Twelve high-risk alpha human papillomavirus (HPV) genotypes cause approximately 690,000 cancer cases annually, with cervical and oropharyngeal cancer being the two most prominent types. HPV testing is performed in laboratory settings for various applications of a clinical, epidemiological, and research nature using a range of clinical specimens collected by clinicians or by individuals (self-collected specimens). Here, we reflect on the importance and justification of using the right test for the right application and provide practical updates for laboratories either participating in or anticipating involvement in HPV testing in three specimen types, namely, urine, blood, and oral specimens, which are considered “alternative” specimens by many. In addition to clinician-collected cervical samples and self-collected cervicovaginal samples, first-void urine is emerging as a credible specimen for HPV-based cervical cancer screening, triage of HPV screen-positive women, monitoring HPV vaccine impact, and HPV testing in groups for which a less invasive sample is preferred. Detection of cell-free DNA (including HPV DNA) in blood has great promise for the early detection of HPV-attributable oropharyngeal cancer (HPV-AOC) and potentially other HPV-driven cancers and as an adjunct prognostic marker in long-term tumor surveillance, including treatment response. The moderate sensitivity of HPV testing in oral rinses or swabs at HPV-AOC diagnosis prevents its use in HPV-AOC secondary prevention but represents a promising prognostic tool in HPV-AOC tertiary prevention, where the HPV persistence in oral rinses throughout treatment may predict early HPV-AOC recurrences and/or the development of secondary HPV-AOC. The increasing sophistication of specific collection devices designed for alternative samples and the enhanced precision of novel molecular technologies are likely to support the evolution of this field and catalyze potential translation into routine practice. Alpha-, Beta-, Gamma-, Mu-, and Nupapillomavirus), several species, and numerous genotypes (https://www.hpvcenter.se/human_reference_clones/)  and are as follows: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, and -59 (Human papillomaviruses (HPVs) have a remarkably stable DNA genome and are classified by the homology of their genome into five genera  . The 12 Most hrHPV infections follow a subclinical course, but some persistent infections are etiologically linked with benign and malignant lesions of the epithelia , 5. HumaIn this review, we reflect on the importance of “tailoring” the HPV test to the application and discuss the following three key and expanding areas in the laboratory detection of HPV: testing of urine, testing of blood, and testing of oral samples. Implications of this expansion for the detection and management of the two most common hrHPV driven cancers, namely, cervical and oropharyngeal, are our focus.Cervical cancer, with 570,000 new annual cases worldwide, is the hrHPV-driven cancer associated with the greatest morbidity and mortality . Because7–Oropharyngeal cancer is the second most common hrHPV-related human cancer, which is traditionally associated with chronic exposure to tobacco, heavy alcohol consumption, and deprivation . HoweverWhile HPV testing is relevant for population-based screening of the healthy population, increasing evidence suggests it may be relevant for individual prognostication in the patient population . To servIn routine clinical laboratory settings, testing for hrHPV is used most frequently as a screening tool in HPV-based primary cervical cancer screening programs . For thiCompared with HPV screening tests, HPV assays required for epidemiology, including vaccine impact monitoring, have different characteristics with respect to analytical performance, HPV genotype range, and the biospecimen applied , 17, 18.in situ hybridization for viral sequences or (more commonly in routine settings) immunohistochemistry for an HPV-associated biomarker (p16INK4a)  . While it of PCR , 22. Nott of PCR –23.In line with this information, when it comes to HPV testing in blood, where virus quantities may be low and labile, highly sensitive PCR-based and alternative amplification approaches confer dividends on performance , 25. DigOral rinse and gargles using sterile saline or alcohol-based mouthwash are used most frequently for sampling the oral cavity or oropharynx for epidemiological and research purposes . Again, As mentioned above, this review summarizes recent evidence and provides updates for the laboratories already participating or anticipating involvement in testing for HPV using the following three specimen types: urine, blood, and oral specimens. The three specimen types targeted by this review are considered “nonstandardized,” “alternative,” or “extragenital” by many; this language is not accepted uniformly, and the main dispute is whether urine is an “extragenital” or “genital” specimen type.To improve equitable access to screening, women in many cervical cancer screening programs or projects are now given the option of providing a self-collected sample –30. Self–Our understanding regarding the use of urine as a valid specimen for testing for HPV has evolved substantially in the last decade . In the When different types of urine specimens were assessed from a cohort of HPV-positive women, substantial differences were observed in the reported copy number of HPV and human DNA when comparing first-void  urine versus the subsequent fractions . It is nThe type of urine specimen or the choice of void fraction used for HPV testing does have a profound impact on the amount of HPV DNA present in the sample and, in turn, the probability of accurate HPV detection. The superiority of first-void urine for HPV testing was confirmed in the first meta-analysis on the subject, published in 2014, but without understanding the underlying reasons and mechanism . SurprisAnother important observation described in pilot experiments is the relative instability of HPV DNA in certain urine samples and the requirement for a preservative in the sample collection/transport media to enhance stability and minimize degradation. Even with well-established DNA extraction protocols, which have been found to garner a high yield and quality of HPV DNA in more cellular samples, significant reductions of HPV DNA in urine specimens have been observed in the absence of a preservative applied immediately after collection of first-void urine . This fivalidation of human papillomavirus assays and collection devices for HPV testing on self-samples (VALHUDES) diagnostic test accuracy study follows a design where the clinical performance of validated PCR-based HPV DNA assays on optimally collected and preserved first-void urine samples is compared with clinician-collected cervical samples, with the latter considered standard of care  self-collected urine samples for HPV testing . In thesAs mentioned, a first-void urine specimen contains female genital secretions, including those harboring locally produced immunoglobulins or immunoglobulins transferred from blood by transudation or exudation . PerhapsIn contrast to women, for whom a first-void urine specimen is a credible alternative to both clinician-collected and self-collected specimens, urine specimens collected from males (including first-void urine) are less appropriate and accurate for the detection of anogenital HPV infections because the anogential secretions “collected” by urination differ substantially due to anatomical differences . ConsequThere are limited data on HPV prevalence, the natural history of HPV-related tumors, and screening options in transgender individuals due to the low acceptance rate of traditional sample collection approaches. A recent pilot study on 200 transgender individuals showed a high acceptance rate (98.5%) of first-void urine self-collected using the Colli-Pee FV-5000 collection device for HPV testing, providing a significant opportunity for natural history, epidemiological, and screening studies in this hard-to-reach population .Blood as a specimen has attracted significant recent attention for the detection and management of HPV-associated disease because it can contain detectable biofragments that may be indicative of underlying or recurrent neoplastic disease , 55. SucINK4a and the HPV PCR status of the pretreatment (solid) biopsy samples in oropharyngeal cancer patients. The detection of cHPV DNA has also been shown to be indicative of disease recurrence posttreatment. In a study of over 100 HPV-AOC patients treated with curative intent, of the 87 patients that had undetectable cHPV DNA, none had recurrent disease during follow-up (median 23 months), whereas the sequential detection of cHPV in two posttreatment samples exhibited a positive predictive value of 94% , and host and viral methylation targets, including CADM1 and EPB41L3 as recently reviewed by Herbst et al. . Most stThere are a smaller but growing number of studies on the use of liquid biopsy for detecting anal cancer; in a study of 57 patients with advanced anal cancer, HPV ctDNA was detected in 91.1% when an HPV-16 genotype-specific HPV assay was employed . In a moCirculating DNA can be single or double stranded and is generally secreted into the blood at an early stage of cancer development through mechanisms that include apoptosis or tumor necrosis  Fig. 2)Fig. 2). After nucleic acid extraction from plasma, the extract may be frozen at −20°C prior to testing, but again, the impact of any longer-term storage on target deterioration should be quantified. The technology is likely to be supported by the increase in the availability of commercial kits, which have been optimized to extract circulating nucleic acid of high quality.Diagnostic targets in liquid biopsies vary and reflect the virus, host, or both; with regard to the virus, the focus has often been only on HPV-16 and/or HPV-18, although there are examples of broader HPV panels . MethylaNGS can detect insertions, deletions, somatic single nucleotide polymorphisms, and gene copy number variations, and it has been applied in an exploratory manner to define biomarkers of interest in cancer patients and, in turn, to create cancer-target “panels,” including for cervical cancer patients . NGS alsMolecular developments in areas such as NGS and ddPCR will clearly help refine the technology and will support quality and consistency. The update of digital minimum information for publication of quantitative digital PCR experiments (dMIQE) is helpful in this regard . AlthougIn the last decade, several investigators have shown that anti-HPV-16 E6 antibody presence in blood may serve as a potential screening, diagnostic, and prognostic marker of HPV-AOC . A pioneAs discussed, strong evidence exists to show that HPV-AOC is a distinct disease and is markedly different from oropharyngeal cancer etiologically associated with tobacco and alcohol at three levels, as follows: epidemiological, clinical, and molecular .INK4a, a tumor suppressor which is upregulated by high-level expression of E7, is critical for cell survival in all HPV-related tumors, whereas it is frequently inactivated in HPV-independent tumors. Therefore, p16INK4a overexpression is a surrogate marker for transcriptionally active HPV infection. In addition to the presence of HPV oncoviral proteins, the most common genetic changes in HPV-AOC are in the phosphoinositide-3-kinase (PI3K) pathway, particularly involving activating mutations and amplifications of the PIK3CA oncogene  between anogenital and oral sites with conflicting results but generally showing low concordance. Also, intraperson hrHPV genotype concordance in males has been reported to be low, although it is higher in males who have sex with males than that in males who have sex with females (only) . SimilarThe key knowledge gaps in the natural history of HPV-AOC that hinder the implementation of preventive measures similar to those used for decades for cervical cancer lie around the existence (or not) of HPV-related preneoplastic lesions that precede HPV-AOC and, if such lesions exist, whether they can be identified early enough to be treated in a safe, effective, and acceptable way to prevent the development of HPV-AOC, as in the case of cervical cancer .Oral rinse and gargle using sterile saline or alcohol-based mouthwash are used most frequently for sampling the oral cavity or oropharynx for epidemiological, research, and clinical purposes , 78. TheAs an alternative to the rinse/gargle biospecimen, if oral lesions are visible, the mucosal surface has been sampled using a swab or brush. Saliva is not considered a robust specimen for HPV testing nor are specimens obtained by swabbing or brushing tonsils or the oropharynx without visible lesions , 78.Relatively few studies have directly compared different sampling approaches in the oral cavity. One study compared oral gargles versus tonsil brushings in a sizeable age-stratified sample of oral/oropharyngeal cancer-free individuals and found that HPV detection was rare when using tonsil brushings from both children and adults . In cont10 PCR-DEIA-LiPA25 system  with an overall HPV positivity of 1.9% versus 8.6%, respectively (10 PCR-DEIA-LiPA25 (65 bp) than that of the Linear Array HPV genotyping test (approximately 450 bp). This difference in analytical performance becomes important in samples with very low viral load  or in samples with partially degraded DNA.In addition to obtaining a robust oral specimen, the choice of the appropriate test for detecting and genotyping HPV in oral samples is clearly important. Because HPV viral load in oral samples is significantly lower than that in genital sites, testing oral specimens requires the use of HPV tests with substantially higher analytical sensitivity . In the ectively . The obsPrimary prevention by HPV vaccination might work equally well in the case of HPV-AOC as for anogenital HPV-related carcinomas but with a potential caveat. Namely, although the US FDA recently also conditionally approved an expanded indication of HPV vaccine for primary prevention of oropharyngeal and other head and neck cancers caused by targeted HPV genotypes, the final vaccine approval for this specific indication is dependent on the results of an ongoing postapproval confirmatory study .As described above, no validated screening strategy for the secondary prevention of HPV-AOC to support early disease detection in the healthy population is routinely available at present . In contINK4a using immunohistochemistry and the presence of HPV DNA or HPV mRNA by either in situ hybridization or PCR-based methods . Detection of cell-free DNA (including HPV DNA) in blood samples is slowly transitioning into routine practice, holding great promise for various applications, including the early detection of HPV-attributable oropharyngeal cancer (HPV-AOC) and potentially other anogenital cancers, as well as an adjunct prognostic marker in long-term tumor surveillance. Anti-HPV-16 E6 antibody presence in blood may serve as a potential screening, diagnostic, and prognostic marker of HPV-AOC, but greater research in this area is warranted. The moderate sensitivity of HPV testing in oral rinses or swabs at HPV-AOC diagnosis prevents its use as a screening or diagnostic tool, but HPV testing in oral rinses represents a promising prognostic tool in the tertiary prevention of HPV-AOC, where persistence in oral rinses through and after treatment may predict early HPV-AOC recurrences and/or the development of secondary HPV-AOC. In addition, HPV testing of oral rinses will continue to be important for natural history and epidemiological studies.In conclusion, three specimen types traditionally considered “alternative” for HPV testing  are shown to have an increasing clinical, epidemiological, and scientific value . The HPV


In North America and in most European countries, Human Papillomavirus (HPV) is responsible for over 70% of oropharyngeal squamous cell carcinomas. The burden of OPSCC, in high-income countries, has been steadily increasing over the past 20 years. As a result, in the USA and in the UK, the burden of HPV-related oropharyngeal squamous cell carcinoma in men has now surpassed that of cervical cancer in women. However, the oncogenic impact of high-risk HPV integration in oropharyngeal squamous cell carcinomas hasn’t been extensively studied. The present study aimed to explore the patterns of HPV integration in oropharyngeal squamous cell carcinomas and to assess the feasibility and reliability of long-read sequencing technology in detecting viral integration events in oropharyngeal head and neck cancers.INK4A positive OPSCC diagnosis and were treated at the McGill University Health Centre, a quaternary center in Montreal. A minimum of 20mg of tumor tissue was used for DNA extraction. Extracted DNA was subjected to Nanopore long-read sequencing to detect and analyze for the presence of high-risk HPV sequences. PCR and Sanger sequencing experiments were performed to confirm Nanopore long-read sequencing readings.A cohort of eight HPV-positive OPSCC pre-treatment patient tumors , were selected. All patients received a p16Nanopore long-read sequencing showed that seven out of eight patient samples displayed either integrated or episomal high-risk HPV sequences. Out of these seven samples, four displayed verifiable integration events upon bioinformatic analysis. Integration confirmation experiments were designed for all four samples using PCR-based methods. Sanger sequencing was also performed. Four distinct HPV integration patterns were identified: concatemer chromosomal integration in a single chromosome, bi-chromosomal concatemer integration, single chromosome complete integration and bi-chromosomal complete integration. HPV concatemer integration also proved more common than full HPV integration events.Long-read sequencing technologies can be effectively used to assess HPV integration patterns in OPSCC tumors. Clinically, more research should be conducted on the prognostication value of high-risk HPV integration in OPSCC tumors using long-read sequencing technology. Oropharyngeal Squamous Cell Carcinoma (OPSCC) is a subtype of Head and Neck Squamous Cell Carcinoma (HNSCC) that typically arises from 4 main subsites: base of tongue (BOT), tonsils, soft palate, and posterior pharyngeal wall . In NortHPV is the most common sexually transmitted infection in the World . As of tVarious studies have shown that the E2 gene is more commonly found in its episomal form. It is most active, early in the infectious process, as it represses highly carcinogenic E6 and E7 genes , 15. HowINK4A positivity (IHC) into the staging of OPSCC tumors , have shown that viral integration of hrHPV strains, namely HPV-16 and HPV-18, was associated with worse overall survival in HPV-positive OPSCC patients –26. SimiINK4A overexpression, a surrogate marker for HPV positivity   . All subjects provided informed consent to participate in this study. All 8 OPSCC samples were collected from the McGill University Health Centre HNSCC biobank. All tumor tissues were obtained at the time of diagnosis prior to treatment initiation. Tumor samples were collected in RPMI media, supplemented with 10% FBS, and stored at -80 °C. Considerations for sample selection included sex, OPSCC subsite and availability of sufficient tumor tissue (>20mg). Clinical information was extracted from the MUHC’s hospital electronic records. HPV status was established using immunohistochemistry (IHC) to assess for p16Genomic DNA was extracted from 20 to 60mg of the tumor tissues using Nanobind® tissue kit (PacBio). The extracted DNA was processed through short read elimination kit . DNA concentration for each sample was measured using Nanodrop 3300 (Thermo Fisher Scientific). Sequencing libraries were constructed using Oxford Nanopore Technologies’ (ONT) ligation sequencing kit (SQK-LSK109). Prepared libraries were subsequently sequenced using the PromethION platform and 1D flow cell, containing protein pore R.9.4.1. Nanopore sequencing results were processed using ONT’s Guppy software (v6.2.7)  into nucleic acid sequences (in fastq format). To generate 10-fold coverage, two libraries were run sequentially on each 1D flowcell platform. PycoQC (v.2.5.2) was used to compute the metrics and perform quality control of the ONT sequencing data.To confirm the presence of HPV-16 sequences, raw nanopore reads, in fasta format, were cross-referenced with the HPV-16 genome (NCBI: GCF_000863945.3) using blat. Upon confirmation of HPV-16 presence, minimap2 (v2.24) was used to align fastq-pass nanopore reads on the human GRCh38 reference genome. Sambamba (v.0.8.1) was then used to convert.sam alignment files to.bam format and to create the.bam index file. Long-read structural variant caller, svim (v.2.0.0), was used to detect all insertions on the human reference genome (GRCh38) found using the minimap2 alignment.bam files previously generated. Detected insertion sequences were then processed using blastn megablast (v.2.10.0+) to solely identify HPV-16 positive insertion reads. The Integrative Genomics Viewer (IGV) was used to load alignment.bam files and ascertain the location of insertion events within the GRCh38 reference genome. As we employed short read elimination kit to enrich our DNA samples for long-length sequences, we applied 1 kb cut off while analysing the long-read sequencing data. Similar methodology was applied to identify HPV-18 positive insertion reads amongst all 8 patient samples and to identify any of the most common 200 HPV subtypes for patient HN0138 (PaVE database).Two sets of primers (forward and reverse) were ordered for each of the following 5 HPV genes: E2, E6, E7, L1, L2  for 1 hour at 37°C. 10ng of the digestion products and appropriate controls were then PCR-amplified using the following primer pairs: E6.2, and GAPDH. To verify the validity of the exonuclease reaction, an in-house plasmid pR26-HPV containing the HPV E6 and E7 genes was linearized using Kpn1  and Xho1  restriction enzymes. The 50ng of linearized and circular plasmids were then digested with Exonuclease V  for 1 hour at 37°C. 10ng of these digested plasmids were then used as a template for PCR amplification with primer pairs E6.2 and E7.2 , 34.5S, NEB fPCR products were assessed using 2% agarose gel electrophoresis. PCR products were extracted using Monarch® DNA Gel Extraction Kit . Purified products were subsequently sequenced using the Sanger sequencing platform. Results from Sanger sequencing data were then compared with ONT’s long-read sequencing data.All eight selected samples were p16-positive oropharyngeal cancers. Four tumor samples belonged to female patients and four belonged to male patients. Subsites included tonsils (4), base of tongue (BOT) (3) and soft palate (1). Patients were between 52 and 72 years of age at the time of diagnosis. Average and median age at diagnosis were respectively 61 and 62 years of age. Five out of eight patients had a significant smoking history (>10 pack-year). Three patients presented with stage I, four presented with stage 2 and one presented with stage 3 HPV-related oropharyngeal cancer. Follow-up durations varied between 6 to 60 months. As of today, none of these patients are deceased Table 1AUpon DNA extraction, samples were submitted for quality testing. All eight samples proved sufficient quantitatively and qualitatively. Table 1BWe designed two sets of reverse and forward primers for the following HPV genes: E2, E6, E7, L1, L2. Similar findings were made across both sets of primers for all five HPV genes. . These findings demonstrated heterogeneous integration patterns at diverse genomic breakpoints  . HoweverINK4A (IHC) as a surrogate marker for HPV positivity (in situ hybridization (ISH), which allows the detection and localization of the viral genome within the cell and qRT-PCR of E6/E7 mRNA transcripts are available, but they are laborious and not feasible in clinical routine laboratories (INK4A (IHC) is routinely used to diagnose HPV even though there is 20 -30% chance of misdiagnosis. These HPV-related OPSCC misdiagnoses sometimes lead to unwarranted treatment de-escalation and result in poorer patient outcomes  displayed hrHPV integration  in HPV-positive OPSCC. In 2017, egration . Overallegration . FurtherC cohort . By demoC cohort .INK4A surrogate marker positivity. Long-read sequencing identified HPV sequences in seven of the eight samples (88%). Similar findings were found across PCR and exonuclease confirmation experiments. Hence, HN0138 was deemed a potential false positive HPV-positive OPSCC diagnosis. However, lack of DNA coverage in a patient presenting with a low viral load cannot be excluded. Presence of HPV-16, either episomal or integrated, was nonetheless confirmed in all seven remaining patient samples , while others extended across multiple chromosomes . Complete HPV integration presented in only one patient (HN0211) as either two HPV concatemers divided amongst two chromosomes (HN0211B1 & HN0211B2) or as a single integration event on a single chromosome (HN0211E). Further studies are warranted to establish whether bi-chromosomal interactions, within a single tumor, are the results of translocation events post-HPV integration or simply the result of linearized concatemers integrating at fragilized chromosomal sites following DDR dysregulation by HPV.In this study, integration events, identified using Nanopore, presented mostly as HPV concatemers. However, these concatemeric events presented in very different ways. Tumors HN0026 and HN0094 presented with single chromosomal integration at intergenic breakpoints, while tumors HN0072 and HN0211 presented with bi-chromosomal integrations at mostly intronic breakpoints. Such findings suggest that there are potentially various ways in which hrHPV strains interact with the human genome at the basal epidermal layer to induce carcinogenesis in HPV-positive OPSCC.Our study looked into 8 HPV-positive OPSCC patient tumors from a single quaternary center in Montreal. Most of our patient samples were relatively recent diagnoses (<24 months). Hence, prognostication and outcome studies could not be performed. Furthermore, more patient samples would be needed, in the future, to truly establish prognostic and outcome values based on patterns of HPV-integration in OPSCCs. Finally, while our DNA coverage is within acceptable limits, access to higher DNA coverage could have further strengthen our data.Identification of HPV integration patterns in OPSCC, using long-read sequencing technology, hasn’t been extensively studied. This study helps confirm that long-read sequencing technology, namely Nanopore, can be effectively and reliably used to assess HPV integration patterns in OPSCC tumors. Four distinct HPV integration patterns were identified: concatemer chromosomal integration in a single chromosome, bi-chromosomal concatemer integration, single chromosome complete integration and bi-chromosomal complete integration. HPV concatemer integration  also proThe datasets presented in this article are not readily available because of patient confidentiality and privacy. Requests to access the datasets should be directed to corresponding author SL.The studies involving humans were approved by Research Ethics Board of McGill University Health Centre . The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.SL: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing. MG: Data curation, Formal Analysis, Methodology, Writing – original draft, Writing – review & editing. AK: Data curation, Investigation, Methodology, Writing – review & editing. GL: Formal Analysis, Software, Writing – review & editing. NG: Resources, Writing – review & editing. AZ: Resources, Writing – review & editing. KR: Resources, Writing – review & editing. MM: Resources, Writing – review & editing. NS: Funding acquisition, Resources, Supervision, Writing – review & editing.


Laryngeal cancer most frequently develops in males aged 60-70 years with a history of tobacco and/or alcohol use, while fewer cases occur in young patients in which tobacco and alcohol are often absent or less significant, highlighting the importance of other etiologies. We present cases of human papillomavirus (HPV)-associated laryngeal cancer in two previously healthy young women. A retrospective case review was carried out for both patients. DNA was extracted from the primary tumors and matched to normal tissue or blood, HPV genotype was determined by PCR and whole exome sequencing was carried out. Genomic results were pooled with laryngeal cancer patients from the cancer genome atlas (TCGA) dataset. The first patient was an 18-year-old female who underwent laryngectomy followed by adjuvant radiation. The second was a 24-year-old female who received chemoradiation. The first patient has remained disease-free for 16 years and the second for two years; both continue to be monitored. One tumor was positive for HPV45 and had mutations in FAT1 and FAT2; the other was positive for HPV31 and had mutations at NOTCH1, MAPK1, and HIST1H2AK. Both tumors had wild-type TP53 alleles. We bring attention to HPV as an etiology of laryngeal carcinoma in young patients, which may have implications for the treatment and prognosis of similar patients. Laryngeal squamous cell carcinoma (LSCC) is one of the most common head and neck cancers, with 184,404 new cases and 88,840 deaths in 2020 worldwide . FurtherHPV has been associated with an increased incidence of all head and neck cancers and is more frequently associated with oropharyngeal squamous cell cancers (OPSCC) . PatientCase 1An 18-year-old female presented with a six-year history of hoarseness and a one-year history of sore throat, difficulty swallowing, and weight loss. She had no other comorbid medical conditions, was a non-drinker, and non-smoker, and had no significant exposure to second-hand smoke. She had no history of acid reflux, no previous HPV diagnosis, no history of high-risk sexual behavior, no previous pap tests were completed, and she had not received a previous HPV vaccine. Although her grandmother had a history of thyroid cancer, there were no other instances of head and neck cancers in the family.Computed tomographic (CT) scan of the head and neck showed a 5.0 cm mass arising below the hyoid bone and extending to the isthmus of the thyroid and the anterior vocal cords with destruction of the thyroid cartilage Figure . There wThe patient underwent a total laryngectomy, bilateral neck dissection, and total thyroidectomy. Pathology described a 1.5 x 1.5 x 1.0 cm invasive squamous cell carcinoma of the right vocal cord that involved the soft tissue anterior to the thyroid and the thyroid capsule, the submucosal layer of the right and left subglottic and supraglottic regions, and suprahyoid soft tissues. There were three positive lymph nodes in the right neck, with the largest measuring 2.0 cm with no extranodal extension. The final pathological stage was IVA (pT4N2bM0) according to the AJCC 8th edition (AJCCV8) staging system.2 IV) given every three weeks with the third cycle omitted due to intolerable nausea and vomiting. Overall, the patient recovered well from surgery without complications and tolerated radiation therapy with only a modest weight loss. The patient has been on surveillance without recurrence for the past 16 years.The patient received postoperative chemoradiation. Radiation therapy consisted of 6600 cGy in 31 fractions using intensity-modulated radiation therapy with three cycles of concurrent cisplatin chemotherapy (100mg/mMolecular testing including genomic sequencing and HPV subtyping (see Appendix for details on methodology). Samples obtained from this patient were positive for HPV45 and confirmed by Sanger sequencing. A total of 312 mutations were identified in the tumor’s whole exome sequences when compared to the patient’s control DNA, including mutations in the FAT1 and FAT2 genes, and the tumor was wild type for TP53.Case 2A 24-year-old female presented with a one-year history of persistent and progressive hoarseness. She had a history of anxiety, depression, tonsillectomy in childhood, and tubal ligation at the age of 21. She had no previous HPV diagnosis, no history of high-risk sexual behavior, no previous pap tests were completed, and she had not received a previous HPV vaccine. There was no known family history of cancer, although she had a 10-pack-year smoking history.The patient underwent nasopharyngoscopy which revealed a lesion with an epicenter of the left posterior half of the true vocal fold extending to the anterior commissure Figure . No subg2 IV) given every three weeks concurrent with external beam radiotherapy 70 Gy in 35 fractions. The patient has experienced no recurrences in the three years after treatment and continues to do well.A panendoscopy and biopsy of the lesion on the left vocal revealed p16-positive squamous cell cancer Figures , 4B. PETMolecular testing including genomic sequencing and HPV subtyping was done (details on Methods can be found in appendices). Samples obtained from this patient were positive for HPV31 by PCR and confirmed by Sanger sequencing and alignment to HPV31 genome sequences in Genbank. A total of 95 mutations were identified in the tumor when compared to the patient’s control DNA, including mutations in the following genes: NOTCH1, MAPK1 (ERK2), and HIST1H2AK, but was wild-type for TP53.Statistical analysesWe have previously examined the LSCC cohort of the TCGA . After rHPV is recognized as an important etiological factor in OPSCC, and HPV-associated OPSCC has a more favorable response to chemoradiation that is now recognized with a separate staging system for these cancers in the 8th edition of the American Joint Committee on Cancer (AJCC) staging system ,10,11. FAlthough HPV status is widely recognized as an etiology and prognostic factor for OPSCC, the same is not clearly recognized for LSCC. A meta-analysis conducted by Torrente et al. showed that approximately 25% of laryngeal cancers may be HPV-related . It seemIt is well recognized that different HPV strains pose different risks for developing malignancy. Although HPV16 is the most common subtype of HPV associated with head and neck cancers, the cases presented in this case report highlight the role of other HPV subtypes in laryngeal carcinoma. Ndiaye et al. conducted a meta-analysis on HPV-associated laryngeal carcinomas and found that HPV16 was the most common subtype associated with these carcinomas worldwide followed by HPV18 and HPV6; however, stratification by age was not reported . InteresIn addition to tumor HPV status being discussed as a prognostic factor in head and neck cancers, the role of the specific HPV subtype on prognosis in HNC has also been explored in the literature to only a limited degree, likely because HPV types may not be routinely collected in pathology testing. There currently is no consensus on the impact of subtypes on prognosis. Bratman et al. found that patients with tumors associated with HPV16 had a better prognosis than those infected with other subtypes, with patients with HPV16-associated tumors having a three-year survival rate of 88% vs. 49% in those associated with other HPV subtypes (p=0.003) . ConversWhile the mutational burden of HPV-negative and HPV-positive head and neck squamous cell carcinomas (HNSCC) are similar, the genomics of the two groups differ in mutational makeup , which iIn addition to ingested carcinogens, the risk of developing laryngeal carcinoma is higher in males and with advancing age. HPV-related head and neck cancers tend to be more common in men than in women; however, it is thought that gender differences in lifestyle and sexual behavior alone are not sufficient to explain the difference in the prevalence of HPV-related head and neck cancers between sexes . It is hAs with other cancers, the risk increases with age, likely due to the accumulation of mutations over time. However, in HPV-associated cervical cancer, it was found that age alone was not an important factor, but rather, the duration of HPV infection and HPV subtype influenced the risk of progressing to cancer . These dThis case report highlights the importance of considering HPV as an etiological factor in laryngeal carcinoma in young patients and emphasizes the importance of considering relatively rare HPV subtypes. The HPV subtypes implicated in both of our cases are included in newer HPV vaccinations, and therefore vaccination at an early age is likely beneficial in preventing HPV-associated laryngeal carcinomas. It is important to consider routinely testing all laryngeal carcinomas for HPV status and subtype, particularly in patients who are younger or do not have a history of carcinogen exposure, to further knowledge about the biology of this disease. Further work assessing tumor HPV status and subtype as a prognostic factor and potential factor in staging is warranted.


An imaginary companion is an invisible or personified entity created by children for themselves. An imaginary companion typically serves as a companion to the child and plays a significant role in their life, especially for only children who may experience more loneliness compared to other children. This research was conducted to investigate the role of an imaginary companion in the lives of only children.The present study was conducted using a qualitative method and a content analysis approach. Through purposeful sampling, a total of 34 preschool and primary school children, aged 6 to 9 years, from schools in Mashhad city, were selected until saturation was reached. They were subjected to semi-structured interviews. After data collection, the data were coded, and then the main and sub-themes were extracted.The research findings were represented in the form of 196 statements, 28 sub-themes, and 9 main themes. The main themes included the role of an imaginary companion in alleviating loneliness, the role of an imaginary companion in amusement, the role of an imaginary companion in emotional regulation, conversations with an imaginary companion, guidance from an imaginary companion for good and bad behaviors, the assistance of an imaginary companion in tasks, helping to generate new scenarios, the advantages of having an imaginary companion, and the disadvantages of having one.Based on the aforementioned findings, it can be concluded that the presence of an imaginary companion can not only support children but also promote creativity and distance them from the virtual space and realities of the real world. Parental awareness of this matter can aid in the child’s growth, fulfill their needs, and, on the other hand, prevent potential harm to children. Significant changes in the form and characteristics of families in Iran are evident . The decResearchers believe that only children experience loneliness and maladjustment due to not having siblings . HoweverBouldin and Pratt  define aAccording to Vygotsky, cognitive development always results from the interaction of humans with each other, especially the interaction of an adult with a child . AccordiPrevious research has reported the prevalence of imaginary companions among children to be as high as 65%, although most studies indicate that between 20 and 35% of children have imaginary companions . In one According to many studies, from single-child families to multi-child individuals, single-friend children experience loneliness, lack of childhood playmates and lack of siblings, and lack of support in youth and growing up .Majors & Baines  believedIn the second half of the 20th century, the world experienced a demographic change. Fertility increased from five children per woman to 2.7 children worldwide . HoweverResearchers have examined and identified several roles that imaginary companions may play in children’s lives , 26. As Studies have shown that having an imaginary companion in childhood has cognitive, social, and emotional benefits . These sConsidering the increase of single-child families in Iran and also the importance of siblings in the growth and development of children, examining the characteristics of single-child children is of particular importance. In Iran, there has not been a valid and organized scientific research with the theme of imaginary companions, and due to the importance of this issue in the lives of single children, this research was done.This research was conducted with the aim of investigating the role of imaginary companions in the lives of single children. In this regard, this research seeks to answer the following questions:What is the role of imaginary companions in the lives of single children?What are the advantages and disadvantages of imaginary companions for only children?The present study was qualitative research. The method used in this research was qualitative content analysis. The research was conducted in a community of 6 to 9-year-old only children with imaginary companions in Mashhad, Iran. The age range of 6 to 9 years was selected based on the findings of Pearson et al.  for thisThe procedure involved obtaining permission from the Ministry of Education and obtaining an ethics code. A participant was selected from the selected classes in schools, and after obtaining parental consent and ensuring the confidentiality of the interviews, the interviews were conducted. The interviews began with the question of whether they have a friend who can only be seen by them and with whom they interact. If the answer was positive (approximately 30 to 40% of the single-child students in each class had an imaginary friend), and their brief explanation matched the correct definition of an imaginary friend, the interview continued with further questions. The interviews with the children were recorded, and each interview lasted approximately twenty minutes to half an hour.To ensure the accuracy and validity of the research data, the interviews were recorded and documented accurately. The entire process of conducting the research was described in detail. The results of the analysis were compared with the raw data to examine the consistency of the findings with the data. Additionally, the obtained results from data analysis were discussed and reviewed with experts to ensure validity and reliability.After the interviews were meticulously coded, both primary and sub-themes were extracted. These steps were conducted in accordance with the guidelines provided by Prasad .The average age of the research participants was 7.5 years. Out of the participants, 18 were girls and 16 were boys. Additionally, 22% of the fathers and 37% of the mothers of the only children had education levels higher than a bachelor’s degree.This study aimed to investigate the role of imaginary companions in the lives of only children. The findings of the research were represented in the form of 196 statements, 28 sub-themes, and 9 main themes. According to the findings, the main roles of imaginary companions included: alleviating loneliness, engaging in play with the imaginary companion, the role of the imaginary companion in emotional regulation, conversing with the imaginary companion, guidance of the imaginary companion in distinguishing between good and bad behaviors, assisting the imaginary companion, and aiding in the absorption of new narratives. Additionally, the advantages and disadvantages of having an imaginary companion were also identified. The main themes will be further explained and described using the sub-themes . Cartoons, games, and dreams contain new and captivating characters that children strive to discover and imitate. Participant 19, a six-year-old boy with working parents, also mentioned, “One night I was asleep, and a baby chick came into my dream and talked to me. Since then, I became friends with it, and we play together. I’m not alone anymore.“The first sub-theme was kindness exhibited by the imaginary companion. The children expressed this by using phrases such as being kind, not bullying, not complaining, and not fighting. For example, participant 16, a six-year-old girl with working parents, stated, “It’s always kind to me. My imaginary friend never bullies me and treats me with kindness.“ The second sub-theme in this section was the reduction in TV and tablet usage. Having an imaginary companion fills the child’s solitude and time, leading them to spend less time on virtual platforms and watching television, or at least reducing their usage. Participant 12, a nine-year-old girl with working parents, mentioned, “It doesn’t hurt, it just makes me watch less TV and play less on the tablet. Even my mom doesn’t know about this friend, and she says I’ve been using the tablet less lately.“ The third sub-theme was controlling the imaginary companion. The children described this by writing the life story of their constructive imaginary friend and being in control of them. Participant 33, a seven-year-old boy with working parents, said, “They don’t bother me because I control them, and I write their story.“ Participant 23, also a nine-year-old boy with working parents, admitted, “I made them myself, so I have control over them. If they bother me, I put them in my toy basket and keep them locked up.“Sometimes, the imaginary companion can cause fear and anxiety in children. For example, participant 9, an eight-year-old girl with a stay-at-home mother, mentioned, “Some of them are very bothersome. They scare me at night with snakes and spiders.“ The second sub-theme was creating a disruptive attachment, expressed through phrases like worrying about the imaginary companion not coming back and feeling down due to the absence of the imaginary companion. Participant 13, a nine-year-old girl with working parents, said, “I get worried when I don’t see them. I keep thinking, what if they never come back? If they don’t come back, where will I find them?“ Participant 27, a nine-year-old boy with a stay-at-home mother, also stated, “I was bothered and depressed when they didn’t come.“ The last sub-theme in this section was the control exerted by the imaginary companion. When the imaginary companion tries to take control of the child’s normative group, it causes distress for the child. As participant 31, an eight-year-old boy with a stay-at-home mother, expressed, “It nags me and keeps telling me to do this and not do that, and it messes with my nerves.“ Participant 29, a nine-year-old boy with working parents, also mentioned, “Sometimes I get upset with it because it tells me what to do and what not to do.“ It’s important to note that while imaginary companions can bring many benefits, including fostering creativity and companionship, these experiences may vary for each child.As mentioned, an imaginary companion plays an important role in the lives of only children, and the research aimed to answer the question: What is the role of an imaginary companion in the lives of only children? The findings indicated that the main roles of an imaginary companion include alleviating loneliness, engaging in play with the imaginary companion, the role of the imaginary companion in emotional regulation, conversing with the imaginary companion, guidance from the imaginary companion regarding good and bad behavior, and assistance in assimilating new scripts. These results align with the findings of Mills , who empRegarding the number of close friends of children with imaginary companions, the findings align with Taylor and colleagues , who sugAdditionally, the findings are consistent with the research of Gleason and colleagues , Majors On the other hand, an imaginary companion can serve as a guide for both good and bad behavior, in line with the findings of Singer and Singer , who higThe latest role expressed by only children in this study regarding imaginary companions was that imaginary companions help attract new scenarios, which is consistent with the findings of Bouldin and Pratt . They stConsidering the findings, it can be said that some children feel lonely due to the absence of siblings, and friends, or the insufficient presence of their mothers.The second question of the study was about the advantages and disadvantages of having an imaginary companion for only children. The findings showed that the advantages of an imaginary companion include their kindness, reduced use of television and tablets, and control exerted by the imaginary companion. The disadvantages of an imaginary companion, according to the findings, include causing fear, disrupting attachment, and controlling the child.The control and kindness of the imaginary companion were benefits mentioned by the only children in this study, which aligns with the findings of McLewin and Muller  and TaylIn our study, a small number of only children mentioned that their imaginary companions may harm them, scare them, make them dependent, or force them to do something, which is also in line with the findings of McLewin and Muller  and SilbExtensive studies show that the imaginary companion is a cross-cultural phenomenon . For exasince the child creates their imaginary companion based on ideals and needs, it should certainly be beneficial and facilitative for them , 36 TherIn general, it can be said that creating an imaginary companion is not exclusive to only children without siblings, but it is more prevalent among them, perhaps because they feel lonelier or receive more attention from their parents. By understanding and examining a child’s imaginary companion, parents can realize the roles and need that the child seeks to fulfill through the imaginary companion. Understanding the advantages of an imaginary companion allows parents to recognize the characteristics and qualities that the child appreciates in themselves, the world, and others. By understanding the disadvantages of an imaginary companion, parents can become aware of any concerns or disturbances in the child’s daily life. Therefore, exploring and understanding the concept of an imaginary companion brings us closer to the child and their world. Parents should consider the characteristics of the imaginary companion and the amount of time the child spends with it. If they notice any abnormality, excessive attachment, or disruption in the child’s daily life, they should take it seriously. Nonetheless, the nature of an imaginary companion is helpful, and if it becomes harmful and detrimental to the child’s daily life, investigating the cause and addressing the issue can be significant.As for the limitations of the study, it should be noted that this research was only conducted on only children in non-governmental schools, and its generalizability to all children may be limited. Additionally, given that the sample included imaginative and creative children, caution should be exercised when relying on statements from younger children. Interviews with unreliable samples were excluded from the study. Therefore, it is suggested that future research be conducted on only children in both governmental and non-governmental schools. Furthermore, it is recommended to include children who have not attended preschool or kindergarten in a study to avoid the disruption or alteration of imaginary companions due to the establishment of social relationships and friendships in these settings.


With green and low-carbon developments in oil fields, an increasing amount of repaired oil tubing is being used as oil and gas transmission pipelines in China. However, due to differences in manufacturing standards between oil tubing and transmission pipelines, there are inevitably some issues during their use. This paper investigates a case of cracking failure in repaired oil tubing used as a gathering and transportation pipeline. The failure occurred after eight months of operation and was characterized by a circumferential crack at the male thread end of the tubing joint. To determine the root cause of the failure, a series of experiments were conducted on the oil tubing. The experiments included visual inspection, chemical composition analysis, mechanical properties testing, hardness testing, metallographic examination, and microstructure analysis. The results revealed that the thread of the cracked tubing was not tightened to the specified position; the connection between the tubing and the coupling was welded in a circumferential direction; and cracks occurred in the heat-affected zone of the weld. Chemical composition, tensile performance, and the Charpy impact of the tubing meet the requirements of API 5CT for P110 material, and no abnormalities were found in the metallographic structure. The microstructure at the weld toe of the fracture is martensite, and the hardness is 476 HV10. Based on the thermal simulation verification test, when the material of the tubing cools from 1200 °C, which is located in the coarse HAZ temperature zone, the base metal transforms into martensite with a little granular bainite, exhibiting its highest hardness value at 371 HV10, which is higher than the allowable hardness for carbon steel and indicates the material has poor weldability. The reasons for the cracking and failure of the tubing are that the P110 repaired tubing has a high carbon equivalent and poor weldability. During the welding process, martensitic structure was formed at the weld toe, and cold cracks appeared in the heat-affected zone, resulting in failure. To avoid the reoccurrence of such failure, recommendations are proposed. Oil wnjection ,5,6 and njection ,8. DurinHowever, during the servicing process, oil tubing is subject to corrosive media and complex loads and is prone to eccentric wear , uniformIn this study, a cracking failure case of repaired oil tubing used as a gathering and transportation pipeline was studied. Such failure cases are rarely reported. Therefore, these research conclusions have great reference value for the application of repaired oil tubing as gathering and transportation pipelines.2S and 2.387% CO2. The leakage region of the oil tubing is the joint, and a circumferential crack occurred at the male thread end, as shown in The studied repaired oil tubing used as a gathering and transportation pipeline leaked after being put into operation for eight months. The oil tubing specification is Φ88.9 mm × 6.45 mm. The material is P110 or C110 according to API 5CT, and the transmission medium is natural gas; the gas transmission volume is about 100,000 cubic meters per day; the service temperature is 15 °C; the operating pressure is 5 MPa. According to the transmission medium composition test results, the medium contains 0.7669% HThe total length of the cracked repaired oil tubing is about 100 cm, including two sections  and coupling. Test samples were taken from the #1 and #2 repaired oil pipes and coupling, and the chemical composition of the samples was analyzed by an ARL4460 direct-reading spectrometer  according to the ASTM A751-20 standard. A tensile test of the #1 and #2 tubing was carried out on longitudinal tensile samples, according to the ASTM A370-20 standard with a UTM5305 material testing machine . The size of the longitudinal Charpy impact test specimen is 5 mm × 10 mm × 55 mm with a V-shaped notch, and the impact test was carried out by a PIT302D impact testing machine  according to the ASTM A370-20 standard at room temperature. The Rockwell hardness test was undertaken on the #1 and #2 tubing rings according to ASTM E18-20 with an RB2002T Rockwell hardness tester . The micro-Vickers hardness test was undertaken on the failed area of the tubing to check whether there is a detrimental phase with the DuraScan 70G5 hardness tester according to the standard ASTM E384-17.The metallographic samples were machined from the #1 and #2 repaired tubing, the coupling, and the failed region, and their microstructure was inspected with an MEF4M metallographic microscope  according to ASTM E45-18a, ASTM E1268-19. The morphology of the cross section of the cracked region was observed under a scanning electron microscope of Tescan Vega . Energy dispersive spectroscopy was adopted to check the element content of the corrosion products on the fracture surface.The weld simulation was conducted on the base metal of the failed #1 repaired tubing on a Gleeble 3500 thermal simulation testing machine  to study the different heat-affected zone (HAZ)s’ microstructure and hardness properties. The dimensions of the sample are Φ4 × 100 mm; the base metal was heated to 850 °C, 950 °C, 1050 °C, 1100 °C, and 1200 °C, respectively, kept for 3 s, then air-cooled to room temperature. And the load was recorded, and the microstructure was observed.The failed repaired tubing at the coupling was cut in half, as shown in The circumferential crack is located near the girth weld of the #1 tubing; the inner surface of the coupling joint is covered with black oil; and there is no obvious corrosion on the coupling and the #1 and #2 tubing. From the cross-section view, there are 20 teeth in total for the #2 male thread tubing, and about 14 teeth for the #1 male thread tubing. Therefore, it can be inferred that the male thread end of the #1 tubing has not been threaded to the specified position. The surface of the fracture is covered with black matter b,c. ObseChemical composition analysis results are shown in The results of the tensile test are shown in Due to the brittleness cracking of the pipes, Charpy impact testing was conducted on the pipe body to assess its fracture toughness. The results of the Charpy impact test are shown in The schematic diagram of the Rockwell hardness test on the pipe shown in The microhardness of the longitudinal section of the crack region was tested with a DuraScan 70G5 micro-Vickers hardness tester  according to the ASTM E384-17 standard . The resThe metallographic structure of the sample was observed under a metallographic microscope. The microstructure of the repaired tubing and coupling was tempered sorbite, and the grain size was 9.5. The microstructure morphology is shown in The microstructure near the fracture and the base metal was further analyzed, as shown in 2S and CO2 contained in the medium, indicating that the crack was corroded by H2S and CO2.The cross-sectional morphology of the fracture was observed under a scanning electron microscope, as shown in The macroscopic analysis of the failed tubing shows that there are welding indications at the connection between the #1 repaired tubing and the coupling; the failure crack is located at the weld, and the fracture has no obvious plastic deformation. The mechanical and chemical composition test results of the failed tubing show that the #1 repaired oil pipe is made of P110, and the #2 repaired tubing is made of C110. The chemical composition and tensile properties of the two tubes meet the standard requirements, and there is no abnormality in the Charpy impact performance.2S and CO2 can be ruled out. From the cross-sectional metallographic view, the crack propagates along the heat-affected zone, so it is inferred that the root cause of the cracking is related to welding.According to the fracture analysis, the crack of the #1 repaired tubing originated from the outer wall, so the failure caused by internal corrosion of HTypically, repair tubing is threaded and not welded. Generally, repaired tubing is used for water and oil transportation or associated gas transmission pipelines with a pressure not greater than 1.5MPa. Since the repaired oil pipe is manually threaded, when it is used as a pipeline, the sealing is difficult to guarantee. When the gas transmission pressure is too high, there may be leakage.According to the information provided by operational personnel, this pipeline leaked during the pressure test and was resealed by welding. Usually, the tendency of the material to form cracks during welding and the performance of the welded joint are used as the main indicators for evaluating the welding performance of the material. The quality of weldability is related to the chemical composition of the material and the welding process used. As for steel, the carbon content has the greatest influence on weldability, so the carbon content in steel is often used as the main indicator for judging the weldability of steel materials. The higher the carbon content, the worse the weldability.Generally, there are several methods for evaluating the weldability of materials, including the implant test, the Y-slit type cracking test, the thermal simulation method, the carbon equivalent evaluation method, and the maximum hardness evaluation method. We employed the carbon equivalent evaluation method and the thermal simulation method to assess the weldability of the tube.The International Institute of Welding (IIW) uses carbon equivalent value (CEV) ,14 to evAccording to Equation (1), the carbon equivalent value of the #1 repaired tubing is calculated as 0.5369%, which indicates that the #1 repaired tubing has poor weldability and is a difficult-to-weld material. According to the cracking tendency Graville diagram ,16 of thExperimental methods for evaluating the susceptibility of weld joints to cold cracking include the Y-slit type cracking test, the tensile restraint cracking test, the restrained butt joint cracking test, the window type restraint cracking test, controlled thermal severity, and an implant test. These methods are based on different restraint conditions and involve observing post-weld cracking. Additionally, thermal simulation is used to study the tendency for weld seam cracking . Due to The welding thermal simulation involves controlling the peak temperature and cooling rate to replicate the welding thermal cycle process in different heat-affected zones. Based on the peak temperature profiles of different welding regions in carbon steel as shown in The thermal simulation test was conducted on the #1 tubing to verify the microstructure of the weld. The two ends of the sample were clamped and were heated to 850 °C, 950 °C, 1050 °C, 1100 °C, and 1200 °C in 10 s intervals, respectively, and then air cooled. The load was recorded during the heating test, and the microstructure and hardness were inspected after the test. The hardness and microstructure of the tubing base metal at different simulated peak temperatures after thermal simulation are shown in According to the International Institute of Welding, the maximum Vickers hardness in the heat-affected zone (HAZ) should not exceed 350HV10, and the presence of martensitic structure should be avoided ,22. FromAccording to the metallographic and hardness results of the fracture section, the microstructure at the weld toe is martensite with a hardness of 476 HV10. Near the weld in the heat-affected zone of the tubing is granular bainite with a hardness of 361 HV10. The microstructure of the base material is tempered sorbite, with a hardness of about 320 HV10. During the welding process, the cooling speed at different positions is different; the microstructure formed is different; and the volume of the microstructure changes differently, so internal stress will be formed. The lath-like martensite structure at the weld toe is a typical hard and brittle structure of carbon steel, which seriously reduces the toughness of the welded joint, leading to cold cracks ,24 in thTo sum up, the thread at the crack failure location was not tightened to the specified position; leakage occurred during the pressure test; and welding was used to repair the leakage. However, the P110 oil pipe has a high carbon equivalent, poor weldability, and martensitic structure formed at the weld toe, leading to the occurrence of cold cracks in the heat-affected zone, resulting in failure.Oil tubing and pipelines are manufactured according to different standards, namely API 5CT and API 5L. These two types of pipes have different service environments and requirements.Oil tubing is primarily used for oil and gas extraction and typically employs threaded connections. Therefore, the API 5CT standard focuses more on the performance and reliability of oil tubing during the extraction process. This standard covers requirements for tubing dimensions, materials, mechanical properties, and chemical composition, ensuring the tubing’s ability to withstand factors such as high pressure, high temperature, and corrosion in harsh oil and gas extraction environments.Pipelines, on the other hand, are primarily used for oil and gas transportation and commonly utilizes welded connections. As a result, the API 5L standard emphasizes the weldability and reliability of pipelines during the transportation process. This standard covers requirements for pipeline dimensions, materials, mechanical properties, and chemical composition, as well as welding performance, ensuring good weldability and suitability for handling pressures, temperatures, and other mechanical demands in the transportation process.In some oil fields in China, old and discarded oil tubing is inspected and repaired for use as transmission pipelines. When using oil tubing as transmission pipelines, it is important to consider that during the installation of pipelines, it is difficult to achieve a specified torque on the pipe threads with wrenches. Consequently, ensuring a certain level of sealing becomes challenging. Therefore, it is not recommended to use repaired oil tubing for transporting hydrogen sulfide gas or in high-pressure gas transmission environments.In the case of pressure testing leaks, considering the higher carbon content and poor weldability of oil tubing, it is advisable to perform welding after preheating and drying, followed by regular inspections to ensure their integrity and to prevent failures.(1)Macroscopic analysis shows that the thread of the cracked tubing is not tightened to the specified position; the connection between the tubing and the coupling is welded in a circumferential direction; and cracks occur in the heat-affected zone of the weld.(2)The results of chemical composition and mechanical testing show that the chemical composition, the tensile performance, and the Charpy impact of the tubing meet the requirements of API 5CT for P110 material; no abnormalities are found in the metallographic structure. The microstructure at the weld toe of the fracture is martensite, and the hardness is 476 HV10.(3)Based on the thermal simulation verification test, when the material of the tubing cools from 1200 °C, which is located in the coarse HAZ temperature zone, the base metal transforms into martensite with a little granular bainite, exhibiting the greatest hardness value of 371 HV10.(4)The reasons for the cracking and failure of the tubing are that the P110 repaired tubing has a high carbon equivalent and poor weldability. During the welding process, martensitic structure is formed at the weld toe, and cold cracks appear in the heat-affected zone, resulting in failure.In this study, repaired tubing was used as a gas transmission pipeline, and after eight months of operation, it cracked and failed. Macro-inspection, chemical composition analysis, mechanical performance tests, microstructure characterization, fracture morphology, and thermal simulation tests were carried out to find the root cause of the failure, and the following conclusions are obtained:(1)It is not suitable to use repaired tubing for medium transportation under the conditions of high-pressure gas transmission.(2)When repaired tubing is used for gathering and transportation construction, special measures should be taken to ensure that the thread is screwed up to the designated position.(3)To weld the repair tubing, the welding consumables could be dried; the base metal and welding consumables could be degreased and cleaned; and the residual stress could be eliminated by heat treatment after welding.To avoid the reoccurrence of such failure, the following recommendations were proposed:


The results of the validation studies indicate that both optimized assays are accurate, specific, and precise over their respective quantifiable ranges. There was a strong linear association between the new and previous versions of both assays. Assay serostatus cutoffs for the redeveloped assays were established based on the bridging studies and, for the HPV-9 cLIA, further refined, based on additional data from HPV vaccine clinical studies so as to align the seropositivity rates between assay versions.Two multiplex immunoassays are routinely used to assess antibody responses in clinical trials of the 9-valent human papillomavirus (9vHPV) vaccine. The HPV6/11/16/18/31/33/45/52/58 competitive Luminex immunoassay (HPV-9 cLIA) and HPV6/11/16/18/31/33/45/52/58 total immunoglobulin G Luminex immunoassay are used for measurements of immunogenicity. Following their initial validation in 2010, both assays were redeveloped, and several parameters were optimized, including the coating concentration of virus-like particles, type of Luminex microspheres, serum sample and reference standard diluent, reference standard starting dilution and titration series, and vendor and concentration of the phycoerythrin-labeled antibodies. Validation studies evaluated the assay performance parameters, including the intra-assay precision (repeatability), intermediate precision, linearity, relative accuracy, and limits of quantitation. In addition, since maintaining a link to the original assays that were used in trials supporting vaccine licensure is critical, the assays were formally bridged to the previous assay versions by using individual patient sera from a 9vHPV vaccine clinical trial (IMPORTANCE Assay modernization is a key aspect of vaccine life cycle management. Thus, new, reoptimized versions of two 9vHPV immunoassays have been developed and validated for use in ongoing and future HPV vaccine clinical trials. These assays are suitable for use in high-throughput testing for HPV antibodies in serum samples. Bridging to previous versions of the assays allows for the continuous monitoring of immune responses across assay versions, including in immunogenicity studies that involve new populations as well as long-term follow-up studies. Human papillomavirus (HPV) is responsible for approximately 690,000 new annual cases of cancer in females and males globally, including cancers of the cervix, head and neck, anus, penis, vagina, and vulva  assays –15. Sinc–esponses –13. Howesponses –. Epitopesponses ––8, 14–16ses ––14–, 10, 16.The 9vHPV vaccine clinical trial program has relied on 2 multiplexed immunoassays to assess anti-HPV antibody responses, and versions of these assays continue to be used in ongoing trials. These immunoassays are the HPV6/11/16/18/31/33/45/52/58 competitive Luminex immunoassay (HPV-9 cLIA), an epitope-specific competitive immunoassay, and the HPV6/11/16/18/31/33/45/52/58 total IgG Luminex immunoassay (HPV-9 IgG-LIA), which is a type-specific direct binding IgG assay. Both the HPV-9 cLIA and HPV-9 IgG-LIA are Luminex immunoassays involving type-specific, yeast-derived VLPs that are coupled to 9 distinct Luminex microspheres, called VLP-coupled microspheres or MS-VLP. Each microsphere has its own distinct fluorescent dye that can be identified following excitation with an infrared laser, which allows for the measurement of multiple HPV types from a single test of a human serum sample.The original versions of the HPV-9 cLIA and HPV-9 IgG-LIA that have been used in clinical trials through 2015 have been described previously , 9, 15. The optimal VLP coupling concentration was determined to be 100 μg/mL for HPV11, 16, 18, 31, 33, 45, 52, and 58 and 200 μg/mL for HPV6, based on the VLP coating saturation points. The optimal VLP coupling concentrations were selected, based on maximizing the dynamic range of the median fluorescence intensity values that were observed over a range of concentrations that were evaluated. Following a comparison with 2 ready-to-use wash buffers, the original 1× phosphate-buffered saline (PBS) + 0.05% Triton X-100 buffer was found to be equivalent to both the 1× PBS Tween 20 and 1× Focus Wash buffers. The 1× PBS Tween 20 wash buffer was selected, given that preweighed packets may be stored at ambient temperature and that the buffer may be prepared by adding distilled water.The significant parameters that were reoptimized for the HPV-9 cLIA and HPV-9 IgG-LIA are summarized in The assay validation data for the HPV-9 cLIA and IgG-LIA are summarized in 10.1128/msphere.00962-21.1TABLE S1aAcceptance limits were based on: (i) experience with respect to acceptable performance for ligand binding assays in general and (ii) the historical performance of the original versions of the HPV-9 cLIA and HPV-9 IgG-LIA. %RSD, percent relative standard deviation; cLIA, competitive Luminex immunoassay; HPV, human papillomavirus; HPV-9 cLIA, HPV6/11/16/18/31/33/45/52/58 cLIA; HPV-9 IgG-LIA, HPV6/11/16/18/31/33/45/52/58 total IgG Luminex immunoassay; IgG, immunoglobulin G; LLOQ, lower limit of quantitation; mAb, monoclonal antibody; mMU, milli-Merck Units; MS-VLP, VLP-coupled microspheres; PE, phycoerythrin; RMSE, root mean square error; SSCO, serostatus cutoff; ULOQ, upper limit of quantitation; VLP, virus-like particle. Download Table S1, DOCX file, 0.02 MB.Summary of validation parameters for the new versions of the HPV-9 cLIA and IgG-LIA. Copyright © 2023 Nolan et al.2023Nolan et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the (i) HPV-9 cLIA precision, linearity, and relative accuracy. In the analysis of interassay precision, which involved a panel of 20 (incurred) human serum samples (each with 12 replicates) from individuals who were vaccinated for all 9 HPV types, the percent relative standard deviation (%RSD) was <20% for all samples with antibody concentrations within the quantifiable range of the assay. The overall precision was <11% for each HPV type. As shown in 2 value was >0.97 for all 648 of the individual dilution curves.In the analysis of linearity, which involved 72 individual test sample dilution curves per HPV type, the slope average was within the range of −1.25 to −0.80 for each sample and HPV type. The RFor each HPV type, the overall estimate of dilution bias was within 1.25-fold per 10-fold dilution. Of the 648 individual estimates of dilution bias that were obtained across the set of runs, samples, and HPV types, 646 were ≤2.0-fold per 10-fold dilution.(ii) HPV-9 IgG-LIA precision, linearity, and relative accuracy. In the analysis of interassay precision, which involved a panel of 20 (incurred) human serum samples (each with 12 replicates) from individuals vaccinated for all 9 HPV types, all samples with antibody concentrations within the quantifiable range of the assay had %RSD < 12%. The overall precision was <8% for each HPV type. As shown in 2 value was >0.99 for all 639 of the individual dilution curves.In the analysis of linearity, which involved 71 individual test sample dilution curves per HPV type, the slope was within the range of −1.20 to −0.90 for each curve and HPV type. The RAll 639 of the individual estimates of dilution bias obtained across the set of runs, samples, and HPV types were ≤2.0-fold per 10-fold dilution. The observed dilution bias was similar across the set of test samples and HPV types, as the concentrations corrected for dilution tended to decrease slightly with increasing dilution. The mean estimated bias was −17.4% to −15.2% per 10-fold dilution across each HPV type.(i) HPV-9 cLIA bridging. Bridging studies were performed to allow for comparison of the results from the optimized assays to historical results obtained using the previous assays. The HPV-type-specific lower limits of quantitation (LLOQs) and upper limits of quantitation (ULOQs) for the new versus previous HPV-9 cLIAs are summarized in For each of the 9 HPV types, the reverse cumulative distribution (RCD) curves for the 2 assay versions overlay one another closely for the month 7 and month 36 postvaccination samples . The ass(ii) HPV-9 cLIA SSCO determination. SSCOs for the reoptimized HPV-9 cLIA were initially established by testing the individual participant serum from Study V503-001 in the bridging study and comparing the results with those obtained from the previous cLIA for the same samples. For each HPV type, the SSCO was determined by balancing the discrepancy between the assay versions in the day 1 and month 36 positivity rates, with greater emphasis being placed on aligning the assays on the day 1 negativity rate. SSCOs were assigned to achieve positivity rates for the day 1 and month 36 samples that were comparable to the corresponding rates from the earlier version of the assay.The monitoring of HPV-9 cLIA data from ongoing testing of clinical study samples indicated that further SSCO refinement was needed to achieve similar rates of HPV18 baseline positivity between the previous and reoptimized cLIAs. When early serum samples from adult female trial participants were measured using the updated cLIA, the baseline positivity rates for HPV18 (41.4%) substantially exceeded the historical HPV18 baseline positivity rates in adult women as well as those rates predicted by the laboratory bridging study (12.7%), whereas the observed baseline positivity rates for the 8 other HPV types either closely approximated or slightly exceeded the expected positivity rates, based on the results of the bridging study. The RCD curves of the anti-HPV18 antibody concentrations in the clinical study baseline samples showed that the vast majority of samples (approximately 87.5%) had antibody concentrations below 59 milli-Merck Units (mMU)/mL in adult female clinical trial participants . Similar10.1128/msphere.00962-21.2FIG S1FIG S1, EPS file, 1.4 MB.HPV18 RCD curves for the baseline samples from the adult female clinical trial participants, using the updated HPV-9 cLIA. HPV, human papillomavirus; HPV-9 cLIA, HPV6/11/16/18/31/33/45/52/58 competitive Luminex immunoassay; mMU, milli-Merck Units; RCD, reverse cumulative distribution. Download Copyright © 2023 Nolan et al.2023Nolan et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the (iii) HPV-9 IgG-LIA bridging. The HPV-type-specific LLOQs and ULOQs for the new versus previous HPV-9 IgG-LIAs are summarized in (iv) HPV-9 IgG-LIA SSCO determination. Based on the selection criteria that are described in Materials and Methods, the SSCOs for the new HPV-9 IgG-LIA were defined  internal reference standard and corresponding concentration units (mMU/mL). The resulting measured antibody concentrations in the cLIA and IgG-LIA for the HPV-16 and HPV-18 international reference standards are listed in Across the evaluable dilutions in the cLIA, the mean concentrations for the HPV-16 and HPV-18 international reference standards in their respective assays were 100.2 and 127.3 mMU/mL, respectively. Given the assigned antibody concentrations for the HPV-16 and HPV-18 international reference standards of 10 IU/mL and 16 IU/mL, respectively, the resulting conversion factor between mMU/mL and IU/mL is 1 mMU/mL, which corresponds to 0.100 IU/mL for the HPV-16 cLIA and 0.126 IU/mL for the HPV-18 cLIA.Across the six evaluable dilutions in the IgG-LIA, the mean concentrations for the HPV-16 and HPV-18 international reference standards in their respective IgG-LIAs were 93.8 and 87.3 mMU/mL, respectively. Using the assigned antibody concentrations for the HPV-16 and HPV-18 international reference standards, the resulting conversion factor between mMU/mL and IU/mL is 1 mMU/mL, which corresponds to 0.107 IU/mL for the HPV-16 IgG-LIA and 0.183 IU/mL for the HPV-18 IgG-LIA.Optimized versions of the HPV-9 cLIA and IgG-LIA have been developed and validated for use in high-throughput testing for HPV antibodies in serum samples. The optimized parameters, including the elimination of ADHS in the assay buffer and increasing the serum sample starting dilution (from 1:4 to 1:10), improved the dilutability of the HPV-9 cLIA. Results of the validation studies indicate that both optimized assays are accurate, specific, and precise throughout the assay quantifiable range for each HPV type.In bridging studies to the prior versions of the HPV-9 cLIA and IgG-LIA, strong positive linear associations between the previous and new assay versions were observed. This allows for continuous immunogenicity assessments in clinical trials of 9vHPV vaccines across assay versions. For example, long-term antibody persistence has been evaluated in long-term follow-up studies –19 acros–20–SSCOs have been established to identify people with serologic response to vaccination. For both optimized assays, SSCOs were initially defined based on the bridging studies, compared with the prior assay versions, using human serum samples from a single clinical study. In the case of the cLIA, the SSCO values were further refined, based on an observed, higher-than-expected HPV18 baseline positivity in actual clinical trial samples from additional studies that were conducted early after the assay reoptimization. This result highlights the importance of including relevant clinical sample panels for SSCO evaluation. Since biological assays, such as cLIA and LIA, are expected to evolve over time as instrumentation and reagents are updated or modernized, cLIA and IgG-LIA performance will continue to be monitored across ongoing and future clinical studies so as to assess the potential need for modification to provide a sustainable immunogenicity endpoint and to enable immunobridging to historical studies.Including assay modernization as a key part of vaccine life cycle management has intrinsic benefits, including increased method robustness, cost reduction, and decreased risk of assay failure . Vaccine20–26–In summary, the HPV-9 cLIA and IgG-LIA have been reoptimized, validated, and bridged to prior versions of the respective assays for use in ongoing and future HPV vaccine clinical trials.The HPV-9  immunoassays utilize the Luminex 200 Instrument System, which is a flexible analyzer that is based on the principles of flow cytometry. The basic principles have been described in detail elsewhere , 9. BrieThe HPV-9 cLIA quantifies antibodies that are specific to dominant neutralizing epitopes. Type-specific, neutralizing monoclonal antibodies are labeled with a PE tag. These tagged antibodies compete with the neutralizing antibodies that are present in the test serum for binding to the corresponding neutralizing epitopes of the VLPs such that the intensity of the fluorescent signal is inversely proportional to the concentration of the anti-HPV neutralizing antibodies. The antibody concentrations are derived from a standard curve that was generated using a reference standard from a pool of serum from individuals immunized against 9 HPV types (the “HPV-9 reference standard”) and calculated using a weighted 4-parameter logistic curve fit. The immune reference serum used as the standard curve was assigned arbitrary values that are expressed in mMU/mL, as previously noted . TherefoThe HPV-9 IgG-LIA quantifies the total HPV-type-specific IgG antibodies. VLP-coupled microspheres are incubated with serum and are then washed to remove unbound antibodies. The captured antibodies are subsequently detected using a human IgG antibody-specific PE-conjugated goat antibody. The magnitude of the PE-derived signal is directly proportional to the serum HPV antibody concentration. The antibody concentrations are derived from a standard curve, using the same reference standards as described above, and are expressed in mMU/mL. Although the same term  is used for the unit of measurement in both assays, the “HPV-9 cLIA mMU/mL” and the “HPV-9 IgG-LIA mMU/mL” are different units of measurement and cannot be directly compared.(i) Optimization of reagents used in both assays. VLP-coupled beads are prepared using specific bead regions that are selected to minimize overlap between the multiplexed beads. Polystyrene beads, which were used in the previous assay versions, are replaced with MagPlex beads (Luminex Corporation) in the current version of the assays. There are several advantages to MagPlex Microspheres over traditional MicroPlex Microspheres, including their 5× increased multiplex capacity, improved percent recovery during handling and wash steps, and ideal design for automated applications  and 1× Focus Wash .The HPV-9 reference standard was initially prediluted in antibody-depleted human serum (ADHS) in previous assay versions. The same reference standard and controls were used for the HPV-9 cLIA and HPV-9 IgG-LIA. To minimize the variability observed within the qualified ADHS batches, the use of ADHS was eliminated in the predilution step.(ii) Redevelopment of the HPV-9 cLIA. To optimize the starting concentration and fold dilution of the HPV-9 reference standard , a 12-point titration curve was created by further dilution of the reference standard to a starting dilution of 1:10. This was followed by 2-fold titration. The serial titration was then adjusted to broaden the range of the standard curve . A starting dilution of 1:20 was also tested using 2-fold, 1.7-fold, and 1.5-fold titration schemes.50) were determined for each PE-mAb, and the reference standard median fluorescence intensity values were compared when testing with PE-mAbs that were prepared at the EC80, EC50, EC40, EC20, and EC10 concentrations.To define the optimal concentrations of the individual PE-labeled mAbs for use in the cLIA, the half maximal effective concentrations  were compared, using HPV16 as a representative HPV-specific mAb. The candidates were tested using 1:5 diluted HPV-9 reference standard and 4 tentative quality-control samples (serum pools from V503-001). The mean interpolated concentrations for HPV16 were calculated, the results from each HPV16 PE-mAb were compared in relation to the expected concentration, and ratios were calculated.(iii) Redevelopment of the HPV-9 IgG-LIA. Reference standard starting dilutions of 1:100, 1:50, 1:40, 1:30, 1:20, and 1:10 were evaluated using a 3-fold titration, and ratios of the observed versus the expected concentrations were calculated for each starting dilution, with the average ratio for each HPV type being used to evaluate the accuracy of the 4-parameter logistic fit. Standard points were considered to be accurate when the ratios were within the range of 0.70 to 1.30.PE-labeled goat anti-human IgG obtained from Jackson ImmunoResearch Laboratories was compared with the PE-conjugate that was used in the previous version of the assay (HP6043), and the optimal working concentrations were determined using HPV reference standards (1:40 and 1:30 starting dilutions with 2-fold titrations). A working concentration of 2.5 μg/mL for the Jackson and HP6043 conjugates was selected for further analysis so as to evaluate the accuracy by determining the ratio of the concentration results (Jackson/HP6043), with a standard point considered to be accurate when the value was within the range of 0.70 to 1.30.2 Solutions Vaccines , San Juan Capistrano, CA, on behalf of Merck & Co. Inc., Rahway, NJ, USA. The validation studies evaluated various performance parameters, including the intra-assay precision (repeatability), intermediate precision, linearity, relative accuracy, and limits of quantitation  Linearity. Assay linearity  was evaluated using 6 individual human serum samples from individuals who were vaccinated for all 9 HPV types. Each of the 6 samples was serially diluted 2-fold, with the number of dilutions being dependent on the antibody concentrations of the undiluted  sample. The highest dilution targeted a concentration below the previously determined assay LLOQ, and the next highest dilution targeted the previously determined LLOQ. Each dilution was treated as an individual sample, and each neat/dilution sample was tested in single by 3 operators on 4 separate days . Two lots of MS-VLP and 2 lots of either pooled 10× mAb-PE conjugate  or goat anti-human IgG-PE conjugate  were evaluated. For each HPV type, a linear regression analysis was performed on the resulting series of measured concentrations for each sample. The acceptance criteria for each HPV type and sample required a slope of −1.25 to −0.80 and an R2 value of ≥0.95 for the HPV-9 cLIA and a slope of −1.20 to −0.90 and an R2 value of ≥0.95 for the IgG-LIA.(ii) Relative accuracy. The relative accuracy, which refers to the ability of the assay to accurately quantitate fractions of a previously measured sample when those samples are spiked into a known negative sample or diluent at specific dilutions, was evaluated by using the results from the data set that was generated to assess the interassay linearity, as described above. Linear regression results were analyzed to estimate the bias per 10-fold dilution. The dilution bias was calculated by regressing the natural logarithm-transformed dilution corrected concentration at each dilution against the natural logarithm-transformed reciprocal dilution. The dilution bias per 10-fold dilution is (10b−1) ×100%, where b is the resulting slope estimate from the regression. For each HPV type, the mean estimate of the dilution bias is essentially obtained by taking the average of the slope estimates over the samples and runs and entering that average into the expression for dilution bias per 10-fold dilution. The assays were considered to be acceptably dilutable if the dilution bias per 10-fold dilution was ≤2.0-fold.(iii) Precision. Precision refers to the degree of agreement among individual test results when a panel of samples spanning the quantifiable range is repeatedly assayed.For both the HPV-9 cLIA and IgG-LIA, intra-assay precision (repeatability) was measured by comparing the results of samples tested under the same conditions . Each of 5 samples from individuals who were vaccinated for all 9 HPV types, with antibody concentrations covering the range of the HPV-9 cLIA or IgG-LIA, were tested 10 times  in a single assay run by each of 2 operators, using the same MS-VLP and conjugate lots. The intra-assay %RSD was calculated for each sample for each HPV type and operator, and the median of these values was calculated to be the overall intra-assay %RSD for each HPV type. The validation acceptance criteria for cLIA and IgG-LIA intra-assay precision required %RSD values of ≤10% and ≤15%, respectively, for each HPV type.Interassay precision (intermediate precision) involves measuring precision under conditions that vary during routine assay testing and performance . To evaluate the interassay precision, a panel of 20 incurred human samples from individuals who were vaccinated for all 9 HPV types, with antibody concentrations across the range of the assay, was tested in single by 3 operators on 4 separate days . Two lots of MS-VLP and pooled mAb-PE conjugate (for the HPV-9 cLIA) or IgG-PE conjugate (for the HPV-9 IgG-LIA) were also incorporated. The interassay %RSD for each sample and the overall interassay %RSD (the median %RSD across the set of samples) were determined for each HPV type.In addition to the 20 incurred samples, the interassay precision was assessed on the results from the serially diluted sample set that was referenced above as part of the linearity analysis. Each dilution of the 6 linearity samples was treated as an individual sample. The interassay %RSD for each sample and overall interassay %RSD (the median %RSD across the set of samples) were determined for each HPV type. For both the cLIA and IgG-LIA, the interassay precision validation acceptance criteria for each HPV type were a %RSD value of ≤30% RSD for ≥80% of the samples within the assays’ quantifiable range and an overall %RSD of ≤25%.(iv) Limits of quantitation. The ULOQ and LLOQ were evaluated using the interassay precision incurred sample and linearity data sets. The test samples that were chosen for these data sets and the dilutions that were used to generate the linearity data set were chosen so as to result in concentrations across the assay range from the ULOQ to below the LLOQ. For each HPV type, the %RSD was calculated for all samples (neat and dilution) with antibody concentrations within the previously determined quantifiable range. The acceptance criteria required the %RSD to be ≤25% within the quantifiable range for each HPV type.2 Solutions Vaccines, using a single lot of VLP-coupled beads, and the concentration results were compared with clinical results obtained using the previous versions of the assays. Summary statistics were calculated, and comparisons between the historical and new results were performed separately for each HPV type and by time of serum collection, post-vaccine dose. Comparisons between assay versions were performed on the natural logarithm-transformed concentrations.Bridging studies were performed to compare the results from the redeveloped assays with historical results that were generated with the previous assay versions , 9 in orFor the purposes of estimating the concentration ratio between assay versions, concentrations reported as <LLOQ were excluded from the analysis. The functional relationship between the assay versions was estimated using the linear statistical relationship model of Tan and Iglewicz , which iP-value that resulted from the test for imbalance in discordant assignments were calculated for each cross-classification comparison.SSCOs were determined during the laboratory bridging study, using the panel of serum samples from the aforementioned study V503-001. For both assays, 2 × 2 cross-classification tables were created for each HPV type by serum collection time point, and they were constructed using the historic SSCO and alternative SSCO values for the updated assays. The negativity rate , agreement rate , and McNemar’s exact SSCOF), which is defined asD1 and ΔM36 denote the difference in the negativity rate between the cLIA versions (redeveloped version minus previous version) at day 1 and month 36, respectively. The expression gives 1.5 times the weight to minimizing the discrepancy on day 1, compared with month 36, and no weight is given to negative ΔM36 values. To select among tied cutoff values, the values that provided for the best balance between the differences in the negativity rates at day 1 and month 36 were selected. The SSCOs were further refined, based on observations using clinical study data that were generated with the revised assays during routine assay testing and following the subsequent re-evaluation of the bridging study so as to better align the seropositivity rates between the assay versions.For the HPV-9 cLIA, the SSCOs for the redeveloped assay were determined by balancing the discrepancy between the assay versions in the day 1 and month 36 positivity rates, with greater emphasis placed on the day 1 negativity rate. Mathematically, the SSCO was the value that minimized the serostatus cutoff function  it must exceed the LLOQ by ≥1 mMU/mL and (ii) the negativity rate for day 1 is within 1.0 percentage point of the negativity rate for the day 1 samples in the prior version of the assay.International reference standards containing HPV-16-specific and HPV-18-specific antibodies were obtained from the National Institute for Biological Standards and Control (NIBSC). On each of 3 days, international reference standards were reconstituted, per vendor instructions, and diluted to the minimum required dilution (MRD) of the respective assay (1:10 cLIA and 1:100 IgG-LIA). The international standards were then serially diluted 1:1.5 across 6 wells of a 96-well plate. On the same plates, MSD’s internal reference standards were diluted to MRD and were further serially diluted, per the respective test methods, across the 12 plate columns (1:1.7 cLIA and 1:2.1 IgG-LIA). Each reference standard was prepared twice, in duplicate, per plate, with two plates being run for each assay on each day. Following the reference standard preparation, the remainder of the assays were executed per the current test methods.