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ORIGINAL ARTICLE
Inter- and intra-laboratory variability of CD4 cell counts in Swaziland
Ganizani Mlawanda, MB ChB, MSc Clinical Epidemiology, Dip HIV Man, DTM&H
School of Health Systems and Public Health, Faculty
of Health Sciences, University of Pretoria, and Royal Swaziland Sugar
Corporation Medical Services Hospitals, Mhlume, Swaziland
Paul Rheeder, MB ChB, MMed, PhD
Jacqui Miot, BPharm, PhD
School of Health Systems and Public Health, Faculty of Health Sciences, University of Pretoria
Corresponding author: G Mlawanda (mlawandag@yahoo.co.uk)
Background. Analytical
variability in CD4 enumeration is well known, but few studies from
southern Africa have quantified the inter- and intra-laboratory
variability in CD4 count measurements. In addition, the possible impact
of time lapse after sample collection on CD4 reliability is not well
understood.
Methods.
A cross-sectional study was conducted at Royal Swaziland Sugar
Corporation Hospital and three laboratories, Lab A (comparator), Lab B
(national reference) and Lab C (rural hospital). Blood from
HIV-infected individuals was collected using routine venepuncture into
separate specimens for each of the three laboratories. The samples were
further subdivided at each laboratory: one was run at 12 hours and the
second at 24 hours after venepuncture. The results of absolute CD4
count and CD4 percentage testing were compared within
(intra-laboratory) and between (inter-laboratory) laboratories.
Results. Among
53 participants, the mean CD4 count at 12 hours was 373 cells/µl, 396
cells/µl and 439 cells/µl, and at 24 hours 359 cells/µl, 389 cells/ µl
and 431 cells/µl, for laboratories A, B and C, respectively. The
coefficient of intra-laboratory variation was 4%, 8% and 20% for CD4
count for laboratories A, B and C, respectively. Comparing 12- and
24-hour measurements, the mean difference (bias) within the
laboratories between the two time points (and limits of agreement,
LOAs) was 14 (-46 to 73), 8 (-161 to 177) and 7 (20 to 33) cells/µl for
labs A, B and C, respectively. Comparing Lab A versus Lab B, lab A
versus Lab C and Lab B versus Lab C, the inter-laboratory bias for the
CD4 count at 12 hours was -32, -64 and -38 cells/µl, respectively. The
corresponding LOAs were -213 to 150, -183 to 55, and -300 to 224,
respectively. At 24 hours, the biases and LOAs were similar to those at
12 hours.
Conclusions.
CD4 counts appeared reliable at all three laboratories. Lab B and Lab C
were clinically interchangeable with the comparator laboratory, Lab A,
but not between themselves. Time to measurement does not affect the
inter-laboratory agreement within 12 and 24 hours.
S Afr J HIV Med 2012;13(2):59-63.
Thirty-five million people are infected by HIV globally, two-thirds of whom live in sub-Saharan Africa.1
Antiretroviral therapy (ART) is a critical intervention for reducing
HIV-related morbidity and mortality, but delivery of ART requires
multiple laboratory investigations.2
In particular, determination of eligibility for ART initiation relies
heavily on CD4 enumeration, and CD4 results are monitored as the major
indicator of response to treatment over time.
The gold standard technique for CD4 enumeration is flow cytometry.3
,
4 Biological
and analytical (laboratory) variations are known to affect CD4
enumeration; biological factors can that influence CD4 results include
haemodilution in pregnancy, seasonal and diurnal variations (lowest at
approximately 12:30 pm, highest at 8:30 pm), surgery, viral infections,
tuberculosis, some intercurrent illnesses, corticosteroids, interferon
and cancer chemotherapy.3
Laboratory variations are known to occur when enumeration techniques different from the gold standard, flow cytometry, are used.3
,
4 In addition, variations are known to be subject to inter-observer differences as well as inter-laboratory differences.5
The time to performing CD4 may also cause variation in final CD4 count;
the World Health Organization (WHO) therefore recommends that all CD4
counts be done within 72 hours from the time of blood collection.3
,
4
In Swaziland and many other parts of southern Africa, blood for CD4
testing is collected from various health centres and then sent to
central laboratories where analysis is done. The time of arrival of
samples differs greatly according to distance from the laboratory, but
the impact of time differences on CD4 results is not well understood.
Clinicians rely on accurate CD4 values, despite this
variability, to make decisions regarding ART initiation and management.
Some previous studies of CD4 variability have produced worrying
results. Sax and Boswell analysed the implication of between-laboratory
variations and found that 58% of CD4 count results had enough variation
to have led to conflicting treatment recommendations.6
Pattanapanyasat and Chimma found CD4 variation between CD4 cell count
results conducted using flow cytometers of different ages in service.7
Various new CD4 enumeration techniques, for example the Guava Easy CD4
and capillary-based CD4, have been compared with gold-standard
techniques and found to be comparable.3
,
4
,
8
Ensuring accurate CD4 counts has become more important
recently, since ART is being initiated at higher CD4 counts, when
clinical signs tend to be less sensitive in detecting immune
suppression.2
In Swaziland, there has been widespread suspicion among HIV clinicians
regarding discrepancies in CD4 count results within and between
laboratories, and concern that these discrepancies may potentially be
large enough to affect decisions to start ART. In order to address this
problem, this study sought to evaluate the intra- and inter-laboratory
variability in CD4 cell enumeration.
Methods
This study was undertaken at HIV clinics at
the Royal Swaziland Sugar Corporation Hospital in Swaziland and three
laboratories, Lab A, Lab B and Lab C (identity of the laboratories
deliberately not disclosed). Lab A was a reputable, internationally
accredited South African laboratory commonly used as standard in
clinical practice across southern Africa. Lab B was the Swazi national
reference laboratory based in the capital city, 250 km away from the
study setting, and had a turnover of 4 000 CD4 enumerations per month.
Lab C was a rural mission hospital laboratory located 80 km from the
study site and had a turnover of 1 700 samples per month. All the three
laboratories used a flow cytometric CD4 enumeration method, and trained
laboratory technicians performed the CD4 tests.
To be eligible, patients had to be adults (>18 years), give
informed consent to the study, and be visiting the health facility for
routine CD4 count. The study included patients regardless of whether
they were on ART or not. After participants’ consent had been
obtained, blood was collected into EDTA tubes, using routine
venepuncture technique, in three aliquots, one each for Lab A, Lab C
and Lab B. The samples were further split into two aliquots at each
respective laboratory, one of which was run at 12 hours and the second
at 24 hours after venepuncture. A reliable transport vehicle ensured
that specimens reached all laboratories within stipulated time.
A sample size of 53 was used. For this type of study, Altman and
Bland recommend a sample size of 30 as ‘minimum acceptable’
and 50 as ‘good’ as it gives a 95% confidence interval (CI)
about ±0.34 s, where s is the standard deviation (SD) of the differences between measurements by the two methods.9
Data were analysed using STATA version 10. For
intra-laboratory variability, the coefficient of variation (CV) and
Bland-Altman (BA) method were used. The BA method was the predominant
technique for inter-laboratory variability. Bland-Altman plots were
generated in Excel Analyze-it. In both cases, for repeatability and
agreement, comparison was based on clinically significant reference
ranges used previously in most studies: 0 - 10% for CV, ±250
cells/µl for CD4 count and 19.5% for CD4 percentage.7
,
8
,
10
,
11 Clinical
impact on antiretroviral therapy (ART) initiation was assessed by Kappa
coefficients with comparison to the standard reference scales.12
Results
Fifty-three participants consented to participate in the study. The mean CD4 count was 373 cells/µl, 396 cells/µl and
439 cells/µl at 12 hours, and 359 cells/µl, 389
cells/µl and 431 cells/µl at 24 hours, for Lab A, Lab B and
Lab C, respectively. Subsequent Wilcoxon sign-rank test revealed some
statistically significant differences in CD4 count between the
laboratories. Table 1 summarises the demographic, clinical and
laboratory characteristics of participants.
Intra-laboratory variability.
The CV for CD4 count for Lab B was low (3.4%) compared with Lab A
(8.5%). This was consistent with intra-laboratory repeatability based
on clinically significant CV range of 0 - 10%. For Lab C the CV was 20.1%, a finding consistent with poor repeatability. For
all three laboratories, the CV of CD4 percentage was even lower: 5.6%,
8.34% and 7.5% for Lab A, Lab B and Lab C, respectively. The results
using the BA method showed that both CD4 count and CD4 percentage were
repeatable, when compared with clinically significant ranges
±250 cells/µl and
±19.5%, for all the laboratories: for CD4 count, the limits of
agreement were -46 cells/ µl to 73 cells/µl for Lab A, -20
cells/µl to 33
cells/µl for Lab B, and -161 cells/µl to 177 cells/µl
for Lab C, as per Fig. 1 and Table 2. The BA plots for Lab A, Lab B and
Lab C had no dispersion suggesting evidence of systematic error.
Inter-laboratory agreement at 12 hours.
For CD4 count, at 12 hours, both Lab C and Lab B could be clinically
interchanged with the comparator, Lab A, based on the limits of
agreement which fell within the clinically significant range (defined
as ±250 cells/µl): -184 cells/µl to 55
cells/µl for Lab C, and -213 cells/µl to 150 cells/µl
for Lab B, which was much wider than for Lab C. When Lab B was compared
for agreement with Lab C, the limits of agreement were -300
cells/µl to 224 cells/µl, which were out of the clinically
significant range, and we therefore concluded that the two laboratories
could not be clinically interchanged. For CD4 percentage all the
laboratories could be clinically interchanged. Compared with the
comparator, Lab A, the limits of agreement for Lab B were -12
cells/µl to 9 cells/µl and -3 cells/µl to 2
cells/µl for Lab C; between Lab B and Lab C the limits were -11
cells/µl to 12 cells/µl. Table 3 summarises the results for
inter-laboratory variability based on BA results at 12 hours and at 24
hours.
Inter-laboratory agreement at 24 hours.
Time to measurement had no significant impact on inter-laboratory
agreement based on the limits of agreement and biases at 24 hours were
similar to those at 12 hours for both CD4 count and CD4 percentage.
When compared with Lab A, the limits of agreement at 24 hours were -205
cells/µl to 135 cells/µl for Lab B and -195 cells/µl
to 66 cells/µl for Lab C. For Lab B/Lab C the limits of agreement
were -265 cells/µl to 191 cells/µl. For CD4 percentage, all
the laboratories were clinically interchangeable. The limits of
agreement were -11% to 9% for Lab A/Lab B, -5% to 3% for Lab A/Lab C
and -10% to 10% for Lab B/Lab C, which were within the reference range,
±19.5%.
Clinical impact on ART initiation. Compared
with Lab A, the percentage agreement for ART eligibility was 81% (i.e.
19% of patients were misclassified) for Lab B and 89% (11% of patients
misclassified) for Lab C. For Lab A/Lab B, 23% eligible patients would
be misclassified and not initiated on ART, as shown in Table 4.
Discussion
In this study we looked at intra- and
inter-laboratory variability, a topic that has been investigated
previously but for which there are few data from southern Africa.5
,
11
,
13
We also analysed the impact of time to measurement on the eventual CD4
result, both within the same laboratory and across participating
laboratories. CD4 count had good repeatability for all the three
laboratories, based on preset clinically significant ranges. Likewise,
CD4 percentage had minimal variation for all the laboratories and even
lower CV, a sign of stronger repeatability than for CD4 count. These
findings concurred with previous intra-laboratory studies.7
,
8
,
10
,
11
Inter-laboratory variability.
Several studies on inter-laboratory and inter-method variability of CD4 count have been published and most show good agreement and interchangeability.7
,
10
,
11 Two studies, however, found significant variations across different laboratories.5
,
13
In this study, inter-laboratory clinical interchangeability results at
12 and 24 hours showed that agreement was independent of time to
measurement. The limits of agreement were similar when time to
measurement was 12 hours or 24 hours. This finding mirrors the WHO
laboratory recommendation that CD4 remains stable within 72 hours from
time of venepuncture.3
,
4 Clinicians using the laboratories in this study should therefore trust equally CD4 results done at 12 hours and 24 hours.
For CD4 percentage, both Lab B and Lab C were in agreement with the
comparator laboratory, Lab A, at 12 and 24 hours with narrower limits
of agreement than for CD4 count. Once again, stability of CD4
percentage and agreement with the comparator laboratory make it a
potentially trustworthy and stable parameter to use in our setting for
possible inclusion in guidelines to determine when to start ART, as
suggested in some previous studies.8
,
10
The degrees of misclassification
in this study were similar to findings from a study by Thakar and
Kumar, which found a kappa factor range of 74% for a CD4 count below
350 cells/µl when two laboratories were being compared.11
Repeating CD4 count measurement and not relying on single CD4 count
results have been known to reduce disease misclassification.6 One shortfall of this use of misclassification as done here is that it does not differentiate between low‐magnitude
inaccuracy, for example a count of 349 cells/µl being
misclassified as >350 cells/µl, which may be reasonably
expected from any test, and high‐magnitude inaccuracy. A study that includes many CD4 values falling close to the defined cut‐off
(as measured by the reference test) will show higher rates of
misclassification by the new test than a study in which the majority of
values lie away from the threshold.4
The clinically significant ranges used in this study were
±250 cells/µl, ±19.5% and CV <10%, because these
were the ranges used in similar studies which had pre-defined ranges.7
,
8
,
10
,
11 The
results of repeatability and agreement therefore relied on this
pre-defined range. However, the choice of clinically significant ranges
is debatable, and a narrower range of ±100 cells/µl could
have changed the interpretation of these results greatly. However, the
magnitude of CD4 count or CD4 percentage variability that can affect
clinical decision making remains poorly defined.13 The author felt that based on the new ART initiation threshold, 350 cells/µl, a range of ±250 cells is reasonable.
In conclusion, CD4 count and CD4 percentage appeared to be
repeatable for all the three laboratories. Lab B and Lab C were
clinically interchangeable with the comparator laboratory, Lab A, for
both CD4 count and CD4 percentage but not between themselves. Time to
measurement does not affect the inter-laboratory agreement within 12
and 24 hours. The clinical implications of inter-laboratory variation
on disease misclassification were comparable to those from previous
studies.
Sources of support and disclosure of funding. A
University of Pretoria RESCOM grant partially covered the laboratory costs during the study.
Competing interests by authors. None.
Research ethics committee approval. Ethics
approval was given by the University of Pretoria Ethics Committee
74/2010 on 21 April 2010, and institutional ethical approval was also
obtained.
Author contributions. Dr
Ganizani Mlawanda conceived the study, and formulated the study design,
data collection, statistical analysis and manuscript design. Prof. Paul
Rheeder and Dr Jacqui Miot were active supervisors throughout from
conception to final manuscript. All authors read and approved the final
manuscript.
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Table 1. Demographic, clinical and laboratory data of participants
Demographic characteristics
Gender (n)
Male
28
Female
25
Age (years) (mean (SD))
37.4 (9.5)
Weight (kg) (mean (SD))
64.8 (12.2)
Clinical characteristics
WHO stage (%)
I
32.1
II
22.6
III
13.2
IV
32.1
On TB treatment (%)
11.3
On ART (%)
47.2
Inpatients (%)
9.4
Outpatients (%)
90.6
Laboratory parameters
Lab A (52 observations)
Mean
25th centile
50th centile
75th centile
CD4 count at 12 h (cells/µl)
373
181
336
539
CD4 count at 24 h (cells/µl)
359
177
323
518
CD4 % at 12 h
17
10
15
22
CD4 % at 24 h
17
10
15
21
Lab B (52 observations)
Mean
25th centile
50th centile
75th centile
CD4 count at 12 h (cells/µl)
396
185
359
568
CD4 count at 24 h (cells/µl)
389
183
346
535
CD4 % at 12 h
18
11
17
24
CD4 % at 24 h
18
10
17
23
Lab C (51 observations)
Mean
25th centile
50th centile
75th centile
CD4 count at 12 h (cells/µl)
439
249
397
611
CD4 count at 24 h (cells/µl)
431
233
396
594
CD4 % at 12 h
18
10
16
22
CD4 % at 24 h
18
10
16
22
Mean time to running CD4 tests (h)
First CD4
Second CD4
Lab A
12.0
24.0
Lab B
12.0
24.0
Lab C
12.0
25.1
Table 2. Intra-laboratory bias and limits of agreement for CD4 count and CD4 percentage at 12 and 24 hours
Limits of agreement
Interpretation
Bias (95% CI)
Lower (95% CI)
Upper (95% CI)
Clinically repeatable?
Absolute CD4 count
Lab A
13.5 (5.0 to 21.9)
-46.0 (-60.6 to -31.5)
73.0 (58.5 to 87.6)
Yes
Lab C
8.2 (-16.0 to 32.4)
-160.5 (-202.2 to -118.9)
176.9 (135.3 to 218.6)
Yes
Lab B
7.0 (3.2 to 10.7)
-19.5 (-25.9 to -13.0)
33.4 (26.9 to 39.9)
Yes
CD4 %
Lab A
0.1 (-0.2 to 0.3)
-1.7 (-2.2 to -1.3)
1.9 (1.5 to 2.4)
Yes
Lab C
-0.3 (-0.7 to 0.1)
-2.9 (-3.5 to -2.2)
2.3 (1.7 to 3.0)
Yes
Lab B
0.1 (-0.3 to 0.5)
-2.8 (-3.5 to -2.1)
3.0 (2.3 to 3.7)
Yes
*Interpretation based on
comparison of limits of agreement with clinically significant range of
CV <10%, and ranges for clinical significance: ±19.5% for
CD4% ±250 cells/µl for CD4 count.7
,
8
,
10
,
11
Table 3. Inter-laboratory bias and limits of agreement for CD4 count and CD4 percentage at 12 and 24 hours
Laboratories
Limits of agreement
Interpretation*
Bias (95% CI)
Lower (95% CI)
Upper (95% CI)
Clinically interchangeable?
CD4 count at 12 h
Lab A/Lab B
-31.5 (-57.6 to -5.5)
-213.3 (-258.2 to -168.4)
150.2(105.3 to 195.1)
Yes
Lab A/Lab C
-64.3 (-81.6 to -47.0)
-183.8 (-213.6 to -154.0)
55.2 (25.4 to 85.0)
Yes
Lab B/Lab C
-38.2 (-75.6 to -0.6)
-300.2 (-364.8 to -235.5)
223.9 (159.2 to 288.5)
No
CD4 % at 12 h
Lab A/Lab B
-1.2 (-2.7 to 0.3)
-11.7 (-14.3 to -9.1)
9.3 (6.7 to 11.9)
Yes
Lab A/Lab C
-0.7 (-1.1 to -0.4)
-3.1 (-3.7 to -2.5)
1.7 (1.1 to 2.2)
Yes
Lab B/Lab C
0.5 (-1.1 to 2.1)
-10.7 (-13.4 to -7.9)
11.6 (8.9 to 14.4))
Yes
CD4 count at 24 h
Lab A/Lab B
-35.6 (-60.0 to -11.1)
-205.7 (-247.6 to -163.7)
134.5 (92.5 to 176.5)
Yes
Lab A/Lab C
8.2 (-16.0 to 32.4)
-195.0 (-227.6 to -162.5)
65.8 (33.3 to 98.3)
Yes
Lab B/Lab C
7.0 (3.2 to 10.7)
-265.0 (-321.3 to -208.7)
191.4 (135.0 to 247.7)
No
CD4 % at 24 h
Lab A/Lab B
-1.2 (-2.5 to 0.2)
-10.5 (-12.8 to -8.2)
9.2 (5.7 to 10.5)
Yes
Lab A/Lab C
-1.1 (-1.6 to -0.5)
-4.9 (-5.8 to -3.9)
2.7 (1.8 to 3.6)
Yes
Lab B/Lab C
0.1 (-1.3 to 1.5)
-9.7 (-12.1 to -7.3)
9.9 (7.4 to 12.3)
Yes
*Interpretation based on
comparison of limits of agreement with clinically significant range of
CV <10%, and ranges for clinical significance: ±19.5% for CD4
% and ±250 cells/µl for CD4 count.7
,
8
,
10
,
11
Table 4. Impact of CD4 variations at ART initiation threshold on treatment decision
Laboratories
Agreement* (%)
Expected agreement (%)
Kappa
Misclassified (%)
Lab A/Lab B
81.1
48.4
0.6
18.9
Lab A/Lab C
88.7
49.9
0.8
11.3
Lab B/Lab C
77.4
50.2
0.6
22.6
*Strength of agreement
according to Byrt’s criteria for assessing Kappa strength:
excellent agreement = 0.93 to 1; very good agreement = 0.81 to 0.92;
good agreement = 0.61 to 0.80; fair agreement = 0.41 to 0.60; slight
agreement = 0.21 to 0.40; poor agreement = 0.01 to 0.20; no agreement
<0.00.12
Lab A
Lab B
Lab C
Fig. 1. Bland Altman plots for intra-laboratory variability of CD4 count for Lab A, Lab B and Lab C.