Accepted Manuscript Title: Evaluation of point-of-care testing of C-reactive protein in forensic autopsy cases Author: Mikiko Soejima Yoshiro Koda PII: DOI: Reference:
S0379-0738(14)00032-2 http://dx.doi.org/doi:10.1016/j.forsciint.2014.01.008 FSI 7483
To appear in:
FSI
Received date: Revised date: Accepted date:
4-9-2013 9-1-2014 15-1-2014
Please cite this article as: M. Soejima, Y. Koda, Evaluation of point-of-care testing of C-reactive protein in forensic autopsy cases, Forensic Science International (2014), http://dx.doi.org/10.1016/j.forsciint.2014.01.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Evaluation of point-of-care testing of C-reactive protein in forensic autopsy cases
ip t
Mikiko Soejima and Yoshiro Koda Department of Forensic Medicine and Human Genetics, Kurume University School of
cr
Medicine, Kurume 830-0011, Japan
us
Correspondence: Yoshiro Koda, MD, PhD, Department of Forensic Medicine and
an
Human Genetics, Kurume University School of Medicine, Kurume 830-0011, Japan. Phone: +81-942-31-7554, fax: +81-942-31-7700, e-mail: [email protected]
M
Short title: Point-of-care CRP Testing in Autopsy
d
Abbreviations: CRP, C-reactive protein; PBS, phosphate buffered saline; one-way
Ac ce p
te
ANOVA, one-way analysis of variance
1
Page 1 of 19
ip t
Evaluation of point-of-care testing of C-reactive protein in forensic autopsy cases
cr
Short title: Point-of-care CRP Testing in Autopsy
us
Abbreviations: CRP, C-reactive protein; PBS, phosphate buffered saline; one-way
Ac ce p
te
d
M
an
ANOVA, one-way analysis of variance
2
Page 2 of 19
ip t
Abstract We assessed the technical performance and robustness of the point-of-care test for
cr
C-reactive protein (CRP) NycoCard CRP for use in forensic autopsy cases. The results
us
of 17 of 39 cadaver blood samples that had CRP in the range effectively measured by
an
the NycoCard (5−120 mg/l) correlated well (r = 0.99) with those of quantitative latex agglutination immunoassay (turbidimetry), and the out-of-range NycoCard results were
M
fully consistent with those obtained by turbidimetry. For the ten sera whose CRP >120
d
mg/l according to NycoCard, a significant correlation (r = 0.98) was observed between
te
values multiplied by the dilution ratio and those of turbidimetry. No significant
Ac ce p
differences were observed after a freeze-thaw procedure. In addition, CRP results using recombinant human CRP spiked with hemoglobin up to 80 g/l were not significantly different from the unspiked results in PBS. The test allows reliable and cost-effective on-site measurement of CRP from a small volume of serum (5 μl) with simple equipment. This semi-quantification method of CRP should be useful for diagnosis during autopsy. Keywords: CRP, point-of-care, autopsy cases, validation
3
Page 3 of 19
ip t
1. Introduction Examinations of biochemical markers are becoming essential in evaluating the
cr
antemortem pathological status [1-4]. One of them, C-reactive protein (CRP), is
us
increased in concentration in blood during acute phase response to tissue injury,
an
infection, or other inflammatory stimuli [5]. Postmortem CRP levels seem to be lower than the corresponding antemortem levels but were not significantly affected by a
M
postmortem interval up to 75 days in many cases [6-9], although a gradual decline
d
during sequential sampling from cadavers was observed [10]. Fujita et al. reported that
te
CRP reflects survival time after trauma and is useful for differentiating acute and
Ac ce p
non-acute death [7]. Several studies suggested that CRP is a useful postmortem marker of sepsis [10], hyperthermia [11], and both alcoholic and diabetic ketoacidosis [6, 8]. Given the usefulness of CRP, the result should ideally be available on site
during the autopsy. In addition, a method that requires only a small amount of specimen is desirable for cases with reduced blood volume. In view of such circumstances, recent studies have demonstrated the usefulness of simple CRP detection kits and liver as alternative source in autopsy cases [6, 12]. The aim of this study was to evaluate the applicability of the NycoCard® CRP
4
Page 4 of 19
point-of-care testing system in forensic autopsy cases through comparison with a
ip t
documented laboratory method. 2. Materials and Methods
cr
2.1 Blood samples
us
This study protocol was approved by the ethical committee of Kurume
an
University School of Medicine. The blood was obtained from the femoral vein, heart, or thoracic cavity when blood was not available from a vessel in 39 autopsy cases
M
performed in our department during two years from October 2011 to August 2013. The
d
postmortem interval for sampling of specimens was estimated to be 8−120 h. The serum
te
was separated by centrifugation at 800 g for 3 min, isolated, and either used
Ac ce p
immediately or stored at -20ºC. Sera whose CRP values exceeded the measurement range of NycoCard (120 mg/l) were diluted eight-fold with phosphate buffered saline (PBS, pH 7.4).
2.2 CRP measurements
CRP of serum of the same origin was quantified by different methods
(NycoCard® CRP test and quantitative latex agglutination immunoassay). The NycoCard® CRP test device together with its dedicated color densitometer, NycoCard Reader II (Axis-Shield, Rodelokka, Oslo, Norway), was used. The test system is based
5
Page 5 of 19
on an immunometric principle, and the measurement is performed by diluting 5 μl of
ip t
serum in the capillary straw in a tube containing about 380 μl of diluent. After mixing 10 sec, 50 μl of this diluted sample was applied to the test device using a dedicated pipet.
cr
After the diluted sample had soaked into the membrane, one drop of the conjugate
us
solution was applied and one drop of the washing solution was applied to remove the
an
excess conjugate. Gold particles coupled to CRP antibodies in the conjugate give the membrane a purple-reddish color proportional to the CRP concentration, and the CRP
M
value is measured as the intensity of the color on the membrane semi-quantitatively by
d
the NycoCard Reader II.
te
Measurement of serum CRP by quantitative latex agglutination immunoassay
Ac ce p
was performed by SRL, Inc. (Tokyo, Japan) by using a Nanopia Wide Range C-Reactive Protein (CRP) reagent kit (Sekisui Medical Co., Ltd., Tokyo, Japan) and JCA-BM 8060 (JEOL Ltd., Tokyo, Japan). 2.3 Evaluation of effect of hemoglobin To evaluate the effect of hemoglobin on the measurement, a solution of
recombinant human CRP (Oriental Yeast Co., Ltd., Tokyo, Japan) at a final concentration of 50 mg/l in 100 μl of PBS (pH 7.4) was spiked with a hemoglobin reference solution (160 g/l) (Alfresa Pharma Corp., Osaka, Japan) to final
6
Page 6 of 19
concentrations of 0, 5, 10, 20, 40, and 80 g/l. Reactions were performed in triplicate,
ip t
and their average and standard deviations were calculated.
cr
2.4 Statistical analyses
us
Linear regression analysis was used to investigate the correlation between the
an
CRP values determined by NycoCard and turbidimetry. To test the effect of freezing and thawing on the CRP value determined by NycoCard, Student’s two-tailed paired t test
M
was used with a 5% level of significance. To evaluate the inhibitory effect of
d
hemoglobin, one-way ANOVA followed by a χ2 test and Bartlett’s test were performed.
te
Statistical analyses were performed using the STATMate IV for Windows software
Ac ce p
(ATMS Co., Ltd., Tokyo, Japan) or Microsoft Excel 2010 (Redmond, WA). 3. Results and Discussion
NycoCard allows measurement of CRP in not only serum but also whole blood. However, the values should be corrected for hematocrit when using whole blood samples. In this study, we used serum because we had no hematocrit data on samples and for comparison with the quantitative latex agglutination immunoassay (turbidimetry), which uses serumAcknowledgements
7
Page 7 of 19
We thank Ms. Katherine Ono for the English editing of this manuscript. This work
ip t
was supported by grants-in-aid for Scientific Research from the Ministry of Education,
cr
Science, Culture and Sports of Japan.
us
.
We measured CRP by turbidimetry and NycoCard on 39 autopsy cases.
an
Turbidimetry gave results for all 39 samples, whereas the NycoCard gave values for 17
M
samples with CRP within the range of 5−120 mg/l by turbidimetry, and the two sets of results were closely related (p = 4.68×10-13, Fig. 1). All sera with a value less than 5
d
mg/l by turbidimetry (n =12) were recorded as 120 mg/l by the NycoCard. Ten samples
Ac ce p
with CRP >120 mg/l were diluted eight-fold and re-evaluated, and the eight-fold values were compared with those of turbidimetry. These results also correlated well (p = 9.45 ×10-7, Fig. 2). Next, we investigated the effects of freezing and thawing on the CRP values of 11 samples. All five samples with CRP values of out-of-range remained unchanged after single freeze-thaw cycle, and no significant differences were detected in sera with CRP concentrations of 5 to 120 mg/l (p = 0.45, n = 6). This result suggests that freezing and thawing does not make a great difference in the CRP concentration. The next step concerned analytical specificity because inhibitory effects due to 8
Page 8 of 19
blood components have sometimes been observed in biochemical examinations.
ip t
Because there was no data on hemoglobin concentration [13], which is an important factor in autopsy samples with hemolysis, we examined the effect of hemoglobin on
cr
CRP values by adding various concentrations to recombinant human CRP at a final
us
concentration of 50 mg/l, but the effect was not statistically significant up to 80 g/l
an
(Table 1).
Suzuki et al. reported a false increase of CRP specific to NycoCard in whole
M
bloods of heated or putrefied autopsy cases or experimental cases [12]. However, the
d
results of our five serum samples from burn cases (two of them were 120 mg/l CRP (n = 10).
Ac ce p
te
d
values were multiplied by eight.
M
Sera were diluted 8-fold with PBS and measured by the NycoCard CRP. The resultant
14
Page 14 of 19
(mg/l)
(mg/l)
0 5
54 56
3 1
10 20 40 80
59 57 61 60
3 1 3 5
p = 0.10
cr
SD
us
Average
Ac ce p
te
d
M
an
Hemoglobin (g/l)
ip t
Table 1 Effect of hemoglobin on measured values
15
Page 15 of 19
i cr us
an
Table 2 Differences between turbidimetry and NycoCard CRP
NycoCard CRP
measurement principle
quantitative latex agglutination immunoassay
solid phase immunoassay using
measurement range specimens
using antibody-coated latex particles 0.1−420 mg/l serum, 500 μl
gold-antibody-conjugates 5−120 mg/l serum or whole blood, 5 μl
M
Turbidimetry (outsourced)
ed
(including dead volume) two days (to obtain result from clinical laboratory testing company, 10-15 min for analysis)
pt
time required
Ac
ce
inhibitors 1. no significant effects (upper limit or range)
bilirubin (50 mg/dl)a ascorbic acid (100 mg/dl)a intralipos (10%)a
bilirubin (85−680 μmol/l)b serum amyloid P component (500 mg/l)b rheumatoid factor (not shown)b
hemoglobin (1000 mg/dl)a
hemoglobin (80 g/l)c intralipid (3–10 g/l)b, d
2. exerted effects a
2 min (handling time)
instructions of Nanopia Wide Range C-Reactive Protein (CRP) reagent kit. reference [13]. c this study. d observed a tendency to overestimate CRP by 10−20% in samples with >10 mg/l CRP by adding Intralipid. b
16
Page 16 of 19
Ac ce p
te
d
M
an
us
cr
ip t
Fig. 1
18
Page 17 of 19
Ac ce p
te
d
M
an
us
cr
ip t
Fig. 2
19
Page 18 of 19
Acknowledgements
ip t
We thank Ms. Katherine Ono for the English editing of this manuscript. This work was supported by grants-in-aid for Scientific Research from the Ministry of Education,
Ac ce p
te
d
M
an
us
cr
Science, Culture and Sports of Japan.
21
Page 19 of 19
S0379-0738(14)00032-2 http://dx.doi.org/doi:10.1016/j.forsciint.2014.01.008 FSI 7483
To appear in:
FSI
Received date: Revised date: Accepted date:
4-9-2013 9-1-2014 15-1-2014
Please cite this article as: M. Soejima, Y. Koda, Evaluation of point-of-care testing of C-reactive protein in forensic autopsy cases, Forensic Science International (2014), http://dx.doi.org/10.1016/j.forsciint.2014.01.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Evaluation of point-of-care testing of C-reactive protein in forensic autopsy cases
ip t
Mikiko Soejima and Yoshiro Koda Department of Forensic Medicine and Human Genetics, Kurume University School of
cr
Medicine, Kurume 830-0011, Japan
us
Correspondence: Yoshiro Koda, MD, PhD, Department of Forensic Medicine and
an
Human Genetics, Kurume University School of Medicine, Kurume 830-0011, Japan. Phone: +81-942-31-7554, fax: +81-942-31-7700, e-mail: [email protected]
M
Short title: Point-of-care CRP Testing in Autopsy
d
Abbreviations: CRP, C-reactive protein; PBS, phosphate buffered saline; one-way
Ac ce p
te
ANOVA, one-way analysis of variance
1
Page 1 of 19
ip t
Evaluation of point-of-care testing of C-reactive protein in forensic autopsy cases
cr
Short title: Point-of-care CRP Testing in Autopsy
us
Abbreviations: CRP, C-reactive protein; PBS, phosphate buffered saline; one-way
Ac ce p
te
d
M
an
ANOVA, one-way analysis of variance
2
Page 2 of 19
ip t
Abstract We assessed the technical performance and robustness of the point-of-care test for
cr
C-reactive protein (CRP) NycoCard CRP for use in forensic autopsy cases. The results
us
of 17 of 39 cadaver blood samples that had CRP in the range effectively measured by
an
the NycoCard (5−120 mg/l) correlated well (r = 0.99) with those of quantitative latex agglutination immunoassay (turbidimetry), and the out-of-range NycoCard results were
M
fully consistent with those obtained by turbidimetry. For the ten sera whose CRP >120
d
mg/l according to NycoCard, a significant correlation (r = 0.98) was observed between
te
values multiplied by the dilution ratio and those of turbidimetry. No significant
Ac ce p
differences were observed after a freeze-thaw procedure. In addition, CRP results using recombinant human CRP spiked with hemoglobin up to 80 g/l were not significantly different from the unspiked results in PBS. The test allows reliable and cost-effective on-site measurement of CRP from a small volume of serum (5 μl) with simple equipment. This semi-quantification method of CRP should be useful for diagnosis during autopsy. Keywords: CRP, point-of-care, autopsy cases, validation
3
Page 3 of 19
ip t
1. Introduction Examinations of biochemical markers are becoming essential in evaluating the
cr
antemortem pathological status [1-4]. One of them, C-reactive protein (CRP), is
us
increased in concentration in blood during acute phase response to tissue injury,
an
infection, or other inflammatory stimuli [5]. Postmortem CRP levels seem to be lower than the corresponding antemortem levels but were not significantly affected by a
M
postmortem interval up to 75 days in many cases [6-9], although a gradual decline
d
during sequential sampling from cadavers was observed [10]. Fujita et al. reported that
te
CRP reflects survival time after trauma and is useful for differentiating acute and
Ac ce p
non-acute death [7]. Several studies suggested that CRP is a useful postmortem marker of sepsis [10], hyperthermia [11], and both alcoholic and diabetic ketoacidosis [6, 8]. Given the usefulness of CRP, the result should ideally be available on site
during the autopsy. In addition, a method that requires only a small amount of specimen is desirable for cases with reduced blood volume. In view of such circumstances, recent studies have demonstrated the usefulness of simple CRP detection kits and liver as alternative source in autopsy cases [6, 12]. The aim of this study was to evaluate the applicability of the NycoCard® CRP
4
Page 4 of 19
point-of-care testing system in forensic autopsy cases through comparison with a
ip t
documented laboratory method. 2. Materials and Methods
cr
2.1 Blood samples
us
This study protocol was approved by the ethical committee of Kurume
an
University School of Medicine. The blood was obtained from the femoral vein, heart, or thoracic cavity when blood was not available from a vessel in 39 autopsy cases
M
performed in our department during two years from October 2011 to August 2013. The
d
postmortem interval for sampling of specimens was estimated to be 8−120 h. The serum
te
was separated by centrifugation at 800 g for 3 min, isolated, and either used
Ac ce p
immediately or stored at -20ºC. Sera whose CRP values exceeded the measurement range of NycoCard (120 mg/l) were diluted eight-fold with phosphate buffered saline (PBS, pH 7.4).
2.2 CRP measurements
CRP of serum of the same origin was quantified by different methods
(NycoCard® CRP test and quantitative latex agglutination immunoassay). The NycoCard® CRP test device together with its dedicated color densitometer, NycoCard Reader II (Axis-Shield, Rodelokka, Oslo, Norway), was used. The test system is based
5
Page 5 of 19
on an immunometric principle, and the measurement is performed by diluting 5 μl of
ip t
serum in the capillary straw in a tube containing about 380 μl of diluent. After mixing 10 sec, 50 μl of this diluted sample was applied to the test device using a dedicated pipet.
cr
After the diluted sample had soaked into the membrane, one drop of the conjugate
us
solution was applied and one drop of the washing solution was applied to remove the
an
excess conjugate. Gold particles coupled to CRP antibodies in the conjugate give the membrane a purple-reddish color proportional to the CRP concentration, and the CRP
M
value is measured as the intensity of the color on the membrane semi-quantitatively by
d
the NycoCard Reader II.
te
Measurement of serum CRP by quantitative latex agglutination immunoassay
Ac ce p
was performed by SRL, Inc. (Tokyo, Japan) by using a Nanopia Wide Range C-Reactive Protein (CRP) reagent kit (Sekisui Medical Co., Ltd., Tokyo, Japan) and JCA-BM 8060 (JEOL Ltd., Tokyo, Japan). 2.3 Evaluation of effect of hemoglobin To evaluate the effect of hemoglobin on the measurement, a solution of
recombinant human CRP (Oriental Yeast Co., Ltd., Tokyo, Japan) at a final concentration of 50 mg/l in 100 μl of PBS (pH 7.4) was spiked with a hemoglobin reference solution (160 g/l) (Alfresa Pharma Corp., Osaka, Japan) to final
6
Page 6 of 19
concentrations of 0, 5, 10, 20, 40, and 80 g/l. Reactions were performed in triplicate,
ip t
and their average and standard deviations were calculated.
cr
2.4 Statistical analyses
us
Linear regression analysis was used to investigate the correlation between the
an
CRP values determined by NycoCard and turbidimetry. To test the effect of freezing and thawing on the CRP value determined by NycoCard, Student’s two-tailed paired t test
M
was used with a 5% level of significance. To evaluate the inhibitory effect of
d
hemoglobin, one-way ANOVA followed by a χ2 test and Bartlett’s test were performed.
te
Statistical analyses were performed using the STATMate IV for Windows software
Ac ce p
(ATMS Co., Ltd., Tokyo, Japan) or Microsoft Excel 2010 (Redmond, WA). 3. Results and Discussion
NycoCard allows measurement of CRP in not only serum but also whole blood. However, the values should be corrected for hematocrit when using whole blood samples. In this study, we used serum because we had no hematocrit data on samples and for comparison with the quantitative latex agglutination immunoassay (turbidimetry), which uses serumAcknowledgements
7
Page 7 of 19
We thank Ms. Katherine Ono for the English editing of this manuscript. This work
ip t
was supported by grants-in-aid for Scientific Research from the Ministry of Education,
cr
Science, Culture and Sports of Japan.
us
.
We measured CRP by turbidimetry and NycoCard on 39 autopsy cases.
an
Turbidimetry gave results for all 39 samples, whereas the NycoCard gave values for 17
M
samples with CRP within the range of 5−120 mg/l by turbidimetry, and the two sets of results were closely related (p = 4.68×10-13, Fig. 1). All sera with a value less than 5
d
mg/l by turbidimetry (n =12) were recorded as 120 mg/l by the NycoCard. Ten samples
Ac ce p
with CRP >120 mg/l were diluted eight-fold and re-evaluated, and the eight-fold values were compared with those of turbidimetry. These results also correlated well (p = 9.45 ×10-7, Fig. 2). Next, we investigated the effects of freezing and thawing on the CRP values of 11 samples. All five samples with CRP values of out-of-range remained unchanged after single freeze-thaw cycle, and no significant differences were detected in sera with CRP concentrations of 5 to 120 mg/l (p = 0.45, n = 6). This result suggests that freezing and thawing does not make a great difference in the CRP concentration. The next step concerned analytical specificity because inhibitory effects due to 8
Page 8 of 19
blood components have sometimes been observed in biochemical examinations.
ip t
Because there was no data on hemoglobin concentration [13], which is an important factor in autopsy samples with hemolysis, we examined the effect of hemoglobin on
cr
CRP values by adding various concentrations to recombinant human CRP at a final
us
concentration of 50 mg/l, but the effect was not statistically significant up to 80 g/l
an
(Table 1).
Suzuki et al. reported a false increase of CRP specific to NycoCard in whole
M
bloods of heated or putrefied autopsy cases or experimental cases [12]. However, the
d
results of our five serum samples from burn cases (two of them were 120 mg/l CRP (n = 10).
Ac ce p
te
d
values were multiplied by eight.
M
Sera were diluted 8-fold with PBS and measured by the NycoCard CRP. The resultant
14
Page 14 of 19
(mg/l)
(mg/l)
0 5
54 56
3 1
10 20 40 80
59 57 61 60
3 1 3 5
p = 0.10
cr
SD
us
Average
Ac ce p
te
d
M
an
Hemoglobin (g/l)
ip t
Table 1 Effect of hemoglobin on measured values
15
Page 15 of 19
i cr us
an
Table 2 Differences between turbidimetry and NycoCard CRP
NycoCard CRP
measurement principle
quantitative latex agglutination immunoassay
solid phase immunoassay using
measurement range specimens
using antibody-coated latex particles 0.1−420 mg/l serum, 500 μl
gold-antibody-conjugates 5−120 mg/l serum or whole blood, 5 μl
M
Turbidimetry (outsourced)
ed
(including dead volume) two days (to obtain result from clinical laboratory testing company, 10-15 min for analysis)
pt
time required
Ac
ce
inhibitors 1. no significant effects (upper limit or range)
bilirubin (50 mg/dl)a ascorbic acid (100 mg/dl)a intralipos (10%)a
bilirubin (85−680 μmol/l)b serum amyloid P component (500 mg/l)b rheumatoid factor (not shown)b
hemoglobin (1000 mg/dl)a
hemoglobin (80 g/l)c intralipid (3–10 g/l)b, d
2. exerted effects a
2 min (handling time)
instructions of Nanopia Wide Range C-Reactive Protein (CRP) reagent kit. reference [13]. c this study. d observed a tendency to overestimate CRP by 10−20% in samples with >10 mg/l CRP by adding Intralipid. b
16
Page 16 of 19
Ac ce p
te
d
M
an
us
cr
ip t
Fig. 1
18
Page 17 of 19
Ac ce p
te
d
M
an
us
cr
ip t
Fig. 2
19
Page 18 of 19
Acknowledgements
ip t
We thank Ms. Katherine Ono for the English editing of this manuscript. This work was supported by grants-in-aid for Scientific Research from the Ministry of Education,
Ac ce p
te
d
M
an
us
cr
Science, Culture and Sports of Japan.
21
Page 19 of 19
