white cloudy suspension Reagent B: white cloudy suspension Reagent C: clear blue liquid

Specimen Preparation Collect blood specimens by venipuncture into evacuated tubes, completely expending the vacuum: Draw pediatric specimens in pediatric tubes to avoid underdrawing.

Specimens must be collected in EDTA with a ratio of 1.0 to 1.5 mg of EDTA per mL of whole blood: Mix blood well by inversion to prevent clotting.

Do not collect specimens in acid citrate dextrose (ACD): ACD might promote phagocytosis of latex spheres.

Label all specimens with a unique patient identifier, date, and time of collection.

Provide on the submission form pertinent medications and disease conditions that might affect the immunophenotyping test. Assure that patient information and test results are accorded confidentiality. Specimen Stability and Transport* Start the assay within six (6) hours of specimen collection. Blood samples should be transported and maintained at 20-25C until processing is begun: Avoid extremes of temperature so that specimens do not freeze or become too hot.

In hot weather, it may be necessary to pack specimens in an insulated container and place inside a second container with an ice pack and absorbent material to maintain ambient temperature. DO NOT REFRIGERATE samples prior to analysis. *NOTES: CD4 deteriorates more rapidly from the surface of T helper cells at 4C than room temperature.

Criteria for Specimen Rejection Hemolysis: Suggests damage to leukocytes as well as RBCs.

Clotted blood: Might result in selective loss of certain subpopulations.

Partial draw: Can cause hypertonic conditions that damage cells.

Temperature extremes Improper specimen labeling Specimen integrity Visually inspect for any other changes that suggest specimen is compromised.

TEST PROCEDURES: 1. Label one 12 x 75 mm tube A+B and one tube C. Label both tubes with specimen ID:

2. IMPORTANT: For each pipetting step, carefully wipe the outside surface of the pipette tip with absorbent tissue before dispensing the contents:

3. Place 100 L of Reagent C into tube labeled C:

4. Place 100 L of whole blood into tube labeled A+B:

5. Add 10 L of Reagent A to the blood in tube A+B. Holding tube vertical, mix gently by hand for 2 minutes immediately after adding Reagent A:

6. Add 10 L of Reagent B to the same test tube. Holding the test tube vertical, mix gently by hand for 2 minutes immediately after adding Reagent B:

7. Add 10 L of the blood-latex spheres mixture from the A+B tube to the C tube. Immediately hold tube C vertical and mix gently by hand for 10 to 15 seconds to lyse red blood cells:

8. Place cover slip on hemacytometer. 9. Load BOTH chambers of the 0.1 mm deep (and one chamber of the 0.2 mm deep) hemacytometer with the sample from tube C: 10. Allow cells to settle in a moisture chamber for 2 minutes. 11. Follow counting procedure listed below.

COUNTING PROCEDURES: CD4 Counting Under the microscope, count the cells that have three or more large latex spheres attached to them as CD4+ lymphocytes. Monocytes should have MY4 (anti-CD14) Cytospheres Monocyte Blocking Reagent on the cytoplasmic membrane and will exhibit a golden brown coloration. Neutrophils can be differentiated by the nuclear stain in the Lysing Reagent and should NOT have latex spheres attached. Count the CD4+ lymphocytes in all 9 squares of BOTH chambers of the 0.1 mm deep (and 9 squares of one chamber of the 0.2 mm deep) hemacytometer:

CD4+ lymphocytes that touch or are intersected by the upper and left-hand boundary lines should be counted in. CD4+ lymphocytes that touch or are intersected by the lower and right-hand boundary lines should NOT be counted. Do NOT count any cell that has Reagent A attached even if CD4 coated latex spheres are attached. Specimens suspected of having tumor cells should NOT be counted using this manual method: These specimens may give falsely elevated CD4 counts.

Hemacytometer Counting Scheme Absolute CD4+ Count Calculations For the 0.1 mm deep hemacytometer: Count BOTH sides of the hemacytometer chamber as outlined above. Multiply the number of CD4+ lymphocytes counted by 7.3.

Example: You count 75 CD4+ lymphocytes in BOTH sides of the 0.1 mm hemacytometer. The absolute count is calculated as follows:

o 75 x 7.3 = 547.5 (or 548 when rounded) CD4+ lymphocytes/mL

Absolute CD4+ Count Calculations For the 0.2 mm deep hemacytometer: Count ONE side of the hemacytometer chamber as outlined above. Multiply the number of CD4+ lymphocytes counted by 7.3.

Example: You count 75 CD4+ lymphocytes in ONE side of the 0.2 mm hemacytometer. The absolute count is calculated as follows:

o 75 x 7.3 = 547.5 (or 548 when rounded) CD4+ lymphocytes/mL

MICROSCOPIC APPEARANCE OF CELLS: CD4+ Lymphocytes COUNT these cells:

CD4-NEGATIVE Lymphocytes Do NOT Count:

Monocytes Do NOT Count:

Neutrophils Do NOT Count:

LIMITATIONS: Limitations of the Assay The reportable range for this manual method is 30 2000 CD4+ cells per mL: If >2000, dilute with native plasma and retest:

o Record calculations and result. o REPORT THAT SPECIMEN WAS DILUTED TO BRING THE SPECIMEN INTO REPORTABLE RANGE. If <30, report as Less than 30 CD4+ cells per mL.

For optimal results, blood samples should be tested within SIX hours of collection: To ensure maximum viability, analyze cells promptly.

Reagents should NOT be diluted, aliquoted or frozen: Use reagents only as packaged.

Reagents are designed for use with fresh whole blood: Do NOT use with fresh, frozen or freeze-dried mononuclear cell preparations.

Do NOT report results from specimens where red blood cells do NOT lyse due to presence of nucleated red blood cells, abnormal protein concentration or haemoglobinopathies. Count cells PROMPTLY after applying to hemacytometer: Prolonged exposure to lysing reagent may cause white blood cell destruction.

QUALITY CONTROL: Use CD-Chex Plus or similar reagents for performance of quality control (QC).

QC should be carried out with every run: A run usually means the 8-hour working day AND whenever you change operator or use a new kit with a different lot number. QC results must be meticulously recorded. Process CD-Chex as you would for other samples: Use either the low or normal control each time.

Other QC procedures can be used: These should be defined and approved prior to use.

If QC results exceed acceptable limits, REJECT all specimens tested in that run: Investigate causes of QC failure and record remedial action. Do NOT test patient specimens until QC results are acceptable.

SAFETY: Procedural Precautions Samples and all materials coming in contact with them should be handled as if capable of transmitting infections and disposed of with proper precautions. Reagent A and Reagent B contain sodium azide. Flush with running water to discard. Reagent C contains acetic acid and crystal violet. Crystal violet may be a mutagen. Wear appropriate laboratory safety equipment when handling. Do NOT use individual kit reagents beyond the expiration date on the label. Do NOT mix reagents from one kit with reagents from any other kit. Incubation times and temperatures other than those specified may give erroneous results. Avoid microbial contamination of reagents or incorrect results may occur. It is harmful if any of the reagents are swallowed. After reagent contact with skin, wash immediately and thoroughly with water.

Laboratory Safety Use universal precautions with all specimens. Establish the following safety practices:

Wear laboratory coats and gloves when processing and analyzing specimens, including counting of cells on the microscope. Never pipette by mouth. Use safety pipetting devices. After working with specimens, remove gloves and wash hands with soap and water.

Dispose of all waste in appropriate containers.

QUALITY ASSURANCE: Assure the overall quality of the CD4+ T-cell testing program by monitoring and evaluating the effectiveness of the laboratory policies and procedures for the pre-analytic, analytic, and postanalytic phases of testing. Practices and processes to be monitored and evaluated include: Methods for collecting, handling, transporting, identifying, processing, and storing specimens. Information provided on test request and result forms. Process for reviewing and reporting of results. Employee training and education including:

o Basic training on CD4 manual count and additional training in hands-on workshops for supervisors. o Attendance at workshops and other programs for personnel. Adherence to appropriate regulations for training and education.

Assurance of satisfactory performance through participation in a performance evaluation program. Satisfactory participation in an established proficiency testing program.

CD4 Module 16: CD4 Counting Using the Partec CyFlow Instrumentation

Purpose: To provide a working knowledge of the principles and procedures behind the CyFlow CD4 count procedure. Pre-requisite Units: CD4 Module 15 Learning Objectives: Describe the principles of the CyFlow Counter and CyFlow SL3 instrumentation. Describe the basic parts and functions of each instrument.

Introduce basic operational procedures. Describe daily and routine maintenance procedures. Describe proper handling and preparation of reagents. Describe proper handling and preparation of patient samples. Discuss data management. Describe quality control and quality assurance procedures.

CONTENT INTRODUCTION: General Description of CyFlow Instruments The CyFlow Counter and CyFlow SL3 are fully-equipped portable/desktop flow cytometers with a solid state laser for green excitation. Can analyze up to three optical parameters including side scatter (SSC) and two fluorescence channels. Perform both fluorescence analysis and true volumetric absolute counting: Does NOT require reference beads or a hematology analyzer for absolute count determination.

CyFlow Counter* *NOTES: Depending on the laboratory, newer or older versions of the CyFlow Counter equipment may be in use. Specifics of operation will vary but basic principles and technology are similar.

CyFlow SL3

Similarities and Differences in CyFlow Counter and CyFlow SL3 Similarities: Analytical capabilities:

o Can measure SSC and two fluorescence parameters. Use an identical sample uptake mechanism to ensure true volumetric absolute counting.

Can be powered by a standard electrical connection, a 12V DC car battery or solar panels.

Differences: Although portable, the CyFlow Counter works best as a laboratory benchtop analyzer.

The CyFlow SL3 is a truly mobile analyzer that can be transported and used in even remote locations. The graphics display and data printer are integrated into the instrument in the CyFlow Counter. The CyFlow SL3 is run from an externally attached desktop or notebook computer with appropriate software.

CyFLOW COUNTER: Parts: Sheath bottle Waste bottle LCD Screen Push button keyboard Tubing system Built-in thermal printer Sample port Power pack Laser Pack Flow cuvette Computer electronic boards Photomultiplier tubes System of pumps

CyFlow Counter Keyboard* *NOTE: This keyboard is for the older version of the CyFlow counter.

CyFlow Counter Keyboard: Function Keys (Old Version)*

GAIN Adjusts the signal gain LIN/LOG Toggles between linear and logarithmic signal gain L-L Adjusts the lower signal level U-L Adjusts the upper signal level FUNC Press to enter the software settings SELECT Used in special functions TEST Turns test signals on/off CURSOR Adjusts the gated area to be included in the count SPEED Adjusts the sample speed CLEAR Clears the histogram DOWN Adjusts value down UP Adjusts value up ENTER Enter CLEAN Initiates cleaning cycle START Start STOP Stop *NOTE: These function keys relate to the older version of the CyFlow Counter.

Function Display on Newer Version of CyFlow Counter CyFLOW SL3: Parts: Sheath bottle with fluid sensor Waste bottle with fluid sensor The counter Complete PC including monitor, keyboard, mouse, and CPU or a laptop computer HP DeskJet (or similar) printer Tubing system Sample port

Interphase cable Power pack Laser pack Flow cuvette Computer electronic boards Photomultiplier tubes System of pumps Micrometer stage for flow cell Optical stage with the parameters SSC, FL1 and FL2 CCD camera with cable for flow monitoring Win TV card for laptops

PREPARATION: Reagent Storage Keep an inventory of all reagents with lot numbers and expiration dates. Rotate stock. Store reagents at 2-8C. Avoid extreme conditions of freezing and temperatures above 37C. Always check for evidence of deterioration.

Specimen Preparation Collect blood specimens by venipuncture into evacuated tubes, completely expending the vacuum: Draw pediatric specimens in pediatric tubes to avoid underdrawing. Mix blood well by inversion to prevent clotting.

Label all specimens with a unique patient identifier, date, and time of collection. Provide on the submission form pertinent medications and disease conditions that might affect the immunophenotyping test. Assure that patient information and test results are accorded confidentiality.

Specimen Stability and Transport* Start the assay within six (6) hours of specimen collection. Blood samples should be transported and maintained at 20-25C until processing is begun: Avoid extremes of temperature so that specimens do not freeze or become too hot.

In hot weather, it may be necessary to pack specimens in an insulated container and place inside a second container with an ice pack and absorbent material to maintain ambient temperature. DO NOT REFRIGERATE samples prior to analysis. *NOTES: CD4 deteriorates more rapidly from the surface of T helper cells at 4C than at room temperature.

TEST PROCEDURES: Two procedures are available for use: 1. 2. Determination of CD4% Determination of absolute CD4

Determination of CD4%* 1. Into each sample and control tube, add:

o 10 L of CD4PE monoclonal antibody o 10 L of CD45PE-CY5 monoclonal antibody o 20 L of well-mixed EDTA whole blood sample 2. 3. 4. 5. Mix and incubate at room temperature in the DARK for 15 minutes. Add 400 L of Buffer 1. Mix and add 400 L of Buffer 2 just before reading. Analyze.

*NOTE: This procedure is used primarily with PEDIATRIC samples. This procedure can be used on both the CyFlow Counter and the CyFlow SL3.

Diagram of Determination of CD4% Procedure

Determination of Absolute CD4* 1. Into each sample and control tube, add:

o 20 L of CD4PE monoclonal antibody o 20 L of well-mixed EDTA whole blood sample 2. 3. 4. Mix and incubate at room temperature in the DARK for 15 minutes. Add 800 L of CD4 buffer. Mix and analyze sample.

*NOTE: This procedure is used primarily with ADULT samples. This procedure can be used on both the CyFlow Counter and the CyFlow SL3.

Diagram of Determination of Absolute CD4 Procedure

True Volumetric Absolute Counting True volumetric absolute counting is achieved mechanically by a series of sensors at the sample uptake port: The upper sensor electrode starts the counting process when the sample meniscus is disconnected. The measurement stops when the lower sensor is disconnected from the sample.

The measuring volume of 200 L is precisely defined by the distance between the electrodes and the inner diameter of the sample tube. The cell concentration (c) is automatically calculated by the software as a function of counted events (N) in the 200 L volume (V). c = N/V Events are shown as counts/mL or counts/ L

Diagram of True Volumetric Absolute Counting Principle* *NOTE: The same mechanism for true volume absolute counting is used on both the CyFlow Counter and the CyFlow SL3.

CyFLOW SL3 OPERATION:

Start-up Procedures 1. 2. 3. 4. 5. 6. 7. Switch on AVR/UPS or other power supply. Fill sheath fluid bottle to 800 mL mark. Discard fluid in waste bottle and rinse with 10% sodium hypochlorite solution. Cork both bottles tightly. Switch on the CyFlow SL3. Switch on the computer and open FloMax software. Verify the template, settings and trigger depending on the analysis to be performed:

o CD4 Absolute o CD4% 8. 9. Clean with cleaning fluid once. Rinse with sheath fluid once.

10. Wait 15 minutes for laser to warm up. 11. Run count check beads: o If count check bead result is acceptable, run samples. o If result is not acceptable, rerun or do troubleshooting.

CyFlow SL3 Data Display:

CyFlow SL3 Shut Down: 1. All cleaning steps must be completed before turning off the instrument:

o Clean with cleaning fluid twice. o Clean with decontamination fluid once. o Rinse with sheath fluid once. 2. 3. 4. Shut down computer. Switch off CyFlow SL3. Switch off power source.

QUALITY CONTROL: Use CD-Chex Plus or similar reagents for performance of quality control (QC). QC should be carried out with every run: A run usually means the 8-hour working day AND whenever you change operator or use a new kit with a different lot number. QC results must be meticulously recorded. Process CD-Chex as you would for other samples: Use either the low or normal control each time.

MAINTENANCE: CyFlow Instrument Maintenance Both the CyFlow Counter and CyFlow SL3 require regular maintenance. Daily cleaning should be done: Before running count check beads as set up for sample run. After each batch run. After each days work.

There are three types of cleaning fluid: Detergent fluid Decontamination fluid Sheath fluid

LABORATORY SAFETY: Use universal precautions with all specimens. Establish the following safety practices: Wear laboratory coats and gloves when processing and analyzing specimens. Never pipette by mouth. Use safety pipetting devices. After working with specimens, remove gloves and wash hands with soap and water.

Dispose of all waste in appropriate containers:

Dispose of waste containing biohazardous materials according to local, regional and national guidelines.

QUALITY ASSURANCE: Assure the overall quality of the CD4+ T-cell testing program by monitoring and evaluating the effectiveness of the laboratory policies and procedures for the pre-analytic, analytic, and postanalytic phases of testing. Practices and processes to be monitored and evaluated include: Methods for collecting, handling, transporting, identifying, processing, and storing specimens. Information provided on test request and result forms. Process for reviewing and reporting of results. Employee training and education including:

o Basic training on theory and operation of CyFlow instruments and additional training in handson workshops for supervisors. o Attendance at workshops and other programs for personnel. Adherence to appropriate regulations for training and education.

Assurance of satisfactory performance through participation in a performance evaluation program. Satisfactory participation in an established proficiency testing program.