The role of fetal hemoglobin in the artificial placenta: A premature ovine model.

INTRODUCTION: A radical paradigm shift in the treatment of premature infants failing conventional treatment is to recreate fetal physiology using an extracorporeal Artificial Placenta (AP). The aim of this study is to evaluate the effects of changing fetal hemoglobin percent (HbF%) on physiology and circuit function during AP support in an ovine model. METHODS: Extremely premature lambs (n = 5) were delivered by cesarean section at 117-121 d estimated gestational age (EGA) (term = 145d), weighing 2.5 ± 0.35 kg. Lambs were cannulated using 10-14Fr cannulae for drainage via the right jugular vein and reinfusion via the umbilical vein. Lambs were intubated and lungs were filled with perfluorodecalin to a meniscus with a pressure of 5-8 cm H2O. The first option for transfusion was fetal whole blood from twins followed by maternal red blood cells. Arterial blood gases were used to titrate AP support to maintain fetal blood gas values. RESULTS: The mean survival time on circuit was 119.6 ± 39.5 h. Hemodynamic parameters and lactate were stable throughout. As more adult blood transfusions were given to maintain hemoglobin at 10 mg/dL, the HbF% declined, reaching 40% by post operative day 7. The HbF% was inversely proportional to flow rates as higher flows were required to maintain adequate oxygen saturation and perfusion. CONCLUSIONS: Transfusion of adult blood led to decreased fetal hemoglobin concentration during AP support. The HbF% was inversely proportional to flow rates. Future directions include strategies to decrease the priming volume and establishing a fetal blood bank to have blood rich in HbF.

Prolonged Severe Acute Respiratory Syndrome Coronavirus 2 Replication in an Immunocompromised Patient.

We describe a case of chronic coronavirus disease 2019 (COVID-19) in a patient with lymphoma and associated B-cell immunodeficiency. Viral cultures and sequence analysis demonstrate ongoing replication of infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for at least 119 days. The patient had 3 admissions related to COVID-19 over a 4-month period and was treated twice with remdesivir and convalescent plasma with resolution of symptoms. The patient's lack of seroconversion and prolonged course illustrate the importance of humoral immunity in resolving SARS-CoV-2 infection. This case highlights challenges in managing immunocompromised hosts, who may act as persistent shedders and sources of transmission.

Reported Awareness and Adoption of 2021 Estimated Glomerular Filtration Rate Equations Among US Clinical Laboratories, March 2022.

This study includes clinical laboratories that participated in the first general chemistry proficiency testing survey in 2022 to assess awareness and adoption of new equations from the Chronic Kidney Disease Epidemiology Collaboration for estimated glomerular filtration rate (eGFR) that eliminated race-adjustment factors, including one based on creatinine and one based on creatinine and cystatin C.

Role of food and aeroallergen sensitization in eosinophilic esophagitis in adults.

BACKGROUND: Evaluation of IgE-mediated food sensitivity is frequently performed for patients with eosinophilic esophagitis (EoE). However, the clinical relevance of identifying IgE-mediated sensitivity to foods in adults is unclear. OBJECTIVE: To determine whether EoE associated with food or aeroallergen sensitivity represents a phenotype of EoE with distinct clinical or biological features. METHODS: A medical record review identified 257 patients with a diagnosis of EoE evaluated in the adult allergy clinic at the University of Wisconsin Hospital and Clinics from 2008 to 2013. Patient records were reviewed to capture measures of disease severity, endoscopy results, pathology reports, allergy testing, medical management and patient-reported outcomes. RESULTS: Evaluation of food sensitization with skin prick testing and/or serum IgE was performed for 93% of patients. Sensitization to at least 1 food was identified in 54% of patients who were more likely to report concomitant asthma, allergic rhinitis, eczema, and/or food allergy compared with nonfood sensitive patients. Aeroallergen sensitivity was identified in 87% of patients tested. Clinical characteristics, including EoE symptoms, disease severity, endoscopic findings, peripheral eosinophilia, and patient-reported outcomes, did not differ between food sensitive and non-food sensitive patients. However, on endoscopy, aeroallergen sensitive patients were more likely to have strictures and less likely to exhibit felinization compared with non-aeroallergen sensitized patients. CONCLUSION: Adults with EoE and IgE-mediated food sensitivity are not phenotypically different than non-food sensitive patients. There is no clear clinical utility in identifying food sensitivity in adults with EoE. Further studies are needed to determine whether aeroallergen sensitivity represents a distinct phenotype of EoE.

P2X7-regulated protection from exacerbations and loss of control is independent of asthma maintenance therapy.

RATIONALE: The function of the P2X(7) nucleotide receptor protects against exacerbation in people with mild-intermittent asthma during viral illnesses, but the impact of disease severity and maintenance therapy has not been studied. OBJECTIVES: To evaluate the association between P2X(7), asthma exacerbations, and incomplete symptom control in a more diverse population. METHODS: A matched P2RX7 genetic case-control was performed with samples from Asthma Clinical Research Network trial participants enrolled before July 2006, and P2X(7) pore activity was determined in whole blood samples as an ancillary study to two trials completed subsequently. MEASUREMENTS AND MAIN RESULTS: A total of 187 exacerbations were studied in 742 subjects, and the change in asthma symptom burden was studied in an additional 110 subjects during a trial of inhaled corticosteroids (ICS) dose optimization. African American carriers of the minor G allele of the rs2230911 loss-of-function single nucleotide polymorphism were more likely to have a history of prednisone use in the previous 12 months, with adjustment for ICS and long-acting ß(2)-agonists use (odds ratio, 2.7; 95% confidence interval, 1.2-6.2; P = 0.018). Despite medium-dose ICS, attenuated pore function predicted earlier exacerbations in incompletely controlled patients with moderate asthma (hazard ratio, 3.2; confidence interval, 1.1-9.3; P = 0.033). After establishing control with low-dose ICS in patients with mild asthma, those with attenuated pore function had more asthma symptoms, rescue albuterol use, and FEV(1) reversal (P < 0.001, 0.03, and 0.03, respectively) during the ICS adjustment phase. CONCLUSIONS: P2X(7) pore function protects against exacerbations of asthma and loss of control, independent of baseline severity and the maintenance therapy.

Uncommon causes of hemoglobin E flags identified during measurement of hemoglobin A1c by ion-exchange high-performance liquid chromatography.

We present 3 cases of discordant results from screening hemoglobin A1c (HbA1c) measured by ion-exchange high-performance liquid chromatography (HPLC) all due to various forms of interference and flagged by the instrument as "suspected hemoglobin E (HbE)." The first case was due to a rare hemoglobin variant, later confirmed to be hemoglobin Hoshida, the second due to "true" heterozygous HbE, and the third a result of analytical artifact causing splitting of the HbA1c peak without an underlying variant hemoglobin. We examine the similarities in these cases along with the laboratory work-up to classify each cause of interference to demonstrate the wide array of potential causes for the suspected HbE flag and why it warrants proper work-up. Because there is no standardized method of reporting out hemoglobin variant interference in HbA1c measurement, we discuss our laboratory's process of investigating discordant HbA1c measurements and reporting results in cases with variant interference as 1 possible model to follow, along with discussing the associated laboratory, ethical, and clinical considerations. We also examine the structure of hemoglobin Hoshida, HbE, and conduct a brief literature review of previous reports.

Protection from asthma in a high-risk birth cohort by attenuated P2X(7) function.

BACKGROUND: Viral illnesses are important factors in both asthma inception and exacerbations, and allergic sensitization in early life further enhances asthma risk through unclear mechanisms. Cellular damage caused by infection or allergen inhalation increases ATP levels in the airways with subsequent purinergic receptor activation. The purinergic receptor P2X(7) can enhance airway leukocyte recruitment to the airways, and P2X(7) knockout mice display a reduced asthma-like phenotype. OBJECTIVE: Based on the P2X(7) knockout mouse, we hypothesized that children with low P2X(7) function would have decreased rates of asthma. METHODS: We used a functional assay to determine P2X(7) pore-producing capacity in whole-blood samples in a birth cohort at high risk for asthma development. The P2X(7) assay was validated with known loss-of-function alleles in human subjects. P2X(7) pore status categorization was used to assess asthma and allergy status in the cohort. RESULTS: Attenuated P2X(7) function was associated with lower asthma rates at ages 6 and 8 years, and the greatest effects were observed in boys. Children with asthma at age 11 years who had low P2X(7) capacity had less severe disease in the previous year. Attenuated P2X(7) function was also associated with sensitization to fewer aeroallergens. CONCLUSION: P2X(7) functional capacity is associated with asthma risk or disease severity, and these relationships appear to be age related.

Retrospective analysis of serum free light chain reference intervals and risk for monoclonal gammopathy suggests different limits than those in international guidelines.

OBJECTIVES: Recent reference interval studies of the serum free light chain (FLC) test using contemporary instruments display divergence with the diagnostic range generally adopted as the international standard. In this study, we perform a retrospective reference interval analysis with risk predictions for monoclonal gammopathy. METHODS: Retrospective laboratory and clinical data for 8,986 patients were included in the study. Reference intervals were generated against a set of inclusion/exclusion criteria for two time periods representing the use of different instruments. The presence of monoclonal gammopathy was established from diagnostic test interpretations and EHR diagnosis codes in the patient problem lists and medical history. RESULTS: The 95% FLC ratio reference intervals were 0.76-2.38 for SPAPLUS®, and 0.68-1.82 for Optilite® instruments. These intervals varied considerably from the current diagnostic range of 0.26-1.65 and mapped approximately to the FLC ratios beyond which risk of monoclonal gammopathy substantially increased. CONCLUSIONS: These findings corroborate recent reference interval studies and support recommendations for independent re-evaluation of intervals by institutions as well as an update of international guidelines.

Urine Protein Immunofixation Electrophoresis: Free Light Chain Urine Immunofixation Electrophoresis Is More Sensitive than Conventional Assays for Detecting Monoclonal Light Chains and Could Serve as a Marker of Minimal Residual Disease.

BACKGROUND: Immunoglobulin monoclonal light chains (MLCs) in serum and urine are markers for monoclonal gammopathy and could serve as markers of minimal residual disease (MRD) in multiple myeloma (MM). Excretion of MLCs in urine is known to result in renal damage and shorter survival in patients with LC-predominant MM. METHODS: Retrospective review of urine immunofixation in 1738 specimens at 3 medical centers was conducted to assess the utility of urinalysis for diagnosis and monitoring of monoclonal gammopathy. We tested 228 stored urine specimens via the modified urine immunofixation method, using antisera to assay free LCs (FLCs). RESULTS: Our review of urine immunofixation results and medical records validated the theory that the only meaningful value-added finding was detection of monoclonal free light chains. Examination of 228 urine specimens using our novel method revealed 18.4% additional positive results. The rate of incremental findings for lambda LCs was nearly 3-fold higher than for kappa LCs. CONCLUSIONS: The new method of urine immunofixation is significantly more sensitive and more efficient than the conventional method for detecting MLCs in urine. The new assay appears to be sensitive enough to prove that MLCs serve as a marker of MRD in MM.

Wild-type SARS-CoV-2 neutralizing immunity decreases across variants and over time but correlates well with diagnostic testing.

Importance: The degree of immune protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants provided by infection versus vaccination with wild-type virus remains unresolved, which could influence future vaccine strategies. The gold-standard for assessing immune protection is viral neutralization; however, few studies involve a large-scale analysis of viral neutralization against the Omicron variant by sera from individuals infected with wild-type virus. Objectives: 1) To define the degree to which infection versus vaccination with wild-type SARS-CoV-2 induced neutralizing antibodies against Delta and Omicron variants.2) To determine whether clinically available data, such as infection/vaccination timing or antibody status, can predict variant neutralization. Methods: We examined a longitudinal cohort of 653 subjects with sera collected three times at 3-to-6-month intervals from April 2020 to June 2021. Individuals were categorized according to SARS-CoV-2 infection and vaccination status. Spike and nucleocapsid antibodies were detected via ADVIA Centaur® (Siemens) and Elecsys® (Roche) assays, respectively. The Healgen Scientific® lateral flow assay was used to detect IgG and IgM spike antibody responses. Pseudoviral neutralization assays were performed on all samples using human ACE2 receptor-expressing HEK-293T cells infected with SARS-CoV-2 spike protein pseudotyped lentiviral particles for wild-type (WT), B.1.617.2 (Delta), and B.1.1.529 (Omicron) variants. Results: Vaccination after infection led to the highest neutralization titers at all timepoints for all variants. Neutralization was also more durable in the setting of prior infection versus vaccination alone. Spike antibody clinical testing effectively predicted neutralization for wild-type and Delta. However, nucleocapsid antibody presence was the best independent predictor of Omicron neutralization. Neutralization of Omicron was lower than neutralization of either wild-type or Delta virus across all groups and timepoints, with significant activity only present in patients that were first infected and later immunized. Conclusions: Participants having both infection and vaccination with wild-type virus had the highest neutralizing antibody levels against all variants and had persistence of activity. Neutralization of WT and Delta virus correlated with spike antibody levels against wild-type and Delta variants, but Omicron neutralization was better correlated with evidence of prior infection. These data help explain why 'breakthrough' Omicron infections occurred in previously vaccinated individuals and suggest better protection is observed in those with both vaccination and previous infection. This study also supports the concept of future SARS-CoV-2 Omicron-specific vaccine boosters.

Transepidermal water loss rises before food anaphylaxis and predicts food challenge outcomes.

BACKGROUNDFood allergy (FA) is a growing health problem requiring physiologic confirmation via the oral food challenge (OFC). Many OFCs result in clinical anaphylaxis, causing discomfort and risk while limiting OFC utility. Transepidermal water loss (TEWL) measurement provides a potential solution to detect food anaphylaxis in real time prior to clinical symptoms. We evaluated whether TEWL changes during an OFC could predict anaphylaxis onset.METHODSPhysicians and nurses blinded to the TEWL results conducted and adjudicated the results of all 209 OFCs in this study. A study coordinator measured TEWL throughout the OFC and had no input on the OFC conduct. TEWL was measured 2 ways in 2 separate groups. First, TEWL was measured using static, discrete measurements. Second, TEWL was measured using continuous monitoring. Participants who consented provided blood samples before and after the OFCs for biomarker analyses.RESULTSTEWL rose significantly (2.93 g/m2/h) during reactions and did not rise during nonreacting OFCs (-1.00 g/m2/h). Systemic increases in tryptase and IL-3 were also detected during reactions, providing supporting biochemical evidence of anaphylaxis. The TEWL rise occurred 48 minutes earlier than clinically evident anaphylaxis. Continuous monitoring detected a significant rise in TEWL that presaged positive OFCs, but no rise was seen in the OFCs that resulted in no reaction, providing high predictive specificity (96%) for anaphylaxis against nonreactions 38 minutes prior to anaphylaxis onset.CONCLUSIONSDuring OFCs, a TEWL rise anticipated a positive clinical challenge. TEWL presents a monitoring modality that may predict food anaphylaxis and facilitate improvements in OFC safety and tolerability.

Rhinovirus-induced IL-1ß release from bronchial epithelial cells is independent of functional P2X7.

Airway epithelial cell defenses to viral infections are often compromised in disease or injury. Danger molecules, including ATP, are released during infection and contribute to nucleotide receptor-dependent inflammatory responses, largely through P2X(7). Although respiratory epithelium has been shown to express a variety of nucleotide receptors, the functional contribution of P2X(7) to the epithelial cell inflammatory response is unclear. We used human donor bronchial epithelial cells (BECs) and primary brushed epithelium to explore responses upon nucleotide and Toll-like receptor stimulation. P2X(7) messenger RNA and protein were observed in unprimed BECs, whereas inflammatory cytokine stimulation increased both messenger RNA and protein. Functional pore activity characteristic of P2X(7) was observed in BECs, and IL-1ß was rapidly released by BECs after Toll-like receptor 3 agonist, polyinosine-polycytidylic acid, priming followed by ATP administration, although no change was observed in IL-18 release. BECs produced more IL-1ß after stimulation with polyinosine-polycytidylic acid than LPS, showing a different preferential response than monocytes. In addition, blockade of nucleotide receptors with oxidized ATP significantly increased human rhinovirus (HRV) recovered 24 hours after infection in BECs, whereas 2'-3'-O-(4-benzoylbenzoyl) ATP treatment of brushed epithelial cells and respiratory cell lines nonsignificantly decreased HRV recovery. IL-1ß release was detected after HRV infection in both BECs and brushed cells, but BzATP did not significantly increase IL-1ß release further. BEC processing of pro-IL-1ß to the mature, cleaved, 17-kD form was confirmed by Western blotting. These results support the expression of functional P2X(7) in human lung epithelium, although its role in epithelial pathogen defense is likely independent of IL-1 family cytokine processing.

Use of Selective Arterial Calcium Stimulation Testing in Identification of Insulinoma in a Patient After Bariatric Surgery.

OBJECTIVE: Insulinomas are rare in the post-bariatric surgery setting. The differential diagnosis for hypoglycemia is broad, requiring laboratory testing to verify endogenous hyperinsulinemic hypoglycemia. Selective arterial calcium stimulation testing (SACST) can help localize abnormal insulin production. We describe a patient with histologically confirmed insulinoma after bariatric surgery diagnosed with the aid of SACST. METHODS: We present a 67 year old woman with a history of Roux-en-Y bypass surgery who presented with endogenous hyperinsulinemic hypoglycemia. Initially, no pancreatic lesion was identified radiologically. We pursued SACST to localize the source of insulin production. RESULTS: The SACST successfully localized the source of hyperfunctioning islet cells to the pancreatic tail with absolute insulin values in a range consistent with insulinoma. Additional radiologic studies showed a small tumor in the pancreatic tail. Pathology showed a well-differentiated neuroendocrine tumor, compatible with insulinoma. CONCLUSION: This case study illustrates the usefulness of SACST for the diagnosis and localization of insulinoma.

Accurate Quantification of Monoclonal Immunoglobulins Migrating in the Beta Region on Protein Electrophoresis.

BACKGROUND: The concentration of monoclonal immunoglobulins (Igs) in neoplastic monoclonal gammopathic manifestations is generally measured by densitometric scanning of the monoclonal peaks on gel or by measuring absorbance at 210 nm in capillary electrophoresis (CE). For monoclonal Igs migrating in the beta region, measurement is complicated by the major beta-region proteins, namely, transferrin and C3. METHODS: C3 interference in densitometry was eliminated by heat treatment of serum, and monoclonal Igs were quantified by densitometry of the residual band. The immunochemical measurement of transferrin was converted to its equivalent densitometric quantity. For monoclonal Ig migrating with transferrin, the contribution of the latter was removed by subtracting the converted transferrin concentration from the combined densitometric quantification of the band. With CE, monoclonal Ig was measured by using immunosubtraction (ISUB) to guide demarcation. RESULTS: The results obtained using the C3 depletion and transferrin subtraction method were lower and yet comparable to the results derived from using CE measurement guided by ISUB. As we expected, the results from both methods were lower than those derived from a perpendicular drop measurement of the peak or via nephelometric assay of the involved isotype. DISCUSSION: Accurate measurement of monoclonal Igs is important for the diagnosis and monitoring of monoclonal gammopathic manifestations. Determination of serum free light chain concentration per gram of monoclonal Ig is an essential measure for the diagnosis of light chain-predominant multiple myeloma. The method described herein improves accuracy of measurements for monoclonal Igs migrating in the beta region, without the need for special reagents or equipment.