Riociguat in patients with sickle cell disease and hypertension or proteinuria (STERIO-SCD): a randomised, double-blind, placebo controlled, phase 1-2 trial.

BACKGROUND: Although nitric oxide based therapeutics have been shown in preclinical models to reduce vaso-occlusive events and improve cardiovascular function, a clinical trial of a phosphodiesterase 5 inhibitor increased rates of admission to hospital for pain. We aimed to examine if riociguat, a direct stimulator of the nitric oxide receptor soluble guanylate cyclase, causes similar increases in vaso-occlusive events. METHODS: This was a phase 1-2, randomised, double blind, placebo-controlled trial. Eligible patients were 18 years or older, had confirmed sickle cell disease documented by haemoglobin electrophoresis or HPLC fractionation (haemoglobin SS, SC, Sß-thalassemia, SD, or SO-Arab), and stage 1 hypertension or proteinuria. Participants were randomly assigned 1:1 to receive either riociguat or matching placebo via a web-based system to maintain allocation concealment. Both treatments were administered orally starting at 1·0 mg three times a day up to 2·5 mg three times a day (highest tolerated dose) for 12 weeks. Dose escalation by 0·5 mg was considered every 2 weeks if systolic blood pressure was greater than 95 mm Hg and the participant had no signs of hypotension; otherwise, the last dose was maintained. The primary outcome was the proportion of participants who had at least one adjudicated treatment-emergent serious adverse event. The analysis was performed by the intention-to-treat. This trial is registered with ClinicalTrials.gov (NCT02633397) and was completed. FINDINGS: Between April 11, 2017, and Dec 31, 2021, 165 participants were screened and consented to be enrolled into the study. Of these, 130 participants were randomly assigned to either riociguat (n=66) or placebo (n=64). The proportion of participants with at least one treatment-emergent serious adverse event was 22·7% (n=15) in the riociguat group and 31·3% (n=20) in the placebo group (difference -8·5% [90% CI -21·4 to 4·5]; p=0·19). A similar pattern emerged in other key safety outcomes, sickle cell related vaso-occlusive events (16·7 [n=11] vs 21·9% [n=14]; difference -5·2% [-17·2 to 6·5]; p=0·42), mean pain severity (3·18 vs 3·32; adjusted mean difference -0·14 [-0·70 to 0·42]; p=0·69), and pain interference (3·15 vs 3·12; 0·04 [-0·62 to 0·69]; p=0·93) at 12 weeks were similar between groups. Regarding the key clinical efficacy endpoints, participants taking riociguat had a blood pressure of -8·20 mm Hg (-10·48 to -5·91) compared with -1·24 (-3·58 to 1·10) in those taking placebo (-6·96 mm Hg (90% CI -10·22 to -3·69; p<0·001). INTERPRETATION: Riociguat was safe and had a significant haemodynamic effect on systemic blood pressure. The results of this study provide measures of effect and variability that will inform power calculations for future trials. FUNDING: Bayer Pharmaceuticals.

Four-factor prothrombin complex concentrate versus andexanet alfa for direct oral anticoagulant reversal.

BACKGROUND: Optimal reversal agent for direct oral anticoagulant (DOAC)-associated major bleeding has not been described. Before the approval of andexanet alfa (AA) in 2018, 4-factor prothrombin complex concentrate (4F-PCC) was recommended by major guidelines. Currently, AA is recommended as the first-line agent by most guidelines. With a paucity of literature comparing the 2 agents, there is clinical value in assessing hemostatic efficacy and safety of the 2 agents. OBJECTIVE: This study aimed to evaluate hemostatic efficacy and safety of AA and 4F-PCC in all DOAC-associated major bleedings. METHODS: A multicenter, retrospective chart review was performed of adult subjects who were admitted for a DOAC-associated major bleeding and received 4F-PCC from February 2018 to May 2019 or AA from May 2019 to September 2021. Some of the exclusion criteria included not receiving a DOAC, receiving multiple reversal agents during the same hospitalization, receiving reversal for any nonmajor bleeding indication, and not receiving the full dose of a reversal agent. The primary outcome was hemostatic efficacy 24 hours after the end of the reversal agent administration. Secondary outcomes included time to administration, hospital mortality, length of stay, need for surgery, and need for additional blood product. Safety outcome was incidence of thrombotic events. RESULTS: There were 99 subjects included in the 4F-PCC group and 84 subjects in the AA group. Hemostatic efficacy was achieved in 69 subjects (69.7%) in the 4F-PCC group and 63 subjects (75%) in the AA group (P = 0.927). In-hospital mortality was seen in 20 subjects (20.2%) in the 4F-PCC group and 10 subjects (11.9%) in the AA group. Thrombotic events were seen in 7 subjects (7.1%) in the 4F-PCC group and 6 subjects (7.1%) in the AA group. CONCLUSIONS: There were no significant differences in hemostatic efficacy, in-hospital mortality, and number of thrombotic events between 4F-PCC and AA.

Biopsychosocial Factors Associated With Pain and Pain-Related Outcomes in Adults and Children With Sickle Cell Disease: A Multivariable Analysis of the GRNDaD Multicenter Registry.

Pain is the primary symptomatic manifestation of sickle cell disease (SCD), an inherited hemoglobinopathy. The characteristics that influence pain experiences and outcomes in SCD are not fully understood. The primary objective of this study was to use multivariable modeling to examine associations of biopsychosocial variables with a disease-specific measure of pain interference known as pain impact. We conducted a secondary analysis of data from the Global Research Network for Data and Discovery national SCD registry. A total of 657 children and adults with SCD were included in the analysis. This sample was 60% female with a median age of 34 (interquartile range 26-42 years) and a chronic pain prevalence of 64%. The model accounted for 58% of the variance in pain impact. Low social (P < .001) and emotional (P < .001) functioning, increasing age (P = .004), low income (P < .001), and high acute painful episodes (P = .007) were most strongly associated with high pain impact in our multivariable model. Additionally, multivariable modeling of pain severity and physical function in 2 comparable samples of registry participants revealed that increasing age and low social functioning were also strongly associated with higher pain severity and low physical functioning. Overall, the results suggest that social and emotional functioning are more strongly associated with pain impact in individuals with SCD than previously studied biological modifiers such as SCD genotype, hemoglobin, and percentage fetal hemoglobin. Future research using longitudinally collected data is needed to confirm these findings. PERSPECTIVE: This study reveals that psychosocial (ie, social and emotional functioning) and demographic (ie, age) variables may play an important role in predicting pain and pain-related outcomes in SCD. Our findings can inform future multicenter prospective longitudinal studies aimed at identifying modifiable psychosocial predictors of adverse pain outcomes in SCD.

Efficacy and Safety of Combined Deferiprone and Deferasirox in Iron-Overloaded Patients: A Systematic Review.

Despite the established efficacy of iron chelation therapy in transfusion-induced iron-overloaded patients, there is no universal agreement regarding the choice of an optimal chelating regimen. Deferasirox (DFX) and deferiprone (DFP) are two oral iron chelators, and combination usage demonstrated effectiveness as an alternative to monotherapies in patients with a limited response to monotherapy. The present systematic review aimed to assess the evidence regarding the outcomes of combined DFP and DFX in iron-overloaded patients. An online search was conducted in PubMed, Scopus, Web of Science, and CENTRAL databases. Interventional and observational studies that assessed the outcomes of combined DFP and DFX in iron-overloaded patients were included. Eleven studies (12 reports) were considered in this meta-analysis. The studies included dual iron chelation strategies for a number of diagnoses. Single-arm studies (n =7) showed a reduction of serum ferritin, which reached the level of statistical significance in three studies. Likewise, most studies reported a numerical reduction in liver iron concentration (LIC) and increased cardiac MRI-T2* values after chelating therapy. Alternatively, comparative studies showed no significant difference in post-treatment serum ferritin between DFX plus DFP and DFX/DFP plus deferoxamine (DFO). The adherence to combination therapy was good to average in nearly 66.7-100% of the patients across four studies. One study reported a poor adherence rate. The combined regimen was generally tolerable, with no reported incidence of serious adverse events among the included studies. In conclusion, the DFP and DFX combination is a safe and feasible option for iron overload patients with a limited response to monotherapy.

Venous thromboembolism prophylaxis practices for patients with sickle cell disease prior to and during the COVID-19 pandemic.

Patients with sickle cell disease (SCD) are predisposed to a hypercoagulable state due to alterations in the coagulation system. Despite concern for the development of venous thromboembolism (VTE) in this population, there are no standardized guidelines for routine thromboprophylaxis. The objective of this study was to assess thromboprophylaxis practices of adult and pediatric treaters of SCD before and during the coronavirus disease of 2019 (COVID-19) pandemic. A cross-sectional electronic survey was distributed to pediatric and adult hematology oncology practitioners through seven SCD-specific interest groups between May 29, 2020, and July 13, 2020. Of 93 total responses, 14% ( N  = 13) reported they only treat patients more than 21 years old; 38.7% ( N  = 36) only treat patients 0-21 years old and 47.3% ( N  = 44) reported they treat both. Our study showed that before the COVID-19 pandemic, 96% of adult practitioners would recommend pharmacologic thromboprophylaxis, mechanical thromboprophylaxis or both for hospitalized adults with thromboprophylaxis, but only 76% of pediatric treaters would recommend any thromboprophylaxis in hospitalized children ( P  < 0.0001), with 24% of pediatric treaters choosing no thromboprophylaxis at all. During the COVID-19 pandemic, pharmacologic thromboprophylaxis specifically was recommended for adults by 94% of treaters and for pediatric patients by 76% of treaters. These findings suggest that despite the lack of evidence-based thromboprophylaxis guidelines in adults and children with thromboprophylaxis, subspecialty treaters routinely provide pharmacologic thromboprophylaxis in their adult patients and will modify their practice in pediatric patients who are considered at a high risk for VTE.

Current Evidence and Rationale to Guide Perioperative Management, Including Transfusion Decisions, in Patients With Sickle Cell Disease.

Sickle cell disease (SCD) is a collection of inherited hemoglobin disorders that results in chronic hemolytic anemia, vaso-occlusion, pain, and end organ damage. Surgery in the SCD population requires careful planning, as perioperative stressors can lead to increased sickling and risk of inducing or further exacerbating vaso-occlusive episodes (VOEs). Additionally, the underlying hypercoagulability and immunocompromised state due to SCD places patients at increased risk of both venous thromboembolism and infection. Judicious fluid administration, temperature regulation, thorough preoperative and postoperative analgesic planning, and preoperative transfusion are all crucial components of decreasing risks of surgery in patients with SCD.

Real-world characteristics of patients with sickle cell disease who initiated crizanlizumab therapy.

OBJECTIVE: To provide real-word evidence of patients with SCD initiating crizanlizumab, their use of other SCD treatments, and crizanlizumab treatment patterns. METHODS: Using IQVIA's US-based, Longitudinal Patient-Centric Pharmacy and Medical Claims Databases patients with a diagnosis of SCD between November 1, 2018, and April 30, 2021, and ≥1 claim for crizanlizumab (date of first claim = index date) between November 1, 2019, and January 31, 2021 who were ≥16 years of age, and had ≥12 months of pre-index data were selected for analysis. Two cohorts were identified based on available follow-up time (3- and 6-month cohorts). Patient characteristics were reported along with pre- and post-index SCD treatments and crizanlizumab treatment patterns (e.g. total doses received, gap-days between doses, days on therapy, discontinuation, and restarts). RESULTS: 540 patients met the base inclusion criteria (345 in the 3-month cohort and 262 in the 6-month cohort. Most patients (64%) were female with a mean (SD) age of 35 (12) years overall. Concomitant hydroxyurea use was observed in 19-39% of patients, while concomitant L-glutamine use was observed for 4-8% of patients. 85% of 3-month cohort patients received at least two doses of crizanlizumab, while 66% of the 6-month cohort received at least 4 doses of crizanlizumab. The median number of gap days between doses was 1 or 2. CONCLUSIONS: 66% of patients who receive crizanlizumab receive at least 4 doses within 6-months. The low median number of gap days suggests high adherence.

Sickle Cell Trevor Thompson Transition Project (ST3P-UP) protocol for managing care transitions: Methods and rationale.

BACKGROUND: Emerging adults with sickle cell disease (EASCD) experience significant challenges transitioning from pediatric to adult care. Acute care utilization increases, quality of life (QOL) declines, with an increased risk of mortality. Currently, there are no practice standards to guide emerging adults through the transition process. We are creating a structured transition education (STE) based program for EASCD by customizing the Six Core Elements (6 CE) of Health Care Transition model and are evaluating the effectiveness of adding peer mentoring (PM). METHODS: The Sickle Cell Trevor Thompson Transition Project (ST3P-UP) is an ongoing multi-site, cluster randomized clinical trial with a target enrollment of 537 EASCD aged 16 to 25 years in pediatric care. Each site (n = 14) comprises a pediatric clinic, adult clinic, and a sickle cell disease (SCD) community-based organization (CBO). Sites are randomized 1:1 to either STE or STE + PM. EASCDs are followed prospectively for 24 months. Rapid cycle plan-do-study-act quality improvement (QI) methods are used to implement the STE. The primary objective is to compare the effectiveness of STE + PM versus STE only at decreasing the number of acute care visits per year over 24 months. The secondary objectives are to compare overall healthcare utilization and patient-reported QOL outcomes at 24 months. CONCLUSION: We aim to demonstrate the feasibility of using a QI approach to implement 6 CE-based practice standards at 14 disparate SCD clinical programs to guide EASCD through the transition process. We hypothesize that adding PM to the STE program will improve acute care reliance, QOL, and satisfaction with transition outcomes.

High Rates of Anxiety Among Adolescents in a Partial Hospitalization Program.

Background: Anxiety disorders are garnering increasing attention for their contribution to high-risk issues and functional impairment. Adolescents are typically admitted to partial hospitalization programs (PHPs) due to high-risk presentations. However, the frequency of anxiety disorders in PHPs is not well-established, in part because anxiety can be overlooked in acute settings due to limited lengths of stay and focus on stabilization. Objective: This study aims to evaluate the frequency and severity of anxiety disorders among a sample of adolescent PHP patients to assess the need for anxiety-specific assessment and interventions in higher acuity settings. Methods: Participants were 158 youths ages 13 to 19 years old (M = 15.49 years, SD = 1.50) who were admitted to an adolescent PHP and their caregivers. Clinician-reported diagnostic information was collected from the youth's electronic medical record, and self- and caregiver-rated severity of anxiety was collected using the Screen for Child Anxiety Related Emotions Disorders (SCARED-C/P). Frequency of anxiety and related disorder diagnoses and self- and caregiver-reported severity were assessed using descriptive statistical methods. Results: 75% of participants were diagnosed with an anxiety disorder (n = 118). On average, participants with anxiety disorders had elevated SCARED-C scores. Youths with depressive disorders had elevated SCARED-C scores even when they did not carry anxiety disorder diagnoses. Caregiver ratings of the youth's anxiety symptoms on the SCARED-P were elevated when youths had anxiety disorders. Conclusions: These findings suggest that anxiety is common in an adolescent PHP setting and support investing in evidence-based assessment and treatment of anxiety in high-acuity settings.

A randomized clinical trial of the efficacy and safety of rivipansel for sickle cell vaso-occlusive crisis.

The efficacy and safety of rivipansel, a predominantly E-selectin antagonist, were studied in a phase 3, randomized, controlled trial for vaso-occlusive crisis (VOC) requiring hospitalization (RESET). A total of 345 subjects (204 adults and 141 children) were randomized and 320 were treated (162 with rivipansel, 158 with placebo) with an IV loading dose, followed by up to 14 additional 12-hourly maintenance doses of rivipansel or placebo, in addition to standard care. Rivipansel was similarly administered during subsequent VOCs in the Open-label Extension (OLE) study. In the full analysis population, the median time to readiness for discharge (TTRFD), the primary end point, was not different between rivipansel and placebo (-5.7 hours, P = .79; hazard ratio, 0.97), nor were differences seen in secondary end points of time to discharge (TTD), time to discontinuation of IV opioids (TTDIVO), and cumulative IV opioid use. Mean soluble E-selectin decreased 61% from baseline after the loading dose in the rivipansel group, while remaining unchanged in the placebo group. In a post hoc analysis, early rivipansel treatment within 26.4 hours of VOC pain onset (earliest quartile of time from VOC onset to treatment) reduced median TTRFD by 56.3 hours, reduced median TTD by 41.5 hours, and reduced median TTDIVO by 50.5 hours, compared with placebo (all P < .05). A similar subgroup analysis comparing OLE early-treatment with early-treatment RESET placebo showed a reduction in TTD of 23.1 hours (P = .062) and in TTDIVO of 30.1 hours (P = .087). Timing of rivipansel administration after pain onset may be critical to achieving accelerated resolution of acute VOC. Trial Registration: Clinicaltrials.gov, NCT02187003 (RESET), NCT02433158 (OLE).

The Evolving Landscape of Drug Therapies for Sickle Cell Disease.

Although sickle cell disease can be cured using allogeneic hematopoietic stem cell transplantation and possibly gene therapy and gene editing, these treatments remain unavailable to most patients. As understanding of the disease pathophysiology increases, progress is being made in developing drug therapies. Hydroxyurea, l-glutamine, crizanlizumab, and voxelotor are currently approved by the US Food and Drug Administration, with multiple others at various stages of testing. With the limited efficacy of individual agents, combinations of agents will likely be required for optimal outcomes. Clinical and surrogate endpoints, other than vaso-occlusive crisis, are increasingly being considered in the evaluation of novel drugs.

Youth Top Problems in an Acute Psychiatric Sample: Describing Consumer-Nominated Treatment Needs in an Adolescent Partial Hospital Setting.

Given the wide range of diagnostic presentations treated in partial hospital programs, finding efficient ways to identify and measure progress on the chief concerns of consumers in these settings is important. The current study uses a self-administered version of the Top Problems Assessment to describe treatment targets identified by youth and their caregivers presenting for care at an adolescent partial hospital setting. Caregiver-youth agreement on these chief concerns upon admission and predictors of agreement were explored. About one-third (34.65%) of caregiver-youth pairs did not match on any target problems. Although anxiety and depression were the most commonly cited top problems in this sample, caregivers and youth exhibited disagreement on these domains. Treatment teams in acute care settings such as a partial hospital program can benefit from careful assessment surrounding the initial goals of treatment as youth and their caregivers may not agree on the referral problems upon entering a program.

The Unique Magnetic Signature of Sickle Red Blood Cells: A Comparison Between the Red Blood Cells of Transfused and Non-Transfused Sickle Cell Disease Patients and Healthy Donors.

Sickle cell disease (SCD) is an inherited blood disorder that affects millions of people worldwide, especially in low-resource regions of the world, where a rapid and affordable test to properly diagnose the disease would be highly valued. Magnetophoresis is a technique that could simultaneously analyze, quantify, and potentially separate the patient's sickle red blood cells (RBCs) from healthy RBCs, but the magnetic characteristics of sickle RBCs have yet to be reported. In this work, we present the single cell magnetic characterization of RBCs obtained from SCD patients. Sufficient single cells are analyzed from patient samples undergoing transfusion therapy and not yet having transfusion therapy (TP and NTP, respectively), such that means and distributions of these single RBC mobilities are created in the form of histograms which facilitated comparison to RBCs from healthy donors (HD). The magnetic characterization is obtained using a technique known as Cell Tracking Velocimetry (CTV) that quantitatively characterizes the RBC response to magnetic and gravitational fields. The magnetic properties of RBCs containing oxygenated, deoxygenated hemoglobin (Hb) and methemoglobin (oxyHb-RBCs, deoxyHb-RBCs, and metHb-RBCs) are further determined. The NTP samples reported the highest magnetic character, especially when compared to oxyHb-RBCs from HD, which implies impaired oxygen binding capabilities. Also, the oxygen-Hb equilibrium curves are obtained to estimate the magnetic character of the cells under intermediate oxygen levels. Our results confirm higher magnetic moment of SCD blood (NTP) under intermediate oxygen levels. These data demonstrate the potential feasibility of magnetophoresis to identify, quantify and separate sickle RBCs from healthy RBCs.

GRNDaD: big data and sickle cell disease.

Big data are used in the pursuit of precision medicine in the general population. Applying these tools to patients with sickle cell disease (SCD) is essential for ensuring that they receive the most appropriate customized therapy for their disease. For these tools to be applied, there must be a large number of willing, fully phenotyped participants in collaborative registries. Working collaboratively to respond to unmet clinical needs and the lack of a large multisite registry, SCD providers at multiple sites developed The Globin Regional Data and Discovery (GRNDaD) registry. The specific goals of GRNDaD are to (1) prospectively obtain high-quality curated data on the evolving population affected by SCD; (2) improve adherence to guideline-based care; (3) provide a platform for ongoing quality improvement across sites; (4) allow for real-time investigation of therapies, and collaborate broadly to address research questions using GRNDaD as a shared platform. GRNDaD's current strength lies in the generous participation of people living with SCD, collaborative investigators, and the opportunity to conduct quality improvement activities across a large number of sites. GRNDaD will serve as the data collection tool for the Health Resources and Services Administration Sickle Cell Treatment Demonstration Program and for the newly established National Alliance for Sickle Cell Centers (https://www.sicklecellcenters.org/). GRNDaD is a robust collaborative registry that providers and researchers will use to identify genetic markers that will help predict outcomes and lead to a better understanding of the natural history of SCD in the modern era of novel therapies.

Potential of cell tracking velocimetry as an economical and portable hematology analyzer.

Anemia and iron deficiency continue to be the most prevalent nutritional disorders in the world, affecting billions of people in both developed and developing countries. The initial diagnosis of anemia is typically based on several markers, including red blood cell (RBC) count, hematocrit and total hemoglobin. Using modern hematology analyzers, erythrocyte parameters such as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), etc. are also being used. However, most of these commercially available analyzers pose several disadvantages: they are expensive instruments that require significant bench space and are heavy enough to limit their use to a specific lab and lead to a delay in results, making them less practical as a point-of-care instrument that can be used for swift clinical evaluation. Thus, there is a need for a portable and economical hematology analyzer that can be used at the point of need. In this work, we evaluated the performance of a system referred to as the cell tracking velocimetry (CTV) to measure several hematological parameters from fresh human blood obtained from healthy donors and from sickle cell disease subjects. Our system, based on the paramagnetic behavior that deoxyhemoglobin or methemoglobin containing RBCs experience when suspended in water after applying a magnetic field, uses a combination of magnets and microfluidics and has the ability to track the movement of thousands of red cells in a short period of time. This allows us to measure not only traditional RBC indices but also novel parameters that are only available for analyzers that assess erythrocytes on a cell by cell basis. As such, we report, for the first time, the use of our CTV as a hematology analyzer that is able to measure MCV, MCH, mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), the percentage of hypochromic cells (which is an indicator of insufficient marrow iron supply that reflects recent iron reduction), and the correlation coefficients between these metrics. Our initial results indicate that most of the parameters measured with CTV are within the normal range for healthy adults. Only the parameters related to the red cell volume (primarily MCV and RDW) were outside the normal range. We observed significant discrepancies between the MCV measured by our technology (and also by an automated cell counter) and the manual method that calculates MCV through the hematocrit obtained by packed cell volume, which are attributed to the artifacts of plasma trapping and cell shrinkage. While there may be limitations for measuring MCV, this device offers a novel point of care instrument to provide rapid RBC parameters such as iron stores that are otherwise not rapidly available to the clinician. Thus, our CTV is a promising technology with the potential to be employed as an accurate, economical, portable and fast hematology analyzer after applying instrument-specific reference ranges or correction factors.

Treatment dilemmas: strategies for priapism, chronic leg ulcer disease, and pulmonary hypertension in sickle cell disease.

Sickle cell disease is a disorder characterized by chronic hemolytic anemia and multiorgan disease complications. Although vaso-occlusive episodes, acute chest syndrome, and neurovascular disease frequently result in complication and have well-documented guidelines for management, the management of chronic hemolytic and vascular-related complications, such as priapism, leg ulcers, and pulmonary hypertension, is not as well recognized despite their increasing reported prevalence and association with morbidity and mortality. This chapter therefore reviews the current updates on diagnosis and management of priapism, leg ulcers, and pulmonary hypertension.

Red blood cells differentiated in vitro using sequential liquid and semi-solid culture as a pre-clinical model.

The in vitro erythrocyte differentiation model remains a strong, clinically relevant tool to model erythroid development in normal and disease related hematopoiesis. This model also has application to developing therapeutics for diseases related to red blood cells such as sickle cell anemia where targeting increased expression of fetal hemoglobin has been a major emphasis. Since the original protocol's publication in 2002, many groups have published modified methodologies to address issues in efficiency of maturation and terminal enucleation, as well as in scalability. While all reports have merit and show efficient enucleation, the methodologies used vary widely in technique and cytokine content. Additionally, despite the strengths in these methods, reproducibility of efficient differentiation to the point of differentiation is difficult. To address these limitations, we developed a streamlined process where total PBMCs are first primed using the original liquid culture expansion phase (published in 2002) before being differentiated with minimal input via standardized, commercially purchased semi-solid medium culture pre-supplemented with erythropoietin. Our data show this methodology to produce similar levels of CD235/CD71 positivity as previous methods but with enhanced CD235 solo positivity and evidence of enucleated cells in comparison with other widely used methods. Given the difficulty and wide variation in in vitro differentiation techniques, we present this methodology as a streamlined methodology for production of mature erythroid cells with minimal input using easily purchased reagents.