Modifications in electrospray tandem mass spectrometry for a neonatal-screening pilot study in Japan

In a neonatal-screening pilot study for inherited disorders in organic acid and amino acid metabolism, we analyzed butyrated acylcarnitines and amino acids in blood spots of more than 20 000 newborns by electrospray tandem mass spectrometry. In order to screen urea cycle disorders, we performed multiple scanning functions with additional stable isotope-labelled internal standards, since such reported functions as neutral loss of m/z 102 or 109 for butyrated amino acids were not sufficient. Arginine levels were measured with arginine-¹³C₆. Hypocitrullinemia for the screening of some urea cycle disorders was detectable by measurement with synthesized citrulline-d₆, although we did not find any such disorders. In the acylcarnitine analysis, we found a patient with propionic acidemia, who has been treated effectively. The increasing false positive rate due to the use of pivalic acid-containing antibiotics in the diagnosis of isovaleric acidemia was a problem in Japan.     

Selective screening for fatty acid oxidation disorders by tandem mass spectrometry: difficulties in practical discrimination

In a selective screening for fatty acid oxidation disorders by tandem mass spectrometry, we tested the diagnostic ratios and acylcarnitine concentrations in sera or blood spots, which were reported to be specific to very long-chain acyl CoA dehydrogenase deficiency, carnitine palmitoyltransferase I deficiency, and carnitine palmitoyltransferase II deficiency. While the acylcarnitine profiles in the majority of these patients were typical in the respective disorders, some overlapping of the indices was observed between these patients and the infants, who showed symptoms mainly related to hypoglycemia but did not have the disorders mentioned above. Although the diagnostic ratio of tetradecenoylcarnitine to dodecanoylcarnitine for very long-chain acyl CoA dehydrogenase deficiency seemed to minimize the overlapping in this study, additional measures including careful assessment of clinical data and enzyme assays may be necessary for the diagnosis in atypical cases.     

Effectiveness of mass screening for endometrial cancer

OBJECTIVE: To investigate the effectiveness of mass screening for endometrial cancer using Endocyte (Laboratoire CCD, Paris, France) endometrial smears. STUDY DESIGN: The study subjects were consecutive patients with documented endometrial cancer diagnosed between January 1, 1989, and December 31, 1997, at 22 hospitals in Japan. One hundred twenty-six cases were detected by mass screening and 1,069 diagnosed in outpatient clinics. We compared the stage of cancer at diagnosis and survival rate of patients in the two groups. RESULTS: Early stage was significantly more frequent in the screening group (P < .001); stage I comprised 88.1% of the screening group as compared with 65.3% of the outpatient group. Well-differentiated adenocarcinoma was significantly more frequent in the screening group (P < .01); grade 1 constituted 74.7% of the screening group as compared with 61.0% of the outpatient group. The five-year survival rate was significantly higher in the screening group than in the outpatient group (94.0% vs. 84.3%, P = .041). The crude hazard ratio (HR) of dying of endometrial cancer for the screening group as compared to the outpatient group was .47 (95% CI .22-.99, P = .048). HR became .96 (95% CI .45-2.08, P = .925) after adjustment for age, study area and cancer stage. CONCLUSION: The results suggest that an endometrial cancer screening program would lead to early detection and improved survival among women with endometrial cancer.     

Evaluation of a mass screening program for lysinuric protein intolerance in the northern part of Japan

Lysinuric protein intolerance (LPI:MIM 222700) is an autosomal recessive disease characterized by defective transport of the dibasic amino acids. We recently reported a local cluster of LPI in the northern part of Japan (Koizumi et al., 2000). Mutational analysis of the LPI patients in this local cluster revealed they were exclusively homozygous for the R410X mutation. The effectiveness of early intervention with citrulline therapy (200 mg/kg per day) and protein restriction (1.5 g/kg per day) was confirmed in these patients. Mass screening was conducted in 4,568 newborn babies between 1999 and 2002, which was estimated to cover 100% of almost all newborns delivered in the screened area. Forty heterozygous newborns were found (0.88%), leading to an estimated incidence of LPI of 1:51,984. The number of people that required screening to detect one case was 51,984, and the cost for mass screening was 30 cents/person (a total of dollars 15,600). This is comparable to, or even less than, the cost of currently screened diseases in Japan. Therefore, we conclude that a mass screening program for LPI can be introduced effectively and economically into an area where an LPI cluster is located as the result of a founder mutation.     

Diagnosis of inborn errors of metabolism using filter paper urine,urease treatment,isotope dilution and gas chromatography–mass spectrometry

This review will be concerned primarily with a practical yet comprehensive diagnostic procedure for the diagnosis or even mass screening of a variety of metabolic disorders. This rapid, highly sensitive procedure offers possibilities for clinical chemistry laboratories to extend their diagnostic capacity to new areas of metabolic disorders. The diagnostic procedure consists of the use of urine or filter paper urine, preincubation of urine with urease, stable isotope dilution, and gas chromatography–mass spectrometry. Sample preparation from urine or filter paper urine, creatinine determination, stable isotope-labeled compounds used, and GC–MS measurement conditions are described. Not only organic acids or polar ones but also amino acids, sugars, polyols, purines, pyrimidines and other compounds are simultaneously analyzed and quantified. In this review, a pilot study for screening of 22 target diseases in newborns we are conducting in Japan is described. A neonate with presymptomatic propionic acidemia was detected among 10,000 neonates in the pilot study. The metabolic profiles of patients with ornithine carbamoyl transferase deficiency, fructose-1,6-bisphosphatase deficiency or succinic semialdehyde dehydrogenase deficiency obtained by this method are presented as examples. They were compared to those obtained by the conventional solvent extraction methods or by the tandem mass spectrometric method currently done with dried filter blood spots. The highly sensitive, specific and comprehensive features of our procedure are also demonstrated by its use in establishing the chemical diagnosis of pyrimidine degradation defects in order to prevent side effects of pyrimidine analogs such as 5-flurouracil, and the differential diagnosis of three types of homocystinuria, orotic aciduria, uraciluria and other urea cycle disorders. Evaluation of the effects of liver transplantation or nutritional conditions such as folate deficiency in patients with inborn errors of metabolism is also described.     

Enzymatic diagnosis of medium-chain acyl-CoA dehydrogenase deficiency by detecting 2-octenoyl-CoA production using high-performance liquid chromatography: a practical confirmatory test for tandem mass spectrometry newborn screening in Japan

Many of the previously described enzymatic assay methods for the diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency have been dependent upon the measurement of radioisotope-labeled co-products or reduction of electron acceptors. We have developed a direct assay method to detect 2-enoyl-CoA production using high-performance liquid chromatography (HPLC). Crude cell lysate prepared from lymphocytes were incubated with n-octanoyl-CoA and ferrocenium hexafluorophosphate. The detection of 2-octenoyl-CoA was significantly reproducible. We applied the assay to samples from four infants suspected to have MCAD deficiency by tandem mass spectrometry (MS/MS) newborn screening conducted in the Hiroshima area of Japan. Three of them were proved to have pathologically reduced residual enzyme activities, although they were associated with various clinical and biochemical phenotypes. In addition, another symptomatic Japanese patient and her presymptomatic sibling who were detected by MS/MS selective screening were successfully diagnosed by our enzymatic assay. These results indicate that the method can be a useful confirmatory test for MS/MS screening of MCAD deficiency.     

Nipple discharge cytology in mass screening for breast cancer

Since 1977 mass screening for breast cancer has been conducted in Miyagi Prefecture, Japan; inspection, palpation and cytologic examination of any nipple discharge are part of the initial screening procedures. Among 149,681 subjects examined, 404 cancer cases and 63 papilloma cases were detected. The nipple discharges from 20,537 women were examined cytologically; of the 61 cancer cases, the smears were positive in 18 cases, suspicious in 7, negative with atypical findings in 12 and negative in 24. Ten of the cancer cases were detected exclusively by the cytologic examination of a nipple discharge. In eight of these ten cancer cases, there was no other initial evidence of the primary tumor. The cytologic diagnosis of discharges without blood from 28 cancer cases was positive or suspicious in 10 cases and negative in 18. Thirty-seven of the papilloma cases were initially detected only by the cytologic examination of a nipple discharge; neither physical examination nor mammography showed any abnormal findings.     

Clinical applications of tandem mass spectrometry: ten years of diagnosis and screening for inherited metabolic diseases

This paper reviews the clinical applications of tandem mass spectrometry (MS–MS) in diagnosis and screening for inherited metabolic diseases in the last 10 years. The broad-spectrum of diseases covered, specificity, ease of sample preparation, and high throughput provided by the MS–MS technology has led to the development of multi-disorder newborn screening programs in many countries for amino acid disorders, organic acidemias, and fatty acid oxidation defects. Issues related to sample acquisition, sample preparation, quantification of metabolites, and validation are discussed. Our current experience with the technique in screening is presented. The application of MS–MS in selective screening has revolutionized the field and made a major impact on the detection of certain disease classes such as the fatty acid oxidation defects. New specific and rapid MS–MS and LC–MS–MS methods for highly polar small molecules are supplementing or replacing some of the classical GC–MS methods for a multitude of metabolites and disorders. New exciting applications are appearing in fields of prenatal, postnatal, and even postmortem diagnosis. Examples for pitfalls in the technique are also presented.     

Gas chromatographic–mass spectrometric screening for organic acidemias using dried urine filter paper: determination of α-ketoacids

There are several organic acid disorders that require information on α-ketoacids, such as maple syrup urine disease or α-ketoadipic acidemia. The recovery, stability and diagnostic availability of α-ketoacids in dried urine filter paper analyzed by GC–MS with oxime-trimethylsilyl derivatization was studied for organic acidemia screening. The recovery of all nine types of α-ketoacids tested, but for phenylpyruvate, 2-ketoadipate, and p-OH-phenylpyruvate, from filter paper samples was acceptable. The stability of pyruvate, branched-chain α-ketoacids, α-ketoadipate and α-ketoglutarate was stable for at least 28 days, although some α-ketoacids such as succinylacetone were unstable. It indicated it was difficult to diagnose only tyrosinemia type 1 among nine specimens from organic acidemia patients tested. The method could be applied to global organic acidemia screening.     

Demands on surgical inpatient services after mass mammographic screening.

The changes in the demand for surgical inpatient care created by mammographic screening for breast cancer were analysed by comparing two counties, one with and one without a mass screening campaign. A comprehensive computerised register of inpatient care in the region was used. The results indicate that population based screening offered to women above 40 years and repeated every two to three years will increase the number of operations required for breast cancer and inpatient days by at least 150% during the initial screening round. During the second round the figures tend to return to previous levels. Of decisive importance for the demands on health service resources are the specificity of screening, the duration of the first screening round, and the age groups included.     

Expected effects of mass screening policies on the frequency of cystic fibrosis homozygotes

We evaluated, by deterministic computer simulation, some effects of a screening programme for carriers of cystic fibrosis mutations. Two different selective regimes (heterozygote advantage and directional selection against recessive homozygotes) and three kinds of response to the screening were simulated. The curves describing the expected decline in the frequency of CF homozygotes allow one to predict some benefits of a screening campaign. In addition, it is shown that a strategy aimed at testing couples, rather than individuals, may become less expensive after only two generations of screening. The main source of uncertainty for a screening programme remains the selection mechanism, namely the existence of some sort of biological advantage for heterozygous carriers of CF mutations. Received: 11 March 1997 / Accepted: 15 May 1997     

A highly cost effective method of mass screening for thalassaemia.

A simple, fast, and reliable two step procedure for the detection of non-alpha-thalassaemias in mass screening programmes is presented. Step 1 consists of a study of red cell morphology and a one tube red cell osmotic fragility tests. This step eliminates the non-thalassaemic samples; the rest are processed through step 2, consisting of determination of red cell indices and haemoglobin studies. Over the past seven years this procedure has been used at this centre in mass screening secondary school students in Latium. Blood samples from 289 763 students were examined, and 6838 cases of thalassaemia detected. It is estimated that 0.35 +/- 0.25% of subjects with thalassaemia escaped detection by this procedure.     

Comprehensive radionuclide lung imaging in preventive mass screening of industrial workers]

The results of a combined roentgenoradionuclide investigation of the lungs were studied in 45 persons at risk of developing chronic nonspecific pulmonary diseases, diagnosed in a mass screening of 3000 workers at an industrial enterprise. The results of fluorographic, roentgenoscopic and roentgenographic investigations of the lungs, roentgenopneumopolygraphy and radionuclide investigation of lung perfusion (pulmoscintigraphy with 99mTc-labeled albumin macroaggregates) were compared. During this combined investigation the signs of nonspecific pulmonary diseases were diagnosed in 40% of the patients, changes of the pulmonary blood flow--in 71%, changes of ventilation--in all the examinees. A conclusion was made of the necessity of additional combined investigation of patients belonging to risk groups, identified during mass screenings.     

Quantitative gas chromatography—chemical ionization mass spectrometry of 2-ketoglutarate from urine as its O-trimethylsilyl-quinoxalinol derivative

Quantitation of 2-ketoglutarate in urine as its O-trimethylsilyl-quinoxalinol derivative by gas chromatography—chemical ionization mass spectrometry is described. This technique, with ammonia as reactant gas, produces no fragmentation and allows only the detection of the protonated molecular ion. It gives the greatest known sensitivity, and could be applied to the determination of urinary 2-ketoglutarate in normal children and in various metabolic disorders, such as dihydrolipoyl dehydrogenase defect, pyruvate carboxylase deficiency and type I glutaric acidemia.     

Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi

The analysis of urinary acylglycines is an important biochemical tool for the diagnosis of many organic acidemias and mitochondrial fatty acid β-oxidation defects. A new rapid analytical method has been developed for quantification of acylglycines in urine by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The method requires a simple sample preparation avoiding derivatization. It has high sensitivity, specificity, and throughput capability, and it requires minimal instrument maintenance. The use of chromatographic separation allows us to identify and quantify isomeric compounds that cannot be solved by appropriate multiple reaction monitoring (MRM) transitions. Urinary concentrations of the different acylglycines were determined using deuterated internal standards. The reference interval for the various metabolites was established using 120 healthy controls. The diagnostic usefulness of the method was demonstrated in three patients with propionic acidemia (PA), one patient with isovaleric acidemia (IVA), two patients with beta ketothiolase deficiency (BKTD), one patient with short branched chain amino acid deficiency (SBCAD), four patients with medium chain acyl-coenzyme A dehydrogenase deficiency (MCADD), one patient with isobutyryl-coenzyme A dehydrogenase deficiency (IBDHD), and one patient with multiple acyl-coenzyme A dehydrogenase deficiency (MADD).     

Liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric analysis of glycine conjugates and urinary isovalerylglycine in isovaleric acidemia

n-Acetylglycine, n-propionylglycine, n-butyrylglycine, isobutyrylglycine, n-valerylglycine, isovalerylglycine, heptanoylglycine, phenylacetylglycine and isovalerylglucuronide were identified based on their liquid chromatographic-atmospheric pressure chemical ionization mass spectra (LC-APCI-MS). We were able to detect the presence of urinary isovalerylglycine in two cases of isovaleric acidemia using LC-APCI-MS. Membrane-filtered urine samples were injected into the LC-APCI-MS system in the negative-ion mode without any further pretreatment, and large amounts of isovalerylglycine were detected as the [M − H]⁻ ion. The urinary excretion of isovalerylglycine appeared to increase after -carnitine therapy. This analytical method is quick and easy and it may be a useful tool in understanding dysfunctional conditions in isovaleric acidemia.     

Control of scabies in a tribal community using mass screening and treatment with oral ivermectin -A cluster randomized controlled trial in Gadchiroli,India

BackgroundScabies is often endemic in tribal communities and difficult to control. We assessed the efficacy of a community-based intervention using mass screening and treatment with oral ivermectin in controlling scabies.Methods/ FindingsIn this cluster randomised controlled trial, 12 villages were randomly selected from a cluster of 42 tribal villages in Gadchiroli district. In these villages, trained community health workers (CHWs) conducted mass screening for scabies. The diagnosis was confirmed by a physician. Six villages each were randomly allocated to the intervention and usual care arm (control arm). In the intervention arm (population 1184) CHWs provided directly observed oral ivermectin to scabies cases and their household contacts. In the usual care arm (population 1567) scabies cases were referred to the nearest clinic for topical treatment as per the standard practice. The primary outcome was prevalence of scabies two months after the treatment. Secondary outcomes were prevalence of scabies after twelve months of treatment and prevalence of impetigo after two and twelve months of treatment. Outcomes were measured by the team in a similar way as the baseline. The trial was registered with the clinical trial registry of India, number CTRI/2017/01/007704.In the baseline, 2 months and 12 months assessments 92.4%, 96% and 94% of the eligible individuals were screened in intervention villages and 91.4%, 91.3% and 95% in the usual care villages. The prevalence of scabies in the intervention and usual care arm was 8.4% vs 8.1% at the baseline, 2.8% vs 8.8% at two months [adjusted relative risk (ARR) 0.21, 95% CI 0.11–0.38] and 7.3% vs 14.1% (ARR 0.49, 95% CI 0.25–0.98) at twelve months The prevalence of impetigo in the intervention and usual care arm was 1.7% vs 0.6% at baseline, 0.6% vs 1% at two months (ARR 0.55, 95% CI 0.22–1.37) and 0.3% vs 0.7% at 12 months (ARR 0.42, 95% CI 0.06–2.74). Adverse effects due to ivermectin occurred in 12.1% of patients and were mild.ConclusionsMass screening and treatment in the community with oral ivermectin delivered by the CHWs is superior to mass screening followed by usual care involving referral to clinic for topical treatment in controlling scabies in this tribal community in Gadchiroli.     

Direct screening of natural product extracts using mass spectrometry

The authors describe first a proof-of-concept experiment to show direct affinity screening using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) is a rapid and informative approach for natural product extract screening. The study used 10 alkaloid-enriched plant extracts and 8 desalted marine extracts spiked with specific inhibitors of bovine carbonic anhydrase II (bCAII; EC4.2.1.1) as a model set. The spiked extracts were incubated with bCAII and then analyzed by ESI-FTICR-MS. The noncovalent complexes were detected, and the specific inhibitors were reidentified in the spiked natural product extracts. There was no interference from the desalted/alkaloid-enriched extracts to the formation of the noncovalent complexes. The method allowed quick identification of the molecular mass of the bound ligand. The authors then applied the screening to identify active compounds in natural product extracts. They employed direct infusion and online size exclusion chromatography (SEC) ESI-FTICR-MS to detect intact target-ligand complex. Eighty-five methanolic plant extracts were screened against bCAII by direct infusion ESI-FTICR-MS and by online SEC-ESI-FTICR-MS. One noncovalent complex was identified from the same plant extract by both methods. The molecular weight of the bound ligand from this extract was determined. Mass-directed purification gave 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2H-chromen-2-one (1) as the active compound. Subsequently, the binding to bCAII was confirmed by ESI-FTICR-MS. The binding specificity was determined by competition experiments between 1 and furosemide, a specific ligand of bCAII.     

Randomised trial of compliance with screening for colorectal cancer.

A randomised trial of compliance with screening for colorectal cancer by means of the haemoccult test was conducted in Farnborough and Basingstoke districts. In each of the 14 participating practices (41 general practitioners) 25 852 men and women aged between 40 and 70 years were randomly allocated by household to one of six groups. The group determined the method of invitation to screening: a letter and the test were sent to the patient, or a letter with an appointment to attend the surgery was sent, or during a routine consultation the general practitioner invited patients to participate, and some patients received an educational booklet about bowel disorders and screening. Of the 17 824 people who were offered screening, 7545 (42%) complied. Compliance was significantly affected by the method of invitation, but not by whether an educational booklet was received, and was highest (57%) in the group that was offered the haemoccult test during a routine consultation (the "opportunistic" approach). In this group the compliance rate achieved by individual general practitioners ranged from 26% to 82%. Compliance was significantly higher in Farnborough, in the older (55-70) age group, in women, and in households in which two or more people were offered screening. The higher compliance in Farnborough may be explained by the higher proportion of older people and by the higher proportion of people living in households of two or more in the population that was offered screening. The fact that the screening programme in Farnborough was offered to the whole community and that the researcher may have acted as a facilitator were probably also important. One per cent of the patients screened had a positive test, and 24 (38%) of the 63 patients who were positive and were investigated in hospital had neoplastic disease. The yield was 1.2 cancers and 1.2 benign adenomas (1 cm or larger in size) per 1000 people screened. This low yield is likely to be a consequence of the relatively young age group screened.     

Neonatal onset of organic acidemia (propionic) diagnosed by tandem mass spectrometry

Propionic acidemia is an autosomal recessive disorder as a result of a deficient activity of propionyl-CoA carboxylase. Propionyl CoA is metabolized by propionyl-CoA carboxylase to methylmalonyl CoA. Propionic acidemia is a major cause of ketotic hyperglycinemia. This disorder is characterized by episodic vomiting, dehydratation, feeding intolerante, lethargy, hypotonia, metabolic acidosis, ketosis and hyperammonemia. The patient presented herein was a full-term female newborn with encephalopathy in the first days of life. She presented hypoglycemia, metabolic acidosis with increased anion gap, ketosis, hyperammonemia, anemia, leukopenia and thrombocytopenia. The brain ultrasonography was normal. The tandem mass expectrometry done by Pediatrix was abnormal, with the acylcarnitine results consistent with an organic acidemia. The parents are consanguineus and have a history of abortus, miscarriage and neonatal death, characteristics suggestive of the presence of genetic defects.