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Duplicate analysis method: a cheaper alternative to commercial IQC materials in limited resource settings for monitoring CD4 testing.

Shete A, Singh DP, Mahajan B, Kokare A, Paranjape R, Thakar M - AIDS Res Ther (2015)

Bottom Line: There was good match between r(2) values and % CV of the laboratories performing both the types of QC methods.Commercially available controls showed limitations such as altered specimen quality leading to difficulties in manual gating and issues with the establishment of laboratory range.Duplicate analysis can serve as a cheaper alternative to commercially available controls for IQC of CD4 testing especially when supplemented with other QC measures for controlling variations caused by reagent, equipment, staff and environment in addition to the successful participation in External Quality Assurance programme.

View Article: PubMed Central - PubMed

Affiliation: National AIDS Research Institute, 73 G Block MIDC Bhosari, Pune, 411026 India.

ABSTRACT

Background: India has a large number of HIV infected patients being followed up at anti-retroviral therapy (ART) centers. The patients are regularly offered CD4 count estimation for deciding their eligibility for ART initiation as well as for monitoring response to ART, making CD4 count estimation a very critical test. Hence, quality control of CD4 testing is utmost important for ultimate success of ART program. As the commercial controls are very expensive, internal quality control (IQC), at present, is being done by duplicate analysis method using previous day samples in most of the laboratories. Hence the study was undertaken to review performance of duplicate analysis method for monitoring daily IQC.

Methods: Quality control (QC) data from 11 Indian laboratories using duplicate analysis and/or commercial controls for IQC of CD4 testing was collected for reviewing information on QC parameters such as precision, accuracy and trend monitoring. Precision was determined by r(2) values and mean % variation for duplicate analysis and coefficient of variation (% CV) for commercial controls. Accuracy was monitored by rate of QC failures for both the types of control and trend monitoring was done by plotting LJ charts for commercial controls and by plotting daily % variation for duplicate analysis.

Results: The laboratories using duplicate analysis for IQC showed good precision with mean % variation ranging from 0.5 to 7.2. There was good match between r(2) values and % CV of the laboratories performing both the types of QC methods. Rates of QC failures were 2.3 for duplicate analysis and 3 per laboratory-year for IMMUNO-TROL controls. Daily trend monitoring showed fluctuation of daily counts around mean in LJ charts and of percent variation around 0% in duplicate analysis method. Commercially available controls showed limitations such as altered specimen quality leading to difficulties in manual gating and issues with the establishment of laboratory range.

Conclusion: Duplicate analysis can serve as a cheaper alternative to commercially available controls for IQC of CD4 testing especially when supplemented with other QC measures for controlling variations caused by reagent, equipment, staff and environment in addition to the successful participation in External Quality Assurance programme.

No MeSH data available.


Related in: MedlinePlus

Representative displays of fresh and stabilized blood samples on FACSCount, FACSCalibur and Cyflow. Equipment displays in row 1 are for fresh blood sample used in duplicate analysis method, row 2 are for the multi-check stabilized blood sample and row 3 are for IMMUNO-TROL controls. a, d, g are from FASCount; b, e, h from FACSCalibur and c, f, i from Cyflow. BD FACSCount CD4/CD3 reagent kit contained anti CD3 PE-Cy5 and anti CD4 PE antibodies. Tricolour reagent used for FACSCalibur analysis contained anti CD45 PerCP, anti CD3 FITC and anti CD4 PE antibodies. The gates shown in the figure are autogates set by the Multiset software used for FACSCalibur analysis. CD4 easy count kit used for Cyflow contained anti CD4 PE antibody for staining, where the gates were set manually.
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Fig1: Representative displays of fresh and stabilized blood samples on FACSCount, FACSCalibur and Cyflow. Equipment displays in row 1 are for fresh blood sample used in duplicate analysis method, row 2 are for the multi-check stabilized blood sample and row 3 are for IMMUNO-TROL controls. a, d, g are from FASCount; b, e, h from FACSCalibur and c, f, i from Cyflow. BD FACSCount CD4/CD3 reagent kit contained anti CD3 PE-Cy5 and anti CD4 PE antibodies. Tricolour reagent used for FACSCalibur analysis contained anti CD45 PerCP, anti CD3 FITC and anti CD4 PE antibodies. The gates shown in the figure are autogates set by the Multiset software used for FACSCalibur analysis. CD4 easy count kit used for Cyflow contained anti CD4 PE antibody for staining, where the gates were set manually.

Mentions: Displays of commercially stabilized blood samples were found to have poor separation of cell populations as compared to fresh blood samples as shown in Fig. 1. The stabilized blood samples have been shown to have altered light scatter and fluorescence staining properties as compared to fresh blood specimens, not satisfying the automatic gating algorithm defined by the instrument software [8, 9]. Hence manual gating was required in case of FACSCalibur for analysis of stabilized blood samples. Setting of manual gates was found to be challenging because of poor separation of populations, requiring intense training of the staff. Such gating was also found to be subjective and unreliable at many times by other investigators also [9]. FACSCount also sometimes failed to acquire these controls, especially IMMUNO-TROL controls, by giving a message of ‘Major tube failure’. Such testing failure due to inability to identify and gate clusters of cells of interest in case of fully automated platforms like FACSCount has also been reported in one of the studies [9]. Interestingly, the displays on Cyflow did not have much problem of poor separation as shown in Fig. 1.Fig. 1


Duplicate analysis method: a cheaper alternative to commercial IQC materials in limited resource settings for monitoring CD4 testing.

Shete A, Singh DP, Mahajan B, Kokare A, Paranjape R, Thakar M - AIDS Res Ther (2015)

Representative displays of fresh and stabilized blood samples on FACSCount, FACSCalibur and Cyflow. Equipment displays in row 1 are for fresh blood sample used in duplicate analysis method, row 2 are for the multi-check stabilized blood sample and row 3 are for IMMUNO-TROL controls. a, d, g are from FASCount; b, e, h from FACSCalibur and c, f, i from Cyflow. BD FACSCount CD4/CD3 reagent kit contained anti CD3 PE-Cy5 and anti CD4 PE antibodies. Tricolour reagent used for FACSCalibur analysis contained anti CD45 PerCP, anti CD3 FITC and anti CD4 PE antibodies. The gates shown in the figure are autogates set by the Multiset software used for FACSCalibur analysis. CD4 easy count kit used for Cyflow contained anti CD4 PE antibody for staining, where the gates were set manually.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4536604&req=5

Fig1: Representative displays of fresh and stabilized blood samples on FACSCount, FACSCalibur and Cyflow. Equipment displays in row 1 are for fresh blood sample used in duplicate analysis method, row 2 are for the multi-check stabilized blood sample and row 3 are for IMMUNO-TROL controls. a, d, g are from FASCount; b, e, h from FACSCalibur and c, f, i from Cyflow. BD FACSCount CD4/CD3 reagent kit contained anti CD3 PE-Cy5 and anti CD4 PE antibodies. Tricolour reagent used for FACSCalibur analysis contained anti CD45 PerCP, anti CD3 FITC and anti CD4 PE antibodies. The gates shown in the figure are autogates set by the Multiset software used for FACSCalibur analysis. CD4 easy count kit used for Cyflow contained anti CD4 PE antibody for staining, where the gates were set manually.
Mentions: Displays of commercially stabilized blood samples were found to have poor separation of cell populations as compared to fresh blood samples as shown in Fig. 1. The stabilized blood samples have been shown to have altered light scatter and fluorescence staining properties as compared to fresh blood specimens, not satisfying the automatic gating algorithm defined by the instrument software [8, 9]. Hence manual gating was required in case of FACSCalibur for analysis of stabilized blood samples. Setting of manual gates was found to be challenging because of poor separation of populations, requiring intense training of the staff. Such gating was also found to be subjective and unreliable at many times by other investigators also [9]. FACSCount also sometimes failed to acquire these controls, especially IMMUNO-TROL controls, by giving a message of ‘Major tube failure’. Such testing failure due to inability to identify and gate clusters of cells of interest in case of fully automated platforms like FACSCount has also been reported in one of the studies [9]. Interestingly, the displays on Cyflow did not have much problem of poor separation as shown in Fig. 1.Fig. 1

Bottom Line: There was good match between r(2) values and % CV of the laboratories performing both the types of QC methods.Commercially available controls showed limitations such as altered specimen quality leading to difficulties in manual gating and issues with the establishment of laboratory range.Duplicate analysis can serve as a cheaper alternative to commercially available controls for IQC of CD4 testing especially when supplemented with other QC measures for controlling variations caused by reagent, equipment, staff and environment in addition to the successful participation in External Quality Assurance programme.

View Article: PubMed Central - PubMed

Affiliation: National AIDS Research Institute, 73 G Block MIDC Bhosari, Pune, 411026 India.

ABSTRACT

Background: India has a large number of HIV infected patients being followed up at anti-retroviral therapy (ART) centers. The patients are regularly offered CD4 count estimation for deciding their eligibility for ART initiation as well as for monitoring response to ART, making CD4 count estimation a very critical test. Hence, quality control of CD4 testing is utmost important for ultimate success of ART program. As the commercial controls are very expensive, internal quality control (IQC), at present, is being done by duplicate analysis method using previous day samples in most of the laboratories. Hence the study was undertaken to review performance of duplicate analysis method for monitoring daily IQC.

Methods: Quality control (QC) data from 11 Indian laboratories using duplicate analysis and/or commercial controls for IQC of CD4 testing was collected for reviewing information on QC parameters such as precision, accuracy and trend monitoring. Precision was determined by r(2) values and mean % variation for duplicate analysis and coefficient of variation (% CV) for commercial controls. Accuracy was monitored by rate of QC failures for both the types of control and trend monitoring was done by plotting LJ charts for commercial controls and by plotting daily % variation for duplicate analysis.

Results: The laboratories using duplicate analysis for IQC showed good precision with mean % variation ranging from 0.5 to 7.2. There was good match between r(2) values and % CV of the laboratories performing both the types of QC methods. Rates of QC failures were 2.3 for duplicate analysis and 3 per laboratory-year for IMMUNO-TROL controls. Daily trend monitoring showed fluctuation of daily counts around mean in LJ charts and of percent variation around 0% in duplicate analysis method. Commercially available controls showed limitations such as altered specimen quality leading to difficulties in manual gating and issues with the establishment of laboratory range.

Conclusion: Duplicate analysis can serve as a cheaper alternative to commercially available controls for IQC of CD4 testing especially when supplemented with other QC measures for controlling variations caused by reagent, equipment, staff and environment in addition to the successful participation in External Quality Assurance programme.

No MeSH data available.


Related in: MedlinePlus