BCR‐ABL1 transcript levels detection, 142 CML patients were monitored for a median time of 24 months. Digital PCR detected BCR‐ABL1 transcripts in the RT‐qPCR undetectable cases. The dPCR analysis of the samples, grouped by the MR classes, revealed a significant difference between MR4.0 and MR4.5 (P = 0.0104) or MR5.0 (P = 0.0032). The clinical and hematological characteristics of the patients grouped according to DMR classes (MR4.0 vs MR4.5‐5.0) were superimposable. Conversely, patients with dPCR values <0.468 BCR‐ABL1 copies/µL (as we previously described) showed a longer DMR duration (P = 0.0220) and mainly belonged to MR4.5‐5.0 (P = 0.0442) classes compared to patients with higher dPCR values. Among the 142 patients, 111 (78%) discontinued the TKI treatment; among the 111 patients, 24 (22%) lost the MR3.0 or MR4.0. RT‐qPCR was not able to discriminate patients with higher risk of MR loss after discontinuation (P = 0.8100). On the contrary, according to dPCR, 12/25 (48%) patients with BCR‐ABL1 values ≥0.468 and 12/86 (14%) patients with BCR‐ABL1 values <0.468 lost DMR in this cohort, respectively (P = 0.0003). Treatment‐free remission of patients who discontinued TKI with a dPCR <0.468 was significantly higher compared to patients with dPCR ≥ 0.468 . In conclusion, dPCR resulted in an improved recognition of stable DMR and of candidates to TKI discontinuation.Treatment‐free remission (TFR) by tyrosine kinase inhibitors (TKI) discontinuation in patients with deep molecular response (DMR) is a paramount goal in the current chronic myeloid leukemia (CML) therapeutic strategy. The best DMR level by real‐time quantitative PCR (RT‐qPCR) for TKI discontinuation is still a matter of debate. To compare the accuracy of digital PCR (dPCR) and RT‐qPCR for  Two decades after the introduction of imatinib (IM), the life expectancy of CML patients treated with TKI has approached to that of the general population3.0 = BCR‐ABL1/ABL1 IS ≤0.1%) to prevent the progression to blastic phase and to obtain a deep molecular response  to gain the opportunity of a treatment discontinuation.The current policy of CML therapy with TKI is to achieve a faster major molecular response (MMR or MRThe results from the Stop IMatinib (STIM) and TWISTER pivotal trials challenged the rule that TKI therapy should be continued lifelong. Since then, both IMAs mentioned above, TFR is one of the most important objectives in CML patients, and recently, TKI discontinuation has become a reality also in clinical practice. In fact, in June 2017, the European Society of Medical Oncology (ESMO) introduced the option of TKI interruption out of the clinical trials too.BCR‐ABL1 transcript by RT‐qPCR, Overall, according to the published data, 50%‐60% of patients with undetectable DMR by RT‐qPCR are expected to lose the DMR.For these reasons, the great majority of patients who undergo TKI discontinuation frequently have a DMR with undetectable levels of Therefore, the RT‐qPCR cannot be considered as an optimal tool neither to select the best candidates for treatment discontinuation nor to design personalized treatment programs, especially in the era of the more potent second‐generation TKI.BCR‐ABL1 transcript quantification by using the Qx100/Qx200 Droplet Digital PCR System (Biorad) and the QuantStudio 3D Digital PCR System (Thermofisher) has been accomplished.In the last years, the digital PCR (dPCR) has emerged as a more sensitive and accurate detection tool of minimal residual disease (MRD) and this increased the interest for its use in the clinical practice.BCR‐ABL1 transcript levels and, possibly, for predicting the patients who are going to relapse after discontinuation of TKI.Although the dPCR is not yet routinely applied for the standard analysis of molecular MRD in CML, preliminary data suggest that it is more sensitive and accurate than RT‐qPCR for monitoring the This study focused on the MRD RT‐qPCR/dPCR comparative monitoring in 142 CML patients treated with TKI and with durable DMR (>2 years), as conventionally assessed by RT‐qPCR, before the enrollment.The aim of this study was to evaluate the reliability and the efficiency of dPCR for a better evaluation of “stable” DMR and for a better selection of the candidates for treatment discontinuation.22.1BCR‐ABL1 transcript, Sokal risk distribution at diagnosis, the first‐ and second‐line TKI treatment, treatment dosage and duration, time to complete cytogenetic response (CCyR), MMR, DMR, and best molecular response (MR).In total, 142 CML patients treated with TKIs  for a median of 99 months (range 14‐215) and with durable (≥2 years) RT‐qPCR DMR  were enrolled into the study, approved by the Ethical Committee of each participating Center. The patients were recruited by 10 Italian Hematologic Centers belonging to the CML GIMEMA  Working Party. The clinical and hematological features of the patients, at the time of the enrollment, included: age, type of Table 4.0 vs the MR4.5‐5.0, and the ones with ≥0.468 vs those with <0.468 BCR‐ABL1 copies/µL, according to the dPCR cutoff value of 0.468 BCR‐ABL1 copies/µL, discriminating the deep molecular responders, as previously reported.The patients were grouped into two pairs of DMR classes: the ones with MR4.0, and 32 (53%) out of 60 cases had MR4.0 undetectable; 82 (58%) had an MR4.5 or MR5.0 and 54 (66%) out of 82 cases were undetectable. On the other hand, according to dPCR results, 31 (22%) out of 142 patients had ≥0.468 BCR‐ABL1 copies/µL, and 111 cases (78%) had <0.468 BCR‐ABL1 copies/µL. . In particular, at the Time point 0, according to RT‐qPCR results, 60 (42%) out of 142 patients had an MRThe median time of concomitant RT‐qPCR/dPCR monitoring was 24 months (range 12‐40). The median follow‐up of the entire cohort is 19 (1‐76).Of note, 111 (78%) out of 142 patients discontinued the TKIs therapy. Fifty (45%) of patients who discontinued TKI therapy were enrolled in different discontinuing trails: ENESTFreedom , ENESTpath , and NP0 in Brescia .Sixty‐one patients (55%) discontinued TKIs therapy out of clinical trials according to the Center policy based on clinical practice and shared patient's decision. All patients were evaluated at the time of discontinuation and after the discontinuation, while 13 cases were monitored also before the discontinuation of treatment Table .During the monitoring period, a total of 556 peripheral blood samples were comparatively analyzed by RT‐qPCR/dPCR.The study was performed according to good clinical practice and Helsinki's Declaration, and the patients gave their informed consent.2.2Conventional RT‐qPCR measurements were carried out at the Reference Laboratory of each participating Center, according to ELN Guidelines.ABL1 as a housekeeping gene.4.0 if ≤0.01% BCR‐ABL1 %IS, MR4.5 if ≤0.0032% BCR‐ABL1 %IS, and MR5.0 if ≤0.001 BCR‐ABL1 %IS. Minimum sum of ABL1 reference gene transcripts, irrespective of whether BCR‐ABL1 was detected or not, 10.000, 32.000, and 100.000 for MR4.0, MR4.5, and MR5.0, respectively.Molecular responses by RT‐qPCR were defined according to the latest laboratory recommendations and using BCR‐ABL1 according to the IS, as recommended by the International Experts Panel's Guide Lines.The participating reference laboratories belonged the Gimema Labnet and accredited by the Gimema Labnet Quality Committee to release the results of RT‐qPCR analysis, since certified for the quantification of 3.0 according to ENESTFreedom and ENESTpath criteria (13 cases) or as “at least two positive RT‐PCR results showing a significant increase , at two consecutive assessments or loss of major molecular response (MMR)” according to what reported by STIM study (98 cases).BCR‐ABL1 transcript by RT‐qPCR and by dPCR at every time point.To the purpose of our study, the loss of the MR was defined as loss of MRThe dPCR analysis was centralized at the Laboratory CREA (Centro di Ricerca Emato‐Oncologico AIL) of the ASST Spedali Civili of Brescia and performed as previously described.Briefly, the cDNA was quantified using a Quant‐iT™ OliGreen® ssDNA kit (Thermofisher Scientific) by Infinite200 (Tecan) and diluted at 50 ng/µL since this quantity resulted the best to obtain a deep quantification and to avoid the saturation of the chip.We prepared 16 μL of reaction mix containing 8 μL of 2X QuantStudio 3D Digital PCR Master Mix (Thermofisher Scientific), 0.8 μL of 20X TaqMan‐MGB‐FAM‐probe assay, 1.1 μL of diluted cDNA, and 6.1 μL of nuclease‐free water (Qiagen). The negative control reaction mix contained 8 µL of 2X QuantStudio 3D Digital PCR Master Mix, 0.8 μL of 20X TaqMan‐MGB‐FAM‐probe assay, and of 7.2 μL nuclease‐free water. One negative control was loaded every thermal cycling run containing samples prepared with the same mix. The reverse transcription negative control reaction mix contained 8 μL of 2X QuantStudio 3D Digital PCR Master Mix, 0.8 μL of 20X TaqMan‐MGB‐FAM‐probe assay, 1.2 μL of reverse transcription blank, and 6.1 μL of nuclease‐free water.BCR‐ABL1 copies/µL of reaction of the replicates.For each sample, we loaded 15 μL of the reaction mixes onto a QuantStudio 3D Digital PCR 20K Chip (Thermofisher Scientific) using the automatic chip loader and the signal was amplified by the following thermocycling profile: 95°C for 8 minutes, 45 cycles at 95°C for 15 seconds, and 60°C for 1 minute, with a final extension step at 60°C for 2 minutes. The amplification was followed by the chips imaging and the secondary analysis performed with the QuantStudio 3D AnalysisSuite Cloud Software. All samples were analyzed twice by different operators and the final results were expressed as means of the number of 2.3t test or Mann‐Whitney and chi‐square were used to compare the continuous and categorical variables, respectively. The t test with Welch's correction has been used in the evaluation of dPCR subgroups as unbalanced sample sizes had to be taken into account . Normality distribution of variables was assessed by a Shapiro‐Wilk test. The type of analysis performed is stated in figures’ and tables’ legends.Clinical and laboratory data were categorized as continuous or categorical variables. The 3.0 or MR4.0 or last control by the Kaplan‐Meier method. Comparison of subgroups was carried out by the log‐rank test. Univariate Cox regression analysis was used to evaluate the association of variables with TFR. Variables included age at diagnosis, sex, previous therapy with IFN, time to MMR and DMR, Sokal class, type of transcript, time to discontinuation, use of frontline second‐ generation TKIs, MR classes considering detectable and undetectable transcript, and dPCR. Multivariable analysis has been performed on dPCR values and, by univariate analysis, the duration of DMR resulted significant.Treatment‐free remission was calculated from the date of TKI withdrawal to the date of loss of MR3.0 after TKI discontinuation. Notably, a cutoff of 0.468 was found to be coincidental with the previous one identified discriminating the deep molecular responders by dPCR.Receiver operator curves analysis was done to recalculate the optimal dPCR cutoff value to discriminate the patients with DMR and lower probability to lose MR3This study aimed to comparatively evaluate the efficiency and the reliability of the RT‐qPCR and dPCR for the recognition of an accurate and “stable” DMR and selection of candidates to TKIs discontinuation.For that purpose, 142 CML patients with a durable RT‐qPCR DMR (≥2 years) were comparatively monitored by RT‐qPCR/dPCR every 3‐4 months for a median time of 24 months (range 12‐40), from the time of enrollment  thereafter (last follow‐up). Furthermore, 111/142 patients underwent treatment discontinuation and were valuable for TFR.BCR‐ABL1 minimal residual disease, as conventionally assessed by RT‐qPCR, were correlated with the dPCR values measured at the same time points.First, the levels of BCR‐ABL1 results were grouped into MR4.0, MR4.5, and MR5.0class (“X axis”). When the same samples were analyzed by dPCR, different levels of BCR‐ABL1 copies were measured (“Y axis”). The numbers of BCR‐ABL1 copies/µL in each MR class resulted heterogeneous and widely dispersed, even the relative medians and range were progressively decreasing moving from the MR4.0 class to MR5.0class. The difference was statistically significant between the MR4.0 class and the MR4.5 (P = 0.0104) or the MR5.0 class (P = 0.0032), but not between the MR4.5 and the MR5.0 classes (P = 0.2021) , while patients with dPCR <0.468 copies/µL presented lower levels of BCR‐ABL1 copies and their median value was of 0.171 (range 0‐0.467) , while patients with dPCR ≥0.468 copies/µL presented higher levels of BCR‐ABL1 copies and had a median value of 0.393 copies/µL (range 0‐2.195). The differences at Time point 0 and the variations during the follow‐up period did not result statistically significant between the two groups.No difference between the monitoring course of MRg Figure D. During4.0 vs MR4.5‐5.0 class, and ≥0.468 vs <0.468 BCR‐ABL1 copies/µL group , grouped into two pairs of classes according to the RT‐qPCR and dPCR results at Time point 0: the MR4.0 vs MR4.5‐5.0) were superimposable, while the patients with dPCR <0.468 had a longer DMR duration (P = 0.0250) and more frequently belonged to MR4.5‐5.0 (P = 0.0442) classes compared to patients with higher dPCR values  and 66/111 (59%) with MR4.5‐5.0 (45/66—68%—presented BCR‐ABL1 transcript undetectable). These patient groups, MR4.0 vs MR4.5‐5.0, did not show any significant statistical difference in clinical and hematological characteristics  patients discontinued the TKI treatment after a median time of 97 months of TKI therapy (range 33‐194). MRD levels assessed by RT‐qPCR at the time of treatment discontinuation identified 45/111 (41%) patients with MRP = 0.0250)  had ≥0.468 and 86/111 (77%) had <0.468. No significant statistical difference was observed in clinical and hematological characteristics, except for a longer overall DMR duration in patients with dPCR <0.468 (76 vs 54 months. 3.0 (10/24) or the MR4.0 (14/24) (Table 4.0 (20%) and 15/66 (23%) were in MR4.5‐5.0 (P = 0.8100); according to dPCR, 12/25 (48%) and 12/86 (14%) had BCR‐ABL1 values ≥0.468 and <0.468, respectively (P = 0.0003)  patients lost the MRP = 0.6510). An additional Kaplan‐Meier analysis was carried out on detectable MR4.0 vs total MR4.5‐5.0 (Supplementary File 3A), on MR4.0‐4.5 detectable and total MR5.0 . In any cases, the consideration of the detectability of the transcript was not statistically significant. On the contrary, a dPCR value below 0.468 BCR‐ABL1 copies/µL at the time of discontinuation highly stratified patients with lower probability to lose MR Indeed, the TFR at 1 and 2 years was 85% and 83% patients presenting dPCR values below the defined cutoff compared to 59% and 52% of patients with higher dPCR values at the time of discontinuation, respectively (P = 0.0017)  and dPCR  resulted significantly predictable of TFR maintenance in univariate analysis. By multivariate analysis, only dPCR retained its significant value  lost MR. On the other hand, 3/13 (23%) presented stable ≥0.468 BCR‐ABL1 copies/µL by dPCR and 2/3 (67%) lost MR (P = 0.1245).In 13 cases, a comparative monitoring of DMR by RT‐qPCR/dPCR was possible for at least 24 months before TKIs discontinuation. In these cases, 10 out of 13 (77%) had stable <0.468 4BCR‐ABL1 molecular levels. Moreover, dPCR is potentially able to improve the recognition of DMR and the selection of the candidates to TKI treatment discontinuation.This study, based on a multicentric cohort of CML patients with durable DMR (≥2 years), shows that the dPCR appears to be more accurate and sensitive than conventional RT‐qPCR for detecting and monitoring the BCR‐ABL1 transcript levels in 142 CML patients having a median duration of TKI treatment and DMR of 8.3 years and 5.9 years, respectively. These long periods could explain why a very high TFR rate (80% at 1 year) was observed in our cohort of patients, in comparison to an expected rate of 50%‐60%, usually reported in the literature.The study reports on a longlasting comparative RT‐qPCR/dPCR monitoring of 4.5 before discontinuation in order to reach the TFR. Moreover, a great uncertainty still remains for the level of MR.Indeed, ESMO recommends at least 5 years of TKI therapy combined with ≥2 years of DMR before TKI discontinuation,4.0 was identified as dissecting the patients with the lowest probabilities to lose the TFR. 5.0 undetectable before IM or nilotinib discontinuation had significantly higher probabilities to maintain the TFR than those in MR4.5. 4.5 undetectable levels of BCR‐ABL1 quantified following the IS system. Considering the pivotal clinical trials concerning TKI discontinuation, in EURO‐SKI study, a threshold of 3.1 years of MRBCR‐ABL1 transcript are additional elements that may play an important role. In fact, because of its low sensitivity, RT‐qPCR could not discriminate the depth of response in patients with undetectable BCR‐ABL1 transcript and a patient with undetectable MR4.0 could belong to MR5.0.It clearly appears that the sensitivity and the detectability of In our experience, the majority of the samples classified as DMR had an undetectable level of transcript, confirming the low RT‐qPCR capability to detect low levels of MRD while the dPCR appeared to overcome this important bias thanks to its high sensitivity and accuracy, allowing a better selection of patients eligible for TKI discontinuation. Our data, according to Takahashi and colleagues,BCR‐ABL1 copies/µL by dPCR, it was evident that both the patients with MR4.0 and those with MR4.5‐5.0 had different levels of MRD .These data suggest that the dPCR may be able to identify a set of optimal and stable molecular responders based on the levels and the stability of MRD  of them lost the MR3.0 or MR4.0.In our cohort of patients, the TFR rate was higher than the one currently reported in the IM and second‐generation TKI discontinuation studies3.0 or MR4.0 loss in the cases with <0.468 BCR‐ABL1 copies/µL by dPCR resulted significantly lower (14%) than one of the cases with ≥0.468 BCR‐ABL1 copies/µL (48%). Indeed, the probability to predict the TFR was 86% for the cases with <0.468 BCR‐ABL1 copies/µL and 52% for the cases with ≥0.468 BCR‐ABL1 copies/µL, and this difference was statistically significant (P=0.0003) .Looking at the clinical and hematological characteristics of these two groups of patients, they resulted comparable, except for the longer duration of DMR  was observed between the rate of MR loss of patients with MR4.0 (detectable or undetectable) vs MR4.5‐5.0 (detectable or undetectable) (Figure Interestingly, these data first showed a potential correlation between the duration and stability of DMR and a measurable reduction of molecular levels of BCR‐ABL1 transcripts and this has to be considered particularly relevant in the era of the more potent second‐generation TKIs. There is general agreement on considering RT‐qPCR not sufficiently adequate to measure low levels of BCR‐ABL1 detection and quantification for a personalized management of CML patients, other efforts have to be performed in the standardization of the procedures and in the alignment of the results generated by the different dPCR platforms.In order to routinely apply dPCR for BCR‐ABL1 transcripts in the cases with undetectable DMR by RT‐qPCR; that patients belonging to the same MR class do not have the same quantities of BCR‐ABL1 transcript when measured by dPCR; and that dPCR may improve the recognition of “stable” DMR, hence contributing to a better selection of candidates to TKIs discontinuation.No firm conclusion can be drawn and, certainly, the higher accuracy, sensitivity, and predictive value of dPCR have to be prospectively confirmed on a larger cohort of cases. However, this study practically tested the potential of dPCR and highlighted that this technique is able to detect measurable quantities of The authors declare no competing financial interests.DR, SB, and MM designed the study; SB, CZ, and EDE performed the dPCR analysis; MM, MF, MD, CC, AI, CB, M. Bonifacio, ET, MT, CD, GG, FS, FDR, M. Bergamaschi, MG, LF, MDD, and EA enrolled the patients and collected the clinical data; NP and EDE performed the statistical analysis; SB, MM, NP, EDE, CZ, and DR wrote the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file.


The likelihood of donating blood changes over the life course, with life events shown to influence entry to and exit from the donor population. While these previous findings provide valuable insights for donor management, blood collection agencies need to be cautious about generalizing findings to other countries as blood donor behaviour is context‐specific. To examine cross‐country variations in donor behaviour, the repeatability of a previous Dutch study on life events and blood donor lapse is examined by using a sample of Danish donors.n = 152 887). Logistic regressions were conducted to examine the association between life events in 2009–2012 and blood donor lapse in 2013–2014.Register data from Statistics Denmark was linked to the Scandinavian Donations and Transfusions database  donors lapsed. Childbirth and losing a job increased the lapsing risk by 11% and 16%, respectively, while health‐related events in the family  decreased the lapsing risk by 5%, 7% and 9%, respectively.Life events are associated with donor lapse of Danish donors. These results are comparable to previous findings from the Netherlands , with two thirds of the associations being in the same direction. Differences between study results were mainly related to effect sizes and demographic compositions of the donor pools. We argue contextual factors to be of importance in blood donor studies. Blood donation and its antecedents are shown to be context‐specific, with contextual factors such as collection practices and cultural differences influencing blood donor attitudes and behaviour To contribute to these country studies and identify more universal and possible context‐specific mechanisms for blood donor behaviour, we compared the association between life events and blood donor lapse between Denmark and the Netherlands by testing the repeatability https://osf.io/9chtq/).In order to test the repeatability of previous findings, we selected data, procedures, measures and statistical analyses based on methodological decisions from the previous study  and non‐medical reasons , were excluded from the sample. To retrieve this information, we used the ineligibility criteria list from the Danish blood bank Blood donor lapse was defined as not making a donation for 24 consecutive months Inclusion of the six life events of interest was based on the previous analyses among blood donors from the Netherlands. Information on the occurrence of health‐related events among family members was retrieved from SCANDAT (i.e. blood transfusion) and SD . Based on CRNs, we matched the donor to parents, children and siblings and then matched these family members to transfusion, hospitalization and death records. Using this information, we created a time‐dependent dichotomous variable representing whether the donor experienced a health‐related event in the family . Information on childbirth was extracted from SD by matching newborn children to their parents using CRNs. We transformed these data such that the individual record of the donor included the CRN of their newborn child and recoded this as a time‐dependent dichotomous variable representing whether a child was born to the donor . Information on donors losing or starting a job was retrieved from SD. Time‐dependent dichotomous variables were created by tracking changes in employment status: from having a paid job to unemployment , and from unemployment to having a paid job .To further explore the association between life events and donor lapse, we included two possible mediating mechanisms in the models. The total number of weekly working hours was included as a proxy for the available time to donate, with a higher number of weekly working hours representing higher costs to donate blood as time becomes a scarcer resource when working hours increase. Having other blood donors in the family was included as a proxy for the influence of social networks. Based on CRNs, we matched the donors to their parents, children, siblings and spouse and then matched the CRNs of these family members to SCANDAT to create a dichotomous variable representing whether the donor knew donors in the family .Sex, age, educational level and total number of previous blood donations were added to the model as control variables. Educational level was coded as lower, medium and higher education according to the International Standard Classification of Education (ISCED) 2011 n = 117 266), unemployed donors for starting a job (n = 7570), employed donors for losing a job (n = 129 836), and all donors for the health‐related events (n = 152 887). We then obtained predicted probabilities for donors who were at risk for the event and did or did not experience the event, keeping the other variables in the model at their means.Analyses were conducted using Stata 15 . Conducting logistic regression analyses, we estimated the association between life events and donor lapse. Donor inclusion requirements included having been ‘at risk’ for experiencing a life event of interest, that is women aged ≤45 and men aged ≤55 for childbirth (ZMediation) As to examine whether childbirth differently affected donor lapse for men and women, we added an interaction term to the model. Mediation analyses (i.e. n = 69 079) lapsed in 2013–2014. Life events occurring most often to the donors were a serious disease in a family member , childbirth  and a blood transfusion for a family member . Table Of the total donor sample, 45·2% (P < 0·001). We found no evidence of a mediating role of knowing other blood donors in the association between a blood transfusion and donor lapse (Table Donors experiencing a blood transfusion for a family member were 5% less likely to lapse than donors who did not experience such an event (OR (95% CI) = 0·91 (0·88–0·94), se Table . These rP < 0·001). Subsequent mediation analysis showed no evidence of knowing other donors as an explaining mechanism for the association between a serious disease and donor lapse. These results were different from previous findings, where no significant difference was found among Dutch donors in their lapsing risk after a serious disease in the family.Donors experiencing a serious disease in the family were 7% less likely to lapse than donors who did not experience a serious disease in the family (OR (95% CI) = 0·88 (0·86–0·90), P < 0·001). As was the case in the other health‐related events, further analysis revealed no evidence of knowing other donors being a mediating factor. The association between a family member's death and donor lapse is comparable to the one found among Dutch donors (i.e. an 8% increase in lapsing risk).Donors having lost one of their family members were 9% less likely to lapse than donors who did not experience a family member's death (OR (95% CI) = 0.84 (0·81–0·87), Since health‐related events were correlated to each other (Appendix C), with people receiving a blood transfusion being more likely to suffer from a serious disease and having a higher mortality risk, we explored their interrelationship in the negative association with blood donor lapse. Table P < 0·001). While childbirth increased the lapsing risk for both men (OR (95% CI) = 1·18 (1·13–1·23), P < 0·001) and women (OR (95% CI) = 1·24 (1·18–1·29), p < 0·001), this association was significantly stronger for women than men. Compared to the study from the Netherlands, effect sizes differ considerably with Dutch donors who got a child being 56% more likely to lapse than donors who did not get a child. Moreover, in the Netherlands no significant difference was found between men and women in their lapsing risk after childbirth.Donors who got a child were 11% more likely to lapse than donors who did not get a child within the same period (OR (95% CI) = 1.21 (1·18–1·25), No difference was found between donors who started a job and donors who stayed unemployed in their subsequent lapsing risk. In the absence of this main effect, we did not perform further mediation analyses and the role of increased working hours in donor lapse. These results differ quite strongly from earlier results in the Netherlands. Starting a job was positively associated with lapse among Dutch donors, with donors who started a job being 22% more likely to lapse compared with donors who stayed unemployed.P < 0·001). Mediation analysis showed that knowing fewer other blood donors was a significant mediator in the model, yet explaining only 3% of the variance in the relation between losing a job and donor lapse . These results repeat previous results from the Netherlands, but effect sizes differ slightly as Dutch donors were 35% more likely to lapse after losing their job, while the same effect was found for the small mediating role of knowing other donors in explaining the association between losing a job and donor lapse.Donors who lost their job were 16% more likely to lapse than donors who stayed at their job during the same period (OR (95% CI) = 1·32 (1·25–1·39), Using longitudinal data from SCANDAT and SD, we conclude that life events related to childbirth and work status, as well as health‐related events in the family, are associated with blood donor lapse. Childbirth and losing a job increased the risk of donor lapse, with childbirth being more detrimental for future blood donations of women than men. In contrast, health‐related events in the family decreased the risk of donor lapse. Once more, the likelihood to donate blood was shown to vary across the donor career, thus illustrating the dynamic nature of blood donor behaviour.For a large part, these results are in line with previous findings from cross‐sectional blood donor studies based on self‐reports. Time constraints due to childbirth and family responsibilities appear to be among the main reasons for donors to discontinue donating blood We also found some discrepancies compared with previous studies. Time constraints due to work are a common self‐reported reason for donors to stop donating blood We found the associations to be comparable between Denmark and the Netherlands. Of the events studied here, two thirds showed the same associative directions with donor lapse, while none of them showed opposite directions. Moreover, half of all confidence intervals showed an overlap between the studies. One might assume that certain  mechanisms are universal factors associated with blood donor behaviour across the donor career in Western high‐income countries. Childbirth constraints a person's available time However, we also found some differences between the study results, mainly related to donor sample compositions and the magnitude of effect sizes. Regarding contextual differences between Denmark and the Netherlands, donor samples might differ as a result of organizational variation in blood collection regimes. Following Healy's categorization Contextual differences other than those exerted by BCAs might contribute to differences in effect sizes. For instance, regulations regarding parental care after childbirth differ significantly between countries. In the Netherlands, fathers have only two days off after childbirth Regarding data differences, donor samples and effect sizes could differ as a result of the sole use of register data in the current study as opposed to the combination of register and survey data in the study from the Netherlands. The current study includes all active donors, while the previous study only included donors participating in both waves of the survey. Analyses showed non‐responders being more likely younger, male, lapsed donors having made a lower number of average donations, possibly explaining why the Danish sample shows a higher proportion of donors with these characteristics. Moreover, register data eliminates the possibility of introducing recall bias and the telescoping effect Although this study provides valuable insights on cross‐country variations in blood donor behaviour, providing more accurate and reliable estimations, our study also has some limitations. Registries typically provide more accurate and complete sources of data, but do not include relevant variables related to subjective perceptions and other individual factors such as the perceived difficulty to plan a donation and talking to other donors. Linking population‐wide registries to results from motivational questionnaires for a subset of the donor population would allow for an even better understanding of donation decisions across the blood donor career, for example understanding interrelationships between various health‐related events and their differences between countries. For now, we used variables serving as proxies for these missing variables, corresponding to other mediators from the Dutch theoretical framework: weekly working hours and the extent to which donors know other blood donors. In this way, we have used the data without introducing too many incongruences.While using different contexts and data in replication studies, it is difficult to pinpoint exactly which differences explain inconsistencies between study results. We acknowledge this limitation. Our aim was to examine the association between life events and donor behaviour in Denmark. The comparison with the Netherlands shows that contextual and data difference could yield various study outcomes. Researchers as well as BCAs need to be aware of such differences and its implications in interpreting international study results.The influence of life events on blood donor behaviour across the life course so far has been studied in the Netherlands, Germany and Denmark. While certainly there are differences between these countries, they also are quite comparable with respect to collection systems as well as socio‐economic circumstances and cultural orientation We suggest future studies to focus on underlying mechanisms explaining the relation between life events and donor behaviour. While the current study and the study from the Netherlands showed social and practical concerns to partially explain why life events impact on donor lapse, the bulk of these associations is still unaccounted for. For instance, neither of the two studies found evidence for social mechanisms playing a role in the relation between health‐related events and donor lapse, nor could we conclude on the different results for starting a job and donor lapse.Further understanding of underlying mechanisms is especially important since the occurrence of specific life events that affect donors' personal resources are increasing (e.g. higher number of labour market transitions due to the rise of temporary contracts) In‐depth studies of these associations would therefore be of practical interest in making evidence‐based decisions on the development of targeted donor retention strategies. For instance, sending postcards to donors who recently got a child might increase engagement between donors and blood banks. Promoting personal donation motivations  might subsequently increase return rates in donors with heightened lapsing risks. Implementing targeted retention strategies require higher levels of personal contact between donors and blood banks. This will become more feasible in the coming years when services such as online donor portals become more easily available, creating opportunities for more intensive information‐sharing, subsequently increasing opportunities for segmented blood donor management.Moreover, exploring work‐home‐donation distances might increase understanding in why people change their donation decision after starting or losing a job, and assist BCAs in deciding on where and when to open their donation locations. Strategically, positioning of collection sites might be effective in recruiting underrepresented groups of young, male and ethnically diverse donors, which is essential in maintaining a sufficient and matching blood supply The authors declare that they have no conflicts of interest relevant to the manuscript submitted to Vox Sanguinis.TP, EM, RB, WK and HU contributed to the study design. TP developed the main conceptual ideas and wrote the manuscript with support from all other authors. TP wrote the code and performed the statistical analyses, based on previous work from SA and with additional analyses by Andreas Stribolt Rigas . All authors provided critical feedback on the interpretation of the outcomes and the final version of the manuscript.


In this article, we present two medicolegal cases illustrating medical and diagnostic pitfalls that can lead to litigation for missed testicular torsion. Testicular torsion (TT) is a urologic emergency with potentially devastating consequences and costs, for providers and patients alike. TT occurs in approximately 4.5 per 100,000 males under the age of 25. While uncommon, TT is the third most common cause of medical malpractice suits in this demographic. As a consequence of varying presentations and physical exam findings, and diagnostic imaging subject to individual interpretation, this time-sensitive diagnosis may be missed by emergency department providers. Delays in diagnosis significantly increases the morbidity associated with TT, and 31.9%–41.9% of such cases result in testicular loss. The average reported settlement for TT malpractice litigation is $60,000. This article discusses two actual malpractice cases involving TT and provides insight and caveats to ensure an optimal evaluation and diagnostic approach to this often-elusive condition. A 16-year-old male arrived at the emergency department (ED) complaining of right lower quadrant abdominal pain with some associated nausea and vomiting. The emergency physician (EP) completed an abdominal exam, obtained labs, an abdominal ultrasound, and a computed tomography (CT) of the abdomen and pelvis. These were all unremarkable. Nevertheless, a surgical consultation was obtained to further evaluate for appendicitis. The surgeon did not feel appendicitis was present, and the patient was discharged. A genital exam was never performed. The following day, the patient returned with right testicular pain. He was immediately taken to the operating room for scrotal exploration and required a right orchiectomy. A lawsuit was initiated for failure to perform a genital exam, and failure to consider testicular torsion (TT) in the diagnosis. Before trial a settlement of $300,000 was reached.A 14-year-old male was taken to the ED after awakening with abdominal pain. Laboratory studies, an abdominal CT, and a scrotal ultrasound were done. The CT was read as suggestive of appendicitis and thus a surgical consultation was obtained. The surgeon did not feel that appendicitis was present. The radiologist reviewed the ultrasound and diagnosed epididymitis. Based on the studies the EP discharged the patient on antibiotics. Three days later the patient awoke with testicle pain and was taken to a different ED where he was diagnosed with TT and received an orchiectomy. A review of the original ultrasound revealed there was decreased blood flow to the testicle. The patient litigated claiming that the diagnosis should have been made on the first visit and the testicle could have been salvaged. The case was solely against the EP and not the radiologist. There was testimony from the EP that he had ordered the “gold standard” test and relied on the interpretation by radiology. After trial, the jury awarded a $500,000 verdict.There is no standard presentation for TT. Testicular torsion presentation can present similarly to epididymitis. A significant number of proven TT cases present with gradual onset discomfort, whereas alternative causes of scrotal pain, such as epididymitis, can present with sudden discomfort in up to 51% of cases.EPs should be hesitant to decide the absence (or presence) of TT based solely on clinical exam. Presence or absence of cremasteric reflexes, scrotal edema/erythema, pain along the upper pole of the testicle or epididymis, enlarged epididymis, transverse lie, Prehn’s sign (pain relief with examiner lifting testicle), and retraction of testicle all fail to give a definitive answer.1The presence of a cremasteric reflex has historically been touted to rule out TT. This unfortunately is not completely true. Several case series, although mostly small, have reported TT with intact cremasteric reflexes.1,6 This is because a scrotal ultrasound can be misread as normal by radiologists.,6 In general, high resolution ultrasonography has a sensitivity of 96% but is not perfect.6A scrotal ultrasound, the “gold standard” test, can be very helpful, although it is not foolproof. Lawyers will argue that “one simple and available test” could have been ordered and made the diagnosis. However, upon review of cases involved in litigation, we found that obtaining an ultrasound did not correlate with a more successful defense.The time window for possible salvage and survival of a torsed testicle is commonly thought to be 6–8 hours.7A review of jury verdict reports in cases of TT was done to identify causes of litigation and factors contributing to verdicts or settlements.2The sudden onset of severe, unrelenting testicular pain is typically held to be diagnostic of TT. This is not the case, however, for a small but significant number of patients with a torsed testicle. The little-recognized fact that TT patients can present with minimal or no pain has proven to be a medicolegal pitfall for EPs.A recent article highlights seven cases of TT and raises a serious liability concern. In all of these patients, there was a period of freedom from pain, or much decreased pain after the initial onset of symptoms (“pain honeymoon”). The diagnosis would be very easy to miss in this clinical scenario. The mechanism is thought to be one of compression of the nerves as they travel in the spermatic the cord with resultant paresthesia and anesthesia.7Missed TT is a frequent source of successful litigation against EPs. There are many traditional paradigms in the areas of history, physical exam, imaging studies, and clinical course that can lead to diagnostic failure. Given these clinical uncertainties and high risk for testicle loss, EPs should routinely document a scrotal exam for young males with lower abdominal pain, have a low threshold for ultrasound imaging with any reasonable suspicion, and use a liberal threshold for urological consultation, if available.Successful litigation for testicular torsion often occurs due to failure to do a genital exam in patients with abdominal pain.Successful litigation for TT often occurs by a failure of the radiologist to notice pathology on scrotal ultrasound. Nevertheless, the EP is held responsible. A urologist should be involved when there is high clinical suspicion for TT in the face of a “negative” ultrasound.Testicular salvage after 24 hours of torsion is still 18%, and physicians should aggressively pursue the diagnosis even in a delayed presentation.The overwhelming majority of malpractice claims were failure of diagnosis, and 2/3 of these cases were diagnosed as epididymitis.not reassure the provider that the diagnosis is not likely.Recent reports described cases of “honeymoon” absence of pain in TT. The improvement of testicular pain or its absence after initial onset should Documented patient informed consent and/or Institutional Review Board approval has been obtained and filed for publication of this case report.


Recent studies reported high diagnostic value of a second generation of cerebrospinal fluid (CSF) Real-Time Quaking-Induced Conversion (RT-QuIC) assay across a wide spectrum of human prions. Here, we applied this improved QuIC (IQ-CSF) for highly efficient detection of TSEs prion protein in goat cerebrospinal fluid. IQ-CSF sensitivity and specificity were evaluated on CSF samples collected at disease endpoint from goats naturally and experimentally infected with scrapie or bovine isolates of C-BSE and L-BSE, respectively. Next, CSF samples collected from L-BSE infected goats during pre-symptomatic stage were also analysed. PrPL-BSE associated seeding activity was detected at early time points after experimental inoculation, with an average time of 439 days before clinical symptoms appeared. Taken together these data are indicative of the great potential of this in vitro prion amplification assay as ante-mortem TSE test for live and asymptomatic small ruminants.Since 2005, two cases of natural bovine spongiform encephalopathies (BSE) have been reported in goats. Furthermore, experimental transmissions of classical (C-BSE) and atypical (L-BSE) forms of BSE in goats were also reported. To minimize further spreading of prion diseases in small ruminants the development of a highly sensitive and specific test for  Sc) form of the cellular prion protein (PrPC)2. PrPSc induces the polymerization and conformational conversion of PrPC to its infectious form5 or, in a variety of in vitro conversion assays6, into alternative forms of the prion protein which are, similarly to PrPSc, partially resistant to digestion with proteases (PrPRes). The pathogenic isoform of the prion protein is therefore a marker associated to TSEs which acts as a seed allowing the ultrasensitive detection of PrPSc using in vitro prion amplification reactions such as Real-Time Quaking Induced Conversion (RT-QuIC)7.Transmissible Spongiform Encephalopathies (TSEs) or prion diseases are fatal neurodegenerative diseases that include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and chronic wasting disease (CWD) in cervids. The infectious agent responsible for these diseases, the prion, appears to be composed primarily of an abnormal, misfolded and oligomeric  and atypical (L-BSE) forms of BSE in goats were also reported11. An important goal to minimize further spreading of small ruminant TSEs is the development of an assay for highly sensitive and intra-vitam detection of prions in infected, but not clinically sick animals. Currently, small ruminant TSEs are definitively diagnosed by post-mortem examinations of brain stem to detect PrPSc 12. RT-QuIC assays can rapidly detect sub-infectious levels of prion seeding activity and have been used successfully to detect multiple human, cervid, ovine, hamster, and mouse prion strains in a variety of biological tissues, such as skin13, cerebrospinal fluid18, saliva19, blood23, and nasal brushings24, showing that this test has the potential of being used for ante-mortem TSE diagnosis. Important studies on human cerebrospinal fluid from patients with sporadic Creutzfeldt-Jakob disease (sCJD) have shown that RT-QuIC has the potential to improve ante-mortem diagnosis for this disease, with a high degree of specificity29. Results obtained by several laboratories with the first generation of this assay (referred to here as previous QuIC-CSF [PQ-CSF]), mainly using full-length (23–231) hamster recombinant PrP (rPrPSen) as the substrate, demonstrated a very high specificity but a suboptimal sensitivity, especially in sCJD subtypes associated with PrPSc type 231. However, Orrù et al.25 recently introduced an improved, second generation RT-QuIC for CSF (referred to here as improved QuIC-CSF [IQ-CSF]) assay which uses a truncated form of hamster rPrPSen (amino acids 90–231) as the substrate, higher incubation temperatures, and the addition of SDS. Initial evaluation of the IQ-CSF assay indicated greater analytical and diagnostic sensitivity, and markedly shorter testing times29.The spread of BSE agent to small ruminants is a major issue in the surveillance of TSEs because BSE passage into a new host may change strain properties, make it difficult to recognize the original strain, and increasing the risk of epidemic spread32. Still, it remains important to understand the time course of prion seeding activity accumulation in cerebrospinal fluid of prion-infected small ruminants in order to develop an early, sensitive, and specific test for ante-mortem and pre-clinical detection of PrPSc. Furthermore, to date there is no information on experimental conditions to amplify goat BSE prion strains by RT-QuIC. Moreover, IQ-CSF conditions have not been previously applied to animal CSF samples. Here, we report for the first time sensitive detection of different small ruminant prion strains in brain and CSF of TSE-infected goats by RT-QuIC assays. Furthermore, intra-vitam detection of preclinical L-BSE-infected goats at early time points is also described. Taken together these data are indicative of the great potential of this in vitro prion amplification assay as ante-mortem TSE test for live and asymptomatic small ruminants.Studies have demonstrated that CSF tests that measure alterations in total PrP levels and prion seeding activity by RT-QuIC, may be useful in the identification of pre-clinical prion casesSc before proceeding to subsequent RT-QuIC analyses, Western Blot (WB) analyses were carried out on brainstem collected from experimental C-BSE and L-BSE infected goats  rPrP4 h Fig. . As prev−8  were positive only by 40 h incubation  amyloid formation (rPrPspon) was observed in only one of the quadruplicate reactions seeded with normal brain tissue dilutions at 70 h (data not shown). Therefore, we chose 60 h as the reaction cut-off.Next, RT-QuIC end-point dilution analysis of three representative clinical TSE-infected goat brain homogenates was performed to evaluate the detection sensitivity for each goat prion strain previously tested. We saw positive reactions with all prion strains using infected brain tissue dilutions down to 10−8 Fig. , althougion Fig. . This seSen substrate. RT-QuIC reactions were seeded with 10−4 to 10−8 brain tissue dilutions and incubated for 60 h using the same experimental conditions described for Ha-S rPrPSen. Both goat BSE prion strains induced conformational conversion of Ha rPrPSen 90–231 giving the fastest response reactions with the L-BSE seed , we found initially that when pure goat CSF was analyzed, positive reactions occurred only after 40 h. However, pure CSF specimens lack SDS that was present in the previously studied brain homogenates, so we then applied RT-QuIC using the combination of the rHaPrPSen 90–231 substrate, 55 °C, and 0.002% SDS (referred to here as improved QuIC-CSF [IQ-CSF] conditions) to an initial panel of CSF samples collected from two symptomatic L-type BSE-infected goats and two patients affected with sCJD  were tested. These measurements revealed equivalent analytical sensitivity between CSF samples collected at clinical onset and more than one year before clinical symptoms appeared. In each case positive reactions occurred until to 0.46 µl of pure CSF seeded Fig. .Sc in each time point before clinical onset, we tested endpoint serial dilutions of these CSF samples collected from 2 L-BSE-infected goats. Finally, using Spearman-Karber analysis of the data, we estimated the dilution of pure CSF required to give 50% positive replicate wells under IQ-CSF conditions (the 50% seeding dose [SD50]). Initial Log SD50 per µl of CSF was measured at 926 dpi with an average value of 0.45. Soon thereafter, seeding activity began a nearly log-linear decrease down to a minimum of 0.2 LogSD50/µl CSF , 1080 dpi, 1200 dpi, and at clinical onset are showed in Fig. CSF Fig. . This lePRNP coding sequence which can delay the incubation period and PrPSc accumulation36. A recent study by Madsen-Bouterse et al.37 revealed that the polymorphisms in caprine PRNP can also affect the sensitivity of PrPSc detection in brain samples by anti-prion mAb-based immunoassays such as IHC and Western blot analysis. It therefore becomes important to have a rapid, sensitive, PRNP polymorphism-independent as well as antibody-independent detection test to diagnose goat TSE. A previous study already demonstrated sensitive and specific detection of classical scrapie prions in goat brains with different genotypes by RT-QuIC38 but to date there is no information on experimental conditions to detect BSE prion seeding activity in small ruminants. We showed that RT-QuIC can be applied successfully to a variety of brain samples from experimental and natural TSE-infected goats including scrapie and BSE. Our findings indicate that by using the Ha-S rPrPSen substrate, RT-QuIC assays can sensitively detect both C-BSE- and L-BSE-associated seeding activity in less than 24 h. Furthermore, conversely to cattle C-BSE33, goat C-BSE prion strain was able to seed conversion of hamster 90–231 rPrPSen substrate. These data confirm that RT-QuIC can also be used in prion strain typing, as different strains show slight differences in RT-QuIC response42.Identification of scrapie or BSE status in goats can be complicated due to the existence of several polymorphisms in the ante-mortem diagnosis. In small ruminants TSE is suspected diagnostically on the basis of clinical examination of symptomatic individuals and is confirmed post-mortem by neuropathological analysis and immunochemical detection of PrPSc in the central nervous system12. One important limitation to this approach is the sensitivity of the detection system, because amounts of PrPSc high enough to be revealed by conventional methods are only present in the brain at late stages of the disease. However, animal transmission studies show that infectivity is present at a relatively early stage of the incubation period and gradually increases as the disease progresses44. Our data indicate that RT-QuIC can amplify otherwise undetectable quantities of PrPSc from CSF of symptomatic and asymptomatic goats. Here, we demonstrate, for the first time, that using recombinant hamster 90–231 PrPSen as substrate in combination with IQ-CSF, the diagnostic sensitivity of RT-QuIC was clearly improved in CSF samples of both natural scrapie and experimental BSE infected goats, allowing rapid and specific prion seeded polymerization in less than 40 hours. One great advantage of this improved assay is that using hamster 90–231 rPrPSen substrate which can be universally applied to various animals with different PrP sequences45 could avoid the potential problem of polymorphism in sheep or goat. Furthermore, the longitudinal study on experimental L-BSE goats showed that as early as two years and half after infection, i.e., at two fifths of the incubation time, it is possible to distinguish infected from non-infected animals by IQ-CSF analysis. In particular, highly efficient detection of PrPL-BSE associated seeding activity was revealed respectively 574, 336 and 407 days prior to onset of clinical signs. Also, the titration of seeding activity in Fig. Sc was maintained during the remainder of the asymptomatic stage. Similar time course in brain PrP deposition was observed from pre-clinical and clinical BSE cases reported by Bannach et al.46. Collectively, these data provide the first indications of the kinetics of prion accumulation in the CSF of prion-infected goats. The observation that PrP seeding activity can be detected in the CSF during early preclinical stages of the disease is consistent with a previous report showing positive RT-QuIC signal in the CSF of intracerebrally scrapie-infected hamsters before the clinical onset47. Clearly, the accumulation time course was highly dependent upon the route of prion inoculation. Indeed, the same previous study already demonstrated that after intralingual scrapie inoculation, seeding activity appeared in hamsters CSF only with the onset of clinical signs, well after higher-level accumulation of seeding activity in CSF of intracerebral challenged animals47. However, data from recent study on sheep CSF of preclinical and clinical naturally occurring scrapie confirmed that alterations in PrP levels and conformation are primary events in the pathology of prion diseases preceding neuronal damage.In fact, one of the main problems related to the management of animal TSEs is the lack of rapid and sensitive tests for ante-mortem CSF test here described is one of few ante-mortem screening methods now available to detect asymptomatic TSE-infected goats48. It could represent a more rapid and sensitive approach to identify TSE-infected flocks, assaying symptomatics and all genetically susceptible animals. Infected animals could be eliminated at an early stage, with a rapid decrease of the potential environmental infection load. Such a surveillance system will also allow decreases the genetic selection pressure, maintaining some genetic variability in the populations, as desired by farmers.Scrapie is still widespread, and the only available eradication measure is the genotype-based eradication program. However, this plan only shows effects in years and requires a high level of compliance by farmers, which is not always the case. Although, furthers studies are necessary to assess the ability of RT-QuIC to detect prion seeding activity in biological samples of more diagnostic interest as blood and excreta, the Overall, our data suggest that CSF prion seeding activity may be related to early prion pathological events and may allow the design of a diagnostic test for preclinical and clinical animals, minimizing the risk of potential exposure to others in the herd.11. The physical and neurological status of the animals was monitored monthly by a board certified veterinary neurologist. For this purpose, a clinical examination protocol, previously used for sheep49 was applied. CSF was collected from the lumbosacral site of each animal, while it was standing, as described by Mayhew et al.50. The animal was sedated with 0.05 mg/Kg xylazine  IV. The collection site (7 × 7 cm) was clipped, surgically prepared and locally anaesthetized with 2.0 ml of procaine hydrochloride . Disposable 21 G 0.80 mm × 50.00 mm hypodermal needles (Terumo ®) were used to collect 5 mL of CSF by gentle syringe aspiration into a sterile tube . In the presence of symptomatology related to the TSE, the animals were euthanized with i.v. enbutramide/mebezonium iodide/tetracaine hydrochloride . After euthanasia, CSF, the whole brain, the entire spinal cord and peripheral nervous tissues were sampled. Each sample was frozen at −80 °C.The analyses with RT-QuIC were carried out on brainstem and CSF samples collected from goats derived from two natural outbreaks of classical scrapie and from caprines intracranially inoculated with 1 ml of a 10% (wt./vol.) brain homogenate from a cow diagnosed with classical (C-BSE) or atypical BSE (L-BSE) as previously describedSc extraction and the following WB analyses were carried out on the brainstem samples collected at necropsy from the experimentally challenged goats. Brain tissue from healthy cattle, C-BSE and L-BSE samples, used for the inoculum, and goats previously confirmed positive or negative for classical scrapie were also analyzed as controls. The analyses were performed as previously reported11. PrPSc was detected using SAF84  monoclonal antibody and an anti-mouse antiserum conjugated with alkaline phosphatase. Reaction was revealed by a chemiluminescent substrate and visualized on Hyperfilm ECL  or by a gel documentation . Image LabTM analysis software (Bio-Rad) was used to analyse blot images.PrP154Q171 polymorph [accession no. AY907689]) prion protein genes were ligated into the pET41 vector (EMD Biosciences). Escherichia coli carrying this vector was grown in Luria broth (LB) medium in the presence of kanamycin and chloramphenicol. rPrPSen expression was induced using Overnight Express Autoinduction system 1 (Novagen) and Bug Buster master mix (Novagen) to isolate inclusion bodies. Following solubilisation of the inclusion bodies in 8M guanidinium-HCl, the denatured protein was purified under 6M guanidinium-HCl denaturing conditions using nickel nitrilotriacetic acid (Ni-NTA) superflow resin (Qiagen) with an AKTA 25 L protein liquid chromatography instrument . The rPrPSen was subjected to on-column refolding using a linear gradient into phosphate buffer and then eluted using an imidazole gradient as previously described14. The purified protein was extensively dialyzed into 10 mM sodium phosphate buffer (pH 5.8). Then, following filtration , a concentration measurement by absorbance at 280 nm was performed and the rPrPSen was stored at −80 °C.Syrian golden hamster residues 90 to 231 (hamster 90–231 or Ha 90–231) and chimeric hamster-sheep , scrapie-infected (n = 2), C-BSE-infected (n = 2), and L-BSE-infected (n = 2) goat brain homogenates (BHs)  were prepared as previously described33 and stored at −80 °C. For RT-QuIC analysis, BHs were serially diluted in 0.1% SDS (Sigma)–N2 (Gibco)–PBS as previously reported14. The RT-QuIC reaction mix was composed of 10 mM phosphate buffer (pH 7.4), 300 mM NaCl, 10 μM ThT, 1 mM EDTA, and 0.1 mg/ml of rPrPSen. Aliquots of this mix (98 μl) were loaded into each well of a black 96-well plate with a clear bottom (Nunc) and seeded with 2 μl of 10−4 to 10−9 brain homogenate dilutions. Normal goat BH dilutions were used as negative controls  and incubated for 70 h at 42 °C in a BMG Labtech FLUOstar plate reader with cycles of 1 min of shaking  and 1 min of rest throughout the incubation. ThT fluorescence measurements  were recorded every 45 min. RT-QuIC reactions were deemed acceptable when the negative controls remained negative for at least 50 h.Normal n = , scrapie25. Briefly, both PQ-CSF and IQ-CSF reactions were run with rPrPSen Ha 90–231 with or without the addition of 0.002% SDS to the reaction mix.Native CSF samples were centrifuged at 1000 × g for 10 min to remove cell debris, using 5417 R Eppendorf centrifuge . Supernatant was transferred to new 1.5-ml tubes and stored at −20 °C. IQ-CSF assays were performed as reported previously for sCJD patients33. Briefly, to compensate for differences between the fluorescence plate readers, data sets were normalized to a percentage of the maximal fluorescence response of the instrument, and the obtained values were plotted against the reaction times. Samples were judged to be RT-QuIC positive using criteria similar to those previously described for RT-QuIC analyses of brain specimens33. A 60 h time point was chosen based on multiple (n = 20) repeat runs in which no spontaneous conversions of the substrate in negative-control seeded reactions were observed up to the experimentally designated time point. Data are displayed as the average of four technical replicates except where indicated. Where multiple biological replicates are displayed, they are represented as the mean ± the standard deviation.RT-QuIC fluorescence readings were analyzed as previously describedAll procedures involving animals and their care were conducted in conformity with national and international laws and policies . The study was approved by the Italian Ministry of Health with authorization number 694/2015-PR of 17th of July 2015.Supplementary Figures


The digital polymerase chain reaction (dPCR) is considered to be the third-generation polymerase chain reaction (PCR), as it yields direct, absolute and precise measures of target sequences. dPCR has proven particularly useful for the accurate detection and quantification of low-abundance nucleic acids, highlighting its advantages in cancer diagnosis and in predicting recurrence and monitoring minimal residual disease, mostly coupled with next generation sequencing. In the last few years, a series of studies have employed dPCR for the analysis of hematologic malignancies. In this review, we will summarize these findings, attempting to focus on the potential future perspectives of the application of this promising technology. IgH) gene derived from a leukemic clone in a background of excess rearranged IgH genes from normal cells [The introduction of the digital polymerase chain reaction (dPCR) in cancer research is quite recent. The history of this third-generation polymerase chain reaction (PCR) technology started in the 1990s with the first attempts to obtain single PCR molecules, applying limiting dilution conditions ,3,4,5,6.al cells . The tecal cells ,9, who wal cells . Since tal cells , the chial cells , the droal cells , the Raial cells , the Cryal cells , the Claal cells , and FORal cells ,16; howeal cells , and alsal cells ,19,20,21An in-depth study of the technical details is not the within the scope of this manuscript, and for this type of examination we cite more specialized reviews ,23. In gIn this literature review we summarize the results obtained from research applying dPCR, and mostly ddPCR, in the field of onco-hematology. In the conclusion, we attempt to envisage what the future of this technology for the study of hematologic malignancies may be, highlighting its strengths and disadvantages, in order to see if there could be further applicability spaces in research and in clinical practice.JAK2 in nearly 90% of PV patients and in around 50% of all ET and MF patients [CALR, present in about 20%–35% of patients affected by ET and primary MF [V617FJAK2 mutation, the major MPN diagnostic criterion [V617FJAK2 mutation by conventional qPCR and 99 of these were evaluated also by ddPCR. The specificity was absolutely concordant between the two methods, but the sensitivity was half a log higher for ddPCR than qPCR [V617FJAK2 mutation, allowing a sensitivity of 0.01%, equitable with that achieved by Kröger et al. with an ARMS qPCR in 2007 [V617FJAK2 mutation. At first, the JAK2 mutation was detected in peripheral blood (PB) samples of 47 patients, and afterwards, among these, 45 of 47 corresponding serum samples showed a very strong correlation between PB and serum. The overall detection sensitivity was 96% and the mutation was detected in all cases where the mutant allele load was above 1%. The observation of a significantly higher allele burden detected in serum compared to PB highlighted the point that ddPCR could be a reliable method for detecting the V617FJAK2 mutation also in serum [dPCR has been applied for the detection of several somatic mutations, both for absolute allele quantification and for rare mutation detection . The mospatients , and CALimary MF . Gene eximary MF . The fir in 2007 ,36,37,38in serum .CALR gene mutations. In 2016, Mensier et al. and Badbaran et al., concomitantly performed ddPCR for the absolute quantification of CALR type 1 and 2 mutations, demonstrating developing ddPCR assays that were able to reach a sensitivity of 0.02%. In the same year, Anelli et al. also described a ddPCR assay for the CALR type 1 and 2 mutation quantification [CALR allelic burden has not yet been established at the disease onset, these reports laid the foundation for ddPCR utility as a reliable method for CALR mutations MRD monitoring [dPCR was tested also for detection of the fication . They defication . Althougnitoring ,42.DNMT3A, TET2, ASXL1, RUNX1, and IDH1/2, shown to be useful in MRD-based prognostication in AML [KIT double gene mutations in core binding factor (CBF)-AML patients, revealing that the double mutations can occur in either the same or different clones and the latter may generate a different sensitivity to CBF-AML treatment [A216VPML gene mutation detection and quantification, linked to trioxide arsenic therapy resistance in acute promyelocytic leukemia (APL) patients. They firstly identified the A216VPML mutation by ddPCR in 5 of 13 APL patients, and then extended the screening to all evaluable serial FU samples, detecting the mutation in all patients showing treatment resistance. They assumed that it may have been present at subclonal level before therapy and then later selected under treatment pressure. These data suggest that ddPCR can be successfully recruited for A216VPML gene mutation screening and that this procedure may allow for the detection of mutant cases earlier than conventional sequencing [Acute Myeloid Leukemias (AMLs) instead, are the most common and severe form of acute leukemia diagnosed in adults. Owing to its heterogeneity, AML is divided into classes associated with different treatment outcomes, disease markers and specific gene expression profiles. AML, unfortunately, frequently relapses after complete remission (CR), and so improved detection and phenotypic characterization of treatment-resistant residual leukemic cells are urgently needed . Parkin n in AML . Insteadreatment . Lastly,quencing .STAT6 gene, the most commonly observed variant, and used it for MRD monitoring [dPCR has been applied also in studies of lymphomas. Classical Hodgkin lymphoma (cHL) accounts for 30% of all lymphomas ; in the nitoring .L265PMYD88 mutation is a hallmark of WM, and its finding can be conclusive for the diagnosis [L265PMYD88 mutation in WM in different tissues  [−5, far superior to the classical PCR method, after analyzing 291 samples (194 baseline samples and 97 FUs) from 148 patients . About 95% of BM and 71% of PB baseline samples were positive for L265PMYD88 [L265PMYD88 detection in WM; instead in relapsed treated patients, where BM analysis should be preferred, considering that PB has a one log lower median mutated:WT ratio compared to BM, ddPCR analysis on plasma ctDNA could represent a promising, less invasive alternative to BM [For the diagnosis of Waldenström Macroglobulinemia (WM), bone marrow (BM) biopsy seems to be mandatory, to define the infiltration of indolent lymphoplasmacytic lymphoma (LPL)  in BM) and monoclonal IgM protein secretion ,62. Howeiagnosis ,64. In 2ve to BM .L265MYD88 gene mutation has been demonstrated to be a diagnostic and prognostic marker also in more than 70% of primary central nervous system lymphomas (PCNSL) [L265PMYD88 detection in PCNSL patients in diagnostic paired tumor-derived DNA and cfDNA samples. They concluded that ddPCR could be employed for non-invasive diagnosis on the liquid biopsy of cfDNA, but not for MRD monitoring in PCNSL [MYD88 mutations (L265P and V217F) in isolated cerebrospinal fluid (CSF)-circulating tumor DNA (ctDNA), in conjunction with evaluating the patient-matched central nervous system (CNS) lymphoma tissue [L265PMYD88 mutation was detected both in formalin-fixed paraffin-embedded (FFPE) tissue from the brain biopsy and in the CSF-ctDNA, highlighting that CSF ctDNA analysis through ddPCR could be a potential minimally invasive approach to diagnosing patients with suspected CNS lymphomas [The  (PCNSL) . A 2017 in PCNSL . In a sia tissue . The MYDymphomas .RHOAG17V gene mutation, an essential element for the appropriate diagnosis of angioimmunoblastic T-cell lymphoma (AITL), a subtype of nodal peripheral T-cell lymphoma (PTCL) [RHOAG17V mutation in 27 of 67 (40.3%) PTCL samples, while ddPCR and the PNA-LNA clamp method both detected the G17V mutation in 31 of 67. Indeed, VAFs calculated with ddPCR and NGS were highly concordant. However, NGS analysis found three other RHOA mutations involving position G17, whose presence could be detected but not appropriately defined with the ddPCR and the PNA-LNA clamp methods, because they showed abnormal signal patterns, different from those of G17V mutated and wild type alleles. The authors concluded that a combination of ddPCR/PNA-LNA clamp methods and NGS is the best way to identify RHOAG17 substitutions and to support the diagnosis of AITL [Tanzima Nuhat et al., in a 2018 study, compared NGS, ddPCR and the peptide nucleic acid-locked nucleic acid (PNA-LNA) clamp method to find which was most efficacious for detecting the a (PTCL) . In thes of AITL .TP53 mutations, the dominant subclonal gene driver of relapse, and revealing uncommon mutations in ATP10A, FAT3, FAM50A and MGA with increased relapse-associated VAFs. In contrast, VAFs of mutations in NOTCH1, SF3B1, POT1, FBXW7, MYD88, NXF1, XPO1, ZMYM3 or CHD2 seemed not to vary at the moment of relapse [Few studies have been performed on Chronic Lymphocytic Leukemia (CLL) testing dPCR assays. CLL is the most common leukemia in adults, characterized by an extremely heterogeneous clinical course, linked to immunogenetic markers associated with different disease prognoses . The fir relapse .c.7541-7542delCT NOTCH1 mutation ddPCR assay, and also analyzing the NOTCH1mut allelic burden, indicated as fractional abundance (FA). They then monitored the FA variation over time in 10 cases. Our data revealed that with the ddPCR method, the incidence of NOTCH1mut in CLL was much higher (53.4%) than previously reported; indeed, in FU analysis, a statistically significant decrease of the NOTCH1mut FA was found from diagnosis after treatment. This same was noted in the relapsed samples as compared to complete or partial remission samples, suggesting that screening by ddPCR might help to identify patients in need of an earlier clinical FU during the “watch and wait” period, and after standard chemotherapy [Minervini et al. carried out a study investigating 88 CLL patients at diagnosis employing a otherapy . A surely important dPCR application area is disease monitoring during patients’ FU to track tumor-associated mutations or mutations arising secondarily in response to and selected by therapy, indicating a drug resistance mechanism, in order to monitor disease progression .non-A, -B, -D) in NPM1 gene, at diagnosis and during FU for MRD monitoring. The dPCR sensitivity was comparable to that of conventional qPCR and enabled MRD diagnostics in all rare NPM1 mutation subtypes tested [IDH1 and IDH2 genes as an MRD indicator to monitor 90 AML patients through both massive parallel sequencing and ddPCR. The study showed that both technologies were appropriate for MRD monitoring, although in some cases ddPCR presented a higher sensitivity [IDH1/2 mutations were also screened in AML patients by Grassi at al. using a new method, the drop-off ddPCR, described below [−3; the researchers screened 60 AML patients, identifying at diagnosis 11 IDH2-mutated cases with a median mutational burden of 13.7%. Parallel SS did not identify mutations in 5/11 cases, likely because of the low mutational burden [KIT mutation as MRD indicator in CBF-AML. They analyzed 20 CBF-AML patients with ddPCR, noting that the KIT gene mutation level during CR in the relapsed patients cluster was statistically significantly higher than that in the remission group. They also monitored the KIT gene mutation level in 10 patients from the initial diagnosis to CR, and found that the degree of remission was significantly lower in patients without recurrence than in patients with recurrence. These data showed that the KIT gene mutation is a helpful marker for MRD monitoring in CBF-AML, and that ddPCR is an effective method for this purpose [Several studies employed dPCR for MRD monitoring in AML. The first report dates back to 2014, when Bacher et al. used dPCR (Fluidigm) to quantify very rare mutations  of circulating BCL2/IGH+ cells at diagnosis or resulting persistently MRD negative during FU, exhibited a significantly better progression-free survival (PFS) when analyzed by ddPCR [MRD assessment is considered to provide valuable prognostic information in various lymphoid malignancies, and is frequently included as a secondary goal in clinical trials ,82,83,84by ddPCR ,88. Veryby ddPCR .BCR-ABL1 fusion gene. This fusion gene is then translated into a BCR-ABL1 oncoprotein that triggers neoplastic transformation of the hematopoietic stem cell. CML is one of the first malignancies for which the principle of targeted therapy has been successfully applied thanks to the use of tyrosine kinase inhibitors (TKI) [ABL1 and BCR-ABL1 fluorescence signals two- and five-fold, respectively, and upgraded the resolution between positive and negative drops [BCR-ABL1 ddPCR assay, that yielded comparable ABL1 and BCR-ABL1 transcript levels to those obtained by qPCR, but showed a switch to a lower molecular response category compared to qPCR adapted to IS [BCR-ABL1 %IS  ddPCR assay. The limit of blanks, limit of detection, and limit of quantification claimed by the manufacturer were confirmed, and the two methods, the QXDx BCR-ABL1 %IS ddPCR assay and the Ipsogen BCR-ABL1 Mbcr IS- qPCR assay, showed a very high correlation (r=0.996) [Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the t translocation, which causes the formation of the chimeric rs (TKI) ,91. The rs (TKI) . To overve drops . Franke ed to IS . Lastly,r=0.996) . BCR-ABL1 MRD [Furthermore, some studies have been carried out to evaluate MRD using dPCR in CML patients. The first dates back to 2011, when Goh et al. compared qPCR with dPCR for this purpose. They screened 62 samples from 43 patients with CML by conventional qPCR, and the analyses were replicated with qPCR (r-qPCR) and/or nanofluidic dPCR . They confirmed the correlation of dPCR with conventional qPCR using samples with various MRD levels. The addition of pre-amplification to r-qPCR and dPCR showed a two–three log improvement compared to conventional qPCR, and 24 of 32 samples negative with qPCR resulted positive with r-qPCR and/or dPCR . These pABL1 MRD .Another important aspect on which dPCR studies have been focused is the discontinuation of tyrosine kinase inhibitors (TKI) therapy for CML patients’ treatment, associated with the prediction of relapse. Some studies have judged dPCR to be a method that could help to prognosticate relapse after TKI discontinuation earlier than qPCR ,99,100. BCR-ABL1 positive cells, in detecting the BCR-ABL1 genomic rearrangement. For the establishment of the BCR-ABL1 junction sequence, Cumbo et al. proposed two alternative approaches: the first one consisted of a first fluorescence in situ hybridization (FISH)-based step followed by SS; the second one employed MinION, a nanopore third generation sequencing technology that the research group consolidated [BCR-ABL1 genomic junction had been defined, ddPCR was employed for patient “personalized monitoring” [Lastly, Cumbo et al. reported a study of MRD monitoring in CML with a DNA-based assay to enhance the sensitivity of detection of olidated ,104,105.itoring” .−4), using ddPCR and NGS for analyzing immunoglobulin/T-cell receptor gene rearrangements as molecular markers [BCR-ABL1 p190 fusion transcript (m-bcr), which account for 20%–30% of adult ALLs [BCR-ABL1+ ALL samples in hematologic and cytogenetic remission were analyzed using the microfluidic dPCR approach (Biomark system from Fluidigm) [BCR-ABL1 transcript, with results at least comparable with those obtained with the conventional qPCR test. However, as compared to the dPCR technologies available today, this system was less sensitive and accurate, as only about 9000 partitions could be generated and the total assay volume was limited to 8 µl; indeed, for each sample the total amount of analyzed RNA was about 20ng [BCR-ABL1+ ALL FU [p190 assay as regards PCR and/or qPCR was, indeed, demonstrated to be able to predict molecular relapse, offering a timely advantage for patient management. In fact, it may allow a rapid change of therapy before hematological relapse [MRD is the strongest prognostic factor in Acute Lymphoblastic Leukemia (ALL), able to predict response to treatment and risk of relapse . However markers . This co markers . These d markers . As regault ALLs ,112,113.luidigm) . They deout 20ng ,114,115.+ ALL FU . With dd+ ALL FU . FU samp relapse ,121,122  relapse .Patients with acute myeloid leukemia benefit from allogeneic hematopoietic stem cell transplantation , which is an established consolidation therapy. However, too often, they suffer relapse after allo-HSCT, that confers poor prognosis, and this remains a major clinical challenge. In this context, the early identification of patients at high risk of relapse, and thus the detection of MRD, is essential, as well as assessment of the successful outcome of the transplant procedure. Therefore, several groups have tested the feasibility of the dPCR method for these purposes .NPM1 , DNMT3A (R882H), MLL-PTD, IDH1 (R132H) and KRAS (G12S) as MRD markers. Indeed, in 15 patients, increasing MC values and positivity for an MRD marker, increased WT1 expression or both, were detected. Furthermore, they determined whether MC or MRD positivity was the first to be detected, highlighting that MC was observed first in six patients and MRD positivity in two patients; in the remaining seven patients they were detected simultaneously [dPCR was employed for chimerism analysis in transplanted patients, the term “mixed chimerism” (MC) meaning the condition of a mixture of genetic profiles resulting after allo-HSCT. A series of studies demonstrated the power of dPCR as a tool for clinical chimerism analysis as compared to established methods based upon Indel and short tandem repeats (STRs) detection ,127,128.dPCR has also been demonstrated to be useful for monitoring platelet engraftment after allo-HSCT. It is normally examined by daily platelet counts, but any necessary platelet transfusions performed in the patient can obscure the detection of platelet engraftment. Using ddPCR, Doescher at al. drew up an experimental assay based on mitochondrial DNA isolated from platelets in order to quantify circulating platelets derived from the stem cell graft, distinguishing them from transfused single-donor apheresis platelets . ConsecuNPM1 MRD-positivity with ddPCR in pre-transplant samples from 51 NPM1-mutated AML patients [NPM1 confers a worse prognosis, independently of other known prognostic markers [In several other studies, dPCR was applied for MRD detection in patients undergoing allo-HSCT. Bill et al. assessed mutated-patients . They ob markers . DNMT3A, IDH1, and IDH2 as MRD markers; these are considered disease founder mutations and their frequent alteration, in particular in DNMT3A, seems to occur very early during leukemogenesis and thus are likely shared by all disease subclones and steadily preserved until the eventual relapse [DNMT3A and IDH1/2 were compared with qPCR analysis for NPM1 mutations, for the WT1 transcript, and for host-specific hematopoietic chimerism, the data seemed be largely concordant. However, as these mutation-specific assays are informative only for specific patients, and the molecular markers might be lost due to molecular disease evolution, the optimal way to tackle MRD is by a multi-target patient-specific approach [In the work conducted by Brambati et al., ddPCR experiments were performed utilizing  relapse ,136,137.BAALC [MN1 genes [BAALC copy numbers were strongly associated with a higher cumulative incidence of relapse within 100 days after HSCT, and shorter OS [MN1 gene expression was associated with a higher cumulative incidence of relapse after HSCT and with a shorter time to relapse. Indeed, AML cases with the highest level of MN1 gene expression presented the highest risk of relapse [BAALC and MN1 expression using ddPCR, together with analysis of the NPM1 mutation and WT1 expression, could be a valid approach to identify AML patients at high risk of early relapse after HSCT. Further prospective studies are needed to confirm their prognostic value [As regards expression analysis, ddPCR was employed in two studies to explore the expression of the BAALC  and MN1 N1 genes . For botN1 genes ,149,150.orter OS . Similar relapse . Thus, tic value ,139. CAR-T (chimeric antigen receptor T) cells transfused in vivo in refractory/relapsed Diffuse Large B-Cell Lymphomas (DLBCL) and primary mediastinal large B cell lymphoma (PMBCL) patients treated with axicabtagene ciloleucel (Axi-cel) CD19-CAR-T cells [CAR regions were designed and tested in duplex reactions, using a reference gene to concomitantly assess cell numbers. Three of these designed assays were demonstrated to be specific and reproducible, with an almost perfect correlation between the CAR target and the reference, and with a LOD of one single CAR-transduced cell, corresponding to a sensitivity of 0.01% for 100 ng genomic DNA. In vivo application to monitor Axi-cel CAR-T numbers in patients showed CAR-T fluctuations, as expected, and the median peak value of Axi-cel CAR-T cells was 11.2/mL at 11.3 days. Indeed, they demonstrated good cell expansion with clinical responses in four of the five patients  on day 30, demonstrating the utility of the ddPCR assay in the clinical management of these patients [Recently, Fehse et al. employed ddPCR also to detect and quantify transgenic -T cells . For thipatients .NOTCH3 isoforms display a prognostic and predictive biomarker potential in Germinal Center B-cell like (GCB) DLBCL [JUN and MYC oncogenic signaling, and that they hypothesized them to be age-dependent and to influence DLBCL development in the murine model [Frequently, dPCR has been employed as a validation tool to corroborate data obtained with other technologies . In reseB) DLBCL . Beheshtne model . ddPCR wne model  and CLL ne model .Often, the dPCR and the NGS are used together ,155. ApaBCR-FGFR1 fusion transcript for MRD monitoring in a rare case of ALL with t/BCR-FGFR1 gene fusion, for which standardized methods are lacking demonstrating that the ddPCR assay was capable of predicting hematologic relapse with a higher sensitivity than the classical PCR method [Furthermore, a series of studies have employed dPCR for other applications . A conveR method .BCR-ABL1 transcript variants. Kjaer et al. analyzed 219 CML patients with either the e13a2 (n = 113) or e14a2 (n = 106) variant, enrolled at three Danish diagnostic centers, and the data obtained suggested that the qPCR assay may underestimate the e14a2 variant compared to the e13a2 variant, since the analysis of qPCR vs. ddPCR values revealed a significant average difference in the bias between the variants (e3a2/e14a2) at the three centers. These data were concomitantly confirmed by Bernardi et al. [dPCR is useful for discriminating transcript variants: some researchers employed the method in a study on CML to establish qPCR efficiency in identifying i et al. ,172. ddPi et al. .Another potential application of dPCR is DNA methylation analysis, which has been explored in several studies of malignancies . In the IDH2 gene mutations in AML patients at diagnosis and follow-up during treatment, obtaining hopeful results [Y641 of EZH2 and D419 of STAT6 in fresh tumor, FFPE, and liquid biopsies in B-cell non-Hodgkin lymphomas, including DLBCL, FL, and lymphoplasmacytic lymphoma. Indeed, they tested multiplexing of these probes with two others designed for two additional lymphoma-related hotspots (L265P of MYD88 and I290R of CCND3), allowing the simultaneous detection of distinct mutations [EZH2 and STAT6 wild-type alleles, capable of highlighting the presence of multiple mutations [Moreover, some authors have recently tried to overcome some limitations of the dPCR approach, such as its employment as a discovery tool, that is limited by the need for a priori knowledge of the mutation to be detected; this limitation restricted the number of alterations screened per reaction, simultaneously increasing the number of probes employed to analyze each known variant allele. In order to face this challenge, an alternative dPCR approach, called drop-off dPCR, was developed; it requires only a single pair of probes to find and quantify all genetic alterations in a targeted region in a single reaction. In this system, the FAM-labeled probe recognizes a reference sequence distant from the target but within the same amplicon, and the HEX-labeled probe binds the target wild-type sequence ,178,179. results . Alcaideutations . Finallyutations .Another alternative application of ddPCR is Somatic Mutation Allelic Ratio Test ddPCR (SMART-ddPCR). In 2015, De Smith et al. employed this method to assess tumor preferential allelic imbalance (PAI), a phenomenon by which a tumor selects single nucleotide polymorphism (SNP) risk alleles and the associated somatic copy number alterations in tumor DNA. This method was successfully applied to test the hypothesis that heritable SNPs associated with childhood ALL may exhibit tumor PAI, comparing results with those obtained from multiplex ligation-dependent probe amplification (MLPA) .Nowadays, when we talk about the personalized treatment of cancer, the need arises to use the most reliable technology, which should be robust and advantageous in terms of the savings of economic resources and time, as well as of ease of use.p210 and p190 BCR-ABL1 transcripts [In the hematologic field, the reported data show dPCR as a rapid and reliable technology for obtaining the absolute quantification of a molecular target at disease diagnosis, during MRD assessment, as well as after HSCT in routine clinical laboratories, without any need for calibrators, standard curve, or laboratory conversion factors. The main advantages of this technology are the high precise and reliable quantification and the excellent reproducibility, as compared to qPCR. As regards the search of somatic mutations, several findings corroborated the feasibility and utility of dPCR, particularly in the context of liquid biopsy as an alternative to BM or tissue biopsy analysis, for example, in the case of cHL and WM. In the context of MRD evaluation, there is a broad consensus that the observation of a complete molecular response (CMR) shortly after the beginning of treatment, such as chemotherapy, plus a TKI for Ph+ ALL, for example, should be interpreted as a strong prognostic factor that guides the choice of transplant option . From thnscripts ,116. HowRegarding its relation with NGS, dPCR is employed for the accurate determination of DNA and RNA throughout NGS workflows, including the final library quantification. Indeed, dPCR is faster and cheaper than conventional NGS, with even higher sensitivity, lower probability of errors, need of less DNA input, and without bioinformatics pipelines . These fBCR-ABL1+, qPCR one-step kits are available, which allow for the analysis of a high number of samples. Simple as the experimental dPCR protocols are, they are still slightly more laborious than qPCR one-step procedures. A solution might be automation, which could be time-saving and have the advantages of greater sensitivity and precision. However, it is unlikely that this will be the key for the worldwide diffusion of dPCR. This technology is still relatively highly expensive in regards equipment and single reactions, so nowadays, qPCR still enjoys greater diffusion in clinical practice, remaining the gold standard for several types of analyses. Only standardization programs will give the proper launch for including dPCR in everyday practice. Despite the clear advantages of this technology, there is still a lack of studies of large cohorts of patients and standardization of procedures. For this purpose, a large standardization study is currently ongoing within the European Scientific Foundation for Laboratory Hemato Oncology (ESLHO). Accordingly, in the future, it will be necessary to schedule further controlled clinical trials and multilaboratory standardization programs to evaluate dPCR as an outcome predictor tool.The choice of the analysis technique should be that of the most precise and sensitive method. These needs justify the efforts spent on the development of the digital technology, considering the high expectations regarding sensitivity and specificity. As regards the hematologic malignancies, several studies conducted in the last few years confirmed the good performance of dPCR. However, we are still far from the goal of stable implementation of this technology in everyday use. The reasons for this probably lie in a reluctance to abandon consolidated procedures, but this is not the only reason. For some kinds of analysis, such as MRD study in CML and ALL