scholarly journals Force-Induced Cooperative Unfolding of Two Distinctive Domains in a Single Gpibalpha Molecule

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3449-3449
Author(s):  
Yunfeng Chen ◽  
Lining Ju ◽  
Jizhong Lou ◽  
Cheng Zhu
Keyword(s):  
Conflicts Of Interest ◽  
Joint Probability ◽  
Md Simulations ◽  
Calcium Flux ◽  
Time Data ◽  
Chi Square ◽  
Biomembrane Force Probe ◽  
Repeat Domain ◽  
A1 Domain ◽  
Intracellular Calcium Flux

Abstract GPIbα, a major member of the GPIb-IX-V complex, initiates a mechano-signaling pathway that leads to platelet intracellular calcium flux when binding to VWF at the A1 domain1. Exactly how this signal is transduced across the membrane is unknown. A recent work identifying the unfolding of a jextamembrane mechanosensitive domain (MSD) (Fig.1 A,C) suggested that this unfolding might play a role in the signal transduction of GPIbα2. Using molecular dynamics (MD) simulations to pull the GPIbα leucine-rich repeat domain (LRRD) from the VWF A1 domain, we observed unfolding of the LRRD (Fig.1 A) (manuscript under review). Here we used a biomembrane force probe (BFP) to study single GPIbα unfolding (Fig. 1E,F). Platelet GPIbα was pulled by A1 coated on the probe through a ramping phase (pink, Fig. 1G) to a clamping phase to wait for bond dissociation under a 25-pN force (red, Fig. 1G). Two unfolding signatures were identified: i) ramped unfolding, featured by a sudden force kink in the ramping phase (dashed circle, Fig. 1G); ii) clamped unfolding, featured by an abrupt force drop in the clamping phase (dashed ellipse, Fig. 1G). Unfolding lengths from both signatures were measured from the force vs. time data (Fig. 1G), based on which the unfolding events of MSD (7-25 nm), LRRD (25-56 nm) or both together (56-80 nm) were differentiated. Intriguingly, LRRD unfolding was only observed in the ramping phase, while MSD could unfold in both ramping and clamping phases. The frequency of observing both LRRD and MSD unfolding in the same cycle was much higher than the product of the frequencies for LRRD and MSD to unfold (the joint probability for both to unfold if they were independent), suggesting that the two GPIbα domains unfolded cooperatively (Fig 2A). Chi-square tests also rejected the hypothesis that MSD and LRRD unfolded independently (p =5.24×10-5). Separating the ramped and clamped unfolding of MSD and evaluating their respective cooperativity with LRRD unfolding demonstrated similar results (Fig 2A). Agreeing with these, both the ramped and clamped unfolding of MSD occurred much more frequently in the presence of LRRD unfolding (Fig 2B, left), and vice versa for the unfolding of LRRD (Fig 2B, right). Cooperative unfolding of LRRD and MSD may play a key role in transducing signals across platelet membrane. References 1 Nesbitt, W. S. et al. The Journal of biological chemistry277, 2965-2972, doi:10.1074/jbc.M110070200 (2002). 2 Zhang, W. et al. Blood125, 562-569, doi:10.1182/blood-2014-07-589507 (2015). Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Identification and Characterization of Integrin alphaIIbbeta3 Intermediate Affinity State Induced By Gpibalpha Mechanotransduction

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 237-237
Author(s):  
Yunfeng Chen ◽  
Lining Ju ◽  
Cheng Zhu
Keyword(s):  
Intermediate State ◽  
Conflicts Of Interest ◽  
Calcium Flux ◽  
High Affinity ◽  
Vascular Surface ◽  
Biomembrane Force Probe ◽  
Force Clamp ◽  
A1 Domain ◽  
Intracellular Calcium Flux

Abstract During arterial haemostasis, platelets tether to and translocate on disrupted vascular surface via binding of GPIbα to the VWF A1 domain, which triggers activation signals that induce intracellular Ca2+ release and up-regulate integrin αIIbβ3 binding capacity1,2. Inhibition of this signaling pathway or blockade of αIIbβ3 binding has a devastating impact on platelet firm adhesion and aggregate formation1,3,4. We used a newly developed switch biomembrane force probe (BFP) assay to characterize the cooperativity between GPIbα and αIIbβ3 on single-molecular level. VWF-A1 and fibronectin fragment (FNIII7-10) were separately coated on two different probes, enabling controlled presentation of two ligands in separate space and time (Fig. A). Pulling GPIbα by A1 on the first probe with a durable force activated the discoid platelet (Fig. A, upper panel), as revealed by an intracellular calcium flux. Upon switching to the FNIII7-10-bearing bead, the A1-triggered platelet was interrogated for the activity of its surface αIIbβ3 (Fig. A, lower panel). A 25-pN force of >2s duration on a single GPIbα-A1 bond was found to activate αIIbβ3 into an intermediate affinity state, as reflected by a ~30% adhesion frequency with a FNIII7-10 bead, significantly higher than binding to low affinity αIIbβ3 on platelets without A1-triggering but much lower than binding to high affinity αIIbβ3 on platelets pre-incubated with ADP or thrombin (Fig. B), two strong platelet-activating agonists. This submaximal activation agreed with the previous immuno-staining findings2. αIIbβ3 activation and outside-in signaling require the sequential engagement of talin and Gα13 to β3 cytoplasmic tail, respectively5,6. Blocking Gα13-β3 engagement by a synthetic peptide mP6 (gift from Xiaoping Du, UIC)6 had no effect on FNIII7-10 binding of resting and A1-triggered platelets, but significantly suppressed the FNIII7-10 binding of platelets pre-incubated with ADP or thrombin by lowering their adhesion frequencies to a level comparable with A1-triggered platelets (Fig. B). The control peptide mP6Scr showed no effect on the ADP or thrombin stimulated platelets (Fig. B). In addition to binding affinity, bond lifetimes were measured by force-clamp experiment. Platelets with no treatment, A1 triggering and ADP stimulation formed αIIbβ3-FNIII7-10 (Fig. C) and αIIbβ3-fibrinogen (Fig. D) bonds with short, intermediate and long lifetimes, respectively. Interactions with fibrinogen exhibited catch-slip bonds regardless of the αIIbβ3 affinity state. By comparison, only high affinity αIIbβ3 exhibited a catch-slip bond with FNIII7-10 as slip-only bonds were observed for FNIII7-10 interactions with intermediate and low affinity αIIbβ3. These data suggest the existence of three distinct integrin states that correspond to the three affinities and force-dependent bond lifetime patterns, in which the intermediate state was induced by the GPIbα mechanotransduction (Fig. E). Gα13 engagement was not required for the intermediate state, but necessary for the high affinity state (Fig. E). References: 1. Nesbitt, W. S. et al. The Journal of biological chemistry277, 2965-2972, doi:10.1074/jbc.M110070200 (2002). 2. Kasirer-Friede, A. et al. Blood103, 3403-3411, doi:10.1182/blood-2003-10-3664 (2004). 3. Savage, B., Saldivar, E. & Ruggeri, Z. M. Cell84, 289-297 (1996). 4. Warwick, S. N. et al. Nature Medicine15, doi:10.1038/nm.1955 (2009). 5. Gong, H. et al. Science327, 340-343, doi:10.1126/science.1174779 (2010). 6. Shen, B. et al. Nature503, 131-135, doi:10.1038/nature12613 (2013). Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

The Study of GPIb-VWF Mediated Early-Stage Platelet Activation Triggering On a Single Cell

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1069-1069
Author(s):  
Lining Ju ◽  
Cheng Zhu ◽  
Miguel A. Cruz ◽  
Yunfeng Chen
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Early Stage ◽  
Morphological Changes ◽  
Morphological Transformation ◽  
Second Stage ◽  
Biomembrane Force Probe ◽  
Interaction Kinetics ◽  
High Level ◽  
A1 Domain

Abstract Abstract 1069 Binding of GPIbα to VWF tethers platelets to disrupted vascular surface during the haemostatic process. The GPIbα –VWF interaction can also trigger outside-in signaling cascade, resulting in platelet activation, characterized by morphological transformation from discoid to a more spiky shape as well as activation of integrin α IIbβ3. Using the adhesion frequency assay with a biomembrane force probe (BFP), we studied signal initiation by repeated brief contacts of a single platelet with a glass bead coated with VWF-A1 domain and/or fibronectin III 7–10 domain (FNIII7–10) in a precisely controlled fashion (Fig. A). Contacting platelets with beads coated VWF-A1 only resulted in adhesion kinetics mediated by GPIbα –VWF interaction kinetics independent of the activation stage of the platelet. Contacting platelets with beads coated FNIII7–10 only resulted in adhesion kinetics that correlated with the activation stage of the platelet. Discoid-shaped platelets yielded low level adhesions mediated by FN interaction with inactive α IIbβ3 (Fig. B, blue). By comparison, spiky-shaped platelets produced high level adhesions mediated by FN interaction with activated α IIbβ3 (Fig. B, red)that was four times stronger than the interaction with inactive α IIbβ3. Contacting platelets with beads coated both VWF-A1 and FNIII7–10 resulted in two-stage adhesion kinetics. The first stage was mediated by GPIbα –VWF binding, which triggered a second stage consisting of an increase in adhesion after a sub-second delay. The second-stage binding coincided with morphological changes characteristic of platelet activation and matched that mediated by FN interaction with activated α IIbβ3. On the other hand, the concurrent calcium imaging showed as the platelet target was brought to the A1 bead in a repeating manner, the recorded calcium fluorescence intensity climbed up as the repeated touches continue (Fig. C). The peak temporally correlates with the morphological change. Our data indicates that binding of VWF-A1 to platelet GPIbα initiates outside-in signaling, leading to rapid irreversible platelet shape changes and calcium mobilization within a few seconds. Disclosures: No relevant conflicts of interest to declare.

Pengaruh merokok terhadap hasil clotting time dan bleeding time pada populasi laki-laki Fakultas Kedokteran UMSU

2019 ◽  
Vol 2 (1) ◽  
pp. 217-222
Author(s):  
Dhio Emerko Ginting ◽  
Fani Ade Irma ◽  
Sri Rezeki Arbaningsih ◽  
Siti Hajar
Keyword(s):  
Bleeding Time ◽  
Clotting Time ◽  
Time Data ◽  
Chi Square ◽  
Cross Sectional

WHO telah menetapkan Indonesia sebagai negara dengan jumlah perokok terbesar ketiga. Ada tiga zat kimia yang paling sangat berbahaya, yaitu tar, nikotin, karbon monoksida yang terkandung dalam rokok. Hasil penelitian di Inggris menunjukkan kepada kita bahwa kurang lebih 50% perokok yang merokok sejak remaja mengalami kematian akibat penyakit yang berhubungan dengan kebiasaan merokok mereka. Penelitian ini bertujuan untuk mengetahui tentang pengaruh asap rokok dengan proses kaskade clotting time. Jenis penelitian ini adalah penelitian analitik dengan menggunakan desain cross-sectional. Penelitian ini menggunakan sampel populasi laki-laki di Fakultas Kedokteran di UMSU yang diwawancara untuk mengetahui bahwa mereka merokok atau tidak, setelah itu, sampel diperiksa clotting time dan bleeding time. Data yang terkumpul dianalisis dengan uji chi-square. Hasil penelitian menunjukkan p = 0,000 bahwa ada hubungan merokok dengan clotting time dan bleeding time pada perokok berat dan sedang. Pada perokok berat dan moderat terdapat hubungan bermakna antara penurunan bleeding time dan peningkatan clotting time dengan merokok. Tidak ada hubungan antara clotting time dan merokok pada perokok ringan.

scholarly journals Making high-dimensional molecular distribution functions tractable through Belief Propagation on Factor Graphs

2021 ◽  
Author(s):  
Zachary Smith ◽  
Pratyush Tiwary
Keyword(s):  
Belief Propagation ◽  
Conformational Dynamics ◽  
Joint Probability ◽  
Md Simulations ◽  
Distribution Functions ◽  
Factor Graph ◽  
High Dimensional ◽  
Enhanced Sampling ◽  
Small Peptides ◽  
Approximate Probability

Molecular dynamics (MD) simulations provide a wealth of high-dimensional data at all-atom and femtosecond resolution but deciphering mechanistic information from this data is an ongoing challenge in physical chemistry and biophysics. Theoretically speaking, joint probabilities of the equilibrium distribution contain all thermodynamic information, but they prove increasingly difficult to compute and interpret as the dimensionality increases. Here, inspired by tools in probabilistic graphical modeling, we develop a factor graph trained through belief propagation that helps factorize the joint probability into an approximate tractable form that can be easily visualized and used. We validate the study through the analysis of the conformational dynamics of two small peptides with 5 and 9 residues. Our validations include testing the conditional dependency predictions through an intervention scheme inspired by Judea Pearl. Secondly we directly use the belief propagation based approximate probability distribution as a high-dimensional static bias for enhanced sampling, where we achieve spontaneous back-and-forth motion between metastable states that is up to 350 times faster than unbiased MD. We believe this work opens up useful ways to thinking about and dealing with high-dimensional molecular simulations.

At Sensor Diagnosis for Smart Healthcare

2018 ◽  
Vol 10 (4) ◽  
pp. 1-13
Author(s):  
Chetna Laroiya ◽  
Vijay Bhushan Aggarwal
Keyword(s):  
Joint Probability ◽  
Sliding Window ◽  
Communication Technologies ◽  
Data Streaming ◽  
Round Trip ◽  
Time Data ◽  
Smart Healthcare ◽  
Body Sensors ◽  
Round Trip Delay

In order to implement IoT-based health-care for improved quality of life, we have to deal with sensor and communication technologies. In this article, the authors propose an approach to analyse real-time data streaming from a patient's surface body sensors, which are to be looked upon in a small sliding window frame. Time series analysis of data from the sensors is effective in reducing the round-trip delay between patient and the medical server. Two algorithms are for the sensor, and odd measures are proposed based on joint probability and joint conditional probability. The proposed algorithms are to be SQL compliant, as traces of at-sensor UDBMS alongside elementary capabilities supports databases with a meagre amount of SQL, which is evident in the literature.

β-Endorphin modulates T-cell intracellular calcium flux and c-myc expression via a potassium channel

1990 ◽  
Vol 27 (2-3) ◽  
pp. 163-171 ◽  
Author(s):  
Christopher J. Hough ◽  
John I. Halperin ◽  
Denise L. Mazorow ◽  
Stephen L. Yeandle ◽  
David B. Millar
Keyword(s):  
T Cell ◽  
Potassium Channel ◽  
Intracellular Calcium ◽  
Calcium Flux ◽  
Intracellular Calcium Flux

Pediatric Precursor-B ALL with BTG1 Deletions Show a Distinct Genomic Profile.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3244-3244
Author(s):  
Esmee Waanders ◽  
Frank N. van Leeuwen ◽  
Eugene Verwiel ◽  
Simon V van Reijmersdal ◽  
Marloes R Levers ◽  
...  
Keyword(s):  
B Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
B Cell Differentiation ◽  
Chi Square ◽  
Genome Wide ◽  
Intron 1 ◽  
Number Variation ◽  
Unselected Cohort

Abstract Abstract 3244 Poster Board III-181 Recent genome-wide profiling studies have revealed that childhood acute lymphoblastic leukemia (ALL) is characterized by recurrent microdeletions, including the cell cycle regulator CDKN2A, the B-cell differentiation genes PAX5, EBF1 and IKZF1 (Ikaros) and the anti-proliferative gene B-cell translocation gene 1 (BTG1). In a previous study, we have shown that BTG1 is an important determinant of glucocorticoid sensitivity (Van Galen et al. Blood/ ASH Annual Meeting Abstracts, 2008). In the present study we have characterized these cases in more detail and elucidated the frequency of recurrent lesions in BTG1 deletion cases. Using locus-specific MLPA screening of an unselected cohort of 305 precursor B-ALL cases, we identified 26 microdeletions (8.5%). All deletions encompassed BTG1 only. We were able to genomically profile 22 diagnosis samples using Affimetrix SNP6.0 arrays. Of these, 12 did not develop a relapse during a minimal of 4,5 years of follow up. The mean number of CNVs was 29.6 of which 10.3 gains and 22.5 losses (median size 512 kb and 115 kb respectively). BTG1 deletions were generally focal, varying in size from 104 kb to 1,4 Mb. In all but one patient the breakpoints at the 5' end of the deletion tightly clustered and subsequent fine-mapping using qPCR revealed that this breakpoint cluster was located within intron 1 of the BTG1 gene. At the 3'end of the deletion, four breakpoint clusters could be identified. Analysis of the copy number variation (CNV) profiles showed that patients with a BTG1 deletion more often harbored a deletion in IKZF1 compared to an unselected cohort of pre-B ALL cases (27% vs 7%, chi-square p=0.042). In contrast, recurrent CNVs like PAX5, EBF1 and CDKN2A/B occur in similar frequencies (23%, 9% and 32% vs 17%, 0% and 23% respectively). In addition, the BTG1 deletion cases that developed into a relapse showed significantly more often a deletion in CDKN2A/B compared to the BTG1 deletion cases that did not develop a relapse (60% vs 8%, p=0.02). Together, these data indicate that pediatric precursor-B ALL carrying BTG1 deletions have distinct genomic profiles, showing increased frequencies of deletions in IKZF1 and CDKN2A. Disclosures No relevant conflicts of interest to declare.

Increasing Hydrophobicity and Disulfide Bridging Between A1 and C2 Subunits Improves Factor VIII Stability.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3176-3176
Author(s):  
Hironao Wakabayashi ◽  
Amy E Griffiths ◽  
Philip Fay
Keyword(s):  
Thermal Stability ◽  
Factor Viii ◽  
Chemical Stability ◽  
Hydrophobic Interaction ◽  
Conflicts Of Interest ◽  
Covalent Modification ◽  
C2 Domain ◽  
Double Mutation ◽  
Fviii Activity ◽  
A1 Domain

Abstract Abstract 3176 Poster Board III-100 Factor VIII (FVIII) consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), while the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. Previously we have generated FVIII mutants with enhanced stability by mutating residues located at A1-A2 or A2-A3 interfaces (Wakabayashi et al, Blood, 112, 2761-9, 2008, Wakabayashi et al, J. Thromb. Haemost. 7, 438-44, 2009). FVIII X-ray structures show close contacts between the Ca2+ binding site contained within the A1 domain and the C2 domain of LC. In this study we mutated residues located at this interface to examine the effects on FVIII(a) stability. Studies assessing FVIII thermal and chemical stability involved monitoring the rates of loss of FVIII activity by FXa generation assay following incubation of FVIII (4 nM) at 57°C or in various concentrations of guanidinium (0-1.2 M). The rate of decay of FVIIIa was monitored over time at 23°C using FXa generation assays following activation of FVIII (1.5 nM) with thrombin. Data were fitted to single exponential decay equations and rates of decay were compared. In one variant, a disulfide bond was introduced between the two domains by a double mutation at Arg121 in A1 and Leu2302 in the C2 domain to Cys (R121C/L2302C). In addition, based on the finding that there is a gap between the methyl groups of Ala108 (A1 domain) and Ala2328 (C2 domain) we mutated Ala108 to Val, Ile, or Leu to examine whether these mutants increase the stability of FVIII by an improved hydrophobic interaction at this site. Significant increases in FVIII thermal stability, up to 4-fold compared with WT, were observed in R121C/L2302C, Ala108Ile, and Ala108Leu. R121C/L2302C and Ala108Ile retained ∼80% FVIII activity as measured by FXa generation assay compared to WT value, however, that of Ala108Leu was ∼25% the WT value. Only Ala108Ile showed an improvement in chemical stability (10% increase in IC50 value as compared with WT FVIII) and FVIIIa decay due to A2 subunit dissociation was similar to WT FVIII (20-40% reduction in FVIIIa decay rate compared to WT). Ca2+ is necessary for FVIII function and EGTA (2 mM) reduced WT FVIII activity by ∼70%. However, EGTA-treated R121C/L2302C FVIII retained ∼100% activity, suggesting that the Ca2+ requirement for FVIII function may be substituted by covalent bonding between the Ca2+ binding region in A1 and C2 subunit. Furthermore, the Ala108Ile variant showed ∼60% activity remaining after EGTA treatment suggesting partial relief of this Ca2+ dependency for stability of the A1-C2 interaction. Next, we tested whether the mutations at the A1-C2 interface can be combined with mutations at A1-A2 or A2-A3 interfaces to generate a FVIII with further improved stability. Previously characterized FVIII variants, designated A domain mutants, showing up to 2-fold increases in thermal stability compared with WT FVIII included Asp519Ala, Asp519Val, Glu665Ala, Glu665Val, Glu1984Ala, and Glu1984Val. In combining those mutations with either R121C/L2302C or Ala108Ile, we obtained variants with >5-fold increases in thermal stability (9/12 mutants), with the Ala108Ile/Glu665Val variant showing the greatest increase (∼10-fold). Most of the mutants (9/12) showed normal FVIII activity values by FXa generation assay (>60%) and 15-30% increases in IC50 values for chemical stability as compared with WT. In addition, the high FVIIIa stability of the A domain mutants was largely preserved in the combined mutations. Collectively, these results suggest that alterations at this A1-C2 contact region by covalent modification or increasing hydrophobic interaction yields improved FVIII stability that can be combined with other high stability mutations to produce additive effects. Disclosures No relevant conflicts of interest to declare.

Cellular Composition and Clonal Evolution of 295 Type III PNH Clones: An Italian Multi-Laboratory Study

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3482-3482
Author(s):  
Elisa Cannizzo ◽  
Domenico Susca ◽  
Anna Maria Triolo ◽  
Catia Lo Pardo ◽  
Francesco Buccisano ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Clonal Evolution ◽  
High Sensitivity ◽  
Red Cells ◽  
P Value ◽  
Physical Parameters ◽  
Cellular Composition ◽  
Chi Square ◽  
Type Iii

Abstract Abstract 3482 Three different types of PNH have been described so far, based upon the presence of hemolysis and bone marrow failure (BMF): a) florid PNH (hemolysis+/BMF-), b) PNH in the context of a BMF syndrome (hemolysis+/BMF+), c) subclinical PNH (hemolysis-/BMF+). In any case, the presence of a PNH clone is a prerequisite and Flow Cytometry (FCM) plays a crucial role in detecting and monitoring it with high sensitivity and specificity. In 2010, an Italian archive of FCM-detected PNH clones (http://int.clonepnh.com/) was created on a multi-laboratory basis. Eighty-one laboratories participated in the project. The aim was twofold: a) to provide a large dictionary of Italian PNH clones; b) based upon stringent rules regarding the compilation of records, to obtain an auto-educational effect on participating laboratories. Here, we describe the cellular composition and the clonal evolution of 295 type III (complete defect of GPI-linked proteins) PNH clones identified during the study. Forty-nine of these clones (16.6%) were accompanied by a PNH-II clone (partial defect of GPI-linked proteins). Hemoglobinuria was the most frequent (27%) reason for testing (RFT), followed by aplastic anemia (AA, 13%), MDS (13%), hemolytic anemia (12%), unexplained cytopenia (UC, 10%), BMF (5%), atypical venous thrombosis (5%), other (15%). Fluorescent Aerolysin (FLAER) was used since 2007, with an increasing % of utilization, from 4% to 60% of cases. CD24 utilization also progressively increased. CD59 was the most used antigen for RBC typing. The most used gating strategies were based upon physical parameters for RBC, CD45 and/or CD33 vs side scatter for granulocytes and monocytes. The 295 clones were categorized into 3 classes according to their size, determined as the percent of PNH cells in peripheral granulocytes: 0–10% (112 clones, or 38%, defined as “small”), 10.1%–70% (69 clones, or 23%, defined as “intermediate”), 70.1%–100% (115 clones, or 39%, defined as “large”). This distribution was significantly different from that expected on the basis of a random distribution within the three classes (i.e. 10%, 60% and 30%): chi square was 51 with a p value <0.0001. Sixty-seven clones were sequentially studied (with a follow up ranging from 3 to 74 months): thirty seven and twelve of them received 2 and 3 determinations, respectively; eight clones received 4 tests while three other clones received 5 tests; two clones received 6 tests, 3 clones received 8 tests, one single clone received 10 tests and another one 14. Twenty of them (30%) showed a change in category (10 increased and 10 decreased). Just 4 clones jumped from “smallest” to “biggest” category or vice versa (one increased and 3 decreased, see Table). Type of change N of cases Median of months to change (range) Increase of 1 category 9 8 (4–74) Decrease of 1 category 7 18 (3–23) Increase of 2 categories (from “small” to “large”) 1 7 (N.A.) Decrease of 2 categories (from “large” to “small”) 3 22 (4–55) The vast majority of clones (287, or 97.3%), typed by analyzing neutrophils, monocytes and red cells, proved trilinear, while 8 clones were composed just by white cells, with red cells completely normal. This is the first multi-laboratory relational database of FCM-detected PNH clones described so far. An auto-educational goal was reached, since general sensitivity increased progressively and reagent choice significantly changed, leading to a stable FCM protocol, consisting of FLAER and CD24 for granulocytes, FLAER and CD14 for monocytes, CD59 for erythrocytes. As regards clonal evolution, the rarity of migration between extreme categories suggests that the belonging to these ones could be sustained by different backgrounds and pressures. Disclosures: No relevant conflicts of interest to declare.

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