Glycine receptors (GlyRs) are anion-permeable pentameric ligand-gated ion channels (pLGICs). The GlyR activation is critical for the control of key neurophysiological functions, such as motor coordination, respiratory control, muscle tone and pain processing. The relevance of the GlyR function is further highlighted by the presence of abnormal glycinergic inhibition in many pathophysiological states, such as hyperekplexia, epilepsy, autism and chronic pain. In this context, previous studies have shown that the functional inhibition of  GlyRs containing the α3 subunit is a pivotal mechanism of pain hypersensitivity. This pathway involves the activation of EP2 receptors and the subsequent PKA-dependent phosphorylation of α3GlyRs within the intracellular domain (ICD), which decrease the GlyR-associated currents and enhance neuronal excitability. Despite the importance of this mechanism of glycinergic dis-inhibition associated with dysfunctional α3GlyRs, our current understanding of the molecular events involved is limited. Here, we report that the activation of PKA signaling pathway decreases the unitary conductance of α3GlyRs. We show in addition that the substitution of the PKA-targeted serine with a negatively charged residue within the ICD of α3GlyRs and of chimeric receptors combining bacterial GLIC and α3GlyR was sufficient to generate receptors with reduced conductance. Thus, our findings reveal a potential biophysical mechanism of glycinergic dis-inhibition and suggest that post-translational modifications of the ICD, such as phosphorylation, may shape the conductance of other pLGICs. To date, a single β subunit and four α subunits (α1–4) has been described. The α subunits share a high degree of sequence identity (≈75%). Nevertheless, they exhibit important differences in their biophysical and pharmacological properties as well as in their distribution along the CNS4.Glycine receptors (GlyRs) belong to the pentameric ligand-gated ion channel (pLGIC) family. GlyRs are anion-permeable channels, allowing the fast influx of chloride and the control of neuronal excitability. An individual GlyR subunit is composed by an extracellular domain (ECD), four transmembrane domains (TM1–4) and an intracellular domain between the TM3 and TM4 domains (ICD)13. The importance of glycinergic inhibition was first recognized in studies using the GlyR antagonist strychnine15. Later, genetic studies found that mutations in the GlyR α1 and β genes are linked to hyperekplexia in humans16. More recent evidence has shown that specific GlyR subunits may play key roles in several diseases. For example, while the α1 subunit has been linked to tumorigenesis and alcohol intoxication18, mutations in the α2 subunit have been linked to autism19. Alterations in the RNA processing of α3 subunits generates hyperactive receptors, which have been related with epilepsy21. In addition, the functional inhibition of spinal dorsal horn α3GlyRs has been shown to be a critical mechanism in inflammation-induced pain hypersensitivity22. Furthermore, α3GlyRs have also been implicated in the modulation of respiratory rhythms23. Collectively, these studies indicate that the glycinergic system may be a promising target for future drug development.In the mammalian CNS, GlyR activity critically controls neurophysiological functions such as motor coordination, respiratory control, muscle tone, as well as pain processing22. The proposed mechanism of α3GlyR-dependent pain sensitization involves the loss of glycinergic inhibition following the activation of neuronal EP2 receptors (EP2-R) by prostaglandin E2 (PGE2)25. EP2-R stimulation increases cAMP and subsequently promote PKA-dependent phosphorylation of the α3GlyR on the S346 residue within the ICD, decreasing the amplitude of glycinergic currents and enhancing the excitability of dorsal horn excitatory neurons. Interestingly, the relevance of α3GlyRs in pain sensitization has been highlighted by the recent characterization of allosteric modulators targeting GlyRs29. These reports have shown that compounds potentiating α3GlyR activity are able to reduce chronic pain symptoms in rodents. These results confirm the key role of α3-containing GlyRs in the analgesic effects of such modulators. However, our current understanding of the molecular events underlying the functional α3GlyR inhibition by PKA-mediated phosphorylation is still very limited.The importance of glycinergic inhibition in chronic pain has been characterized in the superficial dorsal hornHere we report that the activation of PKA signaling pathway decreases the unitary conductance of α3GlyR. In addition, we show that the substitution of the S346 amino acid with a negatively charged residue generate ion channels with lower conductance. Our findings propose an undescribed biophysical framework to understand a mechanism underlying neuronal dis-inhibition. In a broader context, our data suggest that dynamic modifications of the ICD chemical composition shape the conductance of pLGICs.28, EP2-R stimulation with PGE2 (10 μM) decreased the amplitude of glycine-evoked currents . These results support that the stimulation of cAMP production triggers PKA-dependent phosphorylation of α3GlyRs. To study whether a GPCR-independent increase in cAMP levels may reduce the glycine-activated currents, we performed recordings in cells co-expressing α3GlyRs and a light-sensitive bacterial adenylyl cyclase, known as bPAC31. Activation of bPAC with blue light allows the optical triggering of cAMP production in living cells31. Our results showed that the activation of bPAC with blue light gradually diminished the glycine-activated current amplitudes in α3GlyRs by −35.5 ± 4.2% from control . The degree of inhibition was not significantly different from that induced by EP2-R activation  .We first assessed the effects of EP2-R activation on the glycine-activated currents through recombinant α3GlyRs expressed in HEK293 cells. As previously shownnts Fig. . The extst) Fig. . Additiost) Fig. , showed st) Fig. , did notIn order to assess whether the decrease in α3GlyR function triggered by PKA activation is associated with a loss of plasma membrane receptors, we studied cell-surface α3GlyRs under different conditions of PKA activation Fig. . Our res33. The single-channel amplitudes were stable during 12 minutes of recordings   Fig. . Interesst) Fig. . The comst) Fig. . Additiost) Fig. . The α3Gst) Fig. .Figure 236. Nevertheless, other studies have shown that the mutation of charged residues in the ICD modified the conductance of pLGICs, including 5-HT3 receptors, nicotinic acetylcholine receptors and GlyRs39. The results described above suggest that the introduction of a single negative charge at position 346 of α3GlyRs should be able to reduce conductance. To explore this idea, we performed single-channel recordings of point-mutated α3GlyRs in which the key residue for PKA-dependent phosphorylation, serine 346, was mutated to a glutamate (S346E) or to an alanine (S346A)40 40 Fig. . As prev40. Our results suggest that the reduction of the glycine-activated currents through α3GlyRs is mostly related to a decreased ion channel conductance, elicited by the chemical modification of S346 residue  displayed unaltered glycine sensitivity27. Thus, the reduction in the glycine-activated currents after PKA activation appears not clearly connected to changes in the agonist binding site.It is widely established that intracellular phosphorylation mediated by protein kinases is a relevant mechanism of modulation of pLGICsThe results of the present work support the idea that PKA-dependent inhibition of the α3GlyR function is associated to an alteration of the ion channel conductance. This concept is supported by at least three lines of evidence. First, PKA activation using bPAC reduced the unitary conductance of α3GlyRs. Second, a non-hydrolyzable cAMP analog diminished the conductance of α3GlyRs to a similar degree. Third, reduction in the conductance was mimicked by the substitution of the consensus site for PKA-dependent phosphorylation with a phospho-mimetic acidic residue. Interestingly, the S346E α3GlyR construct did not display any noticeable alteration in cell surface expression, open probability, desensitization, or rectification. Collectively, these results suggest that the functional inhibition of a pLGIC elicited by phosphorylation of a serine residue within the ICD is mostly associated with a reduction in the unitary conductance, rather than alterations in the cell surface receptors or agonist binding site.51. We used this feature to address whether the substitution of a single residue within the ICD can affect the conductance of an engineered pLGIC and to determine the relevance of the ECD of α3GlyR for the conductance regulation by the ICD. The comparison of two chimeric receptors, one having the full ICD of α3GlyR (i.e. Lily-α3-ICD) and the other lacking an ICD (i.e. Lily-α3), showed similar proton sensitivities, analogous unitary conductances and comparable open probability. These observations suggest that inclusion of the ICD does not produce striking changes in essential ion channel features. Interestingly, this idea is supported by other reports showing that ICD insertion or replacement between eukaryotic pLGICs and bacterial GLIC channels did not produce alterations in the conductance or ion selectivity53.pLGICs show a modular architecture in which each module  can be interchangeable between receptors to form functional chimeric channels55. Therefore, since the ICD only emerged later in the evolution of eukaryotic pLGICs, one might argue that the ICD has been included merely an accessory domain. Our results showed however that introduction of the S346E mutation into the ICD of the Lily-α3-ICD chimera and of the wild-type α3GlyR produced a significant reduction in the unitary conductance. Together with the results that show a decrease in the conductance after PKA activation, our findings suggest that the phosphorylation state of the S346 residue determines the unitary conductance of α3GlyRs. In addition, our data using chimeric channels showed that the ICD-dependent reduction of the conductance did not requires a glycine-binding site within the ECD. Taken together, our evidences suggest that the contribution of the ICD to the conductance of α3GlyRs appears to be dependent on the amino acid composition of this particular region, rather than on its presence. In a wider context, these results suggest that reversible post-translational modifications targeting a single residue within the ICD of a pLGIC may change the channel conductance, strengthen the idea that the ICD, likely as an independent module, is an important determinant of intrinsic pLGICs properties58.From an evolutionary point of view, these observations are interesting because the overall architecture and functional features of pLGICs are conserved from archaea to metazoan47. These studies have determined that α3-containing GlyRs are expressed in superficial layers of the dorsal horn, contributing to nociceptive processing and chronic pain of inflammatory origin22. An increasing number of studies have suggested in addition that GlyRs are also expressed in supraspinal sites60. Interestingly, recent immunocytochemical and gene expression studies have determined that α3GlyRs are expressed in brainstem, trigeminal ganglia, nucleus accumbens and the hippocampus61. Despite the absence of specific pharmacological tools to identify α3GlyRs, the presence of functional α3-containing GlyRs in several CNS areas has been inferred by using mice lacking α3GlyRs. These studies have found that α3GlyRs participate in both synaptic and tonic glycinergic inhibition27. The unitary conductance regulation of α3GlyRs by the activation of PKA signaling pathway described here provides a plausible regulatory mechanism of glycine-activated chloride influx in these CNS regions. Based on these results, we speculate that impaired chloride conductance may underlie, at least in part, dynamic glycinergic dis-inhibition associated with phosphorylated α3GlyRs, such as inflammatory pain sensitization in the spinal cord and respiratory control by the brainstem23. Phosphorylation may also contribute to the regulation of tonic inhibition strength associated with α3GlyRs in forebrain structures11. Although the present work defined a possible mechanism of glycinergic dysfunction at the biophysical level, whether this phenomenon contributes to the modulation of native GlyRs is still a scientific challenge due to the absence of GlyR subunit-specific inhibitors to study pharmacologically-isolated α3GlyRs in a neuronal context. Further experiments will define whether this regulatory mechanism of α3GlyRs contributes to the control of neuronal activity.The function of GlyRs has been traditionally linked to neuronal inhibition at the level of spinal cordIn conclusion, we demonstrate that the activation of PKA signaling pathway decreases the unitary conductance of α3GlyRs. Our observations point out the importance of the chemical composition of the ICD as a critical determinant for the pLGIC conductance and provide a plausible biophysical mechanism to explain processes of glycinergic dis-inhibition. Although further studies are necessary to understand the structural changes involved, these results may open further possibilities for the design and development of new compounds targeting phosphorylated α3GlyRs, which may recover receptors with unpaired conductance and may have interesting clinical applications.42. The chimeras were then chemically synthesized  and subcloned in a pCDNA3 vector (Invitrogen). The cells were transfected using XfectTM Transfection Reagent  with 0.5–3.0 μg of cDNA plasmids encoding ion channels and 0.5 μg of EGFP. HEK293 cells were used unless otherwise stated. To avoid endogenous proton-gated currents, BHK cells were used in the experiments involving GLIC-GlyR chimeras. In the experiments involving the activation of bPAC or EP2 receptors, 2 μg of each plasmid were used. The photosensitive bacterial adenylyl cyclase (bPAC) was a gift from Dr. Peter Hegemann (Addgene plasmid # 28134)30. The experiments involving optical stimulation of bPAC were performed using mCherry as a transfection marker. The recordings were made 36–48 hours after transfection.The α3L version of the α3GlyR was used in our studies. Mutations were inserted by using the Quick-Change site-directed mutagenesis kit (Agilent). Chimeric receptors composed of the bacterial GLIC channel and α3GlyRs were designed based on previous protocolsPGE2 was purchased from Tocris . All other reagents were from Sigma-Aldrich .42. The patch electrodes (3–4 mΩ) were filled with an intracellular solution containing (in mM): 120 CsCl, 8 EGTA, 10 HEPES (pH 7.4), 4 MgCl2, 0.5 GTP and 2 ATP. The bath solution contained (in mM) 140 NaCl, 5.4 KCl, 2.0 CaCl2, 1.0 MgCl2, 10 HEPES (pH 7.4), and 10 glucose. The recordings were performed with an Axoclamp 200B amplifier  or with a HEKA EPC10 . The signals were acquired using Clampex 10.1 or PatchMaster software. Data analysis was performed off-line using Clampfit 10.1 . The exogenous glycine-evoked currents were obtained using an outlet tube (200 μm ID) of a custom-designed gravity-fed microperfusion system, positioned 50–100 μm from the recorded cell. The agonist was manually applied to the cell using a short pulse (3–4 s). Concentration-response curves were obtained from normalized concentration–response data points. The optical stimulation of bPAC was performed using a fiber-coupled LED emitting blue light (470 nm) and a T-Cube LED Driver . Glycine stocks were prepared daily in high purity distilled water. The stock solutions of PGE2 were prepared in DMSO and kept at −20 °C. Single channel recordings were performed as previously reported42. The recordings were performed in the cell-attached configuration (+60 mV). The patch pipettes (10–20 mΩ) were manually fire-polished in a microforge . The extracellular solution contained (in mM): 20 Na-gluconate, 102.7 NaCl, 2 KCl, 2 CaCl2, 1.2 MgCl2, 10 HEPES, 20 TEA-Cl, 15 sucrose, and 14 glucose, pH 7.4. The pipette was filled with extracellular solution containing glycine (100–200 μM). 8-Br-cAMP (100 μM) was applied to the extracellular solution 15 minutes prior to the recordings. The data were filtered (2-kHz low-pass 8-pole Butterworth) and acquired at 5 kHz using an Axopatch 200B amplifier and a 1322 A Digidata . Data was acquired using pClamp software and analyzed off-line with Clampfit 10.1 . Single-channel conductance (γ) values were determined from the relationship γ = I/(Vm − Vrev), in which I is the current amplitude of single channel events, Vm is the membrane potential and Vrev the reversal potential.The glycine-activated currents were recorded from transiently transfected HEK 293 cells in the whole-cell voltage-clamp configuration (−60 mV). The recordings were performed at room temperature (20–24 °C)2. Subsequently, the cells were fixed with 4% paraformaldehyde  for 15 minutes at 4 °C and blocked with 10% horse serum for 5 min. Epitope visualization was performed by incubating the sample with a secondary antibody Cy3 . Finally, the cells were cover-slipped with Fluorescence Mounting Medium . For quantitative analysis, cells were randomly chosen for imaging using a confocal microscopy . Single stacks of optical sections in the z-axis (1 µm) were acquired, and dual-color immunofluorescent images were captured in simultaneous two-channel mode. The quantification of the fluorescence intensity and number of clusters associated with membrane receptors was carried out off-line using ImageJ software , using previously reported plug-ins and protocols62. Briefly, for each cell analyzed, the relative fluorescence units (URF) of the red channel were obtained by averaging 3 zones with areas of 100 μm2. For the quantification of clusters, the stacks obtained on the Z axis were first placed in a 25 μm × 4 μm box, and then the number of distinguishable points were quantified.To analyze cell surface GlyRs, we used a C-terminal hexa-histidine tagged α3GlyR version. Transfected cells were first washed 3 times with PBS and then incubated with a monoclonal hexa-histidine antibody  for 10 minutes at 37 °C and 5% CO51. The models of Lily-α3 and Lily-α3-ICD subunits were constructed by homology modeling using the structure of the chimera GLIC-α1GlyR called “Lily” [PDB ID: 4 × 5T] as template42. For both chimeric receptors, the section corresponding to α1GlyR in “Lily” was replaced with the sequence of the human α3GlyR from the amino acid 218 (in TM1) to the C-terminal end. In the Lily-α3 model, the ICD was replaced with the sequence SQP42  and energy minimization with a conjugate gradient protocol in the software MacroModel . All images were created with PyMOL 51.The full model of the α3GlyR was created using the structure of the human α3GlyR [PDB ID: 5 TIO] and the ICD predicted ab initio with QUARK, as previously describedAll values were expressed as mean ± SEM. Statistical comparisons were performed using Student t-tests. Multiple comparisons were analyzed with ANOVA followed by a Bonferroni post hoc test. P values less than 0.05 were considered statistically significant. All the statistical analyses and plots were performed with MicroCal Origin 6.0 or 8.0 .Supplementary Information.


Existing techniques of freehand pedicle screw placement primarily focus on various entry points with or without axial trajectory. The objective of this paper is to propose a universal entry point and sagittal and axial trajectory for all subaxial cervical, thoracic and lumbosacral spines freehand pedicle screw placements, and to report the results from a single‐surgeon clinical experience with freehand pedicle screw placement.Two spine vertebrae specimens and 20 cases of three‐dimensional (3D) reconstructions of spine CT images were used for observation of the entry point and sagittal and axial trajectory. The author retrospectively reviewed a total of 610 consecutive patients who underwent open, freehand pedicle screw fixation using a universal entry point and sagittal and axial trajectory for all subaxial cervical, thoracic and lumbosacral spine placements, during an 8‐year period from January 2010 to December 2017. The junction of the lateral margin of the superior articulating process and the transverse process for the thoracic and lumbosacral spines, or lateral mass for the subaxial cervical spine, was determined. The entry point was chosen at 1 mm, 2 mm, and 3 mm (2 mm on average) caudally and medially to this junction for subaxial cervical, thoracic and lumbosacral spines placements, respectively. Both sagittal and axial trajectories were perpendicular to the sagittal and axial planes of the laminae of the isthmus. Among them, 68 patients underwent postoperative computed tomography (CT) scans, including 26 cervical cases, 19 scoliosis thoracic cases, 10 non‐scoliosis thoracic cases, 8 lumbar cases, and 5 sacral cases. Placements of pedicle screws were assessed using CT data and outcome‐based classifications systems.After placing the iron scurf at the junction of the lateral margin of the superior articulating process and the transverse process, the present universal entry point was located at 1 o'clock or 11 o'clock of the pedicle's axial view. After inserting the 2.5 mm Gram needle or the pedicle virtual pin tracts according to the entry point and sagittal and axial trajectory described above, the presented trajectory was located in the pedicle's axial trajectory as in the described technique. A total of 766 pedicle screws were placed in 68 CT scan patients with a 99% accuracy rate in the non‐kyphoscoliosis group and 92% in the kyphoscoliosis group.Freehand pedicle screw placement based on the universal entry point and sagittal and axial trajectory for all subaxial cervical, thoracic and lumbosacral spines can be performed with acceptable safety and accuracy. Pedicle screw fixation is the preferred modality throughout the vertebral column because of its proven effectiveness in stabilizing all three columns of the spine. This advantage has been translated to superior clinical results in certain cases of trauma, instability, deformity, and neoplastic destruction. Therefore, it has become popular during the past decade, first in applications involving the lumbar spine and subsequently in thoracic and cervical spine surgery. However, pedicle screws remain technically demanding to place, particularly in the cervical and thoracic region because of the smaller size and more complex morphology of pedicles. Additionally, complications related to the use of screws are potentially serious, including: screw misplacement, pedicle fracture, screw rupture, bending or loosening, vertebral canal violation, dural tear, vascular or visceral problems, and postoperative neurologic symptoms or pain. Because of this, the freehand pedicle screw insertion techniques and image‐guided techniques have been widely developed to guide placement.Freehand pedicle screw insertion technique relies on tactile feedback of the surgeon and the use of anatomical marks to determine correct screw entry point, without the aid of intraoperative image‐guided systems or explorative laminectomy, with no or limited use of intra‐operative fluoroscopy. While the freehand methods are effective and widely employedAs the free‐hand pedicle screw placement technique relies solely on anatomy, existing techniques primarily focus on various entry points with or without medial angulations Although different entry points at different levels as well as different trajectories have been employed, neither universal entry point nor consistent methods to determine cranial‐caudal  trajectory based on anatomic landmarks have been describedThe anatomic landmark of the junction of the lateral margin of the superior articulating process and the transverse process for thoracic and lumbosacral spines, or lateral mass for the subaxial cervical spine, was determined. The entry point was chosen at 1 mm, 2 mm, and 3 mm (2 mm on average) caudally and medially to this junction for the subaxial cervical, thoracic and lumbosacral spines, respectively . Both sagittal and axial trajectories were perpendicular or orthogonal to the sagittal and axial planes of the laminae of the isthmus Fig. .3 to S1 vertebrae, were used for the present study. Both sides of each vertebra were inserted with a Gram needle 2.5 mm according to the entry point and sagittal and axial trajectory described above. After placing an iron scurf at the junction of the lateral margin of the superior articulating process and the transverse process , the superior, lateral and axial of the Gram needle X‐ray films were obtained for verification of the trajectory.This research has been approved by the IRB of the authorsʼ affiliated institutions. Two spine vertebrae specimens, including the segments from C3 to S1 were used to test the entry point and sagittal and axial trajectory described above. According to the vertebral anatomic structure, the entry point and surface of the laminae of the isthmus were determined with Hyperworks software . After the planes that were orthogonal to the sagittal and axial surface of the laminae of the isthmus were established, the pedicle virtual pin tracts were determined. The 3D image was rotated, and the superior, lateral and axial views were obtained for verification of the trajectory.3D reconstructions of spine CT images were obtained for 20 cases. A 3D model of every two vertebra from C3 to S2, and the age ranged from 5 to 84 years.During an 8‐year period from January 2010 to December 2017, 610 consecutive patients underwent open pedicle screw placement in the subaxial cervical, thoracic and lumbosacral spines. In these 610 patients , the aetiologic diagnosis included cervical trauma (27 cases), thoracolumbar trauma (180 cases), spinal tumor (135 cases), spinal deformity (145 cases), spinal infection (83 cases), and spinal degenerative diseases (43 cases); the distribution of the spine level was from CAll cases were performed by the author (Z.Z.). All pedicle screws were inserted using the freehand technique. After exposure, a straight awl was used to disrupt the cortical bone at the entry point described above. A straight, blunt‐ended gearshift was used to cannulate the pedicle to the desired depth based on the sagittal and axial trajectory described above. A ball‐ended feeler was used to search for breaches. After the typical tapping and probing was repeated, an appropriate size screw was placed. To decrease the operative time, the markers were not used. Intraoperative fluoroscopy was used for initial localization and then again for a final anteroposterior and lateral radiograph.After the surgery and during the following up, standard anteroposterior and lateral films were taken to assess spinal injury, reduction, decompression, internal fixation, and fusion for all 610 patients. Among them, 68 patients underwent postoperative computed tomography (CT) scans, including 26 cervical cases, 19 scoliosis thoracic cases, 10 non‐scoliosis thoracic cases, eight lumbar cases, and five sacral cases. This study was based upon postoperative CT scans because CT scans have been shown to be more sensitive than radiographs in detecting cortical breachesPlacements of pedicle screws were assessed using CT data and outcome‐based classifications modified from previously reported systems as followst test, with P < 0.05 considered to be statistically significant.Statistical analyses were performed using SPSS13.0 software  to evaluate the accuracy of screw placement. The placement of acceptable pedicle screws (Type 0 and 1) and unacceptable pedicle screws (Type 2 and 3) were compared using the Student's After placing the iron scurf at the junction of the lateral margin of the superior articulating process and the transverse process , iron scurf was located at 1 o'clock or 11 o'clock of the pedicle's axial view Fig. D, H, L. t = 8.758, P = 0.000) and non‐kyphoscoliosis group   (Fig. The present choice of anatomic landmarks for the junction of the lateral margin of the superior articulating process, the transverse process or lateral mass, and the laminae of the isthmus was based on them developing from the same vertebral arches in the embryologic stage. There are three primary ossification centers in the typical embryonic vertebrae: one in the center of the centrum and one in each of the vertebra arch halves. The fusion of the fetal vertebral arches to the center occurs well anterior to the pediclesThe present trajectory is located in the pedicle's axial trajectory, which presented several advantages compared to various traditional freehand pedicle screw placement techniques Table 1, 2, 3, Several studies have shown that all reported neurological deficits had medial pedicle perforations of more than 4 mmWe propose several recommendations regarding this technique. Preoperatively, pedicle CT data should be carefully identified, such as the diameter, no medullary cavity or an extremely small medullary cavity, and a developmental disturbance in neurofibromatosis‐1. Intraoperatively, because the present technique relies on pedicle cannulation, care is taken to appreciate the perforation of cancellous bone over the entire length of the pedicle and vertebral body. The blunt‐ended gearshift should advance smoothly with consistent resistance. Abrupt loss of resistance indicates a pedicle breach, while an increase in resistance indicates the gearshift is against the cortical bone, and the trajectory must be re‐evaluated prior to further advancement. During hand drilling, cannulating and tapping, the four walls and the floor of the pedicle track should be carefully probed, with particular attention being paid to the first 15–30 mm of the track and the lateral wall. The surgeon always retreats and repeats the palpation when an abnormal feeling is encountered to get sufficient information about the pedicle walls for decisions of adjustment or abortion. If there is any doubt on the final anteroposterior and lateral radiograph, the screw should be withdrawn, re‐probed, or adjusted if need be. For the lumbar spine, it does not need to be explored the junction of the lateral margin of the superior articulating process and the transverse process for the identification of the entry point, which may increase the bleeding and operation time. It is easy to detect this junction with a straight probe; the entry point is actually located on the superior articulating process. Finally, the assistance of a navigation system can improve the accuracy of pedicle placement, and it plays a key role in pedicles with no medullary cavity or an extremely small medullary cavity if there is high risk of failure of the present technique.The present study has some limitations. First, this was a retrospective study with a relatively small sample size. Second, the data reported here were obtained by a single surgeon. Actually, the same technique was performed regularly by four senior surgeons in our department. They discontinued use of the traditional techniques, and no neurovascular complications were reported due to the use of this technique. The present study excluded these perioperative data. Finally, no cadaveric axial trajectory measurements were performed.It is feasible to employ freehand pedicle screw placement using the universal entry point and sagittal and axial trajectory for all subaxial cervical, thoracic and lumbosacral spines placements, and it is also effective with kyphoscoliosis deformities. While other techniques are effective and widely employed, this particular method may be easier to learn and adopt.


Here we present results from a new approach based on native MS of intact Tau expressed in eukaryotic cells (Sf9). The extent of phosphorylation is heterogeneous, up to ∼20 phosphates per molecule distributed over 51 sites. The medium phosphorylated fraction Pm showed overall occupancies of ∼8 Pi (± 5) with a bell-shaped distribution; the highly phosphorylated fraction Ph had 14 Pi (± 6). The distribution of sites was highly asymmetric . All sites were on Ser or Thr residues, but none were on Tyr. Other known posttranslational modifications were near or below our detection limit . These findings suggest that normal cellular Tau shows a remarkably high extent of phosphorylation, whereas other modifications are nearly absent. This implies that abnormal phosphorylations at certain sites may not affect the extent of phosphorylation significantly and do not represent hyperphosphorylation. By implication, the pathological aggregation of Tau is not likely a consequence of high phosphorylation.Abnormal changes of neuronal Tau protein, such as phosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer's disease. Abnormal phosphorylation is thought to precede aggregation and therefore to promote aggregation, but the nature and extent of phosphorylation remain ill-defined. Tau contains ∼85 potential phosphorylation sites, which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, methodological limitations ( MAPT, Uniprot P10636) is a developmentally regulated protein promoting microtubule-based functions like axonal transport in neurons. The properties of Tau can be altered by various posttranslational modifications (PTMs), most conspicuously by phosphorylation . However, these antibody-based methods were not well-suited for the determination of the occupancy of the P-sites and the overall state of phosphorylation.Initial quantitative assessments of Tau-bound phosphate yielded ∼8 Pn AD-Tau . This wa per Tau  and adulr Tau  resulted in enhanced coverage of the Tau sequence with newly identified P-sites , 13.versus aggregation, an experimental system was needed which enabled high phosphorylation of defined Tau isoforms. This was achieved with the expression of full-length human Tau (2N4R) in transfected Sf9 cells, which yields high protein levels (up to 230 µm in cells) and high diversity of P-sites, as judged by antibody reactivity and MALDI-TOF MS analysis . None of the phosphorylated Tau proteins showed an enhanced tendency for aggregation  and up to 23 observed P-sites. Other MS approaches, designed to identify P-sites in Tau from AD brains and cerebrospinal fluid, revealed ∼30 P-sites but not the overall occupancies  because the detection efficiency varies between phosphorylated and nonphosphorylated peptides. This problem is circumvented by native MS of full-length Tau (E. coli bacteria (B). o, expressed in E. coli, bottom), Tau from the fraction Pm-Tau , and highly phosphorylated fraction Ph-Tau . For Po-Tau, the main signals were recorded between m/z 2500 and 3500. By charge state deconvolution, a series of signals corresponding to charges +16 to +13 can be related to a molecular mass of 45,724 Da . In between, broad peaks are visible for both Pm-Tau and Ph-Tau which can be assigned to a series of charge states +16 to +14 (red squares for Pm-Tau and filled red circles for Ph-Tau). Both series are shifted toward higher m/z compared with the corresponding charge states of the control Tau Po. Further peak series in Po-Tau were attributed to a trimeric complex  of the E. coli periplasmic chaperone protein Skp  copurified with Pm-Tau and Ph-Tau from Sf9 cells.Analysis of tryptic peptides by HPLC-MS/MS reveals sites of protein phosphorylation but does not directly monitor the extent of phosphorylation per molecule  and to m/z 2800 to 3500 (left) were analyzed by charge state deconvolution to generate zero-charge mass spectra revealing further differences between these Tau protein forms (right). For Po-Tau from E. coli (bottom), a sharp peak indicates a molecular mass of 45,724, Da which is 126 Da lower than the molecular mass of unmodified full-length Tau . Taking into account the mass accuracy of ±5 Da, this can be explained by a cleavage of the N-terminal methionine , a broader peak in the zero-charge mass spectrum displays a center mass of 46,883 Da and up to six additional peaks on both sides equally spaced by ∼80 Da. The center mass would correspond to an N-terminally acetylated htau40 carrying 14 ± 6 Pi groups.Native MS spectra in the range of  to 3500 , left wein forms , right.  131 Da; . For Ph-f9 cells , top, a m-Tau was less well-resolved and possibly disturbed by a feature at m/z 2750, which is present in the Ph-Tau spectrum at much lower intensity. A charge state deconvolution results in a center mass of 46,408 ± 400 Da, indicating 8 ± 5 Pi (middle row). Of note, although the native MS spectrum of Pm-Tau was less well-defined, all three charge states of +16 to +14 are clearly shifted toward lower m/z values compared with the Ph-Tau spectrum but remained at higher m/z values compared with the corresponding charge states in the control spectrum of P0-Tau  were detected. In contrast, for control P0-Tau, these two peptides were identified in their nonmodified, nonacetylated N-terminal peptide form. We therefore deduced from the protein sequence (residues 2–441) average masses of 45,718.2 Da for the nonN-terminally acetylated E. coli Tau (P0) and 45,760.2 Da for the N-terminally acetylated form of htau40 (Pm and Ph) in the absence of further modifications.For protein identification and detection of PTMs, the protein samples were subjected to reduction of cysteine residues, alkylation of free thiol groups, and digestion with trypsin, followed by LC–MS/MS analysis. The identified tryptic peptides covered 95% of the Tau sequence and allowed us to detect sites of posttranslational modifications of the Tau proteins. We therefore analyzed our LC–MS/MS data for the following amino acid modifications: acetylation of the protein N terminus, phosphorylation of serine, threonine, or tyrosine, acetylation of lysine, and ubiquitination of lysine. For purified PE. coli P0-Tau, as expected. The P-site analysis of Tau from Sf9 cells confirmed the majority of P-sites found in previous studies . Furthermore, the analysis revealed 10 additional P-sites . No phospho-tyrosines were detected  per Tau molecule, indicating a maximal value of 20 Pi . The heterogenous P-site distribution over the entire length of the Tau chain is generally in line with previous findings . These sites are consistent with the subset of P-sites in Sf9 cells with the highest occupancy (>50%)  purifie0 from E. coli, Pm and Ph-Tau from Sf9 cells) were purified by making use of the heat stability of Tau protein whereby nearly all other proteins are denatured and precipitated, whereas Tau remains in solution because of its hydrophilic nature. The detected small amounts of cellular Tau-binding proteins also passed through the boiling step and were still present in gel filtration column fractions containing the separated Tau proteins. One possibility to explain the presence of these proteins is their high affinity for Tau protein. Thus, the experiments yielded a list of Tau-interacting proteins that might become interesting for analyzing possible Tau interactions in other cell types (see 0-Tau from E. coli and m-Tau and Ph-Tau from Sf9 cells). A substantial part of identified proteins are known for their roles in interacting with RNA or DNA and with chaperones (see “Discussion”).The high sensitivity of LC–MS/MS revealed the presence of tryptic peptides originating from traces of other proteins accompanying Tau through the purification procedure. The recombinant Tau proteins   and the pathological state of Tau . This can be achieved by combining native MS with LC–MS/MS, as described here. We will restrict our discussion to some issues that have received attention in the Tau field during recent years.In the past, the main tools for identifying phosphorylation sites of proteins were based on tryptic cleavage, combined either with TLC of quencing , or MS o-TOF MS) . For the-TOF MS) , 41, 42.-TOF MS) , 43. HowE. coli. Fraction P12 was derived from Sf9 cells after expression of full-length Tau (2N4R), whose mass was consistent with additional ∼12 Pi. Fraction P20 was also from Sf9 cells, with phosphatase inhibitor okadaic acid added during preparation, consistent with additional ∼20 Pi.In previous work , we desci, and PP-Tau , with an overall occupancy of 8 Pi. In addition, the method revealed the individual occupancies of 17 sites, including those of the major Tau antibodies (The same types of fractions were studied by Steen and co-workers  using thtibodies ).Figure 0, Pm, and Ph for zero, medium, and high state of phosphorylation . The common control value is P0-Tau from E. coli (C), which remains without modification, apart from N-terminal processing by removal of Met1. The medium state, reflecting normal kinase/PPase activity in cells (B), is now resolved into a series of peaks centered around a maximum at +8 Pi per molecule, with ∼4 sub-maxima resolved on either side (range +4 to +13 Pi). The high state (A), reflecting normal kinase but inhibited PPase activity, appears as a series of peaks centered around a maximum at +14 Pi, with ∼6 sub-maxima resolved on either side (range +6 to +20 Pi).The apparent discrepancies between the overall occupancies observed in the earlier MALDI-TOF MS and FLEXITau experiments are now superseded by the results from native MS. The fractions are now denoted as P E. coli C, which in cells B, is nowgh state A, refleci for individual fractions and thus +/−5 Pi for mass differences. On the other hand, the FLEXITau method achieved accurate occupancies of individual P-sites. However, sequence coverage was limited (75%) so that the degree of Tau phosphorylation was underestimated  with specific functional P-sites one would expect well-defined stoichiometries of phosphorylation (by specific kinases) whereby assembly or activity are controlled; examples are ribosomes or proteasomes  reveals a high state of phosphorylation. In this scheme, the high phosphorylation of AD-Tau (despite a long postmortem delay) is explained by Tau aggregation which protects them against PPases. Similarly, lowering the temperature (as in anesthesia) decreases PPase activity, with a lesser effect on kinases, causing the impression of hyperphosphorylation , RNAs, or membrane surfaces and the folding of Tau from a “paperclip” to other conformations . Consistent with this, we find that none of the phosphorylation states led to a pronounced increase in Tau aggregation  even thormations . In partrmations .e.g. pT181 by antibody AT270 or pT231 by antibody AT180 , Ser-21205 (AT8 ), and Se205 (AT8 )). Theseversus potential sites was almost twice as large, 41/58 = 71%. Because all Tau domains are largely unstructured and mobile in solution . Examples are cleavage by calpain or caspases  phosphorylation predisposes Tau for aggregation and set off an extended search for kinases of Tau , 59. Howcaspases , 62 or ncaspases . In suppcaspases , 65.32P, followed by phosphopeptide mapping. There was a striking overlap of P-sites with the current results, especially involving the clusters of P-sites listed above. Phosphorylation became enhanced during mitosis, corresponding to the activation of the cell cycle kinase cdc2, a proline-directed kinase. Note that this phosphorylation occurred during a physiological process, without any sign of pathology or Tau aggregation, indicating that the pattern of phosphorylation serves a normal biological role. The same conclusion can be drawn from the analysis of hibernating animals where retraction and regrowth of neuronal networks are accompanied by similar changes in Tau phosphorylation  can be phosphorylated by common Tyr kinases in the brain, ype PXXP , 68, 69.e.g. Lys-174 in Tau), and has been implicated in neuronal damage of aging cells , LC–MS/MS and native MS revealed traces of numerous proteins which remained associated with Tau through the purification , whereas other well-known modifications are present only in minute proportions . The Ser/Thr phosphorylation has a remarkably asymmetric distribution, whereby a low proportion of residues is modified in the acidic N-terminal domain of Tau, in contrast to a high proportion in the basic middle to C-terminal domains, including all of the sites detected by antibodies against Alzheimer's phospho-Tau. This indicates that a substantial degree of phosphorylation (achieved by multiple kinases) is presumably a property of normal soluble Tau in eukaryotic cells, reflecting a phosphorylation tonus dominated by net activity of kinases over phosphatases. By implication, this would be expected to affect many other proteins as well, notably natively unfolded proteins accessible to diverse kinases  in a ratio of 1 g of Sf9 pellet to 10 ml of lysis buffer. This procedure yielded “Pm-Tau” . Next, after centrifugation, the cells were resuspended in lysis buffer and boiled in a water bath at 100 °C for 10 min. By this treatment nearly all proteins were denatured and precipitated, except for Tau, which stays soluble. The cell debris was removed by centrifuging the lysate for 15 min at 16,000 × g. The supernatant containing soluble Tau protein was concentrated in Millipore Amicon Ultra-4 centrifugal filter units (molecular mass cutoff of 3 kDa). This procedure yielded “Ph-Tau” . For further experiments, the fractions containing Tau protein were pooled and concentrated again in Amicon Ultra-4 centrifugal filter units. Finally, the concentrated protein was rebuffered into 200 mm ammonium acetate, pH 7.6.Tau protein  expressed in ribed in . Expressr et al.  or P-Tau . To inc P20-Tau  or PP-Tam ammonium acetate, pH 7.6 were analyzed by nanoflow electrospray ionization MS using a Synapt HDMS (Waters and MS Vision) equipped with a 32,000 m/z range quadrupole. 3 µl of sample were introduced with an in-house manufactured gold-coated capillary needle . The capillary voltage was set to 1.3 kV and the cone voltage to 180 V. The trap cell was filled with argon gas at a flow rate of 1 ml/min, and collisional activation was performed by applying acceleration voltages to the trap cell. The transfer cell was kept at a pressure of about 20% of that in the trap cell and a low acceleration voltage of 5 V. Spectra were calibrated externally using cesium iodide. Charge state spectra were deconvoluted by the program UniDec version 2.7.3 (Purified proteins in 200 mon 2.7.3 .m Tris(2-carboxyethyl)phosphine and alkylation of free thiol groups with 50 mm iodoacetamide/50 mm NH4HCO3 and digested with trypsin overnight at 37 °C. Peptide mixtures were analyzed by nano-HPLC–electrospray ionization–MS/MS using a Q Exactive instrument directly coupled to an UltiMate 3000 RSLCnano HPLC system  equipped with a Nanospray Flex ion source with DirectJunction (Thermo Fisher Scientific). Peptides were washed and preconcentrated on PepMapTM C18 precolumns  and separated using AcclaimPepMapTM RSLC columns  at a flow rate of 250 nl/min and 40–43 °C. For peptide elution, binary solvent systems were used consisting of 0.1% (v/v) formic acid (solvent A) and 0.1% (v/v) formic acid/86% (v/v) acetonitrile (solvent B) with a gradient of 4–42% solvent B in 50 min, 42–95% in 5 min, and 5 min at 95%. The Q Exactive was operated with the following parameters: MS survey scans ranging from m/z 375–1,700 at a resolution of 70,000 (at m/z 200), an automatic gain control of 3 × 106 ions, and a maximum injection time of 60 ms. A TOP12 method was used for higher energy collisional dissociation of precursor peptides (z ≥ 2) in the orbitrap applying a normalized collision energy of 28%, an automatic gain control of 1 × 105 ions, and maximum injection time of 120 ms. The dynamic exclusion time for previously fragmented precursors was set to 45 s.In-solution tryptic digestion of proteins was performed as described previously . In brieE. coli and Spodoptera frugiperda  allowing a maximum of four missed cleavages of trypsin and a mass tolerance of 4.5 ppm for precursor and 20 ppm for fragment ions. Methionine oxidation, acetylation of lysine and protein N terminus, and phosphorylation at serine, threonine, and tyrosine residues were specified as variable modifications, and carbamidomethylation of cysteine was specified as a fixed modification. Additional searches were performed with an extended range of variable modifications including ubiquitination (diglycine at lysine) and O-GlcNAc modification of serine and threonine. However, these modifications were observed only sporadically and in low intensity and borderline significance and were therefore excluded from the final search. The lists of both peptide and protein identifications were filtered applying a threshold for the false discovery rate of <0.01.Data were analyzed using Maxquant software version 1.5.5.1  searchinPXD020985.MS raw data and result files have been deposited to the ProteomeXchange Consortium via the PRIDE repository  and are


Pedicle screw insertion has been known to have several complications even in the most skilled surgical hands. However, injury to the thoracic aorta during pedicle screw insertion is rare, delayed presentation secondary to pseudoaneurysm is even rarer, the pseudoaneurysm formation caused by a series of malpositioned pedicle screws has perhaps not been reported so far.In this paper, we report here a case in which inadvertent injury to the thoracic aorta resulted in pseudoaneurysm, its manifestation was initially vague, resulting in a delayed diagnosis. Delayed aortic pseudoaneurysm or injury can be asymptomatic for a long time. Patients with renewed or continued back pain should alert orthopaedic surgeons regarding the possibility of pseudoaneurysms, regardless of the period that has elapsed after pedicle screw implantation. Delayed aortic pseudoaneurysm or injury can be asymptomatic for a long time. Patients with renewed or continued back pain should alert orthopaedic surgeons regarding the possibility of pseudoaneurysms, regardless of the period that has elapsed after pedicle screw implantation. Pedicle screw insertion is one of the most commonly performed spinal surgical procedures. Given the fact that the instrumentation results in stabilization of all three spinal columns, its use is considered the gold standard while performing fixation for various spinal conditions. Iatrogenic pseudoaneurysm is a rare and dangerous complication during pedicle screw instrumentation surgeriesWe present a case of aortic pseudoaneurysm formation after pedicle screw instrumentation and reviewed the available literature about such injuries. Key learning points, management techniques, and avoidance are discussed.11 to L2 by a computed tomography (CT) examination. The patient complained of severe back pain. There was no neural deficit. The patient underwent thoracolumbar pedicle screw implant surgery using ‘freehand technique’ under fluoroscopic guidance in a local hospital. Eight pedicle screws with contoured rods were placed spanning T11 to L2 after the correction of deformities. The patient tolerated the procedure well and developed no neurodeficits postoperatively. Radiological examination revealed a partial correction of the deformity.A 21‐year‐old man presented to the emergency room (ER) complaining of stomach and serious back pain 2 months after spinal surgical intervention. Two months before his visit, he fell from a 10 m tall building. He was confirmed as suffering fracture and dislocation from TThe patient was presented with a recurrence of pain at the operated site 10 days postoperatively. The pain was a dull ache, non‐radiating, and showed a progressive increase in intensity. This was accompanied by constitutional symptoms like poor appetite and a general feeling of being unwell. In view of the non‐specific nature of the complaints, no further investigation was carried out and the patient was advised to attend regular follow‐ups.11, T12, and L1 levels, which was a disaster. The left side T12 and L1 malpositioned screws had exited the lateral pedicle cortex and were clearly abutting the posteromedial aspect of the descending thoracic aorta (Fig. 11 and both sides of T12 screws also had exited the lateral pedicle cortex and were abutting the wall of the pseudoaneurysm. The left side screw in L2 perforated the lateral pedicle cortex but did not abut the aorta (Fig. in situ so as to avoid any chance of re‐injury. The patient endured the surgery well and had a good recovery in his 4‐month follow‐up examination.In our hospital, enhanced CT and X‐rays were performed after thorough clinical evaluation. The CT scan revealed the malposition of a pedicle screw on both sides at the TAcute aortic injuries caused by pedicle screw implantation have been reported previouslyDelayed aortic injuries have a different clinical presentation from acute ones Table . In acutThe aorta extends along left side of the thoracic and lumbar vertebrae. Quite expectedly, in this case and other studies Table , aortic in vivo bovine study, Faro et al. evaluated histological and biomechanical changes in thoracic aorta wall tissue resulting from intentional severe vessel impingement. During the 12‐month follow‐up period, the authors demonstrated that marked vessel impingement resulted in acute and chronic histopathological changes that significantly compromised vessel wall integrity on biomechanical testingDelayed aortic injuries could present over a wide spectrum and include perforation, pseudoaneurysm formation, and even complete ruptures of arterial walls, which could be fatalet al. reported a case with malpositioned pedicle screws 14 months after surgery. The surgical team re‐intervened and removed the malpositioned screw from the T6 vertebra. Three weeks later, the patient suffered fever and hemorrhagic shock and was diagnosed as having a pseudoaneurysm and bleeding from the aorta. Subsequently, the patient had to undergo re‐exploration anteriorly with vascular surgeryFor malpositioned screws with obvious injured aortic walls, revision surgery is needed. But the potential risk of complete rupture of the aorta should be considered and appropriate strategizing is required. Choi Controversy exists in the accidental finding of asymptomatic patients. Sometimes, screws have slight contact with the aorta. The integrity of the aortic wall may not have been compromised in such cases. When such screws are seen in postoperative imaging in an asymptomatic patient, the surgeon must decide whether it is riskier to revise the screw or to not do any intervention and merely observe it.Foxx retrospectively reviewed 182 consecutive patients, 680 pedicle screws undergoing spinal implants, and found 33 screws were in contact with a major vessel on routine postoperative imaging. Foxx had a mean follow‐up of 44 months and revealed no detectable vessel abnormality in subsequent follow‐ups. He recommends consideration of the overall life expectancy of the patient before contemplating any interventionet al. suggested that it is not mandatory to remove all pedicle screws found to be encroaching on major vascular structures. In cases in which the screw is not revised, regular radiographical follow‐up is required to detect the formation of a delayed pseudoaneurysm or another secondary vascular injuryet al. followed up a patient with malpositioned pedicle screws for 6 months. Unfortunately, the screw penetrated the thoracic aortic wall, and a vascular surgery had to be performedSome surgeons removed only the screws that show higher potential riskDelayed aortic pseudoaneurysm or injury can be asymptomatic for a long time. Patients with renewed or continued back pain should alert orthopedic surgeons regarding the possibility of pseudoaneurysms, regardless of the period that has elapsed after pedicle screw implantation.


The Newborn Screening Technical assistance and Evaluation Program (NewSTEPs) conducts non-regulatory site reviews of state newborn screening programs in the US with the goal of providing comprehensive reports and recommendations to support quality improvements within the system. A detailed coding and qualitative analysis of data extracted from reports of seven programs visited between 2012 and 2017, of thirteen pre-site visit surveys completed by state newborn screening programs, and of information from interviews conducted with three site review experts revealed four common themes that exist across states within the national newborn screening system. These themes include opportunities to implement improvements in: (1) communications inside and outside of the state newborn screening program, (2) education, (3) information technology, and (4) operations. The cross-cutting recommendations provided by NewSTEPs within the comprehensive site review reports may prove valuable for all state programs to consider and to incorporate as quality improvement measures in the absence of a full site review. The analysis of the site review process and recommendations identified important opportunities for improvement, many of which were previously unknown to be common across programs, and also provided affirmation of known challenges. Newborn screening (NBS) is a public health program designed to decrease or eliminate newborn morbidity and mortality from some genetic disorders through pre-symptomatic identification ,2. In thThe Newborn Screening Technical assistance and Evaluation Program (NewSTEPs) provides a comprehensive system of resources and technical assistance for NBS programs in the US. NewSTEPs is a program of the Association of Public Health Laboratories (APHL) and is funded by the US Department of Health and Human Services (HHS), Health Resources and Services Administration (HRSA), and the Maternal and Child Health Bureau (MCHB) . NewSTEPUS state or territorial NBS programs requested a site review from NewSTEPs through a formal process, including a letter of request from an authorized official within the state. NewSTEPs assembled site review teams (SRT) comprised of peer experts from across the nation, spanning areas of expertise within the NBS system including laboratory, follow-up, and clinical specialist experts. Additional site review team members with expertise in information systems were added when needed. To prepare for the site review the SRT: (a) reviewed the information provided by the states online  includinRecognizing that NBS is a complex system requiring many critical partners, once on-site, the SRT meets with public health leadership, state NBS leadership, NBS staff, birthing center staff from at least two birthing facilities, and any clinical specialists who work with the program who are invited by the state program to take part in the site review.  The visiAt the conclusion of the on-site portion, the SRT meets with key personnel to highlight key observations and provide an overview of the next steps. Following the visit, the NewSTEPs SRT provides a comprehensive written report that summarizes findings from the visit including general perceptions, an assessment of the functions of each component of the screening program , needs identified by the SRT, and a list of recommendations for future program changes and growth. NBS programs are encouraged to share the report with programmatic and state leadership to identify opportunities for future resource allocation and quality improvement activities. Approximately 6 months following the site review, NewSTEPs requested that the NBS program complete a follow-up survey online  to identWe reviewed seven full site review reports, thirteen pre-site review documents completed by laboratory and follow-up programs in the seven NBS programs who had site reviews, interviews with three SRT members who had been to multiple states, three completed post-site review surveys, and one poster presented on the results of the site review on two states . Two autThe primary coder reviewed and coded all seven site review reports, of which five were final reports, and two were draft forms. A second coder reviewed four of the seven site review reports early in the process to standardize coding. The two reviewers discussed any coding discrepancies and addressed them through conversation. After analyzing the seven site review reports, the second coder coded all thirteen pre-site review forms to identify any needs not captured in the final reports. No new codes were added. The primary coder then interviewed and coded the responses of three experts—one laboratorian, one follow-up specialist and one clinician—who had been on at least two site reviews. No new codes were added. All reports were re-visited after the coding scheme was finalized. Themes that were mentioned in at least four of the seven site reviews are included in the findings. Finally, the degree of overlap between pre-site visit reports and the site review was calculated by comparing the areas where the SRT noted the most needs to the areas of need noted by the state program staff. NewSTEPs conducted seven site-reviews visited between 2012 and 2017: two were large states , two were medium states , and three were small states . The SRT was on site for 3–5 days per state. The sections of the site review manual were used to organize needs and themes for each program. These sections became the categories, and within each category are the types of needs identified by the SRT. Four main themes emerged that cut across the categories of review: communication inside and outside the state NBS program ; education ; information technology (identified at 6 sites and by 2 experts); and operations . Findings from the site reviews were qualitatively analyzed, and themes are presented. Programmatic changes reported by state NBS programs that resulted from the site review are listed at the end of each theme. Site review reports for all seven programs highlighted the need to improve communication between the laboratory and follow-up staff . Four stThe lack of communication between Laboratory Information Management Systems (LIMS) and short-term follow-up IT systems noted in both the pre-site visit and the site review report causes delays in result reporting. Follow-up staff wait for a report from the laboratory on out-of-range results, and in some cases, need additional information to call out the results to the provider. For example, one reviewer wrote, “Although it takes less than three days to complete all testing, there are four to five days of delays to report out results after all testing are completed. These delays, are caused by limitations on staff and lack of a centralized LIMS system.” The lack of communication between the two information systems also makes it difficult for the laboratory to record the final diagnosis in the laboratory system. The laboratory and follow-up teams began meeting more regularly. One state reported the two teams meet two times a month while another state reported that the teams meet weekly but check in daily for tactical issues.The laboratory supervisor working on Saturday began notifying the follow-up specialist if critical results are anticipated.To address the LIMS issues, one state team reported they are considering options for new LIMS/follow-up program application with the goal of increasing transparency between the two areas and increasing timeliness.In post site-review surveys, states reported the following activities were implemented to improve communication:While states identified internal communication needs, they did not identify communication problems with clinicians or birthing centers on their pre-site review forms. The SRT, however, noted communication problems between the state NBS program and birthing centers and specialists as a top need in the site review reports. The SRT met with at least two birthing centers and any specialists invited by the state NBS program to be part of the site review. Birthing facility staff in four states expressed an interest in receiving information about the number of newborns from their facility identified with a condition through NBS . In one Clinical specialists in five states who met with the SRT expressed their desire to communicate more with the NBS program and be part of the quality assurance process . SpecialWhile a need for better education for birthing providers and facilities only came up in one pre-site review form, it was a strong theme in all seven site review reports . The mosIn addition to the need for up-to-date print materials and training videos, hospital staff in six sites asked for more NBS training. “Staff is very interested in received educational interventions to learn more about NBS and in quality improvement projects around specimen quality, timeliness, and outcomes” (site review report). NBS state policy revisions combined with new birthing providers and facility personnel results in hospital staff without current information. Birthing center staff requested not just more training, but also more consistent training for their staff . For example, many birthing center staff could not recall the last formal training they had received by the state NBS program while others indicated it had been a few years. “They [birthing facility] have not had an in-service or outreach from the state in the past 2–3 years and think it would be helpful to have a visit.” Training topics that came up in more than one state are as follows: (a) when to talk to parents about NBS, (b) how to document parent refusal, (c) that parents could, in fact, refuse NBS, (d) best practices for blood spot specimen collection and drying, (e) when the courier picks up from their facility, and (f) how to follow the critical congenital heart disease (CCHD) screening protocol. Reviewers in two states also noted there was a need to educate birthing providers who provide point-of-care screening why it was important that they report the screen results to the NBS program.Formed a newborn screen advisory committee that had not previously been in place.Published an ongoing newsletter to communicate more regularly with birthing centers.Created and started sending birthing center report cards so birthing centers can track their timeliness for specimen collection, their percentage of unsatisfactory specimens, etc.Began training across the state/region on proper sample collection.Worked with specimen submitters to improve the transit time of their specimens to the state laboratory.Explored hiring an educator who can create and provide hospitals with brochures, collect and share patient stories as part of training staff who will collect the initial NBS, and/or sending feedback to a birthing center when an infant born in their center was identified as having positive newborn screen.Worked with a metabolic specialist to create a rationale document for early collection of NBS specimens for the NICU population.Began a program where birthing facility staff and other submitters tour the state NBS laboratory and the follow-up program so these submitters can see the “big picture”.In post site-review surveys, states reported the following activities were implemented to improve communication between the program with specialists and birthing centers:Information systems and IT needs were identified in the ethics, laboratory system, short-term follow-up, and IT section of the site review reports. In addition to laboratory and follow-up IT systems that could communicate, reviewers identified additional IT needs in six of the seven states . Two sitThe site reviewers identified the need for a better system to record if all newborns received complete screens in 5 of 7 states, including tracking parental refusals, locating missing specimens and documenting infants lost to follow-up. Site-reviewers identified this need, while the pre-site review reports did not. Site reviewers noted, “There is no formal tracking for missing specimens since there is not a way to determine if one is missing,” and “Refusals are documented on the card, but without a known denominator, it is hard to determine how many are refusing.”Three out of seven site review reports identified the need to create a linkage between NBS systems and the state’s electronic birth records to calculate a reliable denominator for quality assurance analysis. Access to the systems for all weekend/holiday/after house program staff via a virtual private network (VPN) in case of an emergency was another need noted in three of the seven states. Additionally, reviewers noted that short-term follow-up programs need to document in an electronic information system rather than relying on a paper system. Reviewers in two state site reviews noted that the state’s reliance on paper documentation slowed down the process and could make response during an emergency difficult if the team had to move off-site. Hired two new LIMS staff to support current LIMS administrator.Upgraded LIMS from outdated to current.Evaluated new LIMS systems to upgrade or select new vendor to meet program’s needs.Developed a provider portal which provides access to medical professionals who could access laboratory reports for their patients.Joined the APHL HIT user group based on the LIMS they are using.Implemented a module that will allow the program to match NBS records with vital records.In post site-review surveys, states reported the following activities were implemented to address IT needs identified in the site review report:The need to strengthen how the state NBS programs are conducting screening and providing follow-up care, as well as the state NBS program resource needs was the final theme identified.Programs completing the pre-site review forms were less likely to identify procedural and regulatory needs than the site reviewers. Specifically, all site review reports identified a need to update policies and rules, while none of the pre-site review forms identified this. A financial assessment of the program, reassessing fees and billings was noted in five site review reports but in only one pre-site review form. Similarly, pre-site review forms did not highlight blood spot retention and storage policies as a need, but five site review reports noted this need. Site review teams noted in a few states there was no continuity of operations plan (COOP) and in several other sites the plan existed, but the NBS program had not recently practiced the plan. The state NBS team noted the low birth weight/prematurity algorithm needed review, including identifying procedures for newborns receiving total parenteral nutrition (TPN). Reviewers noted that programs might miss affected newborns due to collections occurring too early or otherwise in conflict with existing best practices. All seven site review reports noted that the state program needs to annually review their standard operating procedures (SOPs), regulations, and rules . “This yRevised newborn screening regulations.Changed their state policy so that “protein feed for at least 24 hours” is no longer required.Updated their emergency preparedness continuity of operations plan (COOP)Reviewed forms, policies, and processes being used by other states to influence a specimen destruction plan.Had data destruction conversation with LIMS vendor.Developed forms that parents can use to request specimens be destroyed after testing.Explored implementing a newborn screening fee and a merged panel.Worked with state legislature to re-introduce legislation to get the fee changed.Requested and were granted a fee increase.Sought a procurement exemption from the Governor. This led to the state program being able to secure a contract for equipment, reagents, and services for NBS testing in two months instead of two years.In post site-review surveys, states reported the following activities were implemented to improve program standard operating procedures, rules and regulations: Reviewers identified the need for programs to strengthen their sample testing algorithms, cutoffs, and testing processes in six out of seven states . RevieweThe other need identified within the laboratory is to have a process in place to ensure efficient collection of repeat specimens on infants with an unsatisfactory specimen or out-of-range result. In one report, reviewers noted that it could take the state program up to 14 days from the request for a repeat specimen to receipt of that specimen by the lab. Further, reviewers noted that some programs could report critical results more expeditiously with timelier specimen processing and result reviewing. Finally, a challenge identified universally is the hiring, training, and retention of qualified laboratory personnel. Reviewers noted that, “Both the lab and follow-up services are understaffed and have had a hard time retaining strong employees because of the inability of the units to reallocate staff to higher level positions and provide well-deserved raises.” They also noted, “the process to add new positions/human resources within the NBS laboratory is lengthy and requires approval from the Commissioner.” Had senior level staff provide confirmation on qualifying criteria for specimen rejections.Worked with the LIMS provider to configure “test specific unsatisfactory specimen” which will be applied only at the disorder level and not specimen level.Evaluated condition specific cut-offs and conducted new statistical analysis to monitor cut-off changes for several conditions including a plan for surveillance of cut-off variation for state’s presumed positives population on a yearly basis.Hired a Quality Assurance Officer who is located in laboratory administration to ensure transparency in the quality management system.Added a refusal section to the NBS module of the birth certificate registry so hospitals can report refusals to the NBS program.In post site-review surveys, states reported the following activities were implemented to address dried blood spot screening algorithms, cut-offs, and processes:Needs identified in the follow-up program parallel those identified in the laboratory in identifying newborns who were not screened and those who need a repeat screen. Some reviewers noted that programs do not complete active follow-up for newborns with equivocal or unsatisfactory results and that some programs need to develop formal tracking for missing specimens. Further, reviewers noted that there should be a better distinction between the repeat specimen process and the cases lost to follow-up. Finally, site reviewers noted that some follow-up programs could benefit from identifying procedures that are disorder specific following an abnormal result notification. The site reviewers noted in four site review reports that the program needed more follow-up staff, while only one state’s pre-site review forms identified this need. Specific needs included staff who were able to efficiently utilize the LIMS systems to extract information needed for follow-up staff. Further, site reviewers suggested that programs needed specific Quality Assurance personnel to help with identifying areas for improvement within the systems. Similar to laboratory staffing, reviewers noted that retention of follow-up staff was difficult because there was a lack of opportunities for advancement in the NBS programs. Hired new staff.Worked on a follow-up algorithm for unsatisfactory specimens.Created NBS follow-up process maps to outline procedures for initial notification of all abnormal results.Follow-up staff worked with human resources to explore the option of higher title/rank for follow-up staff to increase pay and provide opportunities for promotion.Hired a Quality Improvement specialist.Initiated a quality improvement project to promote voluntary enrollment in Early Intervention after the diagnosis of a hearing loss.Built a module so that hospitals enter CCHD data within the birth certificate registry.In post site-review surveys, one state reported that the site review recommendations played an important role in helping them acquire needed resources. “Lack of follow-up on [for] babies more than 30 days old has provided justification to hire one additional FTE in follow-up for current workload.” . Other activities states reported implementing to improve the follow-up process included: All five of the states who provided input after the site review were able to share many of the activities they have started, and in some cases completed, as a result of the site reviews. Of these five states, two provided some insight into the barriers they face in addressing many of the needs identified by the SRT. Time was identified as a barrier. States needed time to hire and on-board staff to tackle recommendations made by the SRT, and time to procure a contract to resources such as new LIMS. One state also reported they had to delay addressing SRT recommendations because they needed to make changes to their LIMS for at least one new disorder.Another barrier identified by the two states was the rules and/or legislation process. One state mentioned that in order to meet a recommendation, the state would need an administrative rule change to provide tracking and surveillance for point-of-care testing. This same state indicated that the state legislature did not allow the use of dried bloodspots for research so they were unable to meet a SRT recommendation. The time delays due to a slow legislation process was also mentioned as a barrier. One program said they were waiting for a bill to be introduced in state legislation.A final barrier was that the recommendations were “resource intensive”. One state felt that availability of grant funds to purchase needed resources, access to tools that can be used to address the SRT’s resource heavy recommended strategies, and/or connections to other states of similar size/capacity who could share what they have done to meet these needs would help overcome this barrier. This last point is supported by another state who said “the connections provided during the site visit were useful and we have been making contact with other states as well who have been more than willing to share their information” . The goal of this report was to provide an overview of needs and solutions identified in a subset of the NBS programs so that other programs may benefit. When site reviewers identified needs that were not identified by the state program, these insights may help other programs to find challenges that are less readily apparent in a self-assessment. While there is an overlap between the state NBS staff identified needs and those identified by the SRT, there were additional needs identified. “[The site review team] gave very good suggestions on areas where we can make improvements to our program, including areas we hadn’t considered or were even aware were things that other states are doing” . The three areas with the most needs identified by the SRT and the state NBS program prior to the site review include the laboratory system , followed by short-term follow-up , and organizational structure . Birth facility needs ranked second for the most needs identified in a site review report (49 times), but this need was only mentioned once in the pre-site visit form suggesting that sites may be unaware of this need. Agreement between the site review report and pre-site visit form may be by design. The SRT reads the pre-site review forms in preparation for the visit. Further, the pre-site visit form can shape who is part of the SRT. This flexible approach fits within the site review typology posited by Haynes et al.  in which(I)Systematic communication within the program: “there is a need for regular  meetings with laboratory and follow-up staff.” During regularly scheduled meetings, the program could discuss programmatic roles, decisions, processes, and workflows. The programs could also discuss abnormal results and false positive rates. Both laboratory and follow-up staff should take a comprehensive tour of each other’s units.(II)Improving communication between the information systems used by laboratory and follow-up staff: Access between laboratory and follow-up teams’ LIMS systems can support data exchanges which will lead to quicker reporting of screening results and quality assurance efforts.(III)Communication with clinical specialists: Invite clinical specialists to meet with the state program and provide input. The input may include how the program sets cut-offs and the impact NBS has on their clinic’s work.(IV)Communication and education with stakeholders external to the state NBS program: (1) Notify appropriate people at the birthing facility of out-of-range results and unsatisfactory specimens; (2) Communicate with the entire birthing facility staff involved in any part of NBS, those involved in specimen collection through specimen transport; (3) Notify birthing centers and clinicians about new educational materials and simplify the process to access the new materials; and (4) Educate birthing providers who provide point-of-care screening about the importance of reporting results to the NBS program. (V)Information technology: (1) Work with LIMS vendors or other programs using the same platforms to try to maximize the power of the LIMS; (2) Consider an upgrade to the LIMS, if warranted; (3) Track refusals electronically; (4) Connect NBS results with state electronic birth records; and (5) Grant virtual private network (VPN) access to all weekend/holiday/after-hours program staff in case of an emergency. (VI)Standard operating procedures: State NBS programs should review their standard operation procedures (SOPs), regulations, and rules annually. Specifically: (1) Develop processes and procedures to identify newborns not screened, track parent refusals, track repeat screens; and (2) develop a process to ensure efficient collection of repeat specimens following an unsatisfactory initial specimen. (VII)Continuity of operations plan (COOP): Programs without a COOP should talk to other states and NewSTEPs staff to help them identify the components of a COOP. Once the plan is in place, state programs need a procedure to test the plan annually. The whole state NBS system, including birthing facilities, should be part of the testing.The systematic review of the site reviews revealed several common themes. The site review teams proposed several recommendations to help address the identified needs: NBS program site reviews can provide affirmation of the known challenges and needs while uncovering factors that were previously unknown to the program. Although the purpose of site reviews is to provide feedback to the state NBS programs, site review team members also learned from the state NBS programs they reviewed. Through the site review process, reviewers identify needs in their own programs as well as identify practices to strengthen their programs. NewSTEPs.org. The assimilation of lessons learned from site reviews provides other NBS programs insight into systematic challenges within the US. NBS programs who have not had a site review may recognize the needs identified and may find that the recommendations could strengthen their own programs. States can also conduct internal reviews using the NewSTEPs site review manual online  or requeNewSTEPs continually assesses the site review process with each site review team to ensure the reviews are as beneficial and efficient as possible. The reviews will continue to evolve to provide the expertise and technical assistance identified by the site review report, either directly or through connections with other experts that can help them address their needs.