Standards and Guidelines for Clinical Genetics Laboratories
2006 Edition

F1 General
F1.1 A biochemical genetics laboratory is defined as one concerned with the evaluation and/or diagnosis of patients and/or families with inherited metabolic disease, monitoring of treatment, and distinguishing heterozygous carriers from noncarriers by biochemical and/or enzyme analysis of physiological fluids and tissues.
F1.2 Most analyses performed in biochemical genetics laboratories utilize methods similar to those used in clinical chemistry laboratories. Procedures to ensure proper performance and safety of equipment are the same as those utilized in clinical chemistry laboratories. The biochemical genetics laboratory differs from the clinical chemistry laboratory in the extent of interpretation that is necessary to make its results meaningful to the clinician.
F2 Personnel

See Section B for details of policies and requirements for director, supervisor, and staff members of clinical genetics laboratories.

F3 Facilities

See Section C1 for guidelines pertaining to maintenance of facilities and equipment.

F4 Specimens

See Section C2.

F4.1 When appropriate, specimen information should include time relative to the last meal, diet and medications.
F5 Records

See Section C3.

F6 Quality Control/Assurance/Improvement

See Section C4.


Analytical Methods
F7.1 Methods used in biochemical genetics laboratories cover a broad range of techniques and procedures, including ion exchange and gas chromatography, mass spectrometry, and electrophoresis, as well as enzyme and analytical assays that utilize immunologic, spectrophotometric, fluorometric and radiochemical techniques. It is not practical to recommend procedures to be used for particular analyses and analytes, except that procedures used should be consistent with current laboratory practice.
F7.2 Procedures utilized must be capable of providing accurate and, when appropriate, rapid results. This is particularly important for prenatal diagnosis or when the patient is acutely ill. If a rapid result cannot be produced in such circumstances, the referring physician or facility must be notified so as to permit consideration of, and/or plans for, alternative testing.
F7.3 Procedures must be in place to verify normative data used by the laboratory, to document the accuracy and precision of the methods used, and to maintain acceptable performance of each analysis. Such procedures must include appropriate use of internal and external standards and, when possible, analysis of positive and negative patient controls.
F7.4 When possible, the laboratory should establish its own normal, affected and, where appropriate, carrier ranges for each test. These values should be reviewed and updated as necessary. Documentation of the values and when they were established should be available in the laboratory for interpretation of results and for outside review.
F7.5 Quantitative Amino Acid Analysis (Updated November 2003)
F7.5.1 Introduction: The universal importance of amino acids stems from their role in literally all metabolic and cellular functions. Amino acids serve as protein building blocks, as metabolic intermediates, and as a source of energy. By definition, amino acids contain an amino group and a carboxyl group, and may contain another functional group (e.g., sulfhydryl, hydroxyl, or secondary amino- or carboxyl-group). Ion exchange chromatography analysis of amino acids in plasma is the most common diagnostic method to identify defects of amino acid metabolism. Screening methods such as thin layer chromatography that do not result in positive identification of all amino acids of clinical interest can lead to errors. Laboratories that use such methods should make the limitations of the methodology clear and indicate the appropriate follow-up testing in the patient report. The quantitative analysis of a clinical specimen requires three steps:
  1. isolation/separation of the free amino acids from the physiological specimen
  2. separation and identification of the tagged or labeled compounds
  3. quantitation of compounds by comparison to standards
F7.5.2 Background
F7.5.2.1 Clinical Description of Disease (Amino Acidemias and Acidurias): As a group of inborn errors of metabolism, amino acid defects are clinically and biochemically heterogeneous. They are characterized biochemically by the accumulation of pathological amounts of normal metabolites or by the accumulation of metabolites that are not present under physiological conditions but are produced from the activation of alternative pathways in response to the loss of function of a specific gene product (enzyme). The natural history of these disorders is variable and disease-specific.
F7.5.2.2 Prevalence: The birth of a child with an amino acid disorder is not rare. The estimated incidence of phenylketonuria (PKU) alone is 1:12,000, with the combined incidence for all amino acidopathies estimated at 1:6,000. This estimate does not include other inborn errors of metabolism (i.e., organic acid disorders, some urea cycle disorders, and congenital lactic acidemias) that may require amino acid analysis for diagnosis and monitoring of patient treatment.
F7.5.2.3 Mode of Inheritance: The majority of amino acidopathies are inherited as autosomal recessive traits; a few are X-linked.
F7.5.2.4 Specimen Requirements: Plasma is generally the most informative specimen type. Serum may be used but is less ideal. Serum samples are generally left to clot at room temperature, which can lead to artifacts from deamination, conversion of arginine to ornithine by red blood cell arginase, and release of oligopeptides. Urine amino acid analysis is necessary for the diagnosis of some disorders, primarily those involving defective renal transport (e.g., cystinuria, renal Fanconi syndrome). Amino acid concentrations are more variable in urine than in plasma due to factors such as renal function and greater interference from medications. Therefore, urine amino acid analysis should be discouraged as a first tier investigation when screening for an inborn error of metabolism unless a specific disorder is suspected for which plasma is not informative or urine analysis is valuable for the purpose of differential diagnosis. Urine collection should avoid fecal contamination. Urine should be collected without preservatives. Cerebral spinal fluid (CSF) is useful in the diagnosis of several disorders, most notably nonketotic hyperglycinemia. CSF samples are most informative when a plasma sample is collected at the same time and the ratios of amino acid concentrations in CSF to plasma are calculated.
F7.5.2.5 Specimen Processing: Once collected, timely centrifugation and separation of plasma or serum specimens is important to reduce the influence of other blood constituents on the soluble amino acids. Hemolysis should be avoided since red blood cells contain high levels of certain amino acids. Prior to analysis, the plasma or serum specimens must be refrigerated for the short term (< 4 hrs.) or frozen (-20oC) to arrest amino acid degradation. CSF and urine must be frozen immediately to prevent loss of some amino acids. Note that free homocysteine levels in plasma specimens will be reduced over time as homocysteine binds to albumin and other plasma proteins. With timely processing and analysis of specimens, ion exchange chromatography is suitable for detecting the large elevations of homocystine found in homocystinuria. This method is not suitable for detecting moderate elevations of total homocysteine found in hyperhomocysteinemia.
F7.5.2.6 Clinical Indications for Testing: The clinical presentations of different disorders of amino acid metabolism are variable and often non-specific. Onset of symptoms may occur in the neonatal period or as late as adulthood. Amino acid analysis should be considered in many clinical situations, including when any of the following are present:
  1. lethargy, coma, seizures, or vomiting in a neonate
  2. hyperammonemia
  3. ketosis
  4. metabolic acidosis or lactic academia
  5. alkalosis
  6. metabolic decompensation
  7. unexplained developmental delay
  8. polyuria, polydipsia, and dehydration
  9. abnormal amino acid results by newborn screening
  10. previous sibling with similar clinical presentation
  11. clinical presentation specific to an amino acid disorder

F7.5.3 Guidelines
F7.5.3.1 Preanalytical Variables: Treatment of patients with antibiotics such as ampicillin or medications such as cough syrup and some anticonvulsants; nutritional status; and bacterial contamination of specimens may affect results and should be taken into account during interpretation.
F7.5.3.2 Specimen Preparation: Deproteinization of the physiological specimen is necessary. A common laboratory method of specimen preparation is acidification of a known specimen volume with a known small volume of concentrated acid, such as sulfosalicylic acid or trichloroacetic acid (TCA) to precipitate proteins and large molecules, followed by centrifugation, leaving the water soluble amino acids in the supernatant for analysis. Another method is to use low molecular weight cut-off filtration. At least one internal standard is added to the specimen and the pH is monitored and adjusted if necessary. Several methods also employ a microfiltration of the supernatant using a syringe/filter apparatus to protect the column and permit optimum chromatography.
F7.5.3.3 Separation Techniques: Ion exchange chromatography is the most common method of amino acid separation and analysis. Other methods that result in the positive identification of amino acids, such as tandem mass spectrometry, may be used. Tandem mass spectrometry typically is used to measure specific amino acids for newborn screening.

When separating by ion exchange chromatography, derivatization of amino acids is required for detection and can be accomplished either pre-column with o-phthalaldehyde (OPA) or phenylisothiocyanate (PITC) or post-column using ninhydrin. Post-column ninhydrin derivatization is preferred since it involves minimal sample processing and produces more consistent results. Several autosampler/ion exchange/detector configurations are commercially available that allow for positive identification of amino acids by their chromatographic retention time. These instruments have published methodologies that must be validated in the individual laboratory.

F7.5.3.4 Calibration: Quantitation without the use of a reference standard is not acceptable. Quantitation should be based on the performance and regular updating of calibration curves covering the normal range and expected pathological values. The laboratory should determine the linearity of all clinically informative amino acids.
F7.5.3.5 Chromatogram Analysis: Identification of amino acids by ion exchange chromatography relies primarily on chromatographic retention time and retention time relative to an internal standard. If ninhydrin is used for detection, the 440 to 570 nm ratio can be informative for identification of amino acids. Quantitation should be based on the recovery of the internal standard in each specimen compared to the recovery of the internal standard in the quality control amino acid mixture for each run (see F7.5.3.6).
F7.5.3.6 Quality Control: A known control amino acid mixture should be analyzed repeatedly to establish an acceptable range for each analyte. This mixture should be analyzed periodically to verify the instrument. The frequency with which these standards are analyzed depends on the stability and use rate of the derivatization material. If patient samples are run in daily batches, the standard mix should be analyzed with each batch. The internal standard(s) in each specimen serves as a quality control (QC) check for each specimen. A QC program based on the quantitative analysis of normal and abnormal control specimens should be implemented on a regular basis. The use of Westgard rules for clinical specimen analysis further controls the parameters for quality patient diagnosis and reporting (Westgard and Klee, 1999).
F7.5.3.7 Interpretation and Reporting: Patient and specimen information, as contained in Sections C2.4, C2.4.1, and C2.4.2 of these Guidelines, must be included on each report. The phone number of the reporting laboratory is required in case the referring physician has questions. Identification of all relevant amino acids must be listed and the quantity may be listed. When no significant abnormalities are detected, an amino acid analysis could be reported and interpreted in qualitative terms only. When abnormal results are detected, a detailed interpretation should include an overview of the results and their significance, a correlation to available clinical information, elements of a differential diagnosis, and recommendations for additional biochemical testing, including in vitro confirmatory studies (enzyme assay, molecular analysis). It must be recognized that reference values of several amino acids are characteristically age-dependent, thereby requiring that quantitative results be compared to a properly defined age group.

Whenever possible, confirmation of a diagnosis of an amino acid disorder by an independent method, typically by in vitro enzyme assay (blood cells, cultured cells, tissue biopsy) or molecular analysis, is recommended. Interpretations of amino acid results are based upon relative amino acid levels, pattern recognition and correlation of positive and negative findings, rather than on individual abnormal levels alone. Amino acid elevation(s) or overall profiles should be evaluated in the context of clinical findings and/or additional test results.

F7.6 Qualitative Amino Acid Analysis (Updated November 2003)

Qualitative amino acid analysis by thin layer chromatography (TLC) is suitable only for the detection of gross abnormalities. As some disorders may be missed by this method, its use for the purpose of evaluating high-risk patients should be discouraged. Qualitative amino acid analysis must reliably detect conditions in which there are either gross or modest elevations of specific amino acids in blood and/or urine.

F7.6.1 Reports should indicate the method used. If TLC was the method employed, a statement should be added indicating that TLC is suitable only for detection of gross abnormalities, and that quantitative analysis of plasma, urine, or CSF using a more sensitive method is recommended for the diagnosis and monitoring of treatment of conditions characterized by abnormal amino acids in blood and/or urine.
F7.7 Organic Acid Analysis
F7.7.1 Introduction

Organic acids are water-soluble compounds containing one or more carboxyl groups as well as other functional groups (-keto, -hydroxyl). Organic acids are intermediate metabolites of all major groups of organic cellular components: amino acids, lipids, carbohydrates, nucleic acids, and steroids. Gas chromatography mass spectrometry (GC/MS) analysis of organic acids in urine is an important diagnostic method for disorders of organic acid and amino acid metabolism. Gas chromatography alone without positive identification of components of complex profiles is prone to errors and should be discouraged. Three steps are required for the procedure:

1) isolation/separation of the organic acids from the physiological specimen.
2) derivatization of the organic acids to more stable and volatile compounds
3) GC/MS separation and identification of the derivatized compounds.
7.7.2 Background
F7.7.2.1 Clinical Description of Disease (Organic Acidurias): Organic acidurias are a biochemically heterogeneous group of inborn errors of metabolism. They are characterized biochemically by the accumulation of metabolites which are not present under physiological conditions, produced from the activation of alternative pathways in response to the loss of function of a specific gene product (enzyme), or by the accumulation of pathological amounts of normal metabolites. These disorders share a common natural history, which is the occurrence of either acute life-threatening illness in early infancy or unexplained developmental delay with intercurrent episodes of metabolic decompensation in later childhood.
F7.7.2.2 Prevalence: The incidence of individual inborn errors of organic acid metabolism varies from 1 in 10,000 to >1 in 1,000,000 live births. Collectively, their incidences approximate 1 in 3,000 live births. This estimate, however, does not include other inborn errors of metabolism (i.e., amino acid disorders, urea cycle disorders, congenital lactic acidemias) for which diagnosis and monitoring also require organic acid analysis. All possible disease entities included, the incidence of conditions where informative organic acid profiles could be detected in urine is likely to approach 1 in 1,000 live births. It is appreciated that as a group, these defects are under-diagnosed.
F7.7.2.3 Mode of Inheritance: The majority of organic acidemias are inherited as autosomal recessive traits; a few are X-linked.
F7.7.2.4 Specimen Requirements: Organic acids are analyzed in a urine specimen. In rare instances and/or for follow-up or prenatal diagnostic purposes, quantitation of specific metabolites in other physiological fluid specimens is appropriate. Urine specimens require creatinine measurement for standardization of sample preparation (i.e., extraction of a fixed creatinine equivalent) and for determination of relative concentration.
F7.7.2.5 Specimen Processing: Extraction from the physiological specimen and derivatization of the acid(s) is critical for organic acid isolation, identification and quantitation.
F7.7.2.6 Clinical Indications for Testing: Common clinical presentations for organic acidemia patients include lethargy, coma, hypotonia, hypertonia, tachypnea, seizures, ataxia, vomiting, failure to thrive, developmental delay, hepatomegaly and cytopenia. These signs may occur individually or in combination(s). A persistence of metabolic acidosis of unexplained origin and elevated anion gap should be considered strong diagnostic indicators of an organic acidemia. The presence of ketonuria, occasionally massive, provides an important clue toward the recognition of disorders, especially in the neonatal period. Hyperammonemia, hypoglycemia and hyperlacticacidemia are frequent findings, especially during acute episodes of metabolic decompensation. These abnormalities warrant immediate verification by urine organic acid analysis.
F7.7.3 Guidelines
F7.7.3.1 Extraction of Organic Acids: Internal standard(s) is added to the specimen (either a known volume or preferably a fixed creatinine equivalent) and the pH is monitored. Extraction is usually accomplished by extraction with organic solvents (commonly ethylacetate and ether, often used in combination) or anion exchange methods. Oximation of 2-keto acids (required to detect pyruvate, 2-keto glutarate, succinylacetone, branched chain amino acid metabolites) could be performed routinely or as a reflex testing under specific circumstances (lactic aciduria, tyrosiluria, positive DNPH). (See F9 for Chalmers and Lawson, 1982.)
F7.7.3.2 Derivatization: Trimethylsilyl derivatives of organic acids in dried urine extracts are made by addition of N5O5-bis-(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane (BSTFA/TCMS) or similar commercial reagents and allowed to react for variable time (usually 30 minutes) at a temperature from ambient to 80oC. Known alkanes may be added for standardized indexing of the chromatographic separation.
F7.7.3.3 GC/MS Analysis: Different capillary columns are available for organic acid separation and analysis. Several instrument configurations are commercially available that allow for positive identification of compounds by their chromatographic retention time and mass spectra usually obtained from either quadrupole filter or ion trap mass spectrometers in the electron impact ionization mode. Software for data analysis is available and recommended. The use of a computer library of mass spectra for comparison and visualization of the printed spectra is vital for definitive identification and interpretation of each patient specimen.
F7.7.3.4 Calibration: GC/MS analysis allows a biochemical diagnosis of a particular disorder on the basis of identification and, when possible, quantitation of characteristic metabolites. Quantitation without the availability of a reference standard, however, is a questionable practice that should be reduced to a minimum, and clearly indicated as such in reporting. When reference standards are available, quantitation should be based on the performance and regular update of calibration curves covering the normal range and expected pathological values. To calibrate the instrument/column/detector, a mixture of as many known standard compounds as possible should be carried through the entire specimen process. It is also very informative to obtain specimens from diagnosed patients for analysis and comparison.
F7.7.3.5 Chromatogram Analysis: Compound identification is critical to the clinical diagnosis of each genetic defect with abnormal organic acid production. Identification of organic acids relies primarily on evaluation of their mass spectra. Chromatographic retention time and relative retention time to an internal standard could be useful in the correct recognition of isomers.

When appropriate for measurement of specific analytes such as NASP in amniotic fluid or MMA in plasma, isotope-dilution, selected ion mass spectrometry should be used for more accurate analyte quantification.

F7.7.3.6 Quality Control: Tuning of the MS should be checked daily prior to specimen analysis as described for the instrument. A quality control (QC) program based on the quantitative analysis of normal and abnormal controls should be implemented and performed with every batch of patient specimens. Target ranges should be established for each metabolite and used to accept or reject a given QC run. The internal standards in each specimen serve as a QC check for each specimen. The use of Westgard rules for clinical specimen analysis further controls the parameters for quality patient diagnosis and reporting. (See F9 for Westgard and Klee, 1999.)
F7.7.3.7 Reporting: Patient reports must contain appropriate patient and specimen information as contained in Section C 2.4, 2.4.1, and 2.4.2 of these guidelines. The diagnostic specificity of organic acid analysis under acute vs. asymptomatic conditions may vary considerably. Informative profiles may not always be detected in disorders where the excretion of diagnostic metabolites is a reflection of the residual activity of the defective enzyme, the dietary load of precursors, and the anabolic/catabolic status of the patient. In some cases, methods of higher specificity and sensitivity based on the use of stable-isotope labeled internal standards, selected ion monitoring and chemical ionization can effectively overcome the limitations of standard organic acid analysis for the investigation of non-acutely ill patients. An abnormal organic acid analysis is not sufficient to conclusively establish a diagnosis of a particular disorder. Confirmation by an independent method is recommended, typically by in vitro enzyme assay (blood cells, cultured cells, tissue biopsy) or molecular analysis. Patient/specimen results interpretation is based on pattern recognition and correlation of positive and negative findings (for example, ketotic vs. non-ketotic dicarboxylic aciduria), rather than on individual abnormal values.

Identification of all relevant organic acids/compounds must be listed and quantity may be listed (as determined). When no significant abnormalities are detected, an organic acid analysis could be reported and interpreted in qualitative terms only. When abnormal results are detected, a detailed interpretation should include an overview of the results and their significance, a correlation to available clinical information, elements of a differential diagnosis, recommendations for additional biochemical testing and in vitro confirmatory studies (enzyme assay, molecular analysis), name and phone number of reference laboratories who may provide these studies, and a phone number to reach the reporting laboratory in case the referring physician has additional questions. It must be recognized that reference values of several organic acids in urine are characteristically age-dependent, requiring that quantitative results be matched against a properly defined age group.

F7.8 Methods used to screen for mucopolysaccharide (MPS) (glycosaminoglycan) analysis must reliably detect excessive urine MPS (glycosaminoglycan) excretion.
F7.8.1 Qualitative methods for MPS (glycosaminoglycan) analysis must distinguish between increases in heparan sulfate, keratan sulfate, dermatan sulfate and chondroitin sulfates.
F7.8.2 Diagnoses based on urine mucopolysaccharides should be confirmed by enzyme assays on appropriate tissue(s).
F7.9 Methods used to analyze very long chain fatty acids should reliably detect adrenoleukodystrophy, adrenomyeloneuropathy and other disorders of peroxisomes.
F7.10 Diagnostic enzyme assays must reliably distinguish between patients with specific diseases and control subjects. When assays are used to distinguish between heterozygous carriers and noncarriers, data must be available to justify cut-off points. When appropriate, methods should be available to distinguish between enzyme deficiency and pseudodeficiency.
F8 Reports
F8.1 See E8.
F8.2 The report must include an interpretation of the results. When appropriate, this may extend to discussion of the significance of the results and recommendations for further diagnostic procedures. The terms used must be such that the implications of the results are clear to a non-geneticist professional.
F9 References and Resources for Section F

Blau N, Duran M, Blaskovics ME. Physician's guide to the laboratory diagnosis of metabolic diseases. New York: Chapman & Hall Medical, 1996.

Bremer HJ, Duran M, Kamerling JP, Przyrembel H, Wadman SK. Disturbances of amino acid metabolism: clinical chemistry and diagnosis. Baltimore-Munich: Urban & Schwarzenberg, 1981.

Chalmers RA, Lawson AM. Analytical chemistry, biochemistry and diagnosis of the organic acidurias. In Organic Acids in Man. Chapman and Hall, London, 1982.

Goodman SI, Markey SP. Diagnosis of Organic Acidemias by Gas Chromatography-Mass Spectrometry. Alan R. Liss, New York, 1981.

Hoffman G, Aramaki S, Blum-Hoffman E, Nyhan WL, Sweetman L. Quantitative analysis for organic acids in biological samples: batch isolation followed by gas chromatographic-mass spectrometric analysis. Clin Chem 35:587-595, 1989.

Lehotay DC, Clarke JTR. Organic acidurias and related abnormalities. Crit Rev Clin Lab Sci 32:377-429, 1995.

Nyhan WL. Abnormalities in amino acid metabolism in clinical medicine. Norwalk: Appleton-Century-Crofts, 1984.

Slocum RH, Cummings JG. Amino acid analysis of physiological samples. In: Hommes FA, editor. Techniques in diagnostic human biochemical genetics. New York: Wiley-Liss, 1991: 87-126.

Sweetman L. Organic acid analysis. In: Techniques in Diagnostic Human Biochemical Genetics. Hommes FA, ed. Wiley-Liss, New York, 1991:143-176.

Westgard JO, Klee GG. Quality management. In: Tietz Textbook of Clinical Chemistry. Burtis CA and Ashwood EG, eds. 3rd edition. W, B, Saunders, Philadelphia, 1999:384-419.