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. 2020 Aug 10;15(8):e0237543. doi: 10.1371/journal.pone.0237543

Serum albumin is independently associated with higher mortality in adult sickle cell patients: Results of three independent cohorts

Mehdi Nouraie 1,*, Allison E Ashley-Koch 2, Melanie E Garrett 2, Nithya Sritharan 3, Yingze Zhang 1, Jane Little 4, Victor R Gordeuk 5, Mark T Gladwin 1,3, Marilyn J Telen 2, Gregory J Kato 1
Editor: Mary Hamer Hodges6
PMCID: PMC7416942  PMID: 32776978

Abstract

Sickle cell disease (SCD) impacts liver and kidney function as well as skin integrity. These complications, as well as the hyperinflammatory state of SCD, could affect serum albumin. Serum albumin has key roles in antioxidant, anti-inflammatory and antithrombotic pathways and maintains vascular integrity. In SCD, these pathways modulate disease severity and clinical outcomes. We used three independent SCD adult cohorts to assess clinical predictors of serum albumin as well its association with mortality. In 2553 SCD adult participants, the frequency of low (<35 g/L) serum albumin was 5%. Older age and lower hemoglobin (P <0.001) were associated with lower serum albumin in all three cohorts. In age and hemoglobin adjusted analysis, higher liver enzymes (P <0.05) were associated with lower serum albumin. In two of the three cohorts, lower kidney function as measured by Glomerular Filtration Rate (P<0.001) was associated with lower serum albumin. Lower serum albumin predicted higher risk of tricuspid regurgitation velocity ≥ 2.5 m/s (OR = 1.1 per g/L, P ≤0.01). In all three cohorts, patients with low serum albumin had higher mortality (adjusted HR ≥2.9, P ≤0.003). This study confirms the role of serum albumin as a biomarker of disease severity and prognosis in patients with SCD. Albumin as a biomarker and possible mediator of SCD severity should be studied further.

Introduction

Albumin is multifunctional and one of the most abundant plasma proteins. In adults, the normal serum albumin level is 35–50 g/L, accounting for about 50% of the serum proteins [1]. Albumin is vital in human physiology because of its colloidal properties. It also plays a crucial role in maintaining osmotic pressure of extracellular fluids. Albumin has antioxidant properties and scavenges many plasma small molecules, including heme, and many drugs. These characteristics lead to its anti-inflammatory effect. In addition, albumin has also been identified to have emerging biologic roles in maintaining vascular integrity and even as an anti-thrombotic factor [2]. Albumin has a heparin-like activity and reduces platelet aggregation [3].

Serum albumin concentration is determined by its synthesis in the liver, its degradation, and its distribution between the intra- and extra-vascular spaces. The liver of a healthy human of 70 kg produces about 14 g of albumin a day. Albumin is eliminated from the kidney, gastrointestinal tract and through catabolism, and has a total half-life of about 21 days [4] and intravascular half-life of less than 24 hours [1]. A decreased serum albumin level can be caused by low amino acid or energy supply, impaired liver function, increased albumin loss, catabolism or a change in distribution between intravascular and extravascular fluid [5]. Other predictors of decreased serum albumin are age, inflammation, chronic renal disease and general health status. In adults, serum albumin <35 g/L is considered low. Multiple studies suggest that there is a continuous association between lower serum albumin and higher mortality in patients with cancer [6], trauma and recent surgery [7], myocardial infarction (MI) [8], heart failure [9], stroke [10] and sepsis [11]. A recent analysis of healthy adults suggests that serum albumin is one of promising biomarker that can predict biological aging and can predict overall survival [12].

In patients with sickle cell disease (SCD), the presence of HbS in red blood cells initiates many harmful pathways that lead to vaso-occlusion, hemolytic anemia and down-stream reduced nitric oxide (NO) bioavailability, and increased inflammation, oxidative stress and platelet aggregation. In addition, SCD patients are at a higher risk of endothelial damage [13] and have a range of comorbidities, including liver and renal disease, that could affect their serum albumin [13]. Intravascular hemolysis releases free hemoglobin that undergoes oxidation to release free heme, which is partly carried by albumin. However, the role of serum albumin as a potential prognostic factor for mortality has not been examined in SCD. In this study of three independent adult SCD cohorts, we aim to assess the predictors of serum albumin and its prognostic value for mortality in adult SCD patients.

Materials and methods

IRB of University of Pittsburgh and Duke approved the study.

The treatment of pulmonary hypertension and sickle cell disease with sildenafil therapy (Walk-PHaSST)

The Walk-PHaSST study was designed to assemble a large cohort of patients with SCD and to screen their eligibility for a randomized clinical trial of sildenafil via echocardiographic and laboratory screening for pulmonary hypertension. In summary, the study recruited 720 adolescents and adults with SCD at 10 clinical centers in the United States (U.S.) and United Kingdom (UK) between 2007–2009. Each study participant was comprehensively evaluated by collecting clinical, laboratory and echocardiography data [14]. Serum albumin was measured at the time of recruitment for each study subject.

Cooperative study of sickle cell disease (CSSCD)

The CSSCD was a prospective study of the clinical course of SCD in which more than 3700 children and adults were enrolled at 23 clinical centers throughout the continental U.S. between 1978–1988. All acute and chronic complications were documented at the participating centers. Deaths were reported on a form that was completed by the center investigator [15]. Serum albumin was measured at the time of recruitment for each study subject.

Outcome Modifying Genes in Sickle Cell Disease (OMG-SCD)

The OMG-SCD study enrolled over 800 adult patients with SCD to study genetic associations with clinical outcomes from five sickle cell centers in the southeastern U.S. between 2002 and 2015. All the participants’ data included thorough medical histories, clinical assessments, laboratory testing of urine and blood samples, and genotyping [16]. Serum albumin was measured at the time of recruitment for each study subject.

Statistical analysis

For this study, we selected adult (≥18 years) patients with available serum albumin in each cohort. Linear regression was used to investigate the relationship between serum albumin and patient characteristics or clinical measures, adjusted for age and hemoglobin at enrollment. We used the hazard ratio from Cox regression analysis to assess the prognostic value of serum albumin at enrollment on mortality in each cohort, adjusting for age and hemoglobin at enrollment. In these models, the proportional hazard assumption was satisfied. Finally, logistic regression was used to test for association between serum albumin and elevated systolic pulmonary pressure as defined by tricuspid regurgitation velocity (TRV) ≥ 2.5 m/s), adjusting for age, lactate dehydrogenase (LDH), and serum creatinine. We adjusted for multiple hypotheses testing using Simes method [17].

Results

Predictors of serum albumin levels

Walk-PHaSST

As an initial test cohort, this study provided data on 630 adults with SCD (93% Black). In these patients, lower hemoglobin level and older age were associated with lower serum albumin (S1 Table). In age- and hemoglobin-adjusted analysis, higher alkaline phosphatase, kidney dysfunction as measured by serum creatinine or estimated Glomerular Filtration Rate (eGFR) and markers of cardiopulmonary dysfunction, as measured by TRV and NT-proBNP, were associated with lower serum albumin (Table 1). Paradoxically, LDH was associated with higher serum albumin. Lower serum albumin was associated with greater risk of elevated systolic pulmonary artery pressure (adjusted OR = 1.10 per g/L, P < 0.001). Among these patients, 49 (7.8%) had serum albumin <35 g/L at steady state.

Table 1. Age and hemoglobin adjusted association between serum albumin and sickle cell clinical variables in adults with sickle cell disease in test cohorts.
Walk-PHaSST CSSCD
Adjusted beta (SE) or Mean (SE) P value1 Adjusted beta (SE) or Mean (SE) P value1
Female gender, n (%) M = 41.8 (0.25); F = 41.4 (0.23) 0.90 M = 44.3 (0.18); F = 43.7 (0.16) 0.15
SS genotype, n (%) SS/SB0 = 41.6 (0.20); Other = 41.5 (0.40) 0.19 SS/SB0 = 43.9 (0.17); Other = 43.9 (0.17) 0.08
Number of severe pains in last year, n (%) 0.01 (0.36) 0.36 -- --
Chronic transfusion, n (%) N = 41.5 (0.18); Y = 42.0 (0.49) 0.91 N = 43.7 (0.14); Y = 42.1 (1.23) 0.19
History of acute chest syndrome, n (%) N = 41.6 (0.28); Y = 41.6 (0.21) 0.59 -- --
Leg ulcer, n (%) N = 41.9 (0.18); Y = 41.3 (0.38) 0.40 N = 44.1 (0.17); Y = 43.8 (0.17) 0.34
BMI (kg/m2) -0.03 (0.02) 0.14 -0.01 (0.01) 0.15
MCV (fL) -0.01 (0.01) 0.42 -0.008 (0.01) 0.49
White blood cell count (x109/L) 0.04 (0.047) 0.37 -0.07 (0.04) 0.045
Platelet count (x109/L) -0.0002 (0.001) 0.88 -0.0004 (0.001) 0.66
Lactate dehydrogenase (U/L) 0.003 (0.001) <0.001 0.002 (0.001) <0.001
Reticulocyte count (x109/L) 0.03 (0.01) 0.020 -0.02 (0.01) 0.015
Total bilirubin (mg/dL) -0.01 (0.06) 0.87 0.18 (0.05) 0.001
Alanine aminotransferase (U/L) -0.01 (0.008) 0.20 -0.01 (0.003) 0.002
Aspartate aminotransferase (U/L) -0.003 (0.006) 0.55 -0.008 (0.002) 0.002
Alkaline phosphatase (U/L) -0.01 (0.003) <0.001 -0.01 (0.002) <0.001
Creatinine (mg/dL) -0.77 (0.19) <0.001 0.06 (0.18) 0.76
eGFR (mL/min/1.73m2) 0.02 (0.006) <0.001 0.007 (0.004) 0.088
NT-proBNP (pg/mL) -0.0003 (0.0001) <0.001 -0.00004 (0.00004) 0.38
Tricuspid regurgitation velocity, m/sec -2.2 (0.45) <0.001 -- --
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1 P values ≤ 0.015 are significant after correcting for multiple hypothesis testing using False Discover Rate <0.05.

CSSCD

As a validation cohort, data from 1303 adults with SCD (98% Black) from the CSSCD were analyzed. Replicating the findings from Walk-PHaSST, lower hemoglobin and older age were associated with lower serum albumin (S1 Table). In age- and hemoglobin-adjusted analysis, elevated transaminases and alkaline phosphatase were associated with lower serum albumin. Higher total bilirubin and LDH were also associated with higher serum albumin. Kidney function as defined by serum creatinine was not associated with serum albumin (Table 1). In this cohort, 36 (2.8%) had serum albumin <35 g/L.

OMG-SCD

Providing a second validation cohort, the data from 620 adult SCD patients (99% Black) were evaluated from OMG-SCD. As observed in Walk-PHaSST and CSSCD, higher serum albumin was associated with higher hemoglobin and younger age. In this cohort, higher transaminases but not alkaline phosphatase were associated with lower serum albumin. Higher total bilirubin and better kidney function measured by eGFR were associated with higher albumin (Table 2). Lower serum albumin was associated with higher risk of elevated systolic pulmonary artery pressure (adjusted OR = 1.09 per g/L, P = 0.0119). In this cohort, 49 (7.9%) had serum albumin <35 g/L.

Table 2. Age and hemoglobin adjusted association between serum albumin and sickle cell clinical variables in adults with sickle cell disease in validation cohort (OMG).
  Adjusted mean (SE) or beta (SE) P value1
Female gender F = 41.3 (0.27), M = 41.6 (0.29) 0.39
SS genotype SS/SB0 = 41.4 (0.21), other = 41.4 (0.66) 0.99
Hospitalizations for severe pain in last year 0–1 = 41.7 (0.27), 0.036
2–4 = 40.5 (0.40),
>4 = 41.7 (0.50)
Chronic transfusion N = 41.3 (0.21), Y = 41.3 (0.80) 0.97
History of acute chest syndrome N = 41.6 (0.39), Y = 41.2 (0.24) 0.41
Leg ulcer N = 41.5 (0.24), Y = 41.0 (0.44) 0.33
BMI (kg/m2) -0.064 (0.037) 0.08
Current MCV (fL) -0.019 (0.014) 0.18
Current WBC (x109/L) 0.10 (0.05) 0.040
Current Platelet (x109/L) -0.002 (0.001) 0.10
Lactate dehydrogenase (U/L) -0.0007 (0.001) 0.49
Reticulocyte count (x109/L) 0.001 (0.002) 0.53
Total bilirubin (mg/dL) 0.20 (0.08) 0.009
Alanine aminotransferase (U/L) -0.015 (0.007) 0.049
Aspartate aminotransferase (U/L) -0.013 (0.006) 0.022
Alkaline phosphatase (U/L) -0.006 (0.003) 0.08
Creatinine (mg/dL) -0.37 (0.20) 0.07
eGFR (mL/min/1.73m2) 0.021 (0.006) <0.001
NT-proBNP (pg/mL) -0.0003 (0.0011) 0.78
Tricuspid regurgitation velocity (m/sec) -0.65 (0.39) 0.10
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1 P values ≤ 0.001 are significant after correcting for multiple hypothesis testing using False Discover Rate <0.05.

In both Walk-PHaSST and OMG cohorts, higher eGFR was consistently associated with higher serum albumin whereas in CSSCD and Walk-PHaSST cohorts, higher LDH and lower alkaline phosphatase were associated with higher serum albumin.

Serum albumin is associated with higher mortality

Walk-PHaSST

We followed 592 patients for a median of 29 months (IQR: 25–33). There were 21 (3.6%) patients who died over the course of this follow-up period. Serum albumin as a continuous variable (HR = 0.91, 95% CI: 0.85–0.97, P = 0.004) was inversely associated with mortality and low serum albumin (HR = 5.3; 95% CI: 2.0–13.6, P = 0.001) was significantly associated with higher mortality. Probability of survival at 1 and 3 years was 99% and 96% respectively in patients with normal albumin compared to 89% and 86% respectively in patients with low albumin. After adjusting for age and hemoglobin, continuous serum albumin (HR = 0.91, 95% CI: 0.85–0.97, P = 0.014) and low serum albumin (HR = 4.7, 95% CI: 1.7–13.0, P = 0.003) were both significantly associated with mortality (Fig 1A).

Fig 1.

Fig 1

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Low serum albumin (<35 g/L solid line) predicts higher mortality among sickle cell disease patients in a) Walk-PHaSST b) CSSCD and c) OMG-SCD cohorts. Graphs show the age and hemoglobin adjusted survival in each cohort.

CSSCD

One thousand thirty-one patients were followed for a median of 82 months (IQR: 60–89) and 237 (23.0%) patients died during the follow-up period. Continuous serum albumin (HR = 0.94,95% CI: 0.92–0.96, P < 0.001) and low serum albumin (HR = 3.5, 95% CI: 2.1–5.9, P < 0.001) were both significantly associated with mortality. Probability of survival at 1 and 5 years was 97% and 89% in patients with normal albumin compared to 93% and 67% in patients with low albumin. After adjusting for age and hemoglobin, both continuous serum albumin (HR = 0.97, 95% CI: 0.93–0.998, P = 0.039) and low serum albumin (HR = 2.9, 95% CI: 1.5–5.5, P = 0.001) were significantly associated with mortality (Fig 1B).

OMG-SCD

We followed 195 SCD patients for a median of 156 months (IQR: 108–192). In that time, 53 patients died (27.2%). Serum albumin both as a continuous variable (HR = 0.88, 95% CI:0.84–0.93, P < 0.001) and as a dichotomous variable (<35 g/L; HR = 3.8, 95% CI: 2.0–7.2, P < 0.001) was significantly associated with mortality. Probability of survival at 1 and 5 years was 98% and 90% in patients with normal albumin compared to 79% and 63% in patients with low albumin. After adjusting for age and hemoglobin, both serum albumin as a continuous variable (HR = 0.92, 95% CI: 0.87–0.98, P = 0.008) and lower serum albumin (HR = 2.9, 95% CI: 1.4–5.9, P = 0.003) were significantly associated with mortality (Fig 1c).

Discussion

In our study of three independent cohorts of adults with SCD, serum albumin concentration was significantly associated with severity of anemia, as well as liver and renal dysfunction. Low serum albumin was observed in ~5% of patients and predicted a higher estimated systolic pulmonary artery pressure and overall mortality of patients.

Sickle cell disease is a hyper-inflammatory state in which inflammatory markers and reactive oxygen species (ROS) are increased. Serum concentration of pro-inflammatory cytokines, such as IL-6, that are associated with decreased albumin production, is higher in SCD patients [18]. Excessive production of ROS and free heme in SCD may also contribute to a higher rate of albumin catabolism [13].

Acute and chronic complications of SCD impact hepatobiliary function. Sickle cell hepatopathy includes a range of manifestations ranging from liver dysfunction to cholestasis [19]. Liver dysfunction could impact albumin production in SCD patients. In this study, we have been able to replicate an association between decreased hepatobiliary function (as measured by liver transaminases or ALK) and lower serum albumin in three independent SCD cohorts. Liver function can also influence other heme carrying proteins including hemopexin and haptoglobin. We did not measure these proteins in all cohorts but an analysis in the Walk-PHaSST cohort showed that serum albumin and haptoglobin are not correlated (r = 0.06, P = 0.17). Studies in children with severe malnutrition have shown that plasma albumin concentration was modulated by catabolic rates rather than synthesis rates [20, 21]. SCD patient are in a state of hyper-inflammation which could lead to faster catabolic rates.

Renal dysfunction, as measured by higher serum creatinine and lower eGFR, was a strong predictor of low serum albumin in walk-PHaSST and OMG cohorts. Renal insufficiency is a frequent comorbidity in SCD patients, with a prevalence of >25% in adult patients [22]. It is also an important prognostic factor in these patients [23]. Renal tubular loss of albumin and high urine albumin to creatinine ratio is observed in over one third of adults with SCD [24]. Low albumin predicts mortality in patients with chronic kidney disease [25, 26]. Serum albumin is considered a marker of general nutrition and health status in patients with chronic kidney disease. In addition, this prognostic effect could also be explained by the antioxidant effect of albumin and its protection against metabolic acidosis and further kidney damage [27, 28].

In our study, both elevated TRV and NT-proBNP, which are non-invasive markers of elevated pulmonary systolic blood pressure and left ventricular (LV) diastolic dysfunction, are two major prognostic factors in adults with SCD [23]. Both were significantly correlated with low serum albumin. Lower serum albumin has also been associated with worse LV diastolic dysfunction in children with chronic kidney disease [29]. Serum albumin level is an independent predictor of one-year survival in patients with heart failure with preserved ejection fraction [30]. Low-grade albuminuria has been associated with lower LV diastolic function in other clinical settings [31]. The Heart Outcomes Prevention Evaluation study suggests that higher urine creatinine to albumin ratio within the normal range is significantly associated with the increasing prevalence of myocardial dysfunction and mortality [32]. The effect of serum albumin on myocardial function is potentially mediated by multiple mechanisms. Albumin maintains the integrity of the microvasculature of the myocardium and is protective against myocardial edema. Also, it protects against myocyte oxidative stress and inflammation [4, 28, 33]. In addition to such direction protection, this association may be explained by the role of chronic liver disease in the development pulmonary hypertension through portal hypertension [34].

It is also conceivable that low albumin could mediate SCD pathophysiologic mechanisms. It is well known that free heme released during hemolysis is a part of SCD pathophysiology, with increasing evidence that heme induces inflammatory pathways [35]. Heme is hydrophobic and insoluble in plasma and thus is carried by plasma proteins, including haptoglobin, hemopexin and albumin. Serum albumin has a lower affinity for free heme than haptoglobin or hemopexin but can sequester the heme when these two proteins are depleted or saturated [36]. One provocative hypothesis is that low serum albumin provides low heme carrying capacity, potentially increasing the concentrations of free heme that might be taken up by susceptible tissues. Heme response in neutrophils, macrophages, erythroid cells and endothelial cells plays a role in SCD pathophysiology [37].

There are some limitations in our study. We have not measured the nutrition status of our patients, which could provide some insight regarding the cause of the low albumin. In addition, we have not identified the specific cause of mortality. Thus, we cannot speculate whether the low albumin led to specific causes of death or represents a general risk factor for mortality. We also did not examine proteinuria in these cohorts. Serum albumin was comparable between all three cohorts whereas inter-study variation exists in some other clinical variables. This variability could be due to random variation or different patient characteristics including age, disease severity, genetic structure, disease modifying treatments (including hydroxyurea and chronic transfusion) and comorbidities (as well as their treatment). Other factors that can cause inter-study variability are different birth cohorts and time period between CSSCD and the two other studies, differences in the study protocol and laboratory measurements. These differences could limit the generalizability of our findings to any new sickle cell cohort. However, the inclusion of two replication cohorts provides more confidence in our findings. To the best of our knowledge, this is the first report indicating predictors and prognostic impact of serum albumin in SCD patients, despite the ready availability of serum albumin measurement in clinical settings. Understanding the link between inflammation, hypoalbuminemia, and poor outcome in SCD patients could help identify at-risk patients and define novel mechanistic applications of serum albumin in SCD.

There are existing controversies to consider albumin as a supplementary treatment for liver and renal failure as well as volume resuscitation in sepsis [38]. Our findings support the importance of plasma albumin measure and potential benefit of boosting plasma levels of albumin in SCD, especially in SCD endemic countries with high frequency of protein-calorie malnutrition.

Supporting information

S1 Table. Unadjusted correlation between serum albumin and clinical variables in adults with sickle cell disease in test cohorts.

Results are in median (IQR) unless otherwise specified.

(DOCX)

Click here for additional data file. (20.6KB, docx)
S2 Table. Unadjusted correlation between serum albumin and sickle cell clinical variables in adults with sickle cell disease in validation cohort (OMG).

Results are in median (IQR) unless otherwise specified.

(DOCX)

Click here for additional data file. (14.3KB, docx)

Data Availability

University of Pittsburgh and Duke University IRBs restrict data sharing for Walk-Phasst and SCD-OMG to authorized investigators only. The data transfer agreement with NHLBI/BioLincc imposes restrictions on directly sharing data from CSSCD with any other investigator. Data from all three cohorts are regulated by strict data transfer agreement to protect the identity of patients with sickle cell disease. Walk-Phasst and SCD-OMG data are available at https://www.ncbi.nlm.nih.gov/gap/ for authorized investigators. CSSCD data are available at https://biolincc.nhlbi.nih.gov/home/. Walk-Phasst data can be requested from Ms. Nydia Chien (chienn@upmc.edu) and SCD-OMG can be requested from Ms. Radina Simeonova (radina.simeonova@duke.edu) for researchers who meet the criteria for access to confidential data. CSSCD data can be requested from Biolincc (biolincc@imsweb.com).

Funding Statement

M.J. Telen and A.E. Ashley-Koch received grants from Doris Duke Charitable Foundation. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Mary Hamer Hodges

27 May 2020

PONE-D-20-14109

Serum albumin independently predicts higher mortality in adult sickle cell patients: Results of three independent cohorts.

PLOS ONE

Dear Dr. Mehdi Nouraie,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The conclusion could have been bolstered by more references to the literature on albumin synthesis rates in  children with acute-phase protein responses to infection in edematous and non-edematous protein-energy malnutrition. The speculation that hepatic synthetic rates may be affected because of the the association of low albumin with liver enzyme elevation raised concern in the concluding paragraph which should be re-phrased.

Please submit your revised manuscript by June 15th. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Mary Hamer Hodges

Academic Editor

PLOS ONE

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We will update your Data Availability statement to reflect the information you provide in your cover letter.

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We recommend addressing the comments made by Reviewer #1 and resubmitting.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This paper reports on the outcome of analysis on 3 large retrospective cohort of subjects with Sickle Cell Disease. The hypotheses, and justification were clear. The results were clear. Albumin as a negative acute phase protein is a biomarker for worse outcome and this is consistent across a range of clinical models. The conclusion could have been bolstered by reference to the literature where albumin synthesis rates were measured in vivo. So in a model of severe malnutrition in children albumin concentration were modulated by catabolic rates rather than synthesis rates. In fact children with severe malnutrition were able to synthesize a range of positive acute phase proteins at fast enough rate in response to infections. So Sickle cell as an example of inflammatory state may behave similarly. (Morlese JF, Forrester T, Badaloo A, Del Rosario M, Frazer M, Jahoor F. Albumin kinetics in edematous and nonedematous protein-energy malnourished children. Am J Clin Nutr. 1996 Dec;64(6):952-9. doi: 10.1093/ajcn/64.6.952. PMID: 8942422 & Reid, M., Badaloo, A., Forrester, T., Morlese, J.F., Heird, W.C. & Jahoor, F. (2002) The acute-phase protein response to infection in edematous and nonedematous protein-energy malnutrition. Am J Clin Nutr, 76, 1409-1415.). While the authors speculated in the manuscript that hepatic synthetic rates may be affected because of the the association of low albumin with liver enzyme elevation this may not be the case. Within that context I am little concerned about the wording of the concluding paragraph which at a casual glance may seem to be advocating supplemental albumin treatment in SCD and perhaps a clear definite statement that this is not their recommendation should be made

**********

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Reviewer #1: Yes: Marvin E Reid

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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PLoS One. 2020 Aug 10;15(8):e0237543. doi: 10.1371/journal.pone.0237543.r002

Author response to Decision Letter 0

4 Jun 2020

PONE-D-20-14109

Dear Dr. Hodges;

We appreciate your and review comments on our manuscript. Hereby, we are responding to comments and addressing changes in the manuscript:

Comment: The conclusion could have been bolstered by more references to the literature on albumin synthesis rates in children with acute-phase protein responses to infection in edematous and non-edematous protein-energy malnutrition. The speculation that hepatic synthetic rates may be affected because of the association of low albumin with liver enzyme elevation raised concern in the concluding paragraph which should be re-phrased.

Response: We modified the discussion and conclusion to reflect the reviewer’s comments.

Comment: In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Response: The current manuscript is using three different databases. Data from one of these cohort (CSSCD) was received through an agreement with NHLBI/BioLincc. This agreement prohibits investigator form any further data sharing. However, other investigators can apply directly to BioLincc for their own access to the data. The remaining two cohorts are governed by academic institutional IRBs which have their own restrictions on sharing. However, both of the other cohorts are available for access via dbGaP. So, regretfully we are unable to comply within the legal terms of our data use agreement.

Comment: We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Response: This sentence was changed to:

“We did not measure these proteins in all cohorts but an analysis in the Walk-PHaSST cohort showed that serum albumin and haptoglobin are not correlated (r = 0.06, P = 0.17).”

Reviewer #1 comment: This paper reports on the outcome of analysis on 3 large retrospective cohort of subjects with Sickle Cell Disease. The hypotheses, and justification were clear. The results were clear. Albumin as a negative acute phase protein is a biomarker for worse outcome and this is consistent across a range of clinical models. The conclusion could have been bolstered by reference to the literature where albumin synthesis rates were measured in vivo. So in a model of severe malnutrition in children albumin concentration were modulated by catabolic rates rather than synthesis rates. In fact children with severe malnutrition were able to synthesize a range of positive acute phase proteins at fast enough rate in response to infections. So Sickle cell as an example of inflammatory state may behave similarly. (Morlese JF, Forrester T, Badaloo A, Del Rosario M, Frazer M, Jahoor F. Albumin kinetics in edematous and nonedematous protein-energy malnourished children. Am J Clin Nutr. 1996 Dec;64(6):952-9. doi: 10.1093/ajcn/64.6.952. PMID: 8942422 & Reid, M., Badaloo, A., Forrester, T., Morlese, J.F., Heird, W.C. & Jahoor, F. (2002) The acute-phase protein response to infection in edematous and nonedematous protein-energy malnutrition. Am J Clin Nutr, 76, 1409-1415.). While the authors speculated in the manuscript that hepatic synthetic rates may be affected because of the association of low albumin with liver enzyme elevation this may not be the case. Within that context I am little concerned about the wording of the concluding paragraph which at a casual glance may seem to be advocating supplemental albumin treatment in SCD and perhaps a clear definite statement that this is not their recommendation should be made

Response: We cited these references in discussion as:

“Studies in children with severe malnutrition have shown that plasma albumin concentration was modulated by catabolic rates rather than synthesis rates[19, 20]. SCD patient are in a state of hyper-inflammation which could lead to faster catabolic rates.”

Also, we changed the conclusion to:

“There are existing controversies to consider albumin as a supplementary treatment for liver and renal failure as well as volume resuscitation in sepsis [37]. Our findings support the importance of plasma albumin measure and potential benefit of boosting plasma levels of albumin in SCD, especially in SCD endemic countries with high frequency of protein-calorie malnutrition.”

Sincerely yours,

Mehdi Nouraie, MD, PhD

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (23.6KB, docx)

Decision Letter 1

Mary Hamer Hodges

9 Jul 2020

PONE-D-20-14109R1

Serum albumin independently predicts higher mortality in adult sickle cell patients: Results of three independent cohorts .

PLOS ONE

Dear Mehdi Nouraie,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please address the inter-study variability, possible causes and study limitations

Please submit your revised manuscript by 18th July. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Mary Hamer Hodges

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Please address the comments from reviewer #3 regarding the inter-study variability. If no potential cause for this variability can be identified it should be discussed as a weakness of their results.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have addressed my concerns appropriately. The limitations of post-hoc secondary data analyses were addressed satisfactorily.

Reviewer #2: Serum albumin independently predicts higher mortality in adult sickle cell patients:

Results of three independent cohorts is an interesting article all modifications were done as per reviewers recommendations

the manuscript looks much better

Reviewer #3: This paper presents the results for 3 different SSD studies. It was found that low serum albumin was associated with higher mortality across all 3 studies. However, the paper also looks at predictors of low serum albumin in Tables 1 and 2 of the manuscript. There is a large amount of inter-study variability which is not well accounted for. It seems possible that there are significant differences in the enrolled populations of the 3 studies and that these differences may impact the predictors relationship with serum albumin. Please see the attached PDF for my specific comments.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: MARVIN REID

Reviewer #2: Yes: Mohamed A Yassin

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Aug 10;15(8):e0237543. doi: 10.1371/journal.pone.0237543.r004

Author response to Decision Letter 1

12 Jul 2020

Dear Dr. Hodges

Authors appreciate your and reviewers’ invaluable new comments on our manuscript. Hereby, we are addressing the comments and changes in the new version.

Editor Review

Please address the inter-study variability, possible causes and study limitations

Response: We added this statement to study limitation:

“Serum albumin was comparable between all three cohorts whereas inter-study variation exists in some other clinical variables. This variability could be due to random variation or different patient characteristics including age, disease severity, genetic structure, disease modifying treatments (including hydroxyurea and chronic transfusion) and comorbidities (as well as their treatment). Other factors that can cause inter-study variability are different birth cohorts and time period between CSSCD and the two other studies, differences in the study protocol and laboratory measurements. These differences could limit the generalizability of our findings to any new sickle cell cohort.”

Reviewer #3 comments:

This paper presents the results for 3 different SSD studies. It was found that low serum albumin was associated with higher mortality across all 3 studies. However, the paper also looks at predictors of low serum albumin in Tables 1 and 2 of the manuscript. There is a large amount of inter-study variability which is not well accounted for. It seems possible that there are significant differences in the enrolled populations of the 3 studies and that these differences may impact the predictors relationship with serum albumin.

See below for specific comments.

1.1 Specific Comments

1. The term “predicts” in the title is misleading. Many people will interpret the claim that “Serum albumin independently predicts higher mortality” as a statement of that serum albumin has a high predictive value (sensitivity, specificity, AUC, etc) with respect to mortality. However, what is shown is that low serum corresponds to a high hazard ratio. I think “Serum albumin is independently associated with higher mortality” is a more appropriate phrase.

Response: We changed the title to:

“Serum albumin is independently associated with higher mortality in adult sickle cell patients: Results of three independent cohorts”

2. You mentioned you tested serum albumin for normality. Do you mean the residuals from the individual fitted linear models are normal? Serum albumin should not be normal on its own, unless the predictor variables are all insignificant. The residuals are what we expect to be normal. In point of fact, when the sample size is large the ordinary least squares estimates are asymptotically normal even if the residuals are non-normal. The sample sizes in this study are large enough that I think the results are valid even if the residuals are not normal.

Response: We removed this statement from the statistical analysis as it did not impact our analysis.

3. The false discovery rate adjustment method and procedure should be stated.

Response:

We added this statement in statistical method:

“We adjusted for multiple hypotheses testing using Simes method [17]”.

4. The results of table 1 and 2 make drawing conclusions about associations between serum albumin and sickle cell clinical variables challenging. Many of the variables change signs, even significantly, from study to study. For example ARC beta is significant in the WALK study at 0.03. For CCSSD it is significant at -0.02. Then it is insignificant at 0.001 for OMG. There are many examples of this behavior. Inter-study variability of beta and even significance is to expected; however, when a variable is significant and changes signs from study to study

represents a failure in validation. A disclaimer or a summary across all 3 studies of which variables of interest exhibited consistent behavior would help a reader understand how to better interpret Tables 1 and 2.

Response: We added this statement at the end of first result section:

“In both Walk-PHaSST and OMG cohorts, higher eGFR was consistently associated with higher serum albumin whereas in CSSCD and Walk-PHaSST cohorts, higher LDH and lower alkaline phosphatase were associated with higher serum albumin.”

5. Is there any insight into why there is such a significant amount of inter-study variability. Certainly CCSSCD, which ended in 1988, might be difficult to compare to patients collected after the year 2000. Are there differences in Age, hemoglobin, or other variables appearing in tables 1 and 2 between the 3 studies? Perhaps the populations at enrollment are significantly different. If so multivariate models that include the variables in table 1 and 2 might be more consistent.

Response: We added some statements to describe inter-study variability (please see the response to editor comment). In addition, tables 1 and 2 are presenting age and hemoglobin adjusted correlations to partially adjust for baseline differences between three cohorts.

6. In Tables 1 and 2 it is stated that p<0.015 and <0.001 are significant after FDR. However, there are rows in bold with p-values greater than these amounts.

Response: We removed the bold marks to prevent any confusion in these tables.

7. In the discussion it is stated that “ Finally, we also did 203 not correct for multiple testing.” However, in each table the FDR rate is listed. FDR is a multiplicity adjustment method. This is confusing was and FDR used or not?

Response: It was removed from discussion

Sincerely,

Mehdi Nouraie, MD, PhD

Associate Professor of Medicine

University of Pittsburgh

Attachment

Submitted filename: Response to Reviewers_3.docx

Click here for additional data file. (18.2KB, docx)

Decision Letter 2

Mary Hamer Hodges

29 Jul 2020

Serum albumin is independently associated with higher mortality in adult sickle cell patients: Results of three independent cohorts

PONE-D-20-14109R2

Dear Dr. Mehdi Nouraie,

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PLOS ONE

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Reviewer #3: Yes: Adam Lane

Acceptance letter

Mary Hamer Hodges

30 Jul 2020

PONE-D-20-14109R2

Serum albumin is independently associated with higher mortality in adult sickle cell patients: Results of three independent cohorts

Dear Dr. Nouraie:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Mary Hamer Hodges

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Unadjusted correlation between serum albumin and clinical variables in adults with sickle cell disease in test cohorts.

    Results are in median (IQR) unless otherwise specified.

    (DOCX)

    Click here for additional data file. (20.6KB, docx)
    S2 Table. Unadjusted correlation between serum albumin and sickle cell clinical variables in adults with sickle cell disease in validation cohort (OMG).

    Results are in median (IQR) unless otherwise specified.

    (DOCX)

    Click here for additional data file. (14.3KB, docx)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (23.6KB, docx)
    Attachment

    Submitted filename: Response to Reviewers_3.docx

    Click here for additional data file. (18.2KB, docx)

    Data Availability Statement

    University of Pittsburgh and Duke University IRBs restrict data sharing for Walk-Phasst and SCD-OMG to authorized investigators only. The data transfer agreement with NHLBI/BioLincc imposes restrictions on directly sharing data from CSSCD with any other investigator. Data from all three cohorts are regulated by strict data transfer agreement to protect the identity of patients with sickle cell disease. Walk-Phasst and SCD-OMG data are available at https://www.ncbi.nlm.nih.gov/gap/ for authorized investigators. CSSCD data are available at https://biolincc.nhlbi.nih.gov/home/. Walk-Phasst data can be requested from Ms. Nydia Chien (chienn@upmc.edu) and SCD-OMG can be requested from Ms. Radina Simeonova (radina.simeonova@duke.edu) for researchers who meet the criteria for access to confidential data. CSSCD data can be requested from Biolincc (biolincc@imsweb.com).

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