<scp>JAK</scp>2 V617F mutation can be reliably detected in serum using droplet digital <scp>PCR</scp>

Nystrand, Camilla Furlund; Waage, Anders; Jonassen, Christine Monceyron

doi:10.1111/ijlh.12762

Cited by 6 publications

(6 citation statements)

References 28 publications

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“…Our results showed that not only could the JAK2 V617F mutation could be detected in serum by qPCR, but this specimen also presented a mean bias of 4.058% when compared to the paired peripheral blood sample. These results corroborate with the previous description using ddPCR [12], suggesting that the use of serum DNA may allow for increased mutant detection rates. Other serum advantages are: 1) It has less inhibitors than whole blood; 2) it can be directly used in qPCR [14]; and 3) it is more friendly most automated DNA extractions and additional purifications steps are not required to achieve amplifiable DNA [15], allowing increased high-throughput analysis.…”

Section: Discussionsupporting

confidence: 92%

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Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method

et al. 2020

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Detection of the Janus Kinase-2 (JAK2) V617F mutation is a diagnostic criterion for myeloproliferative neoplasms, and high levels of mutant alleles are associated with worse outcomes. This mutation is usually tested on blood DNA by allele-specific qPCR (AS-qPCR) and measured using absolute quantification. However, some automated DNA extractions co-extracts of PCR inhibitors from blood and qPCR absolute quantification need increased efforts in order to maintain standard curves. JAK2 V617F can also be detected in serum using droplet digital PCR (ddPCR), a specimen with less inhibitors and favorable to automated extractions, but ddPCR instruments are not wide available as qPCR thermocyclers. Here, we evaluate whether JAK2 V617F could be accurately quantified by AS-qPCR using the 2-∆∆Cq method on blood DNA and validate the assay using gold-standard molecular diagnostic protocols. Next, we apply the validated method to assess if the mutation could be reliably detected/quantified in serum. JAK2 V617F could be quantified by AS-qPCR using the 2-∆∆Cq method—the assay was highly accurate (bias of 1.91%) compared to a commercial kit, highly precise (total CV% of 0.40%, 1.92%, 11.12% for samples with 93%, 54%, and 2.5% of mutant allele), highly sensitive (limit of detection of 0.15%), and demonstrated a linear detection response from 1.1% to 99.9%. Serum presented a higher mutant allele burden compared to the paired whole blood (mean of 4%), which allows for an increased JAK2 mutant detection rate and favors increased JAK2 V617F high-throughput analysis.

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Section: Discussionsupporting

confidence: 92%

“…The authors also found a higher mutant allele burden in serum compared to paired peripheral blood samples, which may allow for increased mutant detection rates for serum. Reliable results were observed when more than 5 ng of serum DNA input was used in the ddPCR [12].…”

Section: Introductionmentioning

confidence: 99%

Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method

et al. 2020

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“…In contrast, ddPCR allows assessment of the total copy number of target molecules without the need for standard curves, thus facilitating standardization of quantitation. Despite ddPCR being an error-prone method that needs to be standardized, it is less sensitive to PCR inhibitors and therefore more robust than qPCR [28,29]. In addition, ddPCR proved to be an appropriate technique for the detection of mutations present at low allele frequencies because partitioning of the sample reduces wild-type background signals and therefore enhances accuracy of detection.…”

Section: Discussionmentioning

confidence: 99%

Superiority of Droplet Digital PCR Over Real-Time Quantitative PCR for JAK2 V617F Allele Mutational Burden Assessment in Myeloproliferative Neoplasms: A Retrospective Study

et al. 2020

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JAK2 V617F mutational status is an essential diagnostic index in myeloproliferative neoplasms (MPNs). Although widely used for detection of JAK2 V617F mutation in peripheral blood (PB), sensitive real-time quantitative PCR (qPCR) presents some methodological limitations. Recently, emerging alternative technologies, like digital droplet PCR (ddPCR), have been reported to overcome some of qPCR’s technical drawbacks. The purpose of this study was to compare the diagnostic utility of ddPCR to qPCR for JAK2 V617F detection and quantification in samples from MPNs patients. Sensitivity and specificity of qPCR and ddPCR in the detection of the mutation were assessed by using a calibrator panel of mutated DNA on 195 JAK2 positive MPN samples. Based on our results, ddPCR proved to be a suitable, precise, and sensitive method for detection and quantification of the JAK2 V617F mutation.

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“…Previously published detection techniques included; allele-speci c PCR which is sensitive enough to detect 2.5% and another study has 0.01% of sensitivity for mutant DNA (30)(31)(32), droplet digital PCR have sensitivity limit of 96% (33), allele-speci c competitive blocker PCR (ACB-PCR) have 1% (34), an ARMS (35), pyro-sequencing have 5-10%(6, 36), sequencing 10-40% (2,4,15), restriction fragment length polymorphism (RFLP) has 5% (32), high resolution melting curve analysis (HRM) having sensitivity of 0.5-1% (37). Although these approaches are enough sensitive but are not quantitative.…”

Section: Discussionmentioning

confidence: 99%

Development of an in-house real time q-PCR assay for the detection of jak2 mutations in myeloproliferative neoplasms

Umair

Ullah

Khan

et al. 2022

Preprint

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Myeloproliferative Neoplasms (MPNs) are rare heterogeneous hematological disorders usually characterized by one or more lineages of myeloid cells in bone marrow and increase number of normal and abnormal cells. Janus kinase 2 valine to phenylalanine (JAK2-V617F) is usually present in Philadelphia-negative MPNs. Pathogenic mutation in JAK2-V617F cause’s valine to phenylalanine substitution in JAK2 gene on exon-14. Different methods such as Allele-specific PCR (AS-PCR), Amplification refractory mutation system (ARMS-PCR), High resolution melting (HRM) analysis and Molecular beacon probe-based RT-PCR are already available to diagnose JAK2-V617F mutation. In current study, we aimed to develop and optimize real-time PCR assay which will be available locally and be feasible, less expensive and less labor extensive. The DNA was extracted from 128 patients and analyzed on our optimized method using newly designed primers and probe. Standards were generated using in-vitro synthesized sequence (Kinco Biological) and Standard curve was obtained. Predicted sensitivity of the method is at least5% for allele burden of the mutation. The total of 128 MPN patients were included in the present study and 54 (42.1%) were JAK2-V617Fpositive according to the optimized protocols. The study concluded that TaqMan Real time PCR is sensitive, efficient and less expensive for the detection of JAK2-V617F mutation.

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JAK2 V617F mutation can be reliably detected in serum using droplet digital PCR

Abstract: In our study, JAK2 V617F mutation load as low as 1% was reliably detected in serum using ddPCR.

Cited by 6 publications

References 28 publications

Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method

Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method

Superiority of Droplet Digital PCR Over Real-Time Quantitative PCR for JAK2 V617F Allele Mutational Burden Assessment in Myeloproliferative Neoplasms: A Retrospective Study

Development of an in-house real time q-PCR assay for the detection of jak2 mutations in myeloproliferative neoplasms

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