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Guidelines for the Performance of CD4+ T-Cell Determinations in Persons with Human Immunodeficiency Virus Infection
SUGGESTED CITATION Centers for Disease Control. Guidelines for the performance of CD4+ T-cell determinations in persons with human immunodeficiency virus infection. MMWR 1992;41(No. RR-8):(inclusive page numbers).
CIO RESPONSIBLE FOR THIS PUBLICATION: National Center for Infectious Diseases
This document has been developed by CDC to give guidance to laboratories performing lymphocyte immunophenotyping assays in human immunodeficiency virus-infected persons. The recommendations in this document reflect current technology in a field that is rapidly changing. The recommendations apply to laboratory safety, specimen collection, specimen transport, maintenance of specimen integrity, specimen processing, flow cytometer quality control, sample analyses, data analysis, data storage, data reporting, and quality assurance.
The pathogenesis of acquired immunodeficiency syndrome (AIDS) is largely attributable to the decrease in T-lymphocytes bearing the CD4 receptor (CD4+) (1-5). Progressive depletion of CD4+ T-lymphocytes is associated with an increased likelihood of clinical complications. Because of this association, the measurement of CD4+ T-cell levels has been used to establish decision points for initiating Pneumocystis carinii pneumonia prophylaxis (6) and antiviral therapy (7) and for monitoring the efficacy of treatment. CD4+ T-lymphocyte levels are also used as prognostic indicators in patients with human immunodeficiency virus (HIV) disease (8).
The U.S. Public Health Service (PHS) has recommended that CD4+ T-lymphocyte levels be monitored every 3-6 months in all HIV-infected persons (6). The need for CD4+ T-cell testing services has increased and is expected to continue to increase. Moreover, CD4+ T-lymphocyte levels are being considered for inclusion as a criterion for classifying HIV-infected persons by the newly revised CDC HIV classification system and the proposed AIDS surveillance case definition for adults and adolescents. *
The measurement of absolute CD4+ T-cell levels in whole blood is the product of three laboratory techniques: the white blood cell (WBC) count; the percentage of WBCs that are lymphocytes (differential); and the percentage of lymphocytes that are CD4+ T-cells.
The process of measuring the percentage of CD4+ T-lymphocytes in the whole blood sample is referred to as ``immunophenotyping by flow cytometry'' (9-14). Immunophenotyping relies on detecting specific antigenic determinants on the surface of WBCs by antigen-specific monoclonal antibodies that have been labeled with a fluorescent dye or fluorochrome, such as phycoerythrin (PE) or fluorescein isothiocyanate (FITC). The fluorochrome-labeled cells are analyzed by flow cytometry, which categorizes individual cells according to size, granularity, fluorochrome, and fluorochrome intensity. Size and granularity, detected by light scattering, characterize the types of WBCs (granulocytes, monocytes, and lymphocytes). Different fluorochromes are used to distinguish the subpopulations of a given type of WBC.
Flow cytometric immunophenotyping is a highly complex and relatively new technology. As the technology has moved from research laboratories to clinical laboratories, the need for standardization has increased. In response to this need, several sets of guidelines addressing aspects of the CD4+ T-lymphocyte testing process -- in particular, quality control, quality assurance, and reagents for flow cytometric immunophenotyping of lymphocytes -- have been developed (15,16, and National Institute of Allergy and Infectious Diseases (NIAID)/AIDS Clinical Trials Group: Guidelines for hematologic and flow cytometric analysis of ACTG specimens, 1992 **).
To assure the accuracy and reliability of CD4+ T-lymphocyte test results obtained within individual laboratories and to assure the comparability of results between laboratories, standard methods for performing the test, as well as guidelines for quality control and quality assurance, are desirable. This document provides CDC recommendations for immunophenotyping by flow cytometry. The recommendations apply to laboratory safety, specimen collection, specimen transport, maintenance of specimen integrity, specimen processing, flow cytometer quality control, sample analyses, data analysis, data storage, data reporting, and quality assurance.
B. Specimen collection
and flow cytometric immunophenotyping.
2. Collect blood specimens by venipuncture (31) into evacuated tubes containing an appropriate anticoagulant, completely expending the vacuum in the tubes.
3. Draw the appropriate number of tubes:
4. Label all specimens with a unique patient identifier, date, and time of collection.
C. Specimen transport
(32,33). Avoid temperatures less than 10 C and greater than 37 C. In hot weather, it may be necessary to pack the specimen in a container with insulating material around it and place this container inside another that contains a cold pack (ice pack) and absorbent material. This method will help retain the specimen at ambient temperature.
2. Transport specimens to the immunophenotyping laboratory as soon as possible.
3. For transport to locations outside the collection facility but within the state, follow state or local guidelines. One method for packaging such specimens is to place the tube containing the specimen in a leak-proof container, such as a plastic bag, and pack this container inside a cardboard canister containing sufficient material to absorb all the blood 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.
4. For interstate shipment, follow federal guidelines (34) for transporting diagnostic specimens. 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 as well.
5. Obtain specific protocols and arrange appropriate times of collection and transport from the facility collecting the specimen.
D. Specimen integrity
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. Abnormalities in light-scattering patterns will reveal a compromised specimen. b. If blood is hemolyzed or frozen, reject the specimen and request another. c. If clots are visible, reject the specimen and request another. d. If the specimen is greater than 48 hours old before processing, reject the specimen and request another.
E. Specimen processing
Perform the hematologic tests within 6 hours after collecting the blood specimen (29). (See Note under B.1.a.ii.) b. Perform an automated WBC count and differential, counting 10,000 to 30,000 cells (35). If the specimen is rejected or ``flagged'' by the instrument, a manual differential of at least 400 cells can be performed. If the flag is not on the lymphocyte population and the lymphocyte differential is reported by the instrument, the automated lymphocyte differential should be used.
F. Negative and positive controls for immunophenotyping
Use this control with each specimen to determine nonspecific binding of the mouse monoclonal antibody to the cells and to set markers for distinguishing fluorescence-negative and fluorescence-positive cell populations. b. Use a monoclonal antibody with no specificity for human cells but of the same isotype(s) as the test reagents. Note: In many cases, the isotype control may not be optimal for controlling nonspecific fluorescence because of differences in fluorochrome/protein (F/P) ratio and antibody concentration between the isotype control and the test reagents. At this time there is no solution to this problem.
2. Positive methodologic control
3. Positive control for testing reagents
G. Flow cytometer quality control (15)
tightest peaks are produced in all parameters. Note: Some clinical flow cytometers can be aligned and others can be aligned only by qualified service personnel.
2. Calibrate daily. This ensures that the flow cytometer is performing optimally each day and that its performance is the same from day to day.
3. Determine sensitivity daily. The flow cytometer must differentiate between the dim peak and autofluorescence in each fluorescence channel (PE and FITC).
4. Compensate for spectral overlap daily. This step corrects the spectral overlap of one fluorochrome into the fluorescence spectrum of another.
5. Repeat all four instrument quality control procedures whenever instrument problems occur or if the instrument is serviced during the day.
6. Maintain instrument quality control logs, and monitor them continually for changes in any of the parameters. In the logs, record instrument settings as well as peak channels and coefficient of variation (CV) values for optical alignment, calibration, sensitivity, and spectral compensation. Reestablish fluorescence levels for each quality control procedure when lots of beads are changed.
H. Sample analyses
2. Count at least 2,500 gated lymphocytes in each sample. This number assures with 95% confidence that the result is less than or equal to 2% (standard deviation(SD)) of the ``true'' value (binomial sampling). Note: This model assumes that variability determined from preparing and analyzing replicates is less than or equal to 2% SD. Each laboratory must determine the level of variability by preparing and analyzing at least six replicates of the last four tubes in the recommended panel. Measure variability when first validating the methodology used and again when methodologic changes are made.
3. Examine light-scattering patterns on each sample tube. Determine whether lysis or sample preparation, which can affect light scattering, is the same in each sample tube of a patient's specimen. Deviation in a particular tube usually indicates sample preparation error, and the tube should be repeated (a new aliquot of blood is stained and lysed).
lymphocyte gates using forward and side light-scattering patterns and fluorescence staining.
2. Verify the lymphocyte gate by determining the lymphocyte proportion within the gate and the purity of the gate.
3. Set cursors using the isotype control so that less than 2% of cells are positive.
4. Analyze the remaining samples with the cursors set based on the isotype control. Note: In some instances, the isotype-set cursors will not accurately separate positive and negative staining for another sample tube from the same specimen. In such cases, the cursors can be moved on that sample to more accurately separate these populations (Figure 2).
5. Analyze each patient or normal control specimen with light-scattering gates and cursors for positivity set for that particular patient or control.
6. Where spectral compensation of a particular specimen appears to be inappropriate because FITC-labeled cells have been dragged into the PE-positive quadrant or vice-versa (when compensation on all other specimens is appropriate), repeat the sample preparation, prewashing the specimen with phosphate-buffered saline (PBS), pH 7.2, to remove plasma before monoclonal antibodies are added.
7. Include the following analytic reliability checks:
J. Data storage
analyzed. This allows reanalysis of the raw data, including redrawing gates. At a minimum, retain hard copies of the lymphocyte gate and correlated dual histogram data of the fluorescence of each sample.
2. 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.
K. Data reporting
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 (determined from tube 3 in the suggested panel). Similarly, CD8+ T-cells are T-suppressor/cytotoxic cells, and these are positive for both CD3 and CD8 (tube 4 in the panel). It is important not to include other cell types (non-T-cells) in CD4 and CD8 determinations.
2. Report data as a percentage of the total lymphocytes and correct for the lymphocyte purity of the gate. For example, if the lymphocyte purity is 94% and the CD3 value is 70%, correct the CD3 value by dividing 0.70 by 0.94 and then multiply the result by 100 to give a T-lymphocyte value of 74%.
3. Report absolute lymphocyte subset values when an automated complete blood cell (CBC) count (WBC and differential) has been performed from blood drawn at the same time as that for immunophenotyping.
4. Report data from all relevant monoclonal antibody combinations with corresponding reference limits of expected normal values (e.g., CD4+ T-cell percentage, absolute number of CD4+ T-cells). Reference limits for immunophenotyping test results must be determined for each laboratory. See reference 15 for methods for determining these limits.
L. Quality assurance
testing by monitoring and evaluating the effectiveness of the laboratory policies and procedures for the preanalytic, analytic, and postanalytic testing phases:
2. Document all quality assurance activities.
To date, no defined standard exists for immunophenotyping by flow cytometry. This article provides recommendations for laboratory standardization. Proficiency testing and performance evaluation programs are also needed to assess laboratory performance and to measure the level and sources of testing variability between and within laboratories.
Three test components contribute to intra- and interlaboratory variability when lymphocyte populations are measured by immunophenotyping: a) whole blood immunophenotyping preparation and analysis; b) total WBC count; and c) leukocyte differential. Analytic and biologic variability exist in all three test components.
The analytic variability (CV) in determining the WBC count using an automated leukocyte counter ranges from 2.2% to 7.7% and from 9.3% to 17.6% using a hemocytometer. The lymphocyte differential varies from 1.9% to 5.3% for automated counts and from 12.5% to 27% for manual counts (35). Therefore, the variability in the absolute number of lymphocytes in the blood reflects the combined variability of the WBC count and the lymphocyte differential. Biologic variability is even greater: about 10% diurnally and 13% week to week (40).
Estimates of interlaboratory variability (SD) in flow cytometric immunophenotyping results have been derived from proficiency testing data (41; College of American Pathologists (CAP) Flow Cytometry Survey: unpublished data; CDC: Model Performance Evaluation Program (MPEP), unpublished data). An analysis of data from a recent CAP survey of nearly 500 laboratories showed that SD of the percentage of CD4+ T-cells was 3.5% to 5%, regardless of the actual percentage of CD4+ T-cells in the specimen. For duplicate measurements, SD of the percentage of CD4+ T-cells was about 3% when the specimen contained 45% CD4+ T-cells. The results furnished to CDC by 280 laboratories participating in the MPEP for T-lymphocyte immunophenotyping in March 1991 were similar. For samples of CD4+ values in the range of 1% to 16%, SD of the percentage of CD4+ T-cells was about 2.5%; for samples with CD4+ values between 16% and 24%, SD was about 3.4%.
Limited information is available on the degree of interlaboratory variability in CD4+ T-cell counts. In a multicenter proficiency testing study (42) of seven laboratories for the year 1987, interlaboratory CV for the percentage and absolute number of CD4+ T-cells on normal specimens were 6% and 29.4%, respectively. This study has been ongoing and, through rigorous quality assurance and training, CV values have been reduced each year. Subsequently, in 13 laboratories in 1991, CV for the percentage and absolute number of CD4+ T-cells on normal specimens were 4.1% and 8.4%, respectively (Rickman WJ: unpublished data).
Variability in the absolute number of CD4+ T-cells can be reduced in the following ways: a) performing replicate analyses; b) standardizing the test protocol; c) improving technology; and d) increasing the skills and knowledge of testing personnel, including those responsible for specimen collecting and handling. The above methods for reducing variability, if implemented, will increase the reliability of this important laboratory measurement.
Clinicians and others who choose laboratories for testing patient samples need ways of evaluating the performance of flow cytometric immunophenotyping laboratories. Criteria that can be helpful in this process include: a) laboratory accreditation, licensure, or certification by a recognized professional organization or governmental agency; b) laboratory participation in a recognized proficiency testing/performance evaluation program; and c) laboratory use of CDC or other published guidelines for flow cytometry.
The recommendations in this document reflect current technology in a field that is rapidly changing. Revisions in these recommendations will be required as techniques, skills, and knowledge improve. Evaluation of testing practices is a tool that will provide information for making sound changes in these recommendations.
The determination of CD4+ T-cell counts has greatly contributed to the health management of persons infected with HIV, to public health HIV/AIDS prevention programs, and to epidemiologic and clinical research in HIV infection. These recommendations should enhance the ability of laboratories to provide the high quality of testing needed to support these services.
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