Question and Answer Session

Febrary 7, 2006 Presentation:
Tandem Mass Spectrometry Screening For Inborn Errors of Metabolism

Welcome to AACC's Expert Access Live Online program

Tandem Mass Spectrometry Screening For Inborn Errors of Metabolism

This month's expert is Robert E. Grier, PhD. View the presentation and direct your questions to our online expert. AACC would like to thank Bayer HealthCare Diagnostics for making this program possible.

Could you explain Electrospray in more depth?
Washington, DC

Robert E. Grier, PhD: As the small liquid sample (approx. 3 ul) enters the MS/MS, it enters the stream of gas moving through the instrument. The sample is pulled through a cone into the vacuum of the MS/MS. This compression tends to aerosol the sample as an electric charge is applied, producing a positive charge on the surface of the very small droplets. These are moved down the MS/MS path for further processing and analysis.

Can you provide a reference or some calculation that compares the cost of MSMS multiplex analysis versus the one analyte/one disease?
Richmond, CA

Robert E. Grier, PhD: The cost comparison analysis has been performed by several state NBS Programs. The Association of Public Health Laboratories (APHL) has this information. Basically, the comparison is favorable for MS/MS if Phenylketonuria (PKU) and Medium Chain Acyl-CoA Dehydrogenase Deficiency (MCAD) were the only two disorders detected. Having the ability to detect the other disorders does not add to the cost, therefore making the cost effectiveness of MS/MS very favorable. MS/MS comes with a relatively high entry cost/lease for the instruments, but the personnel, reagents, time of preparation and disposables are the same for 35 + disorders identified.

Dear Dr, Could you pls elaborate on the "enzyme treatment & dialysis" which you mentioned in your slides.
Kuala Lumpur, Malaysia

Robert E. Grier, PhD: I am a Ph.D. laboratorian and not a physician. I am referring to treatment of a few of the genetic metabolic disorders that are/can be detected by MS/MS. Enzyme replacement treatment has recently become available for 5 Lysosomal Storage Disorders. Recent work out of the Univ. of Washington (Scott and Gelb) has described an elegant MS/MS protocol to screen newborns for 5 LSD disorders, allowing treatment using the "prepared" enzyme to be administered by injection into the patient. LSD screening has reached the NBS laboratory in the State of New York, but no place else to my knowledge. Dialysis is an acute, clinical treatment for a few of the disorders that can present in the Emergency Room with a life-threatening situation. Dialysis can be used to remove ammonia in the blood stream in urea cycle disorders.

Thank you for your excellent presentation. We are at the process of installing a new amino acid analyzer, we are thinking of Biochrom. This is a didicated amino acid analyzer. However, we also think of establishing a metabolic disorder unit. So we will also look for a system for organic acid, MCAD etc. What is your advise? could you advise me the adress of your instruments suplier or if they have a web site that I could visit? Thank

Robert E. Grier, PhD: My experience would tell you that a metabolic genetic laboratory should have an amino acid analyzer and a GC/MS instrument for organic acids. I will not endorse a specific vendor. There are several good instruments on the market in both categories. Make sure that the computer you acquire with the instrument or that you interface with has the capacity and capability needed for your application. Amino acid analysis is the most utilized instrument for metabolic disorder workup. Geneticists, neurologists, pediatric GI and even general pediatrics physicians need AA analysis for their patients. GC/MS is a sensitive, specific tool to identify / diagnose organic acidemias, fatty acid disorders and urea cycle disorders. My current laboratory also utilizes these instruments to monitor the treatment / clinical status of known affected patients under dietary or medication therapy. Over time, the physican has a record of the metabolites that show the patient's status, e.g. phenylalanine in PKU, branched chain amino acids in Maple Syrup Urine Disease and methylmalonic acid in Methylmalonic Acidemia. Using this information, individual treatment can be modified.

I am almost afraid to ask this question, but how much is start up cost of this instrumentation? Would it be practical in small hospital with 1000 births annually? How much training for techs?
Karasburg, Namibia

Robert E. Grier, PhD: "Fear is not an option." Sorry, bad pun. MS/MS instruments that are set up to perform Newborn Screening retail for approx. $250,000. Lease / purchase choices are available. Training is usually included when you acquire the instrument. I would encourage going to a site that is currently running the instrument for a show-and-tell and perhaps training. There would need to be some site preparation for MS/MS - electrical, vented hood, sample preparation space and small equipment. Newborn screening on-site for a delivery hospital having 1,000 annual births would probably not be cost effective. You should consult with your state NBS Lab as to whether they are currently utilizing MS/MS. If they are, or plan to start shortly, your newborns will receive this testing within your state program. There are a few states who are not currently offering MS/MS. Individual hospitals can provide this screening using one of the two private laboratories in the U.S. that offer this service. (Pediatrix Screening in Pittsburgh and Baylor Medical Center in Dallas)

Any audio part we can hear the presentation? Can we call you to hear the presentation? Thanks.
Los Angeles

AACC Moderator: There is no audio component to this program. The "active" portion of Expert Access is Dr. Grier's presentation and the online Q&A.

How well the marker be measured quantitatively by this method?
Union City, California

Robert E. Grier, PhD: The metabolites identified using MS/MS are "semi-quantitated". Just as with other technologies, if you have the compound of interest, you can analyze it as a standard for comparison to patient samples. Otherwise, comparison to another standard or perhaps labeled compound is performed. Several deuterated amino acids and acylcarnitines are available (see figures in the presentation with "star" compounds). This allows direct or inferred quantitation of the compounds detected. This is certainly good enough for screening and actually is very close to dedicated analysis, e.g. amino acid analyzer quantitation.

In the past, one of the major limitations of MS/MS has been machine down time, especially the autoprep units. Have newer iterations of MS/MS systems addressed this issue?
Boston, Mass.

Robert E. Grier, PhD: MS/MS used for NBS come from two vendors. Their instruments are proven to be very robust for this work. I have used both and do not have a preference. I have seen 100,000 NBS samples run in them with no instrument / part failure. This is with good daily /weekly maintenance / cleaning, trained personnel and a protocol that places "clean" sample preparation as paramount. Both of these systems have autosampling for the MS/MS. Sample prep has some manual handling.

Can you explain what you mean by "no false negative results" and "minimal false positives"
Honolulu, HI

Robert E. Grier, PhD: Based upon several state laboratories and the private laboratories, early report of over a million newborns screened had no false negatives made know to the lab(s). Recently, I am aware of a small number of false negative cases for MCAD and a few organic acidemias. These are clinically thought to be mild cases. The genetic defects screened for by MS/MS usually have elevated, easily identified abnormal levels of the metabolites. The rate of false positives in published literature is approx. 1 per 1,500. To accomplish is, cutoffs for the population the lab is screening need to be established. Ongoing statistical analysis of the population allows for modification of these cutoffs over time. The published incidence for MS/MS disorders is approx. 1 in 4,500 term infants.

Any audio part we can hear the presentation? Can we call you to hear the presentation? Thanks.
Los Angeles

Robert E. Grier, PhD: No, there is no audio portion. My voice might damage your equipment.


Could you share your thoughts about derivatized vs non derivatized methods in particular for C5DC?
Denver, CO

Robert E. Grier, PhD: After 10 years of experience with MS/MS, I come down on the side of derivatization of the sample. This increases the sensitivity of the instrument / detection of metabolites. We are finding newborns with elevated level(s) of metabolite(s) that are not the very high findings we would expect. Workup of these cases is sometimes finding mild clinical manifestations at this age, but unknown as the patient grows and metabolism adjusts. My position is to identify affected genetic disorders in the newborn period as well as possible. I believe that labeled C5DC (carnitine with a 5 carbon dicarboxylic chain attached = an acylcarnitine) is recently available. I would still derivatize.

Is there a problem with a lack of standardization across labs? And if yes can a lab feel confident in their results?
Las Vegas, Nevada

Robert E. Grier, PhD: Standardization is an issue. The CDC sends out proficiency testing twice a year, aimed at the NBS laboratories. I know of laboratories that share abnormal samples between themselves to assess capabilities. This develops comfort, knowledge base for the disorders and experience in reading the MS/MS results. We all know that no two instruments are identical. I encourage monitoring of individual instruments and the group in the same lab over time to asses the inherent variability that must be appreciated in result interpretation. I am involved in a HRSA grant to the Great Lakes Region that is collection data to address this issue. It would appear that some level of standardization is a reasonable goal.

Since you are sceeneing for many analytes in one sample, what kind of quality control materials do you use?
Atlanta, GA

Robert E. Grier, PhD: Internal standards are added to each patient sample in the preparation. This allows QC of each sample, compared to the group it is prepared with and to the collected population data of the lab. A normal sample or spiked sample can be prepared and spotted for a large amount of control material to be used over time. Known affected dried blood spots can be shared with other labs. The CDC prepared and provides proficiency testing for NBS labs twice annually.

Can you please comment on how results are submitted to the physician (as yes/no or with some explanation and recommendation?) Also, have you tried linking MS/MS to LIS (lab info system) or handle the date manually? What is the best way to do it?
Cambridge, MA

Robert E. Grier, PhD: Most state programs report a "normal / unaffected" result as such, with no values for the individual amino acids or acylcarnitines. A positive or affected case is reported as such. Various states and labs include the abnormal metabolite(s) and often give the levels, compared to their normal range(s). Certainly the MS/MS instruments are capable of LIS interface. The amount of data in most state NBS Labs is too great to handle manually. A few states are working toward transmitting screening results to the various birthing centers via internet.

Sorry to be so basic, but is there a reference that you would recommend for individuals interested in learning the basics of MS/MS? General and then possibly getting into test specific
Los Angeles

Robert E. Grier, PhD: The instrument manufacturers have copious literature with basic to sophisticated applications. Waters/MicroMass and Perkin Elmer have NBS materials available.

In one of your final slides you mention the possibiltiy of primary DNA screening for inborn errors of metabolism (I hope you're not putting yourself out of work with this admission). How far along are these tests? Do we know which polymorphisms and mutations cause these disorders? Are there commercial probes for these gene targets?
Reno, NV

Robert E. Grier, PhD: Primary DNA screening is available for several of these MS/MS disorders. Mutations and polymorphisms have been identified. DNA protocols are available for several automated systems. Some private labs are doing this work as requested. However, I have a bias as a biochemist. I believe that function is the way to screen. Does an enzyme have activity - does a metabolite show up, at an abnormal level or as an abnormal metabolite ??? Molecular geneticists have found several disorders with many clinically significant mutations (e.g. Cystic Fibrosis with over 1,000 and counting). New mutations can occur which have not been seen before. While DNA screening would appear a "neat way to go", function - metabolite measurement remains a viable, robust test for the forseeable future.

When C5DC appears elevated on the initial screen, why do the levels decrease on the duplicate retest of the same blood spot. Is there some known interference that would affect the C5DC levels?
Baltimore, MD

Robert E. Grier, PhD: This is a question that I do not have the answer. I certainly have seen this phenomenon and not just with C5DC. This does not occur every time. Possible degradation does not appear likely. If you discover the answer, please communicate with me.

Would you recommend using multiple medium chain length acylcarnitine markers, both alone and ratios, to diagnose MCADD? Do you find that you miss potentially important data by using MRM acquisition? Thanks!
Birmingham, AL

Robert E. Grier, PhD: Those with the most experience with MCAD use several acylcarnitines for interpretation. While the elevation of C8 is the primary marker, C10:1, the C8/C2 ratio, the C8/C10 ratio and perhaps others are very important for the scientist to interpret. The acquisition of data in NBS using MS/MS utilizes several spectra (usually 6, see presentation, toward the end). Certainly one would not want to limit to just MRM.

For NICU babies, why are the first or second specimens normal, but subsequent specimens may give elevations in fatty acid oxidation disorders or organic acidemias?

Robert E. Grier, PhD: Diet. Most all NICU patients receive TPN - total parenteral nutrition - with supplemented levels of amino acids and a good amount of long chain fats. After the initial NBS of NICU patients, most states encourage waiting to do another screen until the patient is off TPN for several days and is usually leaving the unit. As to the first and second screens being normal - NICU patients can have the same risk of being affected with a genetic metabolic disorder as a "normal" infant. Conversely, they do not have an increased risk. They can be identified on their first screen, if affected.

What is your position on non-derivatized MS/MS newborn screening, in light of the newly available C5-DC deuterated internal standard?
Pleasant F. Hooper, M.D.

Robert E. Grier, PhD: Please see previous question and answer.

What sort of quality control do you use for the amino acid and organic acid MSMS assays.
Birmingham, AL

Robert E. Grier, PhD: Please see previous question and answer.

how many newborn factors currently require testing in MI/USA? also, -how much instrument time is requires to get first patient result, and each addn patient result with your proticol?
grand rapids, mi

Robert E. Grier, PhD: I am unclear on your first question. Michigan currently mandates NBS for 11 genetic disorders. It has a Pilot Project for MS/MS that will be transitioned to mandated soon. MS/MS time for each patient sample is approximately 2 minutes. Of that time, 30 seconds is for data acquisition and 1.5 minutes is utilized to flush the MS/MS pathway for the next patient sample. One patient analysis each 2 minutes, auto-sampling of the prepared samples, auto-data acquisition.

CDC send out Proficiency Testing panels 4 times per year. CDC also provides external quality control materials for routine use. Go to for information and to request materials for participation in the Newborn Screening Quality Assurance Program.

Robert E. Grier, PhD: Thank you for the correction and information.

I see in the sample prep that the blood spot size was 3/16". Is this routine newborn screening or follow up testing of initial positives. Most newborn screening is done on a 1/8" blood spot.
madison, Wi

Robert E. Grier, PhD: Various labs should determine the size of the dried blood spot that gives a robust response from the MS/MS following the sample preparation. Most labs have gone to the 3/16" punch for the "material" eluted to match the response they wish to have.

Are ther difficulties in switching analysis for AA & organic acids to analysis for other analytes i.e drugs of abuse and immunosuppressant drug analysis; this would enhance greatly justification for the start-up cost.

Robert E. Grier, PhD: Most applications that I have dealt with were dedicated. However, if you read the journal, Clinical Chemistry, there has been at least one article on MS/MS in each issue for over a year. I believe that MS/MS will become a vital instrument in the hospital chemistry department in the near future. The instrument can analyze/detect a variety of compounds, with variations in the sample prep and setting the "windows" to detect the compounds and fragments.

In calculating the prevalance rate for aminoacidopathies did to include hyperphenylalaninemia and other "non" classical disorders?
madison, Wi

Robert E. Grier, PhD: The quoted incidence rate of 1:4,500 did not include PKU, but did include MCAD. Part of the justification of screening for several very rare disorders is that this detection is included with no additional effort. Screening for a genetic disorder with an incidence of 1:250,000 with an individual assay is not effective, yet MS/MS does this "for free".

Do you have a means for separating compounds that have similar mass and structure such as methylmalonic acid and succinic acid?

Robert E. Grier, PhD: Often the derivatization process will result in a differentiation of the final compound that is detectable. Also, fragmentation of the different structure can give recognizable separation.

What do you use to disolve the AA IS? Methanol only or a mixture with water? And for Ac IS? the same? And, What about the best protocol to extract the blood spots?
Santiago de Compostela. Spain

Robert E. Grier, PhD: To dissolve the amino acids and acylcarnitines for use as internal standards, one can first dissolve them in a small amount of an appropriate buffer and then bring them up to volume in the dried blood spot elution solution. The protocols that I am familiar with use a methanol:water mixture for dried blood spot elution. The best protocol would maximize the elution of both amino acids and acylcarnitines - and Me/H20 comes close.

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