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CORRESPONDENCE •CID 2010:51 (1 November) •1107
Table 1. Mean Coefficients of Variation (CVs) for T Cell Subset Parameters Measured at Baseline and
Daily for Next 3 Days
Parameter
Mean CV (range), % Mean CV
over 3 days, %After 1 day After 2 days After 3 days
Absolute cell count
CD4 5.90 (0.18–14.27) 4.25 (0.00–15.01) 5.06 (0.28–12.13) 5.07
CD8 6.58 (0.10–16.31) 5.37 (0.90–12.30) 5.10 (0.20–20.10) 5.68
CD3 5.87 (0.27–16.09) 4.37 (0.20–12.37) 4.13 (0.13–12.28) 4.79
Percentage of lymphocytes
CD4 3.92 (0.04–8.54) 2.51 (0.05–7.47) 3.57 (0.35–10.14) 3.33
CD8 3.15 (0.19–6.91) 2.30 (0.07–6.97) 3.81 (0.38–12.68) 3.09
CD3 1.88 (0.10–6.01) 1.33 (0.15–3.66) 2.03 (0.06–5.72) 1.75
Total lymphocyte count 6.97 (0.35–15.57) 4.18 (0.05–13.13) 4.09 (0.10–10.51) 5.08
CD4: CD8 ratio 4.70 (0.00–12.45) 3.30 (0.00–11.59) 5.41 (0.00–13.69) 4.47
NOTE. For blood samples of 19 individuals infected with human immunodeficiency virus, individual CVs for a particular
day were calculated from the baseline value and the value determined on that day for each blood sample.
compared with atazanavir-ritonavir (IIP,
4.91 [2]), in the ongoing randomized
comparison of regimens including these
drugs (AIDS Clinical Trials Group pro-
tocol A5257, clinicaltrials.gov identifier
NCT00811954).
Acknowledgments
Potential conflicts of interest. T.J.H. is the re-
cipient of a Bristol-Myers Squibb VirologyFellows
grant. H.R. has served on the data and safety mon-
itoring board for Koronis Pharmaceuticals and
has received honoraria from Roche Diagnostics.
D.R.K. is a consultant to and/or has received re-
search support or honoraria from Abbott, Boeh-
ringer-Ingelheim, Bristol-Myers Squibb, Gilead,
GlaxoSmithKline, Merck, Pfizer, Roche, and ViiV.
Timothy J. Henrich,
1,2
Heather Ribaudo,
3
and Daniel R. Kuritzkes
1,2
1
Division of Infectious Diseases, Brigham
and Women’s Hospital,
2
Harvard Medical School,
and
3
Center for Biostatistics and AIDS Research,
Harvard School of Public Health,
Boston, Massachusetts
References
1. Henrich T, Ribaudo H, Kuritzkes DR. Instan-
taneous inhibitory potential is similar to in-
hibitory quotient at predicting HIV-1 response
to antiretroviral therapy. Clin Infect Dis 2010;
51:93–98.
2. Shen L, Siliciano RF. Achieving a quantitative
understanding of antiretroviral drug efficacy
[letter]. Clin Infect Dis 2010; 51(9):1105–1106
(in this issue).
3. van Leth F, Phanuphak P, Ruxrungtham K, et
al. Comparison of firstline antiretroviral ther-
apy with regimens including nevirapine, efa-
virenz, or both drugs, plus stavudine and la-
mivudine: a randomised open-label trial, the
2NN Study. Lancet 2004;363:1253–1263.
4. Squires K, Lazzarin A, Gatell JM, et al. Com-
parison of once-daily atazanavir with efavirenz,
each in combination with fixed-dose zidovu-
dine and lamivudine, as initial therapy for pa-
tients infected with HIV. J Acquir Immune
Defic Syndr 2004; 36:1011–1019.
5. Ortiz R, Dejesus E, Khanlou H, et al. Efficacy
and safety of once-daily darunavir/ritonavir ver-
sus lopinavir/ritonavir in treatment-naive HIV-
1–infected patients at week 48. AIDS 2008;22:
1389–1397.
6. Eron J Jr, Yeni P, Gathe J Jr, et al. The KLEAN
study of fosamprenavir-ritonavir versus lopi-
navir-ritonavir, each in combination with abac-
avir-lamivudine, for initial treatment of HIV
infection over 48 weeks: a randomised non-
inferiority trial. Lancet 2006; 368:476–482.
7. Walmsley S, Avihingsanon A, Slim J, et al. Gem-
ini: a noninferiority study of saquinavir/rito-
navir versus lopinavir/ritonavir as initial HIV-
1 therapy in adults. J Acquir Immune Defic
Syndr 2009; 50:367–374.
Reprints or correspondence: Dr Daniel R. Kuritzkes, Section
of Retroviral Therapeutics, Brigham and Women’s Hospi-
tal, 65 Landsdowne St, Cambridge, MA 02139 (dkuritzkes@
partners.org).
Clinical Infectious Diseases 2010;51(9):1106–1107
2010 by the Infectious Diseases Society of America. All
rights reserved. 1058-4838/2010/5109-0021$15.00
DOI: 10.1086/656689
T Cell Subset Enumeration
and Weekend HIV Clinics:
Reliable Performance of CD4
Cell Counts after 3 Days
To the Editor—The expansion and im-
provement of services for people infect-
ed with human immunodeficiency virus
(HIV) has led to the introduction of week-
end HIV clinics in many hospitals. In
places where laboratory facilities operate
on weekdays only, blood samples may
wait 2 to 3 days before T cell subset enu-
meration is performed. Guidelines from
the British Committee for Standardisa-
tion in Haematology, published in 1997,
stipulate that CD4 cell counting should
be performed within 18 hours after ven-
esection [1], whereas those from the Cen-
ters for Disease Control and prevention,
published in 2003, recommend testing
within 48 hours [2]. In view of these dif-
fering guidelines, we performed sequen-
tial daily T cell subset enumeration by
flow cytometry for 4 days on a set of
consecutive blood samples received for
CD4 cell counting to determine whether
the samples could provide reliable results
over this time.
Nineteen edetic acid–anticoagulated
blood samples from HIV-infected indi-
viduals were received for CD4 cell count-
ing on the appointed day (the laboratory
is registered with United Kingdom Na-
tional External Quality Assessment Ser-
vice [3]). We used Multitest CD3/8/45/4
kits with TruCount tubes (Becton Dick-
inson) in accordance with the manufac-
turer’s instructions, a method that uses
both single-platform and CD45/side scat-
ter gating technologies [4] that improve
by guest on January 13, 2016http://cid.oxfordjournals.org/Downloaded from
1108 •CID 2010:51 (1 November) •CORRESPONDENCE
the accuracy of CD4 counting [1, 5]. T
cell enumeration was performed once on
the day of venesection (baseline mea-
surement) and once on each sequential
day for a total of 4 days by an experi-
enced operator. Samples were run on a
FACSCanto II flow cytometer (Becton
Dickinson). Blood samples were main-
tained at room temperature in the lab-
oratory and treated the same as routine
clinical samples.
The median age of the 19 subjects was
37 years (range, 23–63 years), and 11 were
male. Mean absolute CD4 cell counts were
422 cells/mL (range, 260–890 cells/mL), and
CD4 counts as a percentage of lympho-
cytes were 23% (range, 26%–35%). Each
day, from day 2 through day 4, the mean
coefficient of variation (CV, calculated
from the baseline value and the value on
that day for each sample) was !7% for all
8 parameters: CD4, CD8, and CD3 lym-
phocyte absolute and percentage counts;
total lymphocyte count; and CD4 :CD8 ra-
tio (Table 1). There was no increase with
time in mean CV from baseline measure-
ment for any parameter ( for all
P1.4
comparisons; Student ttest).
The mean CV from baseline across the
3 days was greater for CD4, CD8, and CD3
absolute lymphocyte counts (5.18%) than
for percentage counts (2.72%). The mean
CV for absolute CD4 was 5.07%, and the
mean CV for CD4 as a percentage of lym-
phocytes was 3.33%. This is no different
from the mean CV for same-day intra-
assay variability for our laboratory of
4.84% for absolute CD4 (Pp.85; Student
ttest) and 4.47% for CD4 as a percentage
of lymphocytes ( ; Student ttest).
Pp.27
The largest mean CV was for CD8 abso-
lute counts (5.68%), and the smallest
mean CV was for CD3 as a percentage of
lymphocytes (1.75%). There was no con-
sistent trend for any parameter with time.
The CVs over the 4-day time course are
consistent with previous flow cytometry
studies that used in-house protocols and
that were published by us and others from
the developed [5] and developing [6]
world. Our findings indicate that T cell
enumeration can be reliably conducted by
flow cytometry 3 days after venesection
and therefore on Mondays after weekend
clinics.
Acknowledgments
Financial support. GlaxoSmithKline (clinical
research fellowship to C.A.M.).
Potential conflicts of interest. All authors: no
conflicts.
Anna E. Seeley,
1
Peter R. Richardson,
1
Timothy Plant,
1
Kaveh Manavi,
2
Sylvie Freeman,
1
Mark T. Drayson,
1
and Calman A. MacLennan
1,2
1
Medical Research Council Centre for Immune
Regulation and Clinical Immunology Service, School
of Immunity and Infection, College of Medicine
and Dental Sciences, University of Birmingham,
and
2
HIV Services, University Hospitals Birmingham
National Health Service Foundation Trust,
Birmingham, United Kingdom
References
1. Guidelines for the enumeration of CD4+ T
lymphocytes in immunosuppressed individu-
als. CD4+ T Lymphocyte Working Party. Mem-
bers of the General Haematology Task Force of
BCSH. Clin Lab Haematol 1997; 19:231–241.
2. Mandy FF, Nicholson JK, McDougal JS.Guide-
lines for performing single-platform absolute
CD4+ T cell determinations with CD45 gating
for persons infected with human immunode-
ficiency virus. Centers for Disease Control and
Prevention. MMWR Recomm Rep 2003;52:
1–13.
3. Whitby L, Granger V, Storie I, et al. Quality
control of CD4+ T lymphocyte enumeration:
results from the last 9 years of the United King-
dom National External Quality Assessment
Scheme for Immune Monitoring (1993–2001).
Cytometry 2002; 50:102–110.
4. Schnizlein-Bick CT, Spritzler J, Wilkening CL,
et al. Evaluation of TruCount absolute-count
tubes for determining CD4 and CD8 cell num-
bers in human immunodeficiency virus–pos-
itive adults. Site Investigators and the NIAID
DAIDS New Technologies Evaluation Group.
Clin Diagn Lab Immunol 2000; 7:336–343.
5. Bergeron M, Nicholson JK, Phaneuf S, et al.
Selection of lymphocyte gating protocol has an
impact on the level of reliability of T cellsubsets
in aging specimens. Cytometry 2002; 50:53–6 1.
6. MacLennan CA, Liu MK, White SA, et al. Di-
agnostic accuracy and clinical utility of a sim-
plified low cost method of counting CD4 cells
with flow cytometry in Malawi: diagnostic ac-
curacy study. BMJ 2007; 335:190.
Reprints or correspondence: Dr Calman A. MacLennan, Med-
ical Research Council Centre for Immune Regulation, Institute
of Biomedical Research, School of Immunity and Infection,
College of Medicine and Dental Sciences, University of Bir-
mingham, Wolfson Dr, Edgbaston, Birmingham, B15 2TT,
United Kingdom (c.maclennan@bham.ac.uk).
Clinical Infectious Diseases 2010;51(9):1107–1108
2010 by the Infectious Diseases Society of America. All
rights reserved. 1058-4838/2010/5109-0022$15.00
DOI: 10.1086/656693
Final Report of the Lyme
Disease Review Panel
of the Infectious Diseases
Society of America:
A Pyrrhic Victory?
To the Editor—Truth in science is es-
tablished through open debate in an in-
dependent process. The scientific process
fails when one side of a debate sets the
rules, controls the arena, and ensures that
its viewpoint prevails. Sadly, this is what
the Infectious Diseases Society of America
(IDSA) has done in the “vindication” of
its beleaguered 2006 Lyme disease guide-
lines described in the final report of the
Lyme Disease Review Panel [1].
The Review Panel was mandated by an
antitrust settlement agreement with the
Connecticut Attorney General, who found
substantial flaws in the IDSA Lyme guide-
lines development process [2]. Yet the
guidelines review was far from indepen-
dent. It was run by the IDSA, which se-
lected the Review Panel members (7 of 8
were members of the IDSA), selected the
chair (a past president of the IDSA), chose
the speakers, and essentially acted as judge
and jury [3, 4].
An ethicist paid by the IDSA screened
panel members for financial conflicts of
interest but failed to consider classic or-
ganizational bias: given the IDSA’s stake
in vindicating its guidelines to reduce po-
tential litigation exposure, maintain its
reputation, and silence competitors, how
could IDSA members be impartial? Se-
lecting panel members with organizational
conflicts of interest while systematically
excluding the physicians who treat the ma-
jority of Lyme disease patients resulted in
a biased review panel [3].
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