831273

research-article2019
DSTXXX10.1177/1932296819831273Journal of Diabetes Science and TechnologyArnold et al

Original Article

Journal of Diabetes Science and Technology

Fingerstick Precision and Total Error

2020, Vol. 14(5) 890­–895
© 2019 Diabetes Technology Society
Article reuse guidelines:
of a Point-of-Care HbA1c Test sagepub.com/journals-permissions
DOI: 10.1177/1932296819831273
https://doi.org/10.1177/1932296819831273
journals.sagepub.com/home/dst

William D. Arnold, PhD1, Kenneth Kupfer, PhD1,

Monica Hvidsten Swensen, MSc2, Kyle S. Fortner, BS1,
Harold E. Bays, MD3, Mathew Davis, MD4,
Leslie J. Klaff, MD, PhD5, and Richard C. San George, PhD1

Abstract
Background: Point-of-care (POC) HbA1c tests hold the promise of reducing the rates of undiagnosed diabetes, provided
they exhibit acceptable analytical performance. The precision and total error of the POC (Afinion™ HbA1c Dx) test were
investigated using whole blood samples obtained by fingerstick and venipuncture.

Methods: Fingerstick samples spanning the assay range were collected from 61 subjects at three representative POC sites.
At each site, six fingerstick samples were obtained from each subject and tested on the POC test across two (Afinion AS100)
instruments. Repeatability, between-operator, and between-instrument components of variance were calculated using
analysis of variance (ANOVA). Four venous samples (low, threshold, medium, and high HbA1c) were measured in duplicate
across three instruments using three reagent lots, twice per day over 20-days. Repeatability, between-run, between-day,
between-lot, and between-instrument components of variance were calculated. These fingerstick and venous blood results,
combined with estimates of imprecision and bias from a prior investigation, allowed for the calculation of the total coefficient
of variation (CV) and total error of the POC test using fingerstick and venous whole blood samples.

Results: The total imprecision ranged from 1.30% to 2.03% CV using fingerstick samples and from 1.31% to 1.64% CV using
venous samples. The total error ranged from 2.87% to 4.75% using fingerstick samples and from 2.93% to 3.80% using venous
samples.

Conclusions: The POC test evaluated here is precise across its measuring range using both fingerstick and venous whole
blood. The calculated total error of the test is well under the accepted quality requirement of ≤6%.

Keywords
diagnosis of diabetes, HbA1c, hemoglobin A1c, point of care, precision, total error

The measurement of HbA1c is an accepted, convenient and
useful means for the diagnosis of diabetes. Despite some
limitations, such as interference from common hemoglobin
variants and conditions associated with increased red blood
cell turnover, HbA1c measurement presents numerous
advantages including greater preanalytical stability, less day-
to-day variability during stress or illness, and does not
require individuals to fast prior to testing.1 The barrier to
evaluating glycemic status could be further reduced by uti-
lizing POC HbA1c methods, which can provide access to
care in disadvantaged communities and offer the potential of
identifying many more underserved individuals with predia-
betes and diabetes.2-4 The advantages and disadvantages of
HbA1c versus fasting plasma glucose or oral glucose toler-
ance testing for the diagnosis of diabetes have been discussed
extensively elsewhere.1
The results of a previous investigation of a POC method,
the Afinion™ HbA1c Dx test (Study A) first suggested that
this method may offer acceptable accuracy and precision
when used by trained laboratory operators.5
1
Abbott Rapid Diagnostics, a division of Abbott Laboratories, San Diego,
CA, USA
2
Abbott Rapid Diagnostics, a division of Abbott Laboratories, Oslo,
Norway
3
L-MARC Research Center, Louisville, KY, USA
4
Rochester Clinical Research, Inc, Rochester, NY, USA
5
Rainier Clinical Research Center, Inc, Renton, WA, USA
Corresponding Author:
Richard C. San George, PhD, Abbott Rapid Diagnostics, a division of
Abbott Laboratories, 9975 Summers Ridge Rd, San Diego, CA 92121,
USA.
Email: rick.sangeorge@alere.com
Arnold et al
After the completion of Study A, there was interest in eval-
uating additional components of variance not assessed in that
study design. In the study reported here (Study B), a direct
evaluation of repeatability, between-operator, and between-
instrument precision is made using fingerstick (capillary)
whole blood samples, the principal sample type used at the
POC. This is a first of a kind study to evaluate components of
variance in fingerstick samples. In addition, precision using
venous whole blood samples was evaluated over 20 days,
consistent with Clinical and Laboratory Standards Institute
(CLSI) Guideline EP05-A3 (Study C).6 Taken together, the
data from these studies (A, B, and C) enable the evaluation of
the total imprecision and total error of this POC test for both
fingerstick and venous whole blood sample types.
Methods
The POC test (manufactured by Alere Technologies AS,
Oslo, Norway; a subsidiary of Abbott Laboratories) is a fully
automated boronate affinity assay for the determination of
the percentage of hemoglobin A1c in human whole blood.
The test cartridge contains all of the reagents necessary for
the determination of %HbA1c. A patient specimen is col-
lected with the integrated sampling device then reinserted
back into the test cartridge, which is then placed in the
Afinion AS100 Analyzer (instrument). The sample is auto-
matically diluted and mixed with a solution that releases
hemoglobin from the erythrocytes. After the hemoglobin is
precipitated, the sample mixture is transferred to a blue
boronic acid conjugate which binds to the cis-diols of gly-
cated hemoglobin. This reaction mixture is soaked through a
filter membrane and all precipitated hemoglobin, glycated
and nonglycated hemoglobin, remains on the membrane.
Excess conjugate is removed with a washing reagent. The
analyzer measures the reflectance of the precipitate on the
membrane as blue (glycated hemoglobin) and red (total
hemoglobin) color intensities. The percentage of total gly-
cated hemoglobin and %HbA1c has been shown to be well
correlated;7 calibration adjusts the measured total glycated
hemoglobin to report the equivalent level of HbA1c. The
POC test is traceable to the International Federation of
Clinical Chemistry and Laboratory Medicine (IFCC)
Reference Method for Measurement of HbA1c,8 and is certi-
fied by the NGSP as having documented traceability to the
Diabetes Control and Complications Trial (DCCT) Reference
Method. The NGSP awards certification to manufacturers for
successfully meeting performance criteria for bias testing.9
In Study A, conducted previously,5 a first-replicate POC
test value (from fingerstick and venous whole blood samples)
was compared against the average of duplicate results from a
venous sample tested on the Tosoh glycohemoglobin test on
the G8 HPLC analyzer at the NGSP Secondary Reference
Laboratory (SRL) at the Diabetes Diagnosis Laboratory,
University of Missouri School of Medicine, Columbia MO.
Passing-Bablok regression equations were calculated and
891
used to estimate the bias of the POC test at HbA1c values of
5.0% (31.1 mmol/mol), 6.5% (47.5 mmol/mol), 8.0%
(63.9 mmol/mol), and 12% (107.7 mmol/mol). Three POC
test cartridge lots were used across sites, and the between-lot
component of variance, as well as repeatability estimates for
low, threshold, medium, and high HbA1c levels, were calcu-
lated as follows. Samples were grouped into the following
four HbA1c interval levels: low (4.00%-5.99% HbA1c, or
20.2-42.0 mmol/mol HbA1c), threshold (6.00%-6.99%
HbA1c, or 42.1-52.9 mmol/mol HbA1c), medium (7.00%-
9.99% HbA1c, or 53.0-85.7 mmol/mol HbA1c), and high
(≥10% HbA1c, or 85.8 mmol/mol HbA1c). For each HbA1c
level the imprecision was separated into repeatability (within-
run) and between-lot components of variance using a two-
factor ANOVA model without nesting based on Type II sums
of squares (MATLAB ANOVAN and SAS PROC MIXED).
In Study B, at three POC study sites having laboratories
operated by trained medical professionals, fingerstick sam-
ples from prospectively enrolled subjects were collected and
tested on the POC test. Each subject signed an informed con-
sent form approved by a central, independent institutional
review board (IRB) prior to any study procedures being con-
ducted. Enrollment included those that met the following
eligibility criteria:
Inclusion criterion:
•• At least 18 years of age
Exclusion criteria:
•• Known to have the hemoglobin variant HbF > 7%
•• Diagnosed with hemophilia
•• Taking glucocorticoid or nicotinic acid medications
•• Diagnosed with iron deficiency, hemolytic anemia,
thalassemias, hereditary spherocytosis, chronic hepatic,
or renal disease
•• Pregnant
•• Received a blood transfusion or cancer chemotherapy
within the prior three weeks
•• Unwilling or unable to sign a written informed con-
sent and comply with study procedures, or is deemed
inappropriate for participation by the site investigator
Each site targeted enrolling five subjects having HbA1c val-
ues within one of the low, threshold, medium, and high levels
as defined above. Fingerstick sample testing was conducted
by using the same lot of POC test cartridges at each of the
three study sites. At each site, for each subject, there were
three test operators who performed testing across two instru-
ments. Each operator collected two fingerstick samples and
tested them, one sample per instrument. The results from the
fingerstick testing were evaluated by using ANOVA method-
ology based on Type II sums of squares (MATLAB ANOVAN
and SAS PROC MIXED), consistent with the methodology
892 Journal of Diabetes Science and Technology 14(5)

Table 1. Summary of Components of Variance Used in the The total CVs, together with the bias estimates from study
Calculation of Total Imprecision, and Study From Which the A, were used to estimate the total error of the POC test at
Estimate Was Obtained. HbA1c values of 5.0% (31.1 mmol/mol), 6.5% (47.5 mmol/
Component of Study where mol), 8.0% (63.9 mmol/mol), and 12% (107.7 mmol/mol)
Sample type variance estimate obtained using fingerstick and venous whole blood samples. The coef-
ficient of variance, bias, and total error are expressed as a
Fingerstick Repeatabilitya A
percentage of the mean, where the mean is expressed in
Between-lot A
NGSP units (%HbA1c).
Between-instrument B
Between-operator B
Between-run C Results
Between-day C
Venous Repeatability C A total of 62 subjects were enrolled across three sites for the
Between-lot C purpose of fingerstick sample testing in Study B. One of the
Between-instrument C 62 subjects had out-of-range HbA1c values and was excluded
Between-run C from analysis. The remaining 61 subjects each provided six
Between-day C fingerstick samples. The population had an average age of
a 60 years, was 56% male, and had HbA1c values evenly dis-
The repeatability estimates from Study A were used in the calculation
of total imprecision using fingerstick samples for all HbA1c levels, except tributed over the measurement range of the POC test.
for the high level, which was estimated from Study B due to larger sample Estimates of between-instrument, between-operator, and
size (15 subjects vs 6 subjects). repeatability components of variance from the testing of
these fingerstick samples are shown in Table 2. Across the
described in CLSI EP05-A3.6 The repeatability (within-run), assay range, the between-instrument imprecision did not
between-operator, and between-instrument components of exceed 0.47% CV, and the between-operator imprecision did
variance were calculated for each HbA1c level by combining not exceed 0.21% CV. While repeatability was the largest
the data from all sites. For each component of variance, the component of variance, it did not exceed 1.39% CV.
CV was calculated as the SD divided by the grand mean of These estimates of fingerstick repeatability were slightly
all the POC test results within the level. smaller than but consistent with those calculated from previ-
In Study C, at the manufacturer’s site, four venous whole ous studies. Table 3 describes the repeatability and between-
blood samples were selected for precision testing, one at lot components of variance that were calculated using prior
each HbA1c level (low, threshold, medium, and high), as duplicate fingerstick measurements in Study A.5
defined for the fingerstick samples. These K2-EDTA- In Study C, four venous whole blood samples, represent-
anticoagulated samples were identified from among deiden- ing low, threshold, medium, and high HbA1c levels, were
tified remnants of specimens from hospital laboratories or tested on the POC test over 20 days. A grand total of 720
fresh samples collected from an IRB approved in-house measurements for each HbA1c level resulted from testing
donation. Each of the four samples was tested on the ana- these samples on each of three cartridge lots and each of
lyzer using each of three reagent lots and each of three instru- three instruments with two runs per day and two measure-
ments. Two replicate results were obtained during each of ments per run. Estimates of the components of variance and
two runs per day over a 20-day period, for a total of 720 total %CV for these venous sample results are shown in
measurements at each HbA1c level. From the results of Table 4. As was the case with fingerstick samples in Study A
Study C, the repeatability (within-run), between-run, and Study B, the repeatability was the single largest compo-
between-day, between-lot, and between-instrument SD and nent of variance for the POC test when using venous whole
CV were calculated (SAS VARCOMP). blood samples.
The total CV of the POC test using fingerstick samples Using the sums of squares of the relevant components of
was calculated based on the sum of squares of six compo- variance, estimates were made of the total imprecision of the
nents of variance: repeatability, between-lot, between-run, POC test when testing fingerstick samples (Table 5). The
between-day, between-instrument, and between-operator total error of the POC test was then estimated according to
(see Table 1). The between-run and between-day compo- the equation:
nents were obtained from the POC test results using venous
samples in Study C, since these components cannot be %TE = % Bias + 1.96 × %CV × (1 + %Bias/100 )
directly estimated using fingerstick samples. The between-
lot and repeatability components were obtained from Study Across the assay range, the total imprecision using finger-
A, because the number of subjects was larger in this study stick samples was 2.03% CV or less, and the total error did
than in Study B. Study B was necessary to estimate the not exceed 4.75% (Table 6). When using venous blood sam-
between-operator and between-instrument components of ples the total CV was 1.64% or less, and the total error was
variance in fingerstick samples. no greater than 3.80%.
Arnold et al 893

Table 2. POC Test Components of Variance Using Fingerstick Whole Blood Samples Estimated in Study B.

Mean HbA1c Between instrument Between operator Repeatability

a b b
HbA1c Level %HbA1c mmol/mol N (M) CV(%) CV(%) CV(%)b

Low 5.33 34.8 15 (90) 0.14 0.00 1.39

Threshold 6.51 47.7 15 (90) 0.30 0.00 1.25
Medium 8.32 67.4 16 (96) 0.00 0.21 1.10
High 12.2 109.8 15 (90) 0.47 0.00 1.14
a
N is the number of subjects, M is the number of test results. bThe CV is expressed as a percentage of the mean in NGSP units (%HbA1c).

Table 3. POC Test Components of Variance Using Fingerstick Whole Blood Samples Estimated in Study A.5

HbA1c level N (M)a Repeatability CV(%)b Between lot CV(%)b

Low 45 (90) 1.52 1.20

Threshold 68 (136) 1.36 0.42
Medium 51 (102) 1.35 0.00
High 6 (12) 0.62 0.07
a
N is the number of subjects, M is the number of test results. bThe CV is expressed as a percentage of the mean in NGSP units (%HbA1c).

Table 4. POC Test Components of Variance Using Venous Whole Blood Samples Estimated in Study C.

Mean HbA1c Repeatability Between run Between day Between lot Between instrument

HbA1c Level %HbA1c mmol/mol %CV %CV %CV %CV %CV Total %CV

Low 5.14 32.7 1.18 0.00 0.60 0.81 0.58 1.64

Threshold 6.55 48.1 1.13 0.00 0.62 0.85 0.00 1.51
Medium 8.06 64.6 0.95 0.20 0.56 0.47 0.49 1.31
High 11.26 99.6 0.89 0.12 0.39 0.88 0.52 1.42

Results using samples from four subjects and 720 test results per subject. The CVs are expressed as a percentage of the mean in NGSP units (%HbA1c).

Table 5. Total Imprecision of the POC Test Using Fingerstick Samples.

%HbA1c (mmol/mol)

Study Variance component 4.00-5.99 (20.2-42.0) 6.00-6.99 (42.1-52.9) 7.00-9.99 (53.0-85.7) ≥10 (85.8)
a
A N (M) 45 (90) 68 (136) 51 (102) 6 (12)
Repeatability %CVb 1.52 1.36 1.35 —
Between-lot %CV 1.20 0.42 0.00 0.07
B N (M)a 15 (90) 15 (90) 16 (96) 15 (90)
Repeatability %CVb — — — 1.14
Between-instrument 0.14 0.30 0.00 0.47
Between-operator 0.00 0.00 0.21 0.00
C Ma 720 720 720 720
Between-run 0.00 0.00 0.21 0.12
Between-day 0.60 0.62 0.56 0.39
TOTAL %CV 2.03 1.58 1.49 1.30

The CVs are expressed as a percentage of the mean in NGSP units (%HbA1c).
a
N is the number of subjects, M is the number of test results. bThe repeatability estimates from Study A were used in the calculation of total imprecision
using fingerstick samples for all HbA1c levels, except for the High level, which was estimated from Study B due to larger sample size.
894 Journal of Diabetes Science and Technology 14(5)

Table 6. Total Imprecision, Bias, and Total Error Estimates for POC Test.

HbA1c level Fingerstick whole blood Venous whole blood

a a
%HbA1c mmol/mol %Bias %CV %TE %Bias %CV %TE
5.0 31.1 −0.800 2.03 4.75 −0.600 1.64 3.80
6.5 47.5 −0.615 1.58 3.69 −0.462 1.51 3.41
8.0 63.9 −0.500 1.49 3.40 −0.375 1.31 2.93
12.0 107.7 −0.333 1.30 2.87 −0.250 1.42 3.03

The CV, bias, and TE is expressed as a percentage of the mean in NGSP units (%HbA1c).
a
Bias relative to the Tosoh Glycohemoglobin test on the G8 HPLC analyzer measured at an NGSP SRL, estimated in Study A.5

Discussion Trial; IFCC, International Federation of Clinical Chemistry and

The results indicate that the POC test evaluated here is
highly precise across the measuring range of the assay when
using fingerstick and venous whole blood samples. The esti-
mated imprecision is well within the <3% CV recom-
mended by the National Academy of Clinical Biochemistry
guidelines for single-method, interlaboratory (between lab-
oratories) HbA1c imprecision.10 The total error of the POC
test, regardless of sample type and HbA1c level, is well
below the criterion of ≤6% total error specified in the FDA
special controls for HbA1c test systems (21 CFR 862.1373)11
and required for NGSP certification.12 Around the diagnos-
tic cut point of 6.5% HbA1c (47.5 mmol/mol), the total
imprecision of the POC test was less than 2% CV, and the
total error was less than 4%.
To our knowledge, there are currently no published stud-
ies evaluating the total imprecision and total error of POC
HbA1c tests using fingerstick samples, however our results
using venous whole blood samples are similar to those from
the most recent College of American Pathologists (CAP)
Survey, which shows the performance of this POC test is
comparable to that of many laboratory methods that are rou-
tinely used in the diagnosis of diabetes.13 These results are
consistent with previous evaluations, where the POC test
evaluated here demonstrated a high degree of accuracy and
precision compared to routine laboratory methods14 and
other POC methods.15,16
The POC test evaluated here has not yet been granted a
CLIA waived categorization, and therefore additional inves-
tigation of its performance in the hands of self-trained, non-
laboratory professionals is warranted. POC HbA1c devices
can offer benefits by providing patient results within min-
utes, facilitating immediate dialogue between the care pro-
vider and patient, and expanding the reach of HbA1c testing
to underserved areas where people with undiagnosed diabe-
tes might be identified.
Abbreviations
ANOVA, analysis of variance; CAP, College of American
Pathologists; CLSI, Clinical and Laboratory Standards Institute; CV,
coefficient of variation; DCCT, Diabetes Control and Complications
Laboratory Medicine; IRB, institutional review board; POC, point of
care; RMS, root mean square; SD, standard deviation; SRL,
Secondary Reference Laboratory.
Acknowledgments
The authors would like to thank David Stokar for his validation of
the ANOVA results and Marta Clendenin for her assistance in pre-
paring this article.
Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of interest
with respect to the research, authorship, and/or publication of this
article: WDA, KK, MHS, KSF, and RCSG are employees of the
study sponsor, Abbott Rapid Diagnostics, a division of Abbott
Laboratories.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article:
Funding was provided by the study sponsor, Abbott Rapid
Diagnostics, a division of Abbott Laboratories.
References
1. American Diabetes Association. Standards of medical care
in diabetes—2018. Diabetes Care. 2018;41(suppl 1):S1-
S156.
2. Leal S, Soto-Rowen M. Usefulness of point-of-care testing
in the treatment of diabetes in an underserved population. J
Diabetes Sci Technol. 2009;3:672-676.
3. Chadee A, Blackhouse G, Goeree R. Point-of-care hemoglo-
bin A1c testing: a budget impact analysis. Ont Health Technol
Assess Ser. 2014;14:1-23.
4. Spaeth BA, Shephard MD, Schatz S. Point-of-care testing for
haemoglobin A1c in remote Australian Indigenous communi-
ties improves timeliness of diabetes care. Rural Remote Health.
2014;14:2849.
5. Arnold WD, Kupfer K, Little RR, et al. Accuracy and preci-
sion of a point-of-care HbA1c test. J Diabetes Sci Technol.
2020;14:883-889.
6. CLSI Guideline EP5-A3. Evaluation of Precision of Quantitative
Measurement Procedures; Approved Guideline. 3rd ed. Wayne,
PA: Clinical and Laboratory Standards Institute; 2013.
Arnold et al
7. Nuttall FQ. Comparison of percent total GHb with percent
HbA1c in people with and without known diabetes. Diabetes
Care. 1998;21:1475-1480.
8. Jeppsson JO, Kobold U, Barr J, et al. Approved IFCC reference
method for the measurement of HbA1c in human blood. Clin
Chem Lab Med. 2002;40:78-89.
9. NGSP. List of NGSP certified methods [Internet]. Columbia
(MO): NGSP; [updated 2019 Feb; cited 2019 Feb 08].
Available from: http://www.ngsp.org/docs/methods.pdf.
10. Sacks DB, Arnold M, Bakris GL, et al. Guidelines and recom-
mendations for laboratory analysis in the diagnosis and man-
agement of diabetes mellitus. Clin Chem. 2011;57:e1-e47.
11. US Department of Health and Human Services. Hemoglobin
A1c test system. [Internet]. Silver Spring, MD: US food and
Drug Adminstration; 2014 Aug 25 [updated 2018 Sep 04;
revised 2018 Apr 01; cited 2019 Feb 08]. Available at: https://
www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch
.cfm?fr=862.1373. Accessed April 2018.
895
12. Rohlfing CL, Parvin CA, Sacks DB, Little RR. Comparing
analytic performance criteria: evaluation of HbA1c certifica-
tion criteria as an example. Clin Chim Acta. 2014;433:259-
263.
13. NGSP. College of American Pathologists (CAP) GH5 survey
data. [Internet]. Columbia, MO: NGSP; [updated 2017 Dec;
cited 2019 Feb 08]. Available at: http://www.ngsp.org/CAP
/CAP17c.pdf. Accessed April 2018.
14. Sobolesky PM, Smith BE, Saenger AK, et al. Multicenter
assessment of a hemoglobin A1c point-of-care device for diag-
nosis of diabetes mellitus. Clin Biochem. 2018;61:18-22.
15. Lenters-Westra E, English E. Evaluation of four HbA1c
point-of-care devices using international quality targets: are
they fit for the purpose? J Diabetes Sci Technol. 2018;12:762-
770.
16. Lenters-Westra E, Slingerland RJ. Three of 7 hemoglobin A1c
point-of-care instruments do not meet generally accepted ana-
lytical performance criteria. Clin Chem. 2014;60:1062-1072.