PKU 550 nm

1. Take a clean 96-well (preferably U bottom) microplate (elution microplate).

2. Add one disk cut from a dried blood spot (3/16 inch or 2 X 1/8inch diameter) per well. Remember to add controls, standards and one blank well.

4. Add 100 microliters of Elution Buffer (TCA 3%) in each well, mix well the contents of each well and place the plate on a plate shaker.

6. While waiting reconstitute one Enzyme vial and one Coenzyme Vial with 20 ml distilled water each. Stable for at least one week refrigerated. Mix 2 parts of Enzyme solution with 2 parts of Coenzyme solution and 1 part of Dilution buffer. You need 100 microliters of this Enzyme-Coenzyme-Dilution buffer mixture for each sample. Please note that you should only mix the quantity you need for the day’s run. The Enzyme-Coenzyme mixture should be discarded if not used within 5 hours. The following table gives the volumes required from each of the three components to run specific number of tests (volumes in ml). We highly recommend the addition of the Dilution buffer just before using the mixture.

# tests	Enzyme	Coenzyme	Dilution buffer	Total volume
50	2	2	1	5
100	4	4	2	10
150	6	6	3	15
200	8	8	4	20
300	12	12	6	30
400	16	16	8	40
500	20	20	10	50

7. Transfer 40 microliters of the TCA eluant in a new microplate at the corresponding wells. Add 100 microliters of the mixture prepared in step 6, per well. Mix well, avoiding the formation of foam. Wait for 30 minutes at room temperature (20-26^oC).

8. Take the Color Reagent and the Color Reagent Booster out of the refrigerator mix one part of Color Reagent Booster with 10 parts color reagent just before using it. Do not pre-warm the mixture. Return the original bottles back to the refrigerator the soonest possible. Avoid exposure to light. Prepare only the quantity you will need for the day.

9. Add 80 microliters of Color Reagent mixture per well. Mix well avoiding the formation of foam.

10. Wait for 10 minutes and measure the microplate at 550 nm, endpoint mode, single measurement. There is no need to wait longer than 15 minutes.

1. Πάρτε ένα καθαρό μικροπλακίδιο των 96 οπών (προτιμητέα τα έχοντα πυθμένα U) το οποίο θα είναι το μικροπλακίδιο έκλουσης (elution microplate).

2. Προσθέσατε έναν χάρτινο δίσκο από δείγμα αποξηραμένης κηλίδας αίματος (διαμέτρου 4,7 χιλιοστών ή 2 X 3,2 χιλιοστών) ανά οπή. Θυμηθείτε να προσθέσετε μάρτυρες, πρότυπα και μία άδεια οπή.

3. Θερμάνετε όλα τα αντιδραστήρια σε θερμοκρασία δωματίου.

4. Προσθέσατε 80 μικρόλιτρα από το Elution Buffer (TCA 3%) σε κάθε οπή, ανακατέψτε καλά τα περιεχόμενα της κάθε οπής και τοποθετείστε το μικροπλακίδιο σε ανακινητή.

5. Αναμείνατε 40 λεπτά σε θερμοκρασία δωματίου (20-26^oC).

6. Ενόσω αναμένετε, προχωρείστε στην ανασύσταση ενός φιαλιδίου ενζύμου Enzyme με 16 ml από το Enzyme Diluent και ενός φιαλιδίου συνενζύμου Coenzyme με 16 ml απεσταγμένου νερού. Τα προκύπτοντα διαλύματα είναι σταθερά επί μία τουλάχιστον εβδομάδα υπό ψύξη. Αναμείξατε 2 μέρη του διαλύματος του ενζύμου Enzyme με 2 μέρη διαλύματος συνενζύμου Coenzyme και 1 μέρος του ρυθμιστικού διαλύματος αραίωσης Dilution buffer. Θα χρειαστείτε 100 μικρόλιτρα από αυτό το μείγμα (Εnzyme-Coenzyme-Dilution buffer) για κάθε εξέταση. Παρακαλούμε σημειώσατε ότι θα πρέπει να παρασκευάζετε μόνον την ποσότητα μείγματος το οποίον πρόκειται να χρησιμοποιήσετε την συγκεκριμένη ημέρα. Το μείγμα Enzyme-Coenzyme είναι άχρηστο εφόσον δεν χρησιμοποιηθεί μέσα σε 5 ώρες από την παρασκευή του. Ο παρακάτω πίνακας δίνει τους όγκους από κάθε αντιδραστήριο οι οποίοι χρειάζονται για συγκεκριμένους αριθμούς εξετάσεων (οι όγκοι δίνονται σε ml). Συνιστάται η προσθήκη του ρυθμιστικού διαλύματος αραίωσης ακριβώς πριν χρησιμοποιήσετε το μείγμα.

# tests	Enzyme	Coenzyme	Dilution buffer	Total volume
50	2	2	1	5
100	4	4	2	10
150	6	6	3	15
200	8	8	4	20
300	12	12	6	30
400	16	16	8	40
500	20 (1 vial)	20 (1 vial)	10	50

7. Μεταφέρατε 40 μικρόλιτρα από κάθε οπή του μικροπλακιδίου έκλουσης στα αντίστοιχες οπές ενός νέου μικροπλακιδίου. Προσθέστε 100 μικρόλιτρα από το μείγμα που φτιάξατε στο βήμα 6 ανά οπή. Ανακατέψτε καλά αποφεύγοντας την δημιουργία φυσαλίδων. Αναμείνατε για 40 λεπτά σε θερμοκρασία δωματίου (20-26^oC).

8. Λίγο πριν περάσει το διάστημα των 40 λεπτών βγάλτε το χρωμογόνο αντιδραστήριο Color Reagent από το ψυγείο (δεν συνιστάται η παραμονή του σε θερμοκρασία δωματίου πέραν του απολύτως αναγκαίου χρόνου). Αναμείξτε 10 μέρη χρωμογόνου με 1 μέρος Color Booster. Πάντα να επιστρέφετε το υπόλοιπο χρωμογόνο αντιδραστήριο στο ψυγείο μετά την χρήση του. Εάν έχουν δημιουργηθεί κρύσταλλοι, ανακατέψτε μέχρι να διαλυθούν.

9. Προσθέστε 80 μικρόλιτρα από το μείγμα του Color Reagent που φτιάξατε στο βήμα 8 ανά οπή. Ανακατέψτε καλά αποφεύγοντας την δημιουργία φυσαλίδων.

10. Αναμείνατε επί 10 λεπτά και μετρείστε το μικροπλακίδιο σε μήκος κύματος 550-570 nm, μία μέτρηση τελικού σημείου. Μην περιμένετε περισσότερο από 10-15 λεπτά.

Υπολογίστε την κλίση και τις τιμές των δειγμάτων

An Enzymatic Colorimetric Assay for the Quantitative Determination of Phenylalanine Levels In Neonates.

Intended Use

The R&D Diagnostics Phenylalanine Kit is an Enzyme Assay (EA) for the quantitative measurement of Phenylalanine concentrations in dried blood spot samples that have been collected onto Schleicher & Schuell (S&S^®) 903^™or 2992 Specimen Collection Paper. This Kit is particularly suitable for use in a newborn screening program to measure Phenylalanine concentrations in newborn infants as an aid to the detection of Phenylketonuria.

Clinical Utility

Phenylketonuria (PKU) is a disorder caused by an inborn error of metabolism. Its rate of incidence is approximately one in 10,000 newborns in the United States.¹ Classical PKU is characterized by a highly elevated blood Phenylalanine concentration which results from the absence of the hepatic enzyme Phenylalanine hydroxylase (EC 1.14.16.1).² The resulting increased circulating concentration of Phenylalanine, if left untreated, can result in a variety of symptoms - the major one being mental retardation.¹ Once diagnosed, treatment with a special diet that restricts dietary Phenylalanine should be initiated promptly. Delays in treatment have been correlated with increases in the severity of the retardation.^3,4,5

Principle of the Assay

The R&D Diagnostics Phenylalanine Kit uses trichloroacetic acid (TCA) to extract Phenylalanine from dried blood spot samples. After extraction, the eluted sample is combined with the enzyme reagent Phenylalanine dehydrogenase (EC 1.4.1.20). This enzyme reagent catalyzes the NAD‑dependent oxidative deamination of Phenylalanine to phenylpyruvate and ammonia. The NADH produced reacts with a color reagent in which a tetrazolium salt gets reduced producing a distinct color. This color is measured colorimetrically and is directly proportional to the concentration of Phenylalanine present in the sample.⁶

Materials Supplied 2000 Tests / Kit

Reagent Description

Phenylalanine Standards and Controls

Human whole blood adjusted to a hematocrit of 55% and containing four standard concentrations of added Phenylalanine, i.e., approximately 2.0, 5.0, 10.0, and 20.0 mg/dl. A zero standard is also included. Please see separate instructions for the recommended use of standards and controls. The two Controls contain low and high concentrations of Phenylalanine. The Standards and Controls are spotted onto Schleicher & Schuell (S&S^®) 903^™ or 2992™ Specimen Collection Paper. Refer to the labels for the exact concentrations of the Standards and the acceptable ranges for the Controls.

Phenylalanine Enzyme

Coenzyme

Expiration: Refer to the expiration date printed on the labels. Stable for 1 month at 2-8º C after reconstituting.

Color Reagent

photosensitive, should be kept wrapped in aluminum foil. Not to be left out of the refrigerator longer then needed.

Color Reagent Booster

TCA

Specimen Collection and Preparation

Refer to NCCLS publication, section LA4-A, “Blood Collection on Filter Paper for Neonatal Screening Programs,” for a complete description of the appropriate blood collection techniques. Infant heel puncture can provide the large drops of blood needed to fill the pre-marked circles on the S&S^®903^™ Specimen Collection Paper. Avoid using excessive pressure at the lancet puncture site and discard the first drop of blood. The collection paper circles must be completely saturated with blood, i.e., blood visible on both sides of the paper. Completely air dry the spotted papers and store them in sealed paper envelopes or containers that will provide protection from moisture, light, heat and contact with other materials. The sample discs should be punched from similar areas on each individual blood spot. Do not punch sample discs from areas that include printed marks or that are near the edges of the blood spot. The dried blood samples can be stored at 2-8° C for a period of at least 4 months.⁷

Materials Required But Not Supplied

Assay Procedure:

2. Add a dried blood spot (3/16 inch or 2 X 1/8inch diameter) controls, standards and one blank per well.

4. Add 100 ml of Elution Buffer (TCA 3%) to each well, mix the contents of each well and place the plate on a plate shaker.

6. Reconstitute one Enzyme Vial and one Coenzyme Vial with 20 ml distilled water each. Stable for at least one week refrigerated.

Mix 2 parts of Enzyme solution with 2 parts of Coenzyme solution and 1 part of Dilution buffer.

7. 100 ml of this Enzyme-Coenzyme-Dilution buffer mixture is needed for each sample. Note this mixture is stable for 5 hours. The following table gives the volumes required for each of the three components to run specific number of tests (volumes in ml). We highly recommend the addition of the Dilution buffer just before using the mixture.

# tests	Enzyme	Coenzyme	Dilution buffer	Total volume
50	2	2	1	5
100	4	4	2	10
150	6	6	3	15
200	8	8	4	20
300	12	12	6	30
400	16	16	8	40
500	20 (1 vial)	20 (1 vial)	10	50

8. Transfer 40 ml of the TCA eluate in a new microplate at the corresponding wells. Add 100 ml of the mixture prepared in step 7, per well. Mix well, avoiding the formation to foam. Incubate 30 minutes at room temperature (20-26^oC).

9. Take the Colour Reagent out of the refrigerator just prior to use. Take out just the quantity you are going to use for the day (assuming 80 microliters / sample). Return the rest of the Colour reagent in the refrigerator. Mix the quantity you have taken with the Color Reagent Booster (10 parts color reagent plus one part Color Reagent Booster).

10. Add 80 ml of Colour Reagent mixture prepared in step 9, per well. Mix well to avoid the formation of foam.

11. After 10 minutes of incubation at room temperature measure the microplate at 550-570 nm (optimal: 550 nm), endpoint mode, single measurement. There is no need to wait longer than 20 minutes.

· The Dilution Buffer and / or the reconstituted Enzyme-Coenzyme-Dilution buffer mixture may appear cloudy. Cloudiness does not affect the assay.

· This assay is to be performed at room temperature (20-26^oC). At higher temperatures (over 28^oC) an abnormally high blank may be observed.

· A High blank may also be observed if the colour reagent stage is prolonged more than 20 minutes.

· The colour reagent is photosensitive. Avoid prolonged exposure to light. It is recommended to keep the bottle wrapped in aluminum foil.

· Caution: The Dilution buffer contains Potassium Hydroxide - Irritant to the skin. In case of contact, rinse with water.

The color reagent in its reduced form [ Blue ] will stain glass or plastic after prolonged contact. It is advisable to discard such material after the test is over, especially when the microplate or other equipment is to be re-used

Typical Standard Curve

The following graph shows the responses (in mOD) of standards with known Phenylalanine content (expressed in mg/dl on the x-axis). Thirteen samples containing different concentrations of phenylalanine were tested in triplicates and the average mOD values were plotted against phenylalanine concentration. The red trend line shows the results of a kit commercially available (samples tested in duplicates).

The R&D Diagnostics Ltd Phenylalanine Screening Kit is intended for use as a tool to screen neonates for elevated levels of phenylalanine. This kit is not to be used for confirmatory testing or to monitor therapy. A definitive clinical diagnosis should not be based on the results of a single test but should be made by the physician only after all clinical and laboratory findings have been evaluated. Another diagnostic procedure performed on a serum sample should be used to confirm the diagnosis of Phenylketonuria.

Precautions

Assay Precautions

1. All blood samples of human origin should be regarded as a potential Biohazard. Handle all blood samples as if capable of transmitting Hepatitis and HIV.

2. The Enzyme Diluent, Color Reagent, Color Reagent Booster, Standards and Controls all contain sodium azide (NaN₃) and Proclin 300 as antimicrobial preservatives. Users should be aware of their toxic properties if absorbed or ingested. Disposal of these reagents should be accompanied by copious flushing with water to avoid accumulation of explosive salts in plumbing systems.

2. The blood spot eluent solution, trichloroacetic acid (TCA), is highly acidic and corrosive. Protective gloves should be worn while using this reagent.

3. Do NOT keep or use the reconstituted Enzyme, Coenzyme, or the combined Enzyme-Coenzyme working solution for any longer than the specified periods of time.

5. Kit components should not be used past the expiration dates printed on the labels.

6. Kit components and test specimens should be at room temperature (18-25º C) before starting the assay. The color reagent is the exception to this rule. Do not warm it before use.

7. Do not use any reagents or solutions that have become cloudy or discolored. This is especially true for the color reagent. The color reagent should be colored yellow. Do not use it if a blue tint is observed.

Quality Control

The reproducibility of the standard curve values and control values should be within defined limits of laboratory acceptability. Commonly used measures of variability are discussed by Westgard, J.O., et al.¹⁰ If the precision of the assay does not correlate with this standard and repetition excludes errors in technique, check the following areas:

REFERENCES

1. Scriver, C.R., Kaufman, S., and S.L.C. Woo. In, The Metabolic Basis of Inherited Diseases I, C.R. Scriver, A.L. Beaudet, W.S. Sly, and D. Valle, Eds., Sixth Edition, McGraw Hill, 1989, p. 495.

3. Waisbren, S.E., Mahon, B.E., Schnell, R.R., and H.L., Levy. 1987 Pediatrics 79:351.

5. Smith, L., Beasley, M.G., and A.E. Ades. 1990 Arch. Dis. Childhood 65:472.

6. Morris, H.C., Miller, J., Campbell, R.S.C., Hammond, P.M., Berry, D.J., and C.P. Price. 1988 J. Antimicrob. 22:93.

7. Wendel, U., Koppelkamm, M., Hummel, W., Sander, J., and U. Langenbeck. 1990 Clin. Chem. Acta. 192:165.