Application of MassARRAY genotyping assay in the diagnosis of neonatal genetic metabolic diseases / 中华检验医学杂志

Objective:To investigate the accuracy, effectiveness and feasibility of MassARRAY genotyping assay in the diagnoses of neonatal genetic metabolic diseases.Methods:This is a retrospective study. From December 2016 to January 2020, newborns were screened by tandem mass spectrometry at the Zhejiang Newborn Screening Center, among which the data of 7 922 suspected positive cases of genetic metabolic diseases were collected. These patients were then tested for the common variants of 27 genetic metabolic diseases by MassARRAY genotyping assay, along with further testing using Sanger or next-generation sequencing used to verify and/or further search for potential variants.Results:A total of 1 408 cases were tested with MassARRAY. Among these, 307 cases were confirmed with certain genetic metabolic diseases. The detection rate of hyperphenylalaninemia was the highest, followed by primary carnitine deficiency, short acyl-coA dehydrogenase deficiency and methylmalonic acidemia. With these cases, the consistency of Sanger sequencing and MassARRAY was 100% (307/307). Another 287 cases were identified as carriers by MassARRAY with a 49.1% (141/287) consistency in reference to Sanger sequencing, mainly involving SLC22A5 and MCCC1 genes. Meanwhile, 50.8% (146/287) of these cases were found to have another variant mainly involving PAH, PTS and ACADS genes. The remaining 814 cases have no variants; 158 cases out of these patients have continuously abnormal amino acids, acyl carnitines, urine organic acid and/or other biochemical indices, and were tested by next-generation sequencing, among which 38% (60/158) were detected with two variants. In this study, a total of 513 patients with genetic metabolic disease were diagnosed, and the detection rate of MassARRAY was 59.8% (307/513). Conclusions:MassARRAY genotyping assay can be used as an early molecular screening method for neonatal genetic metabolic diseases. The detection rate is particularly high in diseases with a high concentration of hotspot variants, such as hyperphenylalaninemia and primary carnitine deficiency. The future application value of MassARRAY should be further improved by continuously optimizing its ability to identify new disease genes and potential variable sites.

The role of extracellular vesicles in human cytomegalovirus infection / 中华检验医学杂志

Extracellular vesicles (EV) are membrane structured vesicles containing proteins, lipids and nucleic acids. EVS produced by virus-infected cells are involved in communication between infected and uninfected cells. EV produced by human cytomegalovirus (HCMV) infected cells can promote the transmission and infection of HCMV and escape the host immune response. It can also activate the body′s immune response against HCMV infection. In-depth study of the mechanism and compositional changes of EV produced by HCMV infected cells will contribute to the prevention, diagnosis, and treatment of HCMV. The current research has achieved some results, but they are not deep enough. Future advancements in EV isolation and identification technologies and the reduction of economic costs will contribute to the extensive development and clinical application of the research.

Etiological detection and epidemiological analysis of children with influenza-like illness / 中华检验医学杂志

Objective:To analyze the status and epidemiological characteristics of respiratory virus infection in children with influenza-like illness in outpatient department, and to provide evidence for the prevention and treatment of children in this area.Methods:Nasopharyngeal swab samples were collected from children who attended the fever clinic of The Children′s Hospital, Zhejiang University School of Medicine due to influenza-like illness from July 2021 to March 2022, and six common respiratory virus nucleic acids were detected by reverse transcription-polymerase chain reaction (RT-PCR). The general information of the children was collected and grouped by gender and age (0-<6 months, 6-<12 months, 1-3< year-old, 3-<6 year-old , and ≥6 year-old), and the chi-square test was used for statistical analysis between the groups to explore the epidemic pattern of respiratory viruses.Results:A total of 739 cases (45.9%, 739/1 609) of respiratory viruses were detected from children with influenza-like illness, including 651 cases (40.5%, 651/1 609) of simple infection and 88 cases (5.5%, 88/1 609) of multiple infections. Respiratory syncytial virus (RSV) was detected in 18.6% (300/1 609), followed by influenza B virus (FluB) in 11.9% (192/1 609), adenovirus (ADV) in 8.3% (134/1 609), parainfluenza virus type 3 (PIV-3) in 7.6% (123/1 609), parainfluenza virus type 1 (PIV-1) in 4.9% (79/1 609), and influenza A virus (FluA) in 0.4% (6/1 609). Multiple infections including double or triple infections, with 81(92.0%, 81/88) cases of double infection and the most common being ADV+RSV (22.7%, 20/88) and 7 (8.0%, 7/88) cases of triple infection. There was a significant difference in the virus detection rate between the age groups (χ2=17.078, P=0.002), with the highest virus detection rate in the 3-<6 years of age group (49.7%, 286/575). Among the detection of simple infection, FluB had the highest detection rate in the ≥ 6 years of age group (26.6%, 98/369), and RSV and PIV-1 had the highest detection rate in the 3-<6 years of age group (20.0%, 115/575 and 5.9%, 34/575). The total monthly virus detection rate increased from 26.8% (37/138) in July to 63.0% (58/92) in January, and decreased to 46.1% (106/230) and 26.8% (37/138) in February and March. The detection rate of RSV was the highest from August to November, the detection rate of FluB was the highest from December to March, the detection rate of ADV increased in December and January, and the detection rate of PIV-3 increased from October to December; the detection rate of PIV-1 did not fluctuate significantly, and FluA was sporadically detected. Conclusions:RSV is the main respiratory virus in children with influenza-like illness. Most respiratory viruses are present as single infections. Multiple infections are more common in double infections. FluB, RSV and PIV-1 infections showed certain age distribution characteristics, especially in children over 3 years of age. The epidemic characteristics of respiratory virus infection show that the epidemic gradually peaks from summer to autumn and winter, and turns into an epidemic decline in spring. RSV was relatively prevalent in autumn, FluB was prevalent in winter and spring, ADV and PIV-3 were prevalent to varying degrees in winter, PIV-1 continued to circulate at a low level, and FluA did not present epidemic characteristics.

Combined application of pathogen-based tests and host reaction-based tests / 中华检验医学杂志

The occurrence development and progression of infectious diseases involves the two aspects: the immune response of both the pathogens and the hosts. At present, pathogen-based testing is the gold standard and the most commonly used method for the diagnosis of infectious diseases. The purpose of the immune system is to identify and eliminate invading pathogens. Testing based on host reactions has become an effective auxiliary means adjunct of traditional pathogen-based tests, with the potential to improve the accuracy and efficiency of diagnosis. The combined application of host-based tests and pathogen-based tests is a new field worth exploring.

Research progress in human cytomegalovirus and its association with TGF-β and Smad signaling pathway / 中华微生物学和免疫学杂志

Human cytomegalovirus (HCMV) is a common herpes virus found in human and can establish lifelong latency in the hosts. In healthy population, HCMV usually results in asymptomatic latent infection. However, the latent HCMV can be activated and cause many serious diseases and even death in people with low immunity. Transforming growth factor-β (TGF-β) is a powerful regulator of many cellular pathways, playing important roles in inflammatory response and cell differentiation, as well as immunomodulatory role in viral infection. The classic Smad signaling pathway is involved in the regulation of many cell activities, including growth, differentiation and apoptosis. This review summarized the progress in the association of HCMV infection with TGF-β and the Smad signaling pathway.

Resistance, molecular epidemiology and virulence gene of non-A-F group serotype isolates of Salmonella enterica enteric subspecies isolated from children / 中华检验医学杂志

Objective:To analyze the anti-drug resistance, molecular epidemiology and virulence gene distribution of non-A-F group serotype isolates of Salmonella enterica enteric subspecis, so as to provide epidemiological basis for clinical diagnosis and treatment.Methods:Serotyping, antimicrobial susceptibility test and whole genome sequencing were performed on 11 isolates of non-A-F group serotype isolates of Salmonella enterica enteric subspecies that were isolated from The Children′s Hospital, Zhejiang University School of Medicine between 2017 and 2020. The serotype, multilocus sequence typing and virulence gene of the whole genome sequencing results were analyzed. Results:In our hospital, the detection rate of non-A-F group serotype isolates of Salmonella enterica enteric subspecies was low (1.13%). Among the 11 strains, there were 3 strains belong to Jangwani serotype, 2 strains of Hvittingfoss serotype, and Wandsworth, Pomona, Kedougou, Urbana, Poona and Kumasi serotypes have 1 strain each. Except for the two multi-drug resistant strains, the other strains were sensitive to most antibiotics, and the MICs were at low levels. A total of 9 ST types were detected in the 11 strains, the 3 Jangwani serotype strains were ST3918, and the other isolates were of different ST types. The phylogenetic tree shown that the three strains of Jangwani serotype were closely related. A total of 103 virulence genes were detected in the 11 strains, including 78 genes related to secretion system, 21 genes related to adherence, 2 genes related to magnesium uptake, 1 gene related to resistance to antimicrobial peptides and 1 gene related to typhoid toxin. Conclusions:The detection rate of the non-A-F group serotype isolates of Salmonella enterica enteric subspecies was low, and the sensitivity of the isolates to common antibiotics was high. The ST types and genetic relationship showed diversity. Clinical laboratory should pay attention to the detection of the non-A-F group serotype isolates of Salmonella enterica enteric subspecies, and the changes in drug resistance and virulence genes of the isolates should be closely monitored.

Prevention and consideration for the biosafety of laboratory testing under epidemic condition / 中华检验医学杂志

Laboratory testing plays an important role in the diagnosis and treatment of patients with Novel Coronavirus pneumonia. However, the lack of understanding of the virus in the early stage led to great difficulties in biosafety protection for clinical laboratories. Based on the latest researches and findings about the virus, this paper provides some personal opinions on the biosafety prevention in clinical laboratorians under epidemic condition for the reference of laboratory workers.

Principles and suggestions on biosafety protection of biological specimen preservation during prevalence of COVID-19 / 浙江大学学报·医学版

Coronavirus disease 2019 (COVID-19) is a grade B infectious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In pace with the spreading of the disease, biosafety risk of the biological specimen preservation in biobanks has been significantly increased and biosafety protection during biological specimen preservation become increasingly important. According to the related national rules and the corresponding guidelines of Chinese Medical Association, this paper introduced the etiology about SARS-CoV-2, epidemiology about COVID-19, and the biosafety protection principles of individuals and biological specimen storage places in the process of personal protection, protection of collection, transport, handling, preservation, detection, post-detection disposal and emergencies of biological specimen. Emphasized to carry out a strict biosafety-risk assessment on biological specimen basing on virus load information, infectivity, and sample type (possible contact transmission, aerosol transmission, and fecal oral transmission).

Application of Region 4 Stork system in the neonatal screening for very long chain acyl-CoA dehydrogenase deficiency by tandem mass spectrometry / 中华检验医学杂志

Objective:To investigate the application feasibility of Region 4 Stork (R4S) system, an international collaborative newborn screening data platform, combined with cut-off value analysis in the neonatal screening for very long chain acyl-CoA dehydrogenase deficiency (VLCADD) by tandem mass spectrometry (MS/MS).Methods:The retrospective study was performed in 2, 040 072 neonates screened by MS/MS in Neonatal Screening Center of Zhejiang Province, China from October 2013 to July 2018. Nine hundred and ten cases were determined and identified as suspected positive VLCADD neonates by traditional cut-off method of tandem mass spectrometry. The original data of these 910 screened neonates were further analyzed by R4S system. Based on clinical diagnosis and ACADL gene test results, the screening efficiency between two methods was statistically compared.Results:The data of 910 suspected VLCADD-positive cases interpreted by cut-off method were further analyzed by R4S system, and the positive interpretation was reduced to 238 cases (including 9 confirmed positive cases). A total of 16 different mutations were found in ACADL gene sequencing among the confirmed children. The screening false positive rate (FPR) declined from 0.44‰ (901/2 040 072) to 0.11‰ (229/2 040 072), the rate of positive predictive value (PPV) increased from 0.99% (9/910) to 3.78% (9/238), and the specificity increased from 99.96% (2 039 162/2 040 063) to 99.99% (2 039 834/2 040 063). There was a statistically significant difference between cut-off method alone and cut-off method combined R4S system analysis (χ2=393.5, P<0.05). Conclusions:The R4S system combined with cut-off method applied in VLCADD neonatal screening by MS/MS can effectively improve screening performance, reduce false positive rate, and has certain value in clinical application.

The roles and mechanisms of circular RNAs in viral infectious diseases / 中华检验医学杂志

Circular RNAs (circRNAs) are covalently linked circular single-stranded RNAs, which have the characteristics like abundance,conservation, stabilization and specificity. It is demonstrated that circRNA plays an important roles in the regulation of gene expression and multiple signal pathways of diseases such as cancer, cardiovascular disease, nervous system disease and autoimmune disease. In recent years, it has been reported that circRNAs are expressed abnormally in viral infectious diseases. CircRNA is expected to be a potential biomarker for viral infectious diseases and a potential target for disease therapy. In this review we briefly introduced the research progression of the roles and mechanisms of circular RNAs in viral infectious diseases and provides the references for future study.

The status and progress of laboratory tests for pediatric allergic disease / 中华检验医学杂志

Allergic diseases posed a serious threat to children′s healthy quality and life safty, and brought heavy economic burden to the society. The clinical diagnosis of allergic diseases is mainly based on the history of disease, the physical examination, the in vivo test and the in vitro test. In addition, some new methods and potential markers have been indicated as the promising strategies for the in vitro tests for the diagnosis of allergic diseases. The study of allergen molecular composition and the screening of new allergen-related markers based on multi-omics are the future development directions of laboratory testing of allergic diseases in pediatrics.

Expert consensus on standardized TORCH laboratory detection and clinical application / 中华检验医学杂志

TORCH, which is considered as a series of pathogens, including the Toxoplasma gondii, Rubella virus, Cytomegalovirus or Herpes simplex virus, often infects the pregnant women to induce the the fetus or newborn infection by transplacental infection or exposure to contaminated genital tract secretions at delivery. Increasing evidence have been confirmed that the infection of TORCH may cause the miscarriage, premature birth, malformed fetus, stillbirth, intrauterine growth retardation, neonatal multiple organ dysfunction and other adverse pregnancy outcomes. For most TORCH-infections cases may lacking the effective treatments during pregnancy, and it is important to achieve the effacing monitoring of TORCH infections before and during pregnancy. The laboratory testing of TORCH has the great significance. However, the consensus opinions still need to improve the the standardization of TORCH testing process and the correct interpretation. Based on the characteristics of the TORCH detection method, this article gives a consensus opinion on the standardized detection and clinical application of TORCH from the laboratory perspective according to the characteristics and types of infection of different pathogens.

Prevention and consideration for the biosafety of laboratory testing under epidemic condition / 中华检验医学杂志

Laboratory testing plays an important role in the diagnosis and treatment of patients with novel coronavirus pneumonia. However, the lack of understanding of the virus in the early stage led to great difficulties in biosafety protection for clinical laboratories. Based on the latest researches and findings about the virus, this paper provides some personal opinions on the biosafety prevention in clinical laboratorians under epidemic condition for the reference of laboratory workers.

Research progress in Gasdermin family proteins and their association with diseases / 中华微生物学和免疫学杂志

Gasdermin family (GSDMs), consisting of six proteins (GSDMA, GSDMB, GSDMC, GSDMD, GSDME and DFNB59) in humans and ten proteins (GSDMA1-3, GSDMC1-4, GSDMD, GSDME and DFNB59) in mice, might be involved in multiple physiological and pathological processes, including epithelial cell development, apoptosis, inflammation, carcinogenesis and immune-related diseases. Recent studies confirmed GSDMD, which containing an N-terminal domain with pore-forming activity and a C-terminal domain with structural autoinhibition, as a crucial substrate of inflammatory caspases in pyroptosis, pioneering a new area for structural and functional research on Gasdermin family proteins. This review will summarize the latest progress in the structures, functions and association with diseases of several Gasdermin family proteins.

Research progress in Gasdermin family proteins and their association with diseases / 中华微生物学和免疫学杂志

Gasdermin family (GSDMs), consisting of six proteins (GSDMA, GSDMB, GSDMC, GSDMD, GSDME and DFNB59) in humans and ten proteins (GSDMA1-3, GSDMC1-4, GSDMD, GSDME and DFNB59) in mice, might be involved in multiple physiological and pathological processes, including ep-ithelial cell development, apoptosis, inflammation, carcinogenesis and immune-related diseases. Recent studies confirmed GSDMD, which containing an N-terminal domain with pore-forming activity and a C-termi-nal domain with structural autoinhibition, as a crucial substrate of inflammatory caspases in pyroptosis, pio-neering a new area for structural and functional research on Gasdermin family proteins. This review will sum-marize the latest progress in the structures, functions and association with diseases of several Gasdermin fam-ily proteins.

The molecular epidemiology of human group A rotavirus infections in children in Hangzhou in 2017 / 中华检验医学杂志

Objective To analyze the infectious status and genotype characteristics of group A rotavirus (RV) in children with diarrhea in Hangzhou city in 2017, and to provide information for the disease surveillance, epidemic control as well as vaccine development.Methods Fecal samples from children with acute diarrhea at Children's Hospital of Zhejiang University were collected from Jan to Dec in 2017. All samples were tested for RV antigen by emulsion technique. The antigen-positive samples were further detected by reverse transcription polymerase chain reaction (RT-PCR) and sequencing to determine the G and P genotypes. The RV positive rates in different genders, ages and months were compared by chi-square test. Results A total of 20895 fecal samples were collected from 12389 male patients and 8506 female patients. The gender ratio was 1.46:1. In 5012 (23.99％) RV antigen positive samples, 2964 (23.92％) were from male patients and 2048 (24.08％) were from female patients. There was no gender difference in RV positive rate (χ2=0.049, P>0.05). In the study, RV could be detected in the whole year. January, February and December were peak months, and the RV positive rates were significantly different in different months (χ2=2654.681, P<0.05). The highest RV positive rate was in 18-24 months age group and the lowest in<6 months age group, respectively. Children under 2 years old accounted for 76.56％RV positive cases, and those under 5 years old accounted for 98.72％ RV positive cases. The RV positive rates were also significantly different in all age groups (χ2=1013.832, P<0.05). A total of 116 samples were selected from each month, following the random stratified sampling principle, for PCR amplification, sequencing, and genotyping according to VP7 (G genotype) and VP4 (P genotype). A total of 4 G genotypes were detected and G9 (85.3％) was the predominant one. In the two detected P genotypes, P[8] (96.6％) was predominant. The 4 G/P combination genotypes were G9P[8] (85.3％), G3P[8] (9.5％), G2P[4] (3.5％), and G1P[8] (1.7％). Conclusions RV was a common pathogen in pediatric patients with acute diarrhea in Hangzhou city in 2017. Children under 5 years old, especially 18 months to 2 years old infants was the main infected population. The study showed RV infection had obvious seasonality and winter was the peak period. The G9 genotype was predominant in G genotypes, P[8] genotype was predominant in P genotypes, and G9P[8] genotype was predominant in G/P combination genotypes, respectively.

The development and application of nucleic acid detection techniques of infectious microorganisms in pediatrics / 中华检验医学杂志

The advance of molecular diagnosis has evolved the field of detection of pathogens in pediatric disease towards the high-throughput ultra-sensitive molecular testing. This review provides an overview and comparison of current molecular tools on the market, including qPCR, multiplex PCR, digital PCR, isothermal amplification, and microfluidic chip, highlighting the future trends of integrated high-throughput molecular testing and automation. These evolutions will maximize the utility of limit patient samples from children and contribute significantly on the diagnosis of infectious disease.

Relationship between gH genotyping and clinical characteristics of children with congenital cytomegalovirus infection / 中华儿科杂志

Objective@#To study the relationship between human cytomegalovirus (HCMV) envelope glycoprotein gene H and clinical features of children with congenital cytomegalovirus infection.@*Methods@#A cohort study was conducted. Newborns diagnosed with congenital cytomegalovirus infection, hospitalized in the Department of Neonatology and Neonatal Intensive Care Unit (NICU) of the Children′s Hospital, Zhejiang University School of Medicine, were included from July 2013 to December 2015.HCMV-DNA gH typing in urine, sputum or blood was conducted. Patients then were divided into gH1 group and gH2 group according to gH genotypes. Patients′ data during hospitalization in newborn and 3-5 years of follow-up were collected.The relationships between gH genotype and clinical manifestations, laboratory examinations, hearing loss and neurological prognosis were analyzed by chi-square test, t test and non-parametric test.@*Results@#A total of 21 cases were enrolled as congenital HCMV infection and followed-up for 3-5 years. Among them, 14 (67%) were gH1 type and 7 (33%) were gH2 type. No mixed infection was found. In the two groups, there were no significant differences in the ratio of males (9/14 vs. 3/7,P=0.397), or birth weight ((2 609±686) vs. (3 021±451) g, t=-1.436, P=0.167). Gestational age of gH1 group was younger than that of gH2 group (38 (29-40) vs. 39(38-40) weeks, Z=-2.18, P=0.029). Moderate to severe hearing loss detected by neonatal auditory brainstem response were found in 40 ears (20 cases). It was higher in gH1 group than that in gH2 group (4/22 vs.0/18, χ2=5.145, P=0.023). In the imaging examination of the nervous system, the Alarcon score of gH1 group was lower than that of gH2 group (0.4±0.3 vs. 1.3±1.1, t=-2.459,P=0.024).No significant statistical difference was found in the probability of motor or language development lag in gH2 group and gH1 group (4/7 vs.4/14, P=0.346).@*Conclusions@#Compared with gH2 infection, gH1 infection in children has a younger gestational age. The major type of hearing loss in neonatal period is gH1 infection. Children with gH2 congenital infections are more likely to suffer from nervous systems damage.

Preliminary application of Region 4 Stork project used in newborn screening by tandem mass spectrometry / 中华检验医学杂志

Objective To investigate the feasibility of Region 4 Stork(R4S)project used for newborn screening by tandem mass spectrometry in China.Methods This retrospective study was performed among 362 822 neonates screened by tandem mass spectrometry from May 2015 to April 2016 in Zhejiang newborn screening center.Infants were grouped by screening result category: 83 true positive cases,360 554 true negative cases and 2 185 false positive cases.Raw data was uploaded into R4S website to perform postanalytical interpretive tools, then results were analyzed with interpretation rules.The comparisons of normal population percentiles were done at five selected percentiles between Zhejiang newborn screening center and R4S project with min-max normalization.Results Compared with cutoff system by using R4S project with interpretation rules,the positive predictive value increased from 3.7%to 8.3%,the specificity increased from 99.40%to 99.75%, and the false positive rate declined from 0.6% to 0.2%. The two cases of true positive hyperprolinemia were reported negative, and one case of β-ketothiolase deficiency was misdiagnosis.Totally 311 638 cases in true negative group were resolved by postanalytical interpretive tools,and the remaining 48 916 cases were excluded with interpretation rules.False positive cases were reduced to 897 cases.Results of percentiles comparison showed that levels of some markers were significantly different between zhejiang newborn screening center and R 4S project.Conclusions R4S project effectively improved the newborn screening performance, whereas leaded to a small number of misdiagnosis and missed diagnosis.Besides,many true negative cases should be excluded with interpretation rules.Optimization should be achieved based on local normal population.(Chin J Lab Med,2018,41:300-304)

Effects of physical and chemical factors in environment for dried blood spot preparation of neonatal screening assay / 临床检验杂志

Objective To investigate the effects of physical and chemical factors in the environment for dried blood sample (DBS) preparation of neonatal screening assay.Methods A total of 60 normal and 120 positive DBS were prepared under control and 10 different conditions.Another 30 normal and 80 positive DBS were prepared under control and 7 different concentration gradients of formaldehyde.The levels of phenylalanine (Phe),glucose-6-phosphate dehydrogenease (G6PD),thyroid stimulating hormone (TSH) and 17α-hydoxyprogesterone (17α-OHP) were tested by time-resolved fluorescence immunoassay or fluorescence assay.Statistical analysis was performed using SPSS 22.0 software.Results Compared with the control group,the results of Phe were not significantly different (P ＞ 0.05) when the samples were dried under the formaldehyde sensitive threshold (4.62 to 6.95 ppm for 18 hours).G6PD levels were significantly lowered when the samples were dried under all the conditions except for fast cold drying (2 to 8 ℃ overnight and formaldehyde condition,0.30 to 0.38 ppm for 4 hours or 0.21 to 0.24 ppm for 18 hours).TSH and 17α-OHP levels were lowered obviously when the samples were dried under the conditions of humidity,UV and formaldehyde condition (TSH:0.32 to 0.52 ppm for 4 hours,0.38 to 0.45 ppm for 18 hours,17α-OHP:4.37 to 4.62 ppm for 4 hours,0.38 to 0.45 ppm for 18 hours).The results of Phe,G6PD,TSH and 17α-OHP were not statistically different with the control group when the samples were dried under the fast cold drying and 2 to 8 ℃ overnight.Conclusion The physical and chemical factors in the environment of DBS preparation should be related to the accuracy of neonatal disease screening closely.The necessary control factors including formaldehyde,ethanol,glacial acetic acid,ultraviolet irradiation,heat,humidity and decoration pollution may exhibit significant effects on the preparation of DBS.Fast cold drying and overnight at 2 to 8 ℃ could be available for DBS preparation.