Peformance Evaluation of Bioporto Diagnostics’ Neutrophil Gelatinase-associated Lipocalin Assay on Automated Clinical Chemistry Analyzer Hitachi 7600

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Peformance Evaluation of Bioporto Diagnostics’ Neutrophil Gelatinase-associated Lipocalin Assay on Automated Clinical Chemistry Analyzer Hitachi 7600

Youkyung Seo, Woonhyoung Lee, and Oh Hun Kwon

Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea

자동화학분석기용 NGAL Test 검사를 이용한 Neutrophil Gelatinase-associated Lipocalin의 분석능 평가

서유경ㆍ이운형ㆍ권오헌 연세대학교 의과대학 진단검사의학과

Received: February 4, 2013 Revision received: June 11, 2013 Accepted: June 12, 2013

Correspondent to: Woonhyoung Lee Department of Laboratory Medicine, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Korea

Tel: +82-2-2228-2449 Fax: +82-2-364-1583 E-mail: [email protected]

Background: Neutrophil gelatinase-associated lipocalin (NGAL) is known to be one of the ideal biomarkers for acute kidney injury providing early information on damage to the kidney.

Methods: We evaluated the performance for precision and the reportable range of the automated NGAL Test (Bioporto Diagnostics, Denmark) assay and compared the values of these tests with widely used point of care test. The reference interval of NGAL was established in Korean adults.

Results: Within run percent coefficient of variation (%CV) and total precision %CV for 2 levels were all within 5%. The reportable range was found to be acceptable for the range of 57.0 - 3182.0 ng/mL (r=0.999). The method comparison was made between Biosite’s assay and Bioporto Diagnostics’ (Passing and Bablok fit, y=1.94x–

65.29; x, Biosite; y, Bioporto; n=31; y range, 250 to 1,308 ng/mL; r

=0.959). The correlation was linear within the limit of 1,500 ng/mL, but not beyond this limit. The 2.5 and 97.5 percentile of the reference range for the samples were 43.2 ng/mL and 124.8 ng/mL, respectively.

Conclusions: Since NGAL Test can be used in automated chemical analyzer, it can not only reduce the man power and time consumed in but also displayed excellent precision and linearity.

Key Words: Acute kidney injury, Neutrophil gelatinase-associated lipocalin, Immunoassay, Turbidimetry

INTRODUCTION

Acute kidney injury (AKI), previously referred to as acute renal failure, can result from decreased renal perfusion; exposure to aminoglycosides under the setting of sepsis; administration of radiocontrast agents in patients receiving angio-

tensin converting enzyme inhibitors; treatment with nonsteroidal antiinflmmatory agents in the presence of congestive heart failure; reperfusion following cardiopulmonary bypass surgery or kidney transplant [1]. It occurs in 5% to 7% of hospitalized patients. In intensive care unit, the prevalence rate of patients with AKI requiring

J Lab Med Qual Assur 2013;35:23-8

hemodialysis is approximately 6% with an associated mortality rate of 60% [2-4].

Risk, Injury, Failure, Loss, End Stage Kidney Disease and the Acute Kidney Injury Network are recent consensus classification systems for AKI.

Both rely on changes in serum creatinine level from baseline reflecting changes in the glomerular filtration rate. However, there is some limitation on creatinine level for AKI. Creatinine elevation is observed within 24 to 48 hours following the onset of AKI. This time constraint of serum creatinine increases the risk for failure in treatment.

Furthermore, when baseline glomerular filtration rate is not known, it will be impossible to accurately determine the degree of decline in renal function.

Definitely, creatinine does not directly reflect cell injury, but rather the delayed functional consequence of the damage [2,5-9].

Several biomarkers including neutrophil gelatinase-associated lipocalin (NGAL), cystatin C, interleukin 18 and kidney injury molecule 1 have been investigated for human populations. Because they are sequential biomarkers, it is likely that the AKI panels will be useful for timing the initial insult and assessing the duration of AKI (analogous to the cardiac panel for evaluating chest pain). It is also likely that the AKI panels will help distinguish between the various types and pathogeneses of AKI [10-12].

Preclinical transcriptome profiling studies identified NGAL to be one of the most upregulated genes in the kidney very early following acute injury. A number of studies have now implicated NGAL as an early diagnostic biomarker for AKI in common clinical situations, such as kidney transplantation, cardiac surgery, and contrast nephropathy. In a recent study of adults under the emergency department setting, a single measurement of NGAL at the time of initial presentation predicted AKI with an outstanding area under the curve of 0.95 and reliably distinguished prerenal azotemia from intrinsic AKI and from chronic kidney disease [3,13-15]. Bioporto Diagnostic’s NGAL assay was developed for application in automated clinical chemistry analyzer. The performance of this reagent is evaluated in this study.

MATERIALS AND METHODS 1. Reagent and Instruments

Plasma NGAL was measured using a particle enhanced turbidimetric immunoassay, NGAL Test (Bioporto Diagnostics, Gentofte, Denmark) on Hitachi 7600 (Hitachi, Tokyo, Japan). The dedicated reagents for the test were used according to the instructions.

2. Samples

The study was carried out with the approval of institutional review board of Severance Hospital.

For the purpose of comparing the method with Triage NGAL Test (Biosite, San Diego, CA, USA) and establishing reference interval, plasma submitted for routine hemoglobin A1c testing in the health promotion center was used. Residual ethylenediaminetetraacetic acid (EDTA) plasma was collected by centrifugation at 3,000 g for 10 minutes.

3. Evaluation 1) Precision

Precision was evaluated by taking measurements 4 times a day over a period of 5 days in accordance with the Clinical and Laboratory Standard Institute (CLSI) EP15 A2 by using two level dedicated quality control materials (200 and 500 ng/mL).

2) Reportable range

EDTA plasma with low NGAL concentration of 57 ng/mL and that with high NGAL concentration of 3,182 ng/mL were proportionately mixed in accordance with CLSI EP6 A. We tested 2 replicates at each of the levels.

3) Method comparison

Comparison was carried out in compliance with the CLSI guideline EP9 A2 [16]. After having examined residual laboratory samples of EDTA whole blood of 39 with Triage NGAL Test, plasma obtained by centrifugation was measured with NGAL Test reagent on Hitachi 7600 automatic chemical analyzer.

4) Reference interval

Total of 129 adults composed of 63 males and 66

Table 1. Precision at different neutrophil gelatinase-associated lipocalin concentration

Quality control Grand mean (ng/mL) S

(ng/mL) CV

(%) S

(ng/mL) CV

(%)

Low 210.6 6.0 2.8 6.9 3.3

High 514.0 7.3 1.4 9.5 1.8

S

, within run standard deviation; CV

, within run percent coefficient of variation; S

, total standard

deviation; CV

, total precision coefficient of variation.

Table 2. Reference intervals of the plasma neutrophil gelatinase-associated lipocalin level in Korean adults according to gender

Lowest (ng/mL) 2.5 Percentile (ng/mL) 97.5 Percentile (ng/mL) Highest (ng/mL)

Male (n=63) 43 44.6 124.5 134

Female (n=66) 35 41.3 124.5 155

Total (n=129) 35 43.2 124.8 155

Fig. 1. Reportable range of the Triage NGAL Test.

NGAL, neutrophil gelatinase-associated lipocalin.

females were included as subjects. Those who had history of administration or underwent surgery during the last 6 months as well as case of chronic disease were excluded by referencing electronic medical record. Other exclusion criteria included glucose level of less than 50 mg/dL or more than 126 mg/dL, aspartate aminotransferase level of more than 36 U/L for male and more than 30 U/L for female, alanine aminotransferase level of more than 50 U/L, Hemoglobin level of less than 11 g/dL, white blood cell level of less than 3,000/mL or more than 15,000/mL, blood urea nitrogen level of less than 5 mg/dL or more than 25 mg/dL, and creatinine level of less than 0.5 mg/dL or more than 1.4 mg/dL [17].

5) Statistical analyses

Analyze it for Microsoft Excel ver. 2.25 was used.

RESULTS 1. Precision

Grand means of low and high level quality control materials were 210.6 and 514.0 ng/mL, respec- tively. Within run percent coefficient of variation (%CV) and total precision %CV were 2.8% and 3.3% at low and 1.4% and 1.8% at high, respectively (Table 1).

2. Reportable Range

The analytical measurement range specified by the manufacturer was 25 to 5,000 ng/mL while that of the NGAL Test reagent on Hitachi 7600 validated in this experiment was 57.0 to 3,182.0 ng/mL. The coefficient of correlation (r) was 0.999 within that range (Fig. 1).

3. Method Comparison

The coefficient of determination (r

) is 0.791

between the Bioporto’s and Biosite’s systems

(Passing and Bablok fit, y=2.29x–179.26; x,

Biosite; y, Bioporto; n=39; y range, 250 to 2,541

ng/mL). But a much better correlation (r

=0.959)

is noted when 8 results with values greater than

1,308 ng/mL for the NGAL Test were excluded

(Passing and Bablok fit, y=1.94x - 65.29; x,

Biosite; y, Bioporto; n=31; y range, 250 to 1,308

ng/mL) (Fig. 2).

(A) (B)

Passing and Bablok (I) fit Passing and Bablok (I) fit

Fig. 2. Passing and Bablok comparison of neutrophil gelatinase associated lipocalin (NGAL) results between NGAL Test and Triage NGAL Test with 39 EDTA specimens (A) and with 31 ones under the value of 1,300 ng/mL (B).

4. Reference Interval

The reference interval was in the range of 43.2 to 124.8 ng/mL, compared with 37 to 106 ng/mL given by the manufacturer. There was no significant difference between male and female in terms of NGAL values (P=0.31) (Table 2).

DISCUSSION

The expression of NGAL by various cell types is subject to extensive regulation. Serum levels of NGAL become elevated in renal tubular injury, bacterial infections systemic disease and acute phase response [18]. In 2003, Mishra et al. [19]

uses transcriptome wide interrogation strategy to identify renal genes that are induced early following renal ischemia, in which protein products might serve as novel biomarkers for AKI. In 2005, they studied children undergoing cardiopulmonary bypass, obtaining the result that the amount of NGAL at 2 hours was the most powerful independent predictor of AKI. For a cutoff value of 50 ug/L, the area under the receiver operating characteristic curve was 0.998, while sensitivity was 1.00, and specificity was 0.98 [20].

Recently, the range of application of NGAL has been broadened to include biomarker for AKI [9,21], clinical effectiveness analysis of dialysis in chronic kidney diseases [22], histological classification for renal cancer [23], and the risk assessment of cardio renal syndrome in AIK patients [24]. There have

been continuous reports of the clinical importance of NGAL as an AKI index along with continued development of technologies for measuring of NGAL.

However, the test thus far displayed some limitation on the clinical application. Pedersen et al. [25] found a batch to batch variation in enzyme linked immunosorbent assay (ELISA) for NGAL and in the effect of temperature on the ELISA plate.

In the study by Dent et al. [26], ELISA tests are referred to as time consuming procedure with short life span. They said that a point of care NGAL monitoring, Triage NGAL Test has good precision of within day %CV of 11% and total precision %CV of 14%. However, its reportable range was found to be below 1,300 ng/mL, thereby placing limitations at higher level of NGAL [26]. In terms of specimen being used, there were difficulties in collecting urine from patients with oliguria. Parikh et al. [27]

stated that urine NGAL was irrelevant to AKI if other factors are controlled.

On the other hand, NGAL Test has a better precision with less than 5% than that of Triage NGAL Test. Also it displayed well maintained linearity up to the concentration of 3,000 ng/mL, making it possible to report more accurate results in measuring high concentration of NGAL. Plasma used in NGAL Test was more reliable than that of urine [27].

The claimed values of precision study were <5% at

200 ng/mL and <3% at 500 ng/mL. The results of

this precision study were acceptable. The coefficient

of determination is improved when 8 results with values greater than 1,308 ng/mL for the NGAL Test were excluded. Although the upper limit of qunatitation of Triage NGAL Test was known as 1,300 ng/mL, results of Triage NGAL Test value greater than 500 ng/mL were not linearly correlated with those of the NGAL Test. Further, it enables measurement with existing automated clinical chemistry analyzer and can be applied usefully in clinical laboratories. Reference intervals of some analytes are said to be gender specific, but was not the case for NGAL in Koreans.

Since NGAL Test reagent can be used in automated chemical analyzer, it can not only reduce the man power and time consumed in but also displayed excellent precision and linearity even at high concentration. Therefore, it is deemed that it will elevate the possibility of utilization of NGAL as various indices as well as prompt diagnosis of AKI.

요 약

배경: Neutrophil gelatinase-associated lipocalin (NGAL)은 급성신손상을 조기에 예측할 수 있는 이상적인 생물학적 표지자로 알려져 있다.

방법: NGAL Test (Bioporto Diagnostics, Denmark) 시약을 Hitachi 7600 (Hitachi, Japan) 자동화학분석기 에 장착하여 정밀도와 보고 가능범위를 평가하고, 흔히 사용 되는 현장검사, Triage NGAL Test (Biosite, USA)와의 상관성을 살펴보았다. 또한 한국인에서의 참고범위를 설정하 였다.

결과: 검사 내 정밀도 변이계수와 총 정밀도는 정도관리물 질 두 농도에서 모두 5% 이내였다. 57.0 - 3,182.0 ng/mL 의 범위에서 우수한 직선성을 보였다(r=0.999). 현장검사 인 Triage NGAL Test와의 상관성 평가에서 상관계수(r

) 는 0.979이었다(Passing and Bablok fit, y=1.94x 65.29; x, Biosite; y, Bioporto; n=31; y range, 250 to 1,308 ng/mL). 본 실험에서 확인한 한국인의 참고범위 는 43.2 - 124.8 ng/mL였다.

결론: NGAL Test검사는 자동화학분석기에서 사용 가능 하여 검사에 소요되는 인력과 시간을 단축시킬 뿐 아니라 높 은 농도에서도 우수한 정밀도와 직선성을 보여, NGAL의 여 러 가지 지표로서의 이용가능성을 높일 것으로 생각된다.

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