Stability of CD4 levels in blood specimens stored in BD Vacutainer CD4 Stabilization Tubes in Buhera District, Zimbabwe Elton Mbofana1, Emmanuel Fajardo2, Steven van den Broucke1, Sandra Simons3, Charlotte van Vyve1, Carol Metcalf2, Helen Bygrave2, Misheck Kuhudzayi4 2 4
Médecins Sans Frontières, South African Medical Unit, Cape Town, South Africa Ministry of Health and Child Welfare, Buhera District, Zimbabwe
Results continued….
Background In Zimbabwe, HIV-infected adolescents and adults with a CD4 count <350 cells/µl are eligible for antiretroviral therapy (ART). Médecins Sans Frontières (MSF) has been supporting HIV health services in the rural Buhera District since 2004. At 22 decentralised clinics, blood samples are collected for CD4 testing only once a week. Hence patients attending the clinic on days that are not blood collection days need to return to the clinic for CD4 testing. Many patients do not return for CD4 testing, and so clinicians are unable to determine their ART eligibility. BD CD4 Vacutainer stabilization tubes may be useful in settings whereCD4 testing is not available on site and it is not possible to transport specimens to the laboratory on the day of the blood draw. We did a field validation study to assess whether blood collected in stabilization tubes and stored at room temperature for several days, gave similar CD4 results to blood collected in EDTA tubes and tested the same day according to the standard method (reference test).
Figure 1: Difference between CD4 in stabilization tube samples and CD4 in the reference sample (cells/µl) 100 50 0 -50 -100
0 (n = 51)
3 (n = 51)
5 (n = 51)
7 (n = 50)
14 (n = 20)
Day 25th centile
Methods The study was conducted in December 2011. Patients attending rural clinics in Buhera District who required a CD4 test were invited to participate. Fiftyone patients consented and provided a sample of blood collected in a standard EDTA Vacutainer tube, and an extra sample of blood collected in a BD CD4 Vacutainer stabilization tube. The samples were taken to one of 2 district laboratories for testing. CD4 testing was done using a BD FACSCount™ cytometer. EDTA tube samples were tested on arrival in the laboratory (Day 0), and the remaining sample was discarded. Stabilization tube samples were tested on arrival in the laboratory, and were stored in racks at room temperature and retested on Days 3, 5, and 7. Twenty of the samples were stored an extra week and retested on Day 14.
median
75th centile
Figure 2: Reliability of CD4 test results using stabilization tube samples compared to the reference CD4 result for identifying patients with a CD4 ≤350 cells/µl 100 80 Percent
3
Médecins Sans Frontières, Murambinda, Zimbabwe Médecins Sans Frontières, Harare, Zimbabwe
CD4 cells/µl
1
60 40 20 0
0 (n = 51)
3 (n = 51)
5 (n = 51)
7 (n = 50)
Day Sensitivity
Specificity
PPV
NPV
Explanation of terms: The sensitivity is the proportion of stabilization tubes samples that had a CD4 result ≤350 cells/µl if the reference CD4 result was ≤350 cells/µl. The specificity is the proportion of stabilization tubes samples that had a CD4 result >350 cells/µl if the reference CD4 result was >350 cells/µl. The positive predictive value (PPV) is the proportion of reference samples that had a CD4 result ≤350 cells/µl if the stabilization tube CD4 result was ≤350 cells/µl. The negative predictive value (NPV) is the proportion of reference samples that had a CD4 result >350 cells/µl if the stabilization tube CD4 result was >350 cells/µl.
Results Conclusions Fifty-one patients participated in the study, of whom 31.4% had a CD4 result ≤350 cells/µl in the reference EDTA specimen. On Day 0, the median CD4 in the reference specimens was 513 cells/µl (interquartile range [IQR]: 279–746 cells/µl). The median difference between the CD4 in the stabilization tube specimen and the reference specimen was -13 cells/µl on Day 0, -10 cells/µl on Day 3, 0 cells/µl on Day 5, +4 cells/µl on Day 7, and +17 cells/µl on Day 14 (Figure 1). (+ denotes that the stabilization tube CD4 result was higher than the reference CD4 result, and – denotes that the stabilization tube CD4 result was lower than the reference CD4 result.) Compared to the reference CD4 result, the result of CD4 tests of stabilization tube samples measured after standing one week at room temperature showed 100% sensitivity, 100% specificity, 100% positive predictive value (PPV), and 100% negative predictive value at a CD4 threshold of 350 cells/µl (Figure 2).
Results of CD4 tests done on stabilization tube samples agreed closely with the reference CD4 result, and remained stable on samples stored in stabilization tubes at room temperature for up to 14 days. These results show that in rural clinics in Buhera District, the use of stabilization tubes is a good alternative to the use of EDTA tubes. Although patients would still need to return to the clinic to receive their CD4 results, use of stabilization tubes would save patients from having to return to the clinic for blood draws as blood samples could be collected on any day of the week.
Contacts: Helen Bygrave (helen.bygrave@joburg.msf.org) Carol Metcalf (carol.metcalf@joburg.msf.org)