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Assess interrater agreement of endoscopic assessment of velopharyngeal (VP) function before and after viewing the video instruction tool (VIT). We hypothesized improvement in interrater agreement using the Golding-Kushner scale (GKS) after viewing the VIT.Prospective study.Multi-institutional.Sixteen fellowship-trained pediatric otolaryngologists who treat velopharyngeal insufficiency (VPI) rated 50 video segments using the GKS before and after watching the VIT. Raters assessed gap size percentage and lateral pharyngeal wall (LPW), soft palate (SP), and posterior pharyngeal wall (PPW) movement. Intraclass correlation coefficient was estimated for these continuous measures. Raters also indicated the presence of a palatal notch, Passavant's ridge, and aberrant pulsations (categorical variables). Fleiss κ coefficient was used for categorical variables. Wilcoxon signed-rank test was performed on the difference between the pre/post individual video ratings.Reliability improved for all continuous variables after watching the instructional video. The improvement was significant for PPW (0.22-0.30, P.001), SP (left: 0.63-0.68, P.001 and right: 0.64-0.68, P = .001), and LPW (left: 0.49-0.54, P = .01 and right: 0.49-0.54, P = .09) but not significant for gap size (0.65-0.69, P = .36). Among categorical variables, agreement on Passavant's ridge significantly improved (0.30-0.36, P = .03).Exposure to a video instruction tool improves interrater agreement of endoscopic assessment of VP function. Significant improvement was observed in our primary end points, specifically posterior pharyngeal wall movement, soft palate movement, and lateral pharyngeal wall movement. There was less impact of the VIT on the interrater agreement of the categorical variables, palatal notch, Passavant's ridge, and aberrant pulsations.

A partir de l’experience au CHU de Quebec, une reflexion sur la sensibilisation et la formation des equipes soignantes a depister la detresse est realisee. Le contexte canadien et quebecois ainsi que l’initiative au CHU de Quebec permettent dans un premier temps d’introduire le depistage comme une strategie et une procedure permettant de favoriser l’acces a des soins de soutien centres sur les besoins de la personne atteinte de cancer. Comme l’implantation de ces nouveaux modeles de soins centres sur la personne se veut systematique, cela necessite a la fois des changements organisationnels ainsi que des changements de comportements des cliniciens. Ces differents enjeux doivent etre pris en consideration dans les programmes de formation. La formation des equipes soignantes et l’integration du depistage a la pratique sont donc discutees a la fois comme un changement organisationnel et a la fois comme un changement de pratique clinique. Le changement organisationnel est decrit comme une serie d’etapes incluant notamment la preparation et la consolidation. Le changement de pratiques implique une modification des comportements des cliniciens et comporte plusieurs obstacles potentiels. Le modele de soins centres sur la personne implique par ailleurs de prendre en compte les preferences de la personne soignee tout en considerant les valeurs de l’ensemble des acteurs impliques dans l’organisation. De plus, ce type de modele necessite des competences a la collaboration interprofessionnelle. La formation des equipes soignantes en matiere de depistage de la detresse va bien au-dela de l’habilitation clinique.

GSK2336805 is an orally bioavailable hepatitis C virus (HCV) inhibitor working through an NS5A-mediated mechanism. This first-time-in-human study was conducted to assess the safety, tolerability, pharmacokinetics, metabolism, and efficacy of GSK2336805 in healthy subjects and subjects infected with HCV genotype 1. We performed a three-part, randomized, double-blind, placebo-controlled study in 46 healthy subjects and 23 HCV-infected subjects. After an overnight fast, healthy subjects received GSK2336805 as 10 mg, 30 mg, 30 mg plus food, and 60 mg in a single dose and 10 mg (7 days), 30 mg (7 days), and 75 mg (14 days) in a once-daily multiple dose. Subjects with HCV received GSK2336805 as a 1- to 120-mg single dose. In subjects with HCV, reductions in HCV RNA were observed within 4 h and a single dose of GSK2336805 of ≥10 mg resulted in a statistically significant ≥2-log reduction in HCV RNA compared with placebo at 24 h postdose. GSK2336805 was readily absorbed in all subjects, and the half-life ( t 1/2 ) was suitable for once-daily dosing. Administration of GSK2336805 with food had no effect on plasma GSK2336805 exposure; however, absorption was delayed, with a median t max (time to maximum concentration of drug in serum) of 4.5 versus 2.0 h. Twenty subjects who received GSK2336805 experienced mild to moderate adverse events; none were serious. GSK2336805 was well tolerated and exhibited rapid, significant antiviral activity after a single dose in HCV-infected subjects. These results support the conduct of further studies evaluating GSK2336805 administered once daily for longer durations in combination with peginterferon, ribavirin, and other direct-acting antivirals. (This study has been registered at ClinicalTrials.gov under registration no. NCT01277692.)

Screening for Distress is suggested as a first step to optimize response to cancer patients’ needs. This change of practice may positively impact coordination of care, a key function of professional cancer navigators. To describe the perceptions of implementing Screening for Distress with professional cancer navigators from the perspective of key actors in Quebec and Nova Scotia. A descriptive pre- and post-implementation study was performed. Interviews were conducted with professional navigators (pivot nurses in oncology from Quebec and cancer patient navigators from Nova Scotia), psychosocial and spiritual oncology staff from Quebec, and health administrators from both provinces, prior to and following the implementation process. Different actors described similar advantages and challenges within both sites. Screening for Distress was perceived as an important change of practice, improving oncology team functioning and patient-centered care. Recommendations to facilitate the implementation process were suggested. This study supports the relevance of involving professional navigators in the implementation process of Screening for Distress.

Tracheal hemorrhage is a common occurrence in pediatric patients with long-term tracheotomies. The majority of these events are related to self-limited etiologies, such as granulation tissue or suction trauma. Tracheo-arterial fistula, however, represents a frequently fatal form of tracheal hemorrhage that may initially be difficult to distinguish from other causes. Previous reports have described the pathophysiology, presentation and management of tracheo-arterial fistula involving the innominate artery. We describe a case of a 21-month-old male with a history of significant congenital cardiac malformations and chronic tracheotomy tube dependence who presented with intermittent, brisk bleeding from the tracheotomy tube. He was ultimately diagnosed with and treated for an arterio-bronchial fistula from a major aorto-pulmonary collateral artery. We review the etiology and management of this disorder.

WR 3 is the brightest very early-type WN star in the sky. Based on several years of time-resolved spectroscopy and precision photometry on various time-scales, we deduce that WR 3 is most likely a single, weak-lined star of type WN3ha (contrary to its current catalogue-type of WN3 + O4), with H lines occurring both in emission and absorption in its wind. This conclusion is confirmed and strengthened via detailed modelling of the spectrum of WR 3. Given the similarity of WR 3 with numerous H-rich WNE stars in the Large Magellanic Cloud and especially the Small Magellanic Cloud, and its location towards the metal-deficient exterior of the Galaxy, we conclude that rotationally induced meridional circulation probably led to the apparently unusual formation of this hot Galactic WN star with enhanced hydrogen. Although we cannot completely rule out the possibility of a binary with a low orbital inclination and/or long period, we regard this latter possibility as highly unlikely.

We present a congenital cystadenoma of the tongue in a neonate, which presented at birth. Cystadenomas are uncommon tumors that form from salivary gland duct tissue and are more commonly seen in adults. This is the youngest case to be reported in the English literature. A review of literature with differential diagnosis and management is presented.

Objectives To assess the speech outcomes and complications in children who had undergone sphincter pharyngoplasty (SP) for management of velopharyngeal insufficiency (VPI). Study design Retrospective chart review. Methods Charts from patients who had sphincter pharyngoplasty between January 1993 and June 1996 were reviewed. Syndrome diagnosis and presence of repaired cleft palate were reviewed. Preoperative speech assessment, videofluoroscopic and nasopharyngoscopic evaluations, age at time of surgery, and postoperative speech assessments were reviewed for all patients. Postoperative videofluoroscopy and nasopharyngoscopy were performed for those patients who had persistent VPI. Obstructive sleep symptoms were also assessed. Results Thirty patients were identified; six patients had no follow-up evaluation, leaving 24 patients included in this study. Average follow-up was 11.7 months (range, 2-35 mo). Velocardiofacial syndrome (VCFS) was the most commonly identified syndrome. Postoperatively, 15 of 24 patients (62.5%) had complete resolution of their VPI; five of 24 (20.8%) had significant improvement; one of 24 (4.2%) had minimal to no change; and three of 24 (12.5%) were hyponasal. Of the six patients with some degree of persistent VPI, three underwent revision surgery. All three patients had complete resolution of their VPI after revision surgery, resulting in an overall success rate of 18 of 24 (75%). Conclusions Sphincter pharyngoplasty has wide application in the management of children with VPI, including those with VCFS. The procedure is readily modified to accommodate an individual patient's needs as determined by preoperative VPI evaluation. A modification of the procedure is described to minimize the risk of postoperative airway obstruction and hyponasality, both regarded as airway complications of sphincter pharyngoplasty.

Objective: To propose a staging system for patients with lymphatic malformations of the head and neck. Design: Retrospective chart review. Patients: Fifty-six patients were treated for lymphatic malformations from 1983 to 1993 at Children's Hospital and Medical Center, Seattle, Wash. The charts were reviewed for anatomic location of the lesion, preoperative and postoperative complications, number of procedures to control disease, long-term sequelae, and persistence of disease. Lesions were characterized as being unilateral or bilateral and suprahyoid and/or infrahyoid. The five patient groups were then compared with respect to the above categories. Results: Preoperative complications reviewed include preoperative infection, respiratory embarassment necessitating airway intervention, and feeding difficulties. Postoperative complications assessed were cranial nerve injury, wound infection, and seroma formation. Long-term sequelae included malocclusion, speech delay, and cosmetic deformity. The rate of persistent disease was also assessed. A staging system was developed based on a progression of extent of disease. Stage I patients (n=12) had unilateral infrahyoid disease and a 17% incidence of complications overall. Stage II patients (n=17) had unilateral suprahyoid disease and a 41% incidence of complications. Stage III patients (n=15) had unilateral suprahyoid and infrahyoid disease and a complication rate of 67%. Stage IV patients (n=5) with bilateral suprahyoid disease had a complication rate of 80%, while stage V patients (n=6) with bilateral suprahyoid and infrahyoid disease had a 100% incidence of complications. Conclusion: Anatomic location of lymphatic malformations of the head and neck can be used to predict prognosis and outcome of surgical intervention. (Arch Otolaryngol Head Neck Surg. 1995;121:577-582)

Objective To explore interrater and intrarater reliability ( R inter and R intra , respectively) of a standardized scale applied to nasoendoscopic assessment of velopharyngeal (VP) function, across multiple centers. Design Multicenter blinded R inter and R intra study. Setting Eight academic tertiary care centers. Participants Sixteen otolaryngologists from 8 centers. Main Outcome Measures Raters estimated lateral pharyngeal and palatal movement on nasoendoscopic tapes from 50 different patients. Raters were asked to (1) estimate gap size during phonation and (2) note the presence of the Passavant ridge, a midline palatal notch on the nasal surface of the soft palate, and aberrant pulsations. Primary outcome measures were R inter and R intra coefficients for estimated gap size, lateral wall, and palatal movement; κ coefficients for the Passavant ridge, a midline palatal notch on the nasal soft palate, and aberrant pulsations were also calculated. Results The R inter coefficients were 0.63 for estimated gap size, 0.41 for lateral wall movement, and 0.43 for palate movement; corresponding R intra coefficients were 0.86, 0.79, and 0.83, respectively. Interrater κ values for qualitative features were 0.10 for the Passavant ridge; 0.48 for a notch on the nasal surface of the soft palate, 0.56 for aberrant pulsations, and 0.39 for estimation of gap size. Conclusions In these data, there was good R intra and fair R inter when using the Golding-Kushner scale for rating VP function based on nasoendoscopy. Estimates of VP gap size demonstrate higher reliability coefficients than total lateral wall, mean palate estimates, and categorical estimate of gap size. The reliability of rating qualitative characteristics (ie, the presence of the Passavant ridge, aberrant pulsations, and notch on the nasal surface of the soft palate) is variable.

To describe the spectrum of pediatric tongue lesions treated surgically at Columbia-Presbyterian Medical Center from January 1990 to December 1999.Retrospective case-series at the pediatric hospital of a tertiary care, academic medical center.Seventeen patients were identified. Their ages ranged from 1 to 132 months (median, 7 months). Eight lesions were located anteriorly: mucous cyst (1), polyp (1), chronic inflammatory mass (1), hamartoma (1), squamous papilloma (2), cavernous hemangioma (1), and vascular malformation (1). Four lesions were located posteriorly: teratoma (1), glial choristoma (1), osseous choristoma (1), and benign epithelial cyst (1). Finally, there were 5 diffuse lesions including macroglossia (4) and massively infiltrating congenital lymphatic malformation (1). Symptoms included respiratory distress (3) and dysarthria (3); all other children were asymptomatic.This series revealed an interesting spectrum of rare solid tumors; compared with other large series, fewer lymphatic and vascular malformations were seen. Presenting symptoms, differential diagnosis, and surgical approach were differentiated according to lesion location.

Objective : To evaluate speech outcomes and complications of sphincter pharyngoplasty and pharyngeal flap performed for management of velopharyngeal insufficiency (VPI). Design : Case series. Setting : Tertiary care children’s hospital. Patients : All patients who underwent pharyngeal flap or sphincter pharyngoplasty from 1990 to 1995. Methods : Perceptual speech analysis was used to assess severity of VPI, presence of nasal air emissions and quality of nasal resonance (hyper, hypo, or normal). Pre-operative measures of velopharyngeal function were based upon nasendoscopy and videofluoroscopic speech assessment. Recommendations for management were made by the attending surgeon. Complications of hyponasality and obstructive sleep symptoms (OSS) were noted. Patient characteristics were compared using univariate analysis. Results : Sixteen patients underwent sphincter pharyngoplasty and 18 patients underwent superiorly based pharyngeal flap. Patients were similar in terms of lateral pharyngeal wall medial motion and palatal elevation. The groups were also similar with regard to VPI severity, though there was a trend for more severe VPI in patients undergoing sphincter pharyngoplasty than pharyngeal flap (50 vs. 33.3%, respectively). Patients with pharyngoplasty had a higher rate of resolution of VPI than those who had pharyngeal flap (50 vs. 22.2%, respectively), although this was not statistically significant. Post-operative hyponasality and obstructive sleep symptoms were present in both groups. However, only patients who underwent PF and had postoperative OSS had obstructive sleep apnea (OSA). Conclusions : There were no detectable anatomic differences between treatment groups implying that treatment selection during the study period was not guided by strict anatomic criteria. Sphincter pharyngoplasty may have a higher success rate with a lower risk of OSS.

1. It was recently proposed that acetylcholinesterase (AChE), in addition to its esteratic activity, has proteolytic activity such that it may cleave theβ-amyloid precursor (β-APP) within theβ-amyloid sequence. The purpose of this paper was to examine further whether AChE or butyrylcholinesterase (BuChE) had associated proteinase activity that was involved in the metabolism ofβ-APP. 2. The ability of various preparations of AChE and BuChE to hydrolyze two synthetic fragments ofβ-APP695 as model substrates containing the normal and aberrant cleavage sites was studied. 3. Digestion of these synthetic substrates with commercial preparations ofElectrophorus electricus AChE indicated the presence of a trypsin-like proteolytic activity cleaving each peptide at the carboxy-terminal side of an internal lysine residue. 4. Purification of the trypsin-like proteinase activity by aminobenzamidine affinity chromatography yielded a preparation that was devoid of AChE activity but retained all of the proteinase activity. 5. Amino-terminal sequence analysis of this preparation showed that the first 13 amino acid residues were identical toβ-pancreatic trypsin. 6. These data indicate that the proteinase activity found in these commercial preparations of AChE is due to contamination with trypsin.

Objective To validate a disease-specific health-related quality of life (HRQOL) instrument for children with obstructive sleep disorders (OSDs). Design Prospective cohort study using a 6-item health-related instrument (OSD-6). Subjects One hundred caregivers of patients with OSDs secondary to adenotonsillar hypertrophy (age range, 2-12 years) from 2 tertiary care, pediatric otolaryngology practices. Intervention The OSD-6 was administered on initial presentation and 4 to 5 weeks after adenotonsillectomy. A subset of patients repeated the OSD-6 within 3 weeks after presentation to assess test-retest reliability. Main Outcome Measures Test-retest reliability, internal consistency, construct validity, and responsiveness to clinical change of the OSD-6 score. Results Test-retest reliability was good (intraclass correlation coefficient = 0.74). Median OSD-6 score was 4.5 (0- to 6-point scale) with higher scores indicating poorer quality of life (QOL). Construct validity was demonstrated by the moderate correlation between OSD-6 score and global adenoid and tonsil-related QOL ( R = −0.62), strong correlation between the OSD-6 change score and change in global adenoid and tonsil-related QOL ( R = −0.63), and the moderate correlation between the change score and parent estimate of clinical change ( R = 0.40). The mean change in OSD-6 score after adenotonsillectomy was 3.0 (95% confidence interval, 2.7-3.4). The mean standardized response was 2.3 (95% confidence interval, 1.9-2.7) indicating the instrument's large responsiveness to clinical change. The change score was very reliable ( R = 0.85). Conclusions The OSD-6 is a reliable, responsive, easily administered instrument. It is valid for detecting change after adenotonsillectomy in children with OSDs.

As an approach to the study of structure-function relationships in the normal and defective forms of human coagulation factor IX, we have begun to develop a series of monoclonal antibodies against specific sites on the protein. Zymogen and activated forms of normal factor IX were used initially as antigen for the preparation of monoclonal antibodies. Recombinant phage were prepared by cloning small (50- to 500-nucleotide) random DNA fragments from the coding region of a factor IX cDNA clone into the expression vector lambda gt11. Immunological screening of these recombinants with mixtures of monoclonal antibodies identified several immunoreactive phage. Further analysis showed that the monoclonal antibody designated IX-30 was generating the positive signals at a frequency of approximately 1/2,500 recombinants. Subcloning and sequence analysis of the inserted DNA in the immunoreactive phage revealed overlapping in-frame insertions, from which it could be inferred that the site in factor IX recognized by IX- 30 is confined to residues 111 through 132 in the light chain. Similar mapping with other monoclonal antibodies should provide additional probes for the protein structure of human factor IX.

SummaryA technique for the detection of von Willebrand factor multimers separated by discontinuous SDS agarose electrophoresis has been developed using non-radioactive com-v pounds. The multimeric patterns were visualized by monospecific anti-human vWF:Ag followed by incubation with biotinylated antibody. After addition of avidin-biotin-peroxidase complex, the peroxidase activitiy was detected by 4-chloro-l-naphthol, giving sharp bands with a clear background.By this method, the differences of vWF : Ag multimers could be easily observed between normal plasma and the plasmas from variant type vWD (IIA, IIB, platelet-type). Large and intermediate multimers were absent in the plasma with vWD type IIA, while only large multimers were absent in the plasma with vWD IIB and platelet-type. The absence of large multimers was also observed in two commercial F VIII preparations having the ratio of vWF/vWF : Ag 0.18 and 0.63. The preparation with the ratio of 0.63 showed the presence of larger intermediate multimers.Electrophoresis in SDS 1.5% agarose gel revealed triplet structure of each small multimer, and a relative increase of the smallest subband was observed in vWD IIA plasma, platelet-type vWD plasma and commercial F VIII preparations.The procedures described are easy and safe to perform and are useful for screening or classifying cases with vWD in general laboratories.

Functional domains of the human factor IX (FIX) molecule may be expressed in E. coli as fusion proteins (FPs) with phage T7 capsid protein 10 (CP 10). Such proteins are synthesized in large amounts and, because of their insolubility can be easily purified Polyclonal antibodies raised to FIX FPs cross react with the native molecule, but the utility of FPs in the production of monoclonal probes of native FIX is unclear. Hence, we have characterized the specificity of murine hybridomas resulting from the immunization with a FP containing amino acids -3 to 49 of human FIX (FP4D) . FP4D proved to be a potent immunogen in mice. Using solid phase ELISA assays, 46% of 2,200 cultures screened reacted with the immunizing antigen. Most of the positive cultures reacted only with FP4D and/or CP 10 and were discarded. Fifty cultures reacted with native FIX and were further studied. None of the FIX reactive cultures inhibited FIX:C activity in clotting assays. Binding of more than half of the cultures to FIX was inhibited by the presence of 5mM Ca++ suggesting a similarity between FP4D and the non Ca++ bound steric state of FIX. Four cultures were subcloned 2X to insure monoclonality. These antibodies were further evaluated for binding to FIX, FP4D, and CP 10 in the presence or absence of Ca++. Three of the four Mabs reacted with FP4D and FIX and showed varying degrees of inhibition by Ca++ only with FIX. One antibody reacted equally with all three proteins in the presence or absence of Ca++. Two of the Mabs were detectable in a solid phase RIA and showed increased radioligand binding in the absence of Ca++. Most Mabs against FP4D appear to detect epitopes which are influenced by the presence of Ca++. Since the FIX amino acid sequence contained in FP4D is gammacarboxylated and capable of binding Ca++ in the native molecule, the epitopes detected by these Mabs may be masked or altered by Ca++ binding. These Mabs may prove to be useful probes in the study of Ca++ binding in the native FIX molecule.

A technique for the detection of von Willebrand factor multimers separated by discontinuous SDS agarose electrophoresis has been developed using non-radioactive compounds. The multimeric patterns were visualized by monospecific anti-human vWF:Ag followed by incubation with biotinylated antibody. After addition of avidin-biotin-peroxidase complex, the peroxidase activity was detected by 4-chloro-1-naphthol, giving sharp bands with a clear background. By this method, the differences of vWF:Ag multimers could be easily observed between normal plasma and the plasmas from variant type vWD (IIA, IIB, platelet-type). Large and intermediate multimers were absent in the plasma with vWD type IIA, while only large multimers were absent in the plasma with vWD IIB and platelet-type. The absence of large multimers was also observed in two commercial FVIII preparations having the ratio of vWF/vWF:Ag 0.18 and 0.63. The preparation with the ratio of 0.63 showed the presence of larger intermediate multimers. Electrophoresis in SDS 1.5% agarose gel revealed triplet structure of each small multimer, and a relative increase of the smallest subband was observed in vWD IIA plasma, platelet-type vWD plasma and commercial FVIII preparations. The procedures described are easy and safe to perform and are useful for screening or classifying cases with vWD in general laboratories.

As an approach to the study of structure-function relationships in the normal and defective forms of human coagulation factor IX, we have begun to develop a series of monoclonal antibodies against specific sites on the protein. Zymogen and activated forms of normal factor IX were used initially as antigen for the preparation of monoclonal antibodies. Recombinant phage were prepared by cloning small (50- to 500-nucleotide) random DNA fragments from the coding region of a factor IX cDNA clone into the expression vector lambda gt11. Immunological screening of these recombinants with mixtures of monoclonal antibodies identified several immunoreactive phage. Further analysis showed that the monoclonal antibody designated IX-30 was generating the positive signals at a frequency of approximately 1/2,500 recombinants. Subcloning and sequence analysis of the inserted DNA in the immunoreactive phage revealed overlapping in-frame insertions, from which it could be inferred that the site in factor IX recognized by IX-30 is confined to residues 111 through 132 in the light chain. Similar mapping with other monoclonal antibodies should provide additional probes for the protein structure of human factor IX.

Factor IX (FIX), a vitamin K dependent coagulation protein, is functionally deficient or absent in patients with hemophilia B. Binding of Ca++ by the gammacarboxyglutamic acid residues (Gla) of FIX is necessary for coagulant activity. Antibodies havebeendes-cribed which selectively bind to FIX in the presence of Ca++ and appear to interact with Ca-H- stabilized epitopes of FIX. One IgM, Kappa murine monoclonal antibody (Mab), 129-1, has been found to react preferentially with FIX in the absence of Ca-H- or with FIX having a reduction in gammacarboxylation. 129-1 was characterized by direct binding ELISA assays, the standard assay buffer (.02M Tris - .15M NaCL pH 7.5) was supplemented with Ca-H- or ED TA to final concentrations to 5mM and lOmM respectively. In the presence of Ca-H-, titers ranged from 10 to 100 as compared to 100 K to 500 K in the presence of EDTA. Control Mab's, FIX-30 and 2D521 showed no such effect. Maximum inhibition occurred ≥2.5 mM Ca-H- concentration. Mg++, Sr-H- and Mn-H- were tested with similar results. Chemically degammacarboxylated FIXa was compared to FIX and FIXa controls in the presence of Ca-H- or EDTA, to determine the importance of Gla residues. In the presence of EDTA, Mab 129-1 had essentially equal reactivity with all these proteins (titers 100 K). In the presence of Ca-H-, binding to FIX and FIXa was inhibited (500 and 1 K respectively). However, the binding to Gla modified FIXa in the presence of Ca-H- was only slightly reduced (10 K), suggesting the importance of Gla residues in the Ca-H- mediated inhibition. FIX was purified from concentrate, coumadinized plasma and culture supernatant (produced in the absence of vitamin K) from the cell line PMN45 using HPLC affinity chromatography with Mab 2D521. Mab 129-1 binding to FIX immunopurified from concentrate and a control biochemically purified FIX protein preparation was significantly higher in the presence of EDTA (titers 500 and 5 K) than in Ca-H- (titers 10 and 10). FIX purified from coumadinized plasma or PMN45 cells showed equal low reactivity with 129-1 in the presence or absence of Ca-H- (titer 10 in all cases). FIX-30 and 2D521 controls showed no such reduction in binding. Therefore, Gla residues may play a role in the binding of 129-1 to FIX.