High Hct Research Articles

Hematological Changes in Chronic Sustained Hypoxia and Chronic Intermittent Hypoxia in a Mouse Model

Hypoxia alters cellular metabolism and chemosensory endocrine regulation. Hypoxia increases production of erythrocytes in order to improve oxygen delivery to tissues. Organisms respond differentially to duration and patterns of hypoxia. Acute hypoxia stabilizes transcription factors' a-subunits of hypoxia inducible factors (HIFs; HIF-1a and HIF-2a) while in chronic hypoxia HIF-2a continues to be augmented, but HIF-1a decreases (1, 2). There are two clinical patterns of hypoxia: chronic sustained hypoxia (CSH) and chronic intermittent hypoxia (CIH). CSH is present in people living at high altitudes, patients with respiratory diseases, and Eisenmenger complex. CIH is characterized by repeated series of hypoxia followed by normoxia exposure and best exemplified by obstructive sleep apnea (OSA). The hematological responses to CSH and CIH has not been compared and studied in detail. We studied 2 separate hypoxic mouse cohorts: 1) CSH (12% O2 for 24 hours/day for 33 days), and 2) CIH emulating human OSA (8% O2 for 30 sec followed by 21% O2 for 90 sec; 8 hours/day for 33 days) and nonhypoxic controls. Hematocrits (Hct) were higher in both CSH and CIH compared to controls. In CSH, Hct increased by 22.0% (p <0.0001) and by 7.6% in CIH (p=0.0115). To evaluate the effect of CSH and CIH on erythron we measured erythropoietin (Epo) and reticulocytes. Reticulocytes were lower in CSH and CIH compared to controls; however, CIH tended to have higher reticulocytes than CSH (p=0.0607). Epo levels decreased in CSH and CIH but not significantly in contrast to increased Epo detected at 3 days after acute hypoxia (2). These Epo levels were reflected by decrease of Epo transcripts in kidney after 33 days of CSH (p=0.0001). In contrast, there was no decrease of Epo kidney transcripts in CIH. Hepcidin (encoded by Hamp) is the principal regulator of iron metabolism; its expression is decreased by hypoxia, iron deficiency, and augmented erythropoiesis. It suppresses erythropoiesis by inhibiting release iron from macrophages. We measured Hamp transcript in the liver. Despite presence of hypoxia, both CSH and CIH had higher Hamp transcripts. These results are consistent with co-existent suppression of erythropoiesis in both CSH and CIH, but more so in CSH. Since inflammation induces hepcidin expression, we measured inflammatory marker transcripts in granulocytes and found increased Il-6 transcripts in both CSH and CIH; Il-1a levels increased only in CSH. As Epo transcripts were decreased in the kidney, we also measured inflammatory markers` transcripts in kidney. The Ccl2, Il-10 and Il-6 transcripts were higher in both CSH and CIH. These results suggest that increased inflammation in CSH and CIH suppresses erythropoiesis via increase of hepcidin. We also measured reactive oxygen species (ROS) in blood cells. The amount of mitochondrial ROS and the number of mitochondrial ROS positive reticulocytes increased in CSH and CIH and correlated with mitochondrial mass. We previously showed that excessive ROS associated with increased mitochondrial mass is due to reduced Bnip3L (the mediator of mitophagy) and decreased catalase upon normoxic return from CSH (2). Bnip3L and Cat (encodes catalase) were also decreased in both CSH and CIH in reticulocytes, which correlated with their increased mitochondrial mass and ROS. We conclude that in CIH there is an increased hematocrit in mouse OSA model which contrasts with human OSA where Hct is not increased. Our data support that the increased Hct occurs in CSH and mouse CIH at the early stages of hypoxia, but that further increases of Hct is prevented by inflammation and hepcidin. The high Hct is further contributed by recent demonstration of prolonged mouse erythrocyte survival in hypoxia (4). Further, in this OSA mouse model increased ROS is restricted to reticulocytes but not present in leukocytes, unlike in human OSA (3). This demonstrates some limitation of using mouse as a model for human OSA, but also some similarities. Excessive ROS in reticulocytes due to increased mitochondrial mass and decreased catalase (due to a decrease of HIF-1a) may explain some features of pathophysiology of CSH and CIH (2). 1. Uchida et al, JBC 2004 2. Song et al, JMM 2015 3. Song et al," Normal Hemoglobin Concentrations in OSA", abstract this meeting 4. Song et al, High Alt Med Biol. 2019 Disclosures No relevant conflicts of interest to declare.

Evaluation of the Performance and Hematocrit Independence of the HemaPEN as a Volumetric Dried Blood Spot Collection Device.

Dried blood spots (DBS) are often used as a less invasive alternative to venous blood sampling. Despite its numerous advantages, the use of conventional DBS suffers from the hematocrit (hct) effect when analyzing a subpunch. This effect could be avoided by using hct-independent sampling devices, of which the hemaPEN is a recent example. This device collects the blood via four integrated 2.74 μL microcapillaries, each depositing the blood on a prepunched paper disc. In this study, we evaluated the technical performance of the hemaPEN devices, using an extensive bioanalytical validation and application on authentic patient samples. An LC-MS/MS method quantifying caffeine and its metabolite paraxanthine in dried whole blood (using the hemaPEN device) was fully validated, meeting all preset acceptance criteria. A comparative analysis of 91 authentic patient samples (hct range: 0.17-0.53) of hemaPEN, 3 mm DBS subpunches, and whole blood revealed a limited hct dependence (≤7% concentration difference over a 0.20-0.50 hct range) for the hemaPEN devices, which we could not attribute to the analytical procedure. Using conventional partial-punch DBS (3 mm punches), concentration differences of ≥25% over this hct range were found. The hemaPEN showed to be robust to the effects of blood sample volume, device lot, analytical operator, and storage stability. The technical performance of the hemaPEN when dealing with patients having a high hct and in cases where a large blood drop is present should be further investigated. Based on the successful validation and application on patient samples, we conclude that the hemaPEN device shows good potential for the volumetric collection of DBS.

Correlation between RBC changes and coagulation parameters in high altitude population

ABSTRACTObjective: To explore the correlations between RBCs indexes and the basic coagulation parameters, and provide data for further studies on high altitude-induced thrombotic disease.Methods: A total of eligible 433 volunteers were divided into different groups according to HGB concentration and HCT, respectively. PT, APTT, TT and Fbg were measured by clotting assays. HGB content, HCT and PLT count were assessed by automated hematology analyzer.Results: APTT and PT were significantly higher in group 4 (high HGB or HCT groups) (p < 0.05 for all comparison) and PLT count was significantly lower in group 4 than in other groups (p < 0.01 for all comparison). APTT and PT showed negative correlations with HGB concentration (r = −0.168 and −0.165 resp.; both p < 0.01), whereas positive correlations were found between APTT and HCT, PT and HCT (r = 0.225 and 0.258, resp.; both p < 0.01). PLT, TT and Fbg showed no correlation with HGB and HCT.Conclusions: HGB and HCT may not correlate with basic coagulation parameters in high altitude population, their predictive value for high altitude-induced thrombotic disease may relatively independent and this remain to be determined in further studies.

Prevalence and associated factors of renal dysfunction and proteinuria in cyanotic congenital heart disease.

Cyanotic nephropathy (CN), seen in 30-50% of patients with congenital cyanotic heart disease (CCHD), affects both tubular and glomerular function, resulting in proteinuria and azotemia. Microalbuminuria is an early marker for glomerular damage and an independent predictor of progressive renal disease. A cross-sectional study was conducted. A total of 116 patients aged 1month to 15years with CCHD at Chiang Mai University Hospital between 2015 and 2016 were assessed and 94 patients were enrolled. To determine the prevalence and associated factors of significant albuminuria in CCHD patients, baseline characteristics, oxygen saturation, surgery, hemoglobin (Hb), hematocrit (Hct), spot urine albumin, urine protein, and creatinine were obtained. Binary logistic-regression modeling was used to identify associated factors. Prevalence of CN in children with CCHD was 58.51% and 92.55% according to albuminuria and proteinuria staging respectively. Prevalence of significant proteinuria, significant albuminuria, and decreased GFR was 88.30%, 41.49% and 31.91% respectively. Participants with significant albuminuria had fewer previous surgeries (p=0.05), a longer waiting time for surgery (p=0.02), enalapril usage (p=0.04), pulmonary hypertension (p=0.03), higher Hct z-score (p=0.03) and lower platelet count (p=0.001) compared with those without significant albuminuria. Using multivariate logistic regression analysis, waiting duration for surgery (p=0.04), Hct >40% (p=0.02), and platelet count <290,000/mm3 (p=0.04) were predictive of microalbuminuria. Cyanotic nephropathy can be detected in the first decade of life with thepresentation of microalbuminuria. High Hct level and low platelet count were identified as a predictor of microalbuminuria, whereas early cardiac surgery decreased the risk of developing significant albuminuria.

Safety and Feasibility of Ultra-Low Dose IL-2 As Graft Versus Host Disease Prophylaxis in Haplo-Identical Stem Cell Transplantation- a Proof of Concept Pilot Study

INTRODUCTIONHaploidentical allogeneic stem cell transplantation (haplo-SCT) incurs a risk of bidirectional immune reactions with either severe acute graft versus host disease (aGVHD) or graft rejection. Induction of immune tolerance with post-graft cyclophosphamide dramatically improves the outcomes of haplo-SCT. However the optimal duration and the combination of systemic immunosuppressive agents in haplo-SCT remain controversial. Ultra-low dose interleukin 2 (ULD IL-2) preferentially expands regulatory T cells (Tregs) and natural killer (NK) cells, promoting both GVHD prevention and graft versus leukemia (GVL) effects. These properties suggest that ULD IL-2 could play a useful role in haplo-SCT. Here we report the outcomes of a pilot study to determine the safety and feasibility of ULD IL-2 as GVHD prophylaxis in haploidentical allo-SCT (14-H-0180, Clinical Trials.gov ID: NCT02226861) METHODSTen subjects with high risk hematological malignancies received a myeloablative conditioning regimens of fludarabine 120mg/m2 (day -10 to day -8) and total body irradiation (TBI; 600-1200 cGy, day -10 to day -6). Thereafter the subjects received donor lymphocyte infusion (DLI) products (2x108 CD3+/kg) on day -6, followed by post-DLI cyclophosphamide 120mg/kg on days -3 and -2. CD 34+ selected, peripheral blood stem cell product was infused on day 0. Sirolimus was initiated on day -1 with goal trough level of 5-12ng/mL until day+60. ULD-IL2 (aldesleukin, 100,000 IU/m2) was given subcutaneously daily for 12 weeks starting day +1. Peripheral blood mononuclear cells (PBMC) and plasma samples were collected at days 14, 28, 60, 84, 100 post-transplant. Multi-color flow cytometry immunophenotyping assay were performed to characterize the subsets of memory T cells, Tregs, NK cells, and monocytes with various functional markers. Plasma levels of biomarkers (ST2, Reg3α, sTNFR1, ANG1, IL-6) were measured using a multiplex microfluidic channel assay. RESULTSThe median age at transplant was 35 years (range 20-66). Most subjects had a high risk EBMT score (median 4, range 2-7) and HCT co-morbidity index (median 4, range 2-7). All subjects achieved successful engraftment (neutrophil >500/uL; median 13 days, platelet >20k/uL; median 15 days) and rapid full donor myeloid and lymphoid chimerism by day 21. At median follow up of 6 month, the overall survival was 71%. One subject died of hepatic veno-occulusive disease (VOD) on day 32 and one subject died of relapse on day 178. All evaluable subjects tolerated ULD-IL2 without significant toxicities. Four subjects experienced either de-novo or rapid exacerbation of acute GVHD after discontinuation of ULD IL-2, resulting in the cumulative incidence of grade II-IV aGVHD of 61% and grade III-IV aGVHD of 36% (Figure A). ULD IL-2 expanded and maintained Helios+FoxP3+Tregs population (pre-transplant, 4.7%±3.1%; day 30, 36.2%±23.1%; day 84, 17.4%±10.6%) as well as CD56brightNK cells population (pre-transplant, 10.7%±13.7%; day 30, 49.7%±10.8%; day 84, 26.1%±6.8%). However on discontinuation of ULD IL-2 both populations decreased to low levels within one week. The timing of aGVHD correlated with a fall of %Tregs in PBMC and a sharp increase of ST2 level in plasma (Figure B). CONCLUSIONULD IL-2 can be safely administered as GVHD prophylaxis after haplo-SCT. Rebound GVHD after discontinuation of ULD IL-2 implies that donor-derived Tregs acquired dependency to exogenous ULD IL-2. Our study is proof of principle that ULD IL-2 induces immune tolerance through Tregs expansion in haplo-SCT, inspiring further clinical and basic researches in human IL-2 biology. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Guidelines on transfusion for fetuses, neonates and older children.

Guidelines on transfusion for fetuses, neonates and older children.

The Prediction of Preeclampsia and Its Association With Hemoglobin and Hematocrit in the First Trimester of Pregnancy

Background: Hypertensive disorders in pregnancy are one of the most serious complications and their early diagnosis is one of the most important goals of prenatal care. Objectives: The objective of this study was to determine the association of first trimester Hemoglobin (Hb) and Hematocrit (Hct) with preeclampsia. Patients and Methods: This descriptive-analytic, prospective study was performed on 1376, less than 12 weeks of gestation, singleton pregnancies, visited for their prenatal care in health and medical clinics of the Qazvin province during years 2013 and 2014. At first, demographic data were recorded in a questionnaire and then all pregnant cases were referred to one of the three reference laboratories for their first trimester routine tests. After hemoglobin and hematocrit date collection, women were categorized in three groups: Hb < 11, Hb ≥ 12.49 and 11 ≤ Hb < 12.49, and based on Hct, two groups: Hct < 38% and Hct ≥ 38. The analysis was done by χ2 (chi-square) and t-test with SPSS 16. Receiver operator characteristics (ROC) curve and Youden’s index were utilized for finding the optimum cut off for each. P values of < 0.05 were considered significant. Results: Preeclampsia incidence was 5.1% in our study. Mean Hb was 12.38 ± 1.69 g/dL in the preeclampsia group and 11.8 ± 1.18 in the non-preeclampsia group, and mean Hct was 37.74 ± 5.15% in the preeclampsia group and 35.45 ± 3.58% in the preeclampsia group and 35.45 ± 3.58% in the non-preeclampsia group, (P = 0.016) (P = 0.001). Furthermore, 43 out of 68 patients with preeclampsia (10.9%) had high hemoglobin (Hb ≥ 12.5 g/dL). We found a significant association between the 1st trimester Hb, Hct and preeclampsia (P 38%) (4.41 - 12.044: CI 95%). According to Youden’s Index, optimum cut-off for 1st trimester Hb was 12.65 and for Hct, this was 38.05%. Conclusions: The association of the 1st trimester high Hb and Hct with preeclampsia was revealed in this study, therefore it could be used as a prediction factor for early preeclampsia diagnosis. Keywords: Preeclampsia; First Trimester; Hemoglobin; Hematocrit

Long-Term Outcomes after Myeloablative Allogeneic Hematopoietic Cell Transplantation for Adult Acute Lymphoblastic Leukemia

Allogeneic hematopoietic cell transplantation (alloHCT) is a curative therapy for high-risk acute lymphoblastic leukemia (ALL). However, long-term outcomes after alloHCT for adult ALL have not been well described. We conducted a retrospective cohort study of 72 consecutive adult ALL patients who underwent a first myeloablative alloHCT at our institution from January 2000-December 2013. Median age at HCT was 38 yrs (range, 18-62), 40 (56%) were male, 18 (38%) had high HCT CI score, 14 (19%) had prior CNS leukemia and 35 (49%) had BCR-ABL+ disease. Donor source was HLA-matched related donor for 50% patients and 90% received PBSC as graft source. All patients were transplanted in CR (72% were in 1st or 2nd CR) and 92% received T-cell replete grafts. Median time from diagnosis to alloHCT was 5 months (range, 2-90). The incidences of grade II-IV and III-IV acute GvHD, chronic GvHD and extensive chronic GvHD were 43%, 13%, 51% and 36%, respectively. The median follow-up for our cohort is 76 months. At 6 years after HCT, probability of overall survival (OS) was 33% (95% CI, 21-44%) and relapse-free survival (RFS) was 30% (95% CI, 19-42%), and the cumulative incidence of relapse was 36% (95% CI, 25-48%) and non-relapse mortality (NRM) was 37% (95% CI, 26-49%). The most common causes of death were relapse (43%) and infection (21%); majority of relapses occurred within the first 2-years post-transplantation. There were no second cancer related deaths. In multivariable analyses, factors significantly associated with OS were HCT CI score (HR 2.69 for high vs. low/int., P=0.002) and CMV status (HR 2.62 for donor+ vs. others, P=0.05). HCT CI score was the only predictive factor for RFS (HR 2.26 for high, P=0.007). We also compared outcomes by BCR-ABL status. BCR-ABL+ patients were older (median age 42 vs. 36 yrs, p=0.02), had low HCT CI score (34% vs 22%, p=0.01), were more likely to be in CR1 (74% vs. 32%, p=0.002), and as a result, proceeded to HCT sooner after diagnosis (median 4 vs 7 months, p=<0.001). For BCR-ABL+ and BCR-ABL- patients, 6 year OS was 41% and 25%, RFS was 40% and 21%, relapse was 27% and 45% and NRM was 38% and 36% (P=NS for all comparisons). Myeloablative alloHCT can provide long-term survival for selected high-risk adult ALL patients. Relapses are relatively uncommon after 2 years post-transplant. Long-term NRM is high in this population and we did not observe a plateau in its incidence until 7.5 years post-transplant, suggesting the need for long-term follow up to prevent and manage late complications of alloHCT. [Display omitted] DisclosuresMajhail:Gamida Cell Ltd.: Consultancy; Anthem Inc.: Consultancy.

Influence of anesthetic variables on short-term and overall survival rates in cats undergoing renal transplantation surgery.

To identify factors associated with short-term (30-day) and overall survival rates in cats that underwent renal transplantation surgery (RTS). Retrospective cohort study. 94 cats that underwent RTS from 1998 through 2010. Data obtained from the medical records pertinent to RTS included cat signalment; anesthetic agents, techniques, and timings; supportive treatment; perioperative physiologic findings; and surgery and warm ischemia times. Associations with short-term and overall survival rates were investigated. Median survival time was 653 days (range, 2 to 4,580 days). Prolonged anesthesia (median, 300 minutes; range, 225 to 445 minutes) reduced overall survival rate but did not influence short-term survival rate. No associations were identified between survival rates and anesthetic agent used, amount and type of fluid administered IV, physiologic abnormalities, and blood product administration. All cats that received μ-opioid receptor antagonists at anesthetic recovery to reverse the effects of μ-opioid receptor agonists survived for at least 30 days. High Hct at the end of anesthesia was also associated with an increase in short-term survival rate. Two cats had an intraoperative hemoglobin oxygen saturation < 90%, and both died within 7 days after surgery. Cats > 12 years old had a lower overall survival rate than did younger cats. Minimization of total anesthesia time, reversal of μ-opioid receptor agonists at the end of anesthesia, and prevention of intraoperative decreases in blood oxygen saturation and postoperative decreases in Hct appeared to help maximize postsurgical survival time in cats undergoing RTS.

Hematocrit-independent recovery is a key for bioanalysis using volumetric absorptive microsampling devices, Mitra™.

A novel microsampling device, Mitra(TM), was evaluated for bioanalysis of E6005 and its O-desmethylated metabolite in human whole blood using an UPLC-MS. A constant volume of blood samples was absorbed onto the tip of Mitra, the analytes were extracted by various solvents and then detected by UPLC-MS. Recovery of the analytes was high in acetonitrile-water (1:1, v/v) but was dependent on hematocrit (Hct) without sonication process, which led to biased accuracy at low and high Hcts. Inclusion of sonication process in extraction improved recovery at high Hct to yield acceptable accuracy across Hcts. Optimization of extraction process to achieve high recovery regardless of Hct is critical in accurate bioanalysis via Mitra.

Peripheral circulation evaluation with near-infrared spectroscopy in skeletal muscle during cardiopulmonary bypass.

We designed a non-invasive, observational, real-time study, using near-infrared spectroscopy (NIRS) to assess the in vivo effects of cardiopulmonary bypass (CPB) on patients' skeletal muscle as well as the effects of hemodilution and hypothermia on tissue oxygen delivery during CPB. The study included 20 consecutive adult patients undergoing open-heart surgery with CPB. Evaluation parameters for peripheral circulation were measured using the NIRO-200NX and recorded every 30 seconds. To assess how hemodilution influences peripheral circulation parameters, we compared data between a group of patients with hematocrit (Hct) values >22% (high Hct group) and those with Hct values ⩽22% (low Hct group). Changes in the concentration of oxygenated hemoglobin (ΔO2Hb, μmol/L), which flows into the skeletal muscle, was an important factor for deciding the tissue oxygenation index (TOI%), showing the tissue oxygen saturation. The low Hct group showed a significant increase in the normalized tissue hemoglobin index (nTHI), showing the percentage change in the amount of initial hemoglobin and TOI compared to the high Hct group. Changes in the concentration of oxygenated hemoglobin (ΔO2Hb, μmol/L) and deoxygenated hemoglobin (ΔHHb, μmol/L) were significantly less in the low Hct group than in the high Hct group, thus, showing good peripheral circulation despite the low hematocrit levels. Our study indicated the presence of a compensatory mechanism in which increased blood flow of the microcirculation is in compensation for the lack of oxyhemoglobin delivery caused by hemodilution.

High blood oxygen affinity in the air-breathing swamp eel Monopterus albus

The Asian swamp eel (Monopterus albus, Zuiew 1793) is a facultative air-breathing fish with reduced gills. Previous studies have shown that gas exchange seems to occur across the epithelium of the buccopharyngeal cavity, the esophagus and the integument, resulting in substantial diffusion limitations that must be compensated by adaptations in others steps of the O2 transport system to secure adequate O2 delivery to the respiring tissues. We therefore investigated O2 binding properties of whole blood, stripped hemoglobin (Hb), two major isoHb components and the myoglobin (Mb) from M. albus. Whole blood was sampled using indwelling catheters for blood gas analysis and determination of O2 equilibrium curves. Hb was purified to assess the effects of endogenous allosteric effectors, and Mb was isolated from heart and skeletal muscle to determine its O2 binding properties. The blood of M. albus has a high O2 carrying capacity [hematocrit (Hct) of 42.4±4.5%] and binds O2 with an unusually high affinity (P50=2.8±0.4mmHg at 27°C and pH7.7), correlating with insensitivity of the Hb to the anionic allosteric effectors that normally decrease Hb-O2 affinity. In addition, Mb is present at high concentrations in both heart and muscle (5.16±0.99 and 1.08±0.19mg ∙ g wet tissue-1, respectively). We suggest that the high Hct and high blood O2 affinity serve to overcome the low diffusion capacity in the relatively inefficient respiratory surfaces, while high Hct and Mb concentration aid in increasing the O2 flux from the blood to the muscles.

Similar Outcomes for Two Anemia Treatment Strategies among Elderly Hemodialysis Patients with Diabetes.

To compare mortality and cardiovascular risk in elderly dialysis patients with diabetes under two clinical strategies of anemia correction: maintaining hematocrit (Hct) between 34.5 and < 39.0% (high Hct strategy), and between 30.0 and <34.5% (low Hct strategy) using intravenous alpha epoetin. Observational data were used to emulate a randomized trial in which diabetic patients who initiated hemodialysis in 2006-2008 were assigned to each anemia correction strategy. Inverse-probability weighting was used to adjust for measured time-dependent confounding. Comparing high with low hematocrit strategy, the hazard ratio (95% confidence interval) was 1.07 (0.83, 1.38) for all-cause mortality and 1.00 (0.81, 1.24) for a composite mortality and cardiovascular endpoint. Among a cohort of elderly hemodialysis patients with diabetes, no differences were found between the low and high hematocrit strategies. A lower target hematocrit - per current Food and Drug Administration (FDA) guidelines - appears to be as safe as higher targets among this population.

Nitric oxide scavenging by cell-free hemoglobin may be a primary factor determining hypertension in polycythemic patients

We tested the hypothesis that hypertension associated with polycythemia vera (PV) may be related to hemoglobin released from erythrocytes (cell-free hemoglobin, fHb). We assessed hematocrit, mean arterial pressure (MAP), blood viscosity, and the level of fHb and nitrite/nitrate (NOx) in the plasma of 73 PV patients and 38 healthy controls. The effect of isovolemic erythrocytapheresis (ECP) on the considered parameters was also studied. From the whole group of PV patients a subset of subjects with normal (normotensive patients, n = 16) and elevated MAP (hypertensive patients, n = 57) can be subtracted.It was found that in comparison with healthy controls, PV patients have significantly (p ≤ 0.01) elevated Hct (0.567 vs. 0.422), blood viscosity (5.45 vs. 3.56 cP), MAP (106.8 vs. 93.8 mmHg), plasma fHb (9.7 vs. 2.8 mg/dL), and NOx levels (34.1 vs. 27.5 μM). Compared with normotensive patients, hypertensive PV patients demonstrated a higher rise in fHb (10.2 vs. 8.0) and plasma NOx levels (35.8 vs. 31.0). In PV patients, fHb positively correlates with MAP (r = 0.489), NOx levels (r = 0.461), hematocrit (r = 0.428), and viscosity (r = 0.393). Blood viscosity positively correlated with hematocrit (r = 0.894), but not with other considered parameters. In PV patients MAP poorly correlated with hematocrit, whereas the correlation between MAP and NOx altered from − 0.325 (healthy control) to + 0.268 (PV patients). ECP procedure was associated with a significant (p < 0.01) reduction of hematocrit, fHb, blood viscosity, and MAP. In the normotensive subgroup of PV patients the ECP procedure did not affect MAP. It can be concluded that accelerated scavenging of nitric oxide by fHb rather than high Hct may be a key factor determining the development of hypertension in PV patients.

High haematocrit in cyanotic congenital heart disease affects how fibrinogen activity is determined by rotational thromboelastometry

Viscoelastometry enables rapid evaluation of coagulopathy in settings such as cardiac surgery but may be influenced by red cell concentration. In order to study the effects of supra-physiological red cell concentrations on viscoelastometry, we compared ROTEM® viscoelastometry and plasma coagulation assay results in high haematocrit (HCT; 0.55-0.76 L/L) blood from patients with cyanotic congenital heart disease (CCHD), and in model high HCT blood (HCT 0.45-0.70 L/L). High HCT blood from CCHD patients (median HCT 0.66 L/L) displayed prolonged clot initiation in the EXTEM® test compared to controls and reduced maximum clot firmness (MCF) in the EXTEM (median 51 mm vs 64 mm in controls) and FIBTEM® (7 mm vs 14 mm) tests. The plasma fibrinogen (Clauss; CF) was similar in CCHD blood to controls (median 2.94 g/L vs 2.49) but the whole blood fibrinogen concentration (WBFC) was reduced (1.27 g/L vs 1.58). The FIBTEM MCF correlated linearly with the CF (r(2)=0.68; p<0.0001) and WBFC (r(2)=0.65; p<0.0001) in control blood but this relationship was maintained only with WBFC in CCHD blood. Model high HCT blood showed abnormal ROTEM test results that were similar to CCHD blood, including reduced FIBTEM MCF (14 mm with HCT 0.32-0.44 vs 6mm with HCT 0.63-0.70). The ROTEM results were HCT dependent but independent of plasma clotting times and fibrinogen concentration. Supra-physiologic HCT causes abnormal ROTEM test results consistent with increased dilution of fibrinogen and coagulation factors in whole blood by red cells. High HCT should be considered during interpretation of ROTEM test results in clinical settings.

The effect of hematocrit on the efficacy of phototherapy for neonatal jaundice

The therapeutic phototherapy action spectrum ranges from 420 to 500 nm. However, a recent report of improved efficacy of fluorescent "turquoise" light (~490 nm) as compared with blue light (~450 nm) underscores the need to define an optimal action spectrum for precision-targeted phototherapy using very narrow wavelength ranges. We used a current semi-empirical model of the optical properties of skin for robust calculations of the fraction of light absorbed by bilirubin at various wavelengths that could be confounded by hemoglobin (Hb), melanin, and skin thickness. Applying assumptions regarding the wavelength dependence of bilirubin photochemistry, "action spectra" were assembled from the calculated values. All the calculated action spectra displayed a peak between 472 and 480 nm (most at 476 nm), which is a significant shift from the well-reported 460 nm absorption peak of bilirubin. Of note, the relative amplitudes of the action spectra showed an inverse relationship with hematocrit (Hct). We speculate that a narrow range of light at 476 nm would be 60% more effective than blue (broadband) fluorescent lamps. Because Hb serves as a major competitor of bilirubin for light absorption, the calculations also predict that the efficacy of phototherapy is dependent on the Hct. A high Hct could reduce therapeutic efficiency.

Defining Incidence, Risk Factors, and Impact on Survival of Central Line-Associated Blood Stream Infections Following Hematopoietic Cell Transplantation in Acute Myeloid Leukemia and Myelodysplastic Syndrome

Central line-associated blood stream infections (CLABSI) commonly complicate the care of patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) after allogeneic stem cell transplantation (HCT). We developed a modified CLABSI (MCLABSI) definition that attempts to exclude pathogens usually acquired because of disruption of mucosal barriers during the vulnerable neutropenic period following HCT that are generally included under the original definition (OCLABSI). We conducted a retrospective study of all AML and MDS patients undergoing HCT between August 2009 and December 2011 at the Cleveland Clinic (N = 73), identifying both OCLABSI and MCLABSI incidence. The median age at transplantation was 52 years (range, 16 to 70); 34 had a high (≥3) HCT comorbidity index (HCT-CI); 34 received bone marrow (BM), 24 received peripheral stem cells (PSC), and 15 received umbilical cord blood cells (UCB). Among these 73 patients, 23 (31.5%) developed OCLABSI, of whom 16 (69.6%) died, and 8 (11%) developed MCLABSI, of whom 7 (87.5%) died. OCLABSI was diagnosed a median of 9 days from HCT: 5 days (range, 2 to 12) for UCB and 78 days (range, 7 to 211) for BM/PSC (P < .001). MCLABSI occurred a median of 12 days from HCT, with similar earlier UCB and later BM/PSC diagnosis (P = .030). Risk factors for OCLABSI in univariate analysis included CBC (P < .001), human leukocyte antigen (HLA)-mismatch (P = .005), low CD34+ count (P = .007), low total nucleated cell dose (P = .016), and non-Caucasian race (P = .017). Risk factors for OCLABSI in multivariable analysis were UCB (P < .001) and high HCT-CI (P = .002). There was a significant increase in mortality for both OCLABSI (hazard ratio, 7.14; CI, 3.31 to 15.37; P < .001) and MCLABSI (hazard ratio, 6.44; CI, 2.28 to 18.18; P < .001). CLABSI is common and associated with high mortality in AML and MDS patients undergoing HCT, especially in UCB recipients and those with high HCT-CI. We propose the MCLABSI definition to replace the OCLABSI definition, given its greater precision for identifying preventable infection in HCT patients.

Guidelines on the management of anaemia and red cell transfusion in adult critically ill patients.

Forward This document aims to summarize the current literature guiding the use of red cell transfusion in critically ill patients and provides recommendations to support clinicians in their day-to-day practice. Critically ill patients differ in their age, diagnosis, co-morbidities, and severity of illness. These factors influence their tolerance of anaemia and alter the risk to benefit ratio of transfusion. The optimal management for an individual may not fall clearly within our recommendations and each decision requires a synthesis of the available evidence and the clinical judgment of the treating physician. This guideline relates to the use of red cells to manage anaemia during critical illness when major haemorrhage is not present. A previous British Committee for Standards in Haematology (BCSH) guideline has been published on massive haemorrhage (Stainsby et al, 2006), but this is a rapidly changing field. We recommend readers consult recent guidelines specifically addressing the management of major haemorrhage for evidence-based guidance. A subsequent BCSH guideline will specifically cover the use of plasma components in critically ill patients.

Correlations Between TET2 Mutation and Clinicohematologic Parameters in Myeloproliferative Neoplasms

AbstractAbstract 1455 Background:Since the acquired somatic mutation, JAK2 V617F, was discovered as a first molecular marker of myeloproliferative neoplasms (MPN), and it has been detected variably in each MPN subtypes. However, JAK2 V617F does not found in all of MPN cases and not necessarily specific to a particular clinicpathologic entity. Recently, mutation of the putative tumor suppressor gene, Ten-Eleven-Translocation-2(TET2), has been identified in MPN patients. However, the frequency of TET2 mutation or its relationship with JAK2 V617F mutation or pathologic function in MPN has not been concluded, yet. The aim of our study was to evaluate the frequency of TET2 in MPN patients, and whether there is any correlation of TET2 mutation with JAK2V617F mutation or the clinicohematologic parameters. Materials and Methods:Total 99 adult MPN patients (18 PV, 62 ET, 11 PMF and 8 MPN unclassified) whose bone marrow cells had been stored from 2007 to 2010 at point of first diagnosis were included in this study. Hematological diagnoses and subtyping were reconfirmed according to the 2008 WHO classification and clinicohematologic datas were collected from patient records. Direct sequencing for TET2(exon3–11) and JAK2 (exons 12 and 14) were performed using an ABI 3730XL DNA analyzer. The JAK2V617F allele burdens were determined by pyrosequencing for samples available and MPL was analyzed by allele-specific PCR. Results:The overall TET2 mutational frequency was 12.1%, and disease-specific mutational frequencies were 22.2% in PV, 9.7% in ET and 18.2% in PMF. The found mutations included 11 mutations, 7 frame-shift (p.Lys95AsnfsX18, p.Gln967AsnfsX40, p.Lys1022GlufsX4, p.Asp1314MetfsX49, p.Gln1534AlafsX43, p.Tyr1618LeufsX4, p.Leu1609GlufsX45), 1 nonsense (p.Gly1735X), 1 missense (Q599R) and 2 splicing mutations (c.3409+1G>T, c.4044+2insT). Those mutations most frequently involved exon 3(four mutations) and exon 11(four mutaions), and rarely intron 3, intron 8 and exon 7. None of the mutations were associated with a karyotypically apparent 4q24 rearrangement. All patients were also screened for JAK2 V617F, and the overall JAK2 V617F positive rate was 68%(94.4% in PV, 69.4% in ET, 45.5% in PMF and 37.5% in MPN, unclassified). All TET2 mutations occurred in JAK2 V617F positive cases. JAK2 exon12 mutation was not found in all patients. MPL W515L was found in one ET patient who also carried JAK2V617F, but not TET2 mutation. Information on JAK2 V617F allele burden was available in 78 patients.Considering all 99 patients, the patient age, hematologic indexes (leukocyte count, neutrophil fraction, lymphocyte fraction, monocyte fraction, Hb, Hct and platelet count), the frequency of organomegaly, marrow fibrosis or thrombotic/hemorrhagic complications were not different according to carrying TET2 mutation. However, TET2 mutation was more frequently found in JAK2 V617F carriers than non-carriers (P=0.008), but JAK2 V617F allele burden did not correlated with the presence of mutant TET2.When analysis was performed for each PV, ET, and PMF (no TET2 mutation in MPN-unclassifiable patients), correlation between TET2 and JAK2 V617F mutational status was not found in each subtypes (P=0.078 in PV, P=0.099 in ET and P=0.182 in PMF). However, the JAK2 V617F allele burden was significantly higher in PMF harboring TET2 mutation than PMF patients did not (88.0 ± 4.3% vs 19.1 ± 28.7%, P=0.034). In statistical analysis for the correlations of clinicohematologic parameters with TET2 mutation in each PV, ET and PMF patients, only a few statistically significant results were identified. The presence of TET2 mutation was correlated with high Hct in PMF (47.4 ± 5.4 vs 25.5 ± 6.2, P=0.037), and TET2 positive ET patients showed relatively higher frequency of organomegaly compared to ET patients without TET2 mutation (50% vs 19.6%, P=0.018). Conclusions:The overall and disease-specific frequencies of TET2 mutation in our study are similar with previous studies, and frame-shift mutation is the most frequent mutation type. There is no specific relationship between JAK2 V617F and TET2 mutation occurrence, but TET2 mutant PMF has higher JAK2 V617F allele burden than non-mutant. TET2 mutation is also associated with a higher Hct in PMF and higher frequency of organomegaly in ET. Larger scale studies involving more MPN patients are needed. Disclosures:No relevant conflicts of interest to declare.

Treatment with a Nitric Oxide-Donating NSAID Alleviates Functional Muscle Ischemia in the Mouse Model of Duchenne Muscular Dystrophy

In patients with Duchenne muscular dystrophy (DMD) and the standard mdx mouse model of DMD, dystrophin deficiency causes loss of neuronal nitric oxide synthase (nNOSμ) from the sarcolemma, producing functional ischemia when the muscles are exercised. We asked if functional muscle ischemia would be eliminated and normal blood flow regulation restored by treatment with an exogenous nitric oxide (NO)-donating drug. Beginning at 8 weeks of age, mdx mice were fed a standard diet supplemented with 1% soybean oil alone or in combination with a low (15 mg/kg) or high (45 mg/kg) dose of HCT 1026, a NO-donating nonsteroidal anti-inflammatory agent which has previously been shown to slow disease progression in the mdx model. After 1 month of treatment, vasoconstrictor responses to intra-arterial norepinephrine (NE) were compared in resting and contracting hindlimbs. In untreated mdx mice, the usual effect of muscle contraction to attenuate NE-mediated vasoconstriction was impaired, resulting in functional ischemia: NE evoked similar decreases in femoral blood flow velocity and femoral vascular conductance (FVC) in the contracting compared to resting hindlimbs (ΔFVC contraction/ΔFVC rest = 0.88±0.03). NE-induced functional ischemia was unaffected by low dose HCT 1026 (ΔFVC ratio = 0.92±0.04; P>0.05 vs untreated), but was alleviated by the high dose of the drug (ΔFVC ratio = 0.22±0.03; P<0.05 vs untreated or low dose). The beneficial effect of high dose HCT 1026 was maintained with treatment up to 3 months. The effect of the NO-donating drug HCT 1026 to normalize blood flow regulation in contracting mdx mouse hindlimb muscles suggests a putative novel treatment for DMD. Further translational research is warranted.