Abstract
Background and Objectives
The human antiglobulin test or Coombs' test is a key tool in the diagnosis and classification of haemolytic anaemia and could present as falsely positive. A retrospective study has described that B‐type natriuretic peptide (NT‐proBNP), a biomarker used for heart failure (HF), could be an additional cause of false positives in the Coombs test.
Materials and Methods
A prospective, observational, single‐centre study was conducted in adult patients diagnosed with HF between October 2024 and March 2025 at a tertiary hospital. The Coombs' test was considered as false positive in cases in which the specificity of the test included immunoglobulin G (IgG), the subsequent elution yielded a negative result, and blood haemolysis parameters were within normal ranges.
Results
Thirty‐six patients were included, of whom nine (25%) had a false‐positive Coombs test. These patients had significantly higher levels of NT‐proBNP (10,825 pg/mL [2587–35,000] vs. 3314 pg/mL [278–11,856], p = 0.0076) and lower glomerular filtration rate (35.3 mL/min/1.73 m2 [23.6–46.3] vs. 63.3 mL/min/1.73 m2 [21.9–90.0], p = 0.0055) compared to patients with a negative Coombs test. No statistically significant associations were found with drugs used concomitantly for HF.
Conclusion
The results suggest that elevated NT‐proBNP could also be a cause of false positives in the Coombs test.
Keywords: B‐type natriuretic peptide, direct antiglobulin test, heart failure, interference
Highlights.
Elevated B‐type natriuretic peptide (NT‐proBNP) levels were significantly associated with false‐positive direct antiglobulin tests (DAT) in patients with heart failure (HF), despite the absence of clinical or laboratory evidence of haemolysis.
NT‐proBNP may represent an additional cause of DAT interference, highlighting that a positive DAT in HF patients does not necessarily indicate autoimmune haemolytic anaemia (AIHA) and should be interpreted cautiously.
Recognizing NT‐proBNP‐related DAT interference could help prevent misdiagnosis and unnecessary or harmful treatments, underscoring the need to incorporate clinical context and biomarkers into diagnostic algorithms and to validate these findings in larger, multi‐centre studies.
INTRODUCTION
The Coombs test or direct antiglobulin test (DAT) is an essential tool for the initial diagnosis of autoimmune haemolytic anaemia (AIHA). It detects antibodies already bound to antigens on the surface of the erythrocyte. However, this diagnosis must be interpreted in the context of other haemolysis parameters and the patient's clinical situation. AIHA can be classified according to the temperature range of the antibody, with warm antibody AIHA (wAIHA) being more common. Approximately 60% of wAIHA cases are idiopathic, while the rest can occur in various clinical contexts, such as lymphoproliferative processes, autoimmune diseases or drug‐mediated diseases [1].
Despite its usefulness, DAT is not free from false positives, with several causes described in the literature, such as poor sample processing, elevated immunoglobulin levels, systemic lupus erythematosus, elevated rheumatoid factor, infections such as human immunodeficiency virus, Epstein–Barr virus, mycoplasma and/or hepatitis C or interference from Wharton's jelly. Even with antibody elution studies, some false positives persist and can lead to unnecessary treatments and clinical risks [2, 3]. There are also studies showing up to 15% of positive DATs in hospitalized patients with acute illnesses in whom there is no evidence of haemolysis or a clear aetiology [4].
Recently, a retrospective study identified B‐type natriuretic peptide (NT‐proBNP) as a possible additional cause of DAT interference. The study showed that patients with a positive DAT specific for immunoglobulin G (IgG) and a negative antibody elution test had elevated NT‐proBNP levels [5].
NT‐proBNP, secreted by cardiomyocytes in response to increased filling pressure and wall stress, is a well‐established biomarker for the diagnosis and stratification of heart failure (HF) [6, 7].
Given that HF is a disease with high prevalence and a high percentage of hospitalizations, exploring the relationship between NT‐proBNP and DAT could improve the interpretation of this test and prevent misdiagnosis. The present study focuses on evaluating this possible association in a real clinical context.
MATERIALS AND METHODS
The main objective was to determine whether elevated NT‐proBNP in patients with HF may be an additional cause of DAT interference. A false positive was defined as the presence of a positive polyspecific DAT with a positive monospecific IgG test and a subsequent negative antibody elution test.
A prospective, descriptive, single‐centre study was designed and conducted in a tertiary hospital. Patients with a confirmed diagnosis of HF according to the latest guidelines of the European Society of Cardiology (ESC, 2021) [8] were included from both the Cardiology and Internal Medicine departments. Patients were included successively from outpatient clinics and the hospital ward. The inclusion period was 6 months, from October 2024 to March 2025. The inclusion criteria were being hospitalized during the study period due to a decompensation of HF or having outpatient follow‐up in specific outpatient clinics for HF. The only exclusion criterion was a positive antibody elution test in the DAT.
All patients included in the study underwent a laboratory profile with complete blood count, renal function, serum NT‐proBNP levels, DAT and haemolysis parameters (reticulocytes, haptoglobin, indirect bilirubin and lactate dehydrogenase). Clinical and laboratory data were obtained from institutional electronic medical records.
The polyspecific DAT was performed using Ortho BioVue System cards (Ortho‐Clinical Diagnostics, USA). The monospecific study was performed with DG Gel DCscan cards (Grifols, Spain), and the antibody elution study was performed with the Elukit kit (Immucor, USA). First, all patients underwent a polyspecific DAT using Ortho BioVue System cards. Those who tested positive had the test repeated using the DG Gel DCscan cards following standard sample processing. With these cards, we repeated the polyspecific test and also included specificities for IgG and complement. All patients tested positive on the Grifols card for polyspecific IgG and none for the complement. Therefore, elution was performed on all of them with a negative result.
Pro‐BNP was determined by the Clinical Analysis service using an immunoassay technique with the Elecsys kit on the Cobas‐e411 analyser (Roche Diagnostics, Switzerland).
Statistical analysis was performed with R (see 4.5.0), using the Rstudio interface. Quantitative variables were expressed as median and interquartile range (IQR) and qualitative variables as frequencies and percentages. The threshold value for statistical significance was set at p < 0.05.
The study was conducted in accordance with international recommendations on clinical research (World Medical Association Declaration of Helsinki) and was approved by the relevant Drug Research Ethics Committee. All patients signed an informed consent form.
RESULTS
Forty‐five patients were asked to participate and nine patients declined to join the study. We finally included 36 patients diagnosed with HF, and DAT and NT‐proBNP were determined in a blood test. No patients were excluded for a positive antibody elution study in the DAT. Nine patients (25%) had a false‐positive DAT, with no clinical or analytical evidence of haemolysis during hospital admission for inpatients and for 1 week for outpatients. The median age was higher in the group with false‐positive DAT (87 years [IQR: 77–90]) compared to the group with negative DAT (79 years [IQR: 71–88]), although this difference was not statistically significant (p = 0.287). With regard to pharmacological treatment, no statistically significant differences were observed between the groups for any of the therapeutic classes analysed. The baseline characteristics and pharmacological treatment used for HF are shown in Table 1.
TABLE 1.
Baseline characteristics by group according to direct antiglobulin tests results.
| False positive DAT (n = 9) | Negative DAT (n = 27) | p‐Value | |
|---|---|---|---|
| Age (years) | 87 (77–90) | 79 (71–88) | 0.287 |
| Gender (%) | 1 | ||
| Male | 4 (44.4) | 14 (51.9) | |
| Female | 5 (55.6) | 13 (48.1) | |
| Type of HF according to LVEF (%) | 0.460 | ||
| Preserved | 4 (44.4) | 17 (63) | |
| Mildly reduced | 1 (11.2) | 1 (3.7) | |
| Reduced | 4 (44.4) | 9 (33.3) | |
| Emergency visits for HF during the previous year (%) | 4 (44.4) | 12 (44.4) | 1 |
| Hospitalization for HF during the previous year (%) | 2 (22.2) | 6 (22.2) | 1 |
| Loop diuretic (%) | 7 (77.8) | 26 (96.3) | 1 |
| Thiazide (%) | 1 (11.1) | 5 (18.5) | 1 |
| iSGLT2 (%) | 6 (66.7) | 23 (85.2) | 0.333 |
| ARNi (%) | 2 (22.2) | 4 (14.8) | 0.627 |
| Beta‐blocker (%) | 9 (100) | 21 (77.8) | 0.303 |
| MRA (%) | 4 (44.4) | 10 (37) | 0.712 |
Abbreviations: ARNi, angiotensin receptor/neprilysin inhibitor; DAT, direct antiglobulin tests; HF, heart failure; iSGLT2, sodium‐glucose cotransporter‐2 inhibitors; LVEF, left ventricular ejection fraction; MRA, mineralocorticoid receptor antagonist.
In the comparative analysis of the analytical parameters, it was observed that patients with false‐positive DAT showed significantly higher levels of NT‐proBNP compared to those with negative NT‐proBNP (10,825 pg/mL [IQR: 2587–35,000] vs. 3314 pg/mL [IQR: 278–11,856], p = 0.0076). Likewise, this group showed a significantly lower estimated glomerular filtration rate (35.3 mL/min/1.73 m2 [IQR: 23.6–46.3] vs. 63.3 mL/min/1.73 m2 [IQR: 21.9–90.0], p = 0.0055). No statistically significant differences were observed in the classic markers of haemolysis, confirming the absence of active haemolysis in both groups. Complete data on analytical parameters are detailed in Table 2.
TABLE 2.
Analytical values according to direct antiglobulin tests results and comparison between groups.
| False positive DAT (n = 9) | Negative DAT (n = 27) | p‐Value | |
|---|---|---|---|
| Hemoglobin (g/dL) | 10.5 (8.1–15.2) | 13.2 (9.1–17.4) | 0.097* |
| Reticulocytes (109/L) |
69.5 (47.6–184) n = 6 |
68.1 (15.9–141) n = 12 |
0.964** |
| Glomerular filtration rate (mL/min/1.73 m2) | 35.3 (23.6–46.3) | 63.3 (21.9–90) | 0.006** |
| Indirect bilirubin (mg/dL) | 0.7 (0.4–2) | 0.9 (0.4–2.4) | 0.264** |
| Haptoglobin (mg/dL) |
134 (30–229) n = 8 |
147 (30–316) n = 25 |
0.557* |
| LDH (U/L) |
473 (264–1578) n = 8 |
407 (264–845) n = 26 |
0.584** |
| NT‐proBNP (pg/mL) | 10,825 (2587–35,000) | 3314 (278–11,856) | 0.008** |
Abbreviations: DAT, direct antiglobulin tests; LDH, lactate dehydrogenase; NT‐proBNP, B‐type natriuretic peptide.
Student's t‐test is performed on the mean of two independent populations.
As it does not follow a normal distribution, a Wilcoxon test has been performed.
Additionally, a descriptive analysis of the percentage of false‐positive results was performed based on NT‐proBNP values distributed by ranges, as shown in Table 3. A trend towards a higher proportion of DAT positivity at higher NT‐proBNP concentrations was observed. The relationship between NT‐proBNP values and DAT results was evaluated using Spearman's test, with a statistically significant correlation (r = 0.45; p = 0.0054).
TABLE 3.
Distribution by B‐type natriuretic peptide ranges and patients with false‐positive DAT.
| NT‐proBNP (pg/mL) | Total number of patients (N = 36) | False positive DAT (N = 9) |
|---|---|---|
| 0–1000 | 7 | 0 |
| 1000–2000 | 3 | 0 |
| 2000–3000 | 2 | 1 (50%) |
| 3000–4000 | 5 | 0 |
| 4000–5000 | 3 | 0 |
| 5000–6000 | 2 | 1 (50%) |
| 6000–7000 | 1 | 0 |
| 7000–8000 | 4 | 2 (50%) |
| 8000–9000 | 3 | 0 |
| 9000–10,000 | 0 | 0 |
| 10,000–11,000 | 1 | 1 (100%) |
| 11,000–12,000 | 2 | 1 (50%) |
| >12,000–30,000 | 3 | 3 (100%) |
Abbreviations: DAT, direct antiglobulin tests; NT‐proBNP, B‐type natriuretic peptide.
DISCUSSION
This study shows the possible association between elevated NT‐proBNP and the occurrence of false positives in DAT in patients diagnosed with HF. The implication of this biomarker in DAT interference represents a relevant contribution to clinical practice, as observed in other molecules already studied, such as the rheumatoid factor [2].
As previously mentioned, 25% of patients with HF included in the study had a false‐positive DAT (with polyspecific and IgG‐specific DAT) with a negative eluate and no evidence of haemolysis. This confirms that a positive DAT does not reflect autoimmune haemolysis. This group of patients had a significantly higher median NT‐proBNP than the group with a negative DAT, as previously reported in the literature [5].
The mechanism by which NT‐proBNP could interfere with the antiglobulin reaction has not been established. As a hypothesis, we could consider that the increase in natriuretic peptide concentration and the deterioration of renal function, in the context of a chronic disease with persistent inflammation, could trigger an alteration in the affinity of the DAT reagents. Protein alteration secondary to renal failure could cause non‐specific reactions or interactions in immunohaematological tests [9].
In addition to the biochemical aspects, it should be noted that advanced HF generates a complex immunological state with endothelial dysfunction, neurohormonal activation and systemic inflammation, leading to a loss of homeostasis that can generate reactivity in laboratory tests. In this regard, NT‐proBNP is not only a marker of haemodynamic overload but also an indirect reflection of the patient's proinflammatory and neurohumoral state [10]. This phenomenon bears some resemblance to other conditions previously described in the literature, such as the presence of rheumatoid factor, a known cause of DAT interference [11].
On the other hand, there are drugs that can interfere with DAT results, causing haemolysis, such as certain antibiotics or anticonvulsants [12]. However, in our study, there was no evidence of haemolysis associated with drugs used in HF. Both groups were mostly taking scheduled furosemide, with no differences observed that would allow the drug to be associated with possible DAT interference.
The main limitation of our study is the small sample size. Secondly, there are variables related to inflammation, such as C‐reactive protein or immunoglobulin values, which could play a role as confounding factors in false‐positive results, which we have not analysed. This would require larger multi‐centre studies analysing other variables of interest, which would allow us to clarify the mechanism of this interference and contribute to improving the diagnostic algorithms for haemolytic anaemia. It is important to describe possible causes of false‐positive DAT results for hospitals that do not have the capacity to perform the complete study with elution techniques, thereby assisting in clinical decision making.
In conclusion, the results of this study suggest that elevated NT‐proBNP concentrations may constitute an additional source of interference in the Coombs test, leading to false‐positive results in patients with HF. This association appears to be more evident in individuals with impaired renal function, pointing to a possible synergistic effect in the context of volume overload and altered biomarker clearance.
Although these findings provide relevant preliminary evidence, additional studies are needed, preferably multi‐centre studies with a larger sample size, to confirm this relationship, evaluate its clinical impact and define cut‐off points that improve the interpretation of the DAT in this population.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
ACKNOWLEDGEMENTS
M.A.C.R. performed the research, collected the data and wrote the paper. A.L.Á. performed the research, wrote the first draft of the manuscript, collected and analysed the data and wrote the paper. S.R.B. performed patient recruitment, data collection and data analysis. P.A.B. performed patient recruitment and data collection. C.J.S. wrote the first draft of the manuscript and performed data collection. E.V.R. performed data collection, contributed essential laboratory techniques and tools and reviewed the manuscript. E.S.C. and A.R. performed data collection and contributed essential laboratory techniques. S.O.Z. reviewed and edited the final paper. J.E.M.B. reviewed the first draft of the manuscript and edited the final paper. L.F.R. performed the research, collected the data and reviewed the final manuscript. J.P.S. performed the research, collected the data and analysed and edited the final paper. M.d.l.E.L.P. performed the research, wrote the first draft and edited the final paper.
Lamilla Álvarez A, Cámara Rodenas MA, Russo Botero S, Aracil Boigues P, Játiva Sáez C, Vidal Rico E, et al. Association between NT‐proBNP and false‐positive Coombs test in patients with heart failure and elevated NT‐proBNP . Vox Sang. 2026;121:1245–1249.
Funding information The authors received no specific funding for this work.
DATA AVAILABILITY STATEMENT
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.
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Associated Data
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Data Availability Statement
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.