External Quality Assessment For Clinical Detection Process Of PML-RAR? Fusion Gene Using Mock Samples With Simulated Clinical Information Of Acute Promyelocytic Leukemia

PML-RAR? fusion gene(FG)detection plays an important role in the management of acute promyelocytic leukemia(APL).PML-RAR? FG molecular testing using reverse transcription quantitative polymerase chain reaction(RT-qPCR)can confirm APL presumptive diagnosis of APL based on morphology,immunophenotype,and/or coagulopathy screen.Sequential measurement of PML-RAR? transcript levels can monitor minmal residual disease(MRD)for documenting disease burden and ultimately confirm molecular remission.RT-qPCR for PML-RAR? FG transcripts is widely used in a routine laboratory setting,hematology molecular laboratories especially.According to PML-RAR? FG detection,total RNA extracted from nucleated leukocytes of clinical samples BM or PB can be divided into three components,including PML-RAR? FG mRNA,CG mRNA and other non-target RNA,which was the largest proportion of total RNA.The bacteriophage MS2 virus-like particles(MS2 VLPs)was stable,nuclease-resistant,and precisely quantify synthetic RNAs.MS2 armored RNAs of PML-RAR? FG L/S N,CG and non-target RNA were made,as which exogenous 23 s rRNA was selected.By mixing different armored RNAs of PML-RAR? FG,CG and 23 s rRNA,we simulated the composition and RNA yield of total RNA of BM nucleated leukocytes to prepare mock leukocyte samples.Different APL clinical cases were designed for different PML-RAR? FG isoform,including clinical testing information at each MRD monitoring point from admission diagnosis to follow-up.According to the clinical information at different MRD monitoring points during the course of APL,different mock leukocyte samples were prepared and used as EQA samples for PML-RAR? FG detection.According to the PML-RAR? clinical detection process,we used strict EQA scoring criteria to highlight the important role of PML-RAR? fusion gene admission screening and qualitative detection in clinical treatment decision-making.We refered to a variety of diagnostic genetics reporting guidelines and recommendations,and established evaluation criteria for the standardized integrity of clinical written reports.All participating laboratories needed to provide clinical treatment plans and treatment adjustments based on APL simulated cases and clinical examination information of EQA samples,in order to examine the willingness and ability of the laboratory to communicate with clinical departments.The laboratories were asked to conduct RNA extraction,leukemia related fusion gene diagnosis screening,qualitative and quantitative test by RT-qPCR,and submit experimental data and clinical reports.The simulated leukocyte samples prepared by us had good adaptability to various RNA extraction methods.The RNA yield of each laboratory is basically consistent with that of real BM samples(2.27-35.70?g),and copy number of control genes is>104,which can satisfy the subsequent PCR experimental testing.As for the PML-RARa FG detection process,30.0%participating laboratories(15/50)were considered as " competent ";8.0%(4/50)participants were classified as " acceptable";62.0%(31/50)laboratories were included in the "improvable" group.The leukemia-related gene fusion screening and quantitative detection performance was significantly better than qualitative detection and clinical reporting.?Diagnostic screening performed well,with only one laboratory showing erroneous results of PML-RAR? isoform identification.?The performance of RT-qPCR qualitative tests was unsatisfactory.51.0%(25/49)of the laboratories had at least one false-positive or false-negative result at the important MRD monitoring point for treatment decision-making.A total of 28 false-positive results and 15 false-negative results were reported.The testing stability of RT-qPCR assay was poor,which showed that the detection ability of different EQA samples was inconsistent in the same laboratory,and the test results were inconsistent between different laboratories.The ability of qualitative and quantitative RT-qPCR detection for PML-RAR? FG rare isoform V was significantly inferior to that for isoform L/S.? The standardized integrity of clinical reportings was poor,and there were few clinical interpretations and further recommendations based on the given APL simulations.Most of clinical laboratory had the willingness to communicate with clinical departments,but the depth needs to be strengthened.In summary,mock leukocyte samples can be used to evaluate PML-RARa fusion gene clinical detection process.The characteristics of clinical BM specimens were successfully simulated in RNA extraction,RT-qPCR detection for control genes and fusion gene.As for different EQA results,clinical laboratories need to strictly perform PCR partition operations to prevent sample contamination,routinely perform internal quality control,and regularly maintain and calibrate PCR instruments to avoid false positive and false negative results.The clinical laboratory also need to actively optimize and validate RT-qPCR Methodology and improve laboratory procedural documents to ensure the stability and accuracy of quantitative test results.The clinical laboratory should pay attention to the clinical information of the test sample and reasonably analyze the test results to obtain the clinical report with professional clinical interpretation.The clinical laboratory needs to continue to participate in external quality assessment and education activities,and strengthen communication with clinicians in order to improve the clinical detection process of the PML-RAR?fusion gene.The main innovations of this study include:(1)APL mock leukocyte samples were prepared using MS2 VLPs,including PML-RAR? fusion gene rare isoform V(2)To design APL clinical case of conventional smooth and extremely recurrence,mock leukocyte samples at different MRD monitoring points were prepared,and were used as PML-RAR? EQA panels.(3)According to clinical testing process,we investigated RNA extraction,diagnostic screening,qualitative and quantitative testing,clinical reports and clinical treatment.(4)We established a strict EQA scoring criterion for the PML-RAR? FG detection process,evaluating diagnostic screening,qualitative and quantitative detection proficiency.(5)we established the scoring criteria for clinical reports and examined the standardized integrity of clinical written reports.(6)APL simulated cases and clinical examination information of each EQA sample were designed to investigate clinical laboratories for clinical response and adjustment.