Outcomes of Neonates Born to Mothers With Severe Acute Respiratory Syndrome Coronavirus 2 Infection at a Large Medical Center in New York City.

Importance: Limited data on vertical and perinatal transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and health outcomes of neonates born to mothers with symptomatic or asymptomatic coronavirus disease 2019 (COVID-19) are available. Studies are needed to inform evidence-based infection prevention and control (IP&C) policies. Objective: To describe the outcomes of neonates born to mothers with perinatal SARS-CoV-2 infection and the IP&C practices associated with these outcomes. Design, Setting, and Participants: This retrospective cohort analysis reviewed the medical records for maternal and newborn data for all 101 neonates born to 100 mothers positive for or with suspected SARS-CoV-2 infection from March 13 to April 24, 2020. Testing for SARS-CoV-2 was performed using Cobas (Roche Diagnostics) or Xpert Xpress (Cepheid) assays. Newborns were admitted to well-baby nurseries (WBNs) (82 infants) and neonatal intensive care units (NICUs) (19 infants) in 2 affiliate hospitals at a large academic medical center in New York, New York. Newborns from the WBNs roomed-in with their mothers, who were required to wear masks. Direct breastfeeding after appropriate hygiene was encouraged. Exposures: Perinatal exposure to maternal asymptomatic/mild vs severe/critical COVID-19. Main Outcomes and Measures: The primary outcome was newborn SARS-CoV-2 testing results. Maternal COVID-19 status was classified as asymptomatic/mildly symptomatic vs severe/critical. Newborn characteristics and clinical courses were compared across maternal COVID-19 severity. Results: In total, 141 tests were obtained from 101 newborns (54 girls [53.5%]) on 0 to 25 days of life (DOL-0 to DOL-25) (median, DOL-1; interquartile range [IQR], DOL-1 to DOL-3). Two newborns had indeterminate test results, indicative of low viral load (2.0%; 95% CI, 0.2%-7.0%); 1 newborn never underwent retesting but remained well on follow-up, and the other had negative results on retesting. Maternal severe/critical COVID-19 was associated with newborns born approximately 1 week earlier (median gestational age, 37.9 [IQR, 37.1-38.4] vs 39.1 [IQR, 38.3-40.2] weeks; P = .02) and at increased risk of requiring phototherapy (3 of 10 [30.0%] vs 6 of 91 [7.0%]; P = .04) compared with newborns of mothers with asymptomatic/mild COVID-19. Fifty-five newborns were followed up in a new COVID-19 Newborn Follow-up Clinic at DOL-3 to DOL-10 and remained well. Twenty of these newborns plus 3 newborns followed up elsewhere had 32 nonroutine encounters documented at DOL-3 to DOL-25, and none had evidence of SARS-CoV-2 infection, including 6 with negative retesting results. Conclusions and Relevance: No clinical evidence of vertical transmission was identified in 101 newborns of mothers positive for or with suspected SARS-CoV-2 infection, despite most newborns rooming-in and direct breastfeeding practices.

Infection prevention and control for labor and delivery, well baby nurseries, and neonatal intensive care units.

During the early months of the COVID-19 pandemic, infection prevention and control (IP&C) for women in labor and mothers and newborns during delivery and receiving post-partum care was quite challenging for staff, patients, and support persons due to a relative lack of evidence-based practices, high rates of community transmission, and shortages of personal protective equipment (PPE). We present our IP&C policies and procedures for the obstetrical population developed from mid-March to mid-May 2020 when New York City served as the epicenter of the pandemic in the U.S. For patients, we describe screening for COVID-19, testing for SARS-CoV-2, and clearing patients from COVID-19 precautions. For staff, we address self-monitoring for symptoms, PPE in different clinical scenarios, and reducing staff exposures to SARS-CoV-2. For visitors/support persons, we address limiting them in labor and delivery, the postpartum units, and the NICU to promote staff and patient safety. We describe management of SARS-CoV-2-positive mothers and their newborns in both the well-baby nursery and in the neonatal ICU. Notably, in the well-baby nursery we do not separate SARS-CoV-2-positive mothers from their newborns, but emphasize maternal mask use and social distancing by placing newborns in isolates and asking mothers to remain 6 feet away unless feeding or changing their newborn. We also encourage direct breastfeeding and do not advocate early bathing. Newborns of SARS-CoV-2-positive mothers are considered persons under investigation (PUIs) until 14 days of life, the duration of the incubation period for SARS-CoV-2. We share two models of community-based care for PUI neonates. Finally, we provide our strategies for enhancing communication and education during the early months of the pandemic.

A review of newborn outcomes during the COVID-19 pandemic.

As the COVID-19 pandemic continues to spread worldwide, it is crucial that we determine populations that are at-risk and develop appropriate clinical care policies to protect them. While several respiratory illnesses are known to seriously impact pregnant women and newborns, preliminary data on the novel SARS-CoV-2 Coronavirus suggest that these groups are no more at-risk than the general population. Here, we review the available literature on newborns born to infected mothers and show that newborns of mothers with positive/suspected SARS-CoV-2 infection rarely acquire the disease or show adverse clinical outcomes. With this evidence in mind, it appears that strict postnatal care policies, including separating mothers and newborns, discouraging breastfeeding, and performing early bathing, may be more likely to adversely impact newborns than they are to reduce the low risk of maternal transmission of SARS-CoV-2 or the even lower risk of severe COVID-19 disease in otherwise healthy newborns.

Relationship-Centered Care in a Novel Dual-Visit Model COVID Nursery Follow-Up Clinic.

The COVID Nursery Follow-Up Clinic at our academic medical center in New York City was established during the COVID-19 pandemic to provide care to infants born to SARS-CoV-2 positive mothers. We describe a novel dual-visit model utilizing telehealth and an in-person visit to provide timely, inclusive and relationship-centered care to the mother/infant couplet in a situation where the mother was unable to come to a traditional in-person visit, but the infant needed medically necessary in-person evaluation.

The prothrombin time/international normalized ratio (PT/INR) Line: derivation of local INR with commercial thromboplastins and coagulometers--two independent studies.

BACKGROUND: The WHO scheme for prothrombin time (PT) standardization has been limited in application, because of its difficulties in implementation, particularly the need for mandatory manual PT testing and for local provision of thromboplastin international reference preparations (IRP). METHODS: The value of a new simpler procedure to derive international normalized ratio (INR), the PT/INR Line, based on only five European Concerted Action on Anticoagulation (ECAA) calibrant plasmas certified by experienced centres has been assessed in two independent exercises using a range of commercial thromboplastins and coagulometers. INRs were compared with manual certified values with thromboplastin IRP from expert centres and in the second study also with INRs from local ISI calibrations. RESULTS: In the first study with the PT/INR Line, 8.7% deviation from certified INRs was reduced to 1.1% with human reagents, and from 7.0% to 2.6% with rabbit reagents. In the second study, deviation was reduced from 11.2% to 0.4% with human reagents by both local ISI calibration and the PT/INR Line. With rabbit reagents, 10.4% deviation was reduced to 1.1% with both procedures; 4.9% deviation was reduced to 0.5% with bovine/combined reagents with local ISI calibrations and to 2.9% with the PT/INR Line. Mean INR dispersion was reduced with all thromboplastins and automated systems using the PT/INR Line. CONCLUSIONS: The procedure using the PT/INR Line provides reliable INR derivation without the need for WHO ISI calibration across the range of locally used commercial thromboplastins and automated PT systems included in two independent international studies.

The cost-effectiveness of computer-assisted anticoagulant dosage: results from the European Action on Anticoagulation (EAA) multicentre study.

BACKGROUND: Increased demand for oral anticoagulation has resulted in wider adoption of computer-assisted dosing in anticoagulant clinics. An economic evaluation has been performed to investigate the cost-effectiveness of computer-assisted dosing in comparison with manual dosing in patients on oral anticoagulant therapy. METHODS: A trial-based cost-effectiveness analysis was conducted as part of the EAA randomized study of computer-assisted dosage vs. manual dosing. The 4.5-year multinational trial was conducted in 32 centres with 13 219 anticoagulation patients randomized to manual or computer-assisted dosage. The main outcome measures were total health care costs, clinical event rates and cost-saving per clinical event prevented by computer dosing compared with manual dosing. RESULTS: Mean dosing costs per patient were lower (difference: euro47) for computer-assisted dosing, but with little difference in clinical event costs. Total overall costs were euro51 lower in the computer-assisted dosing arm. There were a larger number of clinical events in the manual dosing arm. The overall difference between trial arms was not significant (difference in clinical events, -0.003; 95% CI, -0.010-0.004) but there was a significant reduction in events with DVT/PE, suggesting computer-assisted dosage with the two study programs (dawn ac or parma 5) was at least as effective clinically as manual dosage. The cost-effectiveness analysis indicated that computer-assisted dosing is less costly than manual dosing. CONCLUSIONS: Results indicate that computer-assisted dosage with the two programs (dawn ac and parma 5) is cheaper than manual dosage and is at least as effective clinically, indicating that investment in this technology represents value for money.

An international multicenter randomized study of computer-assisted oral anticoagulant dosage vs. medical staff dosage.

BACKGROUND: Increased demand for oral anticoagulants is overwhelming facilities worldwide, resulting in increasing use of computer assistance. A multicenter clinical endpoint study has been performed to compare the safety and effectiveness of computer-assisted dosage with dosage by experienced medical staff at the same centers. METHODS: A randomized study of dosage of two commercial computer-assisted dosage programs (PARMA 5 and DAWN AC) vs. manual dosage at 32 centers with an established interest in oral anticoagulation in 13 countries. The aim was to recruit a minimum of 16,000 patient-years randomized to medical staff or computer-assisted dosage. In total, 13,219 patients participated, 6503 patients being randomized to medical staff and 6716 to computer-assisted dosage. The safety and effectiveness of computer-assisted dosage were compared with those of medical staff dosage. RESULTS: In total, 13,052 patients were recruited (18,617 patient-years). International Normalized Ratio (INR) tests numbered 193 890 with manual dosage and 193,424 with computer-assisted dosage. The number of clinical events with computer-assisted dosage was lower (P = 0.1), but in the 3209 patients with deep vein thrombosis/pulmonary embolism, they were reduced by 37 (24%, P = 0.001). Time in target INR range was significantly improved by computer assistance as compared with medical staff dosage at the majority of centers (P < 0.001). CONCLUSIONS: The safety and effectiveness of computer-assisted dosage has been demonstrated using two different marketed programs in comparison with experienced medical staff dosage at the centers with established interest in anticoagulation. Significant prevention of clinical events in patients with deep vein thrombosis/pulmonary embolism and the achievement of target INR in all clinical groups has been observed. The reliability and safety of other marketed computer-assisted dosage programs need to be established.

Comparison of local International Sensitivity Index calibration and 'Direct INR' methods in correction of locally reported International Normalized Ratios: an international study.

BACKGROUND: It is no longer feasible to check local International Normalized Ratios (INR) by the World Health Organization International Sensitivity Index (ISI) calibrations because the necessary manual prothrombin time technique required has generally been discarded. OBJECTIVES: An international collaborative study at 77 centers has compared local INR correction using the two alternative methods recommended in the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis guidelines: local ISI calibration and 'Direct INR'. METHODS: Success of INR correction by local ISI calibration and with Direct INR was assessed with a set of 27 certified lyophilized plasmas (20 from patients on warfarin and seven from normals). RESULTS: At 49 centers using human thromboplastins, 3.0% initial average local INR deviation from certified INR was reduced by local ISI calibration to 0.7%, and at 25 centers using rabbit reagents, from 15.9% to 7.5%. With a minority of commercial thromboplastins, mainly 'combined' rabbit reagents, INR correction was not achieved by local ISI calibration. However, when rabbit combined reagents were excluded the overall mean INR deviation after correction was reduced further to 3.9%. In contrast, with Direct INR, mean deviation using human thromboplastins increased from 3.0% to 6.6%, but there was some reduction with rabbit reagents from 15.9% to 10% (12.3% with combined reagents excluded). CONCLUSIONS: Local ISI calibration gave INR correction for the majority of PT systems but failed at the small number using combined rabbit reagents suggesting a need for a combined reference thromboplastin. Direct INR correction was disappointing but better than local ISI calibration with combined rabbit reagents. Interlaboratory variability was improved by both procedures with human reagents only.

Heading in football. Part 2: biomechanics of ball heading and head response.

OBJECTIVES: Controversy surrounding the long term effects of repeated impacts from heading has raised awareness among the public and the medical community. However, there is little information about the human response to the impacts and what measures can be taken to alter their effect. The objective of the current study was to gain a better understanding of heading biomechanics through the implementation of a numerical model and subsequent investigation of parameters related to heading technique and ball characteristics. METHODS: A controlled laboratory study was carried out with seven active football players, aged 20-23 years who underwent medical screening and were instrumented with accelerometers mounted in bite plates and electromyographic electrodes on the major neck muscle groups. Balls were delivered at two speeds (6 m/s and 8 m/s) as the subjects demonstrated several specific heading manoeuvres. Photographic targets were tracked via high speed video to measure heading kinematics. One subject demonstrating reasonably averaged flexion-extension muscle activity phased with head acceleration data and upper torso kinematics was used to validate a biofidelic 50th percentile human numerical model with detailed representation of the head and neck. RESULTS: Heading kinematics and subject responses were used with a detailed numerical model to simulate impact biomechanics for a baseline heading scenario. Changes to heading techniques and ball characteristics which mitigated head impact response were identified. CONCLUSION: A numerical model combined with biomechanical measurement techniques is an important tool for parametric investigation of strategies to reduce head impact severity via changes in heading technique or the physical properties of the ball.

Heading in football. Part 1: development of biomechanical methods to investigate head response.

OBJECTIVES: There has been growing controversy regarding long term effects of repeated low severity head impacts such as when heading a football. However, there are few scientific data substantiating these concerns in terms of the biomechanical head response to impact. The present study aimed to develop a research methodology to investigate the biomechanical response of human subjects during intentional heading and identify strategies for reducing head impact severity. METHODS: A controlled laboratory study was carried out with seven active football players, aged 20-23 and of average stature and weight. The subjects were fitted with photographic targets for kinematic analysis and instrumented to measure head linear/angular accelerations and neck muscle activity. Balls were delivered at two speeds (6 m/s and 8 m/s) as the subjects executed several specific forward heading manoeuvres in the standing position. Heading speeds up to 11 m/s were seen when the head closing speed was considered. One subject demonstrating averaged flexion-extension muscle activity phased with head acceleration data and upper torso kinematics was used to validate a biofidelic 50th percentile human model with a detailed head and neck. The model was exercised under ball incoming speeds of 6-7 m/s with parameter variations including torso/head alignment, neck muscle tensing, and follow through. The model output was subsequently compared with additional laboratory tests with football players (n = 3). Additional heading scenarios were investigated including follow through, non-active ball impact, and non-contact events. Subject and model head responses were evaluated with peak linear and rotational accelerations and maximum incremental head impact power. RESULTS: Modelling of neck muscle tensing predicted lower head accelerations and higher neck loads whereas volunteer head acceleration reductions were not consistent. Modelling of head-torso alignment predicted a modest reduction in volunteer head accelerations. Exaggerated follow through while heading reduced volunteer head accelerations modestly. CONCLUSION: Biomechanical methods were developed to measure head impact response. Changing the biomechanics of currently accepted heading techniques will have inconsistent benefits towards the reduction of head loading. Furthermore, mathematical modelling suggested an increased risk of neck loads with one alternative technique. No consistent recommendations can be made on the basis of the current study for altering heading techniques to reduce impact severity.

Heading in football. Part 3: effect of ball properties on head response.

OBJECTIVES: Head impacts from footballs are an essential part of the game but have been implicated in mild and acute neuropsychological impairment. Ball characteristics have been noted in literature to affect the impact response of the head; however, the biomechanics are not well understood. The present study determined whether ball mass, pressure, and construction characteristics help reduce head and neck can impact response. METHODS: Head responses under ball impact (6-7 m/s) were measured with a biofidelic numerical human model and controlled human subject trials (n = 3). Three ball masses and four ball pressures were investigated for frontal heading. Further, the effect of ball construction in wet/dry conditions was studied with the numerical model. The dynamic ball characteristics were determined experimentally. Head linear and angular accelerations were measured and compared with injury assessment functions comprising peak values and head impact power. Neck responses were assessed with the numerical model. RESULTS: Ball mass reductions up to 35% resulted in decreased head responses up to 23-35% for the numerical and subject trials. Similar decreases in neck axial and shear responses were observed. Ball pressure reductions of 50% resulted in head and neck response reductions up to 10-31% for the subject trials and numerical model. Head response reductions up to 15% were observed between different ball constructions. The wet condition generally resulted in greater head and neck responses of up to 20%. CONCLUSION: Ball mass, pressure, and construction can reduce the impact severity to the head and neck. It is foreseeable that the benefits can be extended to players of all ages and skill levels.

European Concerted Action on Anticoagulation. A multicentre calibration study of WHO international reference preparations for thromboplastin, rabbit (RBT/90) and human (rTF/95).

A 10 centre calibration was performed after six years to determine the international sensitivity index (ISI) of rTF/95 relative to RBT/90, and to assess any international normalised ratio (INR) bias compared with the original multicentre calibration. After exclusion of one outlying centre, the follow up calibration gave a mean ISI for rTF/95 of 0.99, which although a small difference, is significantly greater than the mean ISI of 0.94 obtained previously. The change in ISI for international reference preparation (IRP) rTF/95 relative to RBT/90 would lead to a slight bias in INR for human compared with rabbit thromboplastins. At a theoretical INR of 3.0, the INR bias is 6.0%, and this is below the accepted 10% level of clinical relevance. Ongoing stability monitoring of World Health Organisation thromboplastin IRP is advised.

European Concerted Action on Anticoagulation (ECAA): an assessment of a method for ISI calibration of two whole blood point-of-care PT monitor systems based on lyophilized plasmas using whole blood equivalent PT.

Previously, the attempt to simplify the International Sensitivity Index (ISI) calibration of the CoaguChek Mini whole blood point-of-care test prothrombin time (PT) monitor system was successful using lyophilized plasmas from coumarin-treated patients but not with lyophilized artificially depleted plasmas. With the TAS PT-NC monitor system, both types of plasma failed to provide reliable calibrations. The present study assesses a procedure for the ISI calibration of a TAS PT-NC and CoaguChek Mini whole blood point-of-care test PT monitor systems using lyophilized plasmas. Using lyophilized artificially depleted and coumarin plasma calibrations, we have evaluated a correction for the monitor displayed PT. This was based on a 'line of equivalence' derived from the relationship between whole blood and fresh plasma PT with both types of monitor system. With the TAS PT-NC, the use of this 'line of equivalence' resulted in reliable ISI with both lyophilized coumarin and artificially depleted plasmas. There was no significant difference between mean monitor and mean reference International Normalized Ratio (INR) with the artificially depleted plasmas. With the lyophilized coumarin plasma calibrations there was only a small INR difference. Correction with the 'line of equivalence' therefore facilitates calibration of the TAS PT-NC with lyophilized plasmas. With the CoaguChek Mini, the correction based on the 'line of equivalence' did not improve results but was not required with this system.

European Concerted Action on Anticoagulation (ECAA). An assessment of lyophilised plasmas for ISI calibration of CoaguChek and TAS whole blood prothrombin time monitors.

AIMS: The recommended method for the international sensitivity index (ISI) calibration of whole blood point of care testing (POCT) prothrombin time (PT) systems was originally described by Tripodi et al in 1993 but is too complex and demanding. The present European Concerted Action on Anticoagulation (ECAA) study aimed to assess the reliability of simpler ISI calibration using lyophilised plasma samples. METHODS: ISI calibrations using three different types of ECAA lyophilised plasma samples (artificially depleted, individual, and pooled coumarin) were compared with whole blood calibrations on CoaguChek Mini and TAS PT-NC POCT monitors at 10 centres. RESULTS: With CoaguChek Mini systems, lyophilised coumarin plasma samples (both single donation and pooled) gave ISI and international normalised ratio (INR) values comparable to whole blood. With artificially depleted plasma, ISI and INR values were too high. With TAS PT-NC systems, all three types of lyophilised plasma samples gave inaccurate ISI and unreliable INR results, similar to previous ECAA findings with fresh plasma calibrations. CONCLUSIONS: With CoaguChek Mini systems, ISI calibration can be simplified by the use of ECAA lyophilised plasma samples from coumarin treated patients. Further study is needed to devise a simpler calibration method for the TAS PT-NC system.

European Concerted Action on Anticoagulation (ECAA). Minimum number of centres for reliable International Sensitivity Index calibration of CoaguChek and TAS point-of-care whole blood monitors.

INTRODUCTION: Prothrombin time (PT) test systems require multicentre calibration for reliable International Sensitivity Index (ISI). Multicentre calibration of CoaguChek Mini and TAS PT-NC point-of-care test (POCT) systems is less precise than conventional PT testing. The aim of the present study was to determine the number of centres required to give reliable ISI and International Normalised Ratio (INR) with these two POCT whole blood PT monitors. MATERIALS AND METHODS: A simulation study, based on results of a 10-centre calibration exercise, was performed to assess reliability of ISI and INR when the number of centres was reduced from 10 to 2. RESULTS AND CONCLUSIONS: With both systems, the range of ISI and INR deviation increased as the number of centres was reduced. For the CoaguChek Mini, at least five centres were needed for satisfactory INR deviation in 95% of calibrations. With the TAS PT-NC, three centres gave satisfactory INR at this level. The number of centres required for multicentre calibration of these two POCT PT systems is greater than the two proposed by World Health Organisation (WHO) Guidelines for conventional PT testing.

European Concerted Action on Anticoagulation--comparison of fresh plasma and whole blood multicentre ISI calibrations of CoaguChek Mini and TAS PT-NC whole blood prothrombin time point-of-care monitors.

A procedure for using citrated fresh plasmas for International Sensitivity Index (ISI) calibration of two types of whole blood point-of-care test (POCT) prothrombin time (PT) monitor systems has been assessed in a multicentre study. The CoaguChek Mini and TAS PT-NC systems gave higher ISI with whole blood samples than with fresh plasma calibrations. However. there was good agreement between whole blood and fresh plasma monitor system International Normalised Ratio (INR) and the reference INR of target samples. Reliable INR can therefore be obtained with both whole blood and plasma samples on these two POCT systems based on their respective ISI. With the CoaguChek Mini system, the plasma calibration ISI can also be used to derive reliable INR with whole blood PT results. This was not possible with the TAS PT-NC system.

European Concerted Action on Anticoagulation (ECAA): multicentre international sensitivity index calibration of two types of point-of-care prothrombin time monitor systems.

A multicentre modified World Health Organization (WHO)-type international sensitivity index (ISI) calibration has been performed at 10 European Concerted Action on Anticoagulation (ECAA) national laboratories using non-citrated whole-blood on two point-of-care test (POCT) prothrombin time (PT) monitor systems, CoaguChek Mini and TAS PT-NC, using single lots of test cards/strips. The relevant species (human and rabbit) WHO international reference preparations (IRPs) were tested with the manual PT technique on citrated plasma from the same blood donations. The ISI was calculated from the slope of the orthogonal regression line relating log PT (POCT) to log PT (IRP). The mean ISI of the CoaguChek Mini system was 1.75 and 1.13 with the prothrombin time non-citrated Thrombolytic Assessment System (TAS PT-NC). With the CoaguChek Mini system, seven out of 10 calibrations exceeded the current 3% WHO recommended limit for the coefficient of variation (CV) of the slope with conventional PT testing, whereas with the TAS PT-NC system, it was eight out of 10. All the POCT calibrations had a CV of the slope <5%. It is suggested that this level of precision be adopted as the limit of acceptability of calibration of these monitor systems. In these circumstances, the modified WHO-type ISI calibration appeared to be satisfactory for the POCT whole-blood monitors.

European Concerted Action on Anticoagulation (ECAA): International Normalized Ratio variability of CoaguChek and TAS point-of-care testing whole blood prothrombin time monitors.

The object was to assess the variability in displayed International Normalised Ratio (INR) between monitors of the same manufacture using whole blood samples from the same subjects. Two brands of monitor, CoaguChek Mini and the TAS PT-NC were tested. 14 instruments of each brand were tested on the same day at the same laboratory by the same operator using identical blood samples to avoid between-centre differences in samples and operator technique. Whole blood samples from two normal donors and four coumarin-treated patients were tested to assess between-instrument variability of INR. Results have been coded. There was a much wider dispersion of INR on Brand B than on Brand A. One Brand A instrument failed to give a result with one of the two whole blood samples from one patient. One Brand B monitor gave an aberrant result with one of the samples from a normal subject. On both brands of monitor, INR variability appeared to be due mainly to duplication differences rather than between-instrument variability on both normal and coumarin whole blood samples.

The effects of freeze drying and freeze drying additives on the prothrombin time and the international sensitivity index.

AIM: To determine whether freezing, freeze drying protective additives, or freeze drying of plasma samples from patients on coumarin treatment and from normal individuals affects prothrombin times or the international sensitivity index (ISI) calibration. METHODS: The effect of the addition of the protective additives singly and combined on the prothrombin time of coumarin samples and normal samples before and after freeze drying was observed using high and low ISI reference thromboplastins. ISI values were also determined. RESULTS: Freezing caused a prolongation of prothrombin time in the normal plasma samples with both reagents, which was significant with the low ISI human. Prolongation (non-significant) of the prothrombin time in coumarin plasma samples occurred with the human reagent only. Significant prolongation of normal prothrombin time by some of the protective additives before and after freeze drying was observed with both thromboplastins but to a greater extent with the human. Significant prolongation of prothrombin time in coumarin plasma samples was observed, but again was more marked with human thromboplastin. An approximate ISI was determined on the 20 coumarin samples. The only marked ISI change was with the WHO human thromboplastin after freeze drying of plasma, where a decrease from 0.95 to 0.90 was observed, corresponding to a marked prothrombin ratio increase. CONCLUSIONS: Freeze drying additives and the freeze drying procedure prolong normal and coumarin prothrombin times, with low ISI thromboplastin. Less marked prolongations occurred with a high ISI rabbit reagent, coumarin samples showing more significant prolongations. Marked ISI change in freeze dried plasma was only recorded with the low ISI ECAA human reagent. Frozen normal plasma samples cannot be used with confidence for ISI calibrations.

Thermodynamics of the interaction of human immunoglobulin E with its high-affinity receptor Fc epsilon RI.

We have employed isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy to characterize the binding of soluble fragments of IgE (IgE-Fc and Fc epsilon 3-4) to a soluble fragment of the high-affinity receptor Fc epsilon RI alpha-chain (sFc epsilon RI alpha). The thermodynamic parameters for the interaction of IgE-Fc and Fc epsilon 3-4 with sFc epsilon RI alpha, determined using ITC, confirm the earlier conclusion that the C epsilon 2 domain is not involved in the interaction and that the stoichiometry of both complexes is 1:1. For both IgE-Fc and Fc epsilon 3-4, the value of Delta H degrees is -36.9 +/- 4.6 kcal mol-1 at 37.3 degreesC and Delta Cp degrees is -820 +/- 120 cal mol-1 K-1. The temperature at which DeltaS degrees is zero is 284 +/- 1 K, indicating that the entropy contribution to the thermodynamics of association is unfavorable at physiological temperature. Of particular interest is the large value of Delta Cp degrees. The large surface area of IgE and Fc epsilon RI alpha that is implicated in complex formation from previous mutagenesis studies on the two proteins may account in part for the magnitude of Delta Cp degrees. Additional contributions may arise from hydration within the binding site and changes in tertiary structure of the individual components of the complex. However, the CD spectra of IgE, IgE-Fc, and Fc epsilon 3-4 complexes with sFc epsilon RI alpha are merely the sum of the spectra of their individual components, indicating that the secondary structure of the immunoglobulin domain folds are preserved on complex formation. Thus, any change in tertiary structure must be limited to the relative disposition of the immunoglobulin domains C epsilon 3 and C epsilon 4 in IgE and the two immunoglobulin-like domains in the alpha-chain of Fc epsilon RI.