In cases of sudden or unexpected death, autopsy evaluation can include a biochemical analysis to identify deaths caused by fatty acid oxidation (FAO) disorders. Identification of these individuals is important to guide genetic counseling and testing of at-risk family members. Some states mandate postmortem metabolic screening for at least a subset of the population that dies suddenly and unexpectedly.
The thorough investigation of a sudden, unexpected death should include the collection of blood and bile spots on a single filter paper card. The ease of specimen collection, affordability, and availability of direct consultation with laboratory directors and genetic counselors in the Biochemical Genetics Laboratory are key components of the metabolic autopsy protocol at Mayo Clinic.
Fatty Acid Oxidation Disorders—Clinical Background
FAO disorders frequently manifest with sudden and unexpected death at any age and may account for up to 5% of sudden infant deaths.1 FAO plays a major role in energy production when the body’s supply of glucose is insufficient to maintain a normal level of energy (i.e., states of fasting, illness, increased muscular activity).2 During these times, fatty acids are mobilized from adipose tissue, taken up by the liver and muscles, and in a step-wise process oxidized to acetyl-CoA. In the liver, acetyl-CoA is the building block for the synthesis of ketone bodies, which enter the blood stream and provide an alternative substrate for production of energy in other tissues.
Signs and symptoms may vary greatly, frequently leading to life-threatening episodes of metabolic decompensation after a period of inadequate caloric intake and/or intercurrent illness.2 Symptoms vary in severity and may appear at any age, from birth to adult life. Affected patients are at risk for developing a life-threatening episode of metabolic decompensation in response to relatively benign events, like a common viral infection. Once diagnosed, these disorders can be treated by avoidance of fasting, special diets, and cofactor/vitamin supplementation. Sadly, many previously asymptomatic and undiagnosed patients with FAO die at the time of their first acute episode.
The major clinical manifestations associated with individual FAO disorders include:
- hypoketotic hypoglycemia.
- a Reye-like syndrome (an acquired encephalopathy characterized by recurrent vomiting, agitation, and lethargy).
- variable degrees of liver disease and/or failure.
- skeletal myopathy, dilated or hypertrophic cardiomyopathy.
- sudden, unexpected death.
Factors which increase the risk of a metabolic disorder include:
- Family history of sudden infant death syndrome (SIDS) or other sudden, unexplained deaths at any age.
- Family history of Reye syndrome.
- Maternal complications of pregnancy (acute fatty liver of pregnancy, HELLP syndrome, others).
- Lethargy, vomiting, fasting in the 48 hours prior to death.
- Macroscopic findings at autopsy.
- Fatty infiltration of the liver and/or other organs.
- Dilated or hypertrophic cardiomyopathy.
- Allegation of child abuse (excluding obvious cases of trauma/physical harm).
- Autopsy evidence of infection that routinely would not represent a life-threatening event.
Tandem Mass Spectrometry (MS/MS)
The determination of acylcarnitines by tandem mass spectrometry (MS/MS) for the diagnosis of FAO disorders was established in the late 1980s. Diagnostic results are usually characterized by a pattern of significantly elevated acylcarnitine species compared to a normal range. Multiple inborn errors of metabolism can be identified using this method. In recent years, electrospray MS/MS has been applied to the analysis of whole blood and bile spots enabling the postmortem identification of individuals with certain metabolic disorders.1
FAO Disorders Detectable by Acylcarnitine Analysis:
- Short-chain acyl-CoA dehydrogenase (SCAD) deficiency
- Medium/Short-chain 3-hydroxyacyl-CoAdehydrogenase (M/SCHAD) deficiency
- Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency
- Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency and trifunctional protein deficiency
- Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency
- Carnitine palmitoyl transferase deficiency type II (CPT-II)
- Carnitine-acylcarnitine translocase deficiency
- ETF deficiency, ETF-dehydrogenase deficiency (multiple acyl-CoA dehydrogenase deficiency (MADD), glutaric acidemia type II)
In many FAO disorders, characteristic acylcarnitine species accumulate in the blood and bile. Analysis of these metabolites represents the first level of evaluation of a complete postmortem investigation of a sudden and/or unexpected death. If an underlying metabolic disorder is diagnosed, families should seek genetic counseling, which should include a discussion of recurrence risks, the option of future prenatal diagnosis, and testing of at-risk relatives, thereby preventing morbidity and mortality in other affected family members.
Effective postmortem screening depends upon collection of appropriate specimens and result interpretation by experienced laboratory directors. Both blood and bile specimens must be collected in order to detect and independently confirm the largest possible number of disorders. The interpretation of the acylcarnitine profiles is a highly complex task that requires knowledge of normal and abnormal acylcarnitine patterns in living and deceased individuals and in varying tissue types. For example, in comparison to living individuals, the profiles of postmortem blood specimens generally show much higher concentrations of short-chain species. Patients with secondary carnitine deficiency may display uninformative acylcarnitine profiles in blood, but not in bile. Postmortem screening via acylcarnitine analysis will not rule out all FAO disorders, as several conditions are not associated with abnormal acylcarnitine profiles (i.e., carnitine palmitoyltransferase I deficiency, 3-hydroxy-3-methylglutaryl CoA synthase deficiency). Technologically, there are still metabolic disorders that are not detectable with currently available assays.
Specimen Collection and Transport
The required specimens for Postmortem Screening, Bile and Blood Spots (PMSBB) are blood and bile dried on a filter paper card such as newborn screening filter paper cards. These cards are available from Mayo Medical Laboratories (1-800-533-1710) by ordering supply T525, Postmortem Screening Card. If both blood and bile are available, both specimen types should be collected to optimize testing.
Blood-specimen collection is usually from the proximal aorta or by intracardiac puncture. Bile collection is obtained by direct puncture of the gallbladder. After collection, the blood and bile specimens are spotted on a filter paper card, completely filling 2 circles with blood and 2 with bile (25 mL for each circle). At minimum, 1 circle with blood and/or 1 circle with bile should be collected. See photos below for examples of correct and incorrectly collected specimens.
After spotting of the filter paper card, the filter paper card should be allowed to dry at room temperature in a horizontal position for three or more hours. These specimens should not be exposed to heat or direct sunlight. Wet filter paper cards and dried blood and bile specimens should be protected from accidental contact with liquids. Dried blood and bile spots are stable for at least two months when stored at room temperature and indefinitely when stored frozen. Filter paper card specimens should be shipped to Mayo Medical Laboratories (MML).
A clinical history and a summary of the autopsy findings are necessary to provide the most accurate interpretation of the laboratory results, and are used in determining whether additional specimens must be analyzed. The clinical history and autopsy findings should be forwarded either with the dried blood and bile specimens, or as soon as possible after submission of the specimens. Clinical information also may be faxed to (507) 284-9211, to the attention of “Genetic Counselor, Biochemical Genetics Laboratory” with an indication that this is regarding a postmortem screen. Please include the patient’s last and first names, the date of birth, the date of death, and the date the blood and bile spots were submitted to MML.
Original Newborn Screening Card
Retrieval of the original newborn screening card also is recommended whenever possible. The Biochemical Genetics Laboratory at Mayo Clinic can analyze the original newborn blood spots to aid in the postmortem diagnosis of metabolic disorders. Retrieval of the original newborn screening card can be accomplished by faxing or mailing a letter to the laboratory that performs newborn screening for the state in which the decedent was born. The letter must include the current date, decedent’s name, gender, date of birth, place of birth (including hospital, city and state), a request for the specimen to be sent to MML, and the signature of one or both parents. It is helpful to include in the letter that this request is in follow-up to a postmortem screening specimen that was submitted to MML and the date it was sent to MML. A template for this request is available in MML’s online test catalog. A genetic counselor or laboratory director in the Biochemical Genetics Laboratory at Mayo Clinic can provide assistance if you have questions regarding how to locate or contact laboratory personnel affiliated with the laboratory that performed newborn screening on the decedent.
Result Interpretation and Reporting
Inherited metabolic disorders result in the accumulation of abnormal acylcarnitines or increased concentrations of normally detectable species. The quantitative measurements of individual acylcarnitines are not diagnostic by themselves; result interpretation of the postmortem acylcarnitine profile is based primarily on pattern recognition. The pattern of masses and concentrations obtained by MS/MS coincident with acylcarnitine masses yields an interpretive report that is enhanced by any relevant information provided from the clinical or dietary history or from the postmortem examination.
Abnormal results may not be sufficient to conclusively establish a diagnosis of a particular disease. When abnormal results are detected, a detailed interpretation is provided, including an overview of the results and their significance, a correlation to available clinical information provided with the specimen, and possible differential diagnoses. When indicated, we provide recommendations for additional testing, including in vitro confirmatory studies (enzyme assay, molecular analysis) and the name and phone number of key contacts who may offer these studies at Mayo Clinic or elsewhere. If additional decedent specimens are unavailable, molecular genetic analysis of the decedent’s parents may enable the confirmation of a diagnosis in some cases.
How to Order
To order testing, you will need to register on Mayo Medical Laboratories' website and create an account. To set up a new account, please call (800) 533-1710 for a representative to assist you, or follow the steps below to set up an account electronically.
If you already have an account, use the links below to sign in:
Registration takes less than 10 minutes, and new accounts are typically processed and available in one business day. Once registered, sign in and locate the Postmortem Screening, Bile and Blood Spots test in the Test Catalog and click the "Order This Test" button. Follow the prompts to provide billing and contact information. Print the test requisition form to ship with the specimen.
Medical examiners, coroners, and other health practitioners involved with the investigation of sudden, unexpected deaths are encouraged to perform a metabolic autopsy as standard procedure regardless of the age of the decedent. MML provides a low-cost and convenient option for postmortem screening. When a diagnosis is established, referral to a genetics clinic for counseling and facilitation of testing at-risk family members is beneficial.
Consultation with a laboratory director or genetic counselor is strongly encouraged prior to specimen submission. The Biochemical Genetics Laboratory at Mayo Clinic has six laboratory directors and four genetic counselors available for consultation should you have questions regarding the metabolic autopsy protocol, wish to discuss a specific case prior to submission of specimens, or need assistance with result interpretation or are following up on a screen-positive result. These individuals can be reached by contacting Mayo Medical Laboratories at (800) 533-1710.
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