Ionized calcium: analytical challenges and clinical relevance

Introduction

Calcium is the fifth most abundant element in the organism (1). Calcium level depends primarily on dietary intake (2-5). This divalent cation, present in two forms (bound or free) in the body, plays an indispensable role in multiple biological functions, the most important of which is bone mineralization. Indeed, the vast majority of the body calcium (>99%) is stored in bones, as hydroxyapatite crystals, thus responsible for the biomechanical properties of the skeleton (6).

Non-osseous calcium (<1% of body calcium storage) is very finely regulated by a complex interaction between several actors. Its homeostasis is based on a permanent communication between the hormonal system (PTH, 1,25-dihydroxyvitamin D) and the receptors located on three peripheral organs (digestive tract, kidneys and bones) (7,8). This non-osseous calcium thus comprises protein-bound calcium (mainly albumin and globulins in the serum and calmodulin in the cells), free (or ionized) calcium and ionic-bound calcium (complexes of phosphate, carbonate and calcium oxalate), representing respectively about 50%, 40% and 10% of the circulating calcium (Figure 1). This calcium form plays many important roles: from its involvement in several intra- and extracellular signaling pathways, to its implication in nerve impulse transmission and muscle contraction (1,7). This pool of non-osseous calcium is closely regulated in order to maintain total serum calcium level in a physiological window between 2.20 and 2.60 mmol/L. This regulation is even finer regarding ionized calcium levels (between 1.10 and 1.35 mmol/L), given its potentially severe toxicity outside this physiological range. Dehydration, digestive disorders, confusion and arrhythmia mainly in case of hypercalcemia; numbness/tingling in the extremities, muscle spasm, confusion, hallucination and weakness in case of hypocalcemia (9-11).

Figure 1 Body repartition of the different calcium forms.

Calcium is involved in many physiological functions; its dosage and monitoring are essential in many clinical situations. The question about choosing between total or ionized calcium testing has been arising for many years (12-14), and is still debated even though some evidence has recently emerged in favor of routine ionized calcium measurement (15). Indeed, there are a number of factors that influence the value of serum calcium, most notably protein and parathormone serum levels. Thus, the main risk of systematically using the total calcium level is the overestimation of serum calcium status.

The aim of this article is first to discuss about the measurement and estimation of ionized calcium in laboratories, and then to describe its applications in clinical practice.

Ionized calcium and its estimation: an analytical challenge

Calcium is distributed in three forms, an important fraction of it being complexed with proteins (mainly albumin). As previously described, the free form of calcium is the one that plays a major role in the body (13,16-18). This free fraction is very closely modulated by endocrine functions (1,8,16). However, its dosage is limited by constraints which are reducing its routinely use (19,20). Indeed, its precise dosage requires both strict conditions of sample handling as well as taking into account many pre-analytical conditions (21).

Sample handling requirements and ionized calcium measurements

For an accurate determination of the calcium status based on ionized calcium level, it is essential to take all necessary precautions regarding the type of specimen collection and the choice of container collection. The type of tube collection plays a key role in this analysis given the presence of common anticoagulants (EDTA, citrate, oxalate), whose principal function is precisely the ability to complex with free calcium. Not taking this element into account would lead to a significant underestimation of ionized calcium level (22). The International Federation of Clinical Chemistry (IFCC) guidelines recommend heparin as the anticoagulant of choice for the measurement of ionized calcium (23). Heparin chelates a moderate but significant portion of ionized calcium, depending on its type and dosage quantity in the tube. The international recommendations do not particularly prefer sodium/lithium or calcium titrated heparin, as long as the concentrations do not exceed 15 and 50 IU/mL blood respectively (19,21). The assay can thus be performed on whole blood sample with heparin, heparinized plasma or serum.

According to the Clinical Laboratory Standards Institute (CLSI) guidelines, measurement of ionized calcium with plasma should be avoided and use of whole blood specimens should be preferred (in syringes) (24). However, evacuated blood collection tubes containing lithium heparin and gel separator material are used very frequently in clinical laboratories nowadays. The use of heparin lithium tubes has certain advantages, notably that of allowing a rapid result (latency time of approximately 30 minutes for the gel separator tubes), sometimes better corresponding to the clinician’s expectations. The other advantage of lithium heparin tubes is the possibility of using the entire sample volume for analysis. Although not recommended, if the sample has been taken in an evacuated tube, care should be applied to keep it sealed until analysis, in order to avoid any pH change caused by CO₂ leakage. It is also important to treat the samples as soon as possible to limit the potential effects of anaerobic metabolism-induced glycolysis on erythrocytes and leucocytes (25). Of course, the use of a serum sample remains the ideal solution, avoiding in particular any bias related to erythrocytes or heparin but this requires a larger sample volume and a longer process time. Regarding the sample stability, whole blood samples (especially for blood gas analysis) need to be treated very quickly (within half an hour) whereas samples taken into centrifugation to obtain serum/plasma may offer longer stability ranging from several hours (at room temperature) to several days (in refrigerated environment) as long as the tubes remain sealed (26,27). Although many methods, more or less complex, have been described to measure ionized calcium, it seems that its measurement has recently been democratized by the advent of ion-selective electrodes (ISE) (28,29). Indeed, this method currently recommended by the IFCC guidelines is based on a simple principle: the measurement of a potential across a membrane only allowing selective calcium transit, this potential being mathematically correlated to the ionized calcium concentration in the sample tested. To the best of our knowledge, there is currently no large automated instrument for the specific measurement of ionized calcium; however, the vast majority of blood-gas analyzers currently allow a direct ISE assessment of ionized calcium level.

Physiological and pre-analytical issues

In the literature, a number of situations have been identified as potentially responsible for ionized calcium level variations (24,30). We present below the details of the main factors to consider when measuring and interpreting ionized calcium levels.

• Physical activity: by an increase in the lactate level, a drop in pH and bicarbonates, an increase in ionized calcium may occur after a moderate-intensity effort. In a previous work, Ljunghall et al. observed a 5–10% change in ionized calcium levels in healthy volunteers after bicycling exercise (65% of theorical maximal effort) (31).
• Dietary intake: it has been shown that a moderate decrease (approximately 5%) in ionized calcium levels is usually observed within 2 hours of dietary intake (2,3,32). This decrease is probably the consequence of an increased complexation of calcium with proteins and other anions such as phosphates/bicarbonates. Briefly, ionized calcium levels may also be impacted in the same way by all factor which could hinder the calcium-protein complex production (pyruvate, sulfate, beta-hydroxybutyrate).
• Ortho- or clinostatism: indeed, it has been demonstrated that the patient posture at the time of sampling could have an effect on ionized calcium levels, in particular by substantial variations in protein/albumin levels, as well as an increase of both muscle tonus and hydrostatic pressure during the change of position (26,33).
• Circadian rhythm: there is a strong association between circadian rhythm and ionized calcium dosage; indeed, the study of Markowitz et al. revealed significant variations (up to 10%) in ionized calcium levels during a 24 hour-cycle, the nadir being most often reached in the late afternoon. Interestingly, the daily evolution curves for ionized and total calcium levels were not superimposable (34). These differences are probably explained by dietary intakes and variations in acid-base balance during the circadian cycle.
• pH and by extension the ventilatory state: on a biological level, it has been clearly demonstrated that pH fundamentally influences ionized calcium levels by its impact on the complexation with proteins and other anions (16,35-37). Thus, a rise in sample pH promotes the formation of calcium-albumin complexes and causes a decrease in ionized calcium levels. Conversely, a drop in pH may cause an increase in the free fraction of blood calcium and therefore an increase in ionized calcium levels. Thus, the occurrence of hyperventilation-induced respiratory alkalosis a few minutes before sampling may result in a significant decrease of the ionized calcium value (38,39). Overall, there is a strong correlation between ionized calcium and pH, quantified as around 5% calcium level modification for each 0.1 pH unit variation. This relationship is only verified for pH values ranging between 7.20 and 7.60. Given this predictable and well-described relationship, the use of a correction formula when reporting results is discussed (16,40): on the one hand, the effect of pH on ionized calcium is relevant in vivo, so not applying the correction seems consistent if one seeks to measure ionized calcium in the clinical situation where the sample was taken. In other situations where the pH appears distorted by post-analytical manipulation and when the patient has no apparent reason to present any acid-base disorder, this formula may be of interest.
• Other factors potentially affecting the calcium complexing ability: in the first place, we should of course mention albumin, the main protein to which calcium binds and whose variations have a major impact on ionized calcium levels. Briefly, we could also mention here the effects of heparin, fatty acid and bilirubin levels, certain drugs and temperature variations (26).

In conclusion, the realization and interpretation of ionized calcium measurement are challenging as the conditions and constraints that can affect its serum level before, during and after the sampling are numerous and diverse (Figure 2). These elements added to the significant cost of the technique represent all obstacles to its systematic use in current practice (41,42).

Figure 2 Complexity of ionized calcium measurement and interpretation.

The unachieved quest of a perfect calcium estimation formula

Although its dosage is currently more accessible thanks to the ISE technology allowing its determination on blood-gas analyzers, many institutions continue to use total calcium assay in their daily practice, test that is otherwise recommended in the international guidelines (43). In this context, a very large number of formulas have been developed for nearly a century with two main objectives: to estimate the calcium status reported to a normal albumin level or to estimate the ionized calcium level from total calcium and a number of biological parameters such as albumin, protein, pH and anion gap. Historically, the first estimation formula is attributed to the nomogram developed by McLean and Hastings in the 1930s (18). Nevertheless, its tedious application has given way to the most used estimation formula: the Payne correction equation (44). Indeed, this very simple and easy-to-use formula is based on the strong link between total serum calcium and albumin levels (calcium = total calcium − 0.025× albumin +1). According to data from Vancouver Coastal Health and Providence Health Care Laboratories in 2018, total calcium and albumin were ordered concomitantly to biological samples in almost three out of four cases, suggesting that for the vast majority of clinicians, the interpretation of one cannot be fulfilled without the value of the other (45).

The literature concerning these estimation formulas is extremely rich, as illustrated by the work of Dickerson et al. (46). This study evaluated calcium estimation methods in a population of critically ill multiple trauma patients receiving specialized nutrition support. Interestingly, they did a literature review of all available estimation formulas and found 22 different ones, either trying to determine a “corrected” total serum calcium concentration, or to estimate ionized calcium concentration. The results are eloquent, as the set of formulas clearly failed to accurately assess calcium status: Dickerson et al. thus observed a very low average sensitivity of these formulas, for both hypocalcemia and hypercalcemia (25% and 15% respectively). The recent work of Mateu-de Antonio et al. focused on ionized calcium prediction formulas developed in adults, out of any pathology: eleven formulas have been highlighted, including the classic formulas of McLean-Hastings and Payne (47). Again, these formulas showed very poor predictive abilities in two cohorts of hospitalized patients for the detection of hypocalcemia (18% and 26% sensitivity respectively for McLean-Hastings and Payne formulas). In total, at least 30 different formulas have been created or derived from the oldest ones, and the literature has regularly reported their limits in practice (48) (Table 1).

Different hypotheses to explain the limits of theses estimation formulas

The strong correlation between serum calcium and albumin levels is unquestionable, and justifies the common use of these estimation formulas in absence of pH or serum protein abnormalities. Nevertheless, as previously described, the correlation between total and ionized calcium may collapse in the presence of a wide variety of factors: acid-base imbalance, albumin abnormality, protein-bound drugs or fatty acids, or the presence of an excess circulating immunoglobulin for example (20,22,26).

An important hypothesis made about the relation between albumin and calcium is that a decrease of the first induces a decrease of albumin-bound calcium fraction. This would lead to a decrease of total calcium while ionized calcium levels remain normal. In fact, this hypothesis has been dismantled since there is some evidence that in hypoalbuminemic states, the calcium affinity for albumin increases significantly (68). Starting from the Payne formula, the most used in practice, it is not surprising to see a risk of ionized calcium overestimation: indeed, the starting postulate relies on a constant albumin-calcium relationship, regardless of albumin levels. Thus, several studies have since abounded in this direction, even demonstrating that in situations of severe hypo-albuminemia, the correction formula is less efficient than the uncorrected total calcium level (46,63). Payne formula estimation was constructed from 200 patients but was not validated in a second cohort (44). The main goal of this formula was to deduce, in hypoalbuminemic patients, the calcium value it would have been if albumin level was normal. The patients included were not suspected for any ionized calcium abnormality. Recently, Payne himself has addressed a letter to the editor in which he reminds that the formula was not intended to be universally used, being for example not at all suitable to the chronic kidney disease population (69). Moreover, this formula was produced from the data of the time, based on the analytical methods applied to biological measurements at that time, which implies that it should be updated according to the methods used nowadays in clinical laboratories. Another major hypothesis, demonstrated in a recent work, is the need of taking into account the pH in these estimation formulas (53-55). Indeed, it now seems clear that pH is one of the most important adjustment variables when trying to estimate a patient’s calcium status. Indeed, a retrospective study of 5,055 samples confirmed the lack of added value of several correction formulas (including Payne’s) compared to total calcium measurement, in order to accurately estimate the calcium status. Taking into account the pH value has radically improved the prediction performances in our own derived formula (55), especially in patients presenting alkalosis. pH-adjusted formulas are thus rare in the literature, partly explaining the overall poor performance of these estimation equations (53,55). More recently, to overcome this limitation, some authors suggested to also use the anion gap to better estimate the calcium status when ionized calcium is unavailable (70). Moreover, these formulas would be of limited interest since pH determination and ionized calcium measurement require actually the same strict pre-analytical conditions and may be performed under the same conditions on blood-gas analyzers.

Some authors propose to avoid the reproducibility issue of such formulas by producing their own formula directly derived from the measurements carried out in their laboratory (65). On the basis of their work on a cohort of over 4,600 outpatients, James et al. showed a significant improvement in the patients’ calcium status assessment using their own local adjusted equation. Other researchers seem to go further in their reasoning: for Lian et al., the problem lies in the mathematical conception of the regression equations used to define the various estimation formulas (56). Indeed, for them, it would be advisable to take into account, beyond total calcium and albumin levels, other explanatory variables including albumin, ionized calcium value as well sex, age, kidney function, serum phosphate level, and the in- or outpatient status. Although the hypothesis appears convincing, it seems that even a formula derived from its own dataset and resulting from an enlarged number of parameters fails doing better than unadjusted total calcium levels. This may explain why the question of an indisputable formula is still unachieved: this may actually be an unsolvable problem.

Clinical relevance of ionized calcium measurement

We aimed to discern, from the literature data, the situations in which the ionized calcium estimation formulas produce unreliable results, which may negatively impact patient management and justify the systematic use of ionized calcium dosage.

Thyroid, parathyroid and malignancy-related calcium disorders

Calcium regulation relies above all on a complex and integrated regulation by the endocrine system, based on two essential hormones: PTH and active vitamin D. The diagnosis of primary hyperparathyroidism is based on the concomitant presence of abnormally high or normal PTH levels and hypercalcemia with a particular relevance of ionized calcium (71-80). Indeed, the diagnosis of hyperparathyroidism is based primarily on the detection of hypercalcemia, and the frequency of hyperparathyroidism revealed by an isolated increased ionized serum calcium level should make us wary of the sole dosage of total calcium (72-74,76,77,79,80). In a retrospective study of 268 patients treated with parathyroidectomy for primary hyperparathyroidism, ionized calcium levels were found to be more sensitive than total serum calcium levels for the detection of hypercalcemia. In addition, a strong linear relationship between ionized calcium and parathyroid adenoma size has also been demonstrated, a clearer association than with total serum calcium levels (74). In some international guidelines, the dosage of ionized calcium is recommended for the diagnosis of hyperparathyroidism, its routine use being only hampered by its wider accessibility (81). In parallel with hyperparathyroidism, the other major cause of hypercalcemia in humans is malignancy, related to various mechanisms such as bone resorption mediated by PTH related peptide (PTHrp) in case of paraneoplastic syndrome or by the presence of bone metastases (82,83). In the population of cancer patients, studies confirm a significant discrepancy between total and ionized calcium values for the diagnosis of malignancy-related hypercalcemia (84,85). The question of an association between ionized calcium and the cancer-related mortality risk remains debated (84), although several recent prospective studies seem to emphasize this link, especially in prostate and ovarian cancers (86-89). More interestingly, the ionized calcium testing could also detect occult cancer pathologies, as suggested by a prospective study conducted on 6,707 men with more than 15-year follow-up (88). To those situations, we could also add several case reports of pseudo-hypercalcemia (total calcium) related to multiple myeloma, which have led to misdiagnosis and injudicious care (90,91), while ionized calcium levels were normal.

The added value of ionized calcium has also been advanced in the context of hypocalcemia, a frequent and sometimes serious complication of thyroidectomy. Although ionized calcium measurement does not seem to bring any obvious benefit in the detection of postoperative hypocalcemia compared to total serum calcium levels (90), its preoperative and early postoperative values may have predictive capacities regarding the occurrence of symptomatic post-operative hypocalcemia (91,92). This could also apply to all other forms of primary or secondary hypocalcemia, but data are still lacking.

Calcium monitoring and kidney dysfunction

For the follow-up of patients with moderate or advanced chronic kidney disease (including end-stage kidney disease), the monitoring of serum calcium levels is essential. Indeed, these patients are at high cardiovascular risk, mainly mediated by the chronic kidney disease—mineral bone disorder (CKD-MBD) (93,94). This syndrome begins in the early stages of CKD and worsens as kidney function deteriorates. This complex pathology involves the progressive increase in serum phosphorus, PTH and fibroblast growth factor 23 (FGF23) levels in parallel with decreased 1α-hydroxylase activity, calcium uptake and serum calcium levels. This leads to specific abnormalities in the bone turnover, which can reach the stage of adynamic bone disease (95). Clinically, we observe a renal osteodystrophy syndrome, responsible for increased bone fragility, as well as accelerated vessels and soft tissues calcification, thus explaining the high cardiovascular risk in the CKD population (96). This explains the need to closely monitor the mineral metabolism parameters in CKD patients.

Nevertheless, at the most advanced CKD stages, pH modifications and serum albumin fluctuations do not guarantee an adequate estimation of calcium status by total serum calcium measurements (97), although the 2017 ‘Kidney Disease: Improving Global Outcome’ (KDIGO) recommendations do not exclude total calcium monitoring, after albumin adjustment (43). However, reports confirm the relevance of ionized calcium testing for this population. Indeed, there may be a significant risk of misdiagnosing a CKD patient as hypercalcemic with total calcium measurement although ionized calcium level is in the standard range (63,97-100). These results may thus have a direct impact on these patients care, regarding the prescription of calcium or vitamin D supplementation or the use of calcimimetics. Surprisingly, a study of more than 100 CKD stage 3 to 5 patients demonstrated that albumin-adjusted serum calcium levels did not perform better than unadjusted total serum calcium for the detection of ionized calcium abnormalities (100). Similarly, the study of Clase et al. on 50 stable hemodialysis patients emphasized a better agreement between ionized and uncorrected total serum calcium levels than with all formulas for adjusted calcium estimations (63).

After kidney transplantation, calcium monitoring is essential, since hypercalcemia affects more than 10% of this population, even after the first-year post-transplantation (101). This phenomenon is most often related to persistent hyperparathyroidism. For these patients as well, the performance of total calcium dosage seems very limited, in the same way as for CKD patients. Indeed, in a study involving 268 kidney transplant recipients, the diagnosis of hypercalcemia would be missed in 80% of cases if the clinicians would have stuck only to total serum calcium measurements (102). This result is probably related to the high prevalence of metabolic acidosis in the population of kidney transplant patients (10% to 50%, depending on post-transplantation kidney function) (103-105), prevalence much higher than in CKD population before transplantation (106).

Serum calcium levels and cardiovascular outcomes

The extracellular calcium concentration has a direct impact on cells with calcium-sensing receptors on their surface (parathyroid, renal tubule) but also on excitable cells by influencing their membrane potential. This concerns particularly the heart, for which calcium homeostasis is directly involved in myocardial contraction. There is a link between ionized calcium levels and electrocardiogram modifications (107-111), illustrated by few case reports of severe arrhythmias caused by abrupt changes in ionized calcium levels (111,112). Calcium levels also interact with the smooth muscle fibers present in vessel walls, thus explaining the observed link between ionized calcium and blood pressure (113,114). Furthermore, in addition to enhancing calcium deposits in the soft tissues and vascular walls, chronic exposure to high levels of circulating calcium may increase the risk of cardiovascular events (96). This may also be mediated by the association of calcium serum levels and some specific cardiovascular risk factors such as high blood pressure, carbohydrate intolerance and dyslipidemia (115-118). While several studies have shown a link between high levels of total calcium and the occurrence of cardiovascular complications (96,119-121), the work of Ogard et al. on 974 patients followed for nearly 20 years, could not confirm this relationship with ionized calcium levels (122).

Calcium disturbances in critically ill patients

The population of patients admitted to intensive care units (ICU) is very often subject to hypocalcemia (60% to 90% of patients) (123,124), favored by various mechanisms: increased fecal and urinary excretion, vitamin D deficiency and decreased calcium intake, pH modifications, catecholamine-mediated intracellular shift, citrate anticoagulation for continuous veno-venous hemodiafiltration or plasmapheresis, and parenteral nutrition (125-127). Among these critically ill patients, it has been clearly demonstrated that total serum calcium and correction formulas are poor estimators of ionized calcium levels as they failed to accurately classify calcium status in up to 40% of this particular population (46,126,128,129). Although some studies have found a link between the depth of hypocalcemia and the risk of in-hospital or early mortality, the predictive value of ionized calcium levels at ICU admission is still debated (124,130-133). The studies of Choi et al. (133) and Hastbacka et al. (134), including 255 and 993 patients respectively, demonstrated limited predictive capacities with an estimated area under curve inferior to 0.70.

Nevertheless, ionized calcium assay appears to be more strongly associated with mortality risk in some specific ICU patients, such as those experiencing acute kidney injury (135), and those requiring renal replacement therapy (136,137). Moreover, the literature has highlighted some discrepancies between total and ionized calcium levels in patients admitted for acute pancreatitis (138,139) or for severe liver dysfunction requiring transplantation (140). Finally, a recently conducted prospective study on 111 patients hospitalized for hypertensive intracerebral hemorrhage showed that low ionized calcium was an independent risk factor for early expansion of intracerebral hematoma, as well as poor short-term prognosis (141). The use of extracorporeal circulation, and more specifically plasmapheresis, justifies the close monitoring of ionized calcium level, given the risk of its sudden drop due to citrate anticoagulation and the common substitution by fresh plasma (142-144).

Other potential leads for clinical usefulness of ionized calcium testing

The use of ionized calcium dosage has also been the subject of some other sparse studies, which may suggest a broader indication of its measurement. Indeed, in a study involving 1,248 men with fertility disorders, a link was found between ionized calcium levels and semen quality, as well as sex steroid levels (145). Some works have also reported a relationship between ionized calcium levels and the occurrence of preeclampsia (146,147). A study in elderly patients followed for depression also described a link between elevated levels of ionized calcium and white matter hemorrhages detected on brain MRI (148). Finally, the use of the ionized calcium assay also seems useful when administering drugs and chemotherapies known to be associated with ionized calcium disturbances (cisplatin, leucovorin-5-fluorouracil, bisphosphonates for example) (149-151). Some recent reports, however, have drawn attention to the risk of calcium status misinterpretation by ionized calcium testing in patients under Leflunomide treatment, due to analytical interferences (152,153).

Towards a rationale and reasonable prescription of ionized calcium testing in daily practice

In conclusion, the current state of the art seems to insist on the added value of routine ionized calcium measurement for specific populations, such as patients suspected of hyperparathyroidism, suffering from cancer, admitted to intensive care units, or followed for CKD (Figure 3). For the other conditions mentioned above, we currently lack data to justify the broadening of ionized calcium monitoring indications.

Figure 3 Clinical usefulness of ionized calcium measurement, based on level of evidence.

Although its routine use seems increasingly justified in many situations (15), some authors have recently pointed out the risk of drifting to an inappropriate over-prescribing of ionized calcium testing (16,41,154). Indeed, these studies describe the setting of an educational intervention regarding the appropriateness of ionized calcium measurement, which has allowed an up to 75% decrease of its institutional prescription. In connection with these previous works concerning the overuse of ionized calcium, we may distinguish two main situations of interest: first, the specific cases previously detailed and the situations of moderate dyscalcemia. Indeed, the discrepancies between total and ionized calcium almost never concern the situations of frank hyper- or hypocalcemia.

This confirms the need for a thinking on precise clinical indications of ionized calcium testing and their integration into international guidelines (12,13).

Acknowledgments

Funding: None.

Footnote

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/jlpm-20-60). 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.

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