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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 treatingHIV.

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 rangefor 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 no augmentation

Adaptive Immunity:

o Specific

o Second line of defense

o Repeated exposure augmented memory

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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

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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. Innateimmunity is not extremely efficient, but it is effective. For example, HIV needs to bypass the skinto infect someone.

Adaptive/Antigen Specific Immunity

Humoral immunity:

Mediated by antibodies (proteins), which attack foreign substances.

Cell-mediated immunity:

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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 antigenicstimulation.

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.

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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-cellsurface.

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 toantibodies.

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 immuneresponse.

This immune response is designed to fight off and eliminate an infectious organism.

T-B Cells Interaction

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This image shows how a T-helper cell can interact with a B cell. The lymphokines (red dots) aresecreted by the T-helper cell. The lymphokines act upon the B cell inducing the B cell todifferentiate into a plasma cell. The plasma cell releases the antibodies into the circulation tofight 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 theseverity of his/her disease.

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

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*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 theforeign protein. This binding is the first step in the CD8 cytotoxic T-cell destroying the virusinfected 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 oflymphocyte.

The term CD refers to a uniform nomenclature system which has been adopted to identify theseproteins.

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 willexpress different CD molecules.

Over 300 CD molecules have been described.

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*NOTE: You will NOT need to know all 300 CD antigens. Also, it is important to note that CD4 isthe 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 acell.

*NOTE: A chemokine is a substance that will attract cells across a barrier or gradient.

CD Molecules Used for Identifying Different Lymphocyte Subclasses

CD45 All White Blood Cells

CD3 T-cells

CD4 T-helper cells

CD8 T-cytotoxic cells

CD19 B-cells

CD56 Natural killer (NK) cells

CD4 AND HIV INFECTION:

HIV Binding to CD4 and a Chemokine Receptor

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HIV infection is mediated through binding of HIV to CD4 and chemokine receptor molecules. Thehorizontal black line represents the cell membrane. CD4 and the chemokine receptor are part ofthe 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) immunesystem.

HIV suppresses the immune response by infecting and destroying the CD4 positive T-helpercells.

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 statesnotassociated with HIV.

The number of CD4-positive T-cells may be used to stage disease or monitor immunedeterioration in individuals who have HIV infection, documented by antibody, antigen, and ornucleic 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 stagingof HIV infection.

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The number of CD4-positive T-cells is also used to initiate antiretroviral therapy (ART) andtreatment of HIV-related diseases.

The CD4 T cell number can be used as an indicator of anti-retroviral treatmenteffectiveness.

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, diminishingCD4-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-helpercell counts.

In these conditions, there is no progressive decline of CD4 counts overtime as seen inHIV 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 infectionsand HIV disease progression.

CD4 counts of less than 200/L are associated with greater risk for pneumocystisinfections.

CD4 counts

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Clinical Consequences of HIV Infection: Fast Progressors*

*NOTE: Recent studies have identified super fast progressors who have developed full blowAIDS 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

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Normal healthy donors, across the adult age range (18-65 yrs), no medication forapparent illness.

If your region has a blood donor program, specimens obtained from healthy blooddonors may be useful for determining reference ranges.

Test Protocol:

The reference range should be determined using the instrument you will use for patienttesting.

Determine reference ranges for all lymphocyte subclasses that you may use in your HIVmonitoring 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 aninability to test sufficient numbers of healthy children.

Published reference ranges may be used as a guide in the absence of locallydetermined 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 isawake (0800-2200).

This increase may approach 60 CD4-positive T-cells/ L in HIV infected patients andhundreds of cells/ L in healthy controls.

Thus, follow up specimens should be obtained at approximately the same time as the previousspecimen to minimize this circadian variation.

*NOTE: The CD4 cell level varies depending on activeness of an individual. This variation knownas circadian.

RESEARCH STUDIES:

Application #1: HIV Patient Case Study

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CD4 counts in HIV patients are measured periodically to monitor patients immune capabilities.

An HIV infected patient has a CD4 absolute count of75/L on 1 April. On July 1, he has a CD4absolute count of110/L. The April specimen was collected at 0900. The July specimen wascollected 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 absoluteCD4 count. Absolute CD4 numbers will increase from 0900 until 1630. The patient should beredrawn 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 DeterminedUsing MultiSET in Relation to Sex

Parameters

Males (n=107)

MEAN

(95% Cl)

Females (n=107)

MEAN

(95% Cl)

Males and Females (n=214)

MEAN

(95% Cl)

Lymphocytes/ L

2445

(1047,6013)

2427

(1276, 4056)

2436

(1189, 4941)

CD3+ T-cells %67.1

(49.8, 82.3)

70.4

(56.0, 83.0)

68.8

(53.7, 82.6)

CD3+ T-cells/ L1381

(714, 2407)

1629

(881, 3016)

1505

(769, 2798)

CD4+ T-cells %37.6

(24.4, 55.3)

42.6

(31.0, 55.3)

40.1

(27.0, 55.0)

CD4+ T-cells/ L

748

(383, 1321)

940

(514, 1686)

843

(405, 1500)

CD8+ T-cells %25.8

(15.0, 43.5)

24.8

(15.7, 41.0)

25.3

(15.0, 41.0)

CD8+ T-cells/ L526

(222, 1007)

569

(300, 1300)

548

(261, 1033)

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CD4:CD8 ratio1.38

(0.59, 2.43)

1.63

(0.74, 2.92)

1.50

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

*NOTE: Statistically significant value

Comparison of Median Levels of Lymphocyte Subsets among Tanzanian and Swedish HIV

Seronegative Adult MalesTanzanian

(n=107)

Swedish

(n=50)P value

CD4 % 38.0 45.0

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Comparison of lymphocyte subsets among HIV seronegative adults in other studies

12.1% of Tanzanian males compared to 28.5% of Ethiopian males had

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- Discuss the basic capabilities of a flow cytometer, including multiparameter analysis andgating.

- 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 analyticalroutines.

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:

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o The capability of measuring multiple parameters (size, granularity, associated fluorescence) ofeach 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:

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Research Uses

Many, including cell sorting; measures of cell activation such as Ca++ flux, pH changes andmembrane 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 onpurpose 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 absolutecounts.

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:

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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 isobtained.

- In peripheral blood, absolute numbers can be obtained using CBC values or comparison toknown 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:

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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) analysiscan be added for complete documentation of lymphocyte subsets and to aid in assessingaccuracy 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 characteristicsof the cell populations.

o This method is often combined with separate measurement of CD45 vs. CD14 fluorescence ina 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 90light 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 beused to set background autofluorescence levels.

NOTE: Image illustrates CD45 vs. SSC Gating Strategy

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*NOTE: When using the FSC vs. SSC gating strategy, other cells, especially basophils, whichhave 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 thedifferential expression of CD45 on these cells. Therefore, a more accurate CD4 count isobtained.

SUMMARY:

Flow cytometry is a semi-automated method that can be used to determine both the physicalcharacteristics of cells and other characteristics based upon association of cells with analytes thatare 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 betailored 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


- 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. BDFACSCount).

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 immunesuppression.

- So, our goal is to identify and determine the absolute number of CD4 and CD8 positive T-cells per microlitre of blood in HIV infected individuals.

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*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 numbersor CD numbers. Over 330 CD antibodies have been produced to various surface andintracellular molecules. Often the proteins that these antibodies react to are referred to by theirCD number.

For example, the CD4 molecule or CD4 antigen. This chart demonstrates the ontogeny of thelymphoid system, but, more importantly, it demonstrates that the various lymphocytes can beexactly characterized by the CD antigens on their surface. It would only take one if CD4 and CD8were unique to

these two cells. However, that is not the case. You can see here that CD8 antigen is also on NKcells and CD4 is also on monocytes (not shown). So if we add one marker, the T-cell receptordesignated by CD3, then the combination of both markers is now unique to T-helper and T-cytotoxic 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 T-helper cell and, also, the coexpression of both CD3 and CD8 uniquely characterize a cell as a T-cytotoxic cell. Unfortunately, we cannot see these molecules on the cells without an aid. This iswhere 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.

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

- Absolute numbers are derived by comparison to known numbers of reference beads ineach 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 CD8T-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 notinterchangeable.

Storage and Handling of Reagents

Conjugated antibodies will come with specific storage and handling instructions.

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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 onthe surface of cells.

CD4 and CD8 positive T-cells in peripheral blood can be uniquely identified by a combination offluorochrome-labeled CD3 and CD4 antibodies in one tube and anti-CD3 and anti-CD8 antibodiesin 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 notinterchangeable, (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.



- Discuss essential elements of a preventative maintenance plan in an immunophenotypinglaboratory.

- Discuss reasons for performing maintenance on immunophenotyping analyzers andpipettes.

- 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 qualityperformance in the CD4 laboratory. Establishing Standard Operating Procedures (SOPs)regarding maintenance and having technicians follow these guidelines, as well as securingservice contracts is essential to quality assurance. In this module, we will discuss when and howCD4 analyzer maintenance is performed, along with when and how to check pipette precision.

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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 equipmentcontinues 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 alwaysbe documented when performed.

STANDARD OPERATING PROCEDURES (SOPs):

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Maintenance Procedures*

Necessary procedures and documentation

Establish standard operating procedures (SOPs) for equipment maintenance so that thetechnician has specific, clear, and detailed description of how and when to carry outmaintenance.

- These SOPs should be done in a vocabulary appropriate for those persons who are goingto be using them while clearly following the manufacturers instructions.

*NOTE: SOPs for daily, weekly and monthly, as well as annually and bi-annually should beavailable.

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, andanticipate 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 ispurchased or leased.

- This is arranged with the manufacturer; usually on an annual basis (first year is usuallyincluded in purchase as warranty).

*NOTE: When negotiating for purchases and/or leasing, it is a good time to discuss servicecontracts and remember that service is an important factor when deciding on whichanalyzer/vendor to choose. Make sure the services to be provided (how often and what is to becovered under the contract in the way of analyzer parts, reagents, etc.) are clear andunderstandable.

Service Contracts

The manufacturer sends trained representatives for both equipment problems and annual, bi-annual, and preventative maintenance (PM) on the analyzer.

- Annual PMs are necessary and generally arranged and should be scheduled by thelaboratory, so general cleaning and checks may be performed.

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*NOTE: Service contracts differ depending on the manufacturer. It is important to note exactlywhat service and parts will be covered or serviced when instruments are purchased. All settingsare checked to make sure that your analyzer is performing optimally, replacement of lasersand/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 analyzerwhen 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 techniciansknow 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 becomeshectic. Make sure all the following information is readily available so that service/vendor can actimmediately.

MAINTENANCE PROTOCOLS FOR INSTRUMENTS:

Immunophenotyping Laboratory Maintenance Essentials*

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It is vital for equipment to have regular cleaning and maintenance performed to ensure accuracyand precise CD4 results.

It is also necessary to perform regularly scheduled (six months) calibration checks on pipettes toensure they are measuring accurately and with precision.

The daily analyzer cleaning standard procedure takes very little time and can prevent manyproblems.

*NOTE: If you get into a habit of proper cleaning and maintenance, your analyzer will performbetter, 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. Cleaning performed before shut-down of the analyzer.

3. Monthly cleaning, which is a more extensive cleaning cycle.

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


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 fluidicssystem:

- This optimizes the functionality of the fluidics system.

- This also reduces the debris found during the course of analyzing.

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


Rinse the lines when debris is detected, following the general cleaning procedure or followmanufacturers procedure.

Clean with a solution of bleach:

- 5% sodium hypochlorite (home bleach) diluted 1:10 with distilled water. (Important: Followmanufacturers recommendations for bleach strength.)

*NOTE: It is important to read the manufacturers recommended procedures for cleaning andmaintenance. For example, while a *1:10* dilution of 5% bleach is mentioned and used withmany 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 appearsrecommending that you run the cleaning cycle (Note: this message appears after at least 51reagent 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 cansee how simple it is. Go through this reading of the cleaning procedure to demonstrate a typicalcleaning 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 onthe 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 CD4immunophenotyping 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 appropriatecapacity.

Protect the cytometer from ambient dust (cover the instrument, close windows).

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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 wipingdown all other equipment used.

Recording of Maintenance

For quality assurance, recording of all routine daily, weekly, monthly maintenance must be doneto 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

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

Pipette Maintenance:

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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 andcalibration.

*NOTE: The manufactures instructions show how to disassemble the pipette, clean andlubricating it and replacing some parts such as springs and rubber gaskets. Following this pipettemaintenance, 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 theletter.

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 instrumentsis 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.



- 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 aflow cytometry laboratory.

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- 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 thatquality 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 toresult 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 istested for the analyte. If the patient specimen isnt identified, collected or handled properly, thespecimen wont be worthy of testing. You will notice that aspects of analytical included theaccuracy of the result, analytical errors, clerical errors, and these all impact the patient result.

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And the post-analytical aspect includes timely reporting to the patient file for the clinician andrecord 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 storageshould be properly followed.

- Written procedures with criteria for rejection of unacceptable specimens should be madeavailable to all institutions.

*NOTE: Documentation in Requisition logs and corrective action logs is an essential part ofquality assurance.

PROFICIENCY TESTING:

Participation in Proficiency Testing*

Laboratories should enroll, and satisfactorily participate in, a performance evaluation/assessmentprogram:

- If conventional proficiency testing is not available, the laboratory must exercise analternative 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 bechecked against each other at least twice a year for correlation of patient results (bias scatterplots).

*NOTE: Splitting samples with nearest laboratory is an alternative to proficiency testing. Alwaysrun samples as though if they were a patient. This is very important as there is the temptation todo extra steps (i.e., run more than once, etc.). So dont do it (run proficiency specimen more thanonce 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 impacton global HIV management!

A number of regional EQA programmes exist for CD4 T lymphocyte testing, but there are two thatprovide international coverage:

- QASI, Bureau of HIV/AIDS, STD & TB, Ottawa, Canada operated 2-3 times per annum(approx 300 international sites) (contact [email protected]).

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- UK NEQAS for Leukocyte Immunophenotyping, Sheffield, England operated 6 times perannum (2 samples per send out) (approx 500 international sites) ([email protected] 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-cellImmunophenotyping. It will transfer our knowledge of QAP procedures and processes tolaboratories.

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 withmore) drawn from normal, healthy donors.

As you add/gather more control samples, recalculate your references ranges until you have about100 samples.

Set up a system for review.

*NOTE: Care must be taken when establishing normal ranges, especially in highly endemicareas. Obvious outliers should be excluded or further investigated for exclusion.

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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. Ifresults 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 atrandom can be outside reference range limits due to biological factors.

- May be true result - misrepresentation of healthy status (especially true in highly endemicareas).

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 antibodyaliquot.

- Document all these steps on a corrective action log.

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

Exercise #1: Mean, SD Calculation

For 20 different normal adult specimens, the following data was obtained from the BDFACSCount for CD4 absolute values:

DAY

RESULT

DAY

RESULT

DAY

RESULT

DAY

RESULT

1

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750

6

850

11

1100

16

800

2

850

7

1150

12

750

17

1125

3

850

8

1300

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13

950

18

1100

4

1200

9

900

14

1200

19

1150

5

850

10

950

15

1200

20

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900

Question: Determine the reference range to be used by calculating the mean and standarddeviation.

- Mean (average) can be determined by adding the results of the 20 normal controls anddividing by 20.

- Standard deviation can be determined by a complicated formula that is prone to errors dueto 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 bereevaluated 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:

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All data entry results should be verified by a section head, supervisor, or colleague for finalinterpretation and release of results.

In the event that a report has already been sent out and needs correction, a new report is issuedwith 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 limitsMUST 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 theOut of Control Book.

IMPORTANT: Document, Document, Document!!

QUALITY IMPROVEMENT:

Monitor all phases of pre-analytic, analytic, and post-analytic testing continuously for qualityimprovement:

- 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 specifiedpredetermined 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 testresults.

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Recording and monitoring test variables such as temperature, reagents, controls and equipmentfunction allows one to look objectively and retrospectively at parameters vital to the accuracy andprecision of the test.

Documentation allows one to foresee a potential problem before the situation requires correctiveaction and adversely effects patient results.

Levey-Jennings type plots are useful.

These documents must be reviewed daily. Any trends outside the expected range should beinvestigated.

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 budgetpermits) 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.

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- 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 thedecay 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 199626:216.; 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 todetermine 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 typeplot.

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 youshould take?

Answer: Evaluate and rule out problems with:

- Instrument PMT, Compensation, Linearity

- Operator technique skills, procedural

- Product deterioration

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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 accordingto the manufacturers suggested recommendations.

*NOTE: Reagents are very expensive and deterioration of your reagents will result in invalidresults 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 ifthere 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 thanthe 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 fromthe current lot in use.

QC EQUIPMENT:

All equipment in the CD4 laboratory

Should have instruction manuals, as well as SOPs, regarding proper use and maintenancerequirements.

Should be monitored and recorded for quality control procedures, function checks, preventativemaintenance and repairs.

- These should be documented and filed in separate log books.

Cytometers*

Record Serial numbers for each.

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Perform function verification (PMT, Linearity, Compensation, if applicable, on a daily basis).

Perform preventive maintenance (daily and monthly checks, along with bi-annual PreventiveMaintenance checks).

Troubleshooting procedures should be available to all staff.

*NOTE: Follow SOP for performing daily analyzer checks, (mentioned above), which shouldclearly spell out the manufacturers instructions for verifying that the analyzer is functioningappropriately.

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 thephotomultiplier tube voltages and fluorescence compensation are checked and have passed. Ifthis test fails, follow manufacturer's suggested follow-up and be sure to document in a correctiveaction 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 shouldbe 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 maintainquality assurance in the laboratory.

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Pipettes

Improperly calibrated pipettes will affect your assay and should be checked for precision andaccuracy bi-annually.

Pipettes not passing accuracy checks should be cleaned and checked for worn parts, but if youfollow manufacturers instructions and the problem is not apparent, the pipette may be sent to andserviced 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, thelaboratory 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 andunacceptable 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 CD4laboratory.



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*

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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 everystage 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 everystage of laboratory process. This module will point out that when configuring your laboratory it isimportant 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 clinicsserved?

- Must consider specimen transport issues

Infrastructure: Will the infrastructure at the location support the scope of the planned laboratoryactivities?

- Must consider power and water requirements, space issues, type of instrumentation, wastedisposal.

The type of instrumentation should be commensurate with the intended use and with the level ofexpertise of the health care facility.

Laboratories with flow cytometry instruments should be air-conditioned to maintain proper roomtemperature 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 batchage.

Occasional repeat testing requires a system for specimen tracking and temporary storage.

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

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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 eachtest 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 thechange 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 onedirection, from sample processing and preparation to result processing and sample storage for asmall volume, CD4 laboratory.

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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. Noticeworkflow is unidirectional.

Staff Requirements High Volume Testing

This diagram illustrates workstation areas and the number of staff required for each for a highvolume CD4 laboratory.

The CD4 lab testing workstation as well as the result validation and release workstation show thatthey require additional staffing to provide a continuous workflow.

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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 morethan required space for all testing processes. Air-instruments pull air from the room through fansfor cooling, so air should be free from dust, which keep filters on cooling fans from becomingclogged.

Temperature is critical to instruments and test performance. Analyzers may turn off iftemperatures 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-termwithout sagging.

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Bench width should accommodate the entire instrument with sufficient space behind instrument toallow cooling and access for repairs.

*NOTE: Many CD4 analyzers contain delicate lasers and optical sensors. Moving thesesanalyzers, especially high-capacity analyzers, may require service representatives to move toguarantee parts are not damaged. Stable floor and bench top is important to support ananalyzer. 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 bewise 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 ofelectrical supply.

Testing does not allow for interruptions in electrical supply, install adequate uninterrupted powersupply (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

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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. Maintainingstatistical data is critical for improved patient outcomes. There is always pressure for thelaboratory to do more with fewer resources. Demand for CD4 counts will increase. That is why itis 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 CD4counts:

- 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.

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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, bloodrockers, 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 1-2days.

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 maintenanceis an essential part of quality assurance.

Maintenance Record Example

This form is an example of a maintenance record. When utilized it demonstrates service andmaintenance 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 thatyou may be able to possibly anticipate how long a particular part will last so that you can plan forthe next time, and not be left with workflow interruptions.

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Daily Temperature Log Example

Reagents are expensive and will deteriorate, if not stored properly. By maintaining a dailytemperature charts for your refrigeration, you are able to track problems which could leadultimately to result errors.

Routine Centrifuge Quality Control Record Example

Another piece of equipment which must be maintained and documented are centrifuges, usuallybi-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, printingpaper).

Strengthen systems to prevent reagent stock-outages.

Specimens have to be sent to other testing laboratories or discarded if reagent supply isinterrupted 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 toplanning 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 yourinventory is used and when supplies are low. This chart allows for recording of lot numbers andexpiration dates also.

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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 SOPsfor 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 documentingspecimen 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 monitoringand 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 notenough to just analyze and get a result. That result must have a reliable way to be delivered toclinical 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.

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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 laboratorystaff are trained and recertified on safety procedures and universal precautions. Plan ahead andhave in place all of the necessary post-exposure prophylaxis before it is ever needed with SOPsfor 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 internationalguidelines.

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 allinstrument, 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.

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Three levels of training are required for existing staff:

1. Instrument and software operation and applications (provided by instrumentmanufacturer/vendor)

2. CD4 laboratory lab operations and management

3. 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 oflaboratory 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 thischart, you can quickly view all when and what actions are necessary in order to maintain qualitycontrol and quality assurance, maintained in laboratory management.

Conclusions

CD4 counting requires both reliable and competent instrument operation and laboratorymanagement to ensure high quality results, adequate testing resources and short turn-around-time.

CD4 laboratory management and planning are key to sustainable testing services and meetingthe 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 berevised.

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All technical procedures will be checked for compliance with standards of practice as defined inthe 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 operationprocedures (SOPs), policies and work forms are designed, prepared, maintained, placed intoservice, archived and retired from service.

A policy describes a definite course of action or prescribes adherences to proper procedureor 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

AttachmentsCD4 Module 13A: Flow Cytometry Instrumentation Troubleshooting Part A

Purpose: To provide a working knowledge of flow cytometry theory and troubleshooting.


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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 betaken.

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. Itis a good idea to check for these items along with the service engineer so that you becomefamiliar with these features.

**ADDITIONAL NOTE: The reverse-pipette is separate from the analyzer and needs to beplugged in for a minimum of 10 hours. Ask about purchasing extra battery backup or extrapipette, if possible.

Exterior Parts of AnalyzerBecton Dickinson FACSCountTM.JPG

Here are various exterior parts on the front of the analyzer. The fluidics compartment is housedbehind the cover shown on the front of the analyzer.

Instrument Set-Up:*

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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 theFACSCount).

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 discinto 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 theoptical, 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 thedays specimen run.

This involves pinching the connection tubing, removing, filling and replacing the sheath reservoirand turning on the instrument.

*NOTE: Fluidics system begins with the sample probe and, under pressure, once the sample istaken in goes through the fluidics system, passing through the laser intersection and into thewaste receptacle. The sheath fluid reservoir is easily removed to fill by disconnecting the systemfluid connector tubing.

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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 fixedoptical 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 toensure that the cytometer gives acceptably bright fluorescence measurements and thathomogenous 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 establishedspecifications.

Daily Monitoring:

On a daily basis, the lab verifies that the flow cytometers performance is consistent withestablished 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.

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*NOTE: It is critical that patient normal values be established for a given population; both adultand pediatric values must be established due to the big difference in normal values for adult andpediatrics.

Materials for this procedure are patient specimens from patient who are considered to be in ahealthy state. These specimens may come from a patient admitted for a non-critical conditionsuch as routine surgeries or non life threatening diseases. A minimum of twenty specimens mustbe run. For most accurate representative results, 30 specimens should be run over a three-dayperiod. This will permit more variance to be associated with the results; therefore, a morerepresentative of true testing conditions. In order to maximize on specimen availability andeliminate differences, the specimen should be run by all methods at the same time that isautomated and manual. When adequate numbers have been assayed for all CD4 values, amean 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 meanvalue = 1000 and the standard deviation is 100, the range would be 800-1200. These normalvalues should be a standard part of a patient report in order for a physician to evaluate a patientas normal or abnormal. If manual and automated methods normal value were found to differ, thephysician should be alerted when an alternate method was used, and the patient report shouldreflect 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) withnormal 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 inparallel 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 havebeen measurable and known forward-scatter, and fluorescence properties in each channel to beused for sample analysis.

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FACSCountTM reagent uses reference beads that are used by the system software to checkinstrument 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 ofreagent.

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 patientsamples.

*NOTE: Run controls (beads with a normal blood specimen) each day when the instrument isturned on and whenever you open a new lot of reagent. Also, for quality control, after analyzedand 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 andHigh.

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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 stationand pulling the lever down to core the tubes. Then, place tubes in workstation to keep tubesupright, 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 protecttubes from light.

9. Uncap the tubes and pipette *50 L* of fixative solution into each reagent tube, changing tipsbetween 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 fromthe control kit and place them in the control area of the workstation. Add control beads to thereagent tubes just before running the tubes on the instrument. Store reagent tubes in theworkstation until you are ready to add control beads.

12. Vortex the Zero/Low control bead pair and vortex the Zero bead. Pipette 50

cd4 module

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