The REMA Score Revisited: An Optimized Algorithm for Early Prediction of Clonal Mast Cell Disease.

Background The Spanish Network on Mastocytosis (REMA) score was developed to predict clonal mast cell (MC) activation syndromes (MCAS) in patients presenting with anaphylaxis without mastocytosis in the skin (MIS), and guide selection of patients for bone marrow (BM) examination. This study reassessed the diagnostic accuracy of the REMA score after 15 years of use and propose refinements to optimize its predictive value. Methods Globally, 1,204 patients with severe MC mediator-related symptoms without MIS were retrospectively analyzed. All underwent clinical and allergologic evaluation, serum tryptase, BM histology, immunophenotyping, and KIT mutational analysis. REMA scores were calculated and diagnostic accuracy assessed across the score ranges. Multivariable analysis was applied to identify independent predictors and refine prediction. Results Clonal MCAS was diagnosed in 64% of patients. Overall, the REMA score showed excellent sensitivity (87-90%) but limited specificity (34–35%), with the poorest accuracy in the score 0–2 range. In this subgroup, independent predictors were elevated tryptase without hereditary alpha tryptasemia (OR 5.42, 95% CI 2.86–10.28) and insect venom as trigger (OR 2.18, 95% CI 1.28–3.72). KIT p.D816V in blood, although detected in only 29% of all clonal cases, allowed identification of additional low-score patients requiring BM evaluation. A refined algorithm combining these predictors improved specificity to 57% while maintaining sensitivity at 86%. Conclusions The REMA score remains highly sensitive for early detection of clonal MCAS. Incorporation of additional clinical and molecular predictors into an updated diagnostic algorithm enhances specificity and ensures more accurate selection of patients for BM evaluation. TITLE PAGE The REMA Score Revisited: An Optimized Algorithm for Early Prediction of Clonal Mast Cell Disease. Irina Sucre-Adrianza 1, Horacio Caligaris 1,2, Cristina Morales-Cabeza 1,2, Paula Navarro-Navarro 2,3,4,5,6, Laura Sánchez-Muñoz 1,2, Alba Pérez-Pons 2,3,4,5,6, Ana Henriques 1,2, María González-Tablas 2,3,4,5,6, Oscar González-López 2,3,6, María Recuero 7, María Jara-Acevedo 2,3,5,6,8, David González-de-Olano 9, Manuela Mollejo 7, Andrés García-Montero 2,3,5,6, Alberto Orfao 2,3,5,6 and Iván Alvarez-Twose 1,2,5 . 1 Institute of Mastocytosis Studies of Castilla-La Mancha (CLMast)-Spanish Reference Center of Mastocytosis, Hospital Virgen del Valle, Toledo. 2 Spanish Network on Mastocytosis (REMA), Toledo and Salamanca. 3 C ancer Research Center, Department of Medicine, Salamanca 4 Cytometry Service, University of Salamanca, Salamanca. 5 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC; CB16/12/00400), Madrid. 6 Biomedical Research Institute of Salamanca (IBSAL), Salamanca. 7 Pathology Department, Complejo Hospitalario de Toledo, Toledo. 8 Sequencing Service (NUCLEUS-USAL), University of Salamanca, Salamanca 9 Department of Allergy, Hospital Ramón y Cajal, IRYCIS, Madrid, Spain. Corresponding author: Iván Alvarez-Twose, MD, PhD Phone: 0034 925269200 (EXT. 49336) e-mail: [email protected] Short title: Refined REMA score for clonal mast cell disease

mast cell, mast cell activation syndrome, mastocytosis, REMA score, clonal Word count: 3,528 Tables/Figures: 6 (+3 as supplementary material) Funding This study was supported by grants from Instituto de Salud Carlos III (ISCIII; Madrid, Spain) and co-funded by the European Union (grant numbers PI22/01657 and CB16/12/00400 for the Biomedical Research Networking Center Consortium [CIBERONC] program). Disclosure of potential conflict of interest : The authors declare no conflict of interest in relation to this work.

The Spanish Network on Mastocytosis (REMA) score was developed to predict clonal mast cell (MC) activation syndromes (MCAS) in patients presenting with anaphylaxis without mastocytosis in the skin (MIS), and guide selection of patients for bone marrow (BM) examination. This study reassessed the diagnostic accuracy of the REMA score after 15 years of use and propose refinements to optimize its predictive value.

Globally, 1,204 patients with severe MC mediator-related symptoms without MIS were retrospectively analyzed. All underwent clinical and allergologic evaluation, serum tryptase, BM histology, immunophenotyping, and KIT mutational analysis. REMA scores were calculated and diagnostic accuracy assessed across the score ranges. Multivariable analysis was applied to identify independent predictors and refine prediction.

Clonal MCAS was diagnosed in 64% of patients. Overall, the REMA score showed excellent sensitivity (87-90%) but limited specificity (34–35%), with the poorest accuracy in the score 0–2 range. In this subgroup, independent predictors were elevated tryptase without hereditary alpha tryptasemia (OR 5.42, 95% CI 2.86–10.28) and insect venom as trigger (OR 2.18, 95% CI 1.28–3.72). KIT p.D816V in blood, although detected in only 29% of all clonal cases, allowed identification of additional low-score patients requiring BM evaluation. A refined algorithm combining these predictors improved specificity to 57% while maintaining sensitivity at 86%.

The REMA score remains highly sensitive for early detection of clonal MCAS. Incorporation of additional clinical and molecular predictors into an updated diagnostic algorithm enhances specificity and ensures more accurate selection of patients for BM evaluation. ABBREVIATIONS AIM: American Initiative in Mast Cell Diseases. ASO-qPCR: allele specific oligonucleotide quantitative real-time polymerase chain reaction. BM: Bone marrow. BMM: Bone marrow mastocytosis. CNV: Copy number variation. dPCR: digital polymerase chain reaction. FACS: Fluorescence-activated cell sorting. HαT: Hereditary alpha tryptasemia. ISM: Indolent systemic mastocytosis. MC: Mast cell. MCAS: Mast cell activation syndrome. MIS: Mastocytosis in the skin. MMAS: Monoclonal mast cell activation syndrome. nc-MCAS: non-clonal MCAS. NIAID/FAAN: National Institute of Allergy and Infectious Diseases/Food and Allergy and Anaphylaxis Network. PNA: peptide nucleic acid. REMA: Spanish Network on Mastocytosis. SM: Systemic mastocytosis. VAF: Variant allele frequency. WHO: World Health Organization.

Mast cell (MC) activation syndromes (MCAS) comprise a heterogeneous spectrum of disorders characterized by recurrent, often severe, episodes of systemic symptoms resulting from the release of proinflammatory mediators by activated tissue MC with increased serum tryptase levels during acute episodes and response to antimediator therapy (1–3). MCAS are classified into four subtypes (1–3): i) Primary/clonal MCAS, including mastocytosis, mostly with systemic involvement (i.e. systemic mastocytosis –SM–) with or without mastocytosis in the skin (MIS), and monoclonal MCAS (MMAS); ii) Secondary MCAS, triggered by underlying conditions such as allergic diseases; iii) Idiopathic MCAS, where neither primary nor secondary causes are identified; and iv) Mixed (e.g., secondary plus idiopathic) MCAS (2,3). More recently, an additional category —hereditary alpha tryptasemia (HαT)-positive MCAS (4–6)— has been proposed among idiopathic cases, comprising patients harboring increased copy number variation (CNV) of the tryptase-encoding TPSAB1 gene without primary or secondary causes of MC activation. Based on the above, bone marrow (BM) evaluation should be recommended in patients with MCAS to confirm or exclude an underlying clonal disorder. To optimize candidate selection for BM investigation among patients with MC-mediator related symptoms without MIS, the Spanish Network on Mastocytosis (REMA) developed in 2010 (7) a simple predictive tool known as the REMA score, later validated in 2011 (8). This score showed high sensitivity and specificity for predicting SM and MMAS in patients presenting with anaphylaxis (7,8). More recently, a prospective, real-world multicenter study (8) revealed that at least 5% of patients presenting with anaphylaxis harbor an underlying clonal MC disorder which could be reliably identified by the REMA score (9). Herein, we reassessed the accuracy and utility of the REMA score as a screening tool for clonal MC diseases in a large cohort of patients presenting with MC-mediator release symptoms without MIS. Particular emphasis was placed on comparing the diagnostic accuracy of clonal MCAS before and after the validation and systematic implementation of the REMA score in clinical practice, critically analyzing its limitations and drawbacks, and exploring refinements to enhance predictive accuracy. PATIENTS AND METHODS Clinical and allergological evaluation Data from patients referred to the REMA between March 2007 and December 2024, who presented severe MC-mediator release symptoms, predominantly anaphylaxis, in the absence of MIS were retrospectively analyzed. Written informed consent authorizing the use of clinical and laboratory data for research purposes was obtained from each participant, in accordance with the institutional and ethical standards. The diagnostic work-up of patients included: i) assessment of symptoms and triggers of MC-mediator release episodes, ii) dermatologic examination to exclude MIS, iii) measurement of serum baseline tryptase (sBT) levels (ImmunoCAP, Phadia Diagnostics, Uppsala, Sweden), and iv) complete BM examination. Anaphylaxis was defined according to the National Institute of Allergy and Infectious Diseases/Food and Allergy and Anaphylaxis Network (NIAID/FAAN) consensus criteria (10). REMA score The REMA score (7,8) (Table 1) was calculated for each patient based on the most severe MC-mediator release episode. The following clarifications regarding clinical variables in the REMA score are deserved to avoid misinterpretations: i) only the absence of pruritus, urticaria and angioedema scored +1, whereas the presence of any of these symptoms, even if only one, resulted in -2 points; ii) patient-reported sudden aphonia, dysphonia, hoarseness and/or sensation of pharyngeal obstruction were considered surrogates of angioedema and scored -2; and, iii) rare cases of isolated, severe hypotension without dizziness or syncope were scored +3. The total score ranged from -4 to +7 (excluding +6), with values ≥2 indicating a high probability of clonal MCAS (7,8). Bone marrow studies BM studies were performed according to established recommendations (11,12). BM aspirate smears were stained with toluidine blue and May-Grünwald-Giemsa for cytomorphology, while trephine biopsy sections were processed with hematoxylin-eosin, reticulin, tryptase, CD117, CD25 and, since 2013, also with CD30. Multiparameter flow cytometry (4- to 8-color direct immunofluorescence) was performed to evaluate expression of CD45, CD34, CD117, CD25, CD2, IgE and, since 2013, CD30, according to consensus protocols (13,14). Molecular screening for KIT D816V was performed on genomic DNA from whole BM samples and, if negative, from fluorescence-activated cell sorting (FACS)-purified BM MC (15). Between 2007-2015, the peptide nucleic acid (PNA)-mediated PCR (PNA-PCR) method (15,16) was applied to whole BM and, when no KIT mutation had been detected, to FACS-purified BM MC; in turn, from 2016 onwards high-sensitive allele specific oligonucleotide quantitative real-time PCR (ASO-qPCR) was implemented in whole BM samples (17,18), while PNA-PCR was only applied to purified MC whenever ASO-qPCR showed a negative result. In KIT-negative cases, sequencing of the whole KIT gene was performed on FACS-purified BM MC (19). In parallel with BM studies, KIT p.D816V was screened by ASO-qPCR in whole blood and, if negative, in FACS-purified blood myeloid cell populations (e.g., neutrophils, monocytes, eosinophils and basophils) in most patients. Classification of patients Patients were classified into two groups based on clinical and BM findings: clonal MCAS, including SM and MMAS, and non-clonal MCAS (nc-MCAS), according to the World Health Organization (WHO) 2022 diagnostic criteria for mastocytosis (20) and the European Competence Network on Mastocytosis (ECNM)-American Initiative in Mast Cell Diseases (AIM) consensus criteria for MCAS (2). Within SM, only patients with BM mastocytosis (BMM), and indolent SM (ISM) without skin involvement were identified, in the absence of other subtypes of mastocytosis. Patients with recurrent MC-mediator release episodes who did not fulfill criteria for SM, were classified as MMAS when BM MC displayed aberrant phenotype and/or a KIT mutation or, otherwise, as nc-MCAS. HαT genotyping TPSAB1 genotyping was performed on freshly-frozen genomic DNA extracted from blood and/or BM. For patients referred to the REMA prior to 2020, retrospective analyses were carried out on cryopreserved genomic DNA samples stored at the Spanish National DNA Bank Carlos III of the University of Salamanca. CNV analysis of the TPSAB1 gene were performed by digital PCR (dPCR) using the Biomark HD system (Fluidigm), as previously described (5,6). HαT was defined as the presence of ≥ 3 α-tryptase alleles, or 2 α-tryptase alleles in combination with at least 3 β alleles. Statistical methods For continuous variables, median and range values were calculated, while categorical variables were expressed as frequencies. Group comparisons were performed using the Man-Whitney U test and the X 2 test for continuous and categorical variables, respectively. Multivariate logistic regression, restricted to variables showing statistical significance in univariate analyses, was applied to identify independent predictors of clonal MCAS within specific patient subgroups. P-values ≤0.05 were associated with statistical significance. All statistical analyses were performed using the SPSS statistical software package (Version 25.0, IBM Corp, Armonk, NY).

Patient demographic characteristics and subgroups A total of 1,204 adult patients were included, comprising 733 males (61%) and 471 females (39%) with a median (range) age at referral of 53 years (18-83). Based on BM findings, 652 patients (54%) were diagnosed with SM, — 625 BMM and 27 ISM—, 118 (10%) with MMAS and 434 (36%) with nc-MCAS. Male predominated in SM (69%) and MMAS (65%), whereas nc-MCAS was slightly more frequent in females (53%) (p<0.001). Clinical features of MC activation episodes Despite 95% of patients presented with anaphylaxis, only 9% (11% nc-MCAS, 8% SM and 10% MMAS), fulfilled all three MCAS criteria (i.e. typical symptoms, response to antimediator therapy and significant increase of tryptase in acute episodes). The remaining patients systematically fulfilled the first two criteria whereas tryptase during acute episodes showed no elevation (4%) or was not analyzed (87%). The most common presenting symptom was dizziness (84%) followed by syncope (60%), dyspnea (36%), pruritus (27%), flushing (22%), nausea/vomiting (22%), angioedema (18%), palpitations (16%) and urticaria (16%). Less frequent manifestations included abdominal pain (11%), diarrhea (11%), chest pain (7%), cardiac arrest (6%), paresthesias (5%), headache (4%), rhinoconjunctivitis (3%) and fever (<1%). Angioedema, urticaria and pruritus were significantly more frequent in nc-MCAS vs. SM (30% vs. 10%, 30% vs. 5% and 43% vs. 16%, respectively; all p<0.001). Conversely, dizziness (89% vs. 77%, p<0.001), syncope (69% vs. 47%, p<0.001), palpitations (20% vs. 10%, p<0.001), flushing (25% vs. 19%, p=0.03), headache (5% vs. 2%, p=0.029), nausea/vomiting (25% vs. 18%, p=0.014) and paresthesias (6% vs. 3%, p=0.049) were more prevalent in SM (Figure 1A). Patients with MMAS showed intermediate frequencies: angioedema, urticaria and pruritus were higher vs. SM (17% vs. 10%, p=0.030; 19% vs. 5%, p<0.001; 32% vs. 16%, p<0.001, respectively) but lower vs. nc-MCAS (17% vs. 30%, p=0.004; 19% vs. 30%, p=0.013; 32% vs. 43%, p=0.033, respectively), dizziness and syncope were less frequent in MMAS than SM (79% vs. 89% and 54% vs. 69%, respectively; both p=0.001), whereas palpitations were more frequent in MMAS than nc-MCAS (17% vs. 10%, p=0.04) (Figure 1A). Regarding triggers, insect (mostly hymenoptera) venom was significantly more frequent in SM (60%) vs. MMAS (38%) and nc-MCAS (32%) (p<0.001). By contrast, drug-induced episodes were more common in nc-MCAS and MMAS vs. SM (39% and 44% vs. 23%, respectively, p<0.001), while idiopathic reactions predominated in nc-MCAS (52% vs. 33% in MMAS and 32% in SM, p<0.001) (Figure 1B). Serum tryptase and BM histologic features Median (range) sBT levels were significantly (p<0.001) higher in SM (18.8 ng/mL) vs. both MMAS (10.7 ng/mL) and nc-MCAS (15.0 ng/mL); levels also differed significantly between MMAS and nc-MCAS (p=0.002). sBT <20 ng/mL was observed in 53%, 83% and 70% of SM, MMAS and nc-MCAS, respectively (p<0.001 for SM vs. both MMAS and nc-MCAS, and p=0.006 for MMAS vs. nc-MCAS), and sBT<11.5 ng/mL was found in 25%, 52% and 41%, respectively (p<0.001 for SM vs. both MMAS and nc-MCAS, and p=0.03 for MMAS vs. nc-MCAS).Multifocal dense BM MC aggregates were absent in nc-MCAS and MMAS patients, while detected in 42% of SM cases (p<0.001), including 40% of BMM and 83% of ISM (p<0.001). Molecular testing The KIT D816V mutation was detected in 597/621 (96%) BMM, 26/27 (96%) ISM and 69/118 (59%) MMAS patients, but 0/434 nc-MCAS (p<0.001 for all group comparisons). In blood, the mutation was identified in 178/610 (29%) patients, including 49% BMM, 83% ISM and 14% MMAS (p<0.001 for all group comparisons). In 8 additional patients (6 BMM and 2 MMAS), the mutation was detected in FACS-purified myeloid populations despite negative whole-blood results. Other KIT variants were found in 14 BMM (2%), 1 ISM (4%) and 8 MMAS (7%) cases, including D816Y (n=9), D816H (n=6), D816A (n=2), D816T (n=1), I817T (n=1), I817V (n=1), M541L (n=2) and H101Y (n=1). HαT genotypes were more frequent in nc-MCAS (46%) vs. MMAS (38%) and SM (24%), with significant differences between nc-MCAS and SM (p<0.001). Within SM, HαT was more common in ISM than BMM (44% vs. 23%, p=0.05). The distribution of TPSAB1 genotypes in each disease subgroup is shown in Figure S1. REMA score Figure 2A-B shows the distribution of clonal MCAS (SM + MMAS) vs. nc-MCAS patients across each score value of the REMA score. Overall, the median REMA score was significantly higher in SM vs. nc-MCAS and MMAS patients (4 vs. 2 and 2, respectively, p<0.001); although medians overlapped between MMAS and nc-MCAS, distributions showed significantly more homogeneous and slightly higher values in MMAS (p=0.033) (Figure 2B). In more detail, a REMA score ≥2 was observed in 90% SM, 70% MMAS, and 65% nc-MCAS. This corresponded to a sensitivity, specificity and accuracy of 90%, 34% and 64%, respectively, for SM and of 87%, 35% and 68%, respectively, for clonal MCAS (Table S1). Interestingly, the highest sensitivity was observed in patients with HαT (95% for SM and 91% for MC clonality), with sBT >20 ng/mL (92% and 91%) and >40 ng/mL (93% and 92%), and for those with insect- or food-triggered reactions (93%/92% and 95%/91%, respectively) (Table S1). Notably, no significant differences were observed in sensitivity, specificity and accuracy of the REMA score when patients fulfilling vs. those not fulfilling MCAS criteria were separately analyzed (83%, 38% and 64% vs. 88%, 35% and 69%, respectively, p>0.05). Overall, a REMA score >2 was able to correctly classify 79% of patients as clonal MCAS, whereas 75% showing a score <0 were properly classified as nc-MCAS; in turn, in only 48% of cases with score 0-2, the final diagnosis agreed with the score (i.e. clonal MCAS cases for score 2 and nc-MCAS patients for score 0 or 1). Impact of the REMA score on diagnostic accuracy After systematic implementation of the REMA score in 2012, the diagnostic rate of clonal MCAS increased by 13% (66% vs. 53%, p=0.002) (Table 2 and Figure 3A). The impact was greatest in patients with normal sBT, among whom a 24% increase (56% vs. 32%, p=0.03) (Table 2) together with an overall rise in the diagnosis of clonal MCAS with normal sBT levels from 4% to 20% (p<0.001) were observed (Figure 3B). Of note, improved diagnostic rates after (vs. before) implementation of the REMA score were particularly observed among drug-related MCAS patients (+15%, p=0.041) and idiopathic cases (+17%, p=0.005) (Table 2). Notably, among 4,073 referrals to the REMA between 2012 and 2024 due to MC-mediator release episodes without MIS, 3,224 (79%) had a REMA score studies due to different reasons such as detection of KIT p.D816V in blood, markedly elevated sBT (i.e., ≥40 ng/mL), particularly severe clinical presentations (e.g., cardiac arrest), or patient preference. Apart from those few exceptions, the REMA score consistently supported the decision to avoid BM examination, thereby preventing unnecessary invasive procedures in the majority (94%) of referrals. Optimized REMA score-based diagnostic algorithm More detailed analysis of our 2012-2024 cohort of patients confirmed that the REMA score maintains a very high sensitivity for detecting SM and MMAS, but with a substantial reduction of specificity compared with previous reports and the 2007-2012 data, particularly among patients with intermediate scores of 0-2 (Figure 2). In this regard, recent advances in molecular diagnostics, particularly with the implementation of high-sensitive ASO-qPCR and dPCR for KIT p.D816V detection, have also allowed their integration into routine clinical practice (17,18), leading to an improved detection of clonal MCAS. In our series, KIT p.D816V was detected by ASO-qPCR in whole blood at diagnosis in 23%, 23% and 33% of cases with REMA scores <0, 0-2 and KIT p.D816V in blood of 27%, 54% and 56% of clonal MCAS patients classified with the same REMA score ranges of 2, respectively. These findings translated into a specificity and sensitivity of blood-based screening of KIT p.D816V by ASO-qPCR of 100% and 46%, respectively, the latter ranging from 73% in patients with a REMA score 2, respectively (Table S2). Multivariate analysis performed in patients within the less accurate range of the REMA score (i.e., score 0-2) identified two strong predictors of both SM and clonal MCAS: increased sBT (≥11.4 ng/mL) in the absence of HαT OR: 9.13 (95%CI 4.75-17.54), p<0.001; and OR: 5.42 (95%CI 2.86-10.28), p<0.001, respectively and insect venom as a trigger of anaphylaxis OR: 3.72 (95% CI) (2.03-6.80), p<0.001, and OR: 2.18 (95% CI 1.28-3.72), p=0.004, respectively. Interestingly, none of these two factors showed a significant predictive value when analyzed in patients with REMA scores Based on the above findings, we developed a REMA-based diagnostic algorithm designed to identify patients with a higher probability of having clonal MCAS without MIS (Figure 4). According to this new algorithm, BM study is indicated when a patient has: 1) REMA score mutation is detected in blood; and/or 3) REMA score 0-2, provided that either elevated sBT (>11.4 ng/ml) without HαT is present or anaphylaxis is triggered by insect venom. This refined approach enhanced specificity while preserving the high sensitivity of the REMA score for identifying clonal MCAS, showing an overall sensitivity, specificity and accuracy of 86%, 57% and 75%, respectively.

Following its validation in 2011 (8), the REMA score rapidly became a key tool for evaluating patients with MC activation-associated symptoms without MIS, and determining the need for BM study. Prior to this period, patient selection for BM examination lacked standardized criteria and relied mainly on elevated sBT levels and/or the presence of severe MCAS-like symptoms. Our study is based on the largest prospective series reported to date in which the accuracy of the REMA score as a screening tool for clonal MCAS has been evaluated in patients presenting with severe MC mediator-related symptoms without MIS. Overall, our results confirm and extend previous evidence showing that the REMA score retains an excellent sensitivity for predicting MC clonality and mastocytosis (7–9,21) . Implementation of the REMA score has proven particularly valuable for the identification of clonal MCAS patients with normal sBT, among whom the rate of newly-diagnosed cases increased fivefold since the REMA score was introduced in routine clinical practice. However, our findings also revealed a significant reduction in the specificity of the REMA score when compared to previous reports (7,8) , particularly among patients with REMA scores of 0-2. These scores emerged here as the weakest score range of the model and they delineate an intermediate-risk patient subgroup. Such decreased specificity might be due, at least in part, to the fact that initially, once the REMA score was first proposed, many nc-MCAS cases had not undergone BM analyses. Despite this, our observations underscore the need for additional markers to refine risk-stratification of patients who might need to undergo a BM study and avoid overestimating clonal disease based solely on the (highly sensitive) REMA score. In this regard, multivariate analysis based on patients with a REMA score within this gray-zone subgroup of patients (score of 0-2) identified two independent predictors of clonal MCAS: increased sBT ≥11.4 ng/mL in the absence of HαT and insect venom-triggered anaphylaxis. Thus, in line with previous reports, our results suggest that persistent elevation of sBT without HαT (and other causes of hypertryptasemia, such as renal impairment or hematologic disorders), strongly support the specificity of increased sBT as a surrogate marker for an underlying clonal MC disease (22–24) . Likewise, there is a close association in the literature between insect-venom induced anaphylaxis and an underlying clonal MCAS (25–27) . Since 2011, the development of highly-sensitive molecular techniques such as ASO-qPCR (17,18) dPCR (28) , and more recently the ultrasensitive Flow-Super rolling circle amplification (RCA) assay (Flow-SuperRCA) (29) , has revolutionized the molecular diagnosis of MC diseases. These assays detect KIT p.D816V mutation in blood at variant allele frequencies (VAF) as low as <0.01% (ASO-qPCR, dPCR) and <0.001% (Flow-SuperRCA), enabling minimally invasive diagnosis, particularly in patients with low MC burden. As a consequence, they have become now the recommended first-line methods for detecting KIT p.D816V in the diagnostic work-up of MCAS patients (27, 28) . Despite this, our results showed that ASO-qPCR still fails to detect KIT p.D816V in blood of almost half of clonal MCAS cases, supporting the need for even more sensitive molecular assays for improved identification of patients presenting with very low MC loads. In this regard, a recent study by the REMA has shown an increased rate of detection of KIT p.D816V in blood from 39% to 83% in BMM and from 26% to 81% in MMAS, with the use of the Flow-SuperRCA assay, compared to the conventional ASO-qPCR method (29) . This ultrasensitive Flow-SuperRCA approach also allowed reclassification of approximately 15-20% of patients previously considered as nc-MCAS into clonal MCAS (29) . Since this new technology might not be widely available in the short-term, the updated REMA diagnostic algorithm here proposed might represent an appropriate strategy to reliably identify clonal MCAS with high accuracy, until ultrasensitive molecular methods become globally accessible and can be moved up to the first step of the diagnostic algorithms. Thus, here we proposed a refined REMA diagnostic algorithm through the combination of i) KIT p.D816V detection in blood, ii) H α T genotyping, iii) specific (additional) clinical and laboratory variables, and iv) the REMA score. Whereas in the original model a REMA score of ≥2 was considered sufficient to justify a subsequent BM evaluation (7,8) , in the new (revised) algorithm the REMA score threshold is raised from a score ≥2 to a greater score (>2), recommending directly subsequent BM investigations. In turn, BM examination is now also indicated for patients with a REMA score 11.4 ng/ml) in the absence of H α T. Altogether, these modifications enhanced the specificity of the REMA score while preserving its high sensitivity, thereby reducing the rate of false positives and minimizing the risk of overlooking clonal disease. In summary, this study refines the clinical utility of the REMA score as a first-line screening tool for the diagnostic evaluation of patients with suspected clonal MCAS in the absence of MIS. By adjusting score thresholds and integrating new clinical features and molecular biomarkers, we propose a revised REMA score-based diagnostic algorithm with increased specificity and diagnostic accuracy, while retaining an excellent sensitivity. This optimized approach provides a data-based rationale for guiding BM investigations, particularly in challenging scenarios such as patients with normal sBT or those falling into intermediate-risk categories defined by REMA scores of 0-2. Until ultrasensitive molecular assays such as Flow-SuperRCA become widely available, the here proposed REMA diagnostic algorithm represents a robust tool to ensure early and accurate identification of patients with clonal MCAS.

1. Valent P, Akin C, Arock M, Brockow K, Butterfield JH, Carter MC, et al. Definitions, Criteria and Global Classification of Mast Cell Disorders with Special Reference to Mast Cell Activation Syndromes: A Consensus Proposal. Int Arch Allergy Immunol. 2012;157(3):215-25. 2. Valent P, Hartmann K, Bonadonna P, Gülen T, Brockow K, Alvarez-Twose I, et al. Global Classification of Mast Cell Activation Disorders An ICD-10-CM-Adjusted Proposal of the ECNM-AIM Consortium. J Allergy Clin Immunol Pract. 2022;10(8):1941-50. 3. Castells M, Giannetti MP, Hamilton MJ, Novak P, Pozdnyakova O, Nicoloro-SantaBarbara J, et al. Mast cell activation syndrome: Current understanding and research needs. J Allergy Clin Immunol. 2024;154(2):255-63. 4. Lyons JJ, Yu X, Hughes JD, Le QT, Jamil A, Bai Y, et al. Elevated basal serum tryptase identifies a multisystem disorder associated with increased TPSAB1 copy number. Nat Genet. 2016;48(12):1564-9. 5. Lyons JJ. Hereditary Alpha Tryptasemia: Genotyping and Associated Clinical Features. Immunol Allergy Clin North Am. 2018;38(3):483-95. 6. González-de-Olano D, Navarro-Navarro P, Muñoz-González JI, Sánchez-Muñoz L, Henriques A, de-Andrés-Martín A, et al. Clinical impact of the TPSAB1 genotype in mast cell diseases: A REMA study in a cohort of 959 individuals. Allergy. 2024;79(3):711-23. 7. Alvarez-Twose I, González de Olano D, Sánchez-Muñoz L, Matito A, Esteban-López MI, Vega A, et al. Clinical, biological, and molecular characteristics of clonal mast cell disorders presenting with systemic mast cell activation symptoms. J Allergy Clin Immunol. 2010;125(6):1269-1278. 8. Alvarez-Twose I, González-de-Olano D, Sánchez-Muñoz L, Matito A, Jara-Acevedo M, Teodosio C, et al. Validation of the REMA score for predicting mast cell clonality and systemic mastocytosis in patients with systemic mast cell activation symptoms. Int Arch Allergy Immunol. 2012;157(3):275-80. 9. González-de-Olano D, Esteban-López MI, Alonso-Díaz-de-Durana MD, González-Mancebo E, Prieto-García A, Gandolfo-Cano M, et al. Frequency of clonal mast cell diseases among patients presenting with anaphylaxis: A prospective study in 178 patients from 5 tertiary centers in Spain. J Allergy Clin Immunol Pract. 2019;7(8):2924-2926.e1. 10. Sampson HA, Muñoz-Furlong A, Campbell RL, Adkinson NF, Bock SA, Branum A, et al. Second symposium on the definition and management of anaphylaxis: summary report–Second National Institute of Allergy and Infectious Disease/Food Allergy and Anaphylaxis Network symposium. J Allergy Clin Immunol. 2006;117(2):391-7. 11. Valent P, Akin C, Hartmann K, Alvarez-Twose I, Brockow K, Hermine O, et al. Updated Diagnostic Criteria and Classification of Mast Cell Disorders: A Consensus Proposal. HemaSphere. 2021;5(11):e646. 12. Alvarez-Twose I, Morgado JM, Sánchez-Muñoz L, García-Montero A, Mollejo M, Orfao A, et al. Current state of biology and diagnosis of clonal mast cell diseases in adults. Int J Lab Hematol. 2012;34(5):445-60. 13. Sánchez-Muñoz L, Teodosio C, Morgado JMT, Perbellini O, Mayado A, Alvarez-Twose I, et al. Flow cytometry in mastocytosis: utility as a diagnostic and prognostic tool. Immunol Allergy Clin North Am. 2014;34(2):297-313. 14. Morgado JM, Perbellini O, Johnson RC, Teodósio C, Matito A, Álvarez-Twose I, et al. CD30 expression by bone marrow mast cells from different diagnostic variants of systemic mastocytosis. Histopathology. 2013;63(6):780-7. 15. Garcia-Montero AC, Jara-Acevedo M, Teodosio C, Sanchez ML, Nunez R, Prados A, et al. KIT mutation in mast cells and other bone marrow haematopoietic cell lineages in systemic mast cell disorders. A prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients. Blood. 2006;108(7):2366-72. 16. Sotlar K, Escribano L, Landt O, Möhrle S, Herrero S, Torrelo A, et al. One-step detection of c-kit point mutations using peptide nucleic acid-mediated polymerase chain reaction clamping and hybridization probes. Am J Pathol. 2003;162(3):737-46. 17. Kristensen T, Vestergaard H, Moller MB. Improved detection of the KIT D816V mutation in patients with systemic mastocytosis using a quantitative and highly sensitive real-time qPCR assay. J Mol Diagn. 2011;13(2):180-8. 18. Jara-Acevedo M, Teodosio C, Sanchez-Muñoz L, Álvarez-Twose I, Mayado A, Caldas C, et al. Detection of the KIT D816V mutation in peripheral blood of systemic mastocytosis: diagnostic implications. Mod Pathol. 2015;28(8):1138-49. 19. Arock M, Sotlar K, Akin C, Broesby-Olsen S, Hoermann G, Escribano L, et al. KIT mutation analysis in mast cell neoplasms: recommendations of the European competence network on mastocytosis. Leukemia. 2015;29(6):1223-32. 20. Khoury JD, Solary E, Abla O, Akkari Y, Alaggio R, Apperley JF, et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Myeloid and Histiocytic/Dendritic Neoplasms. Leukemia. 2022;36(7):1703-19. 21. Rama TA, Torrado I, Henriques AF, Sánchez-Muñoz L, Jara-Acevedo M, Navarro-Navarro P, et al. Mast Cell Activation Syndromes: Comparison Between Two Scoring Models to Predict for Mast Cell Clonality. J Allergy Clin Immunol Pract. 2023;11(3):908-19. 22. Valent P, Hoermann G, Bonadonna P, Hartmann K, Sperr WR, Broesby-Olsen S, et al. The Normal Range of Baseline Tryptase Should Be 1 to 15 ng/mL and Covers Healthy Individuals With HαT. J Allergy Clin Immunol Pract. 2023;11(10):3010-20. 23. Akin C. How to evaluate the patient with a suspected mast cell disorder and how/when to manage symptoms. Hematol Am Soc Hematol Educ Program. 2022 (1):55-63. 24. Lyons JJ, Greiner G, Hoermann G, Metcalfe DD. Incorporating Tryptase Genotyping Into the Workup and Diagnosis of Mast Cell Diseases and Reactions. J Allergy Clin Immunol Pract. 2022;10(8):1964-73. 25. Alvarez-Twose I, Zanotti R, González-de-Olano D, Bonadonna P, Vega A, Matito A, et al. Nonaggressive systemic mastocytosis (SM) without skin lesions associated with insect-induced anaphylaxis shows unique features versus other indolent SM. J Allergy Clin Immunol. 2014;133(2):520-8. 26. Bonadonna P, Bonifacio M, Lombardo C, Zanotti R. Hymenoptera Allergy and Mast Cell Activation Syndromes. Curr Allergy Asthma Rep. 2016;16(1):5. 27. Bonadonna P, Zanotti R, Pagani M, Caruso B, Perbellini O, Colarossi S, et al. How much specific is the association between hymenoptera venom allergy and mastocytosis? Allergy. 2009;64(9):1379-82. 28. Greiner G, Gurbisz M, Ratzinger F, Witzeneder N, Simonitsch-Klupp I, Mitterbauer-Hohendanner G, et al. Digital PCR: A Sensitive and Precise Method for KIT D816V Quantification in Mastocytosis. Clin Chem. 2018;64(3):547-55. 29. Navarro-Navarro P, González-Tablas M, Pérez-Pons A, Sánchez-Muñoz L, Henriques A, Álvarez-Twose I, et al. Improved diagnostic screening and classification of clonal mast cell diseases by ultrasensitive KIT p.D816V detection. Blood. 2025 [Epub ahead of print].doi: 10.1182/blood.2025029507. 30. Gotlib J, Schwaab J, Shomali W, George TI, Radia DH, Castells M, et al. Proposed European Competence Network on Mastocytosis-American Initiative in Mast Cell Diseases (ECNM-AIM) Response Criteria in Advanced Systemic Mastocytosis. J Allergy Clin Immunol Pract. 2022;10(8):2025-2038. 31. Valent P, Hartmann K, Hoermann G, Reiter A, Alvarez-Twose I, Brockow K, et al. Harmonization of Diagnostic Criteria in Mastocytosis for Use in Clinical Practice: WHO vs ICC vs AIM/ECNM. J Allergy Clin Immunol Pract. 2024;12(12):3250-3260. Supplementary Material File (figure 1. mc-mediator release symptoms and triggers (1).docx) - Download - 234.94 KB File (figure 2. rema score.docx) - Download - 266.56 KB File (figure 3. impact rema score.docx) - Download - 224.67 KB File (figure 4. algorithm.docx) - Download - 70.43 KB File (figure s1. hat.docx) - Download - 193.64 KB File (table 1. rema score (1).docx) - Download - 14.90 KB File (table 2. changes in diagnostic rate.docx) - Download - 16.47 KB File (table s1. rema score efficiency.docx) - Download - 21.88 KB File (table s2. kit d816v in peripheral blood.docx) - Download - 15.67 KB Information & Authors Information Version history Peer review timeline Published Allergy Version of Record19 May 2026Published Copyright This work is licensed under a Non Exclusive No Reuse License.

Authors Metrics & Citations Metrics Article Usage 738views 217downloads Citations Download citation Irina Sucre-Adrianza, Horacio Caligaris, Cristina Morales-Cabeza, et al. The REMA Score Revisited: An Optimized Algorithm for Early Prediction of Clonal Mast Cell Disease.. Authorea. 21 October 2025. DOI: https://doi.org/10.22541/au.176105556.65210861/v1 DOI: https://doi.org/10.22541/au.176105556.65210861/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.