Erythrocytosis: Diagnosis and investigation.

An absolute erythrocytosis is present when the red cell mass is greater than 125% of the predicted. This is suspected when the hemoglobin or hematocrit is above the normal range. An erythrocytosis can be classified as primary or secondary and congenital or acquired. The commonest primary acquired disorder is polycythemia vera. The diagnostic criteria for PV have evolved over time and this is the main diagnosis managed in hematology clinics. There are a variety of rare congenital causes both primary and secondary. In particular in young patients and/or those with a family history a congenital cause is suspected. There remains a larger cohort with acquired erythrocytosis mainly with non-hematological pathology. In order to explore for a cause of erythrocytosis, measurement of the erythropoietin level is a first step. A low erythropoietin level indicates a primary cause and a normal or elevated level indicates a secondary etiology. Further investigation is then dictated by initial findings and includes mutational testing with PCR and NGS for those in whom a congenital cause is suspected. Following this possibly bone marrow biopsy, scans, and further investigation as indicated by history and initial findings. Investigation is directed toward the identification of those with a hematological disorder which would be best managed following guidelines in hematology clinics and referral elsewhere in those for whom there are non-hematological reasons for the elevated hemoglobin.

Anagrelide in essential thrombocythemia: Efficacy and long-term consequences in young patient population.

According to the current treatment recommendations, anagrelide, an oral antiplatelet agent, is recommended as a second-line therapy for patients with high-risk essential thrombocythemia experiencing intolerance or refractoriness to first-line approach, such as hydroxyurea or pegylated interferon alpha-2a. If there is a need for introduction of cytoreductive treatment in young patients with a perspective of lifelong exposure, both the efficacy and long-term outcomes should be known. We present the analysis of 48 young patients, diagnosed with essential thrombocythemia below the age of 60, who were exposed to anagrelide treatment for over 10 years. Our observations show that the highest proportion of complete remissions without adverse events and disease progression is seen in the JAK2-mutated patients. By evaluating the changes in hemoglobin concentration and serum erythropoietin throughout the study, we were able to reveal the development of progressive anemia, resulting from diminished susceptibility to erythropoietin and unrelated to bone marrow fibrosis, in patients harboring CALR mutation. Additionally, occurrence of new bone marrow fibrosis was confirmed in seven JAK2-unmutated patients at the end of the study. In summary, in young patient population, we recommend limiting the use of anagrelide to JAK2-mutated subgroup, reducing exposure time and underline the importance of periodic monitoring for the presence of bone marrow fibrosis.

Characterization of acquired anemia in children by iron metabolism parameters.

Inflammatory states are associated with anemia of chronic disease and acute infection. Hepcidin, a regulator of iron metabolism, is involved in iron pathophysiology during inflammation. We investigated biochemical characteristics in children with anemia from different causes. Four patient groups (n = 38; mean age: 12.44 ± 4.35 years) were studied: (1) inflammatory bowel disease (IBD, 10 patients); (2) iron deficiency anemia (IDA, 12); (3) celiac disease (CD, 8); (4) acute infection (AI, 8). Laboratory measurements were evaluated at diagnosis: blood count, serum iron, transferrin, ferritin, vitamin B12, folic acid, CRP, erythropoietin, hepcidin and soluble transferrin receptor (sTfR). IDA patients had the lowest Hgb (6.9 ± 1.7 g/dL), MCV (63.2 ± 7.2 fL), iron (16.8 ± 13.5 µg/dL), ferritin (4.5 ± 4.5 ng/mL) and hepcidin (3.1 ± 0.8 ng/mL) values, and the highest transferrin and sTfR values. AI patients had the highest ferritin (156.2 ± 124.5 ng/mL), CRP (144.6 ± 94 mg/L) and hepcidin (74.67 ± 12.3 ng/ml) values. Overall, hepcidin levels correlated with CRP and with ferritin (r = 0.83 and 0.85, respectively). Elucidating specific etiology-related biochemical profiles in pediatric patients with anemia from different causes using a combination of laboratory biomarkers, including hepcidin, can help physicians treat the anemia.

Revisiting Diagnostic performances of serum erythropoïetin level and JAK2 mutation for polycythemias: analysis of a cohort of 1090 patients with red cell mass measurement.

We assessed the diagnostic performances of erythropoietin and JAK2 mutations in 1,090 patients with suspected polycythemia who were referred for red cell mass (RCM) measurement. In patients with a high haematocrit and/or haemoglobin level, a low erythropoietin level (<=3·3 mUI/ml) and JAK2 mutation showed comparable positive predictive value (PPV) for true polycythemia (RCM>=125%), 92·1% and 90% respectively. A very-low erythropoietin level (<=1·99 mUI/ml) had a PPV of 100% for polycythemia vera (PV) diagnosis. We confirmed the correlations between RCM, erythropoietin and JAK2 variant allelic frequency in PV patients. This study prompts the need to revisit the role of EPO in PV diagnostic criteria.

Erythropoietin Concentration in Boys With p.His63Asp Polymorphism of the HFE Gene.

The molecular mechanism that regulates iron homeostasis is based on a network of signals, which reflect on the iron requirements of the body. HFE-related hemochromatosis is characterized by excessive intestinal absorption of dietary iron, in particular cases resulting in pathologically high iron storage in tissues and organs. During childhood, HFE gene homozygosity or heterozygosity manifests exclusively in the form of biochemical abnormalities. Because of their mutual link, bioavailable iron and endogenous erythropoietin (EPO) are indispensable for effective erythropoiesis. We analyzed the impact of p.(His63Asp) polymorphism of the HFE gene on erythropoiesis taking into consideration endogenous EPO production in the developmental age. In the study we performed, we observed a significant, strong and negative correlation between the concentration of EPO, hemoglobin, and red blood cell count. A negative trend was also noted on the impact of iron concentration and transferrin saturation on EPO production. In conclusion, this preliminary study demonstrates an impaired impact of endogenous EPO on erythropoiesis in the presence of increased iron content in carriers of p.(His63Asp) (heterozygotes) variant of the HFE gene in developmental age.

Structural Characterization of Polysaccharides Isolated from Panax notoginseng Medicinal Residue and Its Protective Effect on Myelosuppression Induced by Cyclophosphamide.

This study aims to establish the isolation and purification method of polysaccharides from medicinal residue of Panax notoginseng (PPN). The structure and protective effect of PPN on myelosuppression mice were investigated. One neutral polysaccharide (NPPN) and five acidic polysaccharides (APPN I, APPN II-A, APPN II-B, APPN III-A, and APPN III-B) were obtained. The results confirmed that NPPN, APPN I and APPN II-A are glycan with 1, 4 main chains. APPN III-A is a glycan. APPN II-B and APPN III-B are homogalacturonan pectin with 1, 4 main chains. This study demonstrated that NPPN played a bone marrow protective role in myelosuppression mice induced by cyclophosphamide. NPPN could relieve cell cycle arrest, reduce the apoptosis rate of marrow cells, and improve granulocyte-macrophage colony-stimulating (GM-CSF), thermoplastic polyolefin (TPO) and erythropoietin (EPO) serum level, which contributes to promoting the proliferation of hematopoietic cells.

Prediction of Response to Erythropoiesis Stimulating Agents in Low-Risk Myelodysplastic Syndromes.

INTRODUCTION: Erythropoiesis stimulating agents (ESAs) represents the principal treatments for anemia in patients with lower-risk myelodysplastic syndromes (MDS). Pre-treatment erythropoietin (EPO) level and previous blood transfusion requirement are the two major predictors for response to ESAs. However, most evidence was derived from Western countries whereas there have been limited data in patients with Asian background. METHODS: We retrospectively collected data on patients with low-risk MDS who received ESAs. Erythroid response was evaluated according to IWG 2006 criteria. MDS subtypes, r-IPSS, baseline hemoglobin (Hb), ESAs dosage and erythropoietin level were reviewed from medical records. Gene mutations were analyzed in patients' blood or bone marrow at diagnosis by 40-gene myeloid panel targeted sequencing. Clinical and laboratory parameters were compared between erythroid responder and non-responder groups. RESULTS: A total of 47 patients were recruited in the study. The median age at diagnosis of the patients in this cohort was 77 years (IQR, 70-83) and 44.7% were male. The median revised international prognostic scoring system (R-IPSS) score of patients was 2.5. Response rate to ESAs was 46.8% (22/47). Median EPO level in responders was significantly lower than non-responders (27.7 vs. 59.1 U/L, p=0.02). Median ESAs dosage in responder group was 30,000 units per week. Cytogenetic abnormalities were detected in 27.3% and 24% of the responder and non-responder groups, respectively. Of 22 patients with available 40 gene mutation targeted sequencing, ASXL1, IDH2 and TET2 represented the 3 most common mutations and were found in 22%, 22% and 17%, respectively. There were no differences in cytogenetic abnormalities and gene mutations between groups. Patients who responded to ESAs showed a higher 5-year overall survival (OS) compared to non-responders (5-year OS 75% vs. 60.9%; p=0.008). CONCLUSION: We conclude that a low serum EPO level is a predictive factor for responsiveness to ESAs in Asian patients with low-risk MDS.

Understanding increased ferritin levels in pediatric ECMO patients.

Abnormally high serum ferritin levels have been reported during pediatric ECMO, attributed to frequent red blood cell transfusion and suggestive of iron overload. However, the utility of ferritin for diagnosing iron overload is complicated by its response as an acute-phase reactant. In this study, we aimed to assess the utility of ferritin for diagnosing ECMO-related iron overload, with secondary aims of understanding its relationship with inflammation and erythropoiesis. Ferritin was elevated in all pediatric ECMO runs (median 459 ng/ml, IQR = 327.3-694.4). While intermittent elevations in serum iron were observed, all normalized prior to decannulation. Unreported previously, erythropoietin (EPO) remained well above normative values prior to and throughout ECMO runs, despite frequent transfusion and exposure to hyperoxia. Ferritin correlated poorly with serum iron [r(80) = 0.05, p = 0.65], but correlated well with IL-6 [r(76) = 0.48, p < 0.001] and EPO [r(81) = 0.55, p < 0.001]. We suggest that serum ferritin is a poor biomarker of iron overload in ECMO patients, and that future investigation into its relationship with EPO is warranted.

Erythropoietin levels in children with obstructive sleep apnea.

OBJECTIVE: High serum erythropoietin (EPO) levels have been reported in adult patients with obstructive sleep apnea (OSA), however there is a lack of related literature in children with OSA. The main objective of this study was to explore the potential use of EPO as a pediatric OSA biomarker by exploring the relationship between serum EPO levels and the presence of pediatric OSA. METHODS AND MATERIALS: A prospective study was conducted on children (4-12 years old) referred for overnight PSG. Thirty (30) consecutive children with mild. 30 consecutives with moderate, and 30 consecutives with severe OSA (OSA group), as well as 30 consecutive children with AHI≤1 (non-OSA group) were recruited. Morning blood specimens after PSG studies were obtained in order to compare EPO levels. RESULTS: Finally, 115 children included for analysis. Non-OSA group consisted of 29 children (mean age: 6.93 ± 2.10) and OSA-group of 86 children (mean age: 6.78 ± 2.53). Mean EPO values for the non-OSA and OSA groups were 5.46 ± 2.29 mIU/ml and 8.33 ± 4.10 mIU/ml respectively. OSA-group had significant higher EPO levels than non-OSA (P: 0.01) while EPO levels were significantly correlated with AHI (p < 0.001). CONCLUSION: Our study showed that serum EPO levels of children with OSA are significantly higher than those without OSA and correlate significantly with AHI. These results suggest that EPO may be considered as a biomarker candidate for pediatric OSA. Since this may be the first study on the topic further research is needed.

Tracking immature reticulocyte proteins for improved detection of recombinant human erythropoietin (rhEPO) abuse.

Athletes abuse recombinant human erythropoietin (rhEPO) and erythropoiesis stimulating agents to increase hemoglobin mass and improve performance. To evade detection, athletes have developed sophisticated blood doping regimens, which often include rhEPO micro-dosing. Detection of these methods requires biomarkers with increased sensitivity and a sample matrix that is more amenable to frequent testing in the field. We have developed a method to measure two immature reticulocyte proteins, CD71 and ferrochelatase (FECH), and one total erythrocyte protein, Band 3, in dried blood spots (DBS). This method was tested in response to rhEPO administration after low doses, 40 IU/kg, micro-doses, 900 IU, or saline injection in 20 healthy subjects. During administration of low-dose rhEPO, the mean CD71/Band 3 and FECH/Band 3 ratio increased by 412 ± 197% and 250 ± 44%, respectively. The mean response for the current biomarker, RET%, increased by 195 ± 35%. During administration of rhEPO micro-doses, CD71/Band 3 increased to 127 ± 25% on day 35 and 139 ± 36% on day 39, while no increase was observed in RET%. After rhEPO administration, during the washout phase, mean values decreased to a minimum of 64 ± 4% and 64 ± 11% for CD71/Band 3 and RET%, respectively. However, CD71/Band 3 remained below 75% of baseline for at least 4 weeks after rhEPO injection, while RET% returned to baseline levels. The results demonstrate that immature reticulocyte proteins have a larger response to rhEPO administration than the current biomarker, RET%, and can be monitored in the DBS matrix.

Epidemiologic and Genetic Associations of Erythropoietin With Blood Pressure, Hypertension, and Coronary Artery Disease.

[Figure: see text].

Dapagliflozin attenuates diabetic cardiomyopathy through erythropoietin up-regulation of AKT/JAK/MAPK pathways in streptozotocin-induced diabetic rats.

PURPOSE: This study was designed to investigate the mechanism of Dapagliflozin (Dapa) cardioprotection against diabetic cardiomyopathy (DCM). Structural and functional changes in the heart as well as decrease of erythropoietin (EPO) levels were reported in DCM. EPO simultaneously activates three pathways: the Janus-activated kinase-signal transducer and activator of transcription (JAK2/STAT5), phosphatidylinositol-3-kinase-Akt (PI3K/Akt), and extracellular signal-related kinase (ERK/MAPK) cascades, that result in proliferation and differentiation of cardiac cells. METHODS AND RESULTS: DCM was induced by a high fat diet for 10 weeks followed by administration of streptozotocin. After confirmation of diabetes, rats were divided randomly to 5 groups: Group 1; normal control group, Group 2; untreated diabetic group and Groups (3-5); diabetic groups received Dapa daily (0.75 mg, 1.5 or 3 mg/Kg, p.o) respectively for a month. At the end of the experiment, full anaesthesia was induced in all rats using ether inhalation and ECG was recorded. Blood samples were collected then rats were sacrificed and their heart were dissected out and processed for biochemical and histopathological studies. Untreated diabetic rats showed abnormal ECG pattern, elevation of serum cardiac enzymes, decrease EPO levels, downregulation of P-Akt, P-JAK2 and pMAPK pathways, abnormal histological structure of the heart and increase immunostaining intensity of P53 and TNF α in the cardiomyocytes. Dapa in a dose dependent manner attenuated the alterations in the previously mentioned parameters. CONCLUSION: The cardioprotective effect of Dapa could be mediated by increasing EPO levels and activation of P-Akt, P-JAK2 and pMAPK signalling cascades which in turn decrease apoptosis.

Value of erythrocyte sedimentation rate and serum epo levels in evaluating the condition and prognosis of COPD in the elderly.

To investigate the value of erythrocyte sedimentation rate (ESR) and serum EPO levels in evaluating the condition and prognosis of chronic obstructive pulmonary disease (COPD) in the elderly. 120 elderly patients with acute exacerbation of COPD admitted to our hospital from May 2016 to April 2019 were selected. Pulmonary function tests and CT scans were performed on all patients. According to Gold's guidelines for the diagnosis and treatment of chronic obstructive pulmonary disease, patients with different degrees of illness were divided into mild group, moderate group and severe group. The levels of EPO and ESR were measured in the morning after admission and the differences of EPO and ESR between the three groups were compared. The correlation between EPO, ESR and FEV1% of lung function index was analyzed by linear correlation analysis. There was no significant difference in age, sex, BMI index and course of disease among the three groups (P>0.05); ESR and EPO increased in turn among mild, moderate and severe groups, while FEV1% decreased in turn, with significant differences between the two groups (P<0.05). Negative correlation (r values were - 0.33, - 0.49, P<0.05). ESR and serum EPO levels can reflect the severity of COPD in elderly patients to some extent, and are negatively correlated with FEV1% of lung function indicators, which can provide some clues to the condition and prognosis of COPD in elderly patients.

Impact of sodium glucose linked cotransporter-2 inhibition on renal microvascular oxygen tension in a rodent model of diabetes mellitus.

BACKGROUND: The mechanisms whereby inhibitors of sodium-glucose linked cotransporter-2 (SGLT2) exert their nephroprotective effects in patients with diabetes are incompletely understood but have been hypothesized to include improved tissue oxygen tension within the renal cortex. The impact of SGLT2 inhibition is likely complex and region specific within the kidney. We hypothesize that SGLT2 inhibitors have differential effects on renal tissue oxygen delivery and consumption in specific regions of the diabetic kidney, including the superficial cortex, containing SGLT2-rich components of proximal tubules, versus the deeper cortex and outer medulla, containing predominantly SGLT1 receptors. METHODS: We measured glomerular filtration rate (GFR), microvascular kidney oxygen tension (Pk O2 ), erythropoietin (EPO) mRNA, and reticulocyte count in diabetic rats (streptozotocin) treated with the SGLT2 inhibitor, dapagliflozin. Utilizing phosphorescence quenching by oxygen and an intravascular oxygen sensitive probe (Oxyphor PdG4); we explored the effects of SGLT2 inhibition on Pk O2 in a region-specific manner, in vivo, in diabetic and non-diabetic rats. Superficial renal cortical or deeper cortical and outer medullary Pk O2 were measured utilizing excitations with blue and red light wavelengths, respectively. RESULTS: In diabetic rats treated with dapagliflozin, measurement within the superficial cortex (blue light) demonstrated no change in Pk O2 . By contrast, measurements in the deeper cortex and outer medulla (red light) demonstrated a significant reduction in Pk O2 in dapagliflozin treated diabetic rats (p = 0.014). Consistent with these findings, GFR was decreased, hypoxia-responsive EPO mRNA levels were elevated and reticulocyte counts were increased with SGLT2 inhibition in diabetic rats (p < 0.05 for all). CONCLUSIONS: These findings indicate that microvascular kidney oxygen tension is maintained in the superficial cortex but reduced in deeper cortical and outer medullary tissue, possibly due to the regional impact of SGLT-2 inhibition on tissue metabolism. This reduction in deeper Pk O2 had biological impact as demonstrated by increased renal EPO mRNA levels and circulating reticulocyte count.

Low serum erythropoietin levels are associated with fatal COVID-19 cases at 4,150 meters above sea level.

Previous studies suggested that erythropoietin (EPO) may protect against severe COVID-19-induced injuries, ultimately preventing mortality. This hypothesis is based on the fact that, in addition to promoting the increase in red blood cells, EPO is an anti-inflammatory, anti-apoptotic and protective factor in several non-erythropoietic tissues. Furthermore, EPO promotes nitric oxide production in the hypoxic lung and stimulates ventilation by interacting with the respiratory centers of the brainstem. Given that EPO in the blood is increased at high-altitude, we evaluated the serum levels of EPO in critical patients with COVID-19 at "Hospital Agramont" in the city of El Alto (4150 masl) in Bolivia. A total of 16 patients, 15 men, one woman, with a mean age of 55.8 ± 8.49 years, admitted to the Intensive Care Unit were studied. All patients were permanent residents of El Alto, with no travel history below 3000 masl for at least one year. Blood samples were collected upon admission to the ICU. Serum EPO concentration was assessed using an ELISA kit, and a standard technique determined hemoglobin concentration. Only half of the observed patients survived the disease. Remarkably, fatal cases showed 2.5 times lower serum EPO than survivors (2.78 ± 0.8643 mU/mL vs 7.06 ± 2.713 mU/mL; p = 0.0096), and 1.24 times lower hemoglobin levels (13.96 ± 2.56 g/dL vs 17.41 ± 1.61 g/dL; p = 0.0159). While the number of cases evaluated in this work is low, our findings strongly warrant further investigation of EPO levels in COVID-19 patients at high and low altitudes. Our results also support the hypothesis that exogenous EPO administration could help critically ill COVID-19 patients overcome the disease.

Evaluation of beta-2 microglobulin, erythropoietin and tobacco use in polycythemic cases.

In the 2016 WHO classification, hemoglobin and hematocrit thresholds for diagnosing polycythemia vera (PV) have been lowered, increasing the number of consultations for polycythemia investigations. In PV, beta-2 microglobulin (B2m) levels are reportedly increased, whereas erythropoietin (EPO) levels are usually low. Most secondary polycythemia cases (SP) are caused by tobacco use. We decided to analyze the relevance of these three parameters in all patients seen for polycythemia investigations to help differentiate PV from SP cases. A cohort of 257 patients (123 PV; 134 SP) was identified. The median B2m level was higher for PV patients (3.16 vs 1.98 mg/l, p < 0.0001). Increased B2m levels were observed in 83.7% of PV patients (11.9% in SP). The median EPO level was lower in PV patients (4.4 vs 12.3 UI/l, p < 0.0001). Tobacco was used by 42.8% of SP patients (8% in PV, p < 0.0001). Increased B2m, low EPO and no tobacco exposure was predictive of PV (specificity and positive predictive value = 100%). Normal B2m, normal EPO and tobacco exposure was predictive of SP (positive predictive value = 100%). These simple and inexpensive parameters could be used to rapidly differentiate PV from SP cases, before prescribing time-consuming JAK2 V617F mutation analysis by specialists.

Fetal Cytokine Balance, Erythropoietin and Thalassemia but Not Placental Malaria Contribute to Fetal Anemia Risk in Tanzania.

Fetal anemia is common in malaria-endemic areas and a risk factor for anemia as well as mortality during infancy. Placental malaria (PM) and red cell abnormalities have been proposed as possible etiologies, but the relationship between PM and fetal anemia has varied in earlier studies, and the role of red cell abnormalities has not been studied in malaria-endemic areas. In a Tanzanian birth cohort study designed to elucidate the pathogenesis of severe malaria in young infants, we performed a cross-sectional analysis of risk factors for fetal anemia. We determined PM status, newborn red cell abnormalities, and maternal and cord blood levels of iron regulatory proteins, erythropoietin (EPO), cytokines and cytokine receptors. We examined the relationship between these factors and fetal anemia. Fetal anemia was present in 46.2% of the neonates but was not related to PM. Maternal iron deficiency was common (81.6%), most frequent in multigravidae, and interacted with parity to modify risk of fetal anemia, but it was not directly related to risk. Among offspring of iron-deficient women, the odds of fetal anemia increased with fetal α+-thalassemia, as well as these patterns of cord blood cytokines: increased cord IL-6, decreased TNF-RI, and decreased sTfR. The EPO response to fetal anemia was low or absent and EPO levels were significantly decreased in newborns with the most severe anemia. This study from an area of high malaria transmission provides evidence that 1) fetal α+-thalassemia and cytokine balance, but not PM at delivery, are related to fetal anemia; 2) maternal iron deficiency increases the risk that other factors may cause fetal anemia; and 3) fetal anemia has a multifactorial etiology that may require a variety of interventions, although measures that reduce maternal iron deficiency may be generally beneficial.