T E C H N I C A L N O T E

GSP® Neonatal Phenylalanine kit (3308-0010/3308-001B) is a fully automated enzymatic assay for the GSP® system. Optimized for the quantitative determination of phenylalanine concentration in

blood specimens dried on filter paper, the kit is an effective aid in screening newborns for phenylketonuria (Figure 1.). Analytical performance of the GSP Neonatal Phenylalanine kit was determined in verification studies conducted at PerkinElmer, Turku, Finland. In order to establish population distribution data and screening performance for the GSP Neonatal Phenylalanine kit two studies were conducted in newborn screening laboratories. In these studies GSP Phenylalanine kit was compared to manual Phenylalanine kit (NP-1000/-4000).

Introduction

GSP Neonatal Phenylalanine kit 3308-0010/3308-001B

Figure 1. Workflow comparison of manual phenylalanine assay and automated GSP Neonatal Phenylalanine assay

Punch

Punch Result

Load Result

Manual

GSP

Reagent preparation

Reagent preparation

MeasurementPipetting & incubation Transfer step Pipetting &

incubation

Key benefits over existing manual assays:

• Automated assay • Only two hands-on steps for reconstitution• 24 hrs valid calibration curve• Control step to detect missing sample disks• Resorufin step added in the reaction to decrease

the unspecific background fluorescence• All reagents and QC material are bar-coded• Simultaneous screening of any other GSP assays in any order• Reagent on-board stability 4 days / 4 plates

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Figure 2. Content of the GSP Neonatal Phenylalanine kit for 12 plates (3308-0010) Figure 4. Run scenario for GSP Neonatal Phenylalanine assay protocol.

Figure 3. The assay principle of the GSP Neonatal Phenylalanine kit

Full selection of reagents in one kit

Two different sizes of kits are available as 12 plates kit (3308-0010) and a bulk kit for 60 plates [3308-001B] • Neonatal Phenylalanine Calibrators - 7 [15] dried blood spot cassettes each containing 1 set of dried blood spots • Neonatal Phenylalanine Controls - 4 [20] dried blood spot cassettes each containing 3 sets of dried blood spots• Neonatal Phenylalanine Substrate Reagent - 3 [15] vials, lyophilized• Neonatal Phenylalanine Substrate Reagent - 3 [15] vials, lyophilized• Neonatal Phenylalanine Assay Buffer - 3 [15] bottles, ready-for-use• Neonatal Extraction Solution - 1 [5] bottles, ready-for-use• Barcode labels for the plates

Validated assay method

The GSP Neonatal Phenylalanine assay is based on the fluorescent phenylalanine dehydrogenase method, which differs from the fluorescent ninhydrin method used in manual Neonatal Phenyl-alanine kit (NP-1000/NP-4000). In the first reaction phenylalanine dehydrogenase converts phenylalanine in sample to phenylpyru-vate generating NADH. In the presence of NADH, resazurin dye is reduced to fluorescent resorufin, which is measured using an excitation wavelength of 505 nm and an emission wavelength of 580 nm (Figure 3.). Due to longer excitation wavelength than with manual phenylalanine assay, unspecific background fluo-rescence is reduced for improved performance.

GSP Neonatal Phenylalanine kit assay protocol

After punching of the samples and reconstitution of the Phenyl-alanine Substrate and Enzyme Reagents, the assay is fully auto-mated from plate loading to completion.

The assay time for one plate is 2 h 28 min and around 2-3 plates can be processed per hour after an initial 3 hour dwell time (Figure 4.).

• Dispensing Extraction Solution (20 µL/well)• Shaking (30 s) • Incubation (1 h)• Dispensing Assay Buffer (100 µl/well)• Dispensing Substrate Reagent (5 µL/well)• Dispensing Enzyme Reagent (5 µL/well)• Shaking (30 s)• Incubation (1 h)• Shaking (30 s)• Measurement (Phe concentration)• Measurement (Elution Control to check

the sample disk is present in a well)

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Lower limits of detection, measuring range and linearity

The analytical limits were determined in accordance with CLSI document EP17-A2 [2] and linearity was determined in accordance with CLSI document EP06-A [3]. Analytical limits, measuring range and linearity of the GSP Neonatal Phenylalanine kit are summa-rized in Table 2. The Limit of Blank (LoB) is defined as the 95th percentile of a distribution of blank samples (n=150), the Limit of Detection (LoD) is based on 216 determinations of low level samples and the Limit of Quantitation (LoQ) is defined as the lowest concentration with a total CV equal to or less than 24%.

Figure 6. The change of phenylalanine concentration during storage at different temperatures and humidity conditions over time (% of reference without storage at the zero time point).

Table 2. Analytical limits, measuring range and linearity

Specimen stability

The influence of storage time, temperature, and humidity on phenylalanine concentration was studied using several artifi-cial samples spiked with phenylalanine. Phenylalanine concen-trations were measured at different time points after different storage conditions. Storage under desiccated conditions is recom-mended (Figure 6.), [4].

LoB 44.4 µmol/L (0.7 mg/dL)LoD 68 µmol/L (1.1 mg/dL)LoQ 68 µmol/L (1.1 mg/dL)Measuring range from 68 µmol/L to 1200 µmol/L (from 1.1 mg/dL to 19.8 mg/dL) from LoQ to CalFLinearity from 45 µmol/L to 1420 µmol/L (from 0.7 mg/dL to 23.4 mg/dL)

Assay PerformanceCalibration

The primary calibrator of the kit is the 3rd ISNS Reference prepa-ration for Neonatal screening. The calibration unit for phenyl-alanine concentration is defined as µmol/L (or mg/dL) of whole blood. Conversion factor is 1 µmol/L blood= 0.0165 mg/dL blood (1 mg/dL=60.61 µmol/L). The kit calibrators cover the clinically relevant concentration range and the reagents include controls for low (150 µmol/L) and high (700 µmol/L) phenylalanine concen-tration. A typical calibration curve is shown in Figure 5. The cali-bration curve is valid for 24 hours.

Precision

The precision of the assay was determined in accordance with CLSI document EP05-A2 [1] using dried blood spot samples, 3 kit lots and 3 GSP instruments. An analysis of variance approach was used to calculate the variance components. The within-run, within-lot and total variation results for the GSP Neonatal Phenyl-alanine kit are presented in Table 1.

Sample n

Mean phenylalanine concentration µmol/L (mg/dL)

Within lot variation

Between lot variation Total variation

CV% CV% CV%

1 216 57.2 (0.9) 18.6 7.7 20.12 216 69.6 (1.1) 17.4 4.0 17.83 216 93.3 (1.5) 13.5 5.5 14.64 216 131 (2.2) 11.5 5.7 12.85 216 423 (7.0) 9.1 3.0 9.66 216 650 (10.7) 8.2 5.3 9.87 216 1006 (16.6) 8.1 6.7 10.4

Figure 5. A typical calibration curve for GSP Neonatal Phenylalanine assay. The approximate concentration values for controls are shown as red crosses and relevant clinical range is highlighted.

Table 1. Variation of GSP Neonatal Phenylalanine kit using one calibration curve valid for 24 hours. The clinically relevant range is highlighted.

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Fixure 8. Frequency distribution of the PKU concentrations measured in an external study 2 with GSP Neonatal Phenylalanine kit. The x-axis is truncated to 200 µmol/L.

Figure 9. Frequency distribution of the PKU concentrations measured in an internal study with GSP Neonatal Phenylalanine kit and manual Neonatal Phenylalanine kit (NP-1000/-4000). Manual PKU data is shown using HPLC calibration.

Table 4. Range, mean and median values with upper percentiles for the routine and retrospective positive samples using GSP Neonatal Phenylalanine kit of external study 2

Sample type Sample Size

Median (µmol/L)

Mean (µmol/L) Min Max

Upper percentiles (µmol/L blood)

95% 99% 99.5% 99.9%

Routine samples 518 63.0 65.4 24.1 221.0 94.1 114.8 * *

Retrospective positive 37 121 >1400

*Sample size is too small to calculate accurate cut-off value

External study 2

518 routine newborn screening dried blood spot specimens and 37 archived confirmed high phenylalanine specimens were included in an external study 2 conducted in China and the phenylalanine concentration was measured with GSP Neonatal Phenylalanine assay. The frequency distributions of the newborn specimens for PKU concentrations are visualised in Figure 8. and descriptive statistics in Table 4.

Figure 7. Frequency distribution of the phenylalanine concentrations measured in external study site 1 with GSP Neonatal Phenylalanine kit.

Table 3. Range, mean, median values and upper percentiles for the GSP Neonatal Phenylalanine kit (3308-0010/-B) carried out in an external study 1.

External Study 1 Sample Size Mean (µmol/L)

Median (µmol/L) Min Max

Upper percentiles ( µmol/L blood)

99% 99.5% 99.9%

Routine samples 2064 63.5 61.9 8.8 149.0 110.2 113.9 137.0

Retrospective positive 22 152.6* 820.8

Internal study

An internal study comprising of 2212 leftover routine newborn screening DBS samples and 13 confirmed PKU positive newborn specimens was conducted at PerkinElmer, Turku, Finland. Phenylalanine concentration was measured with both the GSP Neonatal Phenyalalanine kit (3308-0010/-B) and manual Neonatal

Platform Median

(µmol/L)Mean

(µmol/L)

Routine samples (n=2212)

Confirmed positive (n=13) Upper percentiles (µmol/L blood)

Min Max Min Max 95% 99% 99.5% 99.9%

GSP PKU 67.9 68.5 0.8 845.1* 191.3 >1400 99.3 113.8 122.9 142.5

Manual PKU (HPLC calibration)

64.7 64.1 18.1 526.3* 202.2 >908 91.7 105.7 122.9 184.3

Manual PKU (ISNS Calibration)

84.6 83.8 23.6 689.6* 264.9 >1189.5 120.0 138.3 160.9 206.7

*This sample was drawn from an HPA patient. The lowest sample result with classical PKU or deficient cofactor was 322.9 µmol/L.

Distribution of resultsInternal and external studies were conducted to produce normal newborn population distribution data for the GSP Neonatal Phenylalanine kit, to assess the screening performance and to compare GSP Neonatal Phenylalanine assay to manual Phenyl-alanine assay (NP-1000/-4000).

Table 5. Range, mean and median values with upper percentiles for the routine and retrospective positive samples using both GSP Neonatal Phenylalanine kit and manual PKU in internal study (excluding confirmed positive PKU specimens).

External study 1

Phenylalanine concentration of 2064 newborn screening speci-mens and 22 archived confirmed high phenylalanine specimens were measured using GSP Neonatal Phenylalanine kit in a Euro-pean newborn screening laboratory. Population range, mean, median and cut-off values corresponding to 99th, 99.5th and 99.9th percentile were calculated. The frequency distributions of the newborn dried blood spot specimens of measured phenylal-anine concentrations are visualised in Figure 7. and descriptive statistics in Table 3.

The 22 positive samples consist of 7 PKU cases with classical phenylketonuria or deficient cofactor (PKU) and 15 hyperphenyl-alaninemia (HPA) cases. All of the 22 retrospective high phenylal-anine specimens were classified as screening positive by the GSP Neonatal Phenylalanine kit by using the higher 99th percentile and 99.5th percentile cut-off values.

Phenylalanine kit (NP-1000/-4000). All the positive samples were classified as screening positive using higher 99.9% percentile. The frequency distribution for routine specimens (excluding confirmed PKU specimens) is shown in Figure 9. and Table 5.

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References[1] CLSI (2004): Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline – Second Edition; CLSI Document EP05-A2. Wayne, PA: Clinical and Laboratory Standards Institute.[2] CLSI (2012): Evaluation of Detection Capability for Clinical Laboratory Measurement procedures; Approved Guideline – Second Edition; CLSI Document EP17-A2. Wayne, PA: Clinical and Laboratory Standards Institute.[3] CLSI (2003): Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline. CLSI document EP06-A. Wayne, PA: Clinical and Laboratory Standards Institute.[4] Adam, B.W., et al. (2011): The stability of markers in dried-blood spots for recommended newborn screening disorders in the United States. Clin Biochem 44, 1445-50.[5] Dhondt J.L., et al. (1998): Preparation of the first European working standard for phenylalanine determination in dried blood spots. J Med Screen 5, 63-66.

Figure 10. Deming regression analysis results between the GSP and manual Phenylalanine methods using GSP Neonatal Phenylalanine as a reference.

Method comparisonGSP Neonatal Phenylalanine kit is calibrated against ISNS Refer-ence preparation for Neonatal Screening [5], whereas manual PKU kit is calibrated against HPLC calibration. The method comparison data from internal verification study shows that there is a level difference between GSP and manual Phenylalanine assays (Figure 10) and no conversion factor between the methods is available.

Result 95% Confidence Iimit

Intercept 16.57 9.05 to 24.09

Slope 0.77 0.74 to 0.81

At GSP Neonatal Phenylalanine concentration levels below 120 µmol/L (or <2 mg/dL) the manual PKU results are ~15 % higher results than the GSP method, whereas at GSP Neonatal Phenyl-alanine concent t are ~15 % lower results than the GSP method.