Diagnostic Confirmation of Clinical Suspicion of IMD
In the case of clinical suspicion of IMD, the diagnosis is commonly based on first-level biochemical tests (available in all clinical biochemistry laboratories), followed by second- level tests (reserved for specialized laboratories) (Table 1), which must be followed, in a specialized clinical environment (clinical center of reference for IMD), by the definitive biochemical classification in genetic biochemistry laboratories. Diagnostic investigations are based on the qualitative or quantitative determination of specific biomarkers of the various diseases in biological fluids, usually plasma, urine, and cerebrospinal fluid (Table 2), followed by the demonstration of cellular enzymatic deficit and subsequent confirmation by genetic analysis.
Table1. First level and second level indicative laboratory investigations for the metabolic diseases diagnosis
Table2. Investigations of biochemical diagnostic assessment for the diagnosis of hereditary metabolic diseases, which can be carried out in the different biological fluids
From the point of view of laboratory medicine, the methodological and technical complexity of the differential diagnostic pathway for IMD has determined the progressive development, in many advanced health systems, of highly specialized laboratory facilities aimed at and integrated to support the clinical centers of reference deputies in the diag nosis and treatment of IMD. Therefore, within the laboratory medicine services, a new specialized branch has been created, which can be defined as genetic biochemistry: a technical and professional environment addressed to “rare diseases” that requires high and specific levels of efficiency, appropriateness, quality assurance, economies of scale, innovation and continuous research, expertise, and qualification of personnel. Below, in accordance with the recommendations contained in the document “Good Laboratory Practices for Biochemical Genetic Testing and Newborn Screening for Inherited Metabolic Disorders”, developed and published in 2012 by the Centers for Disease Control and Prevention, US Department of Health and Human Services, some key features are listed.
– Genetic biochemistry facilities represent a fundamental branch of laboratory medicine, aimed at the evaluation, diagnosis, therapeutic monitoring, clinical management, and, in some cases, the definition of the carrier status for congenital errors of metabolism. The tests performed by a genetic biochemistry laboratory require complex and highly specialized laboratory procedures aimed at the evaluation of enzymatic activities and biomarkers such as amino acids, organic acids, acylcarnitines, fatty acids, glycosaminoglycans, etc., by the use of a wide range of biological samples.
– The laboratory tests falling within the competence of this branch of laboratory medicine are classified as highly complex. They require more stringent criteria from a regulatory point of view (accreditation) regarding the characteristics of the analytical process, the quality management system, and the qualification and expertise of the personnel involved.
Neonatal Screening Process
The diagnosis of IMD is a model of integrated patient management resulting from an articulated and progressive path way. Today, for many IMD, it is also possible to apply the strategy of mass neonatal screening to identify the subject at risk.
The term “neonatal screening” defines secondary preventive medicine programs, activated on a large scale in the first days of life, aiming at the early identification and timely treatment of infants at high risk for certain treatable diseases with a high risk of early mortality and/or severe morbidity in those not diagnosed early. Nowadays, implementing a neo natal screening program is an essential responsibility of the public health system and constitutes a crucial factor in protecting children’s health status. Neonatal screening policies should be guided by an assessment of the overriding interests of the affected individual, with a secondary consideration for the interests of other stakeholders (healthy infants, families, professional areas, and health policy authorities). The screening program and recommendations on the appropriateness of including disease in a neonatal screening program should be based on scientific evidence and broad professional consensus.
In 1963, Robert Guthrie created the first laboratory test (which still today, although considered technically obsolete, bears his name: the Guthrie test) that allowed the semiquan titative measurement of the amino acid phenylalanine (Phe) in a drop of capillary blood, collected by heel prick, and allowed to absorb and dry on a special filter paper (the Guthrie card). This test, technically called the bacterial growth inhibition test, was based on the growth, in a particular agar medium deprived of phenylalanine, of bacterial spores in the deposition zone of a small disc of a few milli meters in diameter, obtained from the blood sample absorbed in the neonatal Guthrie card. The growth halo was proportional to the concentration of Phe in the sample and, through comparison with a series of samples with a known and progressive concentration of Phe (from 2 to 20 mg/100 mL), allowed to attribute, with sufficient accuracy for the use of the test, the value of Phe in the neonatal sample and select, according to a predetermined threshold or cutoff value (usually set at 2–4 mg/100 mL), the infants at risk (moderate or high) of phenylketonuria (PKU) to start the path of diagnostic confirmation and treatment. Because of its sensitivity, low cost, and easy application on a large scale, it allowed to carry out the first mass neonatal screening campaigns to identify newborns affected by PKU, the most common and frequent IMD that, if not treated early after birth with adequate diet therapy, is the cause of severe mental retardation, chronic and highly disabling.
In the following decades, many other diseases, mainly genetic, such as endocrinopathies, congenital errors of metabolism, hemoglobinopathies, and cystic fibrosis, resulted suitable to neonatal screening.
Traditionally, neonatal screening programs use analytes – mainly blood – as indicators or biomarkers of pathology, whose quantitative measurement or qualitative assessment allows, with sufficient efficiency, the selection of subjects at higher risk in the neonatal population. In some programs, laboratory analysis measures substrates accumulated in bio logical fluids by different mechanisms: (1) altered utilization or transformation, from enzymatic deficiency, of a substrate in a biochemical process (PKU: phenylalanine; galactosemia: galactose; congenital adrenal hyperplasia: 17-α-hydroxyprogesterone); (2) mechanical obstruction (cystic fibrosis: immunoreactive trypsin); (3) physiological activation of a feedback (hypothyroidism: TSH). In others, the deficiency or reduction of a substrate indicates a risk situation (hypothyroidism: T4) or, again, the presence of abnormal metabolites absent in normal conditions (hemoglobinopathies).
Finally, screening can be performed by measuring or qualitatively assessing a specific enzyme activity (galactosemia: galactose-1-P-uridyltransferase enzyme activity). In the first days of life, there is a “chronology” of the concentrations of the single biomarkers, which are strongly affected by the bio logical changes occurring in the delicate perinatal period of biochemical adaptation to autonomous life. The time of sample collection for neonatal screening must therefore be appropriately chosen in time windows that ensure, in the presence of pathology, optimal levels for the measurement or evaluation of the analyte in order to achieve the maximum efficiency (sensitivity and specificity) of the system.
Since the 1960s, technological evolution has offered increasing possibilities to expand the laboratory techniques applied to neonatal screening, providing the ability to use an increasing number of biomarkers for disease screening on a large scale. Table 3 highlights the main technologies applicable to neonatal screening. Since the 1990s, multipara metric screening allowing the simultaneous measurement of several analytes has been introduced. It is based on tandem mass spectrometry (MS/MS).
Table3. Main laboratory techniques applicable in DBS samples in neonatal screening programs
Many features make tandem mass spectrometry particularly suitable for the implementation of neonatal screening programs:
– Very high sensitivity: Extremely low blood volume is required
– High analytical speed: About 2–4 min/sample
– Possible automation of the analytical process
– High productivity
– Reduced cost per sample analyzed
Today, the most widespread, although not unique (especially in Europe), organizational model for IMD screening programs is the one developed in 2002 in the United States by the government offices of the Maternal and Child Health Bureau (MCHB), the Health Resources and Services Administration (HRSA), and the United States Department of Health and Human Services (DHHS), in collaboration with the American College of Medical Genetics (ACMG) and the American Academy of Pediatrics (AAP), which has redefined the set of diseases eligible for neonatal screening. They are divided into two panels, defined as:
– Core panel: severe diseases and
– Secondary target panel: conditions with minor clinical impact, which, in the screening process, are part of a differential diagnosis pathway for a condition included in the main panel
The main panel originally included 20 IMDs: nine organic acidemias (OA), five fatty acid oxidation defects (FAO), and six amino acidopathies (AA), all identifiable through the new technologies based on tandem mass spectrometry, which are associated, always in the field of hereditary metabolic diseases, with biotinidase deficiency and classical galactosemia, identifiable by different analytical technologies. The secondary panel includes six other OAs and eight FAOs (detectable by MS/MS technology) and two other nonclassical forms of galactosemia.
Table 4 shows the original description of the main and subpanels.
Table4. ACMG Disease panels for extended neonatal screening
This panel is constantly updated according to new scientific evidence, which makes it possible to include new dis eases according to the predefined criteria. Tandem mass spectrometry has introduced revolutionary progress in the screening and diagnosis of congenital errors of metabolism, allowing many screening laboratories in the world to extend the panels in use, by including ex novo, as new pathologies, fatty acid oxidation defects, and organic acidemias, and by significantly increasing the number of aminoacidopathies selectable with the program. The era of the so-called extended or expanded neonatal screening programs has begun.
Simultaneously, the development of the new technological model, starting from the 2000s, led to a new vision of health policy, which expands the organizational model of neonatal screening programs beyond the laboratory “border,” transforming the single activities related to the selection of a newborn at risk for one of the pathologies included in the screening panel into an articulated systemic health model (“screening system”), sequential and multidisciplinary, which includes all the phases of selection, diagnostic confirmation (biochemical and genetic), taking charge and management at the clinical level (clinical diagnosis, therapy, genetic counseling), final evaluation (epidemiological, eco nomic, efficacy) and all the functions and competences (neonatological, laboratory, clinical specialists) that interact with each other within the screening program and that respond, from a health point of view to two pivotal elements:
– To build an integrated and multidisciplinary service network
– To provide a timely and adequate response to a healthy demand that cannot be postponed
A neonatal screening program achieves its complete objective only when every newborn with a positive test has access to an efficient diagnostic evaluation and every new born with a confirmed diagnosis has access to an appropriate care pathway, chronic, global, and centered on the social binomial “patient-family”.
In more technical terms, neonatal screening can be equated, according to ISO 9000:2005, to a process, which can be defined as a sequence of related or interacting activities that transform an initial situation into a final one by adding value. In the field of public health, neonatal screening is a fundamental step in the evaluation of the health status of every newborn, allowing the newborn with a negative screening to exclude real-risk conditions for diseases included in the panel of pathologies and for the affected new born to be promptly included in the most appropriate therapeutic care pathway. This is the added value of neonatal screening. The concept of the process allows a better understanding of neonatal screening as an action of definition in each newborn of a risk condition (usually classified into three levels: low, intermediate, and high) obtained through the quantitative measurement of biological markers (single or multiple) appropriate to the condition, which must follow the diagnostic confirmation phase. The classic operational f low model of the screening process is represented in Fig. 1, which illustrates the operational algorithm of an expanded neonatal screening program for IMD: the baseline test (amino acid and acylcarnitine analysis) is performed in all neonatal samples; if negative, it concludes the selection process; if positive (with respect to the reference interval, a statistically predefined threshold value in the reference population, or a risk score calculated by dedicated computer algorithms), it determines actions of deepening (control), articulated according to the risk score (low, intermediate, or high risk), and conducted chronologically with a timing defined by the characteristics of the disease and the level of risk. Once this second phase has been completed, all confirmed positives are generally reported to the clinical center of reference for diagnostic confirmation and for taking charge of the newborn at risk. This phase is carried out in concert among the screening laboratory, the laboratory for diagnostic confirmation (facilities that may coincide), and the clinical center. In any case, screening, diagnostic confirmation, and taking charge of the newborn at risk must be concluded, even for pathologies with less severe clinical onset, within the first month of life.
Fig1. Extended neonatal screening: operative-laboratory algorithm. (Copyright EDISES 2021. Reproduced with permission)
This classic design of operational flow is now enhanced by the possibility of performing in the basal sample collected in the first days of life, additional tests, called second-tier tests (2TT). The latter allows for improving the efficiency of the program, increasing especially the specificity and the positive predictivity; it results in a positive impact on both the health system (reduction in the number of required confirmatory tests, reduction of preanalytical and analytical costs) and the social system (reduction of parental anxiety for the outcome of the test).
Figure 2 highlights some examples of second-stage tests (2TT) and subsequent biochemical confirmatory tests in neonatal screening for congenital adrenal hyperplasia, cystic fibrosis, and expanded neonatal screening for IMD.
Fig2. (a) Baseline (T-b) and second instance (2TT) tests for three screening programs. (b) Biochemical tests for diagnostic confirmation. ISC-CAH, congenital adrenal hyperplasia; CF, cystic fibrosis; SNE, extended neonatal screening (Copyright EDISES 2021. Reproduced with permission)
Concerning the current diffusion of extended neonatal screening programs, data from the international literature show a wide diffusion of these new programs in many geo graphical areas of the world with advanced healthcare systems (North America, Europe, Japan, Australasia, and, more recently, Russia and China).
In terms of health economics, the analysis of the overall results of extended neonatal screening programs shows a substantially favorable judgment. According to some authors, in terms of cost-benefit analysis, programs based on MS/MS technology for congenital errors of metabolism can deter mine an overall economic saving if compared to the high costs of care for subjects with long survival diagnosed in the absence of screening. Additionally, the use of MS/MS technology (compared to that of other technologies) can deter mine a higher level of economic savings because of its intrinsic characteristic to detect a panel of diseases in a single test. However, previous evaluations by other authors had instead shown lower economic benefits reserved for programs limited in the number of diseases screened.
Despite the now-prevailing evaluation of the effectiveness of extended neonatal screening programs, the persistence of critical issues or open problems related to the activation of these programs should not be underestimated. The main points of reflection can be synthetically listed:
– The selection of pathologies in which the therapeutic intervention, even if early and started in a presymptomatic period, does not modify the natural history
– inauspicious for morbidity and mortality
– of the disease.
– The identification of biological variants with low clinical “impact” or late-onset pathologies in which there is no evidence of the need or usefulness of therapeutic intervention.
– The inevitable, albeit limited, increase in false-positive newborns (especially in particular categories such as pre mature babies), with potentially negative repercussions (psychological, social, and economic) in the family.
– The risk that, in situations of false negativity (which, although rare, is still present in highly efficient programs), the false sense of security caused by the existence of a targeted screening program will further slowdown the clinical diagnosis of disease.
As already mentioned, the paths of diagnosis and treatment of rare diseases (the pathologies with a prevalence of affected subjects present in a population of less than five cases in 10,000) have today, in the most advanced health systems, significant social and political attention. The technical and applicative potentialities offered by tandem mass spectrometry and the substantial international success of the extended neonatal screening programs (mainly addressed to “rare pathologies”) determine a constant pressure, also in public opinion, for a further expansion of the number of pathologies considered in the screening panels. However, it must be considered that neonatal screening strategies are a tool for the presymptomatic selection of subjects at risk. However, they do not exhaust the entire pathway of diagnosis of hereditary metabolic diseases, as the panels, although large and further expandable, do not cover the entire set of diseases now identified and known.
In conclusion, it is possible to affirm that for IMDs, early postnatal diagnosis is the most effective tool for the prevention or reduction of both the risk of mortality and damage from severe morbidity, considering that for many rare con genital diseases, recent years have greatly improved the approach and the possibility of therapeutic intervention. The introduction of MS/MS technology and the application of molecular biology techniques have revolutionized the neonatal screening, allowing new scenarios of preventive medicine for IMDs.
In such a vast context of applicative potentialities, the temptation may arise in civil society and in the healthcare world (also intended as a response to legitimate social instances of lay groups supporting different pathologies) to adopt a global approach to neonatal screening for an ever- increasing number of congenital pathologies, even outside the criteria of choice based on strong scientific, epidemiological, and health economics evidence. Moreover, it must be considered the possibility that the interests related to the introduction of innovative therapies with high or very high economic value may determine an undue pressure towards operational choices that are effective from the point of view of selection and diagnosis but not supported by subsequent therapeutic interventions that are advantageous for the individual and economically and ethically sustainable for society.
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