Alkaline Phosphatase

Etiology and Epidemiology

Serum ALP levels exhibit age-related variations in healthy individuals. The levels are highest during childhood and puberty due to bone growth and development and then decrease as individuals age. The decline in serum ALP levels within the age group of 15 to 50 is slightly more pronounced in men than in women.[1] These levels increase again during old age, with a notable gender distribution difference. The underlying reasons for these typical fluctuations remain unidentified. Research indicates a positive correlation between body weight and smoking, while an inverse correlation exists with height.[7]

In healthy individuals, the predominant source of the circulating enzyme is the liver and bone. In some individuals, a minor fraction of this enzyme originates from the intestinal tract.[8] Consuming a fatty meal leads to increased serum ALP levels in individuals with blood groups O and B, primarily due to contributions from the intestinal tract. As this elevation can persist for up to 12 hours in the serum, it is advisable to assess serum enzyme levels in a fasting state.[9]

Pathophysiology

Human ALPs (hALP) are tethered to the cell membrane via glycosylphosphatidylinositol. Their release into the serum occurs through the action of specific phospholipase enzymes.[10] These enzymes have a half-life of 7 days, and their clearance from the serum is independent of the bile duct's patency or the liver's functional capacity. However, the precise site where ALP degradation occurs remains unknown. Serum ALP levels may remain elevated for up to one week after the resolution of biliary obstruction.[1]

The liver is the primary source in most patients with elevated enzyme levels. The subsequent most probable contributor is increased osteoblast activity, which is observed in bone disorders or usually during periods of growth. In addition, in the late third trimester, the influx of placental ALP contributes significantly to elevated levels in pregnant women.[11]

The mechanism behind the elevation of ALP in hepatobiliary disorders has been debated. However, research has provided convincing evidence that this increase results from heightened enzyme synthesis rather than a reduction in hepatobiliary excretion of the enzyme.[11] The increase in hepatic enzyme activity directly corresponds to the elevation in serum ALP activity. This is primarily attributed to an upsurge in the translation of the mRNA of ALP—a process facilitated by the increasing bile acid concentration—and increased secretion of ALP into the serum through canalicular leakage into the hepatic sinusoid. The precise mechanism that precipitates its release into circulation has not been elucidated.[1]

Studies indicate that vesicles containing ALP, along with various enzymes attached to the sinusoidal membranes, are detected in the serum of patients with cholestasis. As ALP is newly synthesized as a response to biliary obstruction, its serum level may appear normal in the initial stages of acute biliary obstruction, even when serum aminotransferases have already reached their peak.[12]

Specimen Requirements and Procedure

Blood should be collected after a minimum 8-hour fasting period—both serum and heparinized plasma yield comparable results. Although minor hemolysis is acceptable, it is essential to prevent gross hemolysis. Specific sample storage conditions can lead to an elevation in serum ALP. Notably, enzyme activity substantially increases when refrigerated or frozen sera are warmed.[13]

In fresh serum, ALP activity can increase by as much as 2% within 6 hours at 25 °C. Substantial increases in ALP activity up to 30% are observed upon thawing frozen serum or reconstituting lyophilized specimens. These increases may be due to the release of ALP from complexes with lipoproteins or potentially due to heightened activity of the non-complexed enzyme. For optimal results, it is advisable to analyze ALP specimens on the same day they are collected.[14] Using standard phlebotomy procedures, the specimen can be collected in either a plain red-top tube or a speckled-red-top tube with gel as a serum separator. To prevent contamination, it is essential not to draw tubes containing anticoagulants before using the red-top or speckled-red-top tubes.[15]

Diagnostic Tests

Various clinical methods are available to assess serum ALP levels, distinguished by the substrate used, alkaline pH buffer, and established normal reference values. These tests, in principle, rely on the enzyme's ability to hydrolyze phosphate esters.[16] In the most widely used international method, p-nitrophenol phosphate serves as the substrate, while amino alcohol is used as the buffering agent. The rate at which p-nitrophenol phosphate is released from the substrate serves as a measurable marker of ALP activity, and the outcomes are typically expressed in international units per liter (IU/L).[17] Various methods appear equally effective in identifying abnormal values associated with different clinical conditions. Comparing results obtained from different tests can be straightforward by using multiples of the upper limit of the established normal range.[18]

Electrophoresis, as a standalone method, does not consistently distinguish between the isoenzymes, as the electrophoretic mobility of bone and liver isoenzymes exhibits only slight differences. Therefore, a more dependable approach involves electrophoresis on cellulose acetate in conjunction with heat inactivation. Although not widely accessible, the most accurate method for precisely identifying liver, bone, intestinal, and placental isoenzymes utilizes polyacrylamide gel slab-based separation.[19] 

Testing Procedures

The ALP test is performed on semiautomatic or fully automated analyzers that operate on the principle of photometry. Photometry entails the quantification of light absorption within the ultraviolet (UV), visible (VIS), and infrared (IR) spectrum range. This measurement determines the amount of a particular analyte in a solution or liquid. Photometers utilize a specific light source and detectors capable of converting the light transmitted through a sample solution into an electrical signal proportionate to the light's intensity. These detectors can include photodiodes, photoresistors, or photomultipliers.[20]

Photometry applies Beer-Lambert law to compute coefficients derived from transmittance measurements. A test-specific calibration function establishes a correlation between absorbance and analyte concentration, resulting in highly accurate measurements.[21] The electrophoresis technique is most commonly used for distinguishing between ALP isozymes and isoforms. However, due to potential overlap between the fractions, combining electrophoresis with another separation technique can yield more dependable results.[22] Notably, a direct immunochemical method is currently accessible for measuring bone-related ALP.[23]

The liver fraction exhibits the swiftest migration in electrophoresis, followed by bone, placental, and intestinal fractions. Due to the similarity between liver and bone phosphatases, a distinct demarcation between them is often challenging to achieve. Accurate quantitation using a densitometer can be difficult due to the overlap between the 2 peaks. The liver isoenzyme can be further subdivided into 2 fractions—the major liver band and a smaller fraction, which is referred to as fast liver or α1 liver. The fast liver fraction migrates to the major band anodally, corresponding to the α1 fraction observed in protein electrophoresis.[19]

The major liver fraction is most commonly raised in cases of elevated total ALP levels. Many hepatobiliary conditions lead to elevations of this fraction, typically occurring early in the progression of the disease. The fast-liver fraction has been observed in metastatic liver carcinoma and other hepatobiliary diseases.[24] The fast-liver fraction often indicates obstructive liver disease, but it can also be found without a discernible disease state.[25]

A second approach used to determine the source of an elevated ALP is based on the difference in heat stability.[26] Generally, ALP activity is assessed both before and after subjecting the serum to heating at 56 °C for 10 minutes. If the residual activity after heating is less than 20% of the total activity before heating, it indicates that the ALP elevation is attributed to bone phosphatase. When more than 20% of the activity persists after heating, the elevation is likely due to liver phosphatase. These conclusions are drawn from the observation that the 4 major fractions exhibit varying heat stabilities, with placental ALP being the most heat-resistant, followed by intestinal, liver, and bone fractions in decreasing order of heat stability. Placental ALP, for instance, can withstand heat denaturation at 65 °C for 30 minutes.[11] 

Heat inactivation is an imprecise method for differentiation because inactivation depends on several factors, including precise temperature control, timing, and the requirement for analytical methods sensitive enough to detect even minor residual ALP activity. Moreover, there is a degree of overlap between the heat inactivation profiles of liver and bone fractions, both in liver and bone diseases, further complicating differentiation.[27]

The third approach for distinguishing ALP isoenzymes is based on selective chemical inhibition, with phenylalanine as one of the inhibitors utilized. Phenylalanine demonstrates a significantly greater inhibitory effect on intestinal and placental ALP than liver and bone ALP. Nonetheless, using phenylalanine does not enable the differentiation of placental from intestinal ALP or liver from bone ALP.[28]

In addition to the 4 major ALP isoenzyme fractions, certain abnormal fractions are associated with neoplasms. The most commonly encountered among these are the Regan and Nagao isoenzymes,[29] often referred to as carcinoplacental ALPs due to their similarities to the placental isoenzyme. The Regan isoenzyme has the highest heat stability among all ALP isoenzymes and can withstand denaturation at 65 °C for 30 minutes. The Regan isoenzyme migrates to the same position as the bone fraction, and its activity is inhibited by phenylalanine.[30] The Nagao isoenzyme can be considered a variation of the Regan isoenzyme, with identical electrophoresis, heat stability, and phenylalanine inhibition characteristics.[31] However, Nagao isoenzyme can be inhibited by L-leucine, and it has been identified in metastatic carcinoma of pleural surfaces and adenocarcinoma of the pancreas and bile duct.[32]

Interfering Factors

ALP is inhibited by metal-complexing anticoagulants, as they impede the enzyme by forming complexes with Mg and Zn, and therefore, they should be avoided. Hemolyzed or lipemic specimens should be rejected if they exhibit a high background absorbance. Significantly, bilirubin at concentrations of up to 20 mg/dL does not interfere with ALP measurements.[33] 

The purity of the transphosphorylation buffer is highly critical. In addition, it has been discovered that diethanolamine (DEA) sources can contain notable levels of monoethanolamine (MEA)—a potent inhibitor of ALP. Over time, DEA solutions can degrade during storage, resulting in the concurrent generation of MEA.[34] Certain commercial p-nitrophenol phosphate substrate preparations may contain excessive quantities of p-nitrophenol or inorganic phosphate, or even both. The presence of p-nitrophenol leads to elevated blank absorbance, while inorganic phosphate acts as an inhibitor to ALP.[35]

Serum ALP levels are affected by many factors, including medications, physical conditions, herbal remedies, dietary choices, smoking, alcohol consumption, and pregnancy. Over 1,000 drugs, herbs, and physiological conditions affecting ALP activity have been documented. For example, clofibrate can lower serum ALP activity, reducing all ALP fractions except the liver. This effect may result from an enhanced biliary clearance of ALP.[36] Estrogens, individually or in conjunction with androgens, have been observed to have a suppressive effect on ALP activity. However, separate studies have noted that both estrogens and androgens can elevate serum ALP activity. The reduction in ALP activity observed with estrogen therapy may be attributed to a decreased rate of bone turnover.[37]

Verapamil, commonly prescribed for managing cardiac arrhythmias, angina pectoris, and essential hypertension, has been found to elevate serum ALP levels in hypertensive patients. After administering verapamil to hypertensive patients for a 2-month duration, there is a significant rise in total ALP activity, followed by an increase in bone ALP levels in the serum. These findings indicate that verapamil may affect bone metabolism, secondary to increased parathyroid hormone secretion.[38] Furthermore, any medication that is hepatotoxic or induces cholestasis can lead to a notable elevation in serum ALP, at times to a significant extent.[39]

ALP assays are susceptible to negative interference due to alkali denaturation of hemoglobin, resulting in a negative offset in absorbance readings. Icterus primarily influences chemistry tests through spectrophotometric and chemical interferences. Bilirubin absorbs light within the 400 to 540 nm range, with its peak absorption around 460 nm.[40] Colorimetric assays that rely on primary or secondary absorbance measurements within these wavelength ranges may encounter interference. Notably, unconjugated and conjugated forms of bilirubin may exert different effects on certain assays. In many cases, conjugated bilirubin has been observed to induce a more pronounced interference.[41]

Results, Reporting, and Critical Findings

When ALP is the sole liver biochemical test that shows elevation, while serum aminotransferases remain within the normal range, or when ALP exhibits a disproportionately high increase compared to other liver biochemical tests, it is imperative to concentrate on identifying the cause and origin of this isolated or disproportionate elevation in ALP. For asymptomatic patients with isolated serum ALP elevation, it is crucial to pinpoint the primary source of the abnormality.[10] ALPs derived from the liver, bone, placenta, and intestines have different physicochemical properties.[42]

One approach involves measuring the activity of enzymes, such as 5’-nucleotidase (5NT) and gamma-glutamyl transpeptidase (GGT), which increase in concordance with the liver ALP. These enzymes do not exhibit elevations in bone disorders and demonstrate a strong correlation with hepatobiliary disorders. Serum GGT is highly sensitive to biliary tract diseases but exhibits lower specificity for liver disorders.[43] The 5NT levels may become elevated in pregnant patients. However, in non-pregnant patients, it is relatively specific for liver disorders and demonstrates a strong correlation with serum ALP of hepatic origin. Notably, the absence of an elevated 5NT in the presence of elevated ALP does not necessarily exclude hepatobiliary disease, as they may not rise simultaneously in early or mild hepatic injury cases.[44]

Clinical Significance

The principal clinical significance of measuring serum ALP levels is diagnosing cholestatic liver disease, as some of the most substantial ALP elevations are observed in patients with cholestasis. Usually, elevations 4-fold or more above the upper limit of normal can be found in up to 75% of patients with cholestasis, whether intrahepatic or extrahepatic.[10] The degree of elevation does not help in distinguishing the 2 types of cholestasis. Similar elevations are observed in cases of biliary obstruction due to cancer (cholangiocarcinoma, pancreatic head adenocarcinoma, or ampullary adenocarcinoma), choledocholithiasis, biliary stricture, sclerosing cholangitis, or causes of intrahepatic cholestasis such as primary biliary cholangitis (PBC), drug-induced liver injury, chronic rejection of liver allografts, infiltrative liver diseases (sarcoidosis, amyloidosis, tuberculosis, and liver metastasis), and severe alcoholic hepatitis leading to steatonecrosis.[45] Patients with AIDS may also exhibit notably high levels of ALP, often stemming from cholangiopathy related to opportunistic infections such as cytomegalovirus, cryptosporidiosis, or granulomatous liver involvement from tuberculosis.[46]

Moderate elevation of serum ALP, which can reach up to 4 times the upper limit of normal, is nonspecific, as it can manifest in various liver-related conditions, including cirrhosis, chronic hepatitis, viral hepatitis, congestive heart failure, and ischemic cholangiopathy.[47] Notably, disorders that do not primarily involve the liver, such as intra-abdominal infections, cholestasis of sepsis, Hodgkin lymphoma, myeloid metaplasia, and osteomyelitis, can also lead to a moderate elevation of serum ALP.[10]

Both primary and metastatic cancers elevate serum ALP levels through local bile duct obstruction and an augmented leakage of the liver isoenzyme.[48] Primary extrahepatic cancer does not necessarily have to affect the liver or bone directly. Certain tumors can generate their own ALP in rare instances, such as Hodgkin lymphoma secreting the Regan isoenzyme. Alternatively, they can induce a paraneoplastic effect that causes leakage of the hepatic isoenzyme into the circulation, as seen in Stauffer syndrome due to renal cell carcinoma.[49]

Abnormally low ALP levels can offer clinical insights, particularly in cases of Wilson disease, particularly when it presents in a fulminant form with hemolysis. This is attributed to the displacement of zinc, a cofactor of ALP, by copper in Wilson's disease, a condition characterized by copper overload, resulting in diminished ALP levels.[50] Other factors leading to low ALP levels include zinc deficiency, pernicious anemia, hypothyroidism, and congenital hypophosphatasia.[51]

Transient very high levels of ALP, reaching up to 30 times the upper reference limit, have been documented in children. However, the clinical significance of this finding is unknown. This phenomenon has been termed "transient hyperphosphatasemia" and may involve either the bone or liver isoenzyme.[52] Typically, affected patients are asymptomatic and exhibit an unremarkable medical history, physical examination, and laboratory results. Some patients may have recently experienced mild viral conditions.[53]

Extensive evaluation is usually unnecessary for patients with only a mild elevation of serum ALP, which is less than a 50% increase. These individuals can be clinically monitored with periodic assessments of serum liver biochemical tests. However, when ALP levels are significantly elevated, it is imperative to conduct further evaluation to determine whether the source is hepatic or non-hepatic.[54] The simultaneous elevation of either GGT or 5NT indicates a hepatic source for an elevated ALP level. If the source is non-hepatic, the next step involves assessing potential underlying undiagnosed disorders. Elevated bone ALP levels can be observed in cases of bone metastasis, Paget disease, osteogenic sarcoma, healing fractures, hyperparathyroidism, hyperthyroidism, and osteomalacia. Elevated intestinal fraction of ALP often occurs after a fatty meal and can be hereditary; however, it typically does not necessitate further evaluation.[55] When the liver is suspected as the source, it is crucial to conduct imaging of the biliary tree to distinguish between extrahepatic or intrahepatic cholestasis while also reviewing the patient's medication list.[45]

In most cases, the initial imaging study ordered is right upper quadrant ultrasonography. If the bile duct is dilated, the choice between endoscopic retrograde cholangiopancreatography (ERCP) or magnetic resonance cholangiopancreatography (MRCP) depends on the clinical indication. On the other hand, if the bile duct does not exhibit dilation, the recommended next step is to test for serum antimitochondrial antibody (AMA) to assess for PBC.[56] If the serum AMA level is within the normal range, it becomes essential to evaluate for causes of intrahepatic cholestasis, AMA-negative PBC, sarcoidosis, and other conditions mentioned earlier. In many cases, a liver biopsy is the ultimate diagnostic test used in such situations, as it aids in pinpointing the underlying cause of the elevated serum ALP.[57]

Quality Control and Lab Safety

A quality management system (QMS) is crucial for laboratory tests as it assures the reliability of all aspects of laboratory operations.[58] In the context of laboratories, quality is characterized by the accuracy, reliability, and timeliness of reported test results. A poor QMS can lead to unwarranted treatment or complications, the inability to provide accurate treatment, delayed diagnoses, and unnecessary follow-up diagnostic testing. Implementing a QMS in a laboratory setting effectively ensures that quality control (QC) and quality assurance goals are met and maintained.[59]

QC, often called internal QC, entails monitoring a measurement procedure to verify that results for patient samples meet performance specifications appropriate for patient care. QC also helps identify any error conditions that require correction.[60] When the result for a QC material falls within the acceptable limits of the expected value, it confirms the stability of the measurement procedure, indicating that it is functioning as anticipated. Therefore, results for patient samples can be reported with a high probability that they are suitable for clinical use. If a QC result is not within acceptable limits, it signals a malfunction in the measurement procedure. In such cases, there is a high probability that results for patient samples may not be appropriate for clinical use, thereby necessitating corrective action.[61] If corrective action is indicated, patient sample measurements should be reprocessed once the measurement procedure has been restored to its stable and accurate performance condition. If erroneous results are reported before the error condition is identified, a corrected report must be issued.[62]

QC materials are measured periodically along with patient samples. For non-waived tests, laboratory regulations mandate analyzing at least 2 levels of control materials once every 24 hours. Laboratories have the flexibility to analyze QC samples more frequently if it is deemed necessary to ensure accurate results. QC samples should be assayed after an analyzer's calibration or maintenance to verify the correct method performance. To streamline QC when conducting tests for which the manufacturers' recommendations are less frequent than those mandated by the regulatory agency, for instance, once per month,[63] laboratories can establish an individualized QC plan (IQCP). This involves conducting a risk assessment to identify potential sources of errors in all testing phases and implementing a QC strategy to minimize the likelihood of errors. Patient results can also be incorporated into a statistical QC process to monitor the performance of the measurement procedure.[64]

The Levey-Jennings plot is the most commonly used format for evaluating QC results. This format displays each QC result sequentially over time, enabling a rapid visual performance assessment. Provided that the measurement procedure is carried out under stable conditions in accordance with its specifications, the mean value serves as the target (or expected) value for the QC result, while the standard deviation (SD) lines depict the anticipated imprecision. Assuming a Gaussian (normal) distribution of imprecision, the results should exhibit a uniform distribution around the mean, with outcomes being more frequently observed closer to the mean than at the extremes of the distribution.[65] The conventional method of expressing QC interpretive rules involves using an abbreviation nomenclature popularized in clinical laboratories by Westgard.[66]

External QC, also called external quality assessment (EQA) or proficiency testing (PT), is a monitoring process in which surrogate samples are received from an independent external organization, and the laboratory is not aware of the expected values for these samples. The results for the EQA/PT samples are transmitted to the provider and then compared with results from other laboratories to assess whether a laboratory's measurement procedures align with the expected performance. Notably, the quality of the EQA/PT sample plays a crucial role in interpreting the results.[67] The acceptable performance (accuracy) for ALP assays, defined by the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88), is within the range of the target value ± 30%. The College of American Pathologists (CAP) also utilizes this same acceptability criteria in its surveys.[68] To meet the requirements, a laboratory must attain a score of 80% or higher in each testing event for each analyte and maintain an overall score of 80% or greater across all analytes. Each laboratory is responsible for establishing its acceptability criteria for intra-daily and inter-daily variations, which should be determined based on the specific instrument and quality-control materials in use.[69]

Every clinical laboratory must establish and maintain a comprehensive and formal safety program as its utmost priority. This program is the cornerstone of ensuring the well-being of both patients and laboratory personnel. It offers a structured framework for identifying, mitigating, and managing potential hazards associated with laboratory operations. By adhering to stringent safety protocols, the laboratory significantly reduces the risk of errors, accidents, and exposure to hazardous materials.[70] Barrier protection, including gloves, masks, protective eyewear, and gowns, must be readily available and consistently utilized during blood collection procedures and when handling all patient specimens. Gloves should be disposable, made of nonsterile latex, or constructed from other materials that offer sufficient barrier protection. Phlebotomists must change gloves and dispose of them properly when transitioning between drawing blood from different patients, and they should also wash their hands whenever gloves are changed.[71] 

Facial barrier protection should be used if there is a significant risk of blood or body fluids spatter. Lab technicians should minimize the use of syringes whenever possible and ensure the disposal of needles into rigid containers without manual handling. All sharps should be disposed of appropriately. Furthermore, they should wear protective clothing as an effective barrier against potentially infectious materials. Upon leaving the laboratory, lab technicians should remove their protective clothing. Frequent hand washing is crucial in the laboratory. Employees should wash their hands whenever they exit the laboratory. Notably, warning labels should not be used on patient specimens; all specimens should be treated as potentially hazardous.[72] Biosafety level 2 procedures should be used whenever appropriate. Discarded tubes or infected materials should never be left unattended or unlabeled. Periodic cleaning of the freezer and dry-ice chests is necessary to remove broken ampoules and tubes of biological specimens. Using rubber gloves and respiratory protection during this cleaning process is mandatory.[73]

The Occupational Safety and Health Administration (OSHA) mandates that the hepatitis B vaccine must be offered to all employees at risk of potential exposure as a regular or occasional part of their dutCDC'sThe CDC's Advisory Committee on Immunization Practices recommends that medical technologists, phlebotomists, and pathologists receive the hepatitis B vaccine. Therefore, it is a regulatory requirement that all laboratory employees mentioned above are, at a minimum, provided with the option to receive a free hepatitis B vaccine.[74] 

In 2003, the International Organization for Standardization introduced the ISO 15190 standard to enable medical laboratories of various types to establish policies and procedures that foster a secure working environment within their facilities.[75] This standard is tailored explicitly for safety considerations and covers a broad spectrum of laboratory safety aspects, including managerial requirements and individual personnel's responsibilities, such as fire and radiation safety measures. These safety directives are based on the principles of QMS, which involve clearly defined roles and responsibilities, for instance, the Laboratory Director and Safety Officer, task delegation for employees, and a system of regular audits, continuous assessment, and improvement.[76] 

Enhancing Healthcare Team Outcomes

A healthcare provider may order an ALP test for a routine checkup or if the patient displays symptoms of a bone disorder or liver damage. A thorough evaluation and classification of any aberrant ALP levels and related observations by the patient's healthcare team are pivotal for enhancing healthcare outcomes for the patient.