WHO/BS/2018.2333
ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION
Geneva, 29 October to 2 November 2018
An International Collaborative Study to Establish a
WHO International Reference Reagent for CD4 T cell counting
Luisa de Jesus Saraiva
1
, Janet Sutherland
1
, Richard Stebbings
1
, Jason Hockley
2
, Peter Rigby
2
and
Sandrine Vessillier
1
1
Biotherapeutics Division and
2
Biostatistics Group,
National Institute for Biological Standards and Control,
Potters Bar, Hertfordshire, EN6 3QG, UK.
NOTE:
This document has been prepared for the purpose of inviting comments and suggestions on the
proposals contained therein, which will then be considered by the Expert Committee on
Biological Standardization (ECBS). Comments MUST be received by 8 October 2018 and
should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention:
Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to
the Responsible Officer: Dr Ivana Knezevic at email: [email protected].
© World Health Organization 2018
All rights reserved.
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Dr Ivana Knezevic, Technologies Standards and Norms, Department of Essential Medicines and Health Products, World Health
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WHO/BS/2018.2333
Page 2
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Summary
The World Health Organization (WHO) has recognised the lack of harmonisation in CD4 T cell
counting as an issue for HIV patient care. This report describes the evaluation of a freeze-dried
preparation of pooled human leukocytes, NIBSC code 15/270, for use as a reference reagent for
CD4 T cell enumeration technologies. The material was evaluated by twelve laboratories from
eight different countries. Participants used different CD4 T cell counting technologies all of
which have shown suitable performance through independent peer-reviewed data. These include
single-platform and dual-platform flow cytometry, dedicated CD4 systems and point of care
(PoC) technologies. We found that the vast majority of users employ standard flow cytometers
for their counting. The material worked well in flow cytometry platforms and with the point of
care technology tested. The material could not be read by the dedicated CD4 systems BD FACS
Count and BD FACS Presto and was incompatible with one of the commercial lysis reagents
used. We conclude this material is fit-for-purpose for use with standard flow cytometry platforms
and the point-of-care device Instacount. The issue with the two dedicated CD4 systems will need
to be further investigated with the manufacturer in a follow-up study. Red blood cell lysis
reagents will need to be independently evaluated in each centre to determine suitability. In
addition, we were unable to recruit participants to cover all the technologies used for CD4 T cell
counting. Specifically, it would be important to test the material in the PoC device Alere Pima
CD4 test commonly used in Sub-Saharan Africa. However, we still feel the material has value.
Therefore, we propose that the candidate be established as a WHO reference reagent for CD4 T
cell counting. The IFU will state the technologies for which it has been qualified and the
expected performance of the material, as obtained by participants in this study. A follow-up
study will be organised with manufacturers to allow for testing of the material in a wider breadth
of technologies.
Introduction
Infection with HIV leads to the development of Acquired Immune Deficiency Syndrome (AIDS)
characterized by loss of CD4+ T cells required to mount an effective immune response against
infections. In 2005 the World Health Organisation (WHO) issued an open letter to manufacturers
of CD4+ T cell enumeration technologies emphasizing the need for laboratory monitoring of
immunological parameters to support the clinical monitoring of human immunodeficiency virus-
1 (HIV-1) infected patients. In particular, this letter states that ‘All CD4+ cell enumeration
technologies need to be compatible with a form of external quality assessment programme’.
WHO/BS/2018.2333
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Accurate CD4+ T cell count measurements ensure that patients receive the appropriate anti-
retroviral therapy (ART) against HIV-1 and chemoprophylaxis for opportunistic infections.
The WHO Expert Committee on Biological Standardization (ECBS) endorsed the proposal to
develop a WHO Reference Reagent to serve as a standard for CD4+ cell counting methods at
their 61
st
annual meeting in 2010.
Over 59 international laboratories were invited to participate in the collaborative study in order
to represent the range of methodologies for which the standard would be applicable. Fourteen
competent laboratories representative of the six WHO regions agreed to participate. Twelve
laboratories in four WHO regions returned data. The other two laboratories were unable to
perform the testing due to either moving of their facilities or problems with customs. Any CD4
counting technology that has shown acceptable performance through independent peer-reviewed
data was included in the study design.
Bulk materials, processing and characterization (15/270)
Material 15/270 was prepared from human blood leukocytes isolated from donations to the UK
National Blood Service (NHSBT). The blood was tested by NHSBT by serology and found
negative for antibodies against HIV, HCV, HBsAg and syphilis. The blood was also tested by
NHSBT with NAT for Hepatitis B, Hepatitis C, HIV-1 and HIV-2 (Roche MPX v2.0) and for
Hepatitis E (Roche HEV v1.0). All virology results were negative. Six leukocyte cones and four
whole blood packs were used. Leukocytes were isolated from whole blood by treatment with
ammonium chloride red blood cell lysis buffer and from leukocyte cones by density gradient
centrifugation. The cells were resuspended in complete media containing fixative. After fixation,
the cells were stored in 90%FCS 10%DMSO at -80°C until all donations had been processed. On
the day of filling, the cells were thawed, washed and pooled. The CD4 T cell concentration was
calculated by single-platform flow cytometry using BD Trucount tubes to determine the filling
volume. The cells were suspended in freeze-drying formulation and distributed into ampoules.
The ampoule contents were freeze-dried and sealed under nitrogen. The finished product
characteristics are as follows:
Code number
15/270
Presentation
Sealed, 3 mL glass ampoules
Number of ampoules available
5672
Date filled
February 2016
Mean fill mass
0.5209 g
Precision of fill (CV of fill mass) (=192)
0.16%
Residual moisture (n=12)
0.4%
Mean dry weight
0.01338
Mean oxygen head space (n=12)
0.26%
Microbiological results
Negative
Storage conditions
-20°C
Address of processing facility
NIBSC, Potters Bar, EN6 3QG, UK
Address of custodian
NIBSC, Potters Bar, EN6 3QG, UK
WHO/BS/2018.2333
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Participants
Twelve participants from eight different countries returned data and are listed in Table 1,
alphabetically, by country. The participants are competent laboratories in CD4 T cell counting as
demonstrated by their participation in EQA schemes for CD4 T cell counting (UK NEQAS and
QASI). Each participating laboratory is referred to in the study by a code number. The code
numbers were randomly assigned and do not reflect the order of listing.
Table 1: List of participants in order of country
AUSTRALIA Dr Joseph Manitta
Victorian infectious disease reference laboratory, Melbourne
BELGIUM Dr Luc Kestens
Institute of Tropical Medicine Antwerp, Antwerpen
CANADA Mr Michael Keeney
London Health Sciences Centre, London, Ontario
FRANCE Dr Guillaume Monneret
Hopital E. Herriot, Lyon
INDIA Dr Madhuri Thakar
National AIDS Research Institute, Pune
INDIA Dr P Balakrishnan
YRG Centre for AIDS Research and Education (YRG Care)
NETHERLANDS Dr Markus Beck
University of Twente, Enschede
PORTUGAL Dr Maria Arroz
Hospital S. Francisco Xavier, Lisboa
PORTUGAL Dr Marta Alvim
Instituto Nacional de Saude Doutor Ricardo Jorge, Lisboa
UK Mr Dan Payne
Leicester Royal Infirmary UHL NHS Trust, Leicester
UK Mr David Wilson
Aberdeen Royal Infirmary, Aberdeen
UK Mr Liam Whitby
UK NEQAS for Leucocyte Immunophenotyping, Sheffield
WHO/BS/2018.2333
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Collaborative study for the value assignment of 15/270
The collaborative study was organised by NIBSC. Each laboratory was asked to perform their in-
house method for CD4 T cell counting after reconstitution of each study sample in 1mL of sterile
distilled water. The methodologies used by each laboratory are shown in Table 2. A study
protocol, shown in Appendix 1, and instructions for use were provided with the samples.
Five ampoules were provided to participants. Participants tested one ampoule in eight replicate
runs in order to assess the precision of the assay. The following four ampoules were tested in
single runs. Participants were asked to return CD4+ T cell concentrations expressed as number of
CD4+ T cells per microliter and also as %CD4 of total white blood cells, when applicable to
their routine method.
Table 2: CD4 T cell counting methods used by participants
Laboratory 6 received the samples but was unable to return data due to moving facilities.
Laboratory 7 was unable to receive the samples due to problems with customs.
Type of technology
Instrument
Lab codes
Single-platform flow
cytometry
AQUIOS CL by Beckman Coulter
1 and 14
NAVIOS by Beckman Coulter
2 and 9
BD FACS Calibur
3
BD FACS Canto II
8 and 11
Beckman Coulter FC500
10
Dual-platform flow cytometry
BD FACS Calibur
5 and 12
Dedicated CD4 systems
BD FACS Count system
3
BD FACS Presto system
13
Point of care technologies
InstantCount
4
Statistical analysis
First, precision was assessed. The expected performance for CD4 technologies is a %CV less
than 10% for CD4 counts more than 200 cells/µL (source: guidelines on the WHO website for
‘Multicentre Evaluation of CD4 technologies as part of the WHO Prequalification of Diagnostics
Programme’). Any results showing CVs greater than 10% were deemed to have unacceptable
precision and excluded from value assignment. The remaining CD4 count data was tested using a
Grubbs’ test (Minitab) to detect an outlier value. The %CD4 data was analysed separately with a
Grubb’s test. The data was extrapolated to generate a normal distribution to assign a reference
range covering 99.7% of extrapolated data.
WHO/BS/2018.2333
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Results
Data returned for analysis
Data were contributed by 12 laboratories who performed CD4 T cell counting by their routine
method on 5 ampoules of 15/270. The first ampoule was assessed in 8 replicates to estimate the
precision of the assay (Table 3). The other 4 ampoules were assessed for the purpose of value
assignment of the material (Table 4). Participants using the dedicated CD4 systems BD FACS
Count and BD FACS Presto were unable to assess the material as the software returned an error
code and was unable to analyse the samples. BD was unable to help troubleshoot this problem.
One participant looked only at %CD4 and did not return a CD4 T cell concentration.
General suitability of the reference material
One participant reported incompatibility with Q-Prep, a red blood cell lysis reagent distributed
by Beckman Coulter. There was poor discrimination of the CD4+ T cells from other T cells
when using this reagent but not when using a PBS diluent (Figure 1) or other brands of red blood
cell lysis reagents used in the collaborative study (BD FACS Lysing solution, AQUIOS Lysing
Reagent kit).
Figure 1 – The same sample analysed using either Q-Prep or PBS. One participant noted
poor separation of CD4+ T cells when using their routine in-house method (4 colour flow
cytometry CD45-FITC/CD4-PE/CD8-ECD/CD3-PC5 using Beckman Coulter Q-prep no wash,
single-platform with Flow-count absolute counting beads). (A) Sample treated with Q-prep. (B)
Same sample treated with PBS instead of Q-prep. The x axis depicts fluorescence in the CD3
channel and the y axis fluorescence in the CD4 channel. Note the separation of CD4+ cells from
CD4- cells is poor when Q-prep is used (A) but not when PBS is used (B).
WHO/BS/2018.2333
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Assay validity
Precision assessment is shown in Table 3. Two participants returned %CVs greater than 10%
(12.11% and 22.31%) when analysing one ampoule in eight replicates. The WHO guidelines
state that the expected performance for CD4 technologies is a %CV of less than 10% for CD4
counts more than 200 cells/µL. Therefore, their data was excluded from the value assignment of
15/270. No outliers were found on the remaining data using the Grubb’s test (Minitab).
Value assignment
Value assignment data is shown in Table 4. Laboratory means were used to calculate an overall
mean and standard deviation and a normal distribution was extrapolated from that data. The
overall mean was 336 CD4/µL with a standard deviation of 21.35 CD4/µL. Most laboratories
achieved CVs between 4 and 6% with a maximum of 16%. A reference range spanning 99.7% of
expected normally distributed values would be 272 to 400 CD4/µL which is equal to a distance
of 3 standard deviations from the mean. Individual participant data segregated by assay type is
shown in Figure 2.
Percentage CD4
CD4 percentage is sometimes used as an alternative to CD4 counts, particularly in children under
5 years old (source: ‘Guidelines for the Use of Antiretroviral Agents in Pediatric HIV Infection’,
May 2018, U.S. Department of Health and Human Services webpage). The average percentage
of CD4 T cells among white blood cells in the value assignment exercise was 46%. A reference
range spanning 99.7% of expected normally distributed values would be 40 to 52%. All the
values returned in the study were within this range.
Stability assessment
Accelerated degradation studies were performed at NIBSC with ampoules of 15/270 which had
been stored at -70, -20, +4, +37, +45 and +56°C for 0.5 and 9 months. Four replicate vials were
assessed by single-platform flow cytometry. A brownish colour and resistance to reconstitution
were seen in the samples stored at +56°C for 9 months. This reflects degradation processes such
as Maillard reactions which are irrelevant at the low temperatures that the material is stored but
can be significant at higher temperatures.
Samples stored at higher temperatures showed a higher fluorescence background than samples
stored at -70 to +4°C. The signal to noise ratio between CD4+ T cells and CD4 negative
lymphocytes was used as a measure for degradation. The signal to noise values were normalised
to the storage temperature (-20°C) results and the long-term stability of 15/270 was predicted
using the Arrhenius equation. The estimated percentage loss at -20°C is 0.01% and the estimated
percentage loss at 20°C is 1.59% per month. This equates to an estimate of 2187 years before a
significant drop in CD4 T cell counts at continuous -20°C storage. The material will require a
cold chain for transport which is not a significant issue as it is intended for use mainly by
manufacturers.
WHO/BS/2018.2333
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Discussion
CD4 T cell counting is critical in the monitoring of HIV disease progression. However, there is a
lack of harmonisation in CD4 T cell counting which has been highlighted by the WHO as an
issue in patient care. To help improve international harmonisation of CD4 T cell counting, the
WHO endorsed a project to develop an international reference reagent for CD4 T cell counting.
The plan was to manufacture a stable preparation of leukocytes, to be used as a long-term
reference sample for the comparison of different technologies or the investigation of instrument
performance.
NIBSC manufactured reference material 15/270 from pooled donations from the UK National
Blood Service. The material was sent out for evaluation to laboratories proficient in the counting
of CD4 T cells. Fourteen international laboratories representative of the six WHO regions agreed
to participate. Twelve laboratories returned data. The material worked well in all conventional
flow cytometers and in the PoC device tested. However, participants were unable to analyse it in
the dedicated CD4 systems BD FACS Count and BD FACS Presto which is unfortunate as BD is
one of the largest manufacturers. Two participants used a plug-in offered by BD for automated
counting of CD4 T cells in a standard flow cytometer with no issues which suggests that at least
this automated application recognizes the candidate as ‘like for like’.
In addition, it would be desirable to test the material in a wider range of technologies. The Alere
Pima CD4 test in particular is popular in Sub-Saharan Africa, which suffers the greatest burden
of HIV globally. Unfortunately, we were unable to recruit any participant using this test. The use
of the red blood cell lysis reagent Q-Prep from Beckman Coulter resulted in poor separation of
CD4 T cells from other lymphocytes, which the participant had previously seen with other
stabilised cellular controls as well. However, there were no issues with any of the other red blood
cell lysis reagents used in the study. Expected performance criteria for 15/270 were taken from
the evaluation in single and dual-platform classical flow cytometers and the PoC device
InstantCount and will be included in the Instructions for Use (IFU).
Conclusion and Recommendation
We propose that the candidate 15/270 be established as a WHO reference reagent for use in CD4
T cell counting. The IFU will state that the material has been qualified using single-platform and
dual-platform classical flow cytometers, and the point of care device InstantCount. The expected
performance will be stated in the IFU as:
In the hands of expert laboratories, material 15/270 returned an overall mean of 336 CD4 T
cells/µL, with an intra-laboratory CV between 4 and 6% for most laboratories and a maximum
intra-laboratory CV of 16%. The mean value obtained by an individual laboratory upon repeat
testing is expected to fit within the range of 272-400 CD4 T cells/µL with a maximum CV of
16%.
WHO/BS/2018.2333
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Additionally, the IFU will state the need to validate red blood cell lysis reagents and the %CD4
expected range. A draft IFU can be found in Appendix 2. A follow-up study will be organised by
NIBSC with manufacturers to allow for testing of the material in a wider breath of technologies
including the Alere Pima CD4 test.
Responses from participants
Originally, it was proposed to participants that 15/270 be established as an international
reference reagent for use in selected technologies with a reference range of 272 to 400 CD4 T
cells/µL. A follow-up study would be organised with manufacturers of the technologies that
were unable to be assessed. Eight of the nine laboratories who responded (8/12) agreed with this
proposal. However, one laboratory disagreed. They felt the range was too wide and that this
might be detrimental to the development of adequate CD4 instrumentation. After reflecting on
this comment we revised the proposal to include the expected performance of the material, in
line with that found by the expert laboratories in this study. The participant who had originally
disagreed now supports the revised proposal. One participant felt the material needed more
supporting data and the other (8/12) all agreed with the proposal.
The participant’s comments and response from NIBSC are shown below.
Participant A:
thank you for your email and the update on the reference material. Whilst I appreciate that there
is currently no reference material for CD4 T cell counting I regret to say that I cannot endorse the
use of this material as an international reference.
My decision to not endorse is based on the publication 'Daneau G, Buyze J, Wade D, Diaw PA,
Dieye TN, Sopheak T, Florence E, Lynen L, and Kestens L. CD4 Results with a Bias Larger than
Hundred Cells Per Microliter Can Have a Significant Impact on the Clinical Decision During
Treatment Initiation of HIV Patients. Cytometry Part B 2017 Nov;92(6):476-484'
The manuscript by Daneau et al clearly states that CD4 results with a bias of larger than 100
cells/uL can affect clinical decisions and that new technologies should not have a bias that
exceeds +/- 50 cells/uL. The range for the proposed reference material is 272-400 cells/uL, so
with a mean of 336 cells/uL for the material this gives a bias of +/- 64 cells/uL that would still be
within the reference range and so classified as acceptable. As such, given that the range for the
reference material is greater than the target range proposed for new instrumentation by Daneau et
al I cannot see how the introduction of this material at this time would be beneficial. In fact the
wide range of the material could actually lead to the development of instrumentation that is not
of the required standard required by the laboratory community and this is why I cannot endorse
its use.
I would however suggest an alternative approach. It has been seen that different technologies
give different results with different levels of precision (e.g single platform vs dual platform). So
if it were possible to reassess the material (using single platform technologies only as the gold
standard) then this may give a more favourable outcome. If this were the case I would be happy
to reconsider.
WHO/BS/2018.2333
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NIBSC response:
The participant raises the issue that if the reference reagent were used to validate a new
instrument then in theory a bias greater than 50 CD4 cells/µL between two instruments could be
missed as the data would still be within range (272 to 400 CD4 T cells/µL).
This proposed range reflects a normal distribution extrapolated from laboratory means. However,
the expected variability within one individual laboratory would be much lower so a manufacturer
of a new technology would expect to achieve a much narrower range in their own testing. If you
are taking into account data from various participants, then you have additional sources of
uncertainty than those that apply within one laboratory. Sources of uncertainty might include
differences in cell reconstitution due to pipetting and vial-to-vial variation within a batch,
differences in antibody reagents, data analysis, the use of single or dual platform methods, the
value assigned and variability within a batch of internal reference counting beads, different
operators and instruments, all contributing to data spread. We can describe the expected
variability within one individual laboratory in terms of the %CVs obtained by the participants.
In the value assignment exercise, material 15/270 returned an overall mean of 336 CD4/µL, with
an intra-laboratory CV of 5-6% for most laboratories and not more than 16%. We can state that
the range of values for an individual laboratory should fit within the overall range of 272-400
CD4/µL but respect the %CV conditions described above. This should help prevent the scenario
described above where the reference material is used to justify inadequate instrumentation.
It has been reported in the past that single-platform flow cytometry for CD4 T cell counting
shows better inter-laboratory coefficients of variation than double-platform methods. However, I
would argue two points. Firstly, to evaluate a reference reagent for international use you would
want to include as many relevant technologies as possible, as there are various variables that
might determine what technology end users might use including availability, cost, logistics and
resources. Any CD4 counting technology that has shown acceptable performance through
independent peer-reviewed data was invited to participate in the study. Importantly, the data in
this particular study do not support the exclusion of double-platform methods for value
assignment. The average precision for all methods was 6.52% whereas for single-platform
methods only it was 6.98%. Therefore, whilst I appreciate that the collaborative study has
weaknesses, it does not support the exclusion of non single-platform methods for value
assignment.
Participant B:
I do think that the candidate 15/270 has the potential to be used as a reference material and
should therefore be tested more in depth. But the final conclusion on whether or not it should
indeed be used as a WHO standard requires better statistical significance.
NIBSC response:
The proposal is to establish 15/270 as a reference reagent which is established by WHO in a
situation where the full criteria for an international standard cannot be met. We agree that the
evidence is not strong enough to support its establishment as an international standard. The status
of WHO reference reagent has been assigned to interim standards before, so it is possible to
change the expected performance criteria for 15/270 as we learn more from further testing after
establishment.
WHO/BS/2018.2333
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Acknowledgments
We are deeply thankful to the participants in this collaborative study who have dedicated their
time to this project.
WHO/BS/2018.2333
Page 12
Table 3: Precision assessment. The table shows the precision of the assay as assessed by repeat
testing of one ampoule in eight replicates. Laboratory 6 received the samples but was unable to
return data due to moving facilities. Laboratory 7 was unable to receive the samples due to
problems with customs.
Lab
Precision (n=8)
%CV
1
4.21
2
1.88
3
3.07
3
ERROR
4
5.00
5
9.27
8
1.92
9
3.36
10
Participant only provided %CD4 data
11
12.11
12
2.10
13
ERROR
14
22.31
Table 4: Value assignment. The table shows the laboratory means used in the value assignment
of 15/270, specifically, the calculation of expected means for CD4 counts and %CD4. Some
laboratories were unable to return data or returned incomplete data as described in Table 3.
Laboratories 11 and 14 were excluded from value assignment due to low assay precision.
Lab
CD4 T cells/µL
%CD4
No.vials
Mean
%CV
Mean
%CV
1
359.0
3.89
46.74
2.16
4
2
368.5
3.85
43.68
0.22
4
3
320.5
5.24
47.25
4.69
4
4
345.5
5.83
N/A
N/A
4
5
320.5
4.67
50.5
1.14
4
8
330.8
15.79
44.61
2.17
4
9
335.75
10.67
45.9
0.74
4
12
304.25
5.28
44.93
1.13
4
WHO/BS/2018.2333
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Figure 2 – Participant data in the value assignment exercise: data is shown segregated by
value range on the x axis and number of values in the data in the y axis. For each box, the colour
denotes the method used and the top number is the laboratory code. Each laboratory tested four
ampoules and these are denoted by the small number 1, 2, 3, 4 on the bottom of the box.
Laboratories 11 and 14 (grey) were excluded from value assignment due to low assay precision
in the repeat testing exercise.
WHO/BS/2018.2333
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Appendix 1: Study protocol
An International Reference Reagent for the enumeration of CD4 T cells
COLLABORATIVE STUDY
Study Protocol
April 2016
Study coordinators: Luisa Saraiva, National Institute for Biological Standards and Control, UK
Tel: +44 (0) 1707 641277, Luisa.Sar[email protected]; Sandrine Vessillier, National Institute for
Biological Standards and Control, UK, Tel:+44 (0) 1707 641146, Sandrine.Vessi[email protected]rg
Introduction
This study aims to assess an internationally recognised reference preparation for use as a
comparator sample for the evaluation of CD4 technologies. Participants are competent
laboratories representative of the six WHO regions. Any CD4 counting technology that has
shown acceptable performance through independent peer-reviewed data has been included in the
study.
The results of the study will be combined in a report to the WHO Expert Committee on
Biological Standardization (ECBS) with a recommendation for establishment as a reference
reagent together with any limitations on its use (e.g. suitability only for certain assay methods)
and a consensus unit.
1. AIMS OF THE STUDY
This study sets out to assess specifically:
1. the suitability of the candidate international biological reference reagent for the
enumeration of CD4 T cells in a variety of CD4 T cell counting technologies
2. reference values including a mean and a range expressed as CD4 T cells/μL and
% CD4 to the reference reagent
2. REFERENCE MATERIAL
Participants will be sent four vials of reference material for calculation of CD4 T cell
reference values. Also included is one vial for each method quoted in the participants’
information sheet to be used to assess the precision of the method. Two spare additional
vials are included.
The material is shipped at ambient temperature but please store at cold temperatures
(+4°C or below) upon receipt. Reconstitute material just before use. If testing using more
than one method use the same 4 ampoules for each method. If testing multiple methods is
not feasible on the same day then it is possible to transfer material to new tubes so that it
can be capped and stored at +4°C until use for a maximum of two days.
WHO/BS/2018.2333
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To reconstitute a sample:
1. take ampoule out of storage, place in a tube holder and allow to adjust to room
temperature;
2. break ampoule seal;
3. pipette 1mL of sterile distilled water and allow 5-30 min for rehydration;
4. mix cell suspension well with a 1mL pipette;
5. sample adequate volume for your assay (treat the material as equivalent to a blood
sample at about 1.5x10
6
white blood cells/mL).
3. OUTLINE OF THE STUDY
Repeat 1) and 2) for each method used.
1) Assay precision comparison
Reconstitute 1 ampoule. Perform 8 replicate runs on this specimen using your own assay.
Report CD4 T cells/μL and also %CD4, if applicable to the method used (Table 1).
2) Assessment of reference values
Reconstitute 4 ampoules. Report CD4 T cells/μL and also %CD4, if applicable to the
method used (Table 3). Use the same 4 ampoules for each method.
4. DATA SUBMISSION
A report will be prepared and circulated to all participants. In the data analysis, participating
laboratories will be identified by a laboratory number only. For submission of results and any
further information please email:
Dr Luisa Saraiva
Tel: +44 (0) 1707 641277
Luisa.Saraiva@nibsc.org
Dr Sandrine Vessillier
Tel: +44 (0) 1707 641146
Sandrine.Vessil[email protected]
Deadline for data submission – 31
st
August 2016
WHO/BS/2018.2333
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5. RESULTS
Repeat 1) and 2) for each method used.
Section 1 – Assay precision
Please fill in results in the table below.
Table 1 – Assay precision assessment
Sample
Replicate
CD4 T cells/μL
% CD4
1
1
1
2
1
3
1
4
1
5
1
6
1
7
1
8
Please select the assay method used in the table below:
Table 2 - Method
Method
Select below:
BD FACS Count system
BD FACS Presto system
Alere Pima CD4 test
CyFlow Counter
CyFlow miniPOC
Single-platform flow cytometry with BD FACS Calibur
Single-platform flow cytometry with BD FACS Canto II
Single-platform flow cytometry with AQUIOS CL by Beckman Coulter
Single-platform flow cytometry with NAVIOS by Beckman Coulter
Single-platform flow cytometry with Beckman Coulter FC500 flow cytometer
Dual-platform flow cytometry with Beckman Coulter FC500 flow cytometer
Dual-platform flow cytometry with BD FACS Calibur flow cytometer
Fluorescence imaging on a prototype point-of-care instrument using cell counting
chambers with on-chip sample preparation for immunostaining
Other method, please describe:
Please describe or provide an example of the gating strategy:
WHO/BS/2018.2333
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Section 2 – Assay values
The data in this section will be used to assign target values to the reference reagent. Please fill in results in
the table below. Report CD4 T cells/μL and also %CD4 if applicable to your method.
Table 3 – Assay values
Sample
CD4 T cells/μL
% CD4
1
2
3
4
6. GENERAL SUITABILITY
Please provide any comments on how you feel the material performed in your assay (e.g. did you
find the CD4 level suitable, did you find it easy to use?)
Thank you for helping evaluate our standard!
WHO/BS/2018.2333
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Appendix 2 –Draft Instructions for Use
se
WHO/BS/2018.2333
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