Impact of Anemia on Time to Resolution of Diabetic Ketoacidosis and Hospital Outcomes in Children: A Single-Center Retrospective Cohort Study
Conditions: Diabete Type 1, Ketoacidosis, Diabetic, Pediatrics, Child
Sponsor: Aydin Adnan Menderes University

STUDY PROTOCOL AND STATISTICAL ANALYSIS PLAN 
Official Title 
Impact of Anemia on Time to Resolution of Diabetic Ketoacidosis and Hospital Outcomes in 
Children: A Single-Center Retrospective Cohort Study 
Brief Title 
Hemoglobin Levels and Resolution Time of Diabetic Ketoacidosis in Pediatric Patients 
Unique Protocol ID 
ADUPEDDKAHB2025 
NCT Number 
Pending 
Sponsor 
Aydin Adnan Menderes University 
Principal Investigator 
Aykut Çağlar, MD 
Associate Professor 
Document Date 
January 22, 2026 
 
 
 

1. Study Design 
This is a single-center, retrospective, observational cohort study conducted in a tertiary 
pediatric emergency department and pediatric intensive care unit. 
2. Background and Rationale 
Diabetic ketoacidosis (DKA) remains one of the most serious acute complications of diabetes 
mellitus in children and adolescents. Despite the availability of standardized treatment 
protocols, the time required for biochemical resolution of DKA varies considerably across 
patients. 
Hemoglobin (Hb) is a major non-bicarbonate buffer in human physiology and plays a critical 
role in acid–base homeostasis beyond its primary function as an oxygen carrier. Reduced 
hemoglobin concentration may impair buffering capacity and tissue oxygen delivery, 
potentially contributing to prolonged metabolic acidosis during DKA. Despite this strong 
physiologic rationale, the relationship between anemia and time to DKA resolution has not 
been systematically investigated in pediatric populations. 
This study aims to investigate whether lower hemoglobin levels are associated with a longer 
time to biochemical resolution of DKA in children. In addition, we will standardize 
hemoglobin by age and sex using hemoglobin z-scores to minimize confounding by 
physiologic variation across childhood and adolescence.  
3. Study Objectives and Hypotheses 
Primary Objective 
To evaluate the association between hemoglobin levels measured within 0–24 hours after 
biochemical resolution of diabetic ketoacidosis (DKA) and the time required to achieve 

biochemical resolution of DKA. Primary exposure will be Hb z-score; absolute Hb will be 
assessed in sensitivity analyses. 
Secondary Objectives 
• To assess the association between post-resolution hemoglobin levels and total hospital 
length of stay. 
• To assess the association between post-resolution hemoglobin levels and pediatric 
intensive care unit (PICU) length of stay. 
• To explore the relationship between admission hemoglobin and hematocrit levels, as 
surrogate markers of dehydration severity, and clinical outcomes. 
Hypotheses 
Null Hypothesis (H0): 
• Hemoglobin level measured after biochemical resolution of DKA is not associated 
with the time to resolution of DKA. 
Alternative Hypothesis (H1): 
• Lower hemoglobin levels measured after biochemical resolution of DKA are 
independently associated with a longer time to resolution of DKA. 
4. Study Population 
Inclusion Criteria 
• 
Age 1–18 years 
• 
Diagnosis of DKA according to ISPAD criteria 
• 
First documented DKA episode 

• 
Availability of hemoglobin measurement within 0–24 hours after biochemical 
resolution of DKA 
• 
Documented time of DKA treatment initiation and biochemical resolution 
Exclusion Criteria 
• 
Hyperosmolar hyperglycemic state (HHS) or mixed DKA–HHS 
• 
Known hemoglobinopathies (e.g., thalassemia major, sickle cell disease) 
• 
Evidence of active hemolysis documented in medical records 
• 
Blood transfusion administered before or during the DKA treatment period 
• 
Missing hemoglobin measurement within the predefined post-resolution window 
5. Definitions 
DKA Resolution 
Biochemical resolution of diabetic ketoacidosis (DKA) is defined by the simultaneous 
achievement of the following criteria: 
• 
Venous pH ≥ 7.30, and 
• 
Serum bicarbonate ≥ 15 mmol/L 
Primary Outcome 
Time to DKA resolution: Time, measured in hours, from initiation of DKA treatment to 
biochemical resolution of DKA, as defined above. 
Time Frame: From initiation of DKA treatment until biochemical resolution, assessed up to 7 
days. 
Secondary Outcomes 

Total hospital length of stay: Time, measured in days, from hospital admission to hospital 
discharge. 
 
Time Frame: From hospital admission until hospital discharge, assessed up to 30 days. 
Pediatric intensive care unit (PICU) length of stay: Time, measured in days, from PICU 
admission to PICU discharge. 
Time Frame: From PICU admission until PICU discharge, assessed up to 30 days.6.  
Data Collection 
Data will be extracted retrospectively from electronic medical records using a standardized 
data collection form. Collected variables will include: 
• Demographic characteristics: age and sex 
• Admission clinical and laboratory findings: including venous pH, serum bicarbonate, 
blood glucose, and hematocrit 
• Serial blood gas and biochemical measurements obtained during DKA management 
• Hemoglobin and hematocrit values measured at admission and within 0–24 hours after 
biochemical resolution of DKA 
• Treatment-related timelines: time of DKA treatment initiation and time of biochemical 
resolution 
• Clinical outcomes: time to DKA resolution, PICU length of stay, and total hospital 
length of stay 
To ensure independence of observations and minimize bias related to recurrent events, only 
the first documented DKA episode per patient will be included in the analysis. 
7. Statistical Analysis Plan 

Analysis Population 
All eligible pediatric patients aged 1–18 years who meet the inclusion criteria and have 
available hemoglobin measurements within 0–24 hours after biochemical resolution of 
diabetic ketoacidosis (DKA) will be included in the analysis. To ensure independence of 
observations, only the first documented DKA episode per patient will be analyzed. Expected 
sample size: approximately ~150 patients, based on institutional case volume. 
Exposure Definition 
The primary exposure variable will be post-resolution hemoglobin standardized as an age- 
and sex-adjusted z-score (Hb z-score). 
Hemoglobin z-scores will be calculated using published pediatric reference values providing 
mean and lower limit of normal (−2 SD) hemoglobin concentrations across age groups, with 
sex-specific values applied for adolescents aged 12–18 years. Because the lower limit 
corresponds to −2 standard deviations, the standard deviation (SD) will be derived as: 
SD=(Mean – Lower Limit)/2  
The hemoglobin z-score = (Hb measured – MeanHb reference) / SD reference 
Anemia will be defined as Hb z-score < −2 in secondary and sensitivity analyses. Hemoglobin 
z-scores will be calculated using published pediatric reference values (Lanzkowsky, 2022), 
providing age- and sex-specific mean and −2 SD hemoglobin concentrations. 
Outcome Measures 
The primary outcome is time to biochemical resolution of DKA, defined as the time (in hours) 
from initiation of DKA treatment to achievement of venous pH ≥ 7.30 and serum bicarbonate 
≥ 15 mmol/L. 

Secondary outcomes include pediatric intensive care unit (PICU) length of stay and total 
hospital length of stay, measured in days. 
 
Descriptive Analysis 
Continuous variables will be evaluated for distributional characteristics using histograms and 
Q–Q plots. Normally distributed variables will be summarized as mean ± standard deviation, 
while non-normally distributed variables will be summarized as median and interquartile 
range (IQR). Categorical variables will be presented as frequencies and percentages.  
Primary Analysis 
The association between Hb z-score and time to DKA resolution will be evaluated using 
linear regression analysis, with time to resolution as the dependent variable and Hb z-score as 
the independent variable. 
If time to resolution demonstrates right-skewed distribution, logarithmic transformation will 
be applied. Model assumptions, including linearity, normality of residuals, and 
homoscedasticity, will be assessed using standard diagnostic procedures. 
 
Multivariable Analysis 
To assess the independent association between Hb z-score and time to DKA resolution, 
multivariable linear regression models will be constructed. Covariates will be selected a priori 
based on clinical relevance and biological plausibility and will include: 
• 
Admission venous pH 
• 
Admission serum bicarbonate 

• 
Admission blood glucose level 
• 
Admission hematocrit (as a surrogate marker of dehydration/hemoconcentration) 
• 
Presence of suspected infection (based on clinical diagnosis, inflammatory markers, 
and/or antibiotic initiation) 
Because hemoglobin is standardized by age and sex, age and sex will not be included in the 
primary adjusted model to avoid redundant adjustment; however, age and sex will be added in 
sensitivity analyses to confirm robustness. Collinearity among covariates will be assessed 
using variance inflation factors (VIFs). 
Secondary Analyses 
Associations between Hb z-score and secondary outcomes (PICU length of stay and total 
hospital length of stay) will be analyzed using regression models appropriate to the 
distribution of each outcome. If length-of-stay variables exhibit overdispersion or skewness, 
generalized linear models with appropriate link functions will be considered. 
Sensitivity Analyses 
Sensitivity analyses will be performed to evaluate the robustness of the findings and will 
include: 
1. Modeling hemoglobin as an absolute value (g/dL) instead of z-score 
2. Categorizing hemoglobin status as anemic (Hb z-score < −2) versus non-anemic 
3. Including age and sex as covariates in adjusted models despite z-score standardization 
4. Excluding patients with extreme hemoconcentration (upper quartile of admission 
hematocrit) 
Missing Data 

The extent and pattern of missing data will be evaluated. If missing data account for less than 
10% of observations, a complete-case analysis will be performed. If missingness is 10% or 
greater, multiple imputation using chained equations will be applied under the assumption of 
missing at random. Imputation models will include the outcome, exposure, and all covariates 
used in the primary analysis.  
Statistical Significance 
All statistical tests will be two-sided, and a p-value < 0.05 will be considered statistically 
significant. Statistical analyses will be conducted using R statistical software. 
8. Ethical Considerations 
The study will be conducted in accordance with the Declaration of Helsinki. Ethical approval 
will be obtained from the local institutional review board. 
Due to the retrospective nature of the study and the use of routinely collected clinical data, 
informed consent was waived by the ethics committee. 
9. Data Protection 
All data will be de-identified prior to analysis and stored securely. Access will be restricted to 
study investigators. 
10. Dissemination Plan 
Results will be submitted to a peer-reviewed international journal and presented at national 
and international scientific meetings