Clinica Chimica Acta 433 (2014) 145–149

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Development of a new point-of-care testing system for measuring white blood cell and C-reactive protein levels in whole blood samples Kazuhiko Kotani a,⁎, Takaomi Minami b, Toshiaki Abe c, Junji Sato c, Nobuyuki Taniguchi a, Toshiyuki Yamada a a b c

Department of Clinical Laboratory Medicine, Jichi Medical University, Shimotsuke-City, Tochigi 329-0498, Japan Pediatrics, Jichi Medical University, Shimotsuke-City, Tochigi 329-0498, Japan Eiken Chemical Co., Ltd., Shimotsuga, Tochigi 329-0114, Japan

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Article history: Received 18 November 2013 Received in revised form 31 January 2014 Accepted 7 March 2014 Available online 15 March 2014 Keywords: CRP Granulocyte esterase Inflammation Immunochromatographic assay POCT WBC Leukocyte

a b s t r a c t Background: White blood cell (WBC) count and C-reactive protein (CRP) level are the most common markers of inflammation. There is a growing need for point-of-care testing (POCT) of WBC and CRP, and more advances in convenient devices are required. We developed an analyzer-free POCT system for measuring WBC and CRP using a low volume blood sample. Methods: The POCT-WBC is based on the granulocyte esterase assay, while the POCT-CRP is based on the immunochromatographic assay. These kits were examined for precision as well as correlation with currently used popular commercial automated assays. The correlations were clinically analyzed in children with acute infection (n = 62; mean age 4.2 y). The correlations regarding the monitoring of values were further examined in several follow-up subjects. Results: The POCT-WBC and POCT-CRP kits demonstrated good precision. POCT-WBC exhibited a significantly close correlation with those of the control assay (r = 0.94, p b 0.05). The results of POCT-CRP also exhibited a significantly close correlation with those of the control assay (r = 0.94, p b 0.05). In the follow-up study, the results of the respective kits were similar to those of the control assays. Conclusions: The POCT-WBC and POCT-CRP are promising tools for assessing infection in clinical practice. © 2014 Elsevier B.V. All rights reserved.

1. Introduction White blood cell (WBC) and C-reactive protein (CRP) levels are the most commonly used blood measurements in the diagnosis and therapeutic monitoring of inflammation [1–4]. Patients with acute infection are frequently seen in a variety of settings (e.g., not only hospitals, but also primary care, home care, rural, and general practitioner facilities). Quick medical decision-making is required when treating patients with acute infection, and not all facilities maintain the machines required to measure promptly WBC and CRP [3–7]. Much attention has recently been paid to point-of-care testing (POCT) using portable, table-top, handheld and/or disposable devices operated in non-laboratory environments by non-technical staff to obtain rapid on-site results [7]. There is a growing need for POCT of WBC and CRP [2–5,8]. Some POCT devices for measuring CRP are currently available [2,4, 8–12]. Among these devices, clinical studies have been conducted using the QuikREAD test [4,6,13–17] and Nycocard test [15,17–20], in particular. Most clinical studies have indicated relevant contributions of these tests ⁎ Corresponding author at: Department of Clinical Laboratory Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-City, Tochigi 329-0498, Japan. Tel.: +81 285 58 7386; fax: +81 285 44 9947. E-mail address: [email protected] (K. Kotani).

http://dx.doi.org/10.1016/j.cca.2014.03.004 0009-8981/© 2014 Elsevier B.V. All rights reserved.

to assessing inflammation in clinical practice [6,16–20]. However, these tests generally require the additional use of specific analyzers (even though the analyzers are small relative to the machines as used in hospitals), the samples must be pretreated before measurement in some tests [12], or the reaction after measurement must be checked visually due to the principle of latex agglutination reaction in some tests [9,13]. Although the development of POCT kits for WBC may be behind that of POCT devices for CRP or hemoglobin [3], some POCT devices for WBC, for instance, the HemoCue WBC test [21] and QBC Star test [22], are currently available [3,7,9]. However, no clinical studies of these tests have been reported, and the tests also require the use of specific analyzers. Thus, further advances in the POCT devices for CRP and WBC are still required. Another important point regarding the assessment of inflammation in clinical practice is the simultaneous measurement of WBC and CRP, as these measures have somewhat different characteristics as markers [9]. The WBC level tends to increase during the superacute phase of inflammation, while the CRP level increases in the later stage [1,9,23]. The simultaneous determination of both markers can aid in understanding the phase of inflammation. Furthermore, the increase in WBC during the acute phase depends on that of granulocytes, primarily neutrophils [2], and the tests reflecting of the level of granulocytes (neutrophils) can influence the administration of antibiotics, regardless of the CRP level, since an increase in neutrophils is traditionally considered to be suggestive of bacterial infection [24].

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2. Materials and methods 2.1. POCT-WBC and hs-cRP Details of the principles and procedures of these assays are available from the authors. The results of POCT-WBC are classified into one of three grades: (1+) for a WBC level of b 10000/µl, (2+) for a WBC level of 10000 to 15000/μl, and (3+) for a WBC level of ≥ 15000/μl (the WBC levels are based on a cut-off level for clinical evaluation [25]). The results of POCT-CRP are classified into one of three grades: (−) for a CRP level of b 0.6 mg/dl, (1+) for a CRP level of 0.6 to 5 mg/dl, and (2+) for a CRP level of ≥ 5 mg/dl. 2.2. Precision of POCT-WBC and POCT-CRP The precision of the POCT-WBC and POCT-CRP kits was evaluated for with-in run, between-run and interobserver precision, using blood samples with known concentrations. As for the POCT-WBC, the blood samples were prepared with 3200/μl, 7000/μl and 13,000/μl of neutrophils, respectively. The concentrations of neutrophils were determined with a widely approved automated blood cell analyzer (Horiba Co. Ltd.). As for the POCT-CRP, the blood samples were prepared with 0, 0.6 and 5 mg/dl of CRP, respectively. The concentrations of CRP were determined based on the standardized materials certified by the code of ERM-DA474/ the International Federation of Clinical Chemistry. We tested 10 replicates at each WBC (neutrophil) or CRP level. 2.3. Correlation between the results of control assays and those of POCT-WBC or POCT-CRP kit The WBC level measured using the POCT-WBC was compared with that measured using an automated blood cell analyzer (Coulter LH500; Beckman Coulter, Inc.); this analyzer uses the electric resistance method [25]. The percentages of neutrophils were also determined using this analyzer in some cases. The CRP level measured using the POCT-CRP was compared with that measured with a reagent (Dade Behring. Diagnostics, Inc.) using an automated analyzer (7080; Hitachi High-Technologies Corp.). These control assays are popularly applied in daily practice. Correlations for the respective values were observed using Spearman's rank correlation tests. Between-group differences were observed using the Mann–Whitney U-test. A p b 0.05 was considered significant. The clinical studies were conducted using whole blood samples. First, a healthy control population without any infection (n = 8; mean age 36 y) was tested. Thereafter, the main study was performed in a population of children with acute infection (n = 62; mean age 4.2 y). The subjects visited the Jichi Medical University Hospital due to a chief complaint of a common cold or gastroenteritis, and were diagnosed with an acute infectious disease. The study was approved by the Jichi Medical University Ethics Committee, and all subjects and their parents provided written informed consent. A portion of the subjects repeatedly provided blood samples during multiple visits to the hospital according to the course of the disease. During the follow-up period, the correlations between the levels obtained using the control assays and those obtained using the POCT-WBC and POCT-CRP were investigated.

consistency of the with-in run precision, the between-run precision for 5 consecutive days was also 100%. In addition, the interobserver precision tested by different three operators was 100%. 3.2. Correlation between the results of control assays and those of the POCT-WBC or POCT-CRP In the study consisting of control subjects without any infection, their WBC levels measured using the control assay were all b10,000/μl, and those measured using the POCT-WBC all showed a grade of (1+). In the study of children with acute infection, the WBC level measured using the POCT-WBC corresponded to that measured using the control assay in 58 samples (94%), as shown in Table 1. Four samples showed inconsistent levels between the POCT-WBC and control assay. The WBC level measured using the POCT-WBC was significantly and closely correlated with that measured using the control assay (r = 0.94, p b 0.05; Fig. 1). In the study consisting of control subjects without any infection, their CRP levels measured using the control assay were all b 0.6 mg/dl, and those measured using the POCT-CRP all showed a grade of (−). In the study of those with acute infection, the CRP level measured using the POCT-CRP corresponded to that measured using the control assay in 56 samples (90%), as shown in Table 1. Six samples showed inconsistent levels between the POCT-CRP and control assay. The CRP level measured using the POCT-CRP was significantly and closely correlated with that measured using the control assay (r = 0.94, p b 0.05; Fig. 2). Additionally, we observed the correlation between the levels obtained using the POCT-WBC and POCT-CRP in the study of acute infection. A weak correlation was seen in this population (Table 1). 3.3. Follow-up study of the correlation between the results of control assays and those of the POCT-WBC or POCT-CRP In 4 subjects, we investigated blood samples at least four times during the follow-up period. The levels obtained using the POCT-WBC or POCTCRP were similar to those obtained using the control assays (Fig. 3). 4. Discussion Today, the WBC and CRP levels are crucial markers for the diagnosis and therapeutic monitoring of inflammatory disorders, such as infectious diseases. Under a growing need for POCT of WBC and CRP, some

Table 1 Consistency between the POCT-WBC and CRP levels against automated analyzer (control assay) and against themselves. WBC counts (control assay)

POCT-WBC

POCT-CRP

3. Results 3.1. Precision of the POCT-WBC and POCT-CRP Upon replication, the samples prepared with 3200/μl, 7000/μl and 13,000/μl of neutrophils always corresponded to the semi-quantitative grade of (1 +), (2 +) and (3 +) for WBC, respectively, on the POCTWBC. Similarly, the samples prepared with 0, 0.6, and 5 mg/dl of CRP always corresponded to the semi-quantitative grade of (−), (1+) and (2 +) of CRP, respectively, on the POCT-CRP. In line with the 100% in

POCT-CRP

1+ 2+ 3+ Total

– 1+ 2+ Total

– 1+ 2+ Total

Total

b10,000/μl

10,000 to 15,000/μl

≥15,000/μl

38 0 0 38

3 15 0 18

0 1 5 6

CRP (control assay) b0.6 mg/dl 0.6 to 5 mg/dl

≥5 mg/dl

32 2 0 34

1 15 2 18

0 1 9 10

POCT-WBC 1+

2+

3+

26 6 1 33

11 5 2 18

4 5 2 11

41 16 5 62 Total 33 18 11 62 Total

WBC: white blood cell, POCT: point-of-care testing, CRP: C-reactive protein.

41 16 5 62

K. Kotani et al. / Clinica Chimica Acta 433 (2014) 145–149

Fig. 1. Correlation between the WBC level measured using the POCT-WBC and that measured using an automated analyzer (control assay). WBC: white blood cell, POCT: point-of-care testing.

POCT devices for measuring WBC and CRP are currently available, while advances in these POCT devices are still required. We recently developed new POCT-WBC and POCT-CRP kits. The principal methods of the respective kits would be worth noting. The POCT-WBC is based on the granulocyte esterase assay. The idea of POCT according to a method based on the granulocyte esterase activity was previously reported in POCT using urine and other fluids [26,27]; however, such methods have not been applied to whole blood samples. Because protease inhibitors and elastases present in plasma can hinder the measurement of the WBC-derived activity of granulocyte esterase [28], we integrated a WBC-capture membrane into the POCT-WBC device system in order to capture WBCs in whole blood samples, thereby specifically measuring the WBC-derived esterase activity. On the other hand, the POCT-CRP is based on the immunochromatographic assay as the first application of a POCT system measuring the CRP level. We placed a blood separation membrane in the sample well that allows the device to perform plasma separation in order to test whole blood samples.

(mg/dl)

16

CRP (control assay)

14 12 10 8 6 4 2 0



1+

2+

POCT-CRP Fig. 2. Correlation between the CRP level measured by the POCT-CRP and that measured using an automated blood cell analyzer (control assay). CRP: C-reactive protein, POCT: point-of-care testing.

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These kits also have some advantages in comparison to other POCT for WBC and CRP; i.e., the POCT-WBC and POCT-CRP kits are strip-typed and analyzer-free (thereby, the tests can be performed using a simple process and they can also be used without any maintenance), while the findings obtained using these devices remain only a semi-quantitative evaluation. The present study showed good precision for both kits, as well as good correlations between the results obtained using the kits and those obtained using the control assays in a clinical subject population. These findings suggest that the kits can be used in screening for acute infection as an alternative method to tests. This is useful as quick decision-making is required in patients with acute infection and such patients are frequently seen in various facilities where analyzers used to measure WBC and CRP is not available [3–7]. In the follow-up study of acute infection, the results obtained using the respective kits also showed similar changes to those obtained using the control assays. It may also be possible to use these kits to monitor the disease course. The correlation between the results obtained using the POCTWBC and those obtained using the POCT-CRP was weak in the present study. The inconsistency between these measurements may be possibly due to the fact that the levels of WBC and CRP can differ according to the phase of inflammation [1,9,23]. Obtaining simultaneous measurements of WBC and CRP is meaningful in clinical practice. It would be important to note that the simplicity of our developed kits makes simultaneously measuring the WBC and CRP levels possible at the POCT level. There are several limitations to this study and issues to be considered. Again, the POCT-WBC and POCT-CRP kits provide semiquantitative values classified into 1 of 3 grades. We must acknowledge that this is not suitable in situations requiring quantitative judgment, even though circumstances requiring an exact quantity may be infrequent. In addition, the study population was small and, basically, restricted to children with acute infectious diseases; therefore, the results should be generalized with care. Clinical studies using the POCT devices for CRP have recently focused on reducing the administration of antibiotics and its related costs [4,6,12,14, 16–20,29,30], whereas the present study did not include such an evaluation. Finally, the results obtained using the POCT-WBC and control assay were mostly concordant, and even in the discordant cases, the discordant levels were close in the present study. However, determining the WBC level by measuring the granulocyte esterase activity in the POCT-WBC kit remains to raise questions regarding the rights and wrongs in some cases. The esterase activity of granulocytes (primarily neutrophils [2]) reflects a possible bacterial infection [24], enhancing the meaning of simultaneous measurements of the CRP level [9]. On the other hand, some patients (e.g., those with neutropenia [31]) may not necessarily exhibit a relative action on the POCT-WBC, perhaps resulting in troublesome decision-making. In fact, we examined 4 cases showing a discordant level of the POCT-WBC and also carried out a control assay in this study. In one discordant case ((+ 2) in the POCT-WBC and N15,000/μl in the control assay), the neutrophils (59%) seemed low relative to those observed in other concordant cases (median 82%, range = 56–88%, p N 0.05). In 3 discordant cases ((+ 1) in the POCT-WBC and 10,000 to 15,000/μl in the control assay), the neutrophils (median 34%, range = 15–49%) were significantly lower than those seen in other concordant cases (median 65%, range = 37–80%, p = 0.02). While not all discordant cases displayed neutropenia, the degree of the neutrophils' response to infection could affect the expected concordance with the WBC level. Conflict of interest This study was partly supported by a research grant from Eiken Chemical Co., Ltd.

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Fig. 3. Results of the follow-up study according to the disease course of patients. Left panel: the WBC levels measured using an automated analyzer (control assay: filled square) and as the POCT-WBC (open circle). Right panel: the CRP levels measured using an automated analyzer (control assay: filled square) and the POCT-CRP (open circle). WBC: white blood cell, POCT: point-of-care testing, CRP: C-reactive protein.

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Development of a new point-of-care testing system for measuring white blood cell and C-reactive protein levels in whole blood samples.

White blood cell (WBC) count and C-reactive protein (CRP) level are the most common markers of inflammation. There is a growing need for point-of-care...
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