Abstract
The ability to simultaneously detect JAK2 V617F and MPL W515K/L mutations would substantially improve the early diagnosis of myeloproliferative neoplasms (MPNs) and decrease the risk of arterial thrombosis. The goal of this study is to achieve a point of care testing platform for simultaneous analysis of major genetic alterations in MPN. Here, we report a microfluidic platform including a glass capillary containing polypropylene matrix that extracts genomic DNA from a drop of whole blood, a microchip for simultaneous multi-gene mutation screening, and a handheld battery-powered heating device. The µmLchip system was successfully used for point-of-care identification of the JAK2 V617F and MPL W515K/L mutations. The µmLchip assays were then validated by mutation analysis with samples from 100 MPN patients who had previously been analyzed via unlabeled probe melting curve analysis or real-time PCR. The results from the µmLchip were in perfect agreement with those from the other methods, except for one discrepant result that was negative in the unlabeled probe melting curve analysis but positive in the µmLchip. After T-A cloning, sequences of cloned PCR products revealed JAK2 V617F mutation in the sample. The portable microfluidic platform may be very attractive in developing point-of-care diagnostics for MPL W515K/L and JAK2 V617F mutations.
Highlights
Classical myeloproliferative neoplasms (MPNs), which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are a subclass of hematological malignancies that feature clonally proliferateing blood cells
Because constant temperature is normally required for nucleic acid extraction and amplification, we developed a handheld battery-powered heating device to allow the use of loop-mediated isothermal amplification (LAMP) in remote settings
The results of accuracy analysis were consistent with what we have reported[14]. These results demonstrate that microchip-based LAMP is a highly accurate method for screening for the MPL W515K/L and JAK2 V617F mutations
Summary
Classical myeloproliferative neoplasms (MPNs), which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are a subclass of hematological malignancies that feature clonally proliferateing blood cells. The platform includes a glass capillary containing polypropylene matrix for genomic DNA extraction from a drop of whole blood, a microchip for simultaneous multi-gene mutation screening, and a handheld battery-powered heating device. On the basis of based on our previous work regarding the combination of LAMP and microfluidics, we developed a LAMP microchip for the identification of JAK2 V617F and MPL W515K/L mutation in the peripheral blood of ET and PMF patients. We called it a μmLchip because it is namely a LAMP microchip for simultaneous multi-gene mutation screening. Rapid sample preparation techniques that do not require any additional user interaction are ideal for use of portable microfluidic platforms doctor’s offices, in the field, or at the bed-side
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