Method for interpreting tandem mass spectrometry data for clinical diagnosis

• US 6,455,321 B1
• Filed: 12/16/1999
• Issued: 09/24/2002
• Est. Priority Date: 01/30/1999
• Status: Expired due to Term

• Current Assignee: PerkinElmer Genetics, Inc. (Revvity, Inc.)
• Original Assignee: Neo Gen Screening, Inc. (Mednax Incorporated)
• Inventors: Chace, Donald H.
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): Cole, Monique T.
• Application Number:
  US09/464,132
• Time in Patent Office: 1,013 Days
• Field of Search: 436/86, 436/173, 702/22, 702/23, 702/27
• US Class Current: 436/173
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

A method for interpreting data that is produced after a group of amino acids and acylcarnitines are derivatized from blood spots taken from newborn babies and scanned by a tandem mass spectrometer. Concentration levels of each metabolite, which are directly proportional to the butyl ester fragment after derivatization, are compared to threshold flags for determining a significance of any deviation of the metabolite relative to the flag threshold. The threshold flags are diagnostic limits to the data retrieved from each blood spot. The data includes metabolite concentrations and molar ratios of metabolites with other metabolites. Samples are labeled normal for a disease if the concentration of any of the metabolite concentrations or molar ratio concentration do not deviate from the flag threshold, but, in contrast, the sample must be further evaluated if a value is elevated or deficient to some degree. Thus, as each metabolite fragments at a different mass to charge value (m/z), corresponding data is compared to the respective flag thresholds for determining a next course of action that must be taken to ultimately assist a physician in the diagnosis of a genetic disorder resulting from an elevation or deficiency of the metabolite particular for that disorder.

Claims

1. A method for assisting in a diagnosis of propionic acidemia by interpreting data produced after a dry blood spot on filter paper is derivatized and scanned by a tandem mass spectrometer, comprising the steps of:

acquiring said data, wherein said data includes values comprised of a propionyl carnitine concentration (C3), a molar ratio of an mrm scan of propionyl carnitine and acetyl carnitine (C3mrm/C2), and a molar ratio of propionyl carnitine and palmitoyl carnitine (C3/C16);

applying said values to a propionyl carnitine (C3) decision tree, wherein said decision tree further comprises a C3 flag threshold, a C3mrm/C2 flag threshold, and a C3/C16 flag threshold as diagnostic limits to said C3, said C3mrm/C2, and said C3/C16 respectively;

comparing said values, respectively, with each of said flag thresholds;

identifying whether or not there is an elevation of any of said values above any of said flag thresholds;

interpreting said sample as being normal for said propionyl carnitine, provided there is no said elevation of any of said values;

performing a subsequent re-analysis to obtain mean values, provided any of the following occurs;

i. said C3 is equal to or greater than said C3 flag threshold;

ii. said C3/C16 is greater than or equal to said C3/C16 flag threshold and said C3/C2 is greater than said C3/C2 flag threshold and said C3 is greater than about 2.5 uM;

iii. said C3 is greater than about 4 uM, and either said C3/C16 is greater than said C3/C16 flag threshold or said C3/C2 is greater than said C3/C2 flag threshold;

performing an immediate re-analysis with a preliminary follow-up to obtain priority mean values, provided any of the following occurs;

i. said C3 is greater than about 9.0 uM;

ii. said C3 is greater than about 7.0 uM and said C3/C2 is greater than said C3/C2 flag threshold or said C3/C16 is greater than said C3/C16 flag threshold; and

,

initiating a follow-up protocol based on follow-up criteria utilizing said mean values or said priority mean values for diagnostic purposes provided either said subsequent re-analysis or said immediate re-analysis is performed;

reporting said data to a physician to assist in said diagnosis of propionic acidemia.

2. The method of claim 1, wherein said C3 flag threshold, said C3mrm/C2 flag threshold, and said C3/C16 flag threshold are, respectively, 5.0 uM, 0.3 uM, and 1.75.

3. A method for assisting in a diagnosis of isovaleric acidemia by interpreting data produced after a dry blood spot on filter paper is derivatized and scanned by a tandem mass spectrometer, comprising the steps of:

acquiring said data, wherein said data includes values comprised of an isovaleryl carnitine concentration (C5) and a propionyl carnitine concentration (C3);

applying said isovaleryl carnitine concentration (C5 ) to an isovaleryl carnitine (C5) decision tree, wherein said decision tree further comprises a C5 flag threshold as diagnostic limit to said isovaleryl carnitine concentration (C5);

comparing said C5 to said flag threshold;

identifying whether or not there is an elevation of C5 above said flag threshold;

interpreting said sample as being normal for said isovaleryl carnitine, provided there is no said elevation of said C5;

performing a subsequent re-analysis to obtain a mean value of said C5, provided said C5 is greater than or equal to said C5 flag threshold;

performing an immediate re-analysis with a preliminary follow-up to obtain a priority mean value of said C5, provided any of the following occurs;

i. said C5 is greater than about 2.0 uM;

ii. said C5 is greater than about 1.0 uM and said C3 is greater than about 2.5 uM;

initiating a follow-up protocol based on follow up criteria utilizing said-mean value or

said priority mean value for diagnostic purposes provided either said subsequent re-analysis or said immediate re-analysis is performed; and

,

reporting said data to a physician to assist in said diagnosis of isovaleric acidemia.

4. The method of claim 3, wherein said C5 flag threshold is 0.8 uM.

5. A method for assisting in a diagnosis of hypermethionemia by interpreting data produced after a dry blood spot on filter paper is derivatized and scanned by a tandem mass spectrometer, comprising the steps of:

acquiring said data, wherein said data includes values consisting essentially of a methionine concentration (met) and a molar ratio of methionine and phenylalanine (met/phe);

applying said values to a methionine (met) decision tree, wherein said decision tree further comprises a methionine flag threshold and a met/phe flag threshold as a diagnostic limit to said methionine concentration (met);

comparing said values, respectively, to each said flag threshold;

identifying whether or not there is an elevation of any of said values above any of said flag thresholds;

interpreting said sample as being normal for said hypermethionemia, provided there is no said elevation of any of said values;

performing a subsequent re-analysis to obtain mean values, provided any of the following occur;

i. said met is greater than said met flag threshold;

ii. said met is greater than about 50 uM and said met/phe is greater than said met/phe flag threshold;

performing an immediate re-analysis with a preliminary follow-up to obtain priority mean values, provided any of the following occur;

i. said met is greater than about 150 uM;

ii. said met is greater than about 125 uM and said met/phe is greater than about 1.25;

initiating a follow-up protocol based on follow-up criteria utilizing said mean values or said priority mean values for diagnostic purposes provided either said subsequent re-analysis or said immediate re-analysis is performed; and

,

reporting said data to a physician to assist in said diagnosis of hypermethionemia.

6. The method of claim 5, wherein said methionine flag threshold and said met/phe flag threshold are, respectively, 60 uM and 1.0.

7. A method for assisting in a diagnosis of glutaric acidemia by interpreting data produced after a dry blood spot on filter paper is derivatized and scanned by a tandem mass spectrometer, comprising the steps of:

acquiring said data, wherein said data includes values comprised of a glutaryl carnitine concentration (C5DC) and a molar ratio of glutaryl carnitine and palmitoyl carnitine (C5DC;

C16);

applying said values to a glutaryl carnitine (C5DC) decision tree, wherein said decision tree further comprises a C5DC flag threshold and a C5DC;

C16 flag threshold as diagnostic limits to said glutaryl carnitine concentration (C5DC) and said C5DC;

C16 respectively;

comparing said values, respectively, to each of said flag thresholds;

identifying whether or not there is an elevation of any of said values above any of said flag thresholds;

interpreting said sample as being normal for said glutaric acidemia, provided there is no said elevation of any of said values;

performing a subsequent re-analysis to obtain mean values, provided any of the following occur;

i. said C5DC is greater than said C5DC flag threshold;

ii. said C5DC;

C16 is greater than said C5DC;

C16 flag threshold and said C5DC is greater than about 0.14 uM;

performing an immediate re-analysis with a preliminary follow-up to obtain priority mean values, provided any of the following occur;

i. said C5DC is greater than about 0.4 uM;

ii. said C5DC is greater than about 0.2 uM and said C5DC;

C16 is greater than about 0.2 uM;

initiating a follow-up protocol based on follow up criteria utilizing said mean values or said priority mean values for diagnostic purposes provided either said subsequent re-analysis or said immediate re-analysis is performed; and

,

reporting said data to a physician to assist in said diagnosis of glutaric acidemia.

8. The method of claim 7, wherein said C5DC flag threshold and said C5DC:

C16 flag threshold are, respectively, 0.17 uM and 0.12.

9. A method for assisting in a diagnosis of phenylketonuria by interpreting data produced after a dry blood spot on filter paper is derivatized and scanned by a tandem mass spectrometer, comprising the steps of:

acquiring said data, wherein said data includes values consisting essentially of a phenylalanine concentration (phe), a tyrosine concentration (tyr), and a molar ratio of phenylalanine and tyrosine (phe/tyr);

applying said values to a phenylalanine (phe) decision tree, wherein said decision tree further comprises a phe flag threshold, a tyr flag threshold, and a phe/tyr flag threshold as diagnostic limits to said phe, said tyr, and said phe/tyr molar ratio respectively;

comparing said values, respectively, to each of said flag thresholds;

identifying whether or not there is an elevation of any of said values above any of said flag thresholds;

interpreting said sample as being normal for said phenylketonuria, provided there is no said elevation of any of said values;

performing a subsequent re-analysis to obtain mean values, provided any of the following occur;

i. said phe is greater than said phe flag threshold;

ii. said tyr is greater than said tyr flag threshold;

iii. said phe/tyr is greater than said phe/tyr flag threshold and said phe is greater than 100 uM;

performing an immediate re-analysis with a preliminary follow-up to obtain priority mean values, provided any of the following occur;

i. said phe is greater than about 240 uM;

ii. said phe is greater than about 180 uM and said phe/tyr is greater than said phe/tyr flag threshold;

initiating a follow-up protocol based on follow-up criteria utilizing said mean values or said priority mean values for diagnostic purposes provided either said subsequent re-analysis or said immediate re-analysis is performed; and

,

reporting said data to a physician to assist in said diagnosis of phenylketonuria.

10. The method of claim 9, wherein said phe flag threshold, said tyr flag threshold, and said phe/tyr flag threshold are, respectively, 130 uM, 350 uM, and 2.5.

11. A method for assisting in a diagnosis of Maple Syrup Urine Disease (MSUD) by interpreting data produced after a dry blood spot on filter paper is derivatized and scanned by a tandem mass spectrometer, comprising the steps of:

acquiring said data, wherein said data includes values comprised of a leucine+isoleucine concentration (leu+ile), a valine concentration (val), a molar ratio of leucine and phenylalanine (leu/phe), and a molar ratio of leucine and alanine (leu/ala).

applying said values to a leucine (leu) decision tree, wherein said decision tree further comprises a leu+ile flag threshold, a val flag threshold, a leu/phe molar ratio flag threshold, and a leu/ala molar ratio flag threshold as diagnostic limits to said leu+ile, said val, said leu/phe, and said leu/ala respectively;

comparing said values, respectively, to each of said flag thresholds;

identifying whether or not there is an elevation of any of said values above any of said flag thresholds;

interpreting said sample as being normal for said MSUD, provided there is no said elevation of any of said values;

performing a subsequent re-analysis to obtain mean values, provided any of the following occur;

i. said leu+ile is greater than about 400 uM;

ii. said leu+ile is greater than about 350 uM and said val is greater than said val flag threshold;

iii. said leu+ile is greater than said leu+ile flag threshold and said leu/ala is greater than said leu/ala flag threshold, or said leu/phe is greater than said leu/phe flag threshold and said val is greater than said val flag threshold;

performing an immediate re-analysis with a preliminary follow-up to obtain priority mean values, provided any of the following occur;

i. said leu+ile is greater than about 500 uM;

ii. said leu+ile is greater than about 400 uM and said leu/phe is greater than said leu/phe flag threshold and said leu/ala is greater than said leu/ala flag threshold;

initiating a follow-up protocol based on follow-up criteria utilizing said mean values or said priority mean values for diagnostic purposes provided either said subsequent re-analysis or said immediate re-analysis is performed; and

,

reporting said data to a physician to assist in said diagnosis of MSUD.

12. The method claim 11, wherein said leu+ile flag threshold, said val flag threshold, said leu/phe flag threshold, and said leu/ala flag threshold are, respectively, 325 uM, 300 uM, 8.0, and 2.25.

13. A method for assisting in a diagnosis of citrullinemia by interpreting data produced after a dry blood spot on filter paper is derivatized and scanned by a tandem mass spectrometer, comprising the steps of:

acquiring said data, wherein said data includes values comprised of a citrulline concentration (cit) and a citrulline concentration after an mrm scan (cit[mrm]);

applying said values to a citrulline (cit) decision tree, wherein said decision tree further comprises a cit flag threshold and a cit(mrm) flag threshold as diagnostic limits to said citrulline concentration (cit) and said cit(mrm) concentration respectively;

comparing said values, respectively, to each of said flag thresholds;

identifying whether or not there is an elevation of any of said values above any of said flag thresholds;

interpreting said sample as being normal for said citrullinemia, provided there is no said elevation of any of said values;

performing a subsequent re-analysis to obtain mean values, provided any of the following occur;

i. said cit or said cit(mrm) is greater than said cit flag threshold or said cit(mrm) flag threshold;

performing an immediate re-analysis with a preliminary follow-up to obtain priority mean values, provided any of the following occur;

i. said cit is greater than about 100 uM;

initiating a follow-up protocol based on follow up criteria utilizing said mean values or said priority mean values for diagnostic purposes provided either said subsequent re-analysis or said immediate re-analysis is performed; and

,

reporting said data to a physician to assist in said diagnosis of citrullinemia.

14. The method of claim 13, wherein said cit flag threshold and said cit(mrm) flag threshold are both 55 uM.

15. A method for assisting in a diagnosis of medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency by interpreting data produced after a dry blood spot on filter paper is derivatized and scanned by a tandem mass spectrometer, comprising the steps of:

acquiring said data, wherein said data includes values comprised of an octanoyl carnitine concentration (C8), a molar ratio of octanoyl carnitine and palmitoyl carnitine (C8/C16), a hexanoyl carnitine concentration (C6), a decenoyl carnitine concentration (C10;

1), and a decanoyl carnitine concentration (C10);

applying said values to an octanoyl carnitine (C8) decision tree, wherein said decision tree further comprises a C8 flag threshold, a C8/C16 molar ratio flag threshold, a C6 flag threshold, a C10;

1 flag threshold, and a C10 flag threshold as diagnostic limits to said C8, said C8/C16, said C6, said C10;

1, and said C10 respectively;

comparing said values, respectively, to each of said flag thresholds;

identifying whether or not there is an elevation of any of said values above any of said flag thresholds;

interpreting said sample as being normal for said MCAD deficiency, provided there is no said elevation of any of said values;

performing a subsequent re-analysis to obtain mean values, provided any of the following occur;

i. said C8 is greater than or equal to about 0.4 uM;

ii. said C8 is greater than about 0.3 uM and said C8/C16 is greater than about 0.15;

iii. said C8 is greater than about 0.3 uM and said C6 is greater than about 0.2 uM, or said C10;

1 is greater than about 0.2 uM and said C10 is greater than about 0.3 uM with a low acetyl flag;

performing an immediate re-analysis with a preliminary follow-up to obtain priority mean values, provided any of the following occur;

i. said C8 is greater than about 1.0 uM;

ii. said C8 is greater than about 0.5 uM and said C8/C16 is greater than about 0.35, or said C6 is greater than about 0.3 uM and said C10;

1 is greater than about 0.3 uM;

initiating a follow-up protocol based on follow-up criteria utilizing said mean values or said priority mean values for diagnostic purposes provided either said subsequent re-analysis or said immediate re-analysis is performed; and

,

reporting said data to a physician to assist in said diagnosis of MCAD deficiency.

16. The method of claim 15, wherein said C8 flag threshold, said C8/C16 flag threshold, said C6 flag threshold, said C10:

1 flag threshold, and said C10 flag threshold are, respectively, 0.35 uM, 0.28, 0.16 uM, 0.32 uM, and 0.42 uM.

17. A method for assisting in a diagnosis of VLCAD deficiency by interpreting data produced after a dry blood spot on filter paper is derivatized and scanned by a tandem mass spectrometer, comprising the steps of:

acquiring said data, wherein said data includes values comprised of a saturated myristoyl carnitine concentration (C14), an unsaturated myristoyl carnitine concentration (C14;

1), and a molar ratio of C14;

1 and palmitoyl carnitine (C16);

applying said values to a myristoyl carnitine (C14) decision tree, wherein said decision tree further comprises a C14 flag threshold, a C14;

1 flag threshold, and a C14;

1/C16 flag threshold as diagnostic limits to said C14, said C14;

1, and said C14;

1/C16 respectively;

comparing said values, respectively, to each of said flag thresholds;

identifying whether or not there is an elevation of any of said values above any of said flag thresholds;

interpreting said sample as being normal for said MSUD, provided there is no said elevation of any of said values;

performing a subsequent re-analysis to obtain mean values, provided any of the following occur;

i. said C14 is greater than said C14 flag threshold or said C14;

1 is greater than said C14;

1 flag threshold;

said C14 is greater than about 0.75 uM or said C14;

1 is greater than about 0.65 uM and said C14;

1/C16 is greater than about 0.24;

performing an immediate re-analysis with a preliminary follow-up to obtain priority mean values, provided any of the following occur;

i. said C14 is greater than about 2.0 uM or said C14;

1 is greater than about 1.5 uM;

ii. said C14 is greater than about 1.5 uM and said C14;

1 is greater than about 1.0 uM and said C14;

1/C16 is greater than about 0.3;

initiating a follow-up protocol based on follow-up criteria utilizing said mean values or said priority mean values for diagnostic purposes provided either said subsequent re-analysis or said immediate re-analysis is performed; and

,

reporting said data to a physician to assist in said diagnosis of VLCAD deficiency.

18. The method of claim 17, wherein said C14 flag threshold, said C14:

1 flag threshold, and said C14;

1/C16 flag threshold are, respectively, 0.85 uM, 0.70 uM, and 0.24.

19. A method for assisting in a diagnosis of a Crotonyl co-A carboxylase deficiency elevation by interpreting data produced after a dry blood spot on filter paper is derivatized and scanned by a tandem mass spectrometer, comprising the steps of:

acquiring said data, wherein said data includes values comprised of a C5-OH and C5;

1;

applying said values to a hydroxy-C5 (C5-OH) decision tree, wherein said decision tree further comprises a C5OH flag threshold and a C5;

1 flag threshold as diagnostic limits to said C5OH and said C5;

1 respectively;

comparing said values, respectively, to each of said flag thresholds;

identifying whether or not there is an elevation of any of said values above any of said flag thresholds;

interpreting said sample as being normal for said crotonyl co-A carboxylase deficiency, provided there is no said elevation of any of said values;

performing a subsequent re-analysis to obtain mean values, provided any of the following occur;

said C5OH is greater than or equal to said C5OH flag threshold;

said C5;

1 is greater than or equal to said C5;

1 flag threshold;

performing an immediate re-analysis with a preliminary follow-up to obtain priority mean values, provided any of the following occur;

i. said C5OH is greater than about 3.0 uM;

ii. said C5;

1 is greater than about 1.0 uM;

initiating a follow-up protocol based on follow-up criteria utilizing said mean values or said priority mean values for diagnostic purposes provided either said subsequent re-analysis or said immediate re-analysis is performed; and

,

reporting said data to a physician to assist in said diagnosis of crotonyl co-A carboxylase deficiency.

20. The method of claim 19, wherein said C5OH flag threshold and said C5:

1 flag threshold are, respectively, 0.85 uM and 0.35 uM.