Macro-alkaline phosphatase: a discussion highlighting discrepancy between wet and dry chemistry assays via a unique case report
Case Report

Macro-alkaline phosphatase: a discussion highlighting discrepancy between wet and dry chemistry assays via a unique case report

Taylor Strange1, Mashal Kakakhel1, Dayebgadoh Gerald1, Joseph M. Gosnell1, Renny Varghese2, Alagarraju Muthukumar3, Anthony O. Okorodudu1

1Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA; 2Department of Family Medicine, University of Texas Medical Branch, Galveston, TX, USA; 3Department of Pathology, University of Texas Southwestern, Dallas, TX, USA

Contributions: (I) Conception and design: AO Okorodudu, T Strange; (II) Administrative support: AO Okorodudu; (III) Provision of study materials or patients: R Varghese, AO Okorodudu, A Muthukumar, JM Gosnell; (IV) Collection and assembly of data: JM Gosnell; (V) Data analysis and interpretation: AO Okorodudu, A Muthukumar, T Strange; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.

Correspondence to: Taylor Strange, MD. Department of Pathology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555, USA. Email: tastrang@utmb.edu.

Background: Alkaline phosphatase (ALP) is a key biomarker of hepatobiliary and skeletal disease. Isolated elevations often prompt extensive investigations to rule out various pathologies, which can be invasive and to the detriment of the patient’s physical, financial, and emotional well-being. One benign diagnosis of exclusion for an isolated elevated ALP value is macro-ALP, an immunoglobulin-bound enzyme complex. However, very little literature exists describing this phenomenon, which may prevent its recognition by clinicians and laboratory directors. Additionally, macro-ALP can be difficult to distinguish from other pathologic etiologies, as standard colorimetric assays can only detect the quantity, but not the size, of the ALP enzyme.

Case Description: A 48-year-old male with a history of hypothyroidism and erectile dysfunction demonstrated a persistent 3-year discrepancy in ALP values between our in-house dry chemistry platform (Vitros 7600, QuidelOrtho, microslide technique) and an outside laboratory wet chemistry assay (ARUP Laboratories, Roche Cobas 8000). In-house results were normal (63–91 U/L; reference range, 34–122 U/L), while outside laboratory testing showed persistent elevations (173–242 U/L; reference range, 40–120 U/L). Other markers were normal. Following evaluation by endocrinology and gastroenterology with no concrete diagnosis, our laboratory elected to perform a polyethylene glycol (PEG) precipitation test (Abbott Alinity CI), which confirmed macro-ALP (<40% recovery). The patient remained asymptomatic, and no pathology was identified.

Conclusions: Macro-ALP is a rare cause of isolated ALP elevation. This case highlights a benign presentation in a healthy individual and underscores how methodological differences between wet and dry chemistry assays can yield discrepant results. Awareness of this benign variant and assay-dependent variability can prevent unnecessary investigations.

Keywords: Case report; macroenzyme; alkaline phosphatase (ALP); test methodologies

Received: 20 September 2025; Accepted: 08 December 2025; Published online: 27 January 2026.

doi: 10.21037/jlpm-25-53

Highlight box

Key findings

• Macro-alkaline phosphatase (ALP) may mimic pathology but is benign.

• Discrepant results between wet and dry chemistry assays were observed in this case.

What is known and what is new?

• Macro-ALP is rare and underreported, with most cases linked to comorbid conditions.

• This is, to our knowledge, the first reported case in an otherwise healthy patient.

• Differences between assay platforms may account for discrepant enzyme values.

What is the implication, and what should change now?

• Clinicians should consider macro-ALP when faced with unexplained ALP elevation.

• Greater awareness of assay-specific limitations is needed to avoid unnecessary investigations.

Introduction

Alkaline phosphatase (ALP) is a membrane-bound glycoprotein widely measured in clinical practice as a biomarker of hepatobiliary and skeletal disease (1-4). Elevated ALP values may be physiologic in certain populations (e.g., children, pregnant women) but otherwise often prompt investigation for bone or liver pathology. Common causes include metabolic bone disorders, biliary obstruction, and cholestatic liver disease (5,6) Concurrent testing for gamma-glutamyl transferase (GGT) or 5' nucleotidase can help distinguish hepatic from bone sources (7).

Macro-ALP is a benign variant resulting from immunoglobulin binding, which prolongs enzyme persistence in circulation without clinical consequence (8,9). Because it is rarely reported and poorly recognized, macro-ALP may cause diagnostic confusion and unnecessary evaluations. Published cases are limited, and most describe patients with underlying autoimmune or systemic illness (10-13).

We report, to our knowledge, the first case of macro-ALP identified in a healthy individual. This case is further notable for a persistent discrepancy between wet and dry chemistry assays, underscoring how methodological differences can influence enzyme quantification. We present this article in accordance with the CARE reporting checklist (available at https://jlpm.amegroups.org/article/view/10.21037/jlpm-25-53/rc).

Case presentation

A 48-year-old male followed by endocrinology for hypothyroidism (on liothyronine) and erectile dysfunction [on testosterone replacement therapy and high-dose human chorionic gonadotropin (HCG)] presented with an elevated ALP value from an outside laboratory (QUEST diagnostics, Beckman AU5800) collected for routine screening. His primary physician ordered an additional ALP quantification at our in-house laboratory (via p-nitrophenyl phosphate colorimetric assay, Vitros 7600, QuidelOrtho, Rochester, NY, USA), which returned within normal limits. For confirmation, a blood sample was also sent for isoenzyme evaluation at an additional outside laboratory (ARUP Laboratories, heat inactivation assay for isoenzyme detection, followed by p-nitrophenyl phosphate colorimetric assay, Roche Cobas 8000, Indianapolis, IN, USA), which resulted in an elevated total ALP level with undetectable liver isoenzyme level and a normal bone isoenzyme level. Over the next 3 years (December 2021 to December 2024), in-house ALP values remained normal (63–91 U/L, reference range, 34–122 U/L), while outside laboratory testing reported elevated values (173–242 U/L, reference range, 40–120 U/L) despite these specimens originating from the same phlebotomy event. Outside isoenzyme results at ARUP persistently indicated an undetectable liver isoenzyme, normal bone isoenzyme (39–49 U/L, reference range, 0–55 U/L), and an elevated, heat-stable isoenzyme (133–203 U/L) that could not be identified but was suspected to be placental or germline in origin.

During this timeframe, all other hepatobiliary and skeletal biomarkers were normal. For example, GGT was 24 U/L (reference range, 13–58 U/L), bilirubin 0.7–1.0 mg/dL (reference range, 0.1–1.1 mg/dL), alanine transferase 37–45 U/L (reference range, 5–50 U/L), aspartate transferase 35–39 U/L (reference range, 13–40 U/L), calcium 8.9–9.7 mg/dL (reference range, 8.6–10.6 mg/dL), parathyroid hormone 24.7 pg/mL (reference range, 12.0–88.0 pg/mL), and 25-hydroxyvitamin D 38–46 ng/mL (reference range, 25–80 ng/mL). The patient’s endocrinologist was consulted but did not recommend any further workup. Gastroenterology completed a liver ultrasound that returned within normal limits. The patient ceased his testosterone replacement therapy in hopes that this would lead to a decrease in his ALP values, but the discrepancy persisted (Figure 1).

Figure 1 Our patient’s ALP values, both in-house and outside, alongside testosterone values. For three consecutive years, in-house ALP values (navy line) ranged between 63–91 U/L (reference range, 34–122 U/L). In contrast, values measured at the outside laboratory (ARUP Laboratories, golden line) ranged between 173 and 242 U/L during the same period of time (reference range, 40–120 U/L). Although this patient was initially consuming a testosterone supplement, he discontinued it out of concern for his abnormal ALP values. However, even after discontinuing the supplement and observing his values decrease, his ALP values remained persistently elevated at the outside laboratory. ALP, alkaline phosphatase.

In early 2025, without clarification of etiology from aforementioned specialties, our laboratory advised a polyethylene glycol (PEG) precipitate test via Abbott Alinity CI series equipment (Abbott Laboratories, Lake County, IL, USA) at the University of Texas Southwestern. This PEG precipitation assay was indicative of the presence of a macroenzyme (<40% recovery), and a diagnosis of macro-ALP was made. The patient is currently doing well with no apparent adverse effects of this diagnosis. He expressed relief upon learning that the elevated ALP did not indicate underlying pathology. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Declaration of Helsinki and its subsequent amendments. Written informed consent was waived by the University of Texas Medical Branch institutional review board given the benign nature of the condition and to prevent unnecessary emotional distress to the patient. All identifiable personal details have been removed to ensure patient anonymity.

Discussion

Macroenzymes, including macro-ALP, arise when circulating enzymes bind to other serum proteins (immunoglobulins in the case of macro-ALP) (9). As illustrated in Figure 2, formation of this enzyme-immunoglobulin complex increases the overall molecular size of ALP, creating a bulky macromolecule that is less efficiently cleared by hepatocytes. In addition, immunoglobulin G (IgG) has a substantially longer serum half-life than native ALP (~20 days versus ~7 days), which further prolongs the persistence of the macro-ALP complex in circulation (14,15). The combination of increased molecular weight and delayed clearance results in chronically elevated ALP measurements despite the absence of underlying pathology.

Figure 2 Formation and persistence of macro-ALP. (A) Circulating alkaline phosphatase binds to serum immunoglobulins (most likely IgG) forming a high-molecular weight complex. (B) The increased molecular size reduces hepatocellular uptake and metabolic clearance. (C) Reduced hepatic uptake and increased serum half-life of immunoglobulins results in longer circulation of the macro-ALP complex, leading to persistently elevated levels of ALP in the absence of underlying disease. ALP, alkaline phosphatase; IgG, immunoglobulin G.

Current literature discussing macro-ALP is limited and predominantly restricted to case reports. The earliest reported case of macro-ALP was published in 1979 (16), with few additional reports being submitted since. Most note association of macro-ALP with other autoimmune conditions, such as inflammatory bowel disease (10,11), likely due to the increased prevalence of circulating immunoglobulins within the bloodstream of these patients. The patient in our case, in contrast, had no significant past medical history, making this, to our knowledge, the first case report of macro-ALP without any other comorbidity.

This case illustrates how unrecognized macro-ALP can lead to diagnostic uncertainty, specialist referrals, and costly follow-up testing despite its benign nature. Early recognition could reduce unnecessary imaging, invasive procedures, and patient anxiety, providing both clinical and financial benefits.

Implementing an additional, more specific assay can offer insight in the presence of macro-ALP. For example, in a previous case report (11), using enzyme electrophoresis resolved the initially elevated ALP observed following use of a para-nitrophenyl phosphate assay. In our case, the PEG precipitation assay was a valuable tool. This procedure involves creating a solution with an equal volume of PEG solution (PEG suspended in phosphate buffered saline) and patient serum. When this combination is allowed to mix and is subsequently centrifuged, PEG—a large hydrophilic polymer—forces any macromolecules such as macro-ALP out of solution (Figure 3) (17,18). The remaining ALP is quantified in the supernatant, and recovery is calculated by dividing the supernatant concentration by the total original concentration. If the recovery is less than 40%, it is concluded that macro-ALP is present within the patient’s serum (19). However, it must be noted that PEG precipitation results may be complicated in serum with an excess of large molecules such as immunoglobulins (9).

Figure 3 Description of polyethylene glycol precipitation test. Macroenzyme complexes will bind to the large PEG molecules and subsequently precipitate out of solution. PEG, polyethylene glycol.

With laboratories increasingly relying on high-throughput automated chemistry platforms, awareness of method-dependent discrepancies is crucial for accurate interpretation of liver enzyme results. It is unclear why our in-house laboratory values differed so significantly from those of the outside laboratory. One theorized etiology proposed by our group is that macro-ALP has decreased in functionality. In this scenario, it would be comprehensible that only standard ALP isozymes (e.g., enzymes not bound to immunoglobulin) reacted with the p-nitrophenol in our VITROS assay, as this protocol relies on adequate enzymatic reaction to be detected by the equipment. In contrast, a heat inactivation assay would quantify all ALP isozymes, macro or not, regardless of functionality.

However, a far more likely explanation for the discrepancy is the differences in testing modality—specifically differences in “wet chemistry” and “dry chemistry”.

In general, different modalities will subject specimens to different calibration biases. For example, one study compared two different modalities, one utilizing dry chemistry (VITROS) and one wet chemistry (COBRA) and found that up to 20% of bias could be produced due to different traceability sources utilized (20).

“Wet chemistry” refers to any assay that involves a liquid phase, utilizing techniques such as titration, precipitation, filtration, or ion-selective electrodes. These methods can be either manual or automated, and are generally more cost-effective. In contrast, dry chemistry modalities eliminate the need to put a specimen in solution, which cuts preparation time and improves workflow (21). In our laboratory, ALP is quantified using an automated colorimetric assay within a dry, multilayered cartridge. The layers within each cartridge contain the reagent p-nitrophenyl phosphate, as well as all other necessary reagents and indicators, which allows ALP to react and produce the yellow p-nitrophenol by merely passing through the layers. Dry methods are less susceptible to certain matrix interferences but may underestimate bulky complexes (Figure 4).

Figure 4 Comparison between “dry chemistry” and “wet chemistry” methods. Our laboratory uses multilayered cartridges (A) to measure ALP, in which a sample travels through several dry layers. In contrast, “wet chemistry” mechanisms such as liquid chromatography (B), measure ALP while within a solution. Our team speculates that the multilayered nature of our laboratory’s cartridge might be trapping the large macro-ALP molecules. ALP, alkaline phosphatase.

Wet and dry chemistry modalities will subject specimens to different physical and chemical phenomena. For example, since wet chemistry assays occur in a fully liquid phase, they allow for the free interaction between reagents and all circulating enzyme species, including bulky macro-ALP complexes. In contrast, the aforementioned dry slide platforms require samples to diffuse through multilayered matrices—physical barriers that may partially exclude high-molecular-weight complexes and lead to underestimation of macroenzymatic activity.

This discrepancy has been observed in macro-creatine kinase (CK). In one study, CK activity was significantly lower among dry chemistry assays compared to wet chemistry assays, which was attributed to a macroenzyme (22). The authors hypothesized that a “sieving” effect was taking place when macro-CK was exposed to multiple dry layers for quantitative analysis, thus causing a consistently reduced final laboratory value compared to wet methods.

Clinically, these assay-dependent discrepancies may lead to misinterpretation: dry chemistry may falsely normalize elevations, potentially delaying recognition of macro-ALP, whereas wet chemistry may overstate activity and prompt unnecessary interventions. This case thus highlights a subtle but critical analytical limitation of dry-slide technology, urging clinicians and laboratorians to consider method-specific biases when encountering unexplained enzyme elevations.

This report and analysis has limitations. Because these observations are derived from a single patient, the generalizability of our findings is inherently restricted. Although the discrepant ALP values between wet and dry chemistry platforms are consistent with the expected behavior of high-molecular-weight complexes, this case alone cannot establish a definitive mechanism for assay-dependent differences. The true prevalence of macro-ALP, its behavior across different analytical platforms, and the extent to which specific methodologies underestimate or overestimate activity remain unclear. Larger, systematic evaluations are needed to confirm these observations and to better define the analytical performance of various assay systems in the presence of macroenzymes.

Future work should focus on systematically characterizing how different analytical platforms detect macro-ALP and whether standardized reference procedures can be implemented. Larger studies would clarify the true prevalence of macro-ALP in both healthy and comorbid populations. Moreover, harmonization of assay reporting between wet and dry chemistry systems could prevent misinterpretation and reduce diagnostic burden. Collaborative efforts between laboratorians and clinicians will be essential to ensure benign macroenzymes are correctly identified and distinguished from pathologic causes of enzyme elevation.

Conclusions

In summary, macro-ALP, although rare, may go unrecognized and result in unnecessary testing and procedures. It is critical for both clinicians and laboratory directors to recognize macro-ALP may not always occur in patients with underlying autoimmune illnesses, and thus this variant should also be suspected in relevant cases of incidental findings in healthy individuals. Clinicians should consider macro-ALP in healthy patients with isolated ALP elevation, particularly when discrepant results appear between wet and dry chemistry assays. Additionally, the benefits and limitations of dry chemistry methods need to be kept in check, though the limitations of multilayer dry chemistry cartridges should be considered when interpreting discrepant results, particularly in otherwise healthy patients.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the CARE reporting checklist. Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-53/rc

Peer Review File: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-53/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-53/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Declaration of Helsinki and its subsequent amendments. Written informed consent was waived by the University of Texas Medical Branch institutional review board given the benign nature of the condition and to prevent unnecessary emotional distress to the patient. All identifiable personal details have been removed to ensure patient anonymity.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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doi: 10.21037/jlpm-25-53
Cite this article as: Strange T, Kakakhel M, Gerald D, Gosnell JM, Varghese R, Muthukumar A, Okorodudu AO. Macro-alkaline phosphatase: a discussion highlighting discrepancy between wet and dry chemistry assays via a unique case report. J Lab Precis Med 2026;11:9.

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