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## Complete Summary

#### GUIDELINE TITLE

Guidelines for performing single-platform absolute CD4+ T-Cell
determinations with CD45 Gating for persons infected with human
immunodeficiency virus.

#### BIBLIOGRAPHIC SOURCE(S)

Mandy FF, Nicholson JK, McDougal JS. Guidelines for performing
single-platform absolute CD4+ T-cell determinations with CD45 gating for
persons infected with human immunodeficiency virus. Centers for Disease
Control and Prevention. MMWR Recomm Rep 2003 Jan 31;52(RR-2):1-13. \[68
references\]
[PubMed](http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12583540)

#### GUIDELINE STATUS

This is the current release of the guideline.

### COMPLETE SUMMARY CONTENT

 SCOPE\
 METHODOLOGY - including Rating Scheme and Cost Analysis\
 RECOMMENDATIONS\
 EVIDENCE SUPPORTING THE RECOMMENDATIONS\
 BENEFITS/HARMS OF IMPLEMENTING THE GUIDELINE RECOMMENDATIONS\
 QUALIFYING STATEMENTS\
 IMPLEMENTATION OF THE GUIDELINE\
 INSTITUTE OF MEDICINE (IOM) NATIONAL HEALTHCARE QUALITY REPORT
CATEGORIES\
 IDENTIFYING INFORMATION AND AVAILABILITY\
 DISCLAIMER

### SCOPE

#### DISEASE/CONDITION(S)

Human immunodeficiency virus (HIV)

#### GUIDELINE CATEGORY

Diagnosis\
Management

#### CLINICAL SPECIALTY

Pathology

#### INTENDED USERS

Clinical Laboratory Personnel

#### GUIDELINE OBJECTIVE(S)

-   To supplement previous recommendations published in 1997 (1997
    revised guidelines for performing CD4+ T-cell determinations in
    persons infected with human immunodeficiency virus \[HIV\]. *MMWR*
    1997;46\[No. RR-2\]) that describe dual-platform technology, a
    method in which absolute counts are derived from measurements
    obtained from two instruments---a flow cytometer and hematology
    analyzer

-   To address concerns specific to the implementation of
    single-platform technology for CD4+ T-cell determination in persons
    infected with human immunodeficiency virus, as well as to address
    other general topics, such as laboratory safety and specimen
    handling

#### TARGET POPULATION

Persons infected with human immunodeficiency virus

#### INTERVENTIONS AND PRACTICES CONSIDERED

Single-platform technology absolute CD4+ T-cell determinations with CD45
gating, using three- or four-color monoclonal antibody panels

#### MAJOR OUTCOMES CONSIDERED

-   Effectiveness of single-platform technology (SPT) to provide
    accurate and reliable measures of CD4+T lymphocytes in the
    laboratory

-   Variability in laboratories\' testing practices/procedures

### METHODOLOGY

#### METHODS USED TO COLLECT/SELECT EVIDENCE

Searches of Electronic Databases

#### DESCRIPTION OF METHODS USED TO COLLECT/SELECT THE EVIDENCE

Not stated

#### NUMBER OF SOURCE DOCUMENTS

Not stated

#### METHODS USED TO ASSESS THE QUALITY AND STRENGTH OF THE EVIDENCE

Not stated

#### RATING SCHEME FOR THE STRENGTH OF THE EVIDENCE

Not applicable

#### METHODS USED TO ANALYZE THE EVIDENCE

Review

#### DESCRIPTION OF THE METHODS USED TO ANALYZE THE EVIDENCE

Not stated

#### METHODS USED TO FORMULATE THE RECOMMENDATIONS

Expert Consensus

#### DESCRIPTION OF METHODS USED TO FORMULATE THE RECOMMENDATIONS

These guidelines reflect a consensus of the third national conference on
CD4+ immunophenotyping held on November 14-15, 2001 in Orlando, Florida.
The conference was attended by representatives from public health,
private, and academic laboratories as well as product manufacturers.

#### RATING SCHEME FOR THE STRENGTH OF THE RECOMMENDATIONS

Not applicable

#### COST ANALYSIS

A formal cost analysis was not performed and published cost analyses
were not reviewed.

#### METHOD OF GUIDELINE VALIDATION

Not stated

#### DESCRIPTION OF METHOD OF GUIDELINE VALIDATION

Not stated

### RECOMMENDATIONS

#### MAJOR RECOMMENDATIONS

I.  **Laboratory Safety**

    A.  Use universal precautions with all specimens (CDC, 1988).

    B.  Adhere to the following safety practices (CDC, 1988; CDC, 2001;
        CDC, 1999):

        1.  Wear laboratory coats and gloves when processing and
            analyzing specimens, including reading specimens on the flow
            cytometer.

        2.  Never pipette by mouth. Use safety pipetting devices.

        3.  Never recap needles. Dispose of needles and syringes in
            puncture-proof containers designed for this purpose.

        4.  Handle and manipulate specimens (e.g., aliquot, add
            reagents, vortex, and aspirate) in a class I or II
            biological safety cabinet.

        5.  Centrifuge specimens in safety carriers.

        6.  After working with specimens, remove gloves and wash hands
            with soap and water.

        7.  For stream-in-air flow cytometers, follow the
            manufacturer\'s recommended procedures to eliminate the
            operator\'s exposure to any aerosols or droplets of sample
            material.

        8.  Disinfect flow cytometer wastes. Before adding waste
            materials to the waste container, add a sufficient volume of
            undiluted household bleach (5% sodium hypochlorite) so that
            the final concentration of bleach will be 10% (0.5% sodium
            hypochlorite) when the container is full (e.g., add 100 mL
            of undiluted bleach to an empty 1,000 mL container).

        9.  Disinfect the flow cytometer as recommended by the
            manufacturer. One method is to flush the flow cytometer
            fluidic chambers with a 10% bleach solution for 5\--10
            minutes at the end of the day and then flush with water or
            saline for at least 10 minutes to remove excess bleach,
            which is corrosive.

        10. Disinfect spills with household bleach or an appropriate
            dilution of mycobactericidal disinfectant. **Note**: Organic
            matter will reduce the ability of bleach to disinfect
            infectious agents. National Committee for Clinical
            Laboratory Standards (NCCLS) recommendations regarding how
            to disinfect specific areas should be followed (National
            Committee for Clinical Laboratory Standards, 1991). For use
            on smooth, hard surfaces, a 1% solution of bleach is usually
            adequate for disinfection; for porous surfaces, a 10%
            solution is needed (National Committee for Clinical
            Laboratory Standards, 1991).

        11. Ensure that all samples have been properly fixed after
            staining and lysing but before analysis. **Note**: Some
            commercial reagents employ a single-step, lyse and fix
            method that reduces the infectious activity of
            cell-associated human immunodeficiency virus (HIV) by 3\--5
            logs(Nicholson et al, 1993; Aloisio & Nicholson, 1990);
            however, these reagents have not been evaluated for their
            effectiveness against other agents (e.g., hepatitis virus).
            Cell-free HIV can be inactivated with 1% paraformaldehyde
            within 30 minutes (Cory, Rapp, & Ohlsson-Wilhelm, 1990;
            Lifson, Sasaki, & Engleman, 1986; Martin, Loskoski, &
            McDougal, 1987).

II. **Specimen Collection for Single-Platform Technology**

    A.  Anticoagulant

        1.  Use tripotassium ethylenediamine tetraacetate (K~3~EDTA, 1.5
            ± 0.15 mg/mL blood) or heparin (National Committee for
            Clinical Laboratory Standards, 1989; National Committee for
            Clinical Laboratory Standards, 1992; Nicholson & Green,
            1993), and perform the test within the time frame allowed by
            the single-platform technology (SPT) manufacturer. Because
            acid citrate dextrose is added as a liquid to blood
            collection tubes, its use would make calculating accurate
            final sample volume difficult and is not recommended. With
            this absolute counting technology, use of an accurate sample
            volume is critical.

        2.  Reject specimens that cannot be processed within 72 hours.

    B.  Collect blood specimens by venipuncture (National Committee for
        Clinical Laboratory Standards, 1998) into evacuated tubes
        containing K~3~EDTA anticoagulant, completely expending the
        vacuum in the tubes.

        1.  Use pediatric tubes to obtain specimens from children, and
            ensure that the tube is full.

        2.  Mix the blood well with the anticoagulant to prevent
            clotting.

    C.  Label all specimens with the date, time of collection, and a
        unique patient identifier. Ensure that patient information and
        test results are accorded confidentiality.

III. **Specimen Transport**

     A.  Maintain and transport specimens at room temperature
         (64Â°\--72Â°F \[18Â°\--22Â°C\]) (Paxton & Bendele, 1993; Shield
         et al., 1983; McCoy, Carey, & Krause, 1990; Ekong et al.,
         1993). Specimens should not be exposed to extreme temperatures
         that could allow them to freeze or become too hot. Temperatures
         \>99Â°F (37Â°C) might cause cellular destruction and affect
         flow cytometry measurements (Paxton & Bendele, 1993). In hot
         weather, pack the specimen in an insulated container. If
         necessary, place this container inside another containing an
         ice pack and absorbent material. This method helps retain the
         specimen at ambient temperature. The effect of cool
         temperatures (i.e., \<39Â°F \[4Â°C\]) on CD45 gate-based
         immunophenotyping results is not clear (Paxton & Benedele,
         1993; Ekong et al., 1993).

     B.  Transport specimens to the immunophenotyping laboratory as soon
         as possible.

     C.  For transport to locations outside the collection facility,
         follow state or local guidelines. One method for packaging such
         specimens is to place the tube containing the specimen in a
         leakproof container (e.g., a sealed plastic bag) and to pack
         this container inside a cardboard canister containing
         sufficient material to absorb all the contents should the tube
         break or leak. Cap the canister tightly. Fasten the request
         slip securely to the outside of this canister with a rubber
         band. For mailing, this canister should be placed inside
         another canister containing the mailing label.

     D.  For interstate shipment, follow federal guidelines for
         transporting diagnostic specimens (available at
         [http://www.cdc.gov/od/ohs/biosfty/shipregs.htm](../%20http://www.cdc.gov/od/ohs/biosfty/shipregs.htm)).
         **Note**: Use overnight carriers with an established record of
         consistent overnight delivery to ensure arrival the following
         day. Check with these carriers for their specific packaging
         requirements.

     E.  Obtain specific protocols and arrange appropriate times of
         collection and transport from the facility collecting the
         specimen.

IV. **Specimen Integrity**

    A.  Inspect the tube and its contents immediately upon arrival.

    B.  Take corrective actions if any of the following occur:

        1.  If the specimen is hot or cold to the touch but not
            obviously hemolyzed or frozen, process it but note the
            temperature condition on the worksheet and report form. Do
            not rapidly warm or chill specimens to bring them to room
            temperature because this may adversely affect the
            immunophenotyping results (Paxton & Bendele, 1993).
            Abnormalities in light-scattering patterns may reveal a
            compromised specimen.

        2.  If blood is hemolyzed or frozen, reject the specimen and
            request another.

        3.  If clots are visible, reject the specimen and request
            another.

        4.  If the specimen is received \>72 hours after collection,
            reject it and request another.

V.  **Specimen Processing**

    A.  Perform the test within 48 hours (preferred), but no later than
        72 hours after drawing the blood specimen (Bergeron et al.,
        2002).

    B.  Place the samples on a gentle blood rocker for 5 minutes to
        ensure that the samples are uniformly distributed.

    C.  Pipette blood volumes accurately and in a reproducible manner. A
        reverse pipetting technique is recommended (Refer to information
        box in original guideline document).

    D.  Vortex sample tubes to mix the blood and reagents and break up
        cell aggregates. In addition, vortex samples immediately after
        the lyse/fixation step and before analysis to disperse cells
        optimally.

    E.  Incubate all tubes in the dark during the staining procedure.

    F.  A lyse/no-wash method is required for SPT. Follow directions
        provided by the manufacturer.

    G.  Immediately after processing the specimens, cap the tubes and
        store all stained samples in the dark and under refrigeration
        (39Â°--50Â°F \[4Â°--10Â°C\]) until flow cytometric analysis.
        These specimens should not be stored for longer than 24 hours
        unless the laboratory can demonstrate that scatter and
        fluorescence patterns do not change for specimens for stored
        longer periods.

VI. **Monoclonal Antibody Panels**

    A.  CD45 is required to aid in the identification of lymphocytes.
        Lymphocytes are brightly positive for CD45 and have low
        light-scattering characteristics.

    B.  Monoclonal antibody panels must contain appropriate antibody
        combinations to enumerate CD4+ and CD8+ T-cells and to ensure
        the quality of the results (Refer to Table 1 in original
        guideline document).

        1.  CD4 T-cells are identified as being positive for CD3 and
            CD4.

        2.  CD8 T-cells are identified as being positive for CD3 and
            CD8.

    C.  Three-color monoclonal antibody panels

        1.  Three-color monoclonal antibody panels should fulfill the
            following basic requirements: enumerate CD4+ and CD8+
            T-cells, validate the CD45 gate used, and provide some
            assessment of tube-to-tube variability.

        2.  Three-color monoclonal antibody panels must consist of at
            least two tubes, each with the same lineage marker. For the
            examples described previously, CD3 is the common lineage
            marker in each tube. Differences between replicate CD3
            results should be [\<]{.underline}2%.

        3.  CD19+ B-cell values may be important in assessing immune
            status of pediatric patients.

    D.  Four-color monoclonal antibody panels

        1.  Addition of CD45 to a single tube containing CD3, CD4, and
            CD8 allows the identification of lymphocytes based on CD45
            and side scatter and the enumeration of CD4+ and CD8+
            T-lymphocytes.

        2.  CD19+ B-cell values may be essential for assessing the
            immune status of pediatric patients.

        3.  Use of a second tube containing a natural killer (NK) cell
            marker together with CD3 and CD19 can help to assess the
            recovery and purity of the lymphocytes within the
            CD45/side-scatter gate.

VII. **Negative and Positive Controls for Immunophenotyping**

> **Note**: An isotype control is not needed.

A.  Positive methodologic control

    1.  Use the methodologic control to determine whether procedures for
        preparing and processing the specimens are optimal. Prepare this
        control each time specimens from patients are prepared.

    2.  Use either a whole-blood specimen from a control donor or
        commercial materials validated for this purpose.

    3.  If the methodologic control falls outside established normal
        ranges, determine the reason. **Note**: The purpose of the
        methodologic control is to detect problems in preparing and
        processing the specimens. Biologic factors that cause only the
        whole-blood methodologic control to fall outside normal ranges
        do not invalidate the results from other specimens processed at
        the same time. Poor lysis or poor labeling in all specimens,
        including the methodologic control, invalidates results.

B.  Positive control for evaluating reagents

    1.  Use the positive control to test the labeling efficiency of new
        lots of reagents or when the labeling efficiency of the current
        lot is questioned. Prepare this control only when needed (i.e.,
        when reagents are in question) in parallel with reagent lots of
        known acceptable performance. **Note**: New reagents must
        demonstrate similar results to those of known acceptable
        performance.

    2.  Use a whole-blood specimen or other human lymphocyte preparation
        (e.g., cryopreserved or commercially obtained lyophilized
        lymphocytes or stabilized whole blood).

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VIII. **Flow Cytometer Quality Control**

      A.  Verify optical alignment daily. Usually, clinical flow
          cytometers that are capable of three- and four-color
          immunophenotyping have fixed optical systems, i.e., the
          relative position of the flow cell with respect to the optical
          elements is fixed. In such systems, the instrument operator
          cannot optimize alignment but must verify that the instrument
          meets the manufacturer\'s specifications for optical
          alignment. Regardless of whether the alignment is user
          adjustable, it should be checked with alignment standards,
          such as wide-spectrum fluorescent microfluorospheres with
          measurable light-scatter characteristics. Daily monitoring of
          optical alignment ensures that the cytometer gives acceptably
          bright fluorescence measurements and that homogeneous peaks
          are produced for all parameters to be used in sample analysis
          (National Committee for Clinical Laboratory Standards, 1998).

          1.  Use a stable calibration material (e.g.,
              microfluorospheres labeled with fluorochromes) that has
              measurable and known forward-scatter, side-scatter, and
              fluorescence properties in each channel to be used for
              sample analysis.

          2.  Verify acceptable optical alignment by establishing that
              calibration particles meet manufacturer- or
              laboratory-defined criteria for brightness and
              homogeneity.

          3.  Align stream-in-air flow cytometers daily (at a minimum)
              and stream-in-cuvette flow cytometers (most clinical flow
              cytometers are this type) as recommended by the
              manufacturer.

      B.  Standardize fluorescence and light-scatter signals daily. This
          ensures that the flow cytometer is operating within
          manufacturer- or laboratory-defined acceptance ranges under
          test-specific conditions each day and that its performance is
          consistent from day to day.

          1.  Select machine settings that are appropriate for
              antibody/fluorochrome-labeled, whole-blood specimens.

          2.  Use microfluorospheres or other stable standardization
              material to place the scatter and fluorescence peaks in
              the same narrow range of scatter and fluorescence channels
              each day. Adjust the flow cytometer as needed.

          3.  Retain machine standardization settings for the remaining
              quality control procedures (sensitivity and color
              compensation) and for reading the specimens.

      C.  Determine fluorescence resolution daily. The flow cytometer
          must differentiate between the dim peak and autofluorescence
          in each fluorescence channel (National Committee for Clinical
          Laboratory Standards, 1998).

          1.  Unstained and lysed fresh whole blood is suitable for
              adjusting the photomultiplier tube (PMT) voltages. The
              autofluorescence from the unstained lymphocytes should be
              completely on scale (i.e., \<5% of cells within the
              lymphocyte light-scatter gate fall in channel 0 in each
              fluorescence scale) and should fall within the lower left
              quadrant of the dot plot for every PMT/detector in use.

          2.  Evaluate standardization/calibration material or cells to
              verify that cells with low-level fluorescence can be
              resolved from autofluorescence (e.g., microbeads with
              low-level and negative fluorescence, CD56-labeled
              lymphocytes, or dim cells in CD8-labeled lymphocytes).

          3.  Establish a minimal acceptable distance between peaks;
              monitor this difference, and correct any daily deviations.

      D.  Compensate for spectral overlap daily (See Figure 1 in the
          original guideline document). Compensation is the process of
          correcting for spectral overlap of one fluorochrome into the
          filter window being used to monitor another fluorochrome. In
          most instruments used clinically, this correction is done by
          adjusting the electronic compensation circuits on the flow
          cytometer to place populations not expected to be dual
          positive for two fluorochromes into orthogonal fluorescence
          quadrants with no overlap into the double-positive quadrant.
          At the same time, avoiding overcompensation is essential
          because this may cause dual-positive cells to be incorrectly
          classified as single positive. The following procedures may be
          performed manually, or the software on the flow cytometer may
          perform the spectral compensation automatically.

          1.  Select the compensation control so it will match the
              brightest specimen signal. Use either microbead or
              cellular compensation material containing four populations
              for three-color immunofluorescence (no fluorescence,
              phycoerithrin \[PE\] fluorescence only, fluorescein
              isothiocyanate \[FITC\] fluorescence only, and a
              population that is positive for only the third color) or
              five populations for four-color (the four described
              previously and a population that is positive for only the
              fourth color).

          2.  Analyze this material, and adjust the electronic
              compensation circuits on the flow cytometer to place the
              fluorescent populations in their respective fluorescence
              quadrants with no overlap into the double-positive
              quadrant (Refer to Figure 1 in original guideline
              document). With three fluorochromes, compensation must be
              carried out in an appropriate sequence: FITC, PE, and the
              third color, respectively (Mandy et al., 1992). For
              four-color monoclonal antibody panels, follow the flow
              cytometer manufacturer\'s instructions for four
              fluorochromes. Avoid overcompensation.

          3.  If standardization or calibration particles (microbeads)
              have been used to set compensation, confirm proper
              calibration by using lymphocytes labeled with FITC- and
              PE-labeled monoclonal antibodies and a third-color- or
              fourth-color-labeled monoclonal antibody for three-color
              or four-color panels, respectively (Mandy et al., 1992).
              So that separate cell populations can be recognized
              without overlap, cells in individual tubes may be
              separately stained with each different
              fluorochrome-labeled antibody and then combined in a
              single tube for analysis. These populations should have
              the brightest expected signals. **Note**: Using a
              dimmer-than-expected signal to set compensation can result
              in suboptimal compensation for the brightest signal.

          4.  Reset compensation when photomultiplier tube voltages or
              optical filters are changed.

          5.  Commercially available software can analyze data without
              compensation and perform the compensation automatically.
              When using this software, follow manufacturer\'s
              instructions for this procedure.

      E.  Repeat all four instrument quality control procedures (section
          VIII A--C) whenever instrument problems occur or if the
          instrument has been serviced.

      F.  Maintain instrument quality control logbooks and monitor them
          continually for changes in any of the parameters. In the
          logbook, record instrument settings, peak channels, and
          coefficient of variation (CV) values for materials used to
          monitor or verify optical alignment, standardization,
          fluorescence resolution, and spectral compensation.
          Reestablish target fluorescence levels for each quality
          control procedure when lot numbers of beads are changed or the
          instrument has been serviced.

IX. **Sample Analyses**

    A.  With single-platform absolute count determination, use of the
        lyse/no-wash sample processing is mandatory. The lymphocyte
        population is identified as having bright CD45 fluorescence and
        low side-scattering properties (See Figure 2 in the original
        guideline document). Set the threshold or discriminator as
        recommended by the manufacturer. Adjust side scatter so that all
        leukocyte populations are visible. Draw a gate on the bright
        CD45+ cell population and analyze the cells in that population
        (Nicholson, Hubbard, & Jones, 1996).

    B.  Count at least 2,500 gated lymphocytes in each sample to ensure
        that enough cells and beads have been counted to provide an
        accurate absolute lymphocyte value.

X.  **Data Analysis**

    A.  CD45 gating

        1.  Lymphocytes are identified by being brightly labeled with
            CD45 monoclonal antibody and having low side-scattering
            properties. Two typical examples of a four-color SPT
            analysis based on CD45 gating are illustrated (See Figure 2
            in the original guideline document).

        2.  Establish criteria for cluster identification based on a
            clear definition of lymphocytes that does not include
            basophils (less bright CD45, low side scatter) or monocytes
            (less bright CD45, moderate side scatter). **Note**: Care
            must be taken to include all lymphocytes. CD45 fluorescence
            may be slightly less with B cells than with T cells (the
            major cluster of lymphocytes). NK cells have bright CD45
            fluorescence but have slightly more side-scattering
            properties than the majority of the lymphocytes.

        3.  CD45/side-scatter gates for lymphocytes are assumed to
            contain \>95% lymphocytes. Lymphocyte purity is assumed to
            be high with the CD45/side-scatter gating strategy;
            therefore, correction of lymphocyte subset values is not
            needed (Nicholson, Hubbard, & Jones, 1996).

        4.  If an estimate of lymphocyte recovery is needed (i.e.,
            percentage of total lymphocytes within the CD45/side scatter
            gate), all the B and NK cells must be immunophenotyped as
            well. **Note**: Validation of a CD45/side-scatter gate is
            recommended during its initial use to help determine the
            CD45 and side-scatter characteristics of T, B, and NK cells
            and to ensure their inclusion in the gate.

    B.  Set cursors based on the tube containing CD3/CD4 and CD3/CD8 so
        that the negative and positive cells in the histogram are
        clearly separated.

    C.  Analyze each patient or control specimen with lymphocyte gates
        and cursors for positivity set for that particular patient or
        control.

    D.  Include the following analytic reliability checks, when
        available:

        1.  With single-platform technology (SPT), an additional
            analytical tool can be used to check the accuracy of the
            absolute count; time can be used as a parameter to determine
            how long it takes to obtain a microfluorosphere count that
            represents a unit volume of blood analyzed. Optimally, if
            blood pipetting was performed without noticeable error and
            the beads were accurately added to the tubes, the time
            required to analyze a microliter of whole blood should be
            constant. Follow manufacturer\'s instructions to set time as
            an active parameter. If more or less time is required for a
            sample to accumulate the usual number of microspheres, this
            may indicate a serious counting problem and specimen
            processing should be repeated.

        2.  Optimally, the sum of the percentages of CD3+CD4+ and
            CD3+CD8+ cells should equal the total percentage of CD3+
            cells [+]{.underline} 5%, with a maximum variability of
            \<10%. Note: For specimens containing a considerable number
            of T gamma delta T-cells (Margolick et al., 1991; DePaoli,
            1991), this reliability check may exceed the maximum
            variability.

XI. **Data Storage**

    A.  Store list-mode data for all specimens analyzed. This allows for
        reanalysis of the raw data, including redrawing of gates. At a
        minimum, retain hard copies of the CD45/side-scatter gate and
        correlated dual-histogram data of each sample\'s fluorescence.

    B.  Retain all primary files, worksheets, and report forms for 2
        years or as required by state or local regulation, whichever is
        longer. Data can be stored electronically. Disposal after the
        retention period is at the discretion of the laboratory
        director.

XII. **Data Reporting**

     A.  Report all data in terms of CD designation, with a short
         description of what that designation means. **Note**: CD4+ T
         cells are T-helper cells. The correct cells to report for this
         value are those that are positive for both CD3 and CD4.
         Similarly, CD8+ T-cells are T-suppressor/cytotoxic cells and
         are positive for both CD3 and CD8. Do not include other cell
         types (non-T cells) in CD4 and CD8 T-cell determinations.

     B.  Report lymphocyte subset values as follows:

         1.  Report both percentages and absolute counts.

         2.  With SPT, determine the absolute counts directly from the
             flow cytometers. These calculations are usually handled by
             software that reports calculated results. The following
             formula should be used:

> [No. of events in the bright CD45 region]{.underline}\
> No. of events in the microfluorosphere region
>
> [Total no. of microfluorospheres added]{.underline}\
> Volume of blood added

C.  Report data from all relevant monoclonal antibody combinations with
    corresponding reference limits of expected normal values (e.g., CD4+
    T-cell absolute number and percentage). Reference limits for
    immunophenotyping test results must be determined for each
    laboratory (National Committee for Clinical Laboratory Standards,
    1998). Separate reference ranges must be established for adults and
    children, and the appropriate ranges must be reported for patient
    specimens.

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XIII. **Quality Assurance**

      A.  Ensure the overall quality of the laboratory\'s CD4+ T-cell
          testing by monitoring and evaluating the effectiveness of the
          laboratory policies and procedures for the preanalytic,
          analytic, and postanalytic testing phases. The practices and
          processes to be monitored and evaluated include the following:

          -   methods for collecting, handling, transporting,
              identifying, processing, and storing specimens

          -   information provided on report forms for test requests and
              results

          -   instrument performance, quality control protocols, and
              maintenance

          -   reagent quality control protocols

          -   process for reviewing and reporting results

          -   employee training and education, which should consist of
              the following:

              -   basic training by flow cytometer manufacturers and
                  additional training involving hands-on workshops for
                  flow cytometer operators and supervisors

              -   education of laboratory directors regarding flow
                  cytometric immunophenotyping through workshops and
                  other programs

              -   continuing education regarding new developments for
                  all flow cytometric immunophenotyping personnel
                  through meetings and workshops

              -   adherence to federal and state regulations for
                  training and education

          -   assurance of satisfactory performance. Laboratories must
              fully participate in a performance evaluation program and
              demonstrate acceptable level of performance. When
              proficiency testing programs have been approved by the
              Centers for Medicare & Medicaid Services (formerly, the
              Health Care Financing Administration) as meeting the
              requirements of the Clinical Laboratory Improvement
              Amendments of 1988 (CLIA \'88) (none are currently
              approved for CD4+ T-cell testing), laboratories must
              satisfactorily participate.

          -   review and revision (as necessary or at established
              intervals) of the laboratory\'s policies and procedures to
              ensure adherence to the quality assurance program. All
              staff involved in the testing should be informed of any
              problems identified during the quality assurance review,
              and corrective actions should be taken to prevent
              recurrences.

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      A.  Document all quality assurance activities.

#### CLINICAL ALGORITHM(S)

Not applicable

### EVIDENCE SUPPORTING THE RECOMMENDATIONS

#### REFERENCES SUPPORTING THE RECOMMENDATIONS

[References open in a new
window](http://www.guideline.gov/summary/select_ref.aspx?doc_id=3617)

#### TYPE OF EVIDENCE SUPPORTING THE RECOMMENDATIONS

The evidence supporting the recommendations is stated throughout the
body of the guideline.

### BENEFITS/HARMS OF IMPLEMENTING THE GUIDELINE RECOMMENDATIONS

#### POTENTIAL BENEFITS

The use of guideline recommendations for single-platform technology
(SPT) might foster improved laboratory practices. Outcomes associated
with SPT and CD45 gating include a) increased confidence in results, b)
more reproducible results, c) increased ability to resolve discrepant
problems, d) decreased proportion of unacceptable specimens received for
testing, e) decreased proportion of specimens requiring reanalysis, and
f) fewer incidents that could pose biohazard risks.

#### POTENTIAL HARMS

Not stated

### QUALIFYING STATEMENTS

#### QUALIFYING STATEMENTS

-   Although these guidelines for single-platform technology (SPT) use
    might foster improved laboratory practices, developing comprehensive
    guidelines for every aspect of CD4+ T-cell testing (including some
    laboratory-specific practices) is not possible. Moreover, measuring
    the outcomes associated with the adoption of these guidelines is
    inherently difficult. First, the guidelines lack evaluation
    protocols that can adequately account for the interactions among the
    recommendations. No weight of importance has been assigned for the
    individual recommendations that address unique steps in the testing
    process; hence, the consequences of incompletely following the
    entire set of recommendations are uncertain. Second, because
    published data are not available for every aspect of the guidelines,
    certain recommendations are based on the experience and opinion of
    knowledgeable persons. Recommendations made on this basis, in the
    absence of data, may be biased and inaccurate. Finally, variations
    in testing practices and interactions among the practices (e.g., how
    specimens are obtained and processed, skill of laboratory personnel
    \[such as with pipetting\], testing methods used, test-result
    reporting practices, and compliance with other voluntary standards
    and laboratory regulations) complicate both the development of
    guidelines that will fit every laboratory\'s unique circumstances
    and the assessment of the value of implementing the guidelines.

-   These guidelines for SPT are intended for domestic implementation.
    Several alternative methods are available that require fewer
    reagents and involve more cost-effective gating algorithms. Some of
    these alternative methods may be compatible with current U.S.
    clinical laboratory methods; however, to date they have not been
    validated for domestic applications. As published validation data
    accumulate from multisite studies for methods such as
    Pan*Leuco*gating and primary CD4 gating, these potentially more
    cost-effective options will be considered as alternative or
    substitute methods. In the future, guidelines should be harmonized
    to include all methods that meet domestic performance standards to
    ensure consistent high quality.

### IMPLEMENTATION OF THE GUIDELINE

#### DESCRIPTION OF IMPLEMENTATION STRATEGY

**Evaluation and Validation of a Newly Adopted Single-Platform
Technology in the Laboratory**

When a laboratory adopts the new single-platform technology (SPT),
specimens should be tested in parallel by using both the current and the
new method to characterize any systematic differences in the methods.
Laboratorians should use statistical tools that provide useful
information for the comparison studies. Linear least squares regression
analyses are helpful in establishing good correlations between the new
and established methods. If no error is detected with the new method,
the r^2^ value will approach 1.0. However, regression-type scatter plots
provide inadequate resolution when the errors are small in comparison
with the analytical range and do not characterize the relationship
between the two methods.

A bias scatter plot provides laboratorians with a more useful tool for
determining bias. These simple, high-resolution graphs plot the
differences in the individual measurements of each method (result of old
method\--result of new method) against measurements obtained with one of
the methods (result of old method). Such graphs provide an easy means of
determining if bias is present and distinguishing whether bias is
systematic, proportional, or random/nonconstant. The laboratorian can
visually determine the magnitude of these differences over the entire
range of values. When sufficient values are plotted, outliers or samples
containing interfering substances can be identified. The laboratorian
can then divide the data into ranges relevant to medical decisions and
calculate the systematic error (mean of the bias) and the random error
(standard deviation of the bias) to gain insight into analytical
performance at the specified decision points.

Several detailed guidelines and texts provide additional information
regarding quality goals, method evaluation, estimation of bias, and bias
scatter plots (refer to the original guideline document for relevant
citations). Once a new method is accepted and implemented, the
laboratory will need to confirm or redefine its normal range and should
continue to monitor the correlation between the results and the
patient\'s clinical disease data to ensure that no problems have gone
undetected by the relatively few samples typically tested during method
evaluations.

### INSTITUTE OF MEDICINE (IOM) NATIONAL HEALTHCARE QUALITY REPORT CATEGORIES

#### IOM CARE NEED

Living with Illness\
Staying Healthy

#### IOM DOMAIN

Effectiveness

### IDENTIFYING INFORMATION AND AVAILABILITY

#### BIBLIOGRAPHIC SOURCE(S)

Mandy FF, Nicholson JK, McDougal JS. Guidelines for performing
single-platform absolute CD4+ T-cell determinations with CD45 gating for
persons infected with human immunodeficiency virus. Centers for Disease
Control and Prevention. MMWR Recomm Rep 2003 Jan 31;52(RR-2):1-13. \[68
references\]
[PubMed](http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12583540)

#### ADAPTATION

Not applicable: The guideline was not adapted from another source.

#### DATE RELEASED

2003 Jan 31

#### GUIDELINE DEVELOPER(S)

Centers for Disease Control and Prevention - Federal Government Agency
\[U.S.\]

#### SOURCE(S) OF FUNDING

United States Government

#### GUIDELINE COMMITTEE

Not stated

#### COMPOSITION OF GROUP THAT AUTHORED THE GUIDELINE

*Prepared by*: Francis F. Mandy, PhD (Bureau of HIV/AIDS, STD and TB
Population and Public Health Branch, Health Canada, Ottawa, Ontario,
Canada); Janet K.A. Nicholson, PhD (Office of the Director); J. Steven
McDougal, MD (Division of AIDS, STD, and TB Laboratory Research,
National Center for Infectious Diseases, CDC)

#### FINANCIAL DISCLOSURES/CONFLICTS OF INTEREST

Not stated

#### GUIDELINE STATUS

This is the current release of the guideline.

#### GUIDELINE AVAILABILITY

Electronic copies: Available from the Centers for Disease Control and
Prevention (CDC) Web site:

-   [HTML Format](http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5202a1.htm)

-   [Portable Document Format
    (PDF)](http://www.cdc.gov/mmwr/PDF/rr/rr5202.pdf)

Print copies: Available from the Centers for Disease Control and
Prevention, MMWR, Atlanta, GA 30333. Additional copies can be purchased
from the Superintendent of Documents, U.S. Government Printing Office,
Washington, DC 20402-9325; (202) 783-3238.

#### AVAILABILITY OF COMPANION DOCUMENTS

None available

#### PATIENT RESOURCES

None available

#### NGC STATUS

This NGC summary was completed by ECRI on February 12, 2003.

#### COPYRIGHT STATEMENT

No copyright restrictions apply.

### DISCLAIMER

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Date Modified: 11/3/2008