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Research Letters

Finger-prick blood samples can be used interchangeably with venous samples for CD4 cell counting indicating their potential for use in CD4 rapid tests

MacLennan, Calman Aa,b,c; van Oosterhout, Joep JGd; White, Sarah Aa,e; Drayson, Mark Tb; Zijlstra, Eduard Ed; Molyneux, Malcolm Ea,f

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doi: 10.1097/QAD.0b013e32823bcb03
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Abstract

There is currently a rapid scaling up of antiretroviral therapy (ART) in resource-poor settings, especially in Africa. World Health Organization (WHO) clinical staging is usually employed to assess whether an individual should start ART in such settings [1], but clinical staging does not always correspond to the degree of immunosuppression [2], and patient management could be improved by increased availability of CD4 lymphocyte counts (CD4 cell counts) [3]. The main reasons why CD4 cell counts are not widely used are the high cost of flow cytometry, the ‘gold standard’ method of CD4 cell counting, and a lack of skilled laboratory staff [4,5]. To overcome these problems, a number of simplified non-flow cytometric CD4 cell counting methods have been developed [6–9]. There have been problems with the accuracy of such methods [10], but they have the potential to make affordable CD4 cell counts available for use by staff with minimal training away from major medical centres.

Rapid tests for CD4 cell counts are currently under development [11] and could increase the availability of CD4 cell counting. Such tests could be performed on finger-prick blood samples, enabling them to be conducted where trained phlebotomists are not available or when difficulties are encountered with venous sampling. Finger-prick blood samples are commonly used in resource-poor settings for rapid HIV tests and malaria parasite slides, and so may be more acceptable than venesection.

Studies comparing CD4 cell counts in finger-prick and venous blood are currently lacking. Previous studies of full blood counts in healthy adults using haematological analysers have found mean leukocyte counts that are 7.82% [12] and 8.34% [13] higher in finger-prick blood samples compared with venous blood. The differences were greatest among the large leukocyte subpopulations (principally neutrophils) and were least among small leukocytes (principally lymphocytes).

We therefore conducted a study in HIV-infected adults to estimate the agreement between results obtained using finger-prick and venous blood for absolute CD4 cell counts and CD4 cell counts as a percentage of the total lymphocyte count (%CD4/lymphocyte). The study was conducted in the ART clinic, Queen Elizabeth Central Hospital, Blantyre, Malawi. After informed consent was obtained, simultaneous venous and finger-prick blood samples were taken by three study nurses, experienced in finger-prick and venous blood sampling, from 111 consecutive HIV-infected Malawian adults during September and October 2006. There were no exclusion criteria.

Blood samples were taken simultaneously from the pad of the middle finger and an antecubital vein of the opposite arm. Finger-prick samples were taken using BD Genie lancets (2.0 mm × 1.5 mm; Becton Dickinson, San Jose, California, USA) according to the manufacturer's recommended procedure [14]. This produced a free flow of blood with minimal squeezing of the finger. Venous sampling was performed using a 21G needle and syringe. Finger-prick and venous samples were collected in separate BD Microtainer ethylenediamine tetraacetic acid capillary blood tubes (250 μl per tube). The first drop of finger-prick blood was discarded. One set of samples was rejected, because the finger-prick sample clotted.

Paired blood samples were tested in parallel for CD4 cell counts within 6 h of collection using Multitest CD3/8/45/4 kits with TruCount tubes (TruCount; BD Biosciences, San Jose, California, USA) and full blood counts on a HMX haematological analyser (Beckman Coulter, Fullerton, California, USA). Full blood counts were not obtained on two samples because of insufficient blood. No other data are missing. TruCount is an established flow cytometric CD4 cell counting assay that generates both an absolute CD4 cell count, %CD4/lymphocyte and total lymphocyte count and has excellent repeatability and quality assurance scores [15]. Data were acquired on a FACSCalibur flow cytometer (Becton Dickinson) and analysed using MultiSet software, blinded to the paired result for each sample. The main outcome measure was agreement between finger-prick and venous samples assessed by estimating bias and limits of agreement (bias ±1.96 SD) with 95% confidence intervals (CI) as described by Bland and Altman [16].

The median age of the 110 participants with analysable samples was 33 years (range 20–64). Sixty-nine per cent were women, 5% were pregnant, 26% were taking ART, 14% were on treatment for current bacterial, fungal or tuberculous infection and 15% were febrile (axillary temperature > 37.5°C). At the time of sampling or when started on ART, 21% of participants had WHO clinical stage I HIV/AIDS, 31% stage II, 30% stage III and 18% stage IV. No participants complained about either blood sampling procedure and no adverse events were reported. The median CD4 cell counts on venous blood samples were 277 cells/μl (range 2–1121) for absolute counts and 13.28% (0.16–38.31%) for %CD4/lymphocyte.

Agreement between paired CD4 cell counts was good, with little bias and narrow limits of agreement (Fig. 1). Finger-prick CD4 cell values were higher than venous CD4 cell counts by an average of 6.6 cells/μl (95% CI 1.0, 12.0), with limits of agreement −50.7 cells/μl (95% CI −60.3, −41.2) and 63.7 cells/μl (95% CI 54.2, 73.3). Total lymphocyte counts (by TruCount) were higher in finger-prick blood with a bias of 183 cells/μl, resulting in higher venous compared with finger-prick %CD4/lymphocyte values with a bias of 0.71% (95% CI 0.44, 0.97) and limits of agreement −2.07% (95% CI −2.53, −1.61) and 3.48% (95% CI 3.02, 3.95). Total leukocyte counts were also higher in finger-prick blood compared with venous blood, with a mean difference of 949 cells/μl.

Fig. 1
Fig. 1:
Comparison of CD4 cell counts determined for finger-prick blood and venous blood samples from 110 HIV-infected patients. Solid horizontal lines depict bias and upper and lower limits of agreement. Dashed lines denote 95% confidence intervals for these values. Mean counts are the average of the finger-prick and venous CD4 cell counts for each individual. (a) Absolute CD4 cell counts; (b) CD4 cell count as percentage of the total lymphocyte count.

When expressed as a percentage of the mean value for each cell type count, the biases for the CD4 lymphocyte, total lymphocyte and total leukocyte counts are 2.2, 8.2 and 15.2%, respectively. As previously observed [12,13], these figures suggest that the difference between leukocyte counts in venous and finger-prick blood samples is largely caused by large leukocytes rather than lymphocytes.

The study was conducted under optimum blood sampling conditions. The use of lancets with smaller blades might necessitate excessive finger squeezing to produce a blood sample, leading to dilution of the sample with tissue fluid. Rapid diagnostic tests are, however, likely to require less than the 250 μl blood collected from each finger prick in this study so this may not be a major issue.

These data indicate that provided a careful sampling technique is followed, finger-prick blood samples could be used in place of venous blood samples in HIV-infected African adults for both absolute CD4 cell counts and %CD4/lymphocyte values. This potentially increases the accessibility for CD4 cell counting in resource-poor settings, especially once rapid tests become widely available.

Acknowledgements

The study was approved by the College of Medicine Research and Ethics Committee, University of Malawi. The authors would like to thank nurse Emily Lifa for blood sampling, and the patients and staff at the antiretroviral clinic, Queen Elizabeth Central Hospital, Blantyre, for their assistance with this study.

Sponsorship: This work was supported by a grant from the CD4 Initiative, Imperial College, London, to C.A.M. C.A.M. holds a research fellowship and M.E.M. a programme grant from the Wellcome Trust.

References

1. World Health Organization. Interim WHO clinical staging of HIV/AIDS and HIV/AIDS case definitions for surveillance. African region. Geneva: World Health Organization; 2005. Available at: http://www.who.int/hiv/pub/guidelines/clinicalstaging.pdf. Accessed: 13 September 2006.
2. Kassa E, Rinke de Wit TF, Hailu E, Girma M, Messele T, Mariam HG, et al. Evaluation of the World Health Organization staging system for HIV infection and disease in Ethiopia: association between clinical stages and laboratory markers. AIDS 1999; 13:381–389.
3. World Health Organization. CD4+ T-cell enumeration technologies. Technical information. Geneva: World Health Organization; 2005. Available at: http://www.who.int/diagnostics_laboratory/CD4_Technical_Advice_ENG.pdf. Accessed: 13 September 2006.
4. World Health Organization. Sources and prices of selected medicines and diagnostics for people living with HIV/AIDS. A joint UNICEF,UNAIDS, WHO, MSF project, 6th edition. Geneva: World Health Organization; 2005. Available at: http://www.who.int/HIV/amds/sourcesAug06.pdf. Accessed: 21 May 2006.
5. Harries AD, Schouten EJ, Libamba E. Scaling up antiretroviral treatment in resource-poor settings. Lancet 2006; 367:1870–1872.
6. Loua A, Kestens L, Vanham G, Boel L, Colebunders R, Gigase P, et al. Validity of an ELISA test for CD4+ T lymphocyte count and validity of total lymphocyte count in the assessment of immunodeficiency status in HIV infection. Ann Soc Belg Med Trop 1994; 74:61–68.
7. Mwaba P, Cassol S, Pilon R, Chintu C, Janes M, Nunn A, Zumla A. Use of dried whole blood spots to measure CD4+ lymphocyte counts in HIV-1-infected patients. Lancet 2003; 362:1459–1460.
8. Nouanthong P, Pata S, Sirisanthana T, Kasinrerk W. A simple manual rosetting method for absolute CD4+ lymphocyte counting in resource-limited countries. Clin Vacc Immunol 2006; 13:598–601.
9. Diagbouga S, Chazallon C, Kazatchkine MD, Van de Perre P, Inwoley A, M'Boup S, et al. Successful implementation of a low-cost method for enumerating CD4+ T lymphocytes in resource-limited settings: the ANRS 12-26 study. AIDS 2003; 17:2201–2208.
10. Janossy G. Dried blood spot technology for CD4+ T-cell counting. Lancet 2004; 363:1074.
11. CD4 Initiative– Bill and Melinda Gates Foundation. Imperial College, London. Available at: www.imperial.ac.uk/medicine/cd4. Accessed: 27 February 2007.
12. Daae LNW, Halvorsen S, Mathisen PM, Mironska K. A comparison between haematological parameters in ‘capillary’ and venous blood from healthy adults. Scand J Clin Lab Invest 1988; 48:723–726.
13. Yang Z-W, Yang S-H, Chen L, Qu J, Zhu J, Tang Z. Comparison of blood counts in venous, finger-prick and arterial blood and their measurement variation. Clin Lab Haem 2001; 23:155–159.
14. Becton Dickinson. Successful specimen collection: fingersticks. San Jose, CA, USA: Becton Dickinson; 2003. Available at: http://www.bd.com/vacutainer/pdfs/genie_lancet_specimencollection_fingersticks_VS5755-1.pdf. Accessed: 27 April 2006
15. Schnizlein-Bick CT, Spritzler J, Wilkening CL, Nicholson JKA, O'Gorman MRG. Evaluation of TruCount absolute-count tubes for determining CD4 and CD4 numbers in human immunodeficiency virus-positive adults. Clin Diagn Lab Immunol 2000; 7:336–343.
16. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986; i:307–331.
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