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A Quality Management Systems Approach for CD4 Testing in Resource-Poor Settings

Larry E. Westerman PhD, Luciana Kohatsu PhD, Astrid Ortiz, Bernice McClain, Jonathan Kaplan MD, Thomas Spira MD, Barbara Marston MD, Ilesh V. Jani MD, PhD, John Nkengasong PhD, Linda M. Parsons PhD
DOI: http://dx.doi.org/10.1309/AJCPP7MCHFYLX2FM 556-567 First published online: 1 October 2010

Abstract

Quality assurance (QA) is a systematic process to monitor and improve clinical laboratory practices. The fundamental components of a laboratory QA program include providing a functional and safe laboratory environment, trained and competent personnel, maintained equipment, adequate supplies and reagents, testing of appropriate specimens, internal monitoring of quality, accurate reporting, and external quality assessments. These components are necessary to provide accurate and precise CD4 T-cell counts, an essential test to evaluate start of and monitor effectiveness of antiretroviral therapy for HIV-infected patients. In recent years, CD4 testing has expanded dramatically in resource-limited settings. Information on a CD4 QA program as described in this article will provide guidelines not only for clinical laboratory staff but also for managers of programs responsible for supporting CD4 testing. All agencies involved in implementing CD4 testing must understand the needs of the laboratory and provide advocacy, guidance, and financial support to established CD4 testing sites and programs. This article describes and explains the procedures that must be put in place to provide reliable CD4 determinations in a variety of settings.

Key Words:
  • HIV
  • AIDS
  • CD4 testing
  • Laboratory

Infection with HIV leads to the development of AIDS, which is characterized by the loss of CD4 T cells that are required for proper functioning of a person’s immune system. Assays that detect CD4 T cells in whole blood specimens are relied on to determine the appropriate time to initiate anti-retroviral therapy (ART) in HIV+ people, preferably before clinical signs of disease develop. In industrialized countries, ART regimens are generally started when the CD4 count drops below 350 cells/μL1; in resource-poor countries, the values for initiating therapy differ by country but usually range from 200 to 350 cells/μL. The decision of when to start ART is critical in resource-poor countries in the context of higher death rates and incidence of opportunistic infections.2 In 2006, the World Health Organization recommended that if the CD4 value is between 200 and 350 cells/μL, the decision to begin ART should depend on clinical stage of disease but definitely should be initiated before the CD4 count drops to less than 200 cells/μL.3,4 In November 2009, the World Health Organization updated the recommendation for ART for adults and adolescents.5

CD4 counts are also used as a tool to monitor disease progression and the effectiveness of ART. When a patient is not responding to treatment, CD4 data are used to determine whether to change from first-line to second-line therapy, especially in countries where viral load testing is unavailable. Because of the link between CD4 levels and ART, HIV+ people, either receiving ART or not yet eligible for ART, are usually scheduled for a CD4 count at least once or twice per year, with the possibility that a patient will have 50 or more CD4 tests during his or her lifetime.6 Thus, CD4 testing is a critical laboratory component of HIV and AIDS care and treatment, and the demand for CD4 testing is increasing as more patients are living longer. The improvements in HIV diagnosis, care, and treatment result in a continual rise of the absolute number of people living with HIV who must be monitored.7,8

In resource-poor countries where the prevalence of HIV and AIDS is high, expansion of CD4 testing is a major undertaking. The challenges are many and include selection of appropriate technology, poor laboratory infrastructure, human resource limitations, high cost of instruments and test kits, instrument maintenance, supply chain failures, poor laboratory information management systems, lack of technology appropriate for primary health centers, unreliable specimen transport systems, and insufficient means to ensure quality of testing.6,7,9 In these countries, CD4 testing is not usually performed in health clinics or peripheral laboratories, but most assays are performed in tertiary- and secondary-level clinical laboratories. Many patients must travel long distances for this essential testing or have their specimens transported to a higher level laboratory. To facilitate easy access, CD4 testing should be done as close to the patient as possible, ideally at the primary health care level or with a dedicated specimen referral system that is dependable and timely. As countries move to meet these expanding needs, CD4 testing is being decentralized to an increasing number of test sites. Ensuring the quality of CD4 test results at each of these facilities, whether new or preexisting, is essential to treating HIV-infected people.

This article was developed as a guide to quality assurance CD4 testing in resource-poor settings. We describe and explain quality assurance (QA) criteria that should be established to provide reliable CD4 determinations in a variety of laboratory settings in resource-poor countries. The following topics are included: (1) laboratory environment; (2) human resources; (3) equipment and equipment maintenance; (4) reagent procurement, storage, and inventory; (5) specimen collection and transport; (6) quality monitoring; (7) CD4 testing and reporting; and (8) external quality assessment (EQA). These guidelines should be used by clinical laboratory staff and also by organizations, clinicians, and program managers involved in the management and financial support for CD4 testing. A link between care providers and laboratories will enhance the CD4 testing laboratory’s ability to provide quality results and, in turn, help program staff understand and gain confidence in key laboratory results.7

Laboratory Environment

The environment in which CD4 testing is performed must be conducive to efficient operations that do not compromise the safety of the staff or the quality of the testing process. Most CD4 instruments and assays require well-controlled environmental conditions to ensure optimal operation and accurate test results. Climate control, such as air conditioning, may be necessary to maintain the temperature and humidity within ranges considered acceptable for the CD4 testing platform in use. Improper climate conditions can affect the specimen testing process, reagent quality, and instrument performance and longevity. A temperature monitoring program in which daily temperatures are recorded and reviewed is necessary to ensure not only proper conditions for CD4 testing but also for reagent and supply storage.

Consistent and stable sources of power are required for most CD4 instruments and assays. Uninterruptible power supply units and backup generators should be in place to provide for continuous testing during power interruptions and outages. Power interruptions that cause failures of a testing run result in costly loss of reagents and preparation time. Unstable power supplies can damage or compromise the components of the CD4 instruments.

Adequate and logically organized space should be available for all aspects of the testing process: specimen processing, test setup, CD4 instruments, data recording, and reporting of results and for designated storage spaces for documents, records, reagents, and supplies. The space should be clean and uncluttered, with only essential equipment, documents, supplies, and reagents readily available at the workbench. Clean water is required for hand washing and maintaining a sanitary workplace.

Human Resources

Qualifications

An appropriately trained and well-organized laboratory staff is essential for the successful operation of a CD4 testing facility. The required qualifications for laboratory personnel performing CD4 testing should be determined by each country’s ministry of health or governing body in charge of regulating health care professionals. The qualifications of the laboratory staff must be adequate to perform, interpret, and troubleshoot the CD4 assay.

Training and Competency Assessments

After instrumentation and reagents are available in the laboratory and before performing CD4 testing on patient specimens, laboratory personnel must be well trained and qualified to perform CD4 testing and routine maintenance for the specific instrument in use. The competency of laboratory personnel should be assessed after initial training, and personnel should be approved for testing and instrument maintenance before performing the CD4 assay on patient specimens. Experienced and new testing staff must undergo competency assessments yearly to ensure standardization of performance. All training and initial and annual assessments of competency must be documented, and records must be kept in each staff member’s personnel file. Examples of forms for completion of training and competency assessments are shown in Figure 1 and Figure 2. Personnel who perform suboptimally during competency assessments must be retrained and reassessed before they return to testing patient specimens.

In addition to training specific for CD4 testing, all laboratory staff should be trained annually in general laboratory safety procedures that have been established for the testing facility. The training should include laboratory safety practices, handling blood-borne pathogens, biohazard disposal and waste management, and fire safety. In addition, the following safety practices should always be adhered to during work performed in the laboratory: (1) Wear laboratory coats and gloves when processing and analyzing samples. (2) Never pipette by mouth; instead use safety pipetting devices. (3) Handle specimens with care and open blood tubes carefully to avoid aerosols and splatters. (4) After working with specimens and before leaving the laboratory, remove gloves and wash hands with soap and water. (5) Disinfect the CD4 instrument as recommended by the manufacturer. (6) Disinfect the work area at the start and end of the day. (7) Disinfect the work area periodically throughout the day and as needed for spills. (8) Properly dispose of specimen and waste generated from testing.

Equipment and Equipment Maintenance

For a national laboratory system to minimize variations in test results, improve the operation and efficiency of laboratory networks, and reduce costs in equipment, reagents, and supplies, institutions throughout the country should harmonize CD4 testing equipment, methods, and maintenance procedures for each tier-level of service (provincial, district, or health center).9,10 A wide variety of commercially available technologies exist for CD4 testing, and most are single-platform systems that can derive CD4 counts directly from whole blood specimens by using a dedicated CD4 instrument and reagents, without the additional need for a hematology analyzer. Table 1 provides a list of available CD4 instruments and assays. Included in Table 1 are parameters to be considered before selecting instruments. In addition, the following criteria for selecting test method and equipment should be considered9: (1) in-country approval or validation of the CD4 assay; (2) acceptable precision, accuracy, and performance as reported by an independent assessing agency or in a peer-reviewed article; (3) throughput to meet testing demand and laboratory capacity; (4) stability and storage conditions for reagents; (5) availability of reagents and supplies in country; (6) availability of instrument service and technical assistance; and (7) compatibility of the assay to be monitored by internal QA and EQA programs.

Figure 1

Documentation for CD4 laboratory staff training. QC, quality control; SOP, standard operating procedure.

Figure 2

Documentation of laboratory staff competency to perform CD4 testing. SOP, standard operating procedure.

High-throughput systems that require advanced technical skills to operate, including the EPICS XL-MCL (Beckman Coulter, Brea, CA) and the FACSCalibur (BD Biosciences, San Jose, CA) flow cytometers, may be suitable for tertiary laboratories receiving a high volume of samples per day. Mid- to low-throughput systems, such as the FACSCount (BD Biosciences), Guava PCA (Millipore, Billerica, MA), and CyFlow (Partec, Gorlitz, Germany), have been successfully implemented at district hospitals and some larger primary health care facilities where 20 to 100 samples are tested daily.11 Small peripheral laboratories and health centers may refer specimens to higher level laboratories or, if well-trained technical staff is available, may develop capacity to use smaller, labor-intensive systems for CD4 testing of fewer than 20 samples per day, such as the manual CD4 counting bead assays provided by Beckman Coulter, Invitrogen (Oslo, Norway), and Sysmex (Kobe, Japan). In addition to available assays, new robust and easy-to-use, single-platform, point-of-care CD4 assays are available, and more are being developed and evaluated.10,12 These point-of care assays are anticipated to benefit rural populations with ready access to testing and settings such as clinics for the prevention of mother-to-child transmission, where immediate CD4 results are useful.

Once a national, harmonized approach for the selection and purchase of equipment for a tiered laboratory system has been established, each laboratory receiving new CD4 instruments should perform a verification study to ensure that the instrument is working properly and the assay’s precision and accuracy are within reported limits. If the laboratory is already performing CD4 tests with an established assay, the verification study should compare results obtained with the new technology with those from the original assay. If CD4 testing is new to the laboratory, precision could be estimated by replicate testing of patient specimens and accuracy assessed by comparison of samples from a more experienced laboratory or those from quality control (QC) material and proficiency test panels.10

View this table:
Table 1

Standard operating procedures (SOPs) should be developed for instrument use and maintenance at each level of laboratory according to the manufacturer’s protocol, with adaptations based on the specific situation in the testing facility. SOPs should be available, understandable, and followed by laboratory personnel to ensure standardization of practice and to reduce instrument downtime, testing variations, and errors. SOPs provide not only methods on how to use and maintain an instrument but also procedures on general troubleshooting, guides to other manuals and tools for advance troubleshooting, and information on how to place service calls. Each laboratory must have a plan to perform, implement, and document scheduled and as-needed maintenance. Laboratory personnel must be able to perform simple troubleshooting procedures and be informed of procedures to notify service providers for scheduled preventive maintenance and of instrument or equipment failures.

Daily Maintenance

Performance of daily maintenance procedures is essential to ensuring that the instrument is working properly and any potential downtime is reduced. Performance of the required procedures should be documented on a form that is part of the maintenance SOP to ensure that all steps are performed with each use of the instrument. Free-flowing fluidics of a flow cytometer are vital to obtaining accurate test results. Whole blood specimens used in CD4 assays have a tendency to clog or partially block the tubing of the instrument. It is recommended that cleaning of the instrument be performed before starting CD4 testing, at the end of the test day, and periodically during the day, depending on the workload. In addition, daily maintenance should be performed even on days when testing is not done to prevent deposits from accumulating in the tubing.

Weekly, Monthly, Yearly, and As-Needed Maintenance

As with daily maintenance, performance of scheduled and as-needed maintenance procedures is necessary to ensure that the instrument is working properly and downtime is reduced. A maintenance log similar to the one shown in Figure 3 for scheduled and as-needed maintenance should be developed to ensure that all maintenance procedures are performed and documented. In addition, a calendar should be developed to ensure that all scheduled maintenance is performed when required.

Troubleshooting

Testing failures or instrument malfunctions may occur during routine use of the CD4 instrument. Troubleshooting these failures or malfunctions is necessary before CD4 testing can continue. A corrective action log sheet should be developed to record any problems and error messages that occur during or outside CD4 testing. The corrective action taken to resolve the problem, including advice or service calls from the manufacturer, should be documented. This log should be reviewed periodically to check for trends, and any technical errors that are identified should be immediately addressed. In addition, plans should be made to provide for testing during periods when a primary instrument is not functioning. Potential approaches for backup CD4 testing include having a backup instrument on hand and referring specimens to another laboratory until the primary instrument is back online.

Figure 3

Monthly maintenance log for CD4 instruments.

Service Contracts

Service contracts for all major equipment should be acquired from the manufacturer or the manufacturer’s representative. These contracts are necessary not only to maintain proper functioning of the instrument but also to ensure its expected lifespan. The contract should stipulate that the provider must be responsive for timely and appropriate service. Terms of a service contract must be very specific and include the following: (1) the preventive maintenance procedures to be performed; (2) the schedule for maintenance procedures; (3) whether training will be provided for laboratory personnel or local technicians to perform routine maintenance; (4) the number of free service calls and cost of an unscheduled service call; (5) which parts are included or excluded; (6) availability and immediate contact information of service representatives, including whether they are local or out-of-country; (7) availability of loaned instruments in case of instrument failure; (8) maximum time to respond to and perform a service request; (9) whether troubleshooting help by telephone or e-mail is included; and (10) penalties incurred for faulty service, delays in service, or breaches of the contract.

As described, records of all maintenance, including preventive maintenance performed by a service representative, should be documented in specific log forms or books. Problems or issues that require contacting the service representative should be clearly defined so that unnecessary service calls can be avoided. All direct and backup contact information such as telephone numbers, fax numbers, and e-mail addresses should be readily available.

Reagent Procurement, Inventory, and Storage

Whether ordering from a central supply store or directly from vendors, a protocol to procure supplies and reagents should be developed. To avoid ordering errors, the protocol should include detailed product specifications and be understood and precisely followed by personnel in charge of procurement to ensure that no mistakes, substitutions, or stock-out of reagents and supplies occurs. Stock-outs and improper ordering are a major problem in many settings and often interfere with the ability of a laboratory to continue testing. Included in this protocol should be contact information for vendors or suppliers, examples of forms to be used for procurement, and exact steps in the procurement process. An accurate and up-to-date record should be kept of all orders placed and items received.

Depending on the expiration dates of reagents, a 2- to 3-month supply of reagents and supplies needed for CD4 testing should be available at the testing facility at all times. These commodities should be ordered well in advance to ensure sufficient stock is on hand to handle any possible increases in workload. Consumption rates, workload, and inventory should be constantly monitored to accurately forecast reagent and supply needs. Reagents should be labeled with date of receipt and expiration date and used in order of receipt, with special awareness of reagents with short expiration dates. Ideally, a CD4 instrument or assay should use reagents that do not need cold-chain distribution and storage. However, if cold-chain is required, all reagents must be stored at stated temperatures, and the temperatures of refrigerators and freezers should be monitored and recorded daily. If temperatures are found to be out of range, the quality of any unexpired reagents stored at the suboptimal temperatures must be validated with QC materials before testing patient specimens. There are 2 important caveats concerning use of reagents: (1) reagents that are past their expiration date must never be used for testing patient specimens, and (2) reagents are specific for each instrument and, therefore, must never be used for testing on a different instrument.

Specimen Collection and Transport

Proper specimen collection, temperature of storage and transport, and timing of testing after collection are critical to ensure the quality of the specimen and the accuracy of CD4 test results. Errors in patient identification and in the materials and procedures used to collect blood can lead to specimen rejection or inaccurate test results. For appropriate specimen collection and handling, phlebotomists and nursing staff must follow standardized specimen collection and safety procedures and undergo appropriate primary and refresher training.

Patient Request Form

The specimen and patient should be positively identified by ensuring that the information on the specimen matches that on the request form or patient records. The request form should include patient identifiers, including age and sex of patient, name of submitting health care facility, laboratory tests ordered, collector initials, and date and time of collection Figure 4. A misidentified or mislabeled specimen should not be tested, and the cause of misidentification should be determined so that corrective actions can be implemented to prevent future problems.

Collection of Whole Blood Specimens

Whole blood specimens for CD4 testing are collected with a vacuum blood-drawing tube containing the anticoagulant EDTA. The tube containing EDTA should not be used after the expiration date. To prevent rapid clotting, the blood specimens are best collected by being directly drawn into the EDTA tube. Blood collection with a syringe is acceptable in patients and infants in whom specimens are particularly difficult to obtain. If a syringe is used, whole blood must be immediately placed in the EDTA tube to avoid clotting. The appropriate volume of blood must be added to the tube to mix properly with the anticoagulant. After collection, the tube should be gently inverted to mix the blood and anticoagulant. The smaller tubes specifically developed for pediatric patients should be used to obtain whole blood specimens from children, especially when collecting from infants by using heel or finger sticks. Pediatric collections are more prone to clotting, so the blood must be mixed with the anticoagulant in the tube as soon as it is drawn. All tubes should be labeled immediately after collection with at least 2 patient identifiers and the date and time of collection. The date and time of collection and the collector’s initials should also be written on the request form. Information on the tubes and request form must match and be verified before sending the tubes to the laboratory and at reception at the laboratory.

Figure 4

An example of a CD4 request and report form.

Short-Term Storage of Whole Blood Specimens

To avoid the rejection or discarding of patient samples, specimens should not be collected if they cannot be processed or tested within the time required by the specific testing procedure (usually within 48 hours, but may be longer if blood stabilizers are included in the tube). Whole blood specimens collected in EDTA tubes should be held at ambient temperature, but always less than 30°C. If necessary, the specimens can be kept in an insulated cool box with spacers to ensure that the specimens are not kept too cold and do not come in contact with the cool packs. The specimens must not be refrigerated or frozen, and the stopper on the tube should not be removed until immediately before testing.

Specimen Transport

A reliable specimen transport system, either a commercial courier or dedicated delivery service, must deliver the blood specimens to the testing laboratory within the time limits required by the CD4 assay in use (preferably within 24 to 48 hours). The specimens should be packed in spill-proof, insulated cool boxes with cool packs and spacers. Specimens must not be exposed to extreme conditions because temperatures higher than 37°C could destroy cells and affect test results. The effects of cold (4°C) and hot (>30°C) temperatures decrease CD4 values because of significant losses of the CD4 markers on T cells.13,14 In addition, specimens must be properly packed and transported to minimize shaking.

SOPs on proper labeling, handling, and transport of the blood specimens are to be in place to ensure that the correct specimen is tested, the quality of specimen is optimal, and to provide a safe environment for collectors, transport personnel, and laboratory staff. Procedures and transport logs should be developed to clearly describe how to ship specimens and test results between the collection and the testing facilities. Transporters or couriers should be trained on specimen transport biosafety and be made aware of the need to transport specimens within a proper time to the testing facility.

Commercial shipping of infectious materials from the collection facility to testing locations is subject to national and international regulations. Blood specimens, including from patients infected with HIV, are classified as United Nations (UN) No. 3373 Dangerous Goods, Biological Substances, category B. National requirements for transportation of infectious, dangerous goods should adhere to international regulations such as the International Air Transport Association (IATA) Packing Instruction 650. National transportation regulations should be updated regularly according to IATA infectious substances shipping guidelines that are issued every year.15 For proper labeling, packaging, and transportation of HIV-infected material in each country, protocols should adhere to local postal and courier service regulations for mailing a UN 3373 classified agent according to the current IATA guidance document.

Quality Monitoring

Use of SOPs

SOPs are critical for maintaining consistent testing performance and reducing errors associated with variation in testing. Each testing facility must have up-to-date and practical SOPs for all laboratory activities to ensure the consistency, quality, and integrity of the generated data. Ideally, there should be nationally adopted SOPs for specimen collection, specimen transport, specimen reception and processing, QC, CD4 testing, instrument and equipment maintenance, results reporting, and EQA. To maintain the quality of testing, current SOPs must be readily available in the work areas and accessible to all testing personnel. Laboratory managers, supervisors, and testing personnel must understand and review the SOPs annually. SOPs must be accurate and relevant, and all laboratory personnel must strictly follow the SOPs during the testing process. Any updates to the SOPs must be approved by management and provided to testing personnel. SOPs no longer in use or replaced by newer versions should be removed from the testing site and archived.

Quality Control

Daily testing of QC samples with established target values and acceptable ranges must be done at each CD4 testing laboratory to ensure the quality of CD4 results on patient specimens. The results from daily control sample testing will identify abnormal trends and problems with the instruments and reagents, so that problems can be addressed or corrected as soon as possible. Each testing facility should analyze at least 2 QC samples, one with low CD4 values and another with normal values. A low-level QC sample is needed to determine whether an instrument or assay can accurately report low-level CD4 counts in patient specimens. A normal-level control will represent the CD4 range likely to be seen in uninfected or asymptomatic people. In addition to being tested each day that CD4 testing is performed, QC materials must also be tested after any maintenance or troubleshooting has been performed on the instrument. Table 2 lists some of the more commonly used commercially available QC materials. Not all QC materials can be used on all instruments, so QC materials should be selected on the basis of compatibility with the equipment in use in the testing laboratory.

Monitoring QC Sample Results

As described, QC materials must be tested daily whenever CD4 testing is performed, using the same procedure as that used for patient specimens. Although commercial QC materials have set CD4 ranges, each testing facility should establish its in-laboratory ranges for low and normal CD4 controls. The QC material ranges received from the manufacturer have been derived from multiple laboratories and may be too broad to enable an individual laboratory to detect problems specific to the local situation. To establish in-laboratory ranges, at least 20 measurements of the QC materials should be performed over a period of time. The mean values and standard deviations (SDs) should be calculated and the coefficient of variation percentage plus historical coefficient of variation percentages used to establish new ranges.16 Once these values have been obtained, Levey-Jennings charts should be created for each control; charts should show the mean values surrounded by ±1, ±2, and ± 3 SD Figure 5.

Each day of testing patient specimens, QC results should be entered on the charts, and, ideally, results for each control should fall within ±2 SD from the mean established in each laboratory. Patient results should not be reported if control values are more than ±3 SD from the mean. Levey-Jennings charts should be reviewed monthly to check for and follow up on abnormal trends. Most important, in addition to not reporting patient results if the QC results are out of range, all occurrences of abnormal QC results must be documented, with corrective action performed and documented as necessary.

Calculations of mean and new ranges should be done whenever a new lot number of QC material is acquired. Updates of the mean and ranges can be done periodically and at the end of use of the QC material.16 These cumulative control limit updates provide a good representation of long-term test performance.

Incident Management

A quality management system, with a well-defined SOP, is necessary to identify and correct any errors or variations that affect the quality of the CD4 tests. This system should include a means of reporting any incident that could change the results obtained in the testing procedure. Corrective actions for each incident should also be documented and reviewed on a regular basis to fix any errors or trends.

Review of Documents

A system should be established in the laboratory to retain all logs, records, and reports for a predetermined period. These documents should undergo supervisory review on a regular basis and are an excellent means of identifying potential problems. These documents may include specimen log book or electronic spreadsheet, QC results, equipment maintenance and service records, incident reports, and personnel records.

CD4 Testing and Reporting

Specimen Receipt

A protocol that requires use of a specimen log book or electronic spreadsheet should be developed for specimen receipt. The form should include areas for patient and submitter identifiers, dates of collection and receipt in the laboratory, specimen integrity, tests to be performed, technician initials, and date test result was reported Figure 6. This log book format can be used to monitor delayed or missing information and track turnaround times.

View this table:
Table 2
Figure 5

A and B, Examples of Levey-Jennings charts used to monitor low and normal CD4 control values. Values should fall within ±2 SD from the mean established in each laboratory. Patient results should not be reported if control values are larger than ±3 SD.

On arrival of the specimen in the testing facility, the information on the request form should be checked to ensure that it matches that on the blood tube. When a batch of specimens is received with a shipping list, an immediate check should be made to ensure that all the specimens listed are received, and the sender should be informed if any specimens are missing. If the shipment is complete, the appropriate information on each specimen should be recorded in the log book or electronic spreadsheet. The integrity of the specimen tube and its contents should be inspected immediately on arrival. If the specimen is hot or cold to the touch but not obviously hemolyzed or frozen, it should be noted on the log and report forms, but the specimen should still be tested. If the temperature is not optimal, the specimens should not be rapidly warmed or chilled to bring them to room temperature, since this may adversely affect CD4 results.

A specimen should be rejected and a second specimen requested if any of the following conditions are found: (1) The blood is hemolyzed or frozen. (2) Blood clots are visible. (3) The specimen cannot be processed or tested within the time required by the specific testing procedure (usually within 48 hours, unless blood stabilizer is present). (4) The specimen is not in an appropriate tube containing the anticoagulant EDTA.

Figure 6

An example of a log sheet for tracking incoming specimens and outgoing results.

The health care provider should always be informed as soon as possible about any rejected specimen and given the reason for rejection.

CD4 Testing

SOPs must be developed according to the manufacturer’s protocol and adapted on the basis of the specific situation in each testing facility. The SOP should be clear and understandable and strictly followed by laboratory personnel. This will ensure standardization of the procedure and reduce variations in testing that could cause erroneous CD4 test results.

The test must be performed within the recommended time per the manufacturer’s instructions, usually within 48 hours and rarely more than 72 hours after the blood specimen was obtained. The blood specimen must be well mixed before sampling by placing the specimen on a gentle rocker for 5 minutes or by gently inverting the tube 8 to 10 times. Pipettes should be calibrated and certified at least once, preferably twice, per year. It is essential for accurate results that blood and reagent volumes are pipetted accurately and reproducibly. Inaccurate pipetting is a common reason for erroneous results.

To avoid mistakes, each specimen’s identification number should be checked on the tube and log sheet and accurately entered into the instrument or computer interface. The CD4 instrument should be monitored throughout the procedure to ensure that there are no blockages in the system fluidics and the detection processes are working properly. All CD4 test results must be validated by internal QC, instrument plots or printouts, and supervisory review before releasing results. Repeated testing should be done on specimens with unvalidated results, results with abnormal instrument plots, appearance of error messages, or other conditions as indicated by the SOP.

Reporting of CD4 Results

Daily QC must be within acceptable limits for patient CD4 results to be reported (Figure 5). After supervisory validation of the CD4 test result, the result should be entered on the report form (Figure 4) and rechecked to ensure that it is complete and accurate. The CD4 test result and the date that the report is sent back to the health care facility should be noted on the specimen log form or electronic spreadsheet. The log should be reviewed daily to check on any missing or delayed reports. If possible, all test results from a specimen (eg, CD4, hematology, biochemistry, serology, and viral load) should be compiled and reported to the health care facility. By receiving all results together, the caregiver can make an accurate and informed decision for continued care of the patient. However, the CD4 results should not be delayed by waiting for other tests to be finished but should be reported as soon as possible. A system should be in place to rapidly transport or communicate CD4 results to the health care provider, through a courier service that could transport hard copies of the results or through a networked electronic or telephone-based system.

Document Control

All records should be stored by using an orderly and easy-to-manage system so that results can be readily retrieved. Procedures must be in place to ensure that patient confidentiality is maintained, the records are safe and secure, and a process is in place for disposal of the reports after a defined number of years.

External Quality Assessment

EQA programs monitor the quality of test results at each testing facility. The 3 components of an effective EQA laboratory program are site supervision, retesting of specimens, and proficiency testing (PT). Site supervision of CD4 test sites is usually done by agencies external to the testing facility, such as ministry of health representatives, national reference laboratory staff, implementing partners, or laboratory accreditation organizations. CD4 specimens are not often retested because of the time constraints required to accurately test whole blood specimens. PT is the most widely used approach to monitor the quality of CD4 testing, and each testing facility should participate in an external PT program. The following 4 PT programs provide CD4 panels for testing: (1) United Kingdom National External Quality Assessment Service (http://www.ukneqas.org.uk), (2) Quality Assurance Systems International (http://www.qasidirect.com), (3) College of American Pathologists (http://www.cap.org), and (4) African Regional External Quality Assessment Scheme (http://www.plgcd4.net/G_Afrqas_letter.asp).

All are well-established organizations that charge for enrollment in their programs. Such costs should be included in the budget for a CD4 laboratory. Demonstration of proficiency in these panels provides information to health care providers on the quality of testing in each laboratory and builds confidence in the accuracy of results on patient specimens.17 The performance of a testing facility in a PT program should be reviewed over time by testing facility management and any affiliated external agencies. Oversight of PT performance is necessary to analyze trends that could lead to inaccurate CD4 results and to ensure that management support is provided for correcting technical errors as soon as possible.

Summary

This article provides insight into QA of CD4 testing for resource-poor settings. These QA guidelines are intended for use by clinical laboratory staff, governmental and nongovernmental organizations, clinicians, and program managers involved in provision, management, or financial support of CD4 testing. These guidelines can be used along with more technical guidelines developed for high-resource settings.18

Reliable CD4 counts are essential to determine when to begin or modify ART in HIV+ people. The guidelines list requirements essential for providing accurate and reliable CD4 test results in the areas of laboratory environment; human resources; equipment and equipment maintenance; reagent procurement, storage, and inventory; specimen collection and transport; quality monitoring; CD4 testing and reporting; and EQA. The support of clinicians, laboratory personnel, and program managers is critical for each CD4 testing laboratory’s ability to adhere to these QA guidelines and produce accurate and reliable CD4 results.

Footnotes

  • Supported by the Global AIDS Program, Center for Global Health, US Centers for Disease Control and Prevention.

  • The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the funding agency.

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