Background. Acceptance of dual kidney transplantation (DKT) has proven difficult, due to surgical complexity and concerns regarding long-term outcomes. We herein present a standard technique for ipsilateral DKT and compare outcomes to single-kidney transplant (SKT) recipients. Methods. A retrospective single-center comparison of DKT and SKT performed between February 2007 and July 2013. Results. Of 516 deceased donor kidney transplants, 29 were DKT and 487 were SKT. Mean follow-up was 43 ± 67 months. DKT recipients were older and more likely than SKT recipients to receive an extended criteria graft (p < 0.001). For DKT versus SKT, the rates of delayed graft function (10.3 versus 9.2%) and acute rejection (20.7 versus 22.4%) were equivalent (p = ns). A higher than expected urologic complication rate in the DKT cohort  was reduced through modification of the ureteral anastomosis. Graft survival was equivalent between DKT and SKT groups (p = ns) with actuarial 3-year DKT patient and graft survivals of 100% and 93%. At 3 years, the groups had similar renal function (p = ns). Conclusions. By utilizing extended criteria donor organs as DKT, the donor pool was enlarged while providing excellent patient and graft survival. The DKT urologic complication rate was reduced by modification of the ureteral anastomosis.  Despite increasing wait times for deceased donor transplant candidates, a significant percentage of procured kidneys are not utilized because they are perceived to have inadequate function. According to Scientific Registry of Transplant Recipients (SRTR) data, almost half of extended criteria donor kidneys and one-third of kidneys with serum creatinine greater than 1.5 mg/dL are currently discarded . FurtherWidespread implementation of DKT has however proven difficult, likely due to the complexity of the surgical procedure and concerns regarding poor outcomes. Moreover, while most groups , 6–9 havThis is a single-center retrospective review of all deceased donor kidney transplants performed between February 2007 and July 2013, with particular attention to dual kidney transplants (DKT). Multiorgan and pediatric en bloc transplants were excluded.Donor organs for DKT and single-kidney transplant (SKT) recipients were procured from both donation after brain death (DBD) and donation after cardiac death (DCD) donors.Two categories of kidneys were used for dual transplantation. The first category comprised kidneys from expanded criteria donors (ECD), defined as deceased donors (1) greater than 60 years old or (2) greater than 50 years old and with at least 2 of the following criteria: (a) a history of hypertension, (b) terminal serum creatinine greater than 1.5 mg/dL, or (c) death due to a cerebrovascular accident. The second category consisted of kidneys from standard criteria donors (SCD) that were deemed functionally compromised due to high serum creatinine, poor pump characteristics, or unfavorable histology on biopsy.2, (3) Cr > 2.5 mg/dL, (4) history of longstanding diabetes or hypertension, or (5) glomerulosclerosis between 15 and 50% [All donor kidneys utilized for DKT met the UNOS criteria for dual kidney allocation with at least 2 of the following criteria: (1) age > 60 years, (2) eGFR < 65 mL/min/1.73 m and 50% . All kidKidneys were rejected for DKT if (1) greater than 25% of glomeruli were sclerotic on procurement biopsy, (2) estimated GFR at procurement was less than 50 mLs/min, or (3) at initial gross inspection there were multiple cysts or extensive atherosclerosis extending into the renal arteries.Recipients selected to receive a DKT were matched by donor age and excluded recipients with a body mass index (BMI) greater than 35 or less than 22. All recipients had previously consented to receive an ECD kidney. All patients receiving dual kidney transplants were informed of the risks and benefits of the procedure.The Houston Methodist Research Institute Institutional Review Board approved this retrospective review.One surgical team performed all transplants. The decision to proceed with DKT was made after standard bench preparation of the donor kidneys. A curvilinear incision extending from the symphysis pubis to the anterior superior iliac crest was utilized, similar to but longer in length than the incision used for single-kidney transplantation. This permitted complete dissection of the common internal and external iliac vessels in the extraperitoneal space. The right donor kidney was placed in the superior position. The vein-to-vein anastomosis was performed between the renal vein and the lower cava or common iliac vein. The proximal common iliac artery was used for the end to side arterial anastomosis. For the inferior-positioned donor kidney, the end to side venous anastomosis and end to side arterial anastomoses were performed between the donor vessels and the external iliac vein and artery of the recipient, respectively.Although the vascular anastomoses were standardized, the ureteral anastomoses evolved to address a higher than expected incidence of ureteral stricture. Initially, both ureters were anastomosed to the bladder using the standard Lich-Gregoir technique, as previously described . Over thRecipients of DKT and SKT were treated with the same immunosuppression protocol. Subjects considered at high risk of acute rejection (African Americans (AA) recipients, retransplants, and recipients with PRA > 20%) received a 3-day course of rabbit antithymocyte globulin (rATG) at a dose of 1.5 mg/kg/day. All other subjects received either 2.0 mg/kg of daclizumab or 20 mg of basiliximab for 2 doses. Maintenance immunosuppression consisted of tacrolimus, mycophenolate mofetil (MMF), and prednisone. The dose of tacrolimus was adjusted to maintain a trough level of 8–10 ng/mL for the first 3 months after transplantation and tapered to 5–8 ng/mL thereafter. MMF was given at a dose of 1000 mg twice daily. Methylprednisolone (250 mg) was given on the day of transplantation, tapered to 25 mg by day 5 and then to 5–10 mg by 6 months after transplantation.Patient sera were tested for the presence of de novo donor specific antibody (dnDSA) using a multiplex solid phase bead array  on a Luminex cytometer . Data were analyzed using Fusion software , and the results were recorded as the mean intensity fluorescence (MFI). All beads showing an MFI > 2000 were considered positive. De novo DSA were defined as antibodies directed against the donor HLA that were not present prior to transplant and included HLA A, B, CW, DR, DR51/52/53, DQ, and DP. DSA were tested at 1, 2, 3, 6, 9, and 12 months after transplant or for clinical indication.BK virus testing by polymerase chain reaction (PCR) was performed at 1, 3, 6, 9, 12, 18, and 24 months after transplant or for clinical indication. A viral load of greater than 300 viral copies/mL was considered positive.Renal biopsies were evaluated by light microscopy and immunofluorescence. Electron microscopy examination was performed when glomerular pathology was suspected. Histopathology and classification of rejection were reported according to the Banff 2005 Classification of Renal Allograft Pathology and subsequent updates .p values < 0.05 were considered statistically significant. Statistical analyses were conducted with Stata SE version 13.1  and SAS 9.3 .Categorical characteristics of kidney transplant recipients who received a dual kidney transplant were compared to those with received a single-kidney transplant by Chi square or Fisher's exact tests, where appropriate. Logistic regression was used to analyze continuous covariates. Graft survival and acute rejection were visualized using Kaplan Meier statistics with significance assessed by log rank test. 516 deceased donor kidney transplants were performed at our institution between February 2007 and July 2013, of which 29 were DKT and 487 were SKT. Mean follow-up after transplant was 43.3 ± 65.6 months.Donor and recipient data for DKT and SKT recipients are summarized in p < 0.001). Similarly, DKT recipients were older than SKT recipients, less likely to be highly sensitized, and more likely to have diabetes as the cause of their end stage renal disease (p < 0.05).Donors for DKT were older and more likely to have had a history of diabetes and hypertension, compared to donors for SKT (p < 0.001); however there was no difference between the groups with regard to receipt of DCD organs (p = ns).In the DKT group, 17 of the 29 (58.6%) donors met criteria for ECD classification and 3 donor organs (10.3%) were procured after cardiac death (DCD). Two donors met both ECD and DCD criteria. The use of 12 non-ECD donors for DKT was based principally on a low estimated GFR in 6, unfavorable histology in 4, and poor pump parameters in 2 cases. In the SKT group of 487 patients, 68 (14.0%) organs were from ECD donors, and 46 (9.4%) were procured from DCD donors. No SKT organs met criteria as both ECD and DCD donor. DKT recipients were more likely than SKT recipients to receive an ECD organ (p = ns). The incidence of acute rejection was 20.7% (n = 6) in the DKT group versus 22.4% (n = 109) in the SKT group (p = ns). The incidence of dnDSA was slightly higher in the DKT group, 48.3% (n = 14) versus 31.6% (n = 154) of the SKT recipients (p = ns). The incidence of BK viremia was 27.6% (n = 8) in the DKT group, versus 22.0% (n = 107) in the SKT group (p = ns).The average hospital length of stay after transplant for DKT recipients was 6 days, versus 3 days for SKT recipients. Three of 29 DKT recipients (10.3%) suffered delayed graft function (DGF) versus 45 of 487 SKT patients . All of the urologic complications in the DKT group were due to ureteral strictures, generally occurring at the ureter to bladder anastomosis. Moreover, 3 of the 4 complications occurred in the upper kidney only, and the fourth involved both kidneys. Within the SKT group, 6 complications were due to anastomotic strictures and 4 were caused by urine leaks.Within the first year after transplantation, 4 of 29 DKT patients (14%) developed urologic complication, compared to 10/487 SKT recipients . Specifically, for the DKT group, actuarial patient and graft survivals at 3 years were 100% and 93%, respectively. There were four graft losses in total. One graft was lost at two months after transplant because of recurrent hemolytic uremic syndrome. Another graft loss at 6 months was multifactorial, likely failing due to the combined effects of donor disease, BK viremia, and rejection. A third graft loss at 15 months after transplant was caused by patient noncompliance with immunosuppression therapy, and the fourth graft loss at 5.5 years after transplant was due to chronic allograft nephropathy. There were no patient deaths in this cohort.There was no difference in graft survival between the DKT and SKT groups, as shown in p = 0.001).The mean serum creatinine and estimated glomerular filtration rates  of both DKT and SKT groups are summarized in In this single-center experience, we found that up to 4-years after transplantation, actuarial DKT patient and graft survivals were equivalent to those of SKT. Moreover, despite a greater percentage of ECD kidneys among the DKT cohort, the incidence of DGF, acute rejection, DSA, and BK viremia were also similar between the 2 groups. Importantly, the quality of the donor kidney function was equivalent at all time points studied.There were more urologic complications in the DKT cohort, for the most part due to a greater likelihood of developing a stenosis at the ureter to bladder anastomosis. In this series, most urologic complications occurred with the initial technique (side to side anastomosis of the two ureters and single tunneled ureteroneocystostomy). This was likely due to the fact that the upper transplant kidney ureter was too long. Given the segmental periureteric vascular plexus, the distal ureter may have become compromised due to a limited blood supply, leading to ischemia, stricture, and obstruction. By shortening the length of the upper transplant ureter and using the native ureter, the complication rate declined. But given the small number of cases performed using the revised technique it remains speculative as to whether this problem has been solved. Yet, this technique has been described in previous publications. Wu et al. published a single-case report regarding ipsilateral placement of dual kidneys in a patient who underwent simultaneous nephrectomy for polycystic kidney disease. Similar to our modified technique, a Lich-Gregoir anastomosis was used for the lower kidney, and ureteroureterostomy was formed between the right native ureter and the upper transplant ureter . In anotOur data is generally consistent with previous published reports –6 of duaOur series is novel in two significant ways. Firstly, we did not exclusively utilize ECD kidneys for our DKT, as a number of the kidneys were from donors less than 50 years old with compromised function that would otherwise have been discarded. This is an important point, as most reports of dual kidney transplantation have focused on utilizing exclusively ECD donors or those with reduced GFR. In our series we expanded our acceptance criteria to include any donor with impaired renal function, regardless of age. In this way, we were able to successfully utilize organs from younger donors with biopsy criteria or pump perfusion parameters that resulted in their rejection for use as single kidneys. Secondly, few reports have focused on surgical technique, specifically ureteral complications. The barrier to greater use of this technique remains a technical one: the operating time is longer, the caval anastomosis is more challenging, and potential for ureteral complications is higher. This report provides a detailed description of the surgical procedure and potential complications.In summary, by utilizing marginal quality donor organs as DKT, we have increased the available donor pool while providing excellent function, patient, and graft survival. Ureteral complications may be avoided by shortening the length of the upper transplant kidney ureter. Standardizing this surgical technique resulted in excellent outcomes, providing a means to utilize marginal donor kidneys and address the problem of donor organ shortage.


In the framework of rehabilitation robotics, a major role is played by the human–machine interface (HMI) used to gather the patient’s intent from biological signals, and convert them into control signals for the robotic artifact. Surprisingly, decades of research have not yet declared what the optimal HMI is in this context; in particular, the traditional approach based upon surface electromyography (sEMG) still yields unreliable results due to the inherent variability of the signal. To overcome this problem, the scientific community has recently been advocating the discovery, analysis, and usage of novel HMIs to supersede or augment sEMG; a comparative analysis of such HMIs is therefore a very desirable investigation. In this paper, we compare three such HMIs employed in the detection of finger forces, namely sEMG, ultrasound imaging, and pressure sensing. The comparison is performed along four main lines: the accuracy in the prediction, the stability over time, the wearability, and the cost. A psychophysical experiment involving ten intact subjects engaged in a simple finger-flexion task was set up. Our results show that, at least in this experiment, pressure sensing and sEMG yield comparably good prediction accuracies as opposed to ultrasound imaging; and that pressure sensing enjoys a much better stability than sEMG. Given that pressure sensors are as wearable as sEMG electrodes but way cheaper, we claim that this HMI could represent a valid alternative/augmentation to sEMG to control a multi-fingered hand prosthesis. Although this form of control is reliable, it cannot go very much past such non-dexterous prostheses, e.g., proportional control of two DOFs at best, with switching between DOFs enforced by sequences of cocontractions.Despite decades of research by roboticists, mathematicians, and physiatrists, properly controlling a hand prosthesis by a hand amputee is, still nowadays, an open problem. In the ideal case, proper control means that each single degree of freedom (DOF) of the prosthesis should be independently and proportionally controllable according to the amputee’s intent; for instance, the desire to grab a bottle should immediately result in a stable cylindrical power grasp. So far, this is only enforced on single-DOF grippers such as, e.g., Otto Bock’s Sensorhand Speedi-LIMB Ultra Revolution, RSL Steeper’s BeBionic, and Vincent Systems’s Vincent Evolution 2Azzurra hand, derived from the SmartHand  include, e.g., Touch Bionics’s The community is therefore calling for novel human–machine interfaces (HMIs) to complement, augment, or substitute sEMG, together with the necessity to enforce simultaneous and proportional control using these signals , US (ultrasound imaging), and FSR .2A psychophysical experiment involving ten able-bodied subjects was set up, in order to determine the pros and cons of the three HMIs mentioned above. We gathered fingertip forces from the subjects, while they were induced to apply force patterns using a visual stimulus. At the same time, their sEMG signals, FSR signals, and US images of the forearm were recorded.2.12.1.1FSR400 short by Interlink ElectronicsThe pressure sensing setup comprised a semi-rigid bracelet with ten force-sensing resistors . The choice of these six DOFs of the human hand is motivated by the fact that one of the most dexterous self-powered hand prostheses in the world at the time of the experiment, namely the In the ideal case, data should have been collected with all three devices strapped to the forearm at the same time. However, such an arrangement would have seriously hampered the comfort of the subjects. We rather did a pairwise comparison of the FSR with sEMG and US. This pairwise comparison was done by dividing the data collection into two phases. In the first phase, the sEMG band and the FSR bracelet were affixed to the right forearm of the subject Figure A; wherearepetitions, each repetition consisting of a single-finger flexion for each DOF. During repetitions 1–5, the subjects were required to flex their fingers at 80% maximum force level; the subject was then given a break of 5 min, followed by repetitions 6–10, at 15% of maximum force. This split provided an indication of how the prediction accuracy would behave in a high-force as well as in a low-force scenario.In both phases, the same visual stimuli were administered to the subjects: a 3D hand model of a human hand, flexing the fingers, plus a colored bar denoting the required level of force. A further set of colored bars denoting the force actually applied by the subjects helped ensure that the subjects would exert the required amounts of force to their best. The two phases were identical in terms of the actions that were required by the subject. Each phase comprised ten Ten able-bodied subjects volunteered for the experiment. The entire experimental procedure was conveyed to them in oral and written form, following which their consent was taken in writing. The experiment was approved by the Ethical Committee of DLR. The subjects were seated comfortably on an adjustable office chair for the entire duration. They were given the option to pause or stop the experiment at any point of time.2.3Data obtained from the sEMG, FSR, and FFLS were collected at 50 Hz using a standard analogic-to-digital conversion card. The sEMG signals were filtered by a 1st order low-pass butterworth filter with cutoff frequency at 1 Hz. A gradual drift was observed in the FSR signals, which was remedied by applying a 3rd order high-pass Butterworth filter with cutoff frequency at 0.5 Hz. The filtered signals obtained from the sEMG electrodes and FSRs were directly used, without any further feature extraction phase. The US images were grabbed from the VGA output (at 38 Hz) of the US machine using a standard PCI-bus frame grabber; from each image, 543 local visual features were extracted, denoting the changes in the local levels of gray of each image. For a thorough description of the feature extraction procedure, please refer to Sierra González and Castellini .f (x) = Txw, where x denotes the visual features extracted from each image, and w is a weighting vector, which can be calculated in closed form from a set of  pairs (the training set) previously collected. In particular, if X denotes the training set , and y is a vector collecting the n target (force) values for each collected sample, then w = (TXX + λI)−1TyX. Following the approach tested in the same paper, we applied Ridge Regression to the visual features in order to predict the finger forces. Ridge Regression  = Tϕw(x), where ϕ is a non-linear mapping from the input space  to a higher- (but finite-) dimensional feature space, where linear regression is more likely to succeed. The mapping ϕ is actually a finite approximation of the well-known radial-basis-function kernel; but, as opposed to what happens, e.g., with Support Vector Machines  evaluated. The finiteness of the induced kernel keeps the approach bounded in space and permits the mapping to simply be “plugged in” into the Ridge Regression formula: w = (ϕ(X)Tϕ(X) + λI)−1ϕ(X)Ty. More details about the theory of this method when applied to sEMG can be found in Gijsberts et al.  and the stimulus values – a harder but more realistic choice, since amputees cannot provide any consistent ground truth using sensors. The prediction accuracy of each machine-learning method applied to either regression problem (with forces or stimulus as ground truth) was evaluated by calculating the Root Mean-Squared-Error between the predicted force values and the ground truth obtained from the FFLS sensor, normalized over the target signal range (nRMSE). Notice that this problem represents a hard challenge for any machine-learning method whatsoever: in the most difficult setting, the system is trained on stimulus values but still tested on real force data.The prediction accuracy of each machine-learning method (Ridge Regression and Random Fourier Features in turn) was evaluated by following the stellini : to builLastly, two prediction scenarios were considered, one to ascertain the accuracy obtained by each HMI in general, and another one to test the stability over time of each HMI. For the first scenario, the above cross-validation procedure was used; for the second, we trained on data collected during the first repetition, and subsequently tested on the second, third, fourth, and fifth repetition separately. As the repetitions were always collected in the same chronological order, this would give us an indication of how well the prediction could carry on over time.33.1Figures t-test p-value always larger than 0.05, except for one single case). This confirms that the error values for the FSR obtained in the two experiments are similar, in turn, confirming that the subjects were induced to do approximately the same things during the two experiments. The maximum force exerted averaged over ten subjects and the six degrees of freedom is 22.6 ± 2.1 N.In the graphs, FSR1 and FSR2 denote the two identical experiments performed with FSR, the first time together with sEMG and the second with US. Almost no statistically significant difference in performance is observed  are used as ground truth to train the system Figure A, the erIn the low-forces experiment Figure , the sam3.2Figures From the figures, a clear trend up is evident in the case of sEMG, all the more in the case of the low-forces experiment. In fact, the slope of a linear fit (averaged over the six degrees of freedom) turns out to be 0.0002 ± 0.0014 and −0.0008 ± 0.0004 for FSR, and 0.0079 ± 0.0005 and 0.0092 ± 0.0105 for sEMG. This confirms that FSR enjoys a better stability in time with respect to sEMG.4The experiment described in this paper is a comparative analysis among HMIs for the disabled. Particular care was taken to ensure that the three HMIs under examination were tested in the very same conditions. Since it was infeasible to have the subjects wear the sEMG electrodes, the pressure sensing bracelet and the ultrasound imaging probe all at the same time, we rather tested the HMIs in two pairs, checking later on that the common one  would obtain comparable results in the two experiments.The experimental results show that, overall, all three HMIs can be in principle used in a realistic setting – that is, using an incremental, real-time machine-learning method, in principle trained without the usage of sensors for ground truth, and using a very short training procedure . All three HMIs can potentially enforce simultaneous and proportional control over fingers of a multi-fingered prosthesis. A more detailed discussion of the results follows.4.1repetition-wise, that is, by training on four out of five repetitions, and then testing on the remaining repetition. It can therefore be the case that the ultrasound visual features extracted from each repetition slightly differ from one another (due to probe shift or different levels of muscle activation and shifting from one repetition to the next one). In an initial round of experiments on the same data set performed using random shuffling in cross-validation, error values similar to those obtained in Sierra González and Castellini It is well-known , and the analysis has been performed by finding the optimal machine-learning hyperparamters either by referring to previous literature or by extensive (grid-) search (see Subsection 3 again). In other words, each model found (and the associated prediction accuracy) is the optimal one for the related subject and dataset, stellini , in whicNotice, moreover, that disabled subjects usually have abnormal physical/psychophysical parameters with respect to the healthy population; for example, Parkinson patients present a higher amount of non-voluntary muscle activation than control subjects  to be worn on the skin. The only attempt we are aware of so far is represented by Guo et al. , where oAs far the costs are concerned: cheap as it has become in recent times with respect to the past years , this kind of hardware remains prohibitively expensive to be shipped along with a hand prosthesis, constituting a relevant fraction of the price of the hand itself (hovering around 25.000 EUR). Each sEMG electrode of the kind that we used here is in the range of 150–300 EUR a piece, which makes our array significantly cheaper than an ultrasound device; it must be noted that, probably, even cheaper arrays of sEMG electrodes can be purchased, but in that case, a further signal processing phase  must take place before the signal is available to the machine-learning method, raising the cost of the whole approach. The pressure sensing bracelet, in turn, cost an estimated 50 EUR as it employs force-sensing resistors, which can be bought for <5 EUR apiece , but it also keeps a remarkably stable prediction over time (as opposed to sEMG). It is also cheaper than sEMG, while maintaining full wearability. This does not mean, of course, that pressure sensing is a definitive replacement for sEMG and/or ultrasound. We speculate that this novel HMI will suffer from a number of disturbances once it is used in a real-life application; in particular, its signal will change as the patient swings the forearm (due to the acceleration of the forearm against the sensors harness) and, which is more worrisome, as (s)he lies the forearm on a surface. These problems will need to be countered; one possibility is to fuse this signal with that obtained from sEMG, which will be much less influenced by such artifacts.The weak performance of ultrasound imaging in this experiment, together with its low scores as far as cost and wearability are concerned, pose serious doubts on its use as a competitor HMI. Nevertheless, as we had previously claimed (Sierra González and Castellini, 5Inspired by the recent trend in the rehabilitation robotics community, in this paper we have compared three HMIs for control of multi-fingered prosthetic hands. Surface electromyography, pressure sensing, and ultrasoundimaging were tested on ten intact subjects engaged in a simple, repetitive application of single-finger-force patterns; a state-of-the-art machine-learning method was then used to predict the forces using the signals gathered from the HMIs. Our experimental results indicate that pressure sensing represents a cheaper alternative to sEMG, enforcing prediction accuracy comparable to sEMG, better stability in time, and the same wearability as this more traditional HMI.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Allobates femoralis over several hundred metres, (ii) using a harmonic direction finder with miniature transponders to track these small frogs, and (iii) using a natural river barrier to separate the translocated frogs from any familiar landmarks. We found that homeward orientation was disrupted by the translocation to the unfamiliar area but frogs translocated over similar distances in their local area showed significant homeward orientation and returned to their territories via a direct path. We suggest that poison frogs rely on spatial learning for way-finding in their local area.Among vertebrates, comparable spatial learning abilities have been found in birds, mammals, turtles and fishes, but virtually nothing is known about such abilities in amphibians. Overall, amphibians are the most sedentary vertebrates, but poison frogs (Dendrobatidae) routinely shuttle tadpoles from terrestrial territories to dispersed aquatic deposition sites. We hypothesize that dendrobatid frogs rely on learning for flexible navigation. We tested the role of experience with the local cues for poison frog way-finding by (i) experimentally displacing territorial males of  AmongSpatial memory and its flexibility are expected to vary with a species' mode of locomotion, home-range size, habitat complexity and the variability of the resources being exploited. Overall, amphibians are the most sedentary vertebrates , but Homing after experimental translocations has been observed in many amphibians , but toAllobates femoralis, (ii) using a harmonic direction finder with miniature transponders to track these small frogs in their natural habitat [There are three main challenges to testing the dependence on experience for navigation in the field: (i) finding an animal strongly motived to orient towards a goal, (ii) tracking the movements, and (iii) being able to quantify or exclude the familiarity with a given area. We overcame them by (i) experimentally displacing highly territorial males of a poison frog,  habitat , and , whereas 21 frogs were released at equivalent distances in the centre of an approximately 5 ha river island  with an isolated population of A. femoralis . Both release areas provided comparable and suitable habitats for A. femoralis, but the frogs do not naturally cross the river barrier. Frogs released on the island had an area with a radius of about 100 m to move in before reaching the waterfront .a Shield . Transloa Shield . To testAfter the release, we tracked the individual movement patterns for about 7 days or until the frog returned to the home territory. We localized each frog every 15–60 min during their daylight activity period using a commercially available harmonic direction finder . The position of each frog was recorded on a pocket computer  in ArcPAD (ESRI) using a detailed background map . Experiments were carried out between 1 February 2014 and 22 April 2014 near the field camp ‘Saut Pararé’ in the nature reserve ‘Les Nouragues’, French Guiana.rcGIS 10 (ESRI), after averaging points that were spaced less than 1 m apart (estimated mapping error in the field). We normalized all movement vectors towards the home territory of each frog, so that 0° was the common home bearing. We further described trajectories by a straightness coefficient (SC), defined as the ratio of straight path length to total path length. To test for significance of homeward orientation, we considered only those frogs that moved at least 25 m away from their release point  and used a vector length weighted, second-order Hotelling's test . The vector length corresponds to the SC until the point where the bearing was measured. The island and mainland bearing distributions were compared with Hotelling's two-sample test. For visual representation, we plotted home direction normalized trajectories derived from the linear interpolations of consecutive positions .For each frog, we analysed the X- and Y-coordinates of each position in A3.n = 14, mean vector direction = 354°, 95% CI = 303.5°–30°, Hotelling's F = 14.6, p < 0.001; figure 1a,c), the frogs on the island showed no significantly preferred direction . At 25 m, there was a significant overall difference in bearing distribution of frogs on the mainland from those on the island . On the mainland, seven frogs returned to within a few metres of their capture location and another three moved at least 100 m homewards. All 10 frogs that moved at least 100 m away from the mainland release sites were strongly homeward oriented  and showed direct homing trajectories   .Figure 4.The fact that frogs choose a direct path home when released in their local area but are disoriented in an unfamiliar area demonstrates that experience with local cues is necessary for successful homing. Our findings cannot be solely explained by the use of large-scale gradients or goal-associated cue guidance . They suggest a role of spatial learning.A. femoralis is suggestive of a learned spatial map, but more experiments will be needed to test this hypothesis.True spatial learning requires processing and storing information about distances and directions. It allows novel routes to be created in the familiar area . There aAllobates femoralis relies on the exploitation of multiple, widely dispersed, temporary aquatic deposition sites for successful reproduction. In a recent study, A. femoralis males were found transporting tadpoles up to 180 m away from their territory. In addition, males transported more tadpoles at once when going to more distant deposition sites, which suggests some spatial knowledge of the area [Qualitatively comparable spatial learning abilities have been shown in a broad range of vertebrates ,3. Amphi


Activation of the complement system, a pivotal part of inflammation, occurs at very early stages in the AD brain. Therefore, CSF levels of complement proteins that could be linked to cognitive and structural changes in AD may have diagnostic and prognostic value.Alzheimer’s disease (AD) is characterized by the deposition of tau and amyloid in the brain. Although the core cerebrospinal fluid (CSF) AD biomarkers amyloid β peptide 1–42 .Using xMAP® technology based assays we measured complement 3 (C3) and factor H (FH) in the CSF of 110 controls (CN), 187 mild cognitive impairment (MCI) and 92 AD subjects of the AD Neuroimaging Initiative (ADNI) at baseline. All ADNI participants underwent clinical follow-up at 12 month intervals and MCI subjects had additional visits at 6 and 18 months. The association between CSF biomarkers and different outcome measures were analyzed using Cox proportional hazard models (conversion from MCI to AD), logistic regression models  and mixed-effects models adjusted for age, gender, education, t-tau/AβAlthough no association was found between the complement proteins and clinical diagnosis or cognitive measures, lower levels of C3  and FH  were associated with faster cognitive decline in MCI subjects as measured by the AD Assessment Scale-cognitive subscale (ADAS-Cog) test. Furthermore, lower FH levels were associated with larger lateral ventricular volume (p = 0.024), which is indicative of brain atrophy.Our study confirms a lack of suitability of CSF C3 and FH as diagnostic biomarkers of AD, but points to their modest potential as prognostic biomarkers and therapeutic targets in cognitively impaired patients. Alzheimer’s disease (AD) affects an estimated 34 million people worldwide, a number that is predicted to triple by 2050 due to the aging population . Despite1–42), which correlates inversely with plaque pathology, total tau (t-tau), which is hypothesized to reflect neuroaxonal degeneration, and phosphorylated tau (p-tau181), which may correlate with tangle pathology, have recently been incorporated into the National Institute on Aging guidelines for AD diagnosis [1–42 seems to be an early event, reaching a plateau before the onset of dementia and remaining relatively unchanged thereafter. The increase in CSF tau occurs after Aβ1–42 changes, but still in the preclinical stage of the disease and does not change appreciably over time in cognitively impaired subjects [The cerebrospinal fluid (CSF) biomarkers amyloid β peptide 1–42  and, later in the cascade, complement 5. The resulting active fragments act as proinflammatory and chemotactic anaphylatoxins, opsonins allowing phagocytosis, or anchors for the assembly of the membrane attack complex. The complement system is kept under tight control by soluble and membrane-bound regulators, including factor H (FH), which inhibits C3 convertases in the alternative pathway, and complement 1 inhibitor, which inhibits several proteases of the classical and lectin pathways . The finIn a previous cross-sectional study, we found that CSF levels of C3 and FH were significantly increased in AD patients compared with controls (CN) and that the increase correlated significantly with lower Mini-Mental State Examination (MMSE) scores in AD patients. In the current study, we attempt to validate these observations in a large, independent cohort of well-characterized subjects. In addition, we extend the previous analysis by including patients diagnosed with mild cognitive impairment (MCI) and by analyzing additional clinical and neuroimaging data. Finally, we explore the prognostic potential of CSF C3 and FH levels by analyzing longitudinally collected clinical data.1–42, t-tau and p-tau181 measured in their CSF samples at baseline.Data used in the current study were downloaded on 27 July 2013 from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database . Ethics The same neuropsychological testing battery was applied to all subjects in the ADNI, with visits scheduled every 12 months, except for the MCI subjects who had additional visits at 6 and 18 months. Tests included the MMSE, the Alzheimer’s Disease Assessment Scale – cognitive subscale (ADAS-Cog), the clock drawing test, the Rey Auditory Verbal Learning Test, Digit Span forward and backward, category fluency, the trail-making test, the digit symbol substitution test, the Boston naming test and the logical memory test. Finally, we further characterized the cognitive profile of each subject using the summary composite executive function and memory measures developed by Gibbons and colleagues  and CranCSF samples were obtained in the morning after an overnight fast at ADNI baseline visits. Lumbar puncture was performed with a 20-gauge or 24-gauge spinal needle as described previously   with an Innogenetics kit  according to the manufacturer’s protocol [CSF C3 and FH levels were measured using an xMAP technology-based multiplex human neurodegenerative kit  according to the manufacturer’s overnight protocol with minor modifications. A detailed protocol can be found on the ADNI website . Aβ1–42,protocol . CSF hemprotocol . For detAcquisition of 1.5-T magnetic resonance imaging (MRI) data for the ADNI 1 subjects followed a previously described standardized protocol that included sagittal volumetric three-dimensional magnetization prepared rapid acquisition gradient recalled echo (MPRAGE), with variable resolution around the target of 1.2 mm isotropically. The scans had gone through certain correction methods such as gradwarp, B1 calibration, N3 correction and (in-house) skull-stripping . TheFor the analyses included in the descriptive table Table , one-wayPrevious studies suggest that blood contamination of CSF can significantly affect CSF concentrations of certain proteins . We therAssociations between CSF complement biomarkers and age, gender and APOE ϵ4 presence were tested in linear regression models. FH and C3, but not C3/FH, were associated with both age and gender. None of the CSF complement biomarkers showed a significant association with APOE ϵ4 presence  and a validation set (33%), stratifying by clinical diagnosis. To train a classifier and cross-validate the cutoff values in the discovery set, the subjects were further randomly split 10 times to form training (67%) versus test (33%) sets. The cutoff values of the model were selected in the discovery set using accuracy and the kappa index as performance metrics ,31. The 1–42 ratio, APOE ϵ4 presence and education as covariates was used to study the conversion of MCI to AD for different CSF biomarkers. Standardized values  were used for the biomarker values in order to compare the effect size of the association.A Cox hazards model, with age, gender, t-tau/Aβ1–42 ratio and clinical diagnosis at baseline were included as fixed effects. We include an intercept, follow-up time and squared follow-up time in weeks as random effects. Our model specified the intercept and the regression coefficient for the follow-up time as random effects such that subjects have a unique intercept and slope characterizing their individual trajectories. An interaction between time and clinical diagnosis, between time and t-tau/Aβ1–42 ratio, and between time and the studied CSF biomarker was also included to assess whether these biomarkers were associated with the longitudinal change. A significant interaction between clinical diagnosis and time, for example, would indicate that the slope of change during follow-up is different in CN and MCI subjects. To plot the data we calculated the 25th, 50th and 75th percentile biomarker values for the MCI group and estimated the predicted changes based on the coefficients of the corresponding mixed-effects model .We analyzed the cross-sectional and longitudinal association between CSF biomarkers and different outcome measures using mixed-effects models ,34. A mi1–42 ratio, clinical diagnosis and intracranial volume were included as fixed effects.For the MRI analysis, a mixed-effects model was used, with a nested term inside subjects for the left and right sides for each region of interest. Age, gender and t-tau/AβP < 0.05. In the case of multiple comparisons, the Benjamini–Hochberg correction and the Holms method was applied when a large and lower number of comparisons, respectively, had been performed. Analyses were performed using R v. 3.0.1 [Statistical tests were two-sided and significance was set at v. 3.0.1 .1–42, t-tau and p-tau181 as well as MRI data were included in the current study. Clinical and demographic characteristics of the studied subjects are summarized in Table 1–42, t-tau and p-tau181. The data for CSF Aβ1–42, t-tau and p-tau181 in the AD subjects are in line with published cutoff values from autopsy confirmed cases using the AlzBio3 kit [A total of 389 ADNI 1 participants with CSF C3, FH, AβBio3 kit . Howeverrpartial = 0.81, P < 0.0001) was present after adjusting for age and gender , gender , t-tau/Aβ1–42 ratio , APOE ϵ4 presence  and education  as covariates was used to test the association of the CSF complement biomarkers with conversion of MCI to AD. A weak association between lower levels of C3  and increased conversion was lost after adjustment for multiple comparisons (Padj = 0.14).Of the 160 MCI subjects included in the analysis, 79 converted to AD with a median follow-up time of 158 weeks. A Cox hazards model with age at baseline . Owing to this imbalance in the number of visits, we analyzed the AD subjects and the MCI subjects separately. All of the following analyses were adjusted for age, gender, APOE ϵ4 presence, education and t-tau/Aβn = 256) of the ADNI 1 subjects included in the study also had RBM CSF data available. The RMB CSF panel included C3, but not FH. We explored the association between our CSF C3 measurements and the RBM C3 levels and found a strong correlation between the two , indicating that the two immunoassays performed similarly. As expected, when we repeated the diagnostic and prognostic analyses described above using C3 levels obtained by the RBM assay, very similar, if not identical, results were found, especially with regard to the association between lower levels of C3 and faster cognitive decline of MCI subjects.A subset , consistent with the argument that low CSF FH level is associated with greater brain atrophy.Since brain atrophy detected by MRI is associated with AD severity and correlates closely with changes in cognitive performance , we alsoIn this study, we explored the diagnostic and prognostic value of CSF C3 and FH levels in AD and MCI. In cross-sectional analysis, there were no significant differences in either biomarker or in their ratio between diagnostic groups, and nor were there any correlations with disease severity in AD or MCI subjects as measured by the MMSE, ADAS-Cog, memory summary score or executive function summary score. In the longitudinal analysis of MCI patients, however, low levels of both C3 and FH were modestly associated with an increase in ADAS-Cog scores (more severe cognitive impairment) and validation in an independent and longitudinal cohort is needed. Additionally, there was a significant association of low CSF FH levels with increased lateral ventricular volume, which is indicative of brain atrophy and has been shown to correlate strongly with changes in cognitive tests . StrengtPrevious studies have generally reported elevated levels of complement components in AD CSF, although results are inconsistent. For example, in a recent study  using a 1–42 in AD CSF is hypothesized to be the result of trapping the peptide in plaques, and C3 and FH have both been shown to be present in Aβ plaques [1–42 and C3 or FH, suggesting that the observed decrease in complement biomarkers cannot be readily explained by such a simplistic model. An alternative hypothesis could therefore be that the low CSF levels of C3 and FH in faster progressors may reflect accelerated dysregulation of the complement system in the brain. To this end, many studies have indicated potential involvement of complement system in AD pathogenesis, including observations that: Aβ fibrils activate both the classical and alternative complement pathways in vitro[CR1 genotype [A limitation of our investigation is the potential confounding effect of pharmacotherapy, because subjects were not drug naïve at the time of CSF collection, although the use of many central nervous system-active drugs such as antidepressants or neuroleptics with anti-cholinergic properties, narcotic analgesics and anti-Parkinsonian medications were excluded. The other limitation relates to the fact that the data are correlational without clear mechanistic interpretation. That said, we wish to put forward two hypotheses for discussion. First, our finding of low levels of CSF C3 and FH in MCI patients with accelerated cognitive decline may reflect increased deposition of these complement biomarkers in senile plaques. Decreased Aβ plaques ,42,43. T in vitro-46; inhi in vitro-49; and  in vitro consistegenotype . Thus, iIn summary, our data suggest that CSF C3 and FH levels are prognostic biomarkers of accelerated cognitive decline in MCI, although validation in an independent cohort is needed. Additionally, studies with repeated CSF measurements will shed more light on the utility of CSF C3 and FH levels as AD progression biomarkers. Finally, results obtained in this study should encourage further investigations exploring the mechanisms underlying complement activation, both the classical and alternative cascades, in AD development and progression.1–42: amyloid β peptide 1–42; AD: Alzheimer’s disease; ADAS-Cog: Alzheimer’s Disease Assessment Scale – cognitive subscale; ADNI: Alzheimer’s Disease Neuroimaging Initiative; APOE ϵ4: apolipoprotein E epsilon 4 allele; C3: complement 3; CN: controls; CSF: cerebrospinal fluid; FH: factor H; MCI: mild cognitive impairment; MMSE: Mini-Mental State Examination; MRI: magnetic resonance imaging; p-tau181: tau phosphorylated at threonine 181; RBM: rules-based medicine; t-tau: total tau.AβThe authors declare that they have no competing interests.JBT, AK, LMS, JQT and JZ made substantial contributions to the conception and design of the study and were involved in drafting the manuscript and revising it critically for important intellectual content. AK performed the xMAP assays. JBT, AK and JZ had full access to the entire dataset. LMS and JQT contributed to acquisition and storage of the samples. JBT and AK undertook the statistical analyses. All authors read and approved the final manuscript.is an acknowledgement list for ADNI publications: ADNI infrastructure and site investigators.Click here for fileis the supplemental methods, including subjects, recruitment criteria, CSF sample collection and handling, CSF immunoassay performance and references.Click here for fileis Supplemental Figures S1 and S2 showing associations between CSF FH and hemoglobin, and CSF C3 and FH.Click here for fileis Supplemental Tables S1 to S4 presenting data showing CSF C3 and FH biomarker associations.Click here for file


We performed a randomized, double-blind, cross-over study to assess the neuroregenerative potential of intravenous granulocyte colony-stimulating factor (G-CSF) followed by infusion of mobilized peripheral blood mononuclear cells (mPBMCs) in children with cerebral palsy (CP).18F-fluorodeoxyglucose (FDG) brain positron emission tomography-computed tomography (PET-CT) scanning to evaluate the anatomical and functional changes in the brain.Children with non-severe CP were enrolled in this study. G-CSF was administered for 5 days, then mPBMCs were collected by apheresis and cryopreserved. One month later (M1), recipients were randomized to receive either mPBMCs or a placebo infusion, and these treatment groups were switched at 7 months (M7) and observed for another 6 months (M13). We assessed the efficacy of treatment by evaluating neurodevelopmental tests, as well as by brain magnetic resonance imaging-diffusion tensor imaging (MRI-DTI) and 18F-FDG brain PET-CT scans, there were no significant differences in such changes between the mPBMC and placebo group or between the T1 and T7 group.Fifty-seven patients aged 4.3 ± 1.9 (range 2–10) years and weighing 16.6 ± 4.9 (range 11.6–56.0) kg were enrolled in this study. The administration of G-CSF as well as the collection and reinfusion of mPBMCs were safe and tolerable. The yield of mPBMCs was comparable to that reported in studies of pediatric donors without CP and patients with nonhematologic diseases. 42.6% of the patients responded to the treatment with higher neurodevelopmental scores than would normally be expected. In addition, larger changes in neurodevelopment test scores were observed in the 1 month after G-CSF administration (M0–M1) than during the 6 months after reinfusion with mPBMCs or placebo (M1–M7 or M7–M13). Patients who received G-CSF followed by mPBMC infusion at 7 months (T7 group) demonstrated significantly more neurodevelopmental improvement than patients who received G-CSF followed by mPBMC infusion at 1 month (T1 group). In contrast to the results of neurodevelopment tests, the results of MRI-DTI at the end of this study showed greater improvement in the T1 group. Although we observed metabolic changes to the cerebellum, thalamus and cerebral cortex in the Neurodevelopmental improvement was seen in response to intravenous G-CSF followed by mPBMC reinfusion, particularly to the G-CSF alone even without mPBMC reinfusion. Further studies using a larger number of mPBMCs for the infusion which could be collected by repeated cycles of apheresis or using repeated cycles of G-CSF alone, are needed to clarify the effect of mPBMC reinfusion or G-CSF alone . Children with cerebral palsy (CP) have disabilities both from motor impairment and from related disorders in other functions, including sensation, cognition, communication, vision, and behavior. Although various strategies have been used to improve neurologic impairment in patients with CP, most strategies used to date are complementary therapies, and there is currently no medical treatment that can repair the damaged nervous tissues . Recent Recently, cell therapy has emerged as a potential treatment for patients with CP. Bone marrow (BM)- or cord blood (CB)-derived mesenchymal stem cells (MSCs) have generally been used for cell therapy. In addition, intravenous CB mononuclear cells (CB MNCs) or intrathecal BM MNCs have been also assessed for safety and efficacy in children with CP –12. StudWe hypothesized that mobilized peripheral blood mononuclear cells (mPBMCs) would be a better source of cell therapy for children with CP, if these cells had a similar neuroregenerative potential to BM/CB MNCs. Multipotent precursor cells exist in peripheral blood, and a fraction of elutriated blood cells from normal individuals contains MNCs that have the potential to be MSCs . There aPatients were included in the study if they were between 2 and 10 years of age and had a non-severe type of CP After baseline studies at enrollment (M0), intravenous G-CSF of 10 μg/kg was administered for 5 days. On the fifth day mPBMCs were collected with a single-day course of apheresis and then cryopreserved  to assess gross developmental screening, the pediatric evaluation of disability inventory (PEDI) to assess detailed developmental, the gross motor function classification system (GMFCS) to assess gross motor function staging, the gross motor function measure-88 (GMFM) to assess detailed motor function, the manual ability classification system (MACS) to assess fine motor staging, and the quality of upper extremity skill test (QUEST) to assess fine motor function. The results for each examination tool were evaluated by well-trained physical and occupational therapists, and therapeutic responses were comprehensively assessed by rehabilitation specialists.2, TR/TE = 9000/55 ms, slice thickness = 2 mm. The DTI datasets were transferred to a workstation for processing. Fractional anisotropy (FA) and apparent diffusion coefficient (ADC) values for 18 regions of interest (ROIs) were obtained from the DTI data. All ROIs were set and analyzed by a pediatric neurologist.All patients underwent MRI examination using a 3.0T system . Conventional images including axial T1-weighted, T2-weighted, and fluid-attenuated inversion recovery (FLAIR) were obtained for the anatomical evaluation. DTI data were obtained for the functional evaluation using a single shot echo planar sequence with the following parameters; 15 diffusion gradient directions, maximum b value = 800 s/mm18F-FDG , patients waited for 60 min in a dark room with a dim light before imaging, while 18F-FDG was distributed in brain. PET scans were obtained for 10 min, and images were reconstructed with a 168 × 168 matrix (pixel size = 1.95 × 1.95 mm with a slice thickness of 3.0 mm), and the ordered subset expectation maximum iterative reconstruction algorithm, an 5 mm Gaussian filter, and a 30 cm field of view. Two board-certified nuclear medicine physicians reviewed all three set of individual subject by visual assessment in consensus to check for the differences between the mPBMC group and placebo group, as well as T1 group and T7 group.Brain PET images were acquired using a dedicated PET-CT system  at M0, M7, and M13 to monitor metabolic improvements of the brain. The patients fasted at least 6 h prior to PET-CT scanning. After intravenous injection of U test was used to analyze the neurodevelopment tests. The Wilcoxon rank sum test was used to analyze data from the brain MRI-DTI and PET-CT scans. P values ≤0.05 were considered to be statistically significant.We analyzed the following: safety and yield of G-CSF-mobilized PBMC collection; outcomes at M13; the booster effect of mPBMC infusion (by comparing the mPBMC group and the placebo group); and, outcomes according to mPBMC infusion time . The statistical software SAS for Windows  was used for data analysis. Logistic regression was used to analyze parental assessments, and the Mann–Whitney Fifty-seven patients were enrolled in the current study and their mean age and body weight were 4.3 ± 1.9 years (range 2–10 years) and 16.6 ± 4.9 kg (range 11.6–56.0 kg), respectively. Types of CP were as follows: 31 diplegia, 15 hemiplegia, 11 others . Possible causes of CP were periventricular leukomalacia (n = 37), hypoxic ischemic encephalopathy (n = 2), intracranial hemorrhage (n = 2), and unknown (n = 16). Forty-seven patients for whom complete study data were available were included in this analysis.8/kg, and the TNC count of CD34+ cells was 3.07 ± 2.1 × 106/kg. Pre-freezing and post-thawing cell data were not significantly different between the two groups (T1 and T7), except for the post-thawing TNC counts, where the TNC count was significantly higher in the T7 group (Table G-CSF-mobilized PBMC collection was safe in children with CP. We observed only two cases of fever and one episode of irritability during the G-CSF infusion. Transient hemoglobinuria (n = 3) and abdominal pain n = 1) were reported during the mPBMC infusion, and these were resolved with supportive treatments. The total nucleated cell (TNC) count of mPBMCs was 5.97 ± 1.99 × 10 were repEven before randomization for mPBMC infusion at M1 (i.e. during the one month after G-CSF administration), functional changes were noted by parents of 27 patients from the total group of 47 patients. After randomization, at M13, functional changes were noted by parents of 41 patients in the mPBMC group and by parents of 45 patients in the placebo group, although this difference was not statistically significant. There were also no significant differences in the functional changes recorded by parents at M13 between the T1 and T7 groups  showed overall improvement at M13 in the domains of GMFM, PEDI and QUEST. We defined overall improvement as a score change in GMFM >4 points and/or score changes in PEDI >7 points in at least three items. The clinical characteristics of patients according to their overall therapeutic response are shown in Table In the MRI-DTI scans, there was a trend of increasing FA values and decreasing ADC values over time (M0–M13). However, these trends were not statistically significant, and there were also no significant differences between mPBMC and placebo groups (M1–M7 and M7–M13) in FA or ADC values in any ROIs. To evaluate the effect of mPBMC infusion on the changes in MRI-DTI scans, we analyzed the data according to the time of mPBMC infusion. The FA and ADC values were significantly greater in the T1 group than in the T7 group for the left corona radiata (CRL), left posterior limb of internal capsule (PLL), and the ADC values were significantly decreased in genu ROIs between M0 and M13 Table , and in Although we observed metabolic changes to the cerebellum, thalamus and cerebral cortex in the brain PET-CT, there were no significant differences in the incidence of metabolic changes between the mPBMC and placebo groups, or between the T1 and T7 groups.We have performed a clinical trial using G-CSF and mobilized PBMCs in patients with CP, based upon the following backgrounds. The use of G-CSF in children with CP is ethical and beneficial because it is already proven to be safe in normal volunteer donors , 16, and8/kg, and CD34+ cell counts of 3.07 ± 2.1 × 106/kg. These numbers are sufficient for hematopoietic reconstitution, although the target dose of MNCs or MSCs for cell therapy is yet to be determined. The yield of mPBMCs from children with CP was comparable to the yield from normal pediatric donors and patients with nonhematologic disease, reported in other studies [In the current study, we demonstrated that G-CSF administration and collection/reinfusion of mPBMCs were safe and tolerable in children with CP. A single-day apheresis in children with CP yielded TNC counts of 5.97 ± 1.99 × 10 studies , 19. We We observed that 42.6% of patients showed overall responses in the neurodevelopment tests than would normally be expected. In addition, the largest neurodevelopment score improvements were obtained during 1 month after G-CSF administration followed by mPBMC collection (M0–M1), suggesting that G-CSF alone, irrespective of circulating mPBMCs, can counter neurological impairment in children with CP. We also tried to assess the booster effects of mPBMCs on neurodevelopmental functions. Compared to the placebo group, the group that had a mPBMC infusion at 7 months experienced a greater improvement in neurodevelopmental functions than the group receiving a mPBMC infusion at 1 month. However, it should be pointed out that the higher infused cell doses in the T7 group were associated with neurodevelopmental improvement, because there is, as yet, no evidence that cell dose has an effect on neurologic improvements. In addition, Hara  and HayaGiven these differences between the results of neurodevelopment tests and the changes observed in MRI-DTI scans, the neuroregenerative effect of mPBMC reinfusion may be minimal and/or the additive effect of G-CSF on the central nervous system as well as muscles could be considered. Furthermore, the collection and reinfusion of mPBMCs after G-CSF administration may not be essential because G-CSF could synergize with endogenous circulating mPBMCs. Although we could not demonstrate the effect of G-CSF on muscle and an additional effect of mPBMCs on the changes of FA/ADC values in the MRI-DTI scans, several investigators have found a correlation between ROI-based FA and clinical motor outcome in children with CP –24. TherWe observed a neuroregenerative potential of intravenous G-CSF followed by mPBMC reinfusion. The neurodevelopmental improvement observed may have been caused by G-CSF alone without a contribution from the reinfused mPBMCs. Further studies using higher concentrations of mPBMCs which could be collected by repeated cycles of apheresis or using repeated cycles of G-CSF alone are needed to clarify any benefit of mPBMC reinfusion or G-CSF alone.


Immunoassays are prone to interference by various substances which may cause inaccurate results. This type of interference is difficult to detect analytically.A case of CARDIAC Troponin T Quantitative reader (Roche Diagnostics) assay failure was detected and investigated in order to ascertain the likely cause.Patient whole blood was mixed with cardiac troponin T-positive blood, patient and control sera were denuded of immunoglobulin G by protein A-affinity chromatography and patient sera were mixed with mouse serum. Samples were analysed on a CARDIAC Troponin T Quantitative reader.A mixture of patient whole blood and cardiac troponin T-positive blood resulted in assay failure; removal of immunoglobulin G from patient sera reversed the cardiac troponin T assay failure; the addition of mouse serum as a heterophile antibody blocking agent had no effect.It is proposed that the interference resulting in assay failure may not be because of a heterophile antibody, but rather a result of a circulating autoantibody to cardiac troponin T, which may compete with antibody assay reagents for binding sites. Despite extensive experience with this methodology, however, immunoassays are occasionally subject to interfering substances that compromise their accuracy – indeed, it is estimated that antibody interference affects approximately one in 2000 immunoassay results.A 61-year-old female, with a history of ischaemic heart disease and hypertension, presented to the emergency unit on two occasions 12 days apart with chest discomfort. Repeated attempts by the diagnostic laboratory to obtain cTnT measurements failed, as reflected by the absence of a positive control line on test strips  using protein A-affinity chromatography.6 These samples were analysed for cTnT prior to and after IgG depletion. The CARDIAC Troponin T Quantitative reader is a lateral flow immunoassay, utilising the sandwich principle on a test strip with two murine monoclonal anti-cTnT antibodies.7 Thirdly, in order to exclude the presence of interfering human anti-mouse antibodies (HAMA), mouse serum was added to the patient plasma (1:4) and the mixture was incubated for one hour at room temperature, following which the cTnT was measured. Lastly, to determine whether the automated cTnT assay on the Roche Elecsys E170 analyser was subject to the same interference, dilutions of a known cTnT-positive plasma sample mixed with the patient plasma were assayed for cTnT.Antibody interference was suspected and the following investigation was thus performed. Prior ethics approval was not obtained as the investigation would lead to improvement in this patient’s management. Firstly, a 1:1 mixture of the patient’s sample and a recently-assayed anonymous sample positive for cTnT (both heparinised whole bloods), was assayed for cTnT.7 but despite the presence of additional blocking agent (mouse serum), the control line did not develop, which suggested strongly that the interfering IgG was not an HAMA , both the patient and control samples elicited control lines after IgG depletion (cTnT < 0.03 ng/ml), suggesting that IgG was the interfering substance. Test-strips contain HAMA-blocking antibodies, an HAMA . Dilutio8 rheumatoid factor9 and circulating troponin autoantibodies.11 Recent studies have shown that 15.9% of samples from cohorts of normal blood donors were positive for autoantibodies to troponin T or troponin I and 10.9% were positive for both.12 Autoantibody–antigen macrocomplexes have been well described for a number of biomarkers such as salivary amylase,13 creatine kinase,14 aspartate aminotransferase15 and lactate dehyrogenase,16 but there is no evidence to suggest that these complexes contribute to a pathological process. In contrast, cTn autoantibodies have been shown to contribute to the progression toward heart failure in mice and a similar process may occur in humans.17 Furthermore, it has been proposed that the reduced clearance of immunoglobulin– cardiac troponin complexes from the circulation may result in increased levels of measured troponins.18 This phenomenon does not, therefore, represent assay interference, but rather an analytically-true result, albeit misleading.There is increasing use of cTn measurement in the diagnosis of myocardial injury and any inaccuracy with regard to measured values is more likely to have serious clinical impact than would be the case with immunoassays for many other analytes. Several immunochemical interferences have been well described, including heterophile antibodies,This study shows that the removal of IgG from patient serum reverses the cTnT assay failure and that the addition of mouse serum as a heterophile antibody-blocking agent has no effect. This, together with the quantifiable result of the myoglobin assay (Roche Diagnostics), which uses the same test principle, species of monoclonal antibody and reader as the cTnT assay, suggests that the interference may not be because of a heterophile antibody, but rather because of an autoantibody to cTnT. Such an antibody may compete with the mouse anti-cTnT–gold complex for binding to immobilised cTnT on the test-strip, which normally serves as a test control. In the case of the myoglobin assay, the immobilised control antigen (myoglobin) is different, which may explain why it is unaffected. An autoantibody could also compete with reagent anti-cTnT antibodies for binding sites, thereby preventing the development of a test line .7 and the Elecsys E1708 automated platform but, to the best of our knowledge, this is the first report of cTnT assay failure on the former platform. We conclude that the failure of this assay is a result of putative autoantibodies to cTnT. Assay failure also allowed for the immediate detection of potential interference, but the majority of cases of interference remain undetected at the analytical level. It is crucial, therefore, that any discrepancies between results and the clinical picture be addressed by clinicians with the laboratory, to avoid further potentially-invasive and expensive investigations.There have been several reports of positive interference in the cTnT assay by heterophilic antibodies with the CARDIAC Troponin T Quantitative testA case of assay failure was detected in the CARDIAC Troponin T Quantitative reader. Immunoassays are subject to interference and it is important to investigate anomalous results to exclude such assay interference. After removing IgG from patient serum and re-assaying the cTnT, assay failure was found to reverse. This case highlights the fact that immunoassay interference remains a persistent problem and vigilance is encouraged in order to minimise its impact. Furthermore, in the case of troponins, interference may be caused by circulating autoantibodies to cTn.


Chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS) is a rare syndrome with relapsing brainstem/cerebellar symptoms. To examine the pathogenic processes and investigate potential biomarkers, we analyzed combined materials of brain and cerebrospinal fluid (CSF) by comprehensive methodologies.n = 5) to a neurodegenerative condition, Alzheimer’s disease . Activation of complement was confirmed by immunohistochemistry (IHC) on CLIPPERS brain samples (n = 3) and by ELISA in the CSF. For potential biomarkers, we used biomarker arrays, and compared inflammatory and vessel-associated proteins in the CSF of CLIPPERS (n = 5) with another inflammatory relapsing CNS disease, multiple sclerosis  and healthy subjects .To identify major pathways of perivascular inflammation in CLIPPERS, we first compared the CSF proteome (p < 0.05), eotaxin/CCL11 (p < 0.01), and granulocyte colony-stimulating factor (p < 0.05) in CSF could distinguish CLIPPERS from HS. The quantitative array confirmed elevated concentration of IL-8/CXCL8 and eotaxin/CCL11 compared to HS  besides increased levels of ICAM-1 (p < 0.05) and VCAM-1 (p < 0.001). The increased concentration of VCAM-1 were able to differentiate CLIPPERS from RMS (p < 0.01), and a trend of elevated levels of ICAM-1 and IL-8/CXCL8 compared to RMS was also observed .Two hundred and seven proteins in the CSF discriminated CLIPPERS from AD. The complement cascade, immunoglobulins, and matrix proteins were among the most frequently represented pathways. Pathway analysis of upstream regulators suggested the importance of vascular cell adhesion protein 1 (VCAM1), IFN-γ, interleukin (IL)-1, and IL-10. Differential regulation of more than 10 complement proteins of the 3 complement pathways in the CSF pointed to the role of complement activation. IHC on brain samples confirmed the perivascular complement activation, i.e., deposition of C3bc, C3d, and the terminal C5b-9 complement complex that partially overlapped with accumulation of IgG in the vessel wall. Besides endothelial cell damage, reactivity to smooth muscle actin was lost in the walls of inflamed vessels, but the glia limitans was preserved. The semi-quantitative array indicated that increased level of IL-8/CXCL8 (Complement activation, IgG deposition, and alterations of the extracellular matrix may contribute to inflammation in CLIPPERS. VCAM1, ICAM1, and IL-8 in the CSF may differentiate CLIPPERS from RMS. Chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS) is a rare relapsing disorder with subacute brainstem features, brain MRI displaying multiple punctate or curvilinear foci of gadolinium enhancement, and a clear radiological/clinical response to steroid treatment –3. Sever+ T cells, most of which are also CD4+  and azathioprine (175 mg daily).The 69-year-old woman was diagnosed with CLIPPERS in 2010. The clinical findings have been described previously .The male patient died at the age of 62 years; autopsy data have recently been published .For the examination of CSF proteome, samples from five patients with AD were included . For theThis study was carried out in accordance with the recommendations of Declaration of Helsinki with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the regional ethical committee and the Danish Data Protection Agency (S-20120066).n = 5) from patients #1–4 were used. Amino acid composition was analyzed for an estimation of total protein content, and the samples were treated with protease and phosphatase inhibitors. A pooled sample from patients #1 and #3a served as sample CLIPPERS-1, and a pooled sample from patients #2, #3b, and #4 served as sample CLIPPERS-2. These samples were compared to pooled CSF samples from five patients with AD. Samples were ultracentrifuged, and the supernatant was alkylated and digested  were selected for HCD fragmentation and the fragments were detected at a resolution of 17,500 FWHM. Threshold for ion selection was 1.0e4, the AGC target value 2.0e4, activation time was 0.1 ms, isolation window was 1.5 Da, and normalized collision energy was 29.Peptides from the various fractions were analyzed by a nano-Easy LC (Thermo Fisher Scientific) coupled with a Q-Exactive mass spectrometer . All peptide fractions were re-suspended in 0.1% formic acid (FA) and loaded onto a 2 cm 100 µm inner diameter pre-column using the nano-Easy LC. Peptides were eluted directly onto the analytical column using a gradient of 0–34% buffer B  over 30–90 min depending on the UV intensity of the individual HILIC fractions. All LC–MS/MS runs were performed using an analytical column of 20 cm × 75 µm inner diameter fused silica, packed with C18 material . Mass spectrometry was performed using higher energy collision fragmentation (HCD) fragmentation on a Q-Exactive instrument. MS settings: a full MS scan in the mass area of 400–1,800 Da was performed in the Orbitrap with a resolution of 70,000 FWHM and a target value of 1 × 10N. When obtaining the final list of regulated proteins, we only used the proteins with two or more peptides. For the identification, the searched data were filtered to a threshold of 1% FDR using Percolator.The MS raw files were processed and searched in Mascot and SEQUEST through the Proteome Discoverer 2.1 software (Thermo Fisher Scientific) against the human Uniprot database using the following parameters: precursor mass tolerance of 10 ppm; MSMS mass tolerance of 0.05 Da; enzyme: trypsin and up to two missed cleavages were allowed. For all data iTRAQ and carbamidomethylation was selected as fixed modifications. For the deglycosylated peptides the database search were performed with variable deamidation on Paraffin embedded brainstem biopsy materials from patients #1 and #2 and autopsy material from patients #5 were stained with C9neo  and hematoxylin–eosin (HE). For positive controls in the C9neo stainings, biopsy samples from patients with anti-AQP4 seropositive neuromyelitis optica spectrum disorder (NMOSD) were used. Pathological specimens from the autopsy material (patient #5) were stained for IgG-cig and IgG-smi , GFAP (Ab6 Thermo Fisher Scientific), CD31 , C3bc , and C3d .Twenty-micrometer cryostat sections from formalin-fixed adult Sprague–Dawley rat brains were blocked with 5% goat serum followed by overnight incubation with undiluted CSF from patients #1–3. Following a standard washing procedure , potential IgG binding was visualized with a secondary FITC-conjugated goat-anti-human IgG antibody . Sections were counterstained with 10 µM 4′,6-diamidine-phenylindole dihydrochloride  and coverslipped using ProLong gold antifade (Thermofisher Scientific).Three-micrometer brain stem sections from patient #6 were demasked and blocked with fetal bovine serum and were incubated with undiluted serum IgG (400 µg/ml), from patients #1–3 isolated by protein A affinity chromatography, acidic elution, and dialysis against PBS. Following standard washing procedure, sections were incubated for 2 h with HRP-conjugated rabbit anti-human IgG antibody . After a final rinse, sections were developed using diaminobenzidine dehydrated and coverslipped using Depex mounting medium. Digital images were obtained using a Leica microscope  equipped with a Leica digital camera .Activation products C3bc and soluble terminal complement complex (TCC) were examined in the CSF as described , 20.Cerebrospinal fluid samples from patients #1–4 and three control subjects were applied to the RayBiotech TM Human Inflammation Array C3 as described by the manufacturer.Cerebrospinal fluid samples used in the RayBiotech assay (patients #1–4) were also applied to MesoScale V-PLEX Human Biomarker 37-Plex Kit . We also included CSF samples from seven controls and nine patients with RRMS. All samples were run in duplicate.t-testing to identify regulated proteins. Regulated proteins were exported and used for pathway analysis using the Ingenuity Pathway Analysis program and the STRING program.p-value threshold on 0.05. Mesoscale data were evaluated using one-way ANOVA with multiple comparisons and Tukey correction.The identified and quantified peptides were imported into the Perseus programAltogether, 207 proteins in the CSF proteome and PTMome could discriminate CLIPPERS from AD: 51 proteins were upregulated and 156 proteins were downregulated in the CSF of patients with CLIPPERS  by the C9neo antibody on brain biopsy samples from two patients #1, #2) and autopsy material from another patient (#5) Figure . Proteom, #2 and Damage of the vessel wall was indicated by homogeneous diffuse red staining with HE in autopsy material from patient #5 Figure A. GFAP sTo investigate specific IgG binding to CNS structures, we incubated a pool of four CSF samples from patients #1, #2, and #3a/3b with rat brain tissue. Isolated serum IgG from the same three patients were also incubated with formalin-fixed, paraffin-embedded tissue obtained from patient #5. We could not identify specific IgG staining in any of these experiments (data not shown).The obtained proteomics data suggested the role of cytokines and integrins in network, pathway, and upstream regulator analyses, and we also considered endothelial stress/activation based on the pathology data. Therefore, we next investigated if these molecules may differentiate CLIPPERS from healthy controls and another relapsing inflammatory disease, multiple sclerosis (RMS).We first compared the CSF from CLIPPERS patients to healthy controls by the semi-quantitative RayBiotech array. IL-8, eotaxin (CCL11), and granulocyte colony-stimulating factor (GCSF) could distinguish CLIPPERS from HS Figure .p = 0.06, respectively).Next, we compared CSF of CLIPPERS to CSF obtained from HS and RMS by the quantitative MesoScale V-PLEX Kit Figure . This coMechanisms underlying inflammation in CLIPPERS are largely unknown. To investigate these, we examined the proteome in the CSF of CLIPPERS patients compared to non-inflammatory brain disease AD. AD is a classic neurodegenerative disease of the CNS. Although the role of innate immune responses is well established, adaptive immune-system driven effects, mediated by T and B cells, appear at present to be far less important in AD . There iCerebrospinal fluid proteins that were regulated differently in CLIPPERS compared to AD included the following: (i) proteins related to endothelial damage and activation including molecules involved in leukocyte recruitment  and vascular formation/remodeling ; (ii) serum proteins, which are expected to reach the brain, when endothelial cells are damaged ; (iii) molecules of the perivascular extracellular matrix important in vascular wall formation and remodeling, regulation of coagulation and complement activation, and cell adhesion ; and (iv) molecules related to perivascular infiltrates . Pathway and network analysis also indicated the importance of coagulation, cell adhesion and migration, the complement cascade, ephrin signaling, extracellular matrix-surface interaction, and contactin-mediated cell surface interactions. A network analysis for up-stream regulators of the differentially expressed proteins indicated the importance of several pro- and anti-inflammatory cytokines: IFN-γ, IL-1β, TNF-β, IL-6, IL-4, and IL-10. The differential presence of IgGs in the CSF proteome was unrelated to the presence of OCB; only one patient had temporary OCB in the CSF, and the hierarchical clustering identified similarly significant presence of IgGs and complement in the CSF samples without OCB.We found upregulation of several key members of the complement pathways in CLIPPERS CSF: C4, C4a, C4b, C2, and C2a (lectin and classic pathways); C3, C3a, and C3b ; and C9 . Complement activation products and the terminal complement complex (TCC) could be detected around plaques also in AD , but theThe deposition of complement and IgG may be a consequence of vascular damage due to leakage into the perivascular area; however, the increased levels of soluble TCC in the CSF and perivascular deposition of the MAC in the brain rather suggest local activation. The proteome and pathological data indicated endothelial damage and activation, leakage of serum proteins, vascular inflammation, and fibrosis with dysregulated perivascular extracellular matrix proteins. This may suggest that CLIPPERS is a form of vasculitis. The absence of vessel wall necrosis, granulocyte, or eosinophil infiltration does not support necrotizing vasculitis. Activation of the complement argues against an entirely T cell mediated vasculitis. If complement activation in CLIPPERS were mediated by antibodies, our data would suggest an antigen in the perivascular extracellular matrix. Autoimmunity against laminins has been described in autoimmune diseases –29, pregCytokines were upregulated in the CLIPPERS CSF proteome, or were among upstream regulators, including IFN-γ, IL-1β, and IL-10. VCAM-1 was one of the most upregulated proteins and was overrepresented in networks and pathways. Considering these data that reflect the role of cytokines and the possible role of endothelial stress/activation, we decided to use a broader array screening approach. First, we compared CSF levels of cytokines, chemokines, and biomarkers related to angiogenesis/endothelial stress to HS with a semi-quantitative assay, then we used a quantitative array to compare data also to RMS besides HS.Upregulated VCAM-1 in the CSF could distinguish CLIPPERS from both HS and RMS. Increased concentration of ICAM-1 was also observed compared to HS and as a trend when compared to RMS. These are key molecules in adhesion of lymphocytes and monocytes. Pro-inflammatory cytokines, such as IL-8, increase expression of ICAM-1 and VCAM-1 by endothelial cells . Indeed,This study is limited by the restricted number of patients. However, the number of reported cases worldwide is below 70, and CSF and brain samples are extremely rare. Here, we were able to combine analysis of both CSF and brain material, and in two patients, we even examined paired brain-CSF samples. Confirmation of results on samples from different compartments by other additional experiments and combination of different methods/compartments validating the findings strengthens the validity of our findings.In conclusion, our data strongly suggest that perivascular complement activation is involved in the pathogenesis of CLIPPERS. Proteomics and molecular profiling of the CSF by soluble arrays points to the importance of vessel dysfunction: disruption of the BBB, attraction and adhesion of immune cells to endothelial cells, and angiogenesis. Upregulated VCAM-1, ICAM-1, IL-8, and eotaxin in the CSF may be potential biomarkers in CLIPPERS; nevertheless, their differentiating role from RMS should be confirmed by a higher number of CSF samples. The cause of complement activation in CLIPPERRS is unclear: complement binding, alteration of the ECM, and search for specific antigens may be candidates for further studies.This study was carried out in accordance with the recommendations of Declaration of Helsinki with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the regional ethical committee and the Danish Data Protection Agency (S-20120066).MB and ZI: design and conceptualization, clinical data, analysis and interpretation of the data, drafting the manuscript. AH: performing proteomics, analysis of data. LD: analysis and interpretation of proteomics data. KR, DK, TS, HN, and FP: clinical data and interpretation. PG: examination of complement in the CSF, analysis and interpretation of data, drafting part of the manuscript. RH: indirect immunohistochemistry, analysis and interpretation of data. BC: indirect immunohistochemistry, analysis and interpretation of data, pathology. SH: isolation of IgG. PJ: analysis and interpretation of proteomics data, performing RayBiotech assay. MM: biomarker analysis and interpretation of data. HL: histology, analysis and interpretation of data, drafting part of the manuscript. ML: analysis and interpretation of proteomics.MB reports grant from Lundbeck A/S outside of the submitted work. AH, LD, PG, RH, BK, DK, TS, SH, HN, PJ, and MM report no disclosures. KR reports grants from German Ministry of Education and Research  during the conduct of the study; grants and personal fees from Novartis, personal fees from Bayer Healthcare, personal fees from Biogen Idec, grants and personal fees from Merck Serono, personal fees from Sanofi-Aventis/Genzyme, personal fees from Teva Pharmaceuticals, grants from Guthy Jackson Charitable Foundation, outside the submitted work. FP reports grants and personal fees from various pharmaceutical companies outside the submitted work. HL reports personal fees from Novartis, personal fees from Sanofi Aventis, personal fees from TEVA, personal fees from Roche outside the submitted work. ZI reports grants from Lundbeckfonden, grants from Scleroseforeningen during the conduct of the study; personal fees from Biogen Idec, grants and personal fees from Sanofi Genzyme, personal fees from Novartis, personal fees from Merck Serono outside the submitted work.


The mean CD34+ cell number per microliter in the blood before the first apheresis was 111 for lenograstim, 119 for biosimilar filgrastim, and 124 for filgrastim (p = 0.354); the mean difference was even less significant when comparing CD34+ number per dose of G-CSF per kilogram (p = 0.787). Target doses of CD34+ cells were reached with one apheresis in 87% donors mobilized with lenograstim and in 93% donors mobilized with original and biosimilar filgrastim (p = 0.005). The mobilized apheresis outcomes (mean number of CD34+ cells/kg of donor collected during the first apheresis) was similar with lenograstim, biosimilar filgrastim, and filgrastim: 6.2 × 106, 7.6 × 106, and 7.3 × 106, respectively, p = 0.06. There was no mobilization failure in any of the donors. Biosimilar G-CSF is as effective in the mobilization of hematopoietic stem cells in unrelated donors as original G-CSFs. Small and clinically irrelevant differences seen in the study can be attributed to differences in G-CSF dose and collection-related factors. Active safety surveillance concurrent to clinical use and reporting to donor outcome registry  might help to evaluate the possible short- and long-term complications of biosimilar G-CSF.The World Marrow Donor Organization recommends original granulocyte-colony stimulating factor (G-CSF) for the mobilization of stem cells in healthy unrelated hematopoietic stem cell donors. We report the comparison of a biosimilar G-CSF (Zarzio) with two original G-CSFs (filgrastim and lenograstim) in mobilization in unrelated donors. We included data of 313 consecutive donors who were mobilized during the period from October 2014 to March 2016 at the Medical University of Warsaw. The primary endpoints of this study were the efficiency of CD34+ cell mobilization to the circulation and results of the first apheresis. The mean daily dose of G-CSF was 9.1 μg/kg for lenograstim, 9.8 μg/kg for biosimilar filgrastim, and 9.3 μg/kg for filgrastim ( There are over 27 million registered unrelated hematopoietic stem cell donors worldwide, as reported by the World Marrow Donor Association (WMDA) . The staRecently, biosimilars of G-CSF have been introduced into the stem cell mobilization protocols . The expIn 2015, due to legal issues and changes in market availability, the G-CSF used in mobilization of hematopoietic stem cells in donors and patients at our institution has been first changed from lenograstim to biosimilar filgrastim and later to original filgrastim. As publications that compare biosimilars with both original G-CSFs are limited, we decided to retrospectively analyze the efficiency of mobilization with those three drugs in healthy unrelated hematopoietic stem cell donors.This study included medical data of 313 consecutive donors of both genders aged between 19 and 55 years, who were mobilized from October 2014 to March 2016. The sizes of the donor groups mobilized with original G-CSFs were chosen to resemble the size of the group mobilized with biosimilar filgrastim.The G-CSF formulations used in this study were lenograstim—Granocyte (Chugai), biosimilar filgrastim—Zarzio (Sandoz), and filgrastim—Neupogen (Amgen). We did not report any serious adverse events (SAE) in the donors during this study .Donation data were collected prospectively by our center for scientific purposes. All donors gave written, informed consent allowing the use of their anonymous medical records for research purposes. All procedures were followed in accordance with the ethical standards set by the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2008. The data for this study were collected from medical records of the donors that covered qualification, collection, and, in some instances (when requested by donor center), donor follow-up. The data chosen for the study included individual donor number, type of growth factor, dose of growth factor , type of protocol, days of apheresis, age, sex, weight, height, dose per kilogram, dose per surface area, complete blood count at qualification, complete preapheresis blood count, percent of CD34+ cells in blood, number of CD34+ cells in blood, nucleated cell count in apheresis product/products, percent of CD34+ cells in product/products, CD34+ count , and complete blood count after apheresis.The donors were sent for evaluation and collection by different donor registries. The final clearance was performed by apheresis center—the donors who were qualified had no contraindications according to the WMDA donor medical suitability recommendations. The optimal daily dose for G-CSF was selected based on the donor weight. For lenograstim, the target dose was 7.5–10 μg/kg, and for filgrastim , it was 7.5–10 μg/kg. The daily dose has been rounded to the closest possible with 34-million-IU injections of lenograstim or 30- and 48-million-IU injections of filgrastim. The G-CSF was given 4 days before the donation with the daily dose split into two injections—one given at 8 AM and the second at 8 PM. The donors were trained on how to inject G-CSF and those with no contraindications performed their injections themselves. The access to a nurse who administered injections was provided for donors who objected to self-injections.The apheresis was performed with Spectra Optia cell separator . The separator used software version 4 up to April 20, 2015 and version 11.2 throughout the rest of the studied period. All donors were mobilized with MNC program prior to December 2015; later, cMNC was performed in 30 out of 107 donors in the original filgrastim group. Lenograstim was used until June 2015, biosimilar filgrastim from June to October 2015, and filgrastim was used afterwards. ACD-A was used as coagulation in a proportion of 0.9 AC.p value of <0.05 was considered statistically significant. The center evaluates CD34+ methodology by performing CD34+ enumeration 6–8 times a year with a BD Stem Cell Control Kit . There were no reports of significant discrepancies in CD34+ cell count from transplant centers in the studied period.The donors had one or two aphereses as needed to collect the number of the CD34+ cells required by the transplant center. The CD34+ cell count has been evaluated according to the ISHAGE guidelines  . Statist6 of CD34+ cells per kilogram of body weight of the recipient.If one apheresis provided over 95% of requested stem cells, the second apheresis was not performed. In the case of a second apheresis, the time of the procedure (and the volume of the product) was reduced in selected cases, so as not to mobilize with excess. Mobilization failure was defined as collection of less than 2 × 10The primary endpoints of the study were the efficiency of CD34+ cell mobilization to the circulation (measured as the number of CD34+ cells per microliter prior to the initiation of the first apheresis) and results of the first apheresis. The study was not designed to analyze short- and long-term complications of G-CSF.p = 0.02), and the group mobilized with filgrastim had higher body mass index than the other two groups, 25.6 vs 24.5 and 24.2 (p = 0.018) . The total mean dosage of G-CSF to the first apheresis was different in all groups: 40.7 μg/kg for lenograstim (29.0–52.4), 44 μg/kg for biosimilar filgrastim (36–60), and 42 μg/kg for filgrastim (33–55)—the donors who were mobilized with biosimilar filgrastim received mean higher total doses of G-CSF . After four doses of G-CSF but before the last dose of G-CSF prior to apheresis, the mean white blood cell count was 43.7 g/l for lenograstim (33.8–52.5 interquartile range), 45.2 g/l for biosimilar filgrastim (36.9–52.2 interquartile range (IQR)), and 46.4 g/l for filgrastim (37.3–55.4 IQR) (p = 0.246). The mean CD34+ cell number in the blood before the initiation of apheresis was 111 cells/μl for lenograstim (63–146 IQR), 119 cells/μl for biosimilar filgrastim (83–157 IQR), and 124 cells/μl for filgrastim (73–162 IQR) (p = 0.354).The mean daily dose of G-CSF was 9.1 μg/kg for lenograstim, 9.8 μg/kg for biosimilar filgrastim, and 9.3 μg/kg for filgrastim (p = 0.005). The mean number of collected CD34+ cells × 106 per kilogram of donor: 6.2 for lenograstim, 7.6 for biosimilar filgrastim, and 7.3 for filgrastim (p = 0.06). After correcting for donor gender, the mean number of collected CD34 + cells × 106 per kilogram of recipient did not differ between the groups (p = 0.129) but generally higher collections were reported in men than in women . Additionally, gender had the same effect on collection with all three growth factors (p = 0.847). There were no differences regarding the number of collected CD34+ cells × 106 per kilogram of recipient between donors when MNC or cMNC was used  and those who underwent aphereses using different versions of cell separator software (p = 0.086). There were no mobilization failures, with 100% of donors mobilizing over 2 × 106 CD34+ cells/kg of recipient. The mean difference in the total number of collected CD34+ cells × 106 per kilogram of recipient between different G-CSFs was also not statically significant: 7.7 for lenograstim, 8.5 for biosimilar filgrastim, and 9.4 for filgrastim (p = 0.085). The most important comparisons between mobilization with lenograstim, biosimilar filgrastim, and filgrastim are summarized in Table The mobilization with filgrastim and biosimilar filgrastim was more efficient than that with lenograstim: 93% of donors after one of the filgrastims needed one apheresis for sufficient collection compared to 87% donors after lenograstim  with biosimilar filgrastim. The primary endpoints of our study, mobilization of CD34+ into circulation and results of the first apheresis, have shown no statistically significant differences between original and biosimilar drugs. We observed almost no clinically relevant difference between the drugs—the number of donors needing one apheresis is similar in three groups—with differences related possibly to slight differences in dose, sex distribution, and procedure of cell separation.Previous studies have also shown differences between lenograstim and filgrastim—however, in favor of lenograstim. Ings et al.  have shoThe biggest limitation of this study was the inability to assess the short- and long-term side effects of the studied drugs. On the other hand, the short-term side effects of the drugs will likely not differ significantly . We alsoOur data provide positive evidence for the use of biosimilar G-CSF in healthy unrelated hematopoietic stem cell donors. It is shown that the biosimilar G-CSF does not differ from the original growth factors when the mobilization of the CD34+ positive cells is analyzed. We still need to wait for the results of long-term observations of the donors after biosimilar G-CSF before we can recommend its use as standard in the mobilization of hematopoietic stem cells in healthy unrelated donors.