Predicting MacroB12: Data-Driven Assessment of Pre-PEG B12 Utility and Clinical Caveats

TL;DR

This study addresses the diagnostic challenge of MacroB12, an analytical interference that can mimic or mask true Vitamin B12 status. Using a large retrospective cohort (N=875) and multiple imputation, the authors identify independent predictors of MacroB12, including older age, Rheumatologic/Autoimmune disease, and a paradoxical haematological signature of higher Hb and MCV, alongside a dominant association with pre-PEG B12. ROC analysis yields a moderate discriminative power for pre-PEG B12 (AUC $=0.744$) with an optimal threshold of $1584.0$ pg/mL (sensitivity $71.3\%$, specificity $69.7\%$), underscoring that PEG precipitation remains essential for definitive diagnosis. The findings support a risk-stratified approach integrating clinical context with pre-PEG B12, but emphasize external validation and prospective studies to refine thresholds and mechanistic understanding of MacroB12.

Abstract

MacroB12 interference presents a significant challenge in the diagnosis of Vitamin B12 status, potentially masking true deficiency. To establish robust predictors and quantify the utility of pre-polyethylene glycol (PEG) B12 levels, we conducted a retrospective analysis of 875 individuals with hypercobalaminaemia (greater than 1000 pg/mL), using multiple imputation to handle missing data. Multivariable regression modelling revealed that MacroB12 positivity (less than 30% PEG recovery) was independently associated with a profile suggestive of autoimmunity, including older age (adjusted odds ratio [aOR] 1.03 per year, P<0.001) and a Rheumatologic/Autoimmune diagnosis (aOR 2.96, P=0.01). We also identified a counterintuitive haematological signature, with higher haemoglobin (aOR 1.14, P=0.02) and Mean Corpuscular Volume (aOR 1.04, P=0.01) predicting MacroB12. Receiver Operating Characteristic (ROC) analysis of pre-PEG B12 concentration yielded only moderate discriminatory power (Area Under the Curve [AUC] = 0.744, 95% CI 0.707-0.782). The optimal threshold of 1584.0 pg/mL (sensitivity 71.3%, specificity 69.7%) serves to stratify clinical suspicion, but its performance confirms that it cannot replace definitive confirmatory testing. Our findings define a clinical and laboratory phenotype for suspected MacroB12 but highlight that confirmatory PEG precipitation remains essential for accurate diagnosis, particularly in older patients and those with autoimmune disease.

Figures (6)

• Figure 1: Distinct biochemical and hematological signatures of MacroB12 status reveal paradoxical associations. Comprehensive univariate analysis comparing MacroB12 negative (n=638) and positive (n=237) individuals. Box-and-violin plots show the full data distribution with median (horizontal line), mean (diamond), and interquartile range (box). (A) A forest plot displays effect sizes (Hedges' g for continuous variables, log Odds Ratio for categorical) for all univariate associations. (B) PEG recovery percentages are markedly reduced in MacroB12 positive individuals, defining the analytical interference. (C) Pre-PEG Vitamin B12 concentrations are substantially elevated in the positive group. (D) A scatter plot illustrates the inverse correlation between pre-PEG B12 concentrations and PEG recovery, stratified by MacroB12 status. (E) The age distribution demonstrates a significant shift towards older individuals in the positive cohort. (F) Serum folate levels show no significant association, serving as an important negative control. (G) CRP levels are significantly lower in the positive group (log scale presentation). (H) RDW is modestly but significantly lower in the positive group. (I) MCV is paradoxically higher in MacroB12 positive individuals. (J) Hemoglobin levels are counterintuitively elevated in the positive cohort. P-values from Wilcoxon rank-sum tests are displayed on relevant panels (***P < 0.001; ns, not significant).
• Figure 2: Bivariate and Multivariate Forest Plots of Associations with MacroB12 Status. The figure compares unadjusted and adjusted predictors of positive MacroB12 status, with Odds Ratios (ORs) presented on a logarithmic scale. (A) The Bivariate Analysis (BVA) panel displays unadjusted ORs for each variable, with solid squares representing the point estimate. (B) The Multivariate Analysis (MVA) panel displays adjusted ORs derived from the final logistic regression model, which controlled for all variables shown. Hollow squares represent the point estimate. For both panels, horizontal lines indicate the 95% confidence interval (CI), and the vertical dashed line represents the null effect (OR=1.0). Variables are grouped by clinical and laboratory category. P-values for each association are displayed on the right (***P<0.001, *P<0.05; ns, not significant).
• Figure 3: Bivariate and Multivariate Analysis of Prevalence Ratios for MacroB12 Status. The figure compares unadjusted and adjusted predictors of positive MacroB12 status, with Prevalence Ratios (PRs) presented on a logarithmic scale. (A) The Bivariate Analysis (BVA) panel displays the unadjusted PR for each variable, with solid circles representing the point estimate. (B) The Multivariate Analysis (MVA) panel displays the adjusted PR derived from the final multivariable Poisson regression model, which controlled for all variables shown. Hollow circles represent the point estimate. For both panels, horizontal lines indicate the 95% confidence interval (CI), and the vertical dashed line represents the null effect (PR=1.0). Variables are grouped by clinical and laboratory category. P-values for each association are displayed on the right (***P<0.001, *P<0.05; ns, not significant).
• Figure 4: Diagnostic Performance of Pre-PEG Vitamin B12 and Comparison with Other Markers. (A) Receiver Operating Characteristic (ROC) analysis for pre-PEG B12 concentration. The left plot shows the ROC curve, yielding an Area Under the Curve (AUC) of 0.744 (95% CI 0.707--0.782). The red point indicates the optimal threshold (1584 pg/mL) identified via the Youden index, with a corresponding sensitivity of 71.3% and specificity of 69.7%. The plot on the right displays sensitivity and specificity across the full range of thresholds, with the optimal cut-point marked by a vertical dashed line. (B) Forest plot comparing the AUCs for pre-PEG B12 and other potential predictors. Pre-PEG B12 (highlighted in red) demonstrates superior discriminatory power compared to the other demographic and laboratory markers.
• Figure 5: