Prevention is better than Treatment
R&D DIAGNOSTICS LTD®
MSUDMMR2000 ASSAY
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.
3. Warm up all reagents (except the Color Reagent) to room temperature.
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.
5. Wait 30 minutes at room temperature (20-26oC).
6. While waiting reconstitute one Enzyme and one Coenzyme Vial with distilled water. Each vial should be reconstituted with 20 ml of distilled water. Stable for 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 (20-26oC)
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.
Calculate the slope and the sample values.
INSTRUCTIONS FOR USE (PACKAGE INSERT)
MSUD-MMR2000
R&D DIAGNOSTICS LTD
Leucine and
other branched-chain amino acids Screening Assay
(550 nm)
MANUFACTURER CODE: GRM7 - EDMA CODE: 11.02.01 - PRODUCT FILE No. GR/CA01/GRM7/O/126
FOR IN VITRO DIAGNOSTIC USE ONLY
A kit for the quantitative enzymatic determination of branched-chain amino acids in
human dried blood spot specimens on Schleicher & Schuell 903 paper. Suitable as a
method for screening for inborn errors of leucine and other branched-chain amino acid
metabolism in newborns.
Intended Use
Clinical Background
Principle of the Assay
Kit Contents
Reagents/Materials Required but not Provided
Sample Collection
Assay Procedures
Calculation of Results – Typical standard curve
Limitations of Use
Precautions
Quality control
References
The R&D DIAGNOSTICS LTD MSUD Screening Assay is an enzymatic colorimetric end-point method for the determination of L-leucine and other branched-chain amino acids in dried blood spot specimens taken from newborn human infants as part of a newborn screening program. The test is intended as a screening method for measuring the L-branched-chain amino acid (BCAA) concentrations in newborn blood spot specimens. Elevated results are not diagnostic per se of maple syrup urine disease, but indicate the urgent need for further study of the newborn from which the presumptive
positive specimen was received. The kit is NOT intended for use in monitoring the circulating concentrations of L-BCAAs in MSUD patients, nor to detect ante-natal MSUD or maternal MSUD.
Maple Syrup Urine Disease (MSUD), also known as branched-chain ketoaciduria, is due to a deficiency of the branched-chain ketoacid decarboxylase enzyme (1). The disease is characterized by elevated circulating analyte concentrations, namely L-branched-chain amino acids. MSUD is classified into four major variant forms based on clinical presentation and outcome. Each variant is due to deficient decarboxylation of the branched-chain ketoacids and in each case, all three ketoacids and their respective amino acids are increased in the circulation. In a classical MSUD infant, leucine concentrations reach abnormally high levels within hours of birth regardless of protein intake (2) . Maple Syrup Urine Disease has an incidence of 1 in 225 000 newborns (3). Increased circulating concentrations of L-BCAAs and á-ketoacids, if untreated, cause severe mental and motor retardation1 the severity of which can be lessened by prompt dietary restriction of L-BCAAs (4). However, delayed treatment leads to these chronic symptoms becoming irreversible. The current method for the measurement of circulating L-branched-chain amino acid concentrations is the Guthrie bacterial inhibition assay (BIA) (5), which is semi-quantitative.
The R&D Diagnostics MSUD Kit uses trichloroacetic acid (TCA) to extract Leucine and BCAAs from dried blood spot samples. After extraction, the eluted sample is combined with the enzyme reagent Leucine dehydrogenase (E.C.1.4.1.9) . This enzyme reagent catalyzes the NAD‑dependent oxidative deamination of Leucine and L-BCAAs to á-ketoisocaproate acid. 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 Leucine and BCAA present in the sample (6).
Leucine + NAD+ -------Leu DeH------ > á-ketoisocaproate + NADH
NADH + IEAox + Tetrazolium --------------- > NAD+ + IEAred + Formazan
Leucine Standards and Controls 1 Set
Leucine Enzyme (Lyophilized) 4 x 20 ml
Dilution buffer 1 x 42 ml
Coenzyme (Lyophilized) 4 x 20 ml
Color Reagent 1 x 175 ml
Color Reagent Booster 1 x 17,5 ml
TCA (may not be provided) 4 x 52 ml
Human whole
blood adjusted to a hematocrit of 55% and containing four standard
concentrations of added L-Leucine, i.e., approximately 2.0, 5.0, 10.0, and 20.0
mg/dl (values
may vary). Please see separate instructions for the recommended use of standards
and controls. The two Controls contain low and high concentrations
of LeucineP.
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.
Storage: Dry at 2-8º C
Expiration: Refer to the expiration date printed on the labels
Leucine dehydrogenase lyophilized with buffers and a stabilizer.
Storage: 2-8º C
Expiration: Refer to the expiration date printed on the labels.
Stable for 1 month at 2-8º C after reconstituting.
Lyophilized NAD with buffers and stabilizers.
Storage: 2-8º C
Expiration: Refer to the expiration date printed on the labels. Stable for 1
month at 2-8º C after reconstituting.
A solution of a tetrazolium salt and buffer.
Storage: 2-8º C
Expiration: Refer to the expiration date printed on the label. Extremely
photosensitive, should be kept wrapped in aluminum foil. Not to be left out of the refrigerator longer then needed.
A solution of an Intermediate electron receptor and buffer.
Storage: 2-8º C
Expiration: Refer to the expiration date printed on the label.
A 3.0 % (w/v) solution of trichloroacetic acid (TCA). Storage: 2-8º C
Expiration: Refer to the expiration date printed on the labels.
1. Blood Spot Eluant: 3%(w/v) solution of trichloroacetic acid (TCA) in deionized water.
2. Assay microtitration plates: Flat-bottomed microtitration plates of good optical quality.
3. Elution plates: Millipore MultiscreenTM plates (optional).
4. A Millipore MultiscreenTM Vacuum Manifold (optional).
5. Single, automatic repeating or multi-channel pipettes to deliver volumes in the range 10 to 1000ìl with an accuracy of +/-1.5% over the range 10 to 100ìl.
6. A microtitration plate shaker.
7. A microtitration plate reader plate reader capable of reading absorbances in the range 550-590nm and 690nm in dual wavelength reading mode.
8. A hole punch which produces 1/8” diameter punched discs.
9. Blood collection cards. The minimum pre-printed information required is:
(1) Infants name, mothers name and patient ID number
(2) Date of Birth
(3) Birth weight
(4) Sex
(5) Feeding status
(6) Specimen collection date
(7) Submitters ID and address
(8) Physicians name and telephone number
(9) Testing laboratory name and address
(10) Space for test results
(11) Appropriate number of pre-printed 1/2” (internal diameter) dotted or broken
line circles on attached blood collection paper card
(12) Manufacturer and lot number of collection paper indicated on collection paper
card
(13) Manufacturer or printer listed on the information section
10. Blood collection paper cards:
(1) Blood collection paper cards should be attached to at least 2 copies of the
information section outlined above.
(2) Blood collection paper should be Schleicher & Schuell Grade 903 or
equivalent of a currently approved batch (Contact collection paper
manufacturer for details).
For further details on paper specifications and batch acceptance criteria refer to reference (8) and NCCLS Approved Standard LA4-A2 ‘Blood Collection on Filter Paper for Neonatal Screening Programs’ 2nd Edition (1992), NCCLS, Villanova, PA.
SAMPLE COLLECTION
1.Day of Life First Second Third Fourth Fifth
Age (Hours)
Specimen Quality ( A ) ( B ) ( C )
(24 48) (72 96) (120) hours after Birth
Blood should ideally be collected between the first and second days of life (up to 48 hours) after birth (A and B) above. Results from specimens collected after the second day of life (48 - 120 hours) (C) above should be processed promptly because therapy must be initiated before the tenth day of life.
2 Collect blood from the infant’s heel ONLY using the medial (closest to the body center-line) or the lateral (furthest from the body center-line) portion of the plantar (walking surface). Blood collection from other areas of the infant’s foot, e.g. arch, may result in nerve, tendon or cartilage injury.
3 Fill out the required information on the blood collection form ensuring all copies are legible.
4 To increase local blood flow, warm the skin puncture site using a warm (<42oC; 108oF) moist towel applied to the site for 2-3 minutes. Similarly, holding the infant’s limb below the level of the heart will increase venous flow.
5 Clean the skin with 70%(v/v) isopropyl alcohol. Wipe dry with sterile gauze and allow the skin to air dry.
6 Puncture the heel with a sterile lancet (2.4-2.5mm long) or with an automatic lancet
device. Wipe away the first drop of blood with a sterile gauze.
7 Gently touch one side of the filter paper against a large hanging drop of blood and in one step, allow a sufficient quantity of blood to soak into the paper to completely fill the preprinted circle. Ensure that blood has penetrated and saturated the paper by examining both sides of the paper. Repeat the procedure to fill the required number of preprinted circles on the specimen collection card.
7.1 DO NOT: milk or squeeze the puncture site during collection as this may cause
hemolysis or dilution of the blood with tissue fluids.
7.7.2 DO NOT: apply successive drops of blood to the same preprinted circle. If blood flow diminishes before complete filling of the preprinted circle repeat the sampling in
a new circle.
7.3 DO NOT: Touch or smear the blood spots.
8 Allow the blood spot specimen to air-dry in a suspended horizontal position for a minimum of four hours at ambient temperature (18-22oC; 64-72oF) away from direct sunlight, avoiding contact of the spots with any surfaces.
9 Arrange transport of the collection card to the screening laboratory within 24 hours of collection. L-BCAAs in dried blood spot specimens have been shown to be very stable 3, under a variety of storage conditions. However, it is recommended that a repeat specimen is requested if a period of 10 days or more has elapsed between collection and sample testing. Additionally, the conditions of the collection card storage must be consistent with the stability of the least stable analyte to be
measured in the specimen.
10 Although not the preferred method of blood collection, heparinized capillary tubes have been used to transfer blood from the puncture site to the collection card preprinted circles (Section 6). If this method is used, care must be taken not to scratch or indent the surface of the blood collection paper during transfer from the capillary. The R&D Diagnostics Ltd. MSUD Screening assay has NOT been validated for use with heparinized capillary collected blood.
11 Cord blood should NOT be used as a specimen.
12 For a more detailed treatment of the procedure outlined above the user is referred to NCCLS Approved Standard LA4-A2 ‘Blood Collection on Filter Paper for Neonatal Screening Programs’ Second Edition (1992) NCCLS, Villanova, PA.
1. Take a clean 96-well (preferably U bottom) microplate (elution microplate).
2. Add a dried blood spot (3/16 inch or 2 X 1/8inch diameter) controls, standards and one blank per well.
3. Equilibrate all reagents (except the colour reagent) at room temperature.
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.
5. Incubate 30 minutes at room temperature (20-26oC).
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 (20-26oC).
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. Calculate the slope and the sample values.
Please note the following:
· This assay is to be performed at room temperature (20-26oC). At higher temperatures (over 28oC) 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
Reaction Sequence:
(catalyzed by Leucine dehydrogenase; measured at 340 nm)
(catalyzed by Diaphorase; measured at 550 nm)
The following graph shows the responses (in mOD) of standards with known Leucine content (expressed in mg/dl on the x-axis). Thirteen samples containing five different concentrations of Leucine were tested in triplicates and the average mOD values were plotted against Leucine concentration. The red trend line shows the results of a kit commercially available (samples tested in duplicates)
Limitations
The R&D Diagnostics Ltd MSUD Screening Kit is intended for use as a tool to screen neonates for elevated levels of Leucine and BCAA. 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 MSUD.
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.
4. All of the Kit components used in an assay must be from the same Kit Lot Number.
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.
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.(7) If the precision of the assay does not correlate with this standard and repetition excludes errors in technique, check the following areas:
a. Pipetting and timing devices
b. Instrument calibration
c. Expiration dates on reagent labels and prepared working solutions
d. Storage conditions
e. Temperature control devices
Specificity
1. L-Leucine
The mean recovery of L-leucine in the presence of 20 or 10 mg/dl of the following 15 commonly occurring amino acids was 103.4 % (S.D. 9.9 %)
Hydroxyl-L-Proline, L-Alanine, L-Leucine, L-Arginine, L-Methionine, L-Aspartic Acid, L-Proline, L-Cysteine, L-Serine, L-Glutamic Acid, L-Tryptophan, Glycine, L-Tyrosine, L-Histidine.
Interfering Substances
1 Antibiotics
The mean recovery of L-leucine in the presence of the following antibiotics at concentrations in excess of the upper range of their toxic blood concentrations (where data available) was 104.8 % (S.D. 11.2 %), as compared to an antibiotic-free control.
Amikacin, Metronidazole, Amoxicillin, Nalidixic Acid, Ampicillin, Neomycin, Benzylpenicillin, Nitrofurantoin, Cefotaxime, Oxytetracycline, Cephradine, Penicillin V, Chloramphenicol, Rifampicin, Colisitin, Streptomycin, Clindamycin, Sulfamethoxazole, Erythromycin, Tetracycline, Fusidate, Sodium Tobramycin, Gentamycin, Trimethoprim, Kanamycin Sulfate, Vancomycin, Lincomycin
2 Non-Antibiotics and Metabolites
The mean recovery of L-leucine in the presence of the following compounds at concentrations in excess of the upper range of their toxic or grossly pathological concentrations (where data available) was 101.3 % (S.D. 8.6 %), by comparison to an interferent-free control.
Acetaminophen, Acetylsalicylic Acid, Ascorbic Acid, Caffeine, EDTA, Gentisic acid,
REFERENCES
1. Chuang, D.T., and Shih, V.E. (1995) in The Metabolic Basis of Inherited Diseases I (Scriver, C.R., Beaudet, A.L., Sly, W.S., and Valle, D., eds.) Seventh Edition. McGraw Hill p1239.
2. Westall, R.G., Dancis, J. and Miller, S. (1957) Am. J. Dis. Child. 94: 571
3. Naylor, E.W. (1993) in Laboratory Methods for Neonatal Screening (Therrell, B.L. Jnr. ed.) Apha p115
4. Snyderman, S.E., Norton, P.M., Roithan, E., and Holt, L.E. Jnr. (1964) Pediatrics 34: 454
5. Naylor, E.W. (1980) in Neonatal Screening for Inborn Errors of Metabolism (Bickel, H., Guthrie, R. and Hammersen, G., eds.) Berlin, Springer – Verlag p19
6. Slazyk, W.E. & Hannon, W.H. (1993) Quality Assurance in the Newborn Screening Laboratory in Laboratory Methods for Newborn Screening (Therrell, B.L. ed.) Am. Pub. Health Assoc. 1015 Fifteenth Street N.W. Washington D.C. 20005 p23.
7. Westgard, J.O. & Klee, G.G. (1994) Quality Assurance in Tietz Textbook of Clinical Chemistry (Burtis, C.A. & Ashwood, R. eds.) 3RD Edition, W.B. Saunders, Philadelphia, PA. p548
8. National Committee for Clinical Laboratory Standards (NCCLS)(1984) Internal Quality Control Testing: Principles and Definitions. Publication C24-P. NCCLS, Villanova, PA. Ver. 3 08/02RSC
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Page last edited on 21/03/2006