Prevention is better than Treatment
The following abstracts were presented in Curitiba, Brazil during the 1st Neonatal Screening meeting, held there in November 2001. Reproduced here with the permission of the Author.
G-6-PD DEFICIENCY AND CORRECTION OF THE ENZYME ACTIVITY BY THE HEMOGLOBIN’S CONCENTRATION
Liliana Weber2 and Eurico Camargo Neto1,2.
Centro de Triagem Neonatal1 and Laboratório Nobel RIE2. Porto Alegre, Brazil.
Glucose-6-phosphate dehydrogenase (G6PD) is a cytoplasmamic enzyme present in all cells. It’s main function is the production of reduced phosphated nucleotides (NADPH) in the metabolic way named “pentoses cycle”. The NADPH produced acts as an antioxidant factor to protect the cellular membrane and the G6PD activity is the only source of NADPH in the red blood cells. G6PD deficiency is knwon as the most common enzymopathy in the world, affecting more than 400 million people and is associated with etnic groups. In the Americas a prevalence of 0,5% to 6,9% is estimated and 5% to 25% in Africa, Asia, Midle East and Mediterranean. The clinical symptons are associated to hemolysis induced by “fava beans” or by many drugs used routinely worldwide and can lead to death or to permanent neurologic damage. Using a kit for filter paper samples that measure the G6PD activity in kinectic mode (R&D Diagnostics, Greece), we determined a cutoff of 0.70 mOD/min analysing 1,100 routine samples from our newborn screening program. Tthe G6PD activity was evaluated in 14,219 samples: 568 presented results below the cutoff and new samples were received from 454 children. Low enzyme activity was confirmed in 163 (1.15 from the total), 68 females and 95 males, and 291 false-positives (2,0%) most of all probably due to lost of activity in samples received more than one week after blood collection. After tests in 3,460 new samples, other cutoff was established (2.5 U/g Hb) to report the results considering variations in the hematocrit. As controls we used red cells liophilized from Sigma. Other 6,090 samples were analysed under this new criterion and 377 babies presented enzyme activity below the cutoff and new samples were received from 321. From them, 157 (83 females and 74 males) the G6PD activity ranged between 2.5 to 6.0 U/g Hb (2.6%), compatible with “partial deficiency”, and 98 (38 females and 60 males) with enzyme activity below 2.5 U/g Hb (1.6%), compatible with “profound deficiency”. Both forms need the same care. The correction of the enzyme activity using the hemoglobin concentration in the same procedure showed a new prevalence of the disease: 4.2% for both forms combined.
PERFORMANCE OF A CHEMILUMINESCENT METHOD ADAPTED FOR THE QUANTIFICATION OF TSH IN DRIED BOOD SPOTS
Eurico Camargo Neto1,2 and Jaqueline Schulte2.
Centro de Triagem Neonatal1 and Laboratório Nobel RIE2. Porto Alegre, Brazil
The measurement of TSH is used in our laboratory together T4 with since 1990 as markers for the neonatal screening for congenital hypothyroidism. In 1991, we developed an alternative procedure for the quantification of TSH in filter paper (Clin Chem 1991;36:1796) using a commercial kit developed to measure TSH in serum. The test is used untill now in our routine. The method was standarized and compared with a time-resolved immunofluorimetric assay (DELFIAä), testing simultaneously 4,219 samples obtained by heel sprick in filter paper SS 903, including samples received from the Infant Screening Quality Assurance Program do Centers for Disease Control and Prevention – CDC (Atlanta, USA) and a correlation coeficient of 0.958 was obtained. The cutoff established was 20,0 mIU/L serum equivalent and the patient with congenital hypothyroidism with lower levels of TSH at the screening presented 26,0 mIU/L. ln 10 years we used the method in 834,000 samples and 299 neonates with congenital hypothyroidism were detected (1/2,789), 57 with normal levels of T4 (19%). The retested samples is 0.32% and the false-positive 0,02% with no false-negative results reported untill now. The simultaneous measurement of T4 (RIA) and TSH allowed us the detection of 138 cases of hypoTBGnemia. The TSH method presents good correlation using protocols of incubation of 3 hours or overnight (r=0.995). A new protocol fully automated was developed for the ACS:180ä (Clin Chem 1998;44:2372-73) and the results showed agreement with the routine method.
THIN-LAYER CHROMATOGRAPHY FOR AMINO ACIDS: A LOW COST AND COMPREHENSIVE METHOD
Eurico Camargo Neto1,2, Jaqueline Schulte1, Liliana Weber1 and Rosélia Rubin1.
Centro de Triagem Neonatal1 and Laboratório Nobel RIE2. Porto Alegre, Brazil
The recent use of mass spectometry in tandem brings new perpectives for the neonatal screening for amino acids and organic acidemias. Nevertheless, the high cost of the equipment and the needs of specialized professionals are still limitant aspects for the its adoption in the routine of neonatal screening programs. Besides the quantitative analysis of phenilalnine in filter paper, e use in our routine a thin-layer cromaography for amino acids, with low cost and easy operation, with the possibility to detect, qualitatively, other amino acids (Neto EC et al. Rev Bras Anal Clin 1993;25:81-2). In 880.800 cromatograms we found 66 cases of classical PKU (1/13.345); 162 hyperphenilalaninemias (1/5.440); 9 cases of increased levels of leucine, isoleucine e valine, all confirmaded as leucinosis or Maple Syrup Urine Disease or MSUD (1/98.000); 2.276 transitory tyrosinemias (387); 1 homocistinuria (1/880.800); 55 hypermethioninemias (1/16.000). Other amino acids identified were: OH-proline, tryptofan, aspartic acid, asparagine, lisine and glicine. The follow-up of these patients showed that increased of these amino acids were transitories and that, in almost all cases, were associated to supplemental feed with animal milk. The abnormalitie more frequent was the hypermethionine. Despite the thin-layer chromatography be a qualitative method, its allows the detection of diseases as MSUD and, particularly, the distintion of classical PKU and secondary hyperphenylalalinemia due to the increased of the levels of tyrosine. The sensitive of the method is, at least, 1,0 mg/dL, for all amino acids. Since 1993, the method is submitted to the Infant Screening Quality Assurance Program CDC-Atlanta, USA and, since 1998, to the Australasian Quality Assurance Program, Auckland, New Zealand, showing satisfatory results.
10 YEAR OF NEONATAL SCREENING FOR CONGENITAL HYPERPLASIA
Eurico Camargo Neto1,2, Jaqueline Schulte1, Liliana Weber1 and Rosélia Rubin1.
Centro de Triagem Neonatal1 and Laboratório Nobel RIE2. Porto Alegre, Brazil
The neonatal screening for congenital adrenal hyperplasia (CAH) was introduced in Porto Alegre (Brazil) in 1991, using a RIA test (DPC) for the quantification of the 17-a-OH-progesterona (17-a-OHP) em dried blood on filter paper SS 903. Soon after, two methods began to make part until now of our routine: DELFIA/AutoDELFIA and a RIA kit from ICN, with cutoffs established in 25,0 ng/dL serum equivalent for both kits. Samples with values above this limit are confirmed in serum samples by RIA. In the last 10 years, 553,380 samples were analized and 52 babies were detected with CAH (1/10,645), 29 males (17 salt-loosing, 4 deaths) and 23 females (10 salt-loosing and 3 without ambiguous genitalia). Two cases of non-classical CAH and with late clinical symptons were missing by the neonatal screening. We observed that some normal babies presented transitory high levels of serum 17-a-OHP for undetermined time after the neonatal period. From 406,525 tests (239,463 ICN and 167,062 DELFIA/AutoDELFIA), excluding the prematures babies (<36 weeks), babies with less of 2,5 kg and the true positives, 1,831 babies (0,45%) presented levels of 17-a-OHP higher than 25,0 ng/dL and below 40,0 ng/dL serum equivalent in filter paper. Serum samples were received from 1,719 patients and 1,009 presented results below our cutoff until 1 month of age (9,4 ng/dL) and were classified as “true false-positives” (0,25%). The other 710 samples (0.20%) were distributed as follow: 219 with 17-a-OHP levels between 9,5 e 12,0 ng/mL; 142 between 12,1-15,0 ng/mL; 178 between 15,1-20,0 ng/mL; 124 between 20,1-25,0 ng/mL e 47 above 25,0 ng/mL. The serum levels of 17-a-OHP in all children normalized in most of the cases, in 90 days, and were classified as “transitory increased of 17-a-OHP”. It could be due to the testicular or the ovarian production, or maybe due to heterozygotes patients. All children are still assimptomatics and we must to be care when report results sligthly above the cutoff to avoid unnecessary stress in the families.
CYSTIC FIBROSIS: NEONATAL SCREENING AND MOLECULAR ANALYSIS
Eurico Camargo Neto1,2, Liliana Weber2, Jaqueline Schulte2, Jurema De Mari3, Cristina Castilhos3 and Eduardo Lewis3.
Centro de Triagem Neonatal1, Laboratório Nobel RIE2 and DNA4-Laboratório de Genética e Biologia Molecular3. Porto Alegre, Brazil.
Cystic fibrosis is an autossomic recessive disease, characterized by general disturb of the exocrine functions of the pancreas. The symptons could be greatly diminished by the precocious diagnosis measuring immunoreactive trypsin (IRT) and the DNA analysis of the DF508 mutation using PCR in all samples with IRT above the cutoff (140,0 ng/mL serum equivalent using liquid standards, DELFIA/AutoDELFIA, and 110,0 ng/mL serum equivalent using standards in filter paper). The IRT was quantified in 463,288 samples and 3,557 (0,76%) showed values above the cutoff and were submitted to the DNA analysis for the DF508 mutation and we detected 38 homozygotes in 161 babies with the mutation (1:36 alleles). In this group, 51 heterozygotes for the DF508 mutation had the diagnosis confirmed and 72 are still assymptomatics. The follow-up of these cases allowed the identification of 138 patients with the diagnosis confirmed by sweat tests and/or clinical symptons described by the pediatricians or by the families (1/3,357). The DF508 mutation was identified in 66% of the patients diagnosed. Besides de DF508, 4 other mutations were tested in 17 children with cystic fibrosis and the results were: 2 carriers of the G542X mutation, 1 carrier of the G551D mutation and 1 one compound heterozygote DF508/G542X. The mutations N1303K and R553X were not identified in this group. In a parallel study using Multiplex-PCR in 200 samples with normal IRT, 4 carriers of the DF508 were found and in 200 samples with IRT higher than 140,0 ng/mL serum equivalent 2 homozygotes for the DF508 mutation, 1 one compound heterozygote (DF508/621+1G®T) and 5 carriers of the DF508 mutation. In 5 patients with high levels of IRT in a first sample obtained within one week of life, new samples were requested and received at the maximum with 20 days of life and we found: 2 homozygotes and 3 heterozygotes for the DF508 mutation. We emphasize the importance of the DNA analysis for the DF508 mutation in neonatal screening programs for cystic fibrosis and we understand that to test for IRT in children with more than 30 days of age has the risk to false-negative results.
INCREASED LEVELS OF IRT IN SAMPLES OBTAINED BEFORE 72 HOURS OF LIFE
Eurico Camargo Neto1,2, Victor Nudelman3 and Eurípedes Ferreira3.
Centro de Triagem Neonatal1, Laboratório Nobel RIE2, Porto Alegre, and Hospital Israelita Albert Einstein3, São Paulo, Brazil.
The reference test to screen for cystic fibrosis in neonates is the meaurement of the immunoreactive trypsin (IRT) in Guthrie cards. Due to the particular characteritics of the trypsin molecule, specially when complexed, and the absence of an international reference standard, the results obtained with diferent methodologies are not comparable. The IRT is the test with the higher number of false-positive results in our experience (0.76%, n = 463.228) and could be elevated due to inespecific conditions as perinatal stress, renal insufficiency, intestinal atresia, congenital infections and trisomy of the chromossomes 13 or 18, among other unknow causes. All samples collected in the Hospital Israelita Albert Einstein are obtained before 72 hours of life and has a high false-positive rate of 6% (kit Wallac, liquid standards, cutoff of 140,0 ng/mL serum equivalent). Here we present an statistical analysis of a population of 2,551 neonates, 1,442 females (56,5%) and 1,109 males (43,5%), with IRT tests made in two samples with a mean interval of 12 days from children without clinical symptons, evaluating two variables: gender and age at the collect of the samples that ranged from 24 hours to 20 days of life. The chi-square method was applied to compare gender and the IRT results and no correlation was found between both (c2=0,022; p=0,883). Using the same test, we can’t demonstrate association between the time when the sample was obtained and the false-positive results, after separation of the neonates by age at the time of the blood collection in two groups. Group 1: until 48 hours, 3 to 5 days, 6 to 7 days 8 to 10 days and 11 to 20 days of age. Group 2: until 48 hours and between 3 to 20 days of life. Despite the false-positive results fall with the increase of the age, there is no statitical significance between both variables in each group when compared thebgroups by age. We suggest that the families received informations about the possibility of false-positive results in samples obtained before 72 hours of age and to test again between one to two weeks of live to avoid to label the child as affected for cystic fibrosis.
BIOTINIDASE DEFICIENCY: NEONATAL SCREENING AND MOLECULAR ANALYSIS
Jaqueline Schulte2, Eurico Camargo Neto1,2, Liliana Weber2, Jurema De Mari3, Cristina Castilhos3, Eduardo Lewis3 and Roberto Giugliani3. Centro de Triagem Neonatal1, Laboratório Nobel RIE2 and DNA4-Laboratório de Genética e Biologia Molecular3. Porto Alegre, Brazil.
The biotinidase deficiency is an autossomic recessive disease that can present neurological symptons soon after birth or, if untreated, produce cutaneous abnormalities, convulsuion, mental retardation, coma and death. The disease is classified as “partial deficiency” (10 to 30% of the mean normal enzyme activity) or ‘’profound deficiency” (< 10% of the mean normal enzyme activity) and the combined prevalence is around 1/60,000 worldwide. The treatment is very simple and cheap and is based in the intake of free biotin (10 mg/day). We used the qualitative colorimetric method from Wolf et al in 285,343 samples of blood in filter paper SS 903 and 630 (0,22%) presented low activity. We received new samples in filter paper and serum from 559 patients from all over the country, most of all without not freezed, essencial condition to obtain confiable results. In this group, 30 presented activity below 10% of the mean enzyme activity and 68 with enzyme activity between 10% e 30% (reference range: 4 to 10 mmol/mL/min). In a previous study enrroling 225,136 samples, DNA from 17 children with biotinidase activity below 0,8 mmol/mL/min was sent to Dr. Barry Wolf (Richmond, Virginia, USA) for molecular analysis. In two children mutations associated to profound biotinidase were found and 6 children that presented mutations associated to partial deficiency, suggesting a prevalence of 1/.28,142 for both forms in population studied, a frequency higher the data found in the literature. Others 6 children were classified as heterozygotes. The recall rate was 0,19% and could be due to bad impregnation of the blood in the filter paper, humidity, hot, or delay to receive the samples. Considering that 98 serum samples presented biotinidase activity below than the inferior reference value established, we are making new studies about the stability of the enzyme in serum in diferent conditions and we understand that molecular biology methods must to be considered in neonatal screening for biotinidase deficiency.
GALACTOSEMIA: RESULTS OF 6 YEARS OF NEONATAL SCREENING
Eurico Camargo Neto1,2, Jaqueline Schulte2, Rosélia Rubin2, Liliana Weber2, Eduardo Lewis3, Jurema De Mari3 and Cristina Castilhos3. Centro de Triagem Neonatal1, Laboratório Nobel RIE2 and DNA4-Laboratório de Genética e Biologia Molecular3. Porto Alegre, Brazil.
The classic galactosemia is characterized by the lack of the galactose-1-phosphate-uridil-transferase (GALT), increasing the plasma levels of galactose and galactose-1-phosphato, produts of de degradation of the lactose from the maternal milk. The disease presents a prevalence of 1/40,000-60,000 live births. Its not uncommun the death of patients with galactosemia and, even in patients with early treatment, mental retardation or ovarian disfuntion can be observed. The disease is of dificult identification at birth because there are no specific symptons, and the sistemic signs are anorexia, weight loose, ictericia and hepatosplenomegaly. We tested 287.827 babies for galactosemia using a colorimetric assay from Quantase (Scotland), measuring the levels of galactose and galactose-1-phosphate in samples of blood in filter paper SS 903, and an standard curve from zero to 50 mg/dL. In 597 (0,20%) samples we obtained results of total galactose above the cutoff, 10,0 mg/dL, and a new sample in filter paper was asked with urine to run a thin-layer chromatography for sugars. The lack of galactose-6-phosphate-uridil transferase (GALT) demonstrated in only one patient and a total of 18 had the diagnosis clinically confirmed (2 deaths), suggesting a high prevalence of 1/16,000. In 3 patients with Down syndrome the concentration of total galactose higher than 50,0 mg/dL without clinical confirmation. In a premature babie with hepatosplenomegaly, hepatic test abnormals and anemic, levels of 48,0 mg/dL was found, with lactose in the urine and GALT’s activity borderline. These results, suggesting galactosemia, was desconsidered by the identification of antibodies IgM anti-cytomegalovirus in the neonatal screening for infectious diseases in filter paper and later serum confirmation.
CONGENITAL TOXOPLASMOSIS: A CANDIDATE FOR NEONATAL SCREENING
Eurico Camargo Neto1,2, Jaqueline Schulte2, Rosélia Rubin2, Liliana Weber and Adriana Brites1.
Centro de Triagem Neonatal1 and Laboratório Nobel RIE2. Porto Alegre, Brazil.
Women infected with the Toxoplasma gondii during the pregnancy can transmited the parasite to the fetus. The best method for prevention and control of the infection by the Toxoplasma gondii is uncertain and the effect of the treatment during the pregnancy is not well documented. The infection transmited during the pregnancy can lead to spontaneous abortion, stillbirth, prematures or children at term infected. The infection is not easily recognized at birth or during the neonatal period and can evolve without treatment. The neonatal screening can identify subclinic forms of the infection and the early treatment is believed to prevent late-onset sequelae. We began our study developing an immunoenzymatic assay to detect antibodies IgM anti-Toxoplasma gondii, testing 78,350 samples. After, we adopted in our routine a fluorometric immunoenzymatic capture assay from Labsystems and tested more 209,376 samples for IgM, in the total 287,726 babies. Until now, 118 patients had the diagnosis confirmed (1/2,438): 49 with IgM detected in the babie and in the mother; 16 with IgM only in the babie; 14 with IgM only in the mother and 39 babies with progressive increased of the IgG levels. From these patients, with follow-up since September, 1995, and submited to the conventional treatment, retinal scars were found in 19 (two with one eye blind and one myopic), intracranial calcifications were demonstrated by RX or tomography in 6 and both symptons were identified in 5 children (one treated after 14 months and presenting sequelae). One children presented hepatosplenomegaly (HEM) and lymphoadenopathy, other only with HEM and one children presented malformations, neuromotor retardation and blindness. The others 86 (73%) are, until now, assymptomatics. Around 100 patients with IgM in their serum and/or in their mother’s serums are without the follow-up completed. So, the prevalence of the disease is probably higher than the estimated in this study. We believe that the neonatal screening for congenital toxoplasmosis is justified band that the early treatment can prevent or diminish the sequelae of the disease.
NEONATAL SCREENING FOR HEMOGLOBINOPATHIES USING HPLC AND FIE
Liliana Weber2, Rosélia Rubin2 and Eurico Camargo Neto EC1,2
Centro de Triagem Neonatal1 and Laboratório Nobel RIE2. Porto Alegre, Brazil.
The hemoglobinopathies are considered a public health problem in Brazil, a country characterized by a great mixture of races and 170 million inhabitants, where oficial data estimated the existence of 4 million carriers of the sickle cell trait and 30,000 people with Hb SS, Hb SC, HbSbtal or other heterozygote forms associated with HbS. The main variant to be detected in a public health neonatal screening program is the HbS, that is the responsible for the sickle cell disease. The HbS results from a mutation in the gene that codified the b chain globin, where occurs a substitution of a base in a codon GAG to GTG and, as consequence, the change of a glutamic acid by valine in the position 6 of the peptidic chain. We began our work using an isoletric focusing methodologie - IEF - (Isolab/PerkinElmer) and after we introduced in our routine the high performance liquid chromatography (HPLC, BioRad) to work together. The HPLC is a method standarized for the specific identification of the hemoglobins F, A, S, C, D and E/ A2, the last two not discriminated one from the other because both presented the same retention time in the column. The method classify others hemoglobins as “unknow”. The IEF allows the identification of the same hemoglobins and of other variants through their point isoeletrics (pI), with little discrimination of HbS and HbD. The confirmation of abnormal results was made running the sample by the two methods. In 57,436 samples analysed we found 946 babies with variants of hemoglobins: 634 FS (1,1%), 50 AFS, 169 FC (0,29%), 6 SC, 15 Hb Barts, among several other variants less frequents, as D-Punjab, Philadelphia, Lepore, F-Texas, a or b chain variants or hibrids of both. Considering only the samples in that HbS was identified, and even we can’t afirm the exact number of patients with sickle cell disease as we don’t know how many with results FS will be sick or heterozygote, the falcemic trait is present in, at least, 1/1,147 neonates and one hemoglobin variant is found in each 60 samples (1,65%).
NEONATAL SCREENING FOR MEDIUM-CHAIN ACYL-CoA DEHYDROGENASE (MCAD) USING PCR FOR THE G985A MUTATION
Eurico Camargo Neto1,2, Jurema De Mari3, Cristina Castilhos3, Eduardo Lewis3 and Roberto Giugliani3. Centro de Triagem Neonatal1, Laboratório Nobel RIE2 and DNA4-Laboratório de Genética e Biologia Molecular3. Porto Alegre, Brazil.
The medium-chain acyl-CoA dehydrogenase (MCAD) is a mitochondrial enzyme, essential in the proccess of degradation of fatty acids as energetic source. People with MCAD deficiency, an autossomic recessive genetic disturb, present metabolic alterations associated to infections, hypoglicemia, vomit, diarrhea, concious disturbances, letargy, metabolic acidosis and coma. The first symptons usually appear between 4 and 18 months and 40% of the symptomatic patients can dye until 2 years of age. The disease is also associated to encephalopathy Reye’s Syndrome like and to the Infancy Sudden Death Syndrome and the patients can also present muscular weakness, seizures, cerebral paralysis and growth retardation. A point mutation (change of adenine by guanine) in the position 985 of the cDNA was identified as the most frequent cause of the disease, around 85% to 90% of the positive cases. Studies in US showed a prevalence of 1/7,000 to 1/15,000, considering diferent ethnic groups, and the inclusion of MCAD deficiency in neonatal screening programs has been suggested due to its prevalence and facilities for treatment. Two studies made in Britain populations determined a frequency of homozygotes for the G985A mutation of 1/6,400 and 1/13,400 in 410 and 479 samples, respectively. Nevertheless, the small amount of samples analysed makes these evaluations of low precision. Methodologies of access still restrict to developing countries allow the diagnosis of the MCAD deficiency detecting high levels of octanoyl-carnitine in dried blood spots using tandem mass spectometry, cis-4-decenoate by GC-MS or the detection of hexanglicine in urine using GC-MS and ion-selective monitoring. We tested by PCR 3,488 samples for the G985A mutaton, using dried blood spots. Seven carriers were detected establishing a low carrier frequency of 1/500.
PERFORMANCE OF A FLUOROMETRIC KIT FOR LEUCINE IN FILTER PAPER
Eurico Camargo Neto1,2, Jaqueline Schulte1 and Liliana Weber1.
Centro de Triagem Neonatal1 and Laboratório Nobel RIE2. Porto Alegre, Brazil.
The Maple Syrup Urine Disease (MSUD) is due to the lack of the branch chain a-keto-acids decarboxilase, characterized by the increase of the levels of leucine, isoleucine and valine and their correspondents keto-acids in the blood and urine. The prevalence cited in the literature is 1/250,000, with great variations according to ethnic groups. Patients affected can present symptons since the first week of life, including, vomit, hypotonicity, letargia, coma and death. The survivers usually present motor and/or mental retardation. We evaluated a fluorimetric kit (PerkinElmer) based in the extraction of the branch chain amino acids from filter paper blood spots in a a procedure of 3.5 hours of incubation. The manufacturer suggests a cutoff of 3.9 mg/dL and that all samples with results between 3.2 and 4.5 mg/dL should be retested (gray zone). We tested 860 samples in 10 runs, including controls from the Infant Screening Quality Assurance Program (CDC, Atlanta, USA), with values of zero, 3.0, 7.0 and 11.0 mg/dL and controls from the Australasian Quality Assurance Program, Auckland, New Zealand. The mean value obtained using normal samples previously analysed by a thin-layer chromatography for amino acids was 2.0 mg/dl (SD = 0.904) and the samples from the Quality Assurance Programs showed acceptable results. In 104 samples we detected leucine levels in the “gray zone” suggested by the manufacturer but only one sample presented a value of 4.5 mg/dL, above the cutoff established in our study (mean + 2 SD = 3.8 mg/dL). The sensitivity, estimated testing in triplicate in five runs the 0.4 mg/dL standard, was 0.35 mg/dL, with an CV intra-assay of 3.5% and inter-assay of 7.4%. The controls of 1.8 m/dL and 6.0 mg/dL showed CV intra-assay and inter-assay of 6.6% and 4.8%, respectively. The standard curve, always run in duplicate, has a range from to 0.4 to 16.5 mg/dL, with two standards too close in the high portion of the curve, making at least one unnecessary. The linearity was tested eluting blood from two spots of the 11.4 and 14.9 mg/dL standards and testing them as normal samples. The maximum linear value obtained was 18.0 mg/dL. The test is easy to perform and can be useful to test for MSUD.
NEONATAL SCREENING FOR INFECTIOUS DISEASES
Eurico Camargo Neto1,2 and Rosélia Rubin1
Centro de Triagem Neonatal1 and Laboratório Nobel RIE2. Porto Alegre, Brazil.
Since the development of the Guthrie test for the early diagnosis of phenylketonuria (PKU) in the earlier sixty, several other tratable genetic diseases were included in neonatal screening programs worldwide. Later, other genetic diseases, with prevalence lower than PKU, were also included in neonatal screening programs, using the same dried blood in filter paper (Guthrie cards). The screenings for congenital toxoplamosis and for HIV marked the beginning of a new alternative for the prevention of diseases using the same Guthrie cards: the identification of infectious diseases, so devastating as some genetic diseases, but with short time and potentially benefic treatments. The inclusion of the “TORCH” group in neonatal screening programs was discussed for the first time at the ISNS Meeting, held in Boston in 1996. The WHO estimated that 20 million people are infected by the Tripanozoma cruzi in Latin America, 1% of the live births are infected by the cytomegalovirus (CMV), 4% of the pregnant women in Brazil are infected by the Treponema pallidum and 1% will have a baby with congenital syphilis. We standarized and offer tests for infectious diseases in dried blood spots using filter paper SS 903 and 3,770 samples were analysed. We found, in the children and/or in the mother, 1 case of syphilis, 6 cases of rubella, 2 cases of Chagas’ Disease and 3 cases of cytomegalovirus. Other 118 cases of congenital toxoplasmosis were diagnosed in 287,726 babies screened. Considering that the population that selects a private neonatal screening program is, theoretically, from a higher social class, with more access to informations about hygiene and sexually transmited diseases, the number above probably should be higher if all population could be tested. From 360 pregnant women who were submited to the “triple test” (free estriol, total HCG and AFP) in filter paper and decided to test for infectious diseases we found 1 with syphilis, 6 with rubella, 3 with Chagas’ Disease and 7 with toxoplasmosis. In only 5,378 tests for HIV in neonates, 10 were positives. These data, despite the small amount, show the importance to test for infectious diseases.
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