depar depar depar depar department of health and human ser tment of health and human ser tment of health and human ser tment of health and human ser tment of health and human services vices vices vices vices# Introduction
Obtaining accurate and reliable measures of CD4 + T lymphocytes (CD4 + T cells) is essential to assessing the immune system and managing the health care of persons infected with human immunodeficiency virus (HIV) (1)(2)(3)(4). The pathogenesis of acquired immunodeficiency syndrome (AIDS) is largely attributable to the decrease in the number of T cells that bear the CD4 receptor (5)(6)(7)(8)(9). Progressive depletion of CD4 + T cells is associated with an increased likelihood of severe HIV disease and an unfavorable prognosis (10)(11)(12)(13). Accordingly, the U.S. Public Health Service (PHS) has recommended that CD4 + T-cell levels be monitored every 3-6 months in all HIVinfected persons (14). Measurement of CD4 + T-cell levels has been used to establish decision points for initiating prophylaxis for Pneumocystis carinii pneumonia and other opportunistic infections and for initiating and monitoring antiretroviral therapy (15)(16)(17)(18)(19)(20). CD4 + T-cell levels are also a criterion for categorizing HIV-related clinical conditions according to CDC's classification system for HIV infection and surveillance case definition of AIDS among adults and adolescents (21).
Single-platform technology (SPT) is designed to enable determinations of both absolute and percentage lymphocyte subset values using a single tube. Until recently, most absolute T-cell numbers were derived from three measurements determined with two different instruments, a hematology analyzer and a flow cytometer (dual-platform technology ). Hence, the CD4 + T-cell number is the product of three laboratory measurements: the white blood cell count, the percentage of white blood cells that are lymphocytes (differential), and the percentage of lymphocytes that are CD4 + T cells (determined by flow cytometry). In 1997, CDC published guidelines addressing concerns related to DPT (22); those guidelines remain appropriate for laboratories performing CD4 + T-cell counts with this technology.
On November 2001, a third national conference on CD4 + immunophenotyping was held in Orlando, Florida, to discuss scientific and technologic advances in the development and production of reagents, instrumentation, and software that have occurred since publication of the 1997

# Guidelines for Performing Single-Platform Absolute CD4 + T-Cell Determinations with CD45 Gating for Persons Infected with Human Immunodeficiency Virus
MMWR January 31, 2003
guidelines. The conference was attended by representatives from public health, private, and academic laboratories as well as product manufacturers. These guidelines reflect a consensus of that conference, reviewed by attendees, and specifically related to the performance of SPT. Development of new guidelines was driven by advances in knowledge and experience with new approaches to enumerate CD4 + T cells. First, a gating strategy for identifying lymphocytes using CD45 fluorescence and side-scattering characteristics is now the preferred method for identifying lymphocytes accurately and reproducibly. Second, three-or four-color flow cytometry has been demonstrated to be superior to two-color methods for measuring CD4 + and CD8 + T-cell counts (23). Finally, the availability of Food and Drug Administration (FDA)-approved commercial microfluorosphere counting reagents for SPT has resulted in decreased interlaboratory variability (24,25). Consequently, SPT is the preferred method in an increasing number of laboratories (4).

# Recommendations

# I. Laboratory Safety
A. Use universal precautions with all specimens (26). B. Adhere to the following safety practices (27)(28)(29):
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. NCCLS recommendations regarding how to disinfect specific areas should be followed (30).
For use on smooth, hard surfaces, a 1% solution of bleach is usually adequate for disinfection; for porous surfaces, a 10% solution is needed (30). 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 HIV by 3-5 logs (31,32); 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 (33)(34)(35). 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.

# II. Specimen Collection for Single-Platform Technology

# 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 (39). 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 (44). 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 (Box). 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 (39-50F ) 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. Use of SPT to obtain absolute CD4 counts requires accurate and precise measurement of blood and beads. Reverse pipetting technique is recommended for dispensing these products.

# VI. Monoclonal Antibody Panels

# Testing Pipetting Precision
The precision of pipetting should be evaluated periodically (e.g., monthly) to ensure the accuracy of results. Retain all records of this evaluation procedure for quality assurance purposes.
- Using the reverse pipetting technique, pipette 10 replicates of blood and record the weights. Select a volume normally used in the performance of the assay. - Using the reverse pipetting technique, pipette 10 replicates of bead suspension and record the weights (this applies to methods in which the beads must be pipetted into the tubes). - Calculate the mean, standard deviation, and coefficient of variation (CV). The CV for replicates should be <2% (Table 2).

# Testing Pipetting Accuracy
The following procedure can be used to test the pipette and how accurately it measures volume. Water is used because the weight of 1 L of water is 1 g.
- Using the reverse pipetting technique, pipette 10 replicates of distilled water and record the weight. (100 L of water should weigh 0.1000 grams.) (Table 2) - Calculate the mean, standard deviation, and CV. The CV must be <2% (range: 0.098-0.102).

# Procedures
The following information is consolidated from operational instruction manuals from several pipette manufacturers. Complete information and more detailed instructions are contained in specific pipette instruction manuals; some of these are available online. Read the manufacturer's manual carefully before beginning the pipetting procedure.
- Select the desired volume (with manual pipettes, higher volumes should be set first; if adjusting from a lower to a higher volume, first surpass the desired volume and then slowly decrease the volume until the required setting is reached). - If applicable, select the desired mode (e.g., reverse pipette). This is recommended for optimal precision and reproducibility. - Reverse pipetting can be done with a manual pipette by pressing the control button slightly past the first stop when aspirating, taking up more liquid than will be dispensed, then pressing the control button only to the first stop when dispensing. A small volume will remain in the tip after dispensing. - Select an appropriate tip (usually color matches the color of the control button).

# Prerinsing
The following procedures will help ensure optimal precision and accuracy.
- Volumes >10 L: Prerinse pipette 2-3 times for each new tip (this involves aspirating and dispensing liquid several times).
Reasons for prerinsing include the following: -to compensate for system pressure, for slight differences in temperature between pipette and liquid, and for properties of the liquid; -to clear the thin film formed by the liquid on the inside of the pipette. Without prerinsing, retention of a thin film on the inside wall of the tip would cause the first volume to be too small. The thickness and nature of this film, and therefore the potential source of error, will vary depending on the nature of the liquid being pipetted. - Volumes <10 L: Do not prerinse pipette, but rinse tip after dispensing to ensure that the whole volume was dispensed. For smaller volumes, prerinsing is not recommended because the dispensed volume would be too great.

# Filling
- Make sure tip is securely attached.
- Hold pipette upright.
- When aspirating, try to keep the tip at a constant depth below the surface of the liquid. - Glide control button slowly and smoothly (electronic pipettes perform this step automatically). - When pipetting viscous liquids (e.g., whole blood), leave the tip in the liquid for 1-2 seconds after aspirating before withdrawing it. - After liquid is in the tip, never lay the pipette on its side.

# Dispensing
- Hold the tip at an angle, against the inside wall of the vessel/ tube if possible. - Glide control button slowly and smoothly (electronic pipettes perform this step automatically).

# Other Recommendations
- To ensure optimal performance, the temperature of the pipetted solution and the pipette and tips should be the same (volume errors may occur because of changes in air displacement and viscosity of the liquid). Do not pipette liquids with temperatures >70 o C. - Volume errors may also occur with liquids that have a high vapor pressure or a density/viscosity that differs greatly from water. Water is most commonly used to calibrate pipettes and to check inaccuracy and imprecision. A pipette could possibly be recalibrated for liquids with densities that vary greatly from that of water. - Pipettes should be checked regularly for precision and accuracy.
- Regular maintenance (e.g., cleaning) should be performed either by the user or a service technician according to manufacturer's instructions.   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 (Figure 2). 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 (47). 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

# Evaluation and Validation of a Newly Adopted SPT in the Laboratory
When a laboratory adopts the new 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 ap-proach 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 (50)(51)(52).
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) (50). 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 (50)(51)(52).
Several detailed guidelines and texts provide additional information regarding quality goals, method evaluation, estimation of bias, and bias scatter plots (50)(51)(52)(53)(54). 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.

# Discussion
More than 1.6 million CD4 + T-cell measurements are performed yearly by the approximately 600 testing laboratories in the United States (55). This figure is based on the reported number of tests performed annually by laboratories participating in CDC's Model Performance Evaluation Program (MPEP) for T-lymphocyte immunophenotyping in 1996. These measurements are performed with flow cytometers using either multiplatform technology or SPT. SPT was introduced for clinical application in 1996, and its wide-scale implementation is relatively new. In 2000, results of two independent multicenter studies studies of SPT were reported (24,25). Those and subsequent reports on SPT and CD45 gating (56)(57)(58)(59)(60) have increasingly encouraged adoption of these improved testing practices (61,62). The resulting 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 propor-tion of unacceptable specimens received for testing, e) decreased proportion of specimens requiring reanalysis, and f ) fewer incidents that could pose biohazard risks (61).
Although these guidelines for 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 , testing methods used, testresult 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.
The first CDC recommendations for laboratory performance of CD4 + T-cell testing (63) were written so as not to impede development of new technology or investigations into better ways to assess the status of the immune system in HIVinfected persons. Developments in the technology have resulted in an assay that is technically less complicated and more accurate. These single-platform methods are now being implemented in as many as one fourth of the laboratories in the United States (MPEP data). In addition, other T-cell phenotypic markers are being investigated as prognostic indicators or markers of treatment efficacy, alone and in combination with other cellular markers (64,65).
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 PanLeucogating ( 66) and primary CD4 gating (67,68), these potentially more costeffective 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.

# Introduction
In 2001, the QuantiFERON  -TB test (QFT) (manufactured by Cellestis Limited, Carnegie, Victoria, Australia) was approved by the Food and Drug Administration (FDA) as an aid for detecting latent Mycobacterium tuberculosis infection (1). This test is an in vitro diagnostic aid that measures a component of cell-mediated immune reactivity to M. tuberculosis. The test is based on the quantification of interferon-gamma (IFN-) released from sensitized lymphocytes in whole blood incubated overnight with purified protein derivative (PPD) from M. tuberculosis and control antigens.
Tuberculin skin testing (TST) has been used for years as an aid in diagnosing latent tuberculosis infection (LTBI) and includes measurement of the delayed type hypersensitivity response 48-72 hours after intradermal injection of PPD. TST and QFT do not measure the same components of the immunologic response and are not interchangeable. Assessment of the accuracy of these tests is limited by lack of a standard for confirming LTBI.
As a diagnostic test, QFT 1) requires phlebotomy, 2) can be accomplished after a single patient visit, 3) assesses responses to multiple antigens simultaneously, and 4) does not boost anamnestic immune responses. Compared with TST, QFT results are less subject to reader bias and error. In a CDCsponsored multicenter trial, QFT and TST results were mod-erately concordant (overall kappa value = 0.60). The level of concordance was adversely affected by prior bacille Calmette-Guerin (BCG) vaccination, immune reactivity to nontuberculous mycobacteria (NTM), and a prior positive TST (2). In addition to the multicenter study, two other published studies have demonstrated moderate concordance between TST and QFT (3,4). However, one of the five sites involved in the CDC study reported less agreement (5).
Limitations of QFT include the need to draw blood and process it within 12 hours after collection and limited laboratory and clinical experience with the assay. The utility of QFT in predicting the progression to active tuberculosis has not been evaluated.
This report provides interim recommendations for using and interpreting QFT results based on available data. As with TST, interpretation and indicated applications of QFT differ between those persons at low risk and those at increased risk for LTBI. This report should assist public health officials, health-care providers, and laboratorians who are responsible for TB control activities in the United States in their efforts to incorporate QFT testing for detecting and treating LTBI.

# QFT Performance, Interpretation, and Use
Tuberculin testing is performed for persons who are 1) suspected as having active TB; 2) at increased risk for progression to active TB; 3) at increased risk for LTBI; or 4) at low risk for LTBI, but are tested for other reasons (Table 1).

# QFT Performance
Aliquots of heparinized whole blood are incubated with the test antigens for 16-24 hours.- The antigens included in the test kits are PPD from M. tuberculosis (tuberculin)  and PPD from Mycobacterium avium (avian sensitin). The kits also include phytohemaglutinin (a mitogen used as a positive assay control), and saline (negative control or nil). After incubation, the concentration of IFN- in the separated plasma is determined by enzyme-linked immunosorbent assay (ELISA).
QFT results are based on the proportion of IFN- released in response to tuberculin as compared with mitogen, or (tu-berculin -nil) / (mitogen -nil)  100 = percentage tuberculin response.  The difference in the amount of IFN- released in response to tuberculin as compared with avian sensitin is expressed as (avian -nil) -(tuberculin -nil) / (tuberculin -nil)  100 = percentage avian difference. A computer program is available from the test manufacturer that performs these calculations and interprets the test results. 

# QFT Interpretation
Interpretation of QFT results (Table 2) is stratified by estimated risk for infection with M. tuberculosis, in a manner simi-- Additional technical information is available from the manufacturer at http:// www.cellestis.com.  PPD from M. tuberculosis is referred to by the manufacturer and in FDA documents as human PPD.

# Using QFT for Persons at Increased Risk for LTBI
QFT can aid in detecting M. tuberculosis infections among certain populations who are at increased risk for LTBI (6). These populations include recent immigrants (i.e., immigrated within the previous 5 years) from high-prevalence countries where tuberculosis case rates are >30/100,000, injection-drug users, residents and employees of prisons and jails, and healthcare workers who, after their preemployment assessment, are considered at increased risk for exposure to tuberculosis. For these populations, a percentage tuberculin response of >15 should be considered a positive QFT result.

# Using QFT for Persons at Low Risk for LTBI
CDC discourages use of diagnostic tests for LTBI among populations at low risk for infection with M. tuberculosis (6). However, initial testing is occasionally performed among certain population groups for surveillance purposes or where cases of active, infectious tuberculosis might result in extensive transmission to highly susceptible populations. These populations include military personnel, hospital staff and health-care workers whose risk of prior exposure to TB was low, and U.S.-born students at higher education institutions (e.g., as a requirement for admission to U.S. colleges and universities). For these populations, a percentage tuberculin response of >30 should be considered a positive QFT result.

# Recommendations
The highest priority of targeted tuberculin testing programs remains one that identifies persons at increased risk for TB who will benefit from treatment for LTBI. Following that principle, targeted tuberculin testing should be conducted among groups at risk for recent infection with M. tuberculosis and those who, regardless of duration of infection, are at increased risk for progression to active TB. The role of QFT in targeted testing has not yet been defined, but QFT can be considered for LTBI screening as follows:
- initial and serial testing of persons with an increased risk for LTBI (e.g., recent immigrants, injection-drug users, and residents and employees of prisons and jails); - initial and serial testing of persons who are, by history, at low risk for LTBI but whose future activity might place them at increased risk for exposure, and others eligible for LTBI surveillance programs (e.g., health-care workers and military personnel); or - testing of persons for whom LTBI screening is performed but who are not considered to have an increased probability of infection (e.g., entrance requirements for certain schools and workplaces). Before QFT testing is contemplated, arrangements should be made with a qualified laboratory. Those arrangements should include quality assurance and collection and transport of blood within the required 12 hours.
Confirmation of QFT results with TST is possible because performance of QFT does not affect subsequent QFT or TST results. The probability of LTBI is greatest when both the QFT and TST are positive. Considerations for confirmation are as follows:
- When the probability of LTBI is low, confirmation of a positive QFT result with TST is recommended before initiation of LTBI treatment. LTBI therapy is not recommended for persons at low risk who are QFT-negative or who are QFT-positive but TST-negative. - TST can also be used to confirm a positive QFT for persons at increased risk for LTBI. However, the need for LTBI treatment when QFT is positive and the subsequent TST is negative should be based on clinical judgment and perceived risk. - Negative QFT results do not require confirmation, but results can be confirmed with either a repeat QFT or TST if the accuracy of the initial test is in question.

# Contraindications
Because of insufficient data on which to base recommendations, QFT is not recommended for - evaluation of persons with suspected tuberculosis. Active tuberculosis is associated with suppressed IFN- responses, and in prior studies, fewer persons with active TB had positive QFT results than TST results. The degree of suppression appears to be related to the severity of disease and the duration of therapy. Studies are under way that compare the sensitivity of QFT and TST among persons with untreated active TB. - assessment of contacts of persons with infectious tuberculosis, because rates of conversion of QFT and TST af-ter a known exposure to M. tuberculosis have not been compared, and concordance of QFT and TST after exposure and with serial LTBI screening have not been studied. - screening of children aged <17 years, pregnant women, or for persons with clinical conditions that increase the risk for progression of LTBI to active TB (e.g., human immunodeficiency virus infection). Further studies are needed to define the appropriate use of QFT among these persons. - detection of LTBI after suspected exposure (i.e., contact investigation after a resident or employee is diagnosed with active TB or a laboratory spill of M. tuberculosis) of persons participating in longitudinal LTBI surveillance programs. The approach of using QFT for initial screening, followed by QFT and TST 3 months after the end of the suspected exposure, has not been evaluated. - confirmation of TST results because injection of PPD for TST might affect subsequent QFT results. Although QFT is not recommended for confirmation of TST results, QFT can be used for surveillance <12 months after a negative TST, if the initial QFT is negative. - diagnosis of M. avium complex disease.

# Conclusions
These interim recommendations are intended to achieve a high rate of acceptance and completion of testing for LTBI among groups who have been identified for targeted testing. Testing programs using TST or QFT should only be implemented if plans are also in place for the necessary follow-up medical evaluation and treatment (e.g., chest radiograph or LTBI treatment) of persons who are diagnosed with LTBI and quality laboratory services are ensured.