Lab Clin Med Research Articles

Incidence of Typical Neutrophil Count with Fy(a-b-) Status Among Hematology Referrals for Neutropenia at an Urban Safety-Net Hospital

Typical neutrophil count with Fy(a-b-) status, known variously as Duffy null phenotype or Benign Ethnic Neutropenia, causes asymptomatic neutropenia without clinical sequelae.[i][ii] Fy(a-b-) phenotype is correlated to homozygosity of a single nucleotide polymorphism in the promoter region of atypical chemokine receptor 1 (ACKR1; formerly known as DARC), a decoy chemokine receptor, and may protect against malaria infection.[iii][iv] This genotype is enriched in malaria-endemic regions.[v] 25% to 50% of people of African ancestry in the United States are thought to have Fy(a-b-) status and are often erroneously identified as having pathologically low neutrophil counts. We performed a retrospective chart review of new neutropenia referrals to Hematology Clinic at Boston Medical Center (BMC) to evaluate diagnostic patterns for typical neutrophil count with Fy(a-b-) status. Overall, 103 new referrals for evaluation of neutropenia were made to BMC Hematology Clinic from 1/2020 to 2/2022, of which 78 were included for further analysis. Demographic and laboratory data are summarized in the accompanying Table. Typical neutrophil count with Fy(a-b-) status was identified as the primary etiology for neutropenia in 64.1% (50 of 78 referrals), of which 41 (82%) identified as African American or Black or were born in an African or Caribbean country. Of patients suspected of having Duffy null phenotype, 66% (33 of 50 patients) underwent confirmatory blood bank testing, and 97% (32 of 33) of these patients were found to have Fy(a-b-) status. Average cost of laboratory visit was calculated for each new referral using the Outpatient Price Transparency Tool from BMC Billing Department, which is based on chargemaster prices for the hospital. Patients with typical neutrophil count with Fy(a-b-) status on average had lower, but not negligible, cost of workup ($363.82 vs $689.46; p<.005 by unpaired student's T test assuming unequal variance) and were statistically less likely to have a follow up appointment (p=0.039 by unpaired student's T test assuming unequal variance) compared to those without. Though this phenotype reflects normal variation of white cell count in non-White populations, it is a "...label that illustrates how common phenotypes seen in non-White individuals continue to be considered nonstandard in the medical community." Workup of referrals that have typical neutrophil count with Fy(a-b-) status is not currently recommended, unless they have a change in their baseline white cell count or new, atypical features concerning for a secondary etiology.[vi] In our study, asymptomatic referrals were the majority and represent an unnecessary cost burden on an overtaxed healthcare system. Fy(a,b) antigen testing provides a low-cost alternative to hematology referral for confirmation of Fy(a-b-) status for patients or providers who require reassurance. Expanded use of serological Fy(a,b) antigen testing by primary care providers for patients with ancestry from malaria-endemic regions and chronic, asymptomatic neutropenia could reduce cost of care and referrals to Hematology clinic. [i] Ortiz MV, Meier ER, Hsieh MM. Identification and Clinical Characterization of Children With Benign Ethnic Neutropenia. J Pediatr Hematol Oncol. 2016 Apr;38(3):e140-3. doi: 10.1097/MPH.0000000000000528. PMID: 26925714; PMCID: PMC5102334. [ii] Merz LE, Achebe M. When non-Whiteness becomes a condition. Blood. 2021 Jan 7;137(1):13-15. doi: 10.1182/blood.2020008600. PMID: 33181819. [iii] Reich D et al. Reduced neutrophil count in people of African descent is due to a regulatory variant in the Duffy antigen receptor for chemokines gene. PLoS Genet. 2009 Jan;5(1):e1000360. doi: 10.1371/journal.pgen.1000360. Epub 2009 Jan 30. PMID: 19180233; PMCID: PMC2628742. [iv] Miller LH, Mason SJ, Clyde DF, McGinniss MH. The resistance factor to Plasmodium vivax in blacks. The Duffy-blood-group genotype, FyFy. N Engl J Med. 1976 Aug 5;295(6):302-4. doi: 10.1056/NEJM197608052950602. PMID: 778616. [v] Haddy TB, Rana SR, Castro O. Benign ethnic neutropenia: what is a normal absolute neutrophil count? J Lab Clin Med. 1999 Jan;133(1):15-22. doi: 10.1053/lc.1999.v133.a94931. PMID: 10385477. [vi] Gibson C, Berliner N. How we evaluate and treat neutropenia in adults. Blood (2014) 124 (8): 1251-1258. https://doi.org/10.1182/blood-2014-02-482612 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Alcohol exacerbated biochemical and biophysical alterations in liver mitochondrial membrane of diabetic male wistar rats – A possible amelioration by green tea

Alcohol exacerbated biochemical and biophysical alterations in liver mitochondrial membrane of diabetic male wistar rats – A possible amelioration by green tea

Тромботические события при редких формах генетических тромбофилий

Цель исследования: изучить лабораторный фенотип редких мутаций Лейдена [FVL А(1961)A] и гена протромбина [FII А(20210)A] при их клинической реализации в виде тромбозов. Материалы и методы. Проведено многоцентровое проспективное наблюдательное исследование, включающее 80 носителей редких генетических форм тромбофилий: FVL А(1961)A (n = 31), FII А(20210)A (n = 10) и компаунд FII G(20210)A + FVL G(1691)A (n = 39). У всех пациентов исследована резистентность фактора Va к активированному протеину С (APC-R) по нормализованному отношению (НО) и уровень активности протромбина в плазме крови в состоянии физического здоровья и в случае эпизода тромботического события. Результаты. Определена ассоциативная связь развития тромботических событий со снижением НО при оценке APC-R как у гомозиготных носителей мутации Лейден (Ме = 0,35; 95% ДИ = 0,31–0,37 по НО), так и гетерозиготных компаундов (Ме = 0,43; 95% ДИ = 0,42–0,45 по НО). Заключение. Лабораторный мониторинг пациентов с редкими формами генетических тромбофилий позволяет выделить группу высокого тромбогенного риска, нуждающуюся в пролонгированной тромбопрофилактике. Objectives: to study the laboratory phenotype of rare Leiden mutation [FVL A(1961)A] and the prothrombin gene mutation [FII А(20210)A] implementating clinically as thrombosis. Patients / Methods. A multicenter prospective observational study included 80 carriers of rare genetic kinds of thrombophilia: FVL A(1961)A (n = 31), prothrombin FII А(20210)A (n = 10) and the compound FII G(20210)A + FVL G(1691)A (n = 39). All patients were tested for the factor Va resistance to activated protein C (APC-R), normalized ratio (NR), and the plasma prothrombin activity (%) in healthy conditions and in the case of thrombotic event. Results. An associative relation between the development of thrombotic events and a decrease in NR was determined when assessing APC-R, both in homozygous carriers of the Leiden mutation (Me = 0.35; 95% CI = 0.31–0.37 in NR) and heterozygous compounds (Me = 0.43; 95% СI = 0.42–0.45 in NR). Conclusions. Laboratory follow up for patients with rare kinds of genetic thrombophilias allows to identify a group at high thrombogenic risk requiring prolonged thromboprophylaxis.

The inflammatory cytokine, interleukin‐1‐beta, drives central immune cell infiltration, changes in sympathetic nerve activity and blood pressure

IntroductionThe area postrema (AP) is a brain region that lacks a blood brain barrier allowing blood‐borne factors to access the brain. Since the AP is connected to numerous brain regions that play a key role blood pressure regulation, we hypothesised that blood‐borne inflammatory factors such as interleukin‐1 beta (IL‐1β) might play a role in the development of hypertension. In support of this hypothesis, plasma Interleukin‐1 beta is increased in hypertensive patients (Dalekos et al., 1997, J Lab Clin Med,129: 300–8). Furthermore, the receptors for IL‐1β are abundantly expressed in the AP. Here we test whether IL‐1β can act on the AP to cause an increase in blood pressure and renal sympathetic nerve activity (RSNA) in rats.MethodsBlood pressure and renal sympathetic nerve activity (RSNA) were recorded and analyzed after microinjection of different doses of IL‐1β into the AP (10ng and 25ng) of anaesthetised rats. In order to show that IL‐1β, specifically causes an increase in blood pressure, specific IL‐1 receptor blocking antibodies (IL‐1Ra) were injected into the AP prior to a 25ng IL‐1β injection. The AP was extracted for fluorescent activated cell sorting (FACS).ResultsExperiments showed that microinjection of 25ng IL‐1β caused a significant (p&lt;0.01; Mann‐Whitney test) increase in both systolic blood pressure (19.6 ± 3.6 mmHg) and heart rate (55 ±10 bpm) compared with control injections of saline (SBP: 5.47 ± 1.58; HR 12 ± 6). Pre‐treatment with IL‐1Ra attenuated the response to IL‐1β, and was comparable with saline controls. IL‐1β also caused an increase in RSNA (137% ± 43%) and FACS revealed an increase in immune cell infiltration. Immunohistochemistry also revealed that IL‐1β injection results in Fos activation in the AP.ConclusionsIL‐1β injection appears to activate neurons in the area postrema which leads to an increase in blood pressure. By providing new insight into the actions of IL‐1β in the brain, we show that IL‐1β might be a potential target for future treatments for hypertension.Support or Funding InformationAustralian Research Council (FT170100363)National Health and Medical Research Council (GNT 1079680)Rebecca L Cooper Medical Research Foundation

Characterization of drug-neutralizing antibodies in patients with Fabry disease during infusion

Characterization of drug-neutralizing antibodies in patients with Fabry disease during infusion

The spleen dictates platelet destruction, anti-platelet antibody production, and lymphocyte distribution patterns in a murine model of immune thrombocytopenia.

For many years, splenectomy has been used to treat immune thrombocytopenia (ITP), and this procedure benefits approximately two-thirds of the treated patients. Although splenectomy may raise platelet counts, antibody-coated platelets and cytotoxic T lymphocytes appear to persist or can change over time. To better understand how the spleen may affect anti-platelet immune responses, we used a murine model of ITP demonstrating both antibody-mediated and T lymphocyte-mediated thrombocytopenia. Mice with severe combined immunodeficiency (SCID) were either splenectomized or not and transfused with splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61(+) platelets. Platelet counts and anti-platelet antibody levels were performed weekly. After 4weeks, the mice were sacrificed, and lymphoid organs were harvested and examined by flow cytometry to quantify CD4(+)CD25(+)FoxP3(+) Tregs and conventional cross-presenting XCR1(+) and tolerizing SIRPα+ dendritic cells. The results indicate that compared with control non-splenectomized mice, thrombocytopenia was improved and anti-platelet antibody production was significantly diminished in all splenectomized mice that received immune splenocytes. Splenectomized SCID mice also had a marked reduction in Tregs in the thymus together with an increased proportion of both thymic dendritic cell subsets that correlated with increased platelet counts. Of interest, although splenectomy diminished anti-platelet antibody production and raised platelet counts, marrow megakaryocyte densities were still significantly reduced in mice that received immune splenocytes. These results suggest that the spleen in murine ITP not only is the primary site responsible for platelet destruction, but it also controls, to a significant extent, the antibody response against platelets and the migration patterns of lymphocyte subsets.

Adjunct Therapies In Type 1 Diabetes

The arrival of insulin in 1921 initiated a revolution in the treatment of type 1 diabetes (T1D), as it was the first rational treatment in such patients ( 1. Banting F.G. Best C.H. Internal secretion of pancreas. J Lab Clin Med. 1922; 7: 465-480 Google Scholar ). The dramatic improvements in the clinical condition of these patients led to the most rapid award of a Nobel Prize in medicine in 1923 ( 2. Rosenfeld L. Insulin: discovery and controversy. Clin Chem. 2002; 48: 2270-2288 Crossref PubMed Scopus (176) Google Scholar ). It was expected that all problems of this condition would thus be solved. However, as defined in the article by Bode and Garg in this issue of Endocrine Practice, problems with the management of T1D continue, including the unpredictable variability of blood glucose concentrations, diabetic ketoacidosis (DKA), and long-term micro-angiopathic and macro-angiopathic complications. In addition, a recent study based on the Swedish database demonstrated a marked and alarming increase of mortality in this patient population when compared normal subjects ( 3. Lind M. Svensson A.M. Kosiborod M. et al. Glycemic control and excess mortality in type 1 diabetes. N Engl J Med. 2014; 371: 1972-1982 Crossref PubMed Scopus (539) Google Scholar ). Clearly, we need to improve upon the current standards of care, as only 20% of this population is at the hemoglobin A1c (HbA1c) goal of <7%. Furthermore, 40 to 50% of patients with T1D now have metabolic syndrome, necessitating the use of adjuvant therapies that have complementary benefits of weight loss, insulin dose reduction, and blood pressure lowering, in addition to glycemic control ( 4. KuhadiyaND, DhindsaS, GhanimH, MehtaA, et al. Liraglutide as additional treatement to insulin in patients with type 1 diabetes mellitus: a randomized placebo-controlled clinical trial. AACE 2014 Late Breaking Abstract. Google Scholar ).

Subclinical pulmonary congestion is prevalent in nephrotic syndrome.

In patients with nephrotic syndrome (NS), the lung is considered an organ protected from the risk of edema. However, data on objectively measured lung water in NS patients is lacking. Here we measured lung water by an ultrasound (US) technique as well as by transthoracic impedance in 42 asymptomatic patients with active NS, in 14 stage G5D CKD patients on chronic hemodialysis, and in 21 healthy individuals. In patients with active NS, the median number of US-B lines (a metric of lung water) after 5 min in a supine position was significantly higher (12; interquartile range: 7-25) compared with that in healthy individuals (4; 2-9) but similar to that in hemodialysis patients (23; 10-39). The difference between NS patients and healthy individuals was significantly amplified (16; 10-35 vs. 4; 2-9) after 60 min of supine resting and significantly attenuated after 5 min of standing (10; 7-25 vs. 3; 1-6). Posture-dependent changes in lung water in patients with active NS were significantly accentuated compared with both hemodialysis patients and healthy individuals. After NS remission, the number of US-B lines was significantly reduced to 5 (4-18) at 5 min and to 6 (5-22) at 60 min approaching the normal range. Lung congestion in patients with active NS was confirmed by transthoracic impedance. Thus, asymptomatic pulmonary congestion is pervasive in patients with NS. A clinical trial is needed to assess the utility of lung US for the management of patients with NS.

Commonly Used Clinical Intravenous Fluid Formulations Differentially Affect Sickle Red Blood Cell Stiffness and Transit Time

Background:Disruption of red blood cell (RBC) volume regulation and water homeostasis is a major component of sickle cell pathophysiology. As such, hydration is a mainstay of prevention and treatment of vaso-occlusive crises (VOC) in patients with sickle cell disease (SCD). However, evidence for guiding clinicians' choice for intravenous (IV) fluid hydration in the acute setting is lacking (Okomo, Coch Data Syst Rev, 2012). Pediatric hematologists typically discourage rapid infusion of hypotonic fluid given the risk of hyponatremia, although such fluids as 5% dextrose with 34meq/L or 77meq/L sodium are often used after stabilizing various acute clinical situations relevant to SCD. Although altered sodium concentration and osmolarity have been shown to affect erythrocyte swelling and rheology, dextrose-containing fluids used clinically such as D5 ¼ and D5 ½ normal saline (NS), have not been studied (Reinhart et al., Mic Res, 2015; Hijiya et al., J Lab Clin Med, 1991). To those ends, we sought to investigate the effect of different clinically relevant IV fluid formulations on normal and sickle RBC stiffness and deformability.Methods:Fresh blood was obtained from healthy volunteers and patients with sickle cell anemia (SS) on hydroxyurea and not transfused for at least 100 days. Sterile, clinical grade fluids stored at room temperature were used for the experiments (Baxter, Figure 1A). Our laboratory has previously published a description of a microfluidic device comprised of multiple parallel microchannels 5μm wide recapitulating the in vivo geometry of capillaries (Rosenbluth et al., Lab Chip, 2008). This microvasculature-on-a-chip enables measurements of single-RBC transit times, which correlate with cell stiffness (Figure 1B,C). For the transit time experiments, a master silicon wafer was used to mold the microfluidic channels out of polydimethylsiloxane (PDMS) silicone. Centrifuged RBCs were washed with phosphate-buffered saline (PBS) and then diluted to 0.5% hematocrit (HCT) in the various fluids prior to flow. Cell suspensions were perfused into a microfluidic device pre-coated with 2% bovine serum albumin (BSA) at an average linear flow rate of 0.50 mm s-1 in the smallest channels with a syringe pump (Harvard Apparatus) and then imaged at 20x at 20 frames per second. Images were recorded for future analysis. For the experiments assessing RBC shape, washed RBCs were diluted with PBS to 0.5% HCT and imaged at 40x in plastic wells at time 0. PBS was removed and 100 μL of each clinical fluid formulation was added to the wells and images were then obtained over time. RBC circularity was calculated using custom-written scripts in Matlab (Figure 1D).Results:RBC transit times in both healthy and sickle blood were affected by osmolarity and the various solute concentrations (Figures 2A,B). Transit times of sickle RBCs in all IV fluid formulations were significantly higher, over 10 times, than that of RBCs from healthy donors. Transit times for both healthy and sickle donors were least in the D5 ¼ NS solution. Of note, sickle RBC transit time was greatest in the NS solution. Sickle RBC circularity also changed with solute concentration and osmolarity with statistical significance (Figure 2C). Cells with the highest change in circularity from baseline were also those exposed to the D5 ¼ NS solution.Conclusion:Our results suggest the stiffness of sickle RBCs is affected by different formulations of clinical IV fluids. Increased transit time of sickle RBCs in NS through our device may in part be explained by the decreased circularity, indicating that RBCs adopt more irregular shapes in this fluid. This, in turn, could lead to increased propensity of microchannel obstruction. Although the exact mechanisms are unclear, this begs the question of whether NS is an appropriate choice for initial fluid resuscitation for VOC and other SCD-related complications as it could exacerbate the already high stiffness and shape irregularity of sickle RBCs, further increasing microvascular occlusion. As these in vitro results have significant clinical implications, ongoing experiments involve investigating how these fluid-dependent effects may alter sickle RBC adhesion in 'endothelialized' microfluidic devices, how different oxygen tensions affect these fluid-mediated effects, potential differences on and off of hydroxyurea, and the underlying mechanisms of this IV fluid formulation-dependent effect. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Global Contributions to the Understanding of DNA Repair and Skin Cancer

Living organisms exposed to sunlight have developed mechanisms to repair the damage that UVR induces in their DNA. These DNA repair systems are present in most life forms including bacteria, yeast, rodents, and mammals. Three rare human genetic diseases have defects in the DNA repair system called nucleotide excision repair (NER). Patients with xeroderma pigmentosum (XP) have defective NER and a more than 10,000-fold increased frequency of skin cancer, whereas patients with Cockayne syndrome (CS) or with trichothiodystrophy (TTD) have NER defects without increase in cancer. These are strikingly different clinical phenotypes (Figure 1). The dramatic differences in their manifestations reflect the complexity of DNA repair and the broad scope and central role of DNA repair on preservation of diverse biological functions. This article will review the major milestones in our understanding of DNA repair and skin cancer (Table 1). These include recognition of the clinical diseases, understanding the types of damage sunlight causes in the DNA, recognition of DNA repair in different organisms, discovery of defective DNA repair in humans, cloning of multiple human DNA repair genes, and identification of UV-type mutations in skin cancer.

Homozygosity for the V122I Mutation in Transthyretin Is Associated with Earlier Onset of Cardiac Amyloidosis in the African American Population in the Seventh Decade of Life

Individuals heterozygous for the V122I mutation in transthyretin (TTR) tend to develop cardiac amyloidosis, often after the seventh decade of life. Although homozygotes have been reported, these have typically been single case reports. We report a cohort of 13 V122I homozygotes. TTR gene sequencing results from the Mayo Clinic Molecular Genetics Laboratory between September 2004 and January 2013 were reviewed; 177 heterozygotes and 13 homozygotes for the V122I alteration were identified. Detailed clinical history was available for the 24 heterozygotes seen at Mayo Clinic. We compared age at onset of disease for this group to homozygotes, both alone and pooled with the 11 homozygotes from the literature. Individuals with homozygous V122I manifested symptoms a mean of 10 years earlier than heterozygotes (63.8 ± 5.7 versus 72 ± 8.1 yrs, P = 0.0002). Further, males were significantly overrepresented in both heterozygous and homozygous individuals. There was a trend for an even higher male bias in the homozygous group. All 24 homozygotes were African American, whereas four of the heterozygotes were reported as white. Two novel V122I compound heterozygotes were also identified, with clinical presentation in the late fifth or early sixth decade of life. This study is the largest homozygous V122I cohort reported and demonstrates association with earlier age at onset. It also highlights the uncertain penetrance, particularly with respect to sex.

THU0021 DNA Methylation of Peripheral Blood Cell Subpopulations in Patients with Early Rheumatoid Arthritis before and after Methotrexate Treatment

BackgroundEpigenetic regulation influences gene expression and can affect disease course and treatment response. Global DNA methylation of T cells from patients with RA is decreased1. This hypomethylation could favour inflammation...

Pharmacodynamics of Platelet Inhibition with Aspirin: Feasibility for Detection of Suboptimal Platelet Inhibition

AbstractAbstract 4361 Introduction:Patients with diabetes mellitus and or metabolic syndrome may or may not be maximally protected from future vascular events with aspirin. To be maximally protected at least 90% of platelets should be inhibited by aspirin (aspirinated). Since platelet acetylation by aspirin is limited to the first 30 minutes after ingestion, in conditions associated with increased platelet production the percentage of aspirinated platelets could be diluted to less than 90% with the usual daily dosing. We studied the pharmacodynamics of aspirin inhibition of platelets to evaluate the feasibility of developing a metric that would detect suboptimal aspirin inhibition of platelets. Methods:The slope of the platelet prostaglandin agonist (PPA) stimulated light aggregometry curve was used to gage the degree of platelet inhibition with aspirin.(Schwartz J. Lab Clin Med 2002; 139:227) Seven normal subjects were studied. After observed ingestion of 325mg of aspirin, the degree of platelet inhibition was studied at the following post aspirin time points; 0, 8hrs, 24hrs and 48hrs. Ex-vivo dilution curves were constructed using time 0 as 100% non-aspirinated and 1hr post aspirin as 100% aspirinated under the assumption that over 24hrs 10% of platelets are newly formed and non-aspirinated. Statistical evaluations were performed for each subject studied using both within subject comparisons and considering the group as a whole, between-subject comparisons.Results: Within group comparisons:A linear regression analysis comparing the in-vivo PPA aggregometry slopes with the PPA slopes obtained via ex-vivo dilutions demonstrated that the ex-vivo dilution curves for a particular subject reliably predicted the degree of aspirin inhibition observed in-vivo for that subject at the following time points with their corresponding percentages of aspirinated platelets; 8hrs (97% aspirinated) 24hrs (90% aspirinated) and 48hrs (80% aspirinated) (r2=0.6, p< 0.001). In addition, a direct in group comparison showed no differences between the in-vivo PPA slopes and those obtained with ex-vivo dilutions with 8 hr. and 48 hr. time points (p=0.16 and p=0.31, respectively), but a difference was observed at the 24 hr. time point (p=0.02). Entire group comparisons: Platelet inhibition with aspirin was highly variable among subjects. When analyzed as a group, the ex-vivo dilution curves were not predictive of the slopes obtained in-vivo. The PPA slopes observed in-vivo were significantly different from those obtained from the ex-vivo dilution curves at all of the time points (p<0.05). Conclusions:1. In normal subjects the ex-vivo dilution curve for a particular subject reliably predicts the degree of platelet inhibition observed in-vivo for that subject. 2. The variation in aspirin inhibition among subjects is large and suggests that the development of a metric to detect suboptimal platelet inhibition with aspirin is only possible if the individual subjects serve as their own control. Disclosures:No relevant conflicts of interest to declare.

Effects of Preoperative Intravenous Erythropoietin Plus Iron on Outcome in Anemic Patients After Cardiac Valve Replacement

Preoperative anemia is a risk factor for postoperative morbidity and in-hospital mortality in cardiac surgery. However, it is not known whether treatment of anemia before cardiac surgery by administering recombinant human erythropoietin (rhEPO) plus iron improves postoperative outcomes and decreases red blood cell transfusions in these patients. In 1998 a collection of consecutive data for patients who underwent valve replacement was initiated and the inclusion criterion was anemia. Treatment with rhEPO was given at a dose of 500 IU/kg/day every week for 4 weeks and the fifth dose 48 hours before valve replacement. During each rhEPO session, patients received intravenous iron sucrose supplementation. The intervention cohort (2006 to 2011) included 75 patients and the observation cohort was composed of 59 patients who did not receive any treatment (1998 to 2005). Multivariable logistic regression analysis showed that administration of combined therapy was independently associated with decreased postoperative morbidity (odds ratio [OR] 0.13, 95% confidence interval [CI] 0.03 to 0.59 p = 0.008) and in-hospital mortality (OR 0.16, 95% CI 0.28 to 0.95 p = 0.04) after adjusting for logistic European System for Cardiac Operative Risk Evaluation score, type of intervention, time of cardiopulmonary bypass, and year of surgery. Individually, this treatment also decreased postoperative renal failure (OR 0.23, 95% CI 0.06 to 0.88, p = 0.03). Rate of red blood cell transfusion decreased from 93% in the observation cohort to 67% in the intervention cohort as did days of hospitalization (median, 15 days, 10 to 27, versus 10 days, 8 to 14, respectively, p = 0.01 for all comparisons). In conclusion, administration of intravenous rhEPO plus iron in anemic patients before valve replacement improves postoperative survival, decreases blood transfusions, and shortens hospitalization.

Deletion of the epidermal growth factor receptor in renal proximal tubule epithelial cells delays recovery from acute kidney injury

To determine the role of epidermal growth factor receptor (EGFR) activation in renal functional and structural recovery from acute kidney injury (AKI), we generated mice with a specific EGFR deletion in the renal proximal tubule (EGFRptKO). Ischemia–reperfusion injury markedly activated EGFR in control littermate mice; however, this was inhibited in either the knockout or wild-type mice given erlotinib, a specific EGFR tyrosine kinase inhibitor. Blood urea nitrogen and serum creatinine increased to a comparable level in EGFRptKO and control mice 24 h after reperfusion, but the subsequent rate of renal function recovery was markedly slowed in the knockout mice. Twenty-four hours after reperfusion, both the knockout and the inhibitor-treated mice had a similar degree of histologic renal injury as control mice, but at day 6 there was minimal evidence of injury in the control mice while both EGFRptKO and erlotinib-treated mice still had persistent proximal tubule dilation, epithelial simplification, and cast formation. Additionally, renal cell proliferation was delayed due to decreased ERK and Akt signaling. Thus, our studies provide both genetic and pharmacologic evidence that proximal tubule EGFR activation plays an important role in the recovery phase after acute kidney injury.

Control of iron metabolism – Lessons from neonatal hemochromatosis

When hepatologists begin their investigations at the scene of liver damage, iron is certainly on the list of ‘‘usual suspects’’ to be rounded up. Hemochromatosis, a well-known cause of chronic liver disease, is the most prevalent genetic disorder in adults [1]. Serum iron parameters are therefore included in most guidelines on how to investigate patients with elevated transaminases or chronic liver disease [2]. The hemochromatosis (HFE) genotype is an important piece of evidence in patients with liver disease and elevated transferrin saturation with hyperferritinemia, because homozygosity for the C282Y allele of HFE is considered sufficient to diagnose hemochromatosis under these circumstances [1]. Compound heterozygosity for C282Y and H63D has been almost removed from the watch-list and is currently considered a mere risk factor for liver diseases – sufficient to cause hemochromatosis or even cirrhosis only in combination with other health burdens. Hence, a partner in crime for HFE mutation must be found in patients with liver disease and C282Y/H63D compound heterozygosity [3]. If there is evidence for iron overload in the absence of hemochromatosis-associated HFE genotypes and the hepatopathologist finds iron confined to hepatocytes, sharp investigators are well advised to search for the culprit in the genes encoding transferrin receptor 2 (TFR2) and ferroportin (SLC40A1) or even to hunt for combined mutations in these genes if a patient presents with severe iron overload or early-onset disease [4]. If the patient is adolescent or before the 3rd decade of life and presents with heart failure or hypogonadism in addition to – often overlooked – liver disease, other genes will be committed in custody. Mutations in HJV and HAMP, which respectively encode hemojuvelin and hepcidin, will be sufficient to identify juvenile hemochromatosis [5]. Through the power of genetics, iron-related diseases are now subject to close scrutiny. In addition, genetic studies have greatly advanced our understanding of hemochromatosis. This progress is reflected in the proposed re-classification of human iron

Epitope Mapping of a Monoclonal Antibody to Factor VIII That Inhibits Factor IXa:Factor VIIIa Interaction and Thrombin Activation of Factor VIII

Abstract 1177Factor VIII (FVIII) circulates in plasma as a noncovalent heterodimer consisting of a heavy chain (HC, A1-a1-A2-a2-B domains) and a light chain (LC, a3-A3-C1-C2 domains) in a noncovalent complex with von Willebrand factor (wVF). Thrombin (IIa) cleaves FVIII between the A1-a1/A2 domains at Arg372, A2-a2/B domains at Arg740 and B-a3/A3 domains at Arg1689 generating FVIIIa that consists of an A1-a1/A2-a2/A3-C1-C2 heterotrimer. FVIIIa increases the efficiency of Factor IXa (FIXa) catalyzed activation of Factor X (FX) in a Ca2+ and phospholipid (PL) dependent manner. The A3-C1-C2 segment of FVIIIa plays an important role in FIXa:FVIIIa interaction. Here, we describe a series of experiments to map the epitope of a monoclonal antibody (mAb) that is reported to inhibit FVIII clotting activity in a one stage clotting assay (Brown et al; J Lab Clin Med, 101: 793–805, 1983).The binding of mAb to FVIII, B-domain deleted FVIII and isolated LC was assessed using surface plasmon resonance. In these experiments, mAb captured on a protein A/G coupled CM5 sensor chip served as the ligand, and FVIII and its isolated fragments served as the analytes. The Kd of binding of LC (∼40 nM) was similar to FVIII and the B-domain deleted FVIII. No binding was observed for isolated A1 and A2 domains. Further, in plasma based inhibition assays, the Kd of binding of mAb to FVIII-vWF complex and to FVIII was ∼30 nM. This suggests that the mAb epitope does not significantly overlap with the vWF binding site in the acidic a3 region of LC. Western blot analysis confirmed that the mAb is specific for the LC of FVIII. Moreover, IIa-cleaved LC starting at residue 1690 gave only a weak signal and FXa-cleaved LC starting at residue 1721 did not react with the mAb in Western blots. These data suggest that the epitope for this mAb spans the IIa-cleavage site in the LC. Consistent with these observations, the A3-C1-C2 fragment but not the C1-C2 fragment expressed in COS cells reacted with the mAb. To further define a part of the epitope in the IIa-cleaved LC, twelve A3 domain deletion fragments were constructed and expressed in E. coli. Western blot analysis of these fragments restricted the partial epitope to 1690–1710 residues of the IIa-cleaved LC. In additional experiments, the mAb did not inhibit mouse, rabbit or canine plasma FVIII in a one stage clotting assay. It did however inhibit porcine plasma FVIII with ∼40 nM Kd, sheep plasma FVIII with ∼ 68 nM Kd, and bovine plasma FVIII with ∼300 nM Kd. Analysis of the sequence alignment of residues 1680 to 1710 of FVIII from each species indicated that residues 1681 to 1694 of human FVIII most likely constitute the epitope of this mAb. The dissimilarity and the charge differences in amino acids suggest that residues Asp1681, Glu1684, Asn1685, and Ser1687 on the N terminal side and Lys1693 on the C terminal side of the IIa-cleavage site Arg1689-Ser1690 may be important for this epitope.Fluorescence energy transfer (FRET) experiments indicated that the mAb inhibits FIXa interaction with the IIa-cleaved LC consisting of A3-C1-C2 domains. In these experiments, A3-C1-C2 subunit was labeled with acrylodan (fluorescence donor) and FIXa was labeled with fluorescein-Phe-Phe-Arg-chloromethylketone (fluorescence acceptor). In the presence of FIXa, the acrylodan fluorescence was quenched indicating a biomolecular complex formation. Addition of 1.2 μM mAb abolished the acrylodan fluorescence quenching suggesting inhibition of the FIXa:LC interaction. Notably, the mAb did not inhibit activation of FX by FIXa/Ca2+/PL and FXa-cleaved FVIIIa (instead of IIa-cleaved FVIIIa). This suggests that the mAb inhibits FIXa:LC interaction by a steric hindrance and not by a direct blockage of the FIXa:LC interactive sites.In summary, the mAb inhibits clotting by preventing FVIII activation by IIa. The epitope of the mAb appears to be restricted to residues 1681–1694 of FVIII. Notably, in some of the hemophilia A patients, the epitope of the inhibitory antibodies is confined to the IIa-cleavage site including the a3 acidic domain of LC. To locate the epitope for such antibodies, one of the approaches used was to construct porcine and human FVIII hybrids. Our strategy may represent a simplified approach to locate the epitope of similar antibodies in hemophilia A patients. Such antibodies may bind strongly to LC and weakly to IIa-cleaved LC. Further, these antibodies may not bind to FXa-cleaved LC or A1/A2 subunits. Disclosures:No relevant conflicts of interest to declare.

Formation of Procoagulant Platelets in Heparin-Induced Thrombocytopenia (HIT) Follows a Unique Signaling Pathway

Abstract 197HIT is a drug-induced, immune-mediated thrombocytopenia and thrombosis disorder associated with considerable morbidity and mortality. HIT is associated with generation of procoagulant platelet microparticles (Warkentin et al., Blood 1994; 84:3691), one of the cardinal features of procoagulant platelets, along with a phosphatidylserine-positive (PS+) external plasma membrane and surface retention of procoagulant proteins from plasma or released from platelet γ-granules. Other names for procoagulant platelets include COAT, or coated, platelets (Dale, J Thromb Haemostas 2005; 3:2185). To date, virtually all of the work on procoagulant platelets has used collagen or convulxin signaling via ITAM-associated GPVI, combined with thrombin signaling via GPCRs PAR1/PAR4. Batar and Dale reported the formation of coated platelets as a result of dual platelet FcγRIIa and thrombin stimulation; they used crosslinking of an anti-FcγRIIa antibody or anti-platelet glycoprotein antibodies which also engaged FcγRIIa (J Lab Clin Med 2001; 138:393). We report here the procoagulant activation of human and FcγRIIa transgenic (tg) mouse platelets stimulated by the HIT immune complex (IC) and thrombin (thr). HIT IC consist of ultralarge complexes of heparin and platelet factor 4 (hep/PF4) bound by HIT-like monoclonal antibody KKO. Washed human platelets, wild-type mouse platelets, or FcγRIIa tg mouse platelets were stimulated with the HIT IC + thr. Controls include convulxin (100 ng/ml) + thr, HIT IC alone, or thr alone as the stimulus. Human platelets and FcγRIIa-tg platelets, but not wild-type mouse platelets, develop a 40 to 60% PS+ population in response to HIT IC (100 ug/ml KKO)+ thr (0.5 U/ml), but not to either alone, as assessed by flow cytometry using labeled Annexin V. Unlike cvx + thr, which consistently shows a broad distribution of PS+ platelets in our hands and in the literature, HIT IC-stimulated platelets reproducibly show one discrete PS+ subpopulation (n = 6 per group). Preincubation of platelets with novel specific Syk inhibitor PRT318 (Reilly et al., Blood 2011; 117:2241) completely prevented PS+ platelet formation. FcγRIIa-tg platelets null for Akt2 showed a ∼50% reduction in the formation of PS+ platelets. Studies with FcγRIIa-tg mice null for Cal-DAG GEFI are ongoing. Understanding the unique pattern of procoagulant platelets generated by HIT IC + thr and the signals which produce it may lead to new diagnostic tests and therapeutic interventions. Disclosures:No relevant conflicts of interest to declare.

Exhaled Carbon Monoxide as a Marker of Hemolysis In Transgenic Mouse Models of Sickle Cell Anemia.

AbstractAbstract 1642Sickle cell disease is characterized by recurring episodes of hemolysis and painful vasoocclusion, which leads to ischemia-reperfusion injury and organ damage. Recently, the roles of heme-induced oxidative stress, nitric oxide consumption, endothelial cell activation, and inflammation in sickle cell disease have been recognized, in part because of the development of transgenic murine models of this condition. These mouse models provide insights into the pathophysiology of human sickle cell disease and the development of new therapeutic approaches. However, hemolytic rates and disease severity vary considerably between mouse models. Ferrous (Fe2+) hemoglobin (Hb) is easily oxidized in circulation to ferric (Fe3+) Hb, which readily releases free heme. Free heme is degraded by heme oxygenase which produces carbon monoxide (CO) as a byproduct. Endogenous CO production has been used as a measure of in vivo hemolysis in humans and mice, but there have been no reports of endogenous CO production in sickle mouse models. VCO was measured in sickle mouse models using a previously described rebreathing technique t (Levitt et al. J Lab Clin Med 113:241-247). Expired CO levels were highest in Hb-S knock-in Townes-SS sickle mice, almost 7-fold higher than normal control Townes-AA mice (p<0.05). Expired CO also was significantly (p<0.05) higher in HbS-BERK mice compared to normal control HbA-BERK. Injection of phenylhydrazine or hemin into normal C57BL/6 mice also raised expired CO levels 24 hours after injection (p<0.05). Expired CO levels were not significantly higher than controls in the New York, S+S-Antilles, het-BERK and Townes-AS models. These data confirm that hemolytic rates differ markedly between models. Additional data will be presented on the effects of inhaled CO on expired CO and the hemolytic rate in the Townes-SS model. Disclosures:Belcher:Sangart: Consultancy, Research Funding. Vercellotti:Sangart: Consultancy, Research Funding.

Diabetic Platelets Are Not Resistant to Aspirin Inhibition

AbstractAbstract 3324A daily aspirin in patients with myocardial infarctions (MI) effectively reduces the risk of occlusive vascular disease by 25%. This protective effect is reduced or absent in diabetics possibly because aspirin induced inhibition of platelet function is diminished. We tested the hypothesis that the amount of aspirin induced inhibition of platelets in diabetics is decreased. Methods:Each subject was evaluated at 2 time points: off-aspirin and 2-hours after observed ingestion of a 325mg aspirin tablet. Platelets were stimulated in a light aggregometer with platelet prostaglandin agonist (PPA). The slope of the PPA aggregation curve decreases with aspirin inhibition and is a sensitive measure of the amount of aspirin induced platelet inhibition (Schwartz J. Lab Clin Med 2002;139:227). Light aggregometry with arachidonic acid was used to verify that the patients had or had not taken aspirin. Maximal aspirin induced decrease in platelet function was defined as the difference between the PPA aggregation curve slopes at the 2 time points, off-aspirin and 2-hours post aspirin ingestion. 187 adult subjects (62% male 38% female) divided into four groups were studied, MI patients without diabetes n=98, diabetics with an MI n=41, diabetics without an MI n=9, and normal subjects n=39. Results:Comparisons of the PPA slopes of the 4 groups showed that diabetics had higher slopes than non-diabetics. Platelets from diabetics with and without an MI had significantly (p<0.05) increased off aspirin platelet reactivity (PPA slope) compared to normals. At the 2 hr time point patients with diabetes and an MI had a significantly higher PPA slope than the MI without diabetes group. Maximum aspirin induced decrease in platelet function was negatively correlated with age (r=-0.32, p=0.002). When controlled for age, there was no difference in the maximum decrease in platelet function among the 4 groups. Conclusions:1. Platelets from diabetic patients with and without MI have increased platelet reactivity to PPA. 2. The maximum decrease in platelet function observed in diabetic patients with and without an MI was similar to that observed in MI patients without diabetes and in normals. This suggests that the decreased protection from future vascular events observed with aspirin in diabetic patients is not because of an intrinsic resistance to aspirin inhibition of platelets. Disclosures:No relevant conflicts of interest to declare.