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The quality and quantity of compounds affected by viral inactivation methods in dried blood spots

  • Ming Wang , Chaowen Yu , Shi Tang , Zhihong Liao , Kexing Wan and Shan Liu EMAIL logo
Published/Copyright: November 13, 2023
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Journal of Laboratory Medicine
From the journal Journal of Laboratory Medicine Volume 47 Issue 6

Abstract

Objectives

The aim is to evaluate the effect of viral inactivation methods on the quality and quantity of compounds in dried blood spots (DBS).

Methods

Three effective and common inactivation methods were selected via the literature search, including: heating at 56 °C for 30 min, irradiation with UVC for 30 min, and surface wetting with 70 % ethanol. The concentration and clinical predicting significance of hormones, amino acids, and acylcarnitines from DBS were assessed, and the quality and quantity of extracted deoxyribonucleic acid (DNA) from DBS were evaluated.

Results

Compared to control, we found that there was no significant difference on hormones concentration in the DBS treated by heating at 56 °C for 30 min (thyroid stimulating hormone p=0.36, 17-hydroxyprogesterone p=0.52). And heating at 56 °C for 30 min had a minimal changed coefficient of variation on the concentration of amino acids and acylcarnitines. All three inactivation methods slightly changed the yield of DNA extraction, but did not affect the quality of the DNA. Importantly, the three inactivation methods wouldn’t change the clinical predicting significance of above-compounds mostly, especially heating at 56 °C for 30 min.

Conclusions

Considering the minimal effect on the quality and quantity of various compounds, the contaminated DBS could be pretreated by the three inactivation methods, as temporary emergency inactivation methods, especially heating at 56 °C for 30 min.

Keywords: DNA extraction; dried blood spots; metabolites; viral inactivation methods

Introduction

Newborn screening (NBS) aims to identify and treat inherited metabolic disorders (IMDs) at an early stage, thus, to reduce infant’s morbidity and mortality [1, 2]. Accompanying with used dried blood spots (DBS) [3], advanced detective technology (e.g. liquid chromatography-tandem mass spectrometry [4]) and increased consciousness of prevention diseases, more and more inherited metabolic diseases have been screened in neonates including: hypothyroidism, phenylketonuria, thalassemia [5] and severe combined immunodeficiency [6] etc. As a result, in Denmark’s 10-year experience, the proportion of newborns presenting with a salt-wasting congenital adrenal hyperplasia crisis was reduced from 29 to 10 % after the introduction of screening [7]. Totally, NBS improves the survival rate and quality of life of newborns with IMDs.

The process of NBS includes education, sample collection, laboratory diagnostic tests, follow-up, management and intervention. Until now, a comprehensive quality management system (CQMS) has basically been established for the whole NBS process [8], to improve the outcome of the neonates with IMDs. However, biosafety protection has not been included in the current CQMS. Notably, hepatitis B virus (HBV) [9], hepatitis C virus (HCV) [10], even the currently prevalent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [11] are common person-to-person transmission pathogens via contaminated objects or medical devices and maintain stability for a long periods in inanimate surfaces. And the viral infectivity could be remained partly in dried blood spots, such as HBV [12]. Considering the increased possibility of transmission being due to multiple individuals working together to complete the entire NBS process, the prevention of person-to-person transmission must be given more attention.

According to the expert consensus in a pandemic situation [13], the contaminated DBS must be labeled with biosafety hazard signs and then tested under appropriate biosafety conditions, even thoroughly disinfected during the epidemic. In fact, the routine NBS laboratory is only under biosafety level 1 (BSL-1) conditions in some laboratories in China and other developing countries, which can’t meet the need of biosecurity for person-to-person transmission pathogens, such as the biosafety level 3/biosafety level 2 (BSL-3/BSL-2) agents [14]. If contaminated DBS is present in routine clinic practice, how laboratory diagnostic tests can be performed safely under BSL-1 conditions is still unclear.

In previous reports, heat, ethanol, 1-propanol, 2-propanol, and ultraviolet (UV) have been reported to alter the stability of pathogens and disinfect virus completely/partly [9, 10, 15]. However, inactivation methods also affect the quality and/or quantity of the compounds in DBS. According to data by Carreon E et al. [16], most acylcarnitines and amino acids lost 50 % of their initial concentration after 8 days’ exposure at 37 °C/45 °C with humidity >70 %. Additionally, 70 % ethanol could improve the amplification efficiency of low levels of HIV DNA in DBS, but not medium or high levels [17]. Thus, we will investigate the effect of inactivation methods on the quality and quantity of compounds from DBS.

In this study, a literature review was conducted to uncover the most common and effective methods for inactivating the viruses on the surface of DBS. Then, the selected methods were applied to DBS, the compounds in the spots (including: hormones, enzymes, amino acids, acylcarnitine and deoxyribonucleic acid (DNA)) were detected, and detective values were compared with control group to evaluate the changes caused by inactivation methods. The results allowed us to determine a better inactivation method with a least impact on various compounds in contaminated DBS.

Materials and methods

Selection viral inactivation methods for DBS

To find better inactivation methods for person-to-person transmission viruses, literature search in PubMed.gov was conducted using the query: (virus) AND ((contaminated objects) OR (inanimate surfaces or objects)) AND ((inactivation) OR (disinfection)). Then, the only inactivation methods that were provided in the literature with clear inactivated conditions and inactivated efficacy evaluation, were considered as the candidate methods. The most effective and common methods were selected for further investigation.

Specimens

All DBS were remained specimens, which were used for routine clinical neonatal inherited metabolic disease screening and were prepared from October 2022 to November 2022. And remained specimens were stored at 4 °C and <30 % humidity, according to the literature [13] and expert consensus [18]. According to the cutoff value for suggesting inherited metabolic disease, five negative specimens with normal level of metabolite and 10 positive specimens with abnormal level of metabolite (including: amino acid, carnitine, thyroid stimulating hormone (TSH), 17-hydroxyprogesterone (17ɑ-OH), and so forth) were collected. Given six detective methods for all compounds which needed to be detected in the study, totally 90 specimens were used. Every specimen was punched into 96-well microtiter plate at 3.2 mm, and then the spots were divided into four groups (control and three different inactivated groups), three replicates for every group. Totally 180 spots were detected by each detection method. Informed consent was obtained when dried blood spots were collected from all individuals included in this study. The study was approved by the Ethics Committee of the Children’s Hospital of Chongqing Medical University, Approval Number: (2022) IRB (STUDY) NO. 536.

Detective HBV DNA copy number and HBV antigen

Serum from the patients with HBV was dripped into filter paper, dried, and punched into 96-well microtiter plate at 3.2 mm and then treated by different inactivation methods. According to the manual instructions, DNA was acquired from DBS (TianGen Biotech Co, Ltd, Beijing, China) and HBV DNA copy number was detected by polymerase chain reaction (Sansure Biotech Co, Ltd, Hunan, China). HBsAg were detected by HBsAg Quantitative Reagent Kit on ARCHITECT i2000SR (Abbott, IL, USA).

Identification for hormone concentration

The 96-well microtiter plate with pretreated DBS was performed on 1235 automatic immunoassay system (PerkinElmer, USA) for TSH or genetic screening processor (GSP) (PerkinElmer, USA) for 17ɑ-OH, and the fluorescence intensity was detected. Then, the absolute concentration was determined by standard curve. All solution was provided by Fenghua Biotechnology Co., Ltd (GuangZhou, China) or PerkinElmer (Turku, Finland), respectively.

Identification for enzyme activity

GSP (PerkinElmer, The USA State) was used to analyze the activity of glucose-6-phosphate dehydrogenase (G6PD), according to GSP user manual and GSP workstation user manual. All solution was provided by PerkinElmer (Turku, Finland).

Identification for amino acid and carnitine concentration

According to the instruction of Neobase™ non-derivatized MSMS kit provided by PerkinElmer (Turku, Finland), amino acid and carnitines were extracted from DBS. Then, the extraction was analyzed in the API 3200 LC/MS/MS system (AB Sciex, USA). Finally, the concentration was calculated according to the standard curve. According to the literature [19], bias±15 % and CV≤15 % were used as quality control levels for the accuracy and precision of the liquid chromatography-tandem mass spectrometry (LC-MS/MS).

DNA extraction and assessment

According to the manual instructions of TianGen Biotech Co, Ltd (Beijing, China), DBS, GAS buffer and proteinase K were added into a centrifuge tube, mixed sufficiently and shaken in an oscillator at 900 rpm for 45 min. Then, the collected supernatant, magnetic bead buffer and GHC buffer were added into a new centrifuge tube, mixed and shaken in an oscillator at 900 rpm for 10 min. Finally, magnetic bead adsorption was used to capture and purify the DNA. The DNA yield was measured using an UV spectrophotometer at wavelengths of 260 and 280 nm. The presence of high molecular weight DNA and the quality of the DNA were also checked by agarose gel electrophoresis (0.8 %) [20].

Statistical analysis

All data was presented as the mean of at least three independent experiments. Differential compounds were analyzed by Wilcoxon matched pairs test, between control and different inactivated groups. DNA yield was compared among different groups by one-way ANOVA (and non-parametric). For all statistical analyses, GraphPad Prism 5.0 software was used with a significance level of p-value <0.05.

Results

Determination of viral inactivation methods

According to the query, 177 articles were retrieved from PubMed.gov. After further filtering, only 15 articles were thoroughly reviewed for the candidate inactivation methods. As shown in Table 1, common viruses could be effectively inactivated by thermal inactivation, UV irradiation and solvent/detergent (including: ethanol, propanol, etc.), and different inactivation methods were used for different viruses. Overall, the minimum condition includes: heating at 55.6 °C for 30 min, exposure to 30 % ethanol for 1 min, 30 % 1-propanol for 1 min and 60 % 2-propanol for 1 min, and irradiation with UVC 3.5 mJ/cm2. Considering that the selected methods could inactivate more viruses and could be accepted by clinical diagnostic laboratories due to low cost, easy purchase and convenient operation, three effective and common methods including: heating at 56 °C for 30 min, irradiation with UVC for 30 min, and surface wetting with 70 % ethanol were selected for further investigation.

Table 1:

The effectiveness of different inactivation methods by literature research.

Author Virus Condition Effectiveness
Kim YI et al. (2020) SARS-CoV-2 56 °C 30 min Complete inactivation
65 °C 15 min
Leclercq I et al. (2014) MERS-CoV 56 °C 25 min 99.99 % inactivation
65 °C 15 min
Jonges M (2010) Influenza virus 55.6 °C 30 min Complete inactivation
Li K et al. (2010) Hepatitis C virus 56 °C 40 min Complete inactivation
60 °C 8 min
65 °C 4 min
Xiling G et al. (2021) SARS-CoV-2 30 % ethanol 1 min Complete inactivation
40 % ethanol 0.5 min
Than TT et al. (2019) Hepatitis B virus 30 % 1-propanol 1 min Complete inactivation
60 % 2-propanol 1 min
60 % ethanol 1 min
Doerrbecker J et al. (2011) Hepatitis C virus 50 % ethanol 5 min Complete inactivation
Krilov LR et al. (1993) Respiratory syncytial virus 70 % ethanol 5 min Complete inactivation
Eterpi M et al. (2009) Adenovirus 70 % ethanol 1 min 90 % inactivation
Gidari A et al. (2021) SARS-CoV-2 UVC 10.25–23.71 mJ/cm2 99.99 % inactivation
Griffiths A et al. (2020) SARS-CoV-2 UVC 7.641 mJ/cm2 for wet virus and 4.245 mJ/cm2 for dry virus >99.99 % inactivation
Bedell et al. (2016) MERS-CoV UVC 37.2 mJ/cm2 >99.999 % inactivation
Ruetalo N et al. (2021) SARS-CoV-2 UVC 3.5 mJ/cm2 Complete inactivation
Li K et al. (2010) Hepatitis C virus UVC 54 mJ/cm2 Complete inactivation
Boogaard I et al. (2007) Respiratory syncytial virus UVC 4.05 mJ/cm2 Complete inactivation

To identify the inactivation method’s effect, the DBS with HBV(+) DNA plasma was detected HBV-DNA copy number and the DBS with HBV(+) serum was detected HBsAg. The copy number of HBV DNA in DBS (6.39 × 103 copy number/mL) was lower than in HBV(+) DNA plasma (4 × 104 copy number/mL), and the concentration of HBsAg (about 80 IU/mL) in DBS was also lower than in serum (about 650 IU/mL) (Supplementary Figure 1). Further, compared to the control, heating at 56 °C for 30 min gained 60.75 % HBV DNA load, while irradiation with UVC for 30 min lost 46.49 % HBV DNA load, surface wetting with 70 % ethanol lost 6.78 % HBV DNA load. On the other hand, HBsAg quantitative results showed that heating at 56 °C for 30 min lost 33.23 % HBsAg, irradiation with UVC for 30 min lost 57.48 % HBsAg and surface wetting with 70 % ethanol lost 99.90 % HBsAg (Supplementary Figure 1). These data showed that DBS still maintained the HBV DNA and HBsAg partly, and the three inactivation methods could decrease the copy number of HBV DNA and the concentration of HBsAg partly.

The effect of inactivation methods on hormones concentration

TSH and 17α-OH, as routine clinic screening markers, were detected before and after inactivation by fluorometric analysis. The decreased concentration was observed in the most of the samples. The concentration of TSH (Figure 1A) and 17α-OH (Figure 1B), had no significant difference between the control and the group after heating at 56 °C for 30 min only (p=0.36, p=0.52 respectively). And according to the cutoff value, heating changed the clinical predicting significance of TSH (0/15) and 17α-OH (2/15) minimally.

Figure 1: The comparison of hormones concentration among different groups. The concentration of (A) TSH and (B) 17ɑ-OH were detected by fluorometric analysis, between control group and pretreated groups with inactivation methods. Con., concentration; TSH, thyroid stimulating hormone; 17ɑ-OH, 17-hydroxyprogesterone; Ctrl, control; H, heating at 56 °C for 30 min; UV, irradiation with UVC for 30 min; Eth, surface wetting with 70 % ethanol. All p-Value were calculated by Wilcoxon matched pairs test. Red line indicates the cutoff value.
Figure 1:

The comparison of hormones concentration among different groups. The concentration of (A) TSH and (B) 17ɑ-OH were detected by fluorometric analysis, between control group and pretreated groups with inactivation methods. Con., concentration; TSH, thyroid stimulating hormone; 17ɑ-OH, 17-hydroxyprogesterone; Ctrl, control; H, heating at 56 °C for 30 min; UV, irradiation with UVC for 30 min; Eth, surface wetting with 70 % ethanol. All p-Value were calculated by Wilcoxon matched pairs test. Red line indicates the cutoff value.

The effect of inactivation methods on amino acids concentration

To investigate more metabolites, LC-MS/MS was used to detect more amino acids. Except for leucine and proline, the concentration of other amino acids was decreased after heating at 56 °C for 30 min, and all coefficients of variation (CV) for 11 amino acids were less than 15 %. However, except for alanine and ornithine, the concentration of other amino acids was increased after irradiation with UVC for 30 min, and the concentration of all amino acids was increased after wetting the surface with 70 % ethanol (Table 2 and Supplementary Table 2), the number of amino acids with CV >15 % is 3 and 5, respectively.

Table 2:

The effect of inactivation methods on amino acids detected by liquid chromatography-tandem mass spectrometry.

Index 56 °C 30 min UVA 30 min 70 % ethanol
CV, % p-Value CV, % p-Value CV, % p-Value
ALA −5.16 % 0.01 −9.73 % 0.04 11.17 %
ARG −3.20 % 10.27 % 76.76 %
CIT −5.33 % 25.44 % 67.38 %
GLY −2.91 % 0.46 % 8.75 %
LEU 0.59 % 10.60 % 16.38 % 0.04
MET −12.46 % 31.44 % 19.67 %
ORN −1.72 % −3.23 % 1.89 %
PHE −0.06 % 21.37 % 13.11 %
PRO 1.44 % 3.00 % 5.74 %
TYR −0.72 % 8.89 % 16.41 %
VAL −0.37 % 6.29 % 5.87 %

  1. CV=(the value detected after inactivation ways−the value detected after inactivation ways)/the value detected after inactivation ways. p-Value is calculated by Wilcoxon matched pairs test. CV, coefficient of variation; ALA, alanine; ARG, arginine; CIT, citrulline; GLY, glycine; LEU, leucine; MET, methionine; ORN, ornithine; PHE, phenylalanine; PRO, proline; TYR, tyrosine; VAL, valine.

The effect of inactivation methods on acylcarnitine concentration

Meanwhile, heating at 56 °C for 30 min down-regulated the most acylcarnitine concentration, both irradiation with UVC for 30 min and surface wetting with 70 % ethanol up-regulated all acylcarnitine concentrations by LC-MS/MS (Table 3 and Supplementary Table 3). CV <15 % for all acylcarnitines was observed only in the group heated at 56 °C for 30 min. Therefore, the role of heating on different lengths of acylcarnitines was further analyzed (Figure 2). As shown in Figure 2, the acylcarnitines were divided into short-chain acylcarnitine (C4–C6), medium-chain acylcarnitine (C8–C12), and long-chain acylcarnitine (C14–C18), the average CV of concentration was maximal for medium-chain acylcarnitine, and the average CV was minimal for long-chain acylcarnitine (Figure 2A). The average CV for alkenyl-carnitine was consistent with acylcarnitine (Figure 2B).

Table 3:

The role of inactivation ways on acylcarnitines detected by liquid chromatography-tandem mass spectrometry.

Index 56 °C 30 min UVA 30 min 70 % ethanol
CV, % p-Value CV, % p-Value CV, % p-Value
C0 0.39 % 6.33 % 10.68 %
C2 −1.15 % 39.79 % 35.63 % 0.04
C3 −4.66 % 41.60 % 59.27 % 0.01
C4 0.07 % 16.78 % 0.01 17.60 % <0.01
C5 −0.90 % 19.98 % <0.01 16.89 % 0.02
C6 6.23 % 0.03 14.28 % 0.01 8.81 %
C8 −6.62 % 75.59 % 39.53 %
C10 −10.11 % 19.27 % 16.29 %
C12 −6.98 % 21.79 % 45.94 %
C14 0.98 % 5.55 % 16.73 %
C16 −0.27 % 7.49 % 23.91 % 0.03
C18 −2.11 % 3.55 % 11.72 %

  1. CV=(the value detected after inactivation ways−the value detected after inactivation ways)/the value detected after inactivation ways. p-Value is calculated by Wilcoxon matched pairs test. CV, coefficient of variation.

Figure 2: The difference of CV among different length of acylcarnitine. The DBS after heating 56 °C 30 min was detected the concentration of acylcarnitine, and the CV was compared to the control. (A) The changed CV among different acylcarnitine. (B) The changed CV among different alkenyl-carnitine. (C) The changed CV among different acyl-carnitine esters derived from hydroxylated acids. CV, coefficient of variation; acyl, acylcarnitine; C4–C6 contains C4, C5 and C6; C8–C12, contains C8, C10 and C12; C14–C18, contains C14, C16 and C18; C4:1–C6:1 contains C5:1; C8:1–C12:1 contains C8:1, C10:1, C10:2, and C12:1; C14:1–C18:1 contains C14:1, C14:2, C16:1, C18:1 and C18:2; C (16–18):1OH contains C16:1OH and C18:1OH; C (14–18)OH contains C14OH, C16OH and C18OH.
Figure 2:

The difference of CV among different length of acylcarnitine. The DBS after heating 56 °C 30 min was detected the concentration of acylcarnitine, and the CV was compared to the control. (A) The changed CV among different acylcarnitine. (B) The changed CV among different alkenyl-carnitine. (C) The changed CV among different acyl-carnitine esters derived from hydroxylated acids. CV, coefficient of variation; acyl, acylcarnitine; C4–C6 contains C4, C5 and C6; C8–C12, contains C8, C10 and C12; C14–C18, contains C14, C16 and C18; C4:1–C6:1 contains C5:1; C8:1–C12:1 contains C8:1, C10:1, C10:2, and C12:1; C14:1–C18:1 contains C14:1, C14:2, C16:1, C18:1 and C18:2; C (16–18):1OH contains C16:1OH and C18:1OH; C (14–18)OH contains C14OH, C16OH and C18OH.

The effect of inactivation methods on the quality and quantity of extracted DNA

Then, the effect of the inactivation methods on DNA extraction was evaluated. The quality and quantity of DNA from DBS were compared among the groups treated with different inactivation methods. The heating, irradiation and ethanol couldn’t affect the high molecular weight DNA and degrade or break it (Figure 3B). However, heating could increase the yield of DNA, whereas irradiation and ethanol decreased the yield of DNA, compared to the control (Figure 3A and B and Supplementary Table 1).

Figure 3: The assessment of DNA quality. (A) Comparison for the yield of DNA among different groups. (B) The presentation image for DNA agarose electrophoresis. Ctrl, control; H, heating at 56 °C for 30 min; UV, irradiation with UVC for 30 min; Eth, surface wetting with 70 % ethanol.
Figure 3:

The assessment of DNA quality. (A) Comparison for the yield of DNA among different groups. (B) The presentation image for DNA agarose electrophoresis. Ctrl, control; H, heating at 56 °C for 30 min; UV, irradiation with UVC for 30 min; Eth, surface wetting with 70 % ethanol.

In conclusion, the three inactivation methods including heating at 56 °C for 30 min, irradiation with UVC for 30 min and surface wetting with 70 % ethanol slightly affected the quality and quantity of DNA; heating at 56 °C for 30 min was a better inactivation method for hormone, amino acids and acylcarnitine detection.

Discussion

Although the infection rate of bacteria is higher than that of virus, the survival duration of different bacterial species on inanimate surfaces varies significantly. According to the results of Vonberg RP et al. [21], the most common Gram-negative species other than A. baumannii (survival time >4 weeks) were inactivated in less than two days, S. aureus and E. faecium had longer survival times. In addition, Leptospira kirschneri did not survive on a drying solid surface, but did survive in diluted dog urine with a slightly alkaline or acidic environment [22]. Thus, our study only evaluated viral infection, being attributing to the relative stability of viruses on common touch inanimate surfaces [9–11, 23].

Nowadays, the screening diseases for newborn is more and more, which is resulting from exploring more sensitive and specific biomarkers. Overall, biomarkers can be divided into five types: vitamins [24, 25], hormones [26, 27], enzymes [26, 28], nucleic acids [5, 6, 29], amino acids and acylcarnitine [16, 30, 31]. Hormones, amino acids and acylcarnitine are still the most common biomarkers for routine clinical screening, especially in developing countries. Recently, nucleic acid detection has been applied to neonatal screening for the diseases such as thalassemia [5], severe combined immunodeficiency [6], and type for the diseases such as Duchenne muscular dystrophy [29]. Therefore, the effect of inactivation methods on hormones, amino acids, acylcarnitine and DNA was discussed in our study. Further studies are needed for other biomarkers.

We demonstrated that the concentration of most metabolites in DBS was decreased after heating, which might be caused by enzyme inactivity [32], amino acid degradation [33] and thermal hydrolysis [34] by heating at 56 °C for 30 min. However, the increased yield of DNA might be correlated with an increased digested amount from DBS, which was due to a more appropriate elution temperature at 56 °C. For irradiation, the literature [35, 36] suggested that UVC could damage DNA slightly, which was consistent with our results. And UVC-induced oxidative damage [37] might also be responsible for the reduced amino acid concentrations. Since ethanol was not only disinfectant but also fixative, it would reduce the digested amount of all compounds in dried blood spot, which explained the decreased concentration after ethanol treatment partly.

For enzyme, the activity of only G6PD was detected in our study (Supplementary Figure 2). Heating, trypsin, organic solvent, protease and thermolysin, 4.0 M urea could inactivate enzyme [38–40]. And the range of temperature, enzyme and chemical solvent is different for inactivating different enzyme. In the study, the data indicated that ethanol solution caused an abrupt decrease in the activity of G6PD. Although, irradiation changed the activity of G6PD slightly (p=0.20), the detective value around cutoff value was lacking. More inactivation methods and samples were needed to investigate the effect of inactivation methods on enzyme activity.

Based on CV <15 % as a quality standard, all amino acids and acylcarnitine were in control after heating at 56 °C for 30 min. For other treatments, one or more amino acids (Table 2) and acylcarnitine (Table 3) could not meet the standard. Meanwhile, heating also affected the concentration and clinical predicting significance of TSH (Figure 1A) and 17α-OH (Figure 1B) minimally. The data suggested that heating at 56 °C for 30 min might is a better treatment for detecting amino acids, acylcarnitine and hormone.

DNA was eluted from the magnetic rod using deionized water, which may have contributed to the decreased value of OD260/OD280 for the extracted DNA (Supplementary Table 1). However, the yield and quality of DNA were acceptable, with no significant difference among groups for DNA yield (p=0.26) (Supplementary Table 1) and no tailing and small fragments (size<100 bp) on agarose gel electrophoresis (Figure 3B). Given that concentration of approximately 10 ng/μL is optimal for PCR amplification [41], therefore, the DNA yield could be further improved by punching more DBS for extraction.

Conclusions

The changed concentration of metabolites in DBS after inactivation treatment did not affect the clinical predicting significance in most cases, and the CV of DNA yield and quality was acceptable for further PCR amplification and analysis. Therefore, heating at 56 °C for 30 min, irradiation with UVC for 30 min and surface wetting with 70 % ethanol could be used to pretreat the contaminated DBS, especially heating at 56 °C for 30 min.

Corresponding author: Shan Liu, Center for Clinical Molecular Medicine & Newborn Screening, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Engineering Research Center of Stem Cell Therapy, The Children’s Hospital of Chongqing Medical University, 136 Zhongshan Er Road, Yuzhong District, Chongqing 400014, P.R. China, Phone & Fax: 86-023-63621942, E-mail:

  1. Research ethics: The study was approved by the Ethics Committee of the Children’s Hospital of Chongqing Medical University, Approval Number: (2022) IRB (STUDY) NO. 536.

  2. Informed consent: Informed consent was obtained when dried blood spots collected from all individuals included in this study.

  3. Author contributions: The study was designed by Shan Liu and Chaowen Yu; experiments were performed by Ming Wang, Shi Tang, Zhihong Liao and Kexing Wan; data were analyzed and interpreted by Ming Wang and Shan Liu; manuscript was written and revised by Shan Liu.

  4. Competing interests: Authors state no conflict of interest.

  5. Research funding: None declared.

  6. Data availability: The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/labmed-2023-0099).

Received: 2023-03-27
Accepted: 2023-10-12
Published Online: 2023-11-13
Published in Print: 2023-12-15

© 2023 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

  1. Frontmatter
  2. Original Articles
  3. Proof of concept: stabilized whole blood material suitable for external quality assessment of near-patient testing devices
  4. Predictive value of combined serum IL-6 with UREA on severity of neonatal pneumonia: an observational study
  5. Performance evaluation of the automated body fluid analysis of the new Sysmex XR haematology analyser
  6. The quality and quantity of compounds affected by viral inactivation methods in dried blood spots
  7. Short Communication
  8. Second generation of soluble transferrin receptor assay – consequences for the interpretation of the ‘Thomas plot’
  9. Acknowledgment
  10. Acknowledgment
https://doi.org/10.1515/labmed-2023-0099
Keywords for this article
DNA extraction; dried blood spots; metabolites; viral inactivation methods
Creative Commons
BY 4.0

Articles in the same Issue

  1. Frontmatter
  2. Original Articles
  3. Proof of concept: stabilized whole blood material suitable for external quality assessment of near-patient testing devices
  4. Predictive value of combined serum IL-6 with UREA on severity of neonatal pneumonia: an observational study
  5. Performance evaluation of the automated body fluid analysis of the new Sysmex XR haematology analyser
  6. The quality and quantity of compounds affected by viral inactivation methods in dried blood spots
  7. Short Communication
  8. Second generation of soluble transferrin receptor assay – consequences for the interpretation of the ‘Thomas plot’
  9. Acknowledgment
  10. Acknowledgment
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