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Test ID ALLOI Allo-isoleucine, Blood Spot

Specimen Required

Supplies: Card - Blood Spot Collection (Filter Paper) (T493)


Preferred: Blood Spot Collection (Filter Paper) (T493)

Acceptable: Local newborn screening card, Whatman Protein Saver 903 filter paper, PerkinElmer 226 (formerly Ahlstrom 226) filter paper, Munktell filter paper, Postmortem Screening Card

Specimen Volume: 2 Blood spots

Collection Instructions:

1. Do not use device or capillary tube containing EDTA to collect specimen.

2. At least 1 spot should be complete and unpunched.

3. An alternative blood collection option for a patient 1 year of age or older is a fingerstick. For infants younger than 1 year, a heel stick should be used. See How to Collect Dried Blood Spot Samples via fingerstick.

4. Include type of feeding information on the collection card.

5. Let blood dry on filter paper at ambient temperature in a horizontal position for a minimum of 3 hours.

6. Do not expose specimen to heat or direct sunlight.

7. Do not stack wet specimens.

8. Keep specimen dry.

Additional Information:

1. For collection instructions, see Blood Spot Collection Instructions

2. For collection instructions in Spanish, see Blood Spot Collection Card-Spanish Instructions (T777)

3. For collection instructions in Chinese, see Blood Spot Collection Card-Chinese Instructions (T800)

Useful For

Evaluation of newborn screening specimens that test positive for branched-chain amino acids elevations


Follow-up of patients with maple syrup urine disease

Genetics Test Information

Second-tier test for abnormal newborn screen and follow-up of patients with maple syrup urine disease.

Method Name

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Portions of this test are covered by patents held by Quest Diagnostics

Reporting Name

Allo-isoleucine, BS

Specimen Type

Whole blood

Specimen Minimum Volume

1 Blood spot

Specimen Stability Information

Specimen Type Temperature Time Special Container
Whole blood Ambient (preferred) 59 days FILTER PAPER
  Frozen  59 days FILTER PAPER
  Refrigerated  59 days FILTER PAPER

Reject Due To

Blood spot shows layering
Serum rings
Multiple applications
Insufficient specimen

Clinical Information

Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by the deficiency of the branched-chain-ketoacid dehydrogenase (BCKDH) complex. The BCKDH complex is involved in the metabolism of the branched-chain amino acids (BCAA): isoleucine (Ile), leucine (Leu), and valine (Val). Classic MSUD presents in the neonate with feeding intolerance, failure to thrive, vomiting, lethargy, and maple syrup odor to urine and cerumen. If untreated, it progresses to irreversible intellectual disability, hyperactivity, failure to thrive, seizures, coma, cerebral edema, and possibly death.


MSUD is a panethnic condition but is most prevalent in the Old Order Mennonite community in Lancaster, Pennsylvania with an incidence there of 1 in760 live births. The incidence of MSUD is approximately 1 in 185,000 live births in the general population.


Newborn screening includes the measurement of BCAA (Leu, Ile, and Val), which are elevated in MSUD. However, unaffected infants receiving total parenteral nutrition frequently have increased levels of BCAA, a situation that often triggers unnecessary follow-up investigations. Abnormal concentrations of allo-isoleucine (Allo-Ile) are pathognomonic for MSUD. The determination of Allo-Ile (second-tier testing) in the same newborn screening specimens that reveals elevated BCAA allows for positive identification of patients with MSUD and differentiation from BCAA elevations due to dietary artifacts, reducing the occurrence of false-positive newborn screening results.


Treatment of MSUD aims to normalize the concentration of BCAA by dietary restriction of these amino acids. BCAA are essential amino acids, which require frequent adjustment of the dietary treatment. Dietary monitoring is accomplished by regular determination of BCAA and Allo-Ile concentrations.

Reference Values

Allo-isoleucine: <2 nmol/mL

Leucine: 35-215 nmol/mL

Isoleucine: 13-130 nmol/mL

Valine: 51-325 nmol/mL


An interpretive report will also be provided.


Allo-isoleucine is nearly undetectable in individuals not affected by maple syrup urine disease (MSUD). Accordingly, its presence is diagnostic for MSUD, and its absence is sufficient to rule-out MSUD.


No significant cautionary statements

Supportive Data

In a blinded study containing specimens obtained from maple syrup urine disease (MSUD) cases (n=16), non-MSUD patients treated with total parenteral nutrition (n=19), and healthy controls (n=541), this assay correctly identified all MSUD and non-MSUD cases.

Clinical Reference

1. Chace DH, Kalas TA, Naylor EW: Use of tandem mass spectrometry for multianalyte screening of dried blood specimens from newborns. Clin Chem. 2003 Nov;49(11):1797-1817. doi: 10.1373/clinchem.2003.022178

2. Simon E, Fingerhut R, Baumkotter J, Konstantopoulou V, Ratschmann R, Wendel U: Maple syrup urine disease: Favorable effect of early diagnosis by newborn screening on the neonatal course of the disease. J Inherit Metab Dis. 2006 Aug;29(4):532-537. doi: 10.1007/s10545-006-0315-y

3. Strauss KA, Puffenberger EG, Carson VJ: Maple syrup urine disease. In: Adam MP, Ardinger HH, Pagon RA, et al. eds. GeneReviews [Internet]. University of Washington, Seattle; 2006. Updated April 23, 2020. Accessed October 5, 2022. Available at

4. Frazier DM, Allgeier C, Homer C, et al: Nutrition management guideline for maple syrup urine disease: An evidence- and consensus-based approach. Mol Genet Metab. 2014 Jul:112(3)210-217. doi: 10.1016/j.ymgme.2014.05.006

5. Blackburn PR, Gass JM, Vairo FPE, Farnham KM, Atwal HK, Macklin S, Klee EW, Atwal PS. Maple syrup urine disease: mechanisms and management. Appl Clin Genet. 2017 Sep 6;10:57-66. doi: 10.2147/TACG.S125962

Method Description

This method quantifies valine (Val), allo-isoleucine (Allo-Ile), leucine (Ile), and leucine (Leu) using stable isotope-labeled internal standards (IS): d8-Val, d10-Allo-Ile, and d3-Leu. Branched-chain amino acids (BCAA) are extracted from a 3/16-inch dried blood spot (DBS) using a methanol:water (50:50) solution containing the IS. The filter plate containing the DBS and the IS are placed on an orbital shaker for 30 minutes at ambient temperature. The blood spot eluate is centrifuged into a 96-well round-bottom plate, dried under nitrogen, and reconstituted in aqueous mobile phase. BCAA are separated and detected by liquid chromatography-tandem mass spectrometry in positive selected reaction monitoring mode. Chromatography is performed using an Applied Biosystems AAA C18 (4.6x150 mm) column, with mobile phases 0.1% formic acid:0.01% heptofluorobutyric acid (HFBA):water and 0.1% formic acid:0.01% HFBA:acetonitrile. Total analysis time is 15 minutes including column re-equilibration.(Oglesbee D, Kramer K, Lacey J, et al: Second-tier test for quantification of alloisoleucine and branched-chain amino acids in dried blood spots to improve newborn screening of maple syrup urine disease [MSUD]. Clin Chem. 2008 Mar;54(3):542-549. doi: 10.1373/clinchem.2007.098434; Stroek K, Boelen A, Bouva MJ, et al. Evaluation of 11 years of newborn screening for maple syrup urine disease in the Netherlands and a systematic review of the literature: Strategies for optimization. JIMD Rep. 2020 May 13;54(1):68-78. doi: 10.1002/jmd2.12124)

Day(s) Performed

Monday through Friday

Report Available

2 to 4 days

Test Classification

This test was developed, and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.

CPT Code Information


LOINC Code Information

Test ID Test Order Name Order LOINC Value
ALLOI Allo-isoleucine, BS 94571-7


Result ID Test Result Name Result LOINC Value
27453 Interpretation 46743-1
27457 Allo-isoleucine 94572-5
27458 Leucine 47679-6
27459 Isoleucine 47671-3
27460 Valine 47799-2
27455 Reviewed By 18771-6
Mayo Clinic Laboratories | Pediatric Catalog Additional Information: