Biochemical Tests for Diagnosing and Evaluation Stages of Chronic Kidney Disease

Abstract

The primary biochemical markers for the diagnosing and evaluating stages of chronic kidney disease (CKD) are serum creatinine and the estimated glomerular filtration rate (GFR). For diagnosing CKD, the GFR needs to be <60 mL/min/1.73 m² for more than 3 months. Less frequently used endogenous marker for estimating the GFR is cystatin C. Alternatively, exogenous markers that can be used include inulin, iotalamat, and iohexol if clarity is not achieved with creatinine or cystatin C. Globally, urinary albumin and albumin-creatinine ratio are the recommended tests from a spot collection to estimate the kidney damage. Urinary protein estimate’s use is declining, especially 24-hour collections. There are several other markers discussed in brief that may be a useful adjunct in identifying causes and likely management strategies for CKD. Finally, pitfalls of the primary methodologies for the above tests are provided to guide readers in better understanding the results and their use in patient care decisions.

1. Introduction

Chronic kidney disease (CKD) is a significant health issue, with a global prevalence approaching 10% [1]. It is a condition associated with long-term morbidity and mortality linked to its severe impact on the cardiovascular system. In the healthcare setting, many resources are allocated toward the monitoring and treatment of CKD. These can be in the form of medical practitioner consults, allied health input, medications, dialysis, and biochemical monitoring of progression and complications.

Biochemical tests are pivotal to the diagnosis, monitoring, and management of CKD. Increased understanding of the array of methods for individual tests and the ways best to interpret result for each test can be critical in optimizing decisions for patient care. Additionally, understanding the deficiencies or pitfalls of each test with preanalytical collection and handling processes and analytical performance, including interferences, may be very useful in better understanding the accuracy of results and empowering clinical staff to interact with pathology experts and subsequently deliver improved patient care decisions.

Biochemical testing and monitoring can be used for assessing renal dysfunction, the progression of CKD staging, biochemical and endocrine sequelae of CKD, and optimizing therapy and dialysis. This chapter will endeavor to provide an overview of the biochemical analytes employed in CKD monitoring and provide laboratory considerations in the understanding of each.

2. Creatinine

Creatinine is the end metabolic product of creatine in muscle, and the amount of creatinine in the serum and urine is related to muscle mass and renal excretion. It is consistently converted into creatinine at ~2% per day by an irreversible process, freely filtered at the glomerulus, and not reabsorbed by the renal tubules, which makes it a good endogenous marker for kidney impairment detection. Its use in kidney function evaluation has limitations, which include age, sex, race, and body weight. Hence, the development of the estimated glomerular filtration rate (eGFR) and the measured glomerular filtration rate (mGFR) has been adopted. GFR estimation or measurement is utilized to determine the amount of blood passing through the kidneys per minute, and its amount can be indicative of kidney function from the clearance of endogenous biomarkers (creatinine and cystatin C) or exogenous substances, which include urine isotope collections, such as inulin, iotalamat, and iohexol, used for measuring the GFR more accurately. Although they may provide superior accuracy, they are invasive, impractical, time consuming, and expensive, including the need for specialized chromatographic test methods. The eGFR minimizes the effect of age and sex compared to a solitary creatinine level and, thus, is one of the best indicators of renal function in CKD. There are also formulas that adjust for race. Most of the formulas utilize creatinine or cystatin C, and any potential errors with both the analytes will also be propagated by the eGFR calculation. The limitations of these formulas include that they can only be used in adults greater than 18 years old, and an alternative formula, e.g., Schwarz formula, is a potential eGFR calculator that can be used in a pediatric population, although care may be needed in its application to certain ethnic groups [2]. The KDIGO guideline for CKD based on the GFR is <60 mL/min per 1.73 m² (Table 1) [3].

GFR categories	GFR result
Normal to increased	≥90 mL/min per 1.73 m^2
Mildly reduced	68–89 mL/min per 1.73 m^2
Moderately reduced	30–59 mL/min per 1.73 m^2
Severely reduced	15–29 mL/min per 1.73 m^2
Kidney failure	<15 mL/min per 1.73 m^2

Table 1.

KDIGO-suggested GFR categories [3].

In an effort to standardize a creatinine result in a patient with a known gender and age, the laboratory will provide an eGFR, which provides an estimated GFR based on formulas derived from experimental models. This eGFR is mostly calculated currently using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI), which has the advantage over its predecessor (the Modification of Diet in Renal Disease (MDRD) formula) in that it can be used across the whole range of GFR, while the MDRD formula is applicable to patients with established kidney disease. This is important, as it allows for more accurate stratification of the stage of early kidney disease (stages 1 and 2). Both the formulas have inherent limitations, such as being unable to be applied to pediatric patients (less than 18-year-old) and being inaccurate at the extremes of body size and age, but the CKD-EPI has been shown to be a superior formula for estimating eGFR [4].

The measuring methods are divided into chemical and enzymatic. The chemical methods are primarily based on alkaline picrate, which was first described by Jaffe in 1886; hence, the methods are commonly referred to as Jaffe methods. Creatinine reacts with picrate in an alkaline solution to form the picrate-creatinine complex and has an orange-red color, which is measured at 490–520 nm.

This reaction is not specific for creatinine; hence, many method variations have been introduced over time. The chemical methods started as end point, which are now almost nonexistent, and they have been superseded by kinetic assays in order to minimize noncreatinine chromogens causing interferences. These noncreatinine chromogens have a different rate of color development, allowing for the separation of the rate of color development from creatinine from the interferents. Additionally, the reaction incubation temperature has influence on the reaction of the interferents, and temperatures of 25--41°C have been used to minimize interferences [5]. They are relatively inexpensive and the most widely used, but they are liable to a number of interferents, including: a) positive interferents: ascorbic acid, pyruvate, protein, glucose, creatine, various cephalosporins, acetoacetate, and fluorescein and b) negative interferents: bilirubin, hemoglobin F, and dopamine [6]. The bacterial contamination of urine samples can cause the production of substances that inhibit the Jaffe reaction.

The enzymatic methods are increasingly used in laboratories as they become more economical to improve accuracy, but they are not immune to interferences. Enzymatic methods are typically used in point-of-care (POC) devices [7]. There are three commonly used enzymatic methods: a) creatininase (creatinine amidohydrolase), which is the least used due to poor kinetics, reduced sensitivity, and poor precision; b) creatininase and creatinase are far more frequently used, and to overcome ascorbic acid interference, ascorbate oxidase is included in the reagent; and c) creatinine deaminase (creatinine iminohydrolase) that can be affected by endogenous ammonia in small amounts, for example, from protein deamination and release red cell chromogens when blood is left uncentrifuged at room or higher temperature. The bacterial contamination of urine samples can cause protein deamination.

Isotope dilution-mass spectrometry (IDMS) is the reference method of creatinine measurement. All the Jaffe and enzyme methods are standardized against the IDMS method. High-performance liquid chromatography (HPLC) methods exist, and more recently, a combination of IDMS and HPLC methods has been reported to improve turnaround time and accuracy. These methods are not practical for routine and rapid turnaround usage.

The differences between methods, calibrators, and patient samples (noncommutability) limit the transference of results between analytical methods; thus, it is important that result interpretation is based on the reference ranges provided for the method. This includes not only creatinine results but eGFR results as well.

3. Urinary albumin and total protein

The measurement of urine protein leak in urine is pivotal to the detection, diagnosis, prognosis, and treatment of kidney disease. Albumin is an abundant protein in urine; hence, either total protein or albumin along with creatinine is routinely used as biochemical diagnostic markers. Proteinuria/albuminuria due to diabetes and/or hypertension is an early marker of kidney disease and is a leading cause of chronic kidney disease and cardiovascular incidents. In general, current guidelines recommend the measurement of an early morning random urine collection for albumin-creatinine ratio (ACR). It is calculated by dividing the albumin concentration in milligrams by creatinine concentration in millimoles. Persistent increased albumin in the urine (two positive tests over 3 or more months) is the principal marker of kidney damage.

Although the 24-hour collections have been the “gold standard,” alternative methods for detecting protein excretion, such as urinary ACR, correct for variations in urinary concentration due to hydration as well as provide more convenience than that of timed urine collections. The spot specimen correlates well with 24-hour collections in adults. Australian recommendations were published [8] (Table 2) as part of the standardization process for primary care by a consensus working group, and these recommendations were adopted by Kidney Australia in 2013 on the limits for urinary albumin-creatinine ratio alone. The study estimated that about six million individuals (~25%) have one or more of the major risk factors for CKD and that about 1.4 million (6%) Australian adults have CKD [8]. Soon after this the KDIGO recommendations were adopted.

Sex	ACR for males (mg/mmol)	ACR for females (mg/mmol)	24-hour urinary albumin (mg/day)
Normal	<2.5	<3.5
Microalbuminuria	2.5–25	3.5–35	30–300
Macroalbuminuria	>25	>35	>300

Table 2.

Categories of albuminuria in Australia in 2012.

The most recent KDIGO guideline for CKD based on the markers of kidney damage is that the disease duration must be >3 months, which also suggested new terminology. The suggested limits of albumin and protein excretion rates AER and PER and ACR and PCR values are presented in Table 3 [3].

Albuminuria and proteinuria categories	AER, PER, ACR, and PCR results
Normal	AER <10 mg/d; ACR <10 mg/g (<1 mg/mmol)
Mild	AER 10–29 mg/d; ACR <10 mg/g (1.0–2.9 mg/mmol)
Normal to mild	AER <30 mg/d; ACR <30 mg/g (<3 mg/mmol); PER <150 mg/d; PCR <150 mg/g (<15 mg/mmol)
Moderate	AER 30–300 mg/d; ACR 30–300 mg/g (3–30 mg/mmol); PER 150–500 mg/d; PCR 150–500 mg/g (15–50 mg/mmol)
Severe	AER >300 mg/d; ACR >300 mg/g (>30 mg/mmol); PER >500 mg/d; PCR >500 mg/g (>50 mg/mmol)
Nephrotic syndrome	AER >2200 mg/d; ACR >2200 mg/g (>220 mg/mmol); PER >3500 mg/d; PCR >3500 mg/g (>350 mg/mmol)

Table 3.

KDIGO suggested albuminuria and proteinuria categories.

In our tertiary hospital from 2010 to 2020, there has been 62% reduction in timed protein, 40% reduction in random protein requests, and 72% reduction in timed albumins requests, while there has been 22% increase in random urine requests. Time urine advantage is that it accounts for day night variations as well as positional or exercise variations.

Patients need to be provided with clear instruction for sample collections to mitigate any preanalytical problems, such as avoid collection if there is any bleeding (menstrual or urine), and other physiological contaminations intended (e.g., topping up with water) or unintended.

Albumin in the urine is present in variable structural forms, and it is not always as a single intact polypeptide molecule, but there is fragmented form, and it may include highly carbonylated form. The modification is by proteolysis during passage through the urinary tract, and chemical modification during specimen storage leads to the formation of albumin fragments [9]. The different molecular forms of albumin are not observed in the samples of healthy individuals or from all the patients, but these changes reflect the kidney diseases processes. These patient-specific albumin differences can lead to differences in results between assays based on the technology type of the assay and the specificity of the immunoassay. Albumin methods for urinary albumin can be: a) immunonephelometric using a specialized equipment (nephelometer) and primarily and infrequently used in large specialized laboratories; b) immunoturbidimetric assays are the most commonly used and can be set up on all the general chemistry high-throughput automated analyzers; c) point-of-care assays, where rapid turnaround of results or no laboratory exists; d) HPLC assays; and e) sensor or emerging assays. A review of the Royal Colleague of Pathologist Australasia Quality Assurance programs for 2021 showed that for albumin, 92% of the participating laboratories utilize immunoturbidimetric methods, 4.9% immunonephelometric, and only 3% other methods. For total protein, 49% utilize benzathonium chloride and 49% the pyrogallol red-molybdate methods. Almost all of the testing for urinary proteins and albumin is performed by automated methods on general chemistry analyzers.

Measuring albumin is the preferred method for defining and staging CKD. The urine of a healthy individual contains up to 150 mg of protein in total per day of which approximately 20 mg is albumin.

3.1 Urinary albumin methods

The immunoassay-based methods use either monoclonal or polyclonal antibodies. Currently, the methods are not standardized. The antibodies target specific epitopes on the albumin molecule or fragments. The target antibody epitopes can vary from method to method, and combined with the molecular complexity of urine albumin, this can lead to diminished correlation in results between methods. It is, therefore, of fundamental importance that results should be interpreted with caution if the patient is using more than one pathology service; ideally, the same method is suggested for the best management outcomes. This will minimize misclassifications of the albuminuria category for CKD. The methods are sufficiently sensitive and specific to measure concentration levels down to or below 5 mg/L and total precision of <5% (correlation coefficient), which allows for identifying patients with increased albumin excretion. Immunoturbidimetric methods measure only immunoreactive albumin and are unable to detect fragmented albumin; thus, they can underestimate albumin in biological samples. Most of the pathology laboratories will provide albumin results in less than 3 hours from the receipt of samples.

In contrast, the immunonephelometric assay has showed a marked reduction of 53 ± 4% when carbamylated and glycated albumin was present [10]. Very high albumin concentration in the sample can lead to an underestimation of albumin concentration because it exceeds the antialbumin antibody concentration, a large amount of albumin cannot bind its antibody, and the obtained value is low, which is known as the “hook effect.”

ELISA provides faster results and uses a small amount of antialbumin antibodies; however, competitive ELISA uses only one antibody and is less specific for albumin compared with sandwich ELISA. In sandwich ELISA, two antialbumin antibodies are required, offering better specificity but making the method expensive. ELISA methods are semiautomated and, therefore, not completely free from human analytical error [11].

There are numerous POC strip-based methods now available using different technologies and methodologies such as:

1. Use of immunoturbidimetric reaction.

2. Use of dye reagents on strips for detecting albuminuria (or proteinuria) at the POC.

   Reagent strip-based methods can also be applied for the quantitative estimation of protein or albumin by reflectance spectrophotometry. Result accuracy can be influenced due to interferences from, e.g., high urine protein hematuria, soaps, dyes, and some drugs. In a study, Pugia et al. showed 10–15% false positives when the albumin was <20 mg/L [12].

3. Immunochemical methods applied on the strip using monoclonal enzyme-labeled antialbumin antibodies or gold-nanoparticle-labeled antialbumin antibodies. Alternatively, polyclonal antialbumin antibodies may be used to detect the presence of albumin in the urine sample by the latex agglutination inhibition test [11].

   Dye-binding assay reports albumin result greater than 20 mg/L as positive, whereas immunologically immunoturbidimetry assays report results from 5 to 150 mg/L. Increasingly, the ones that provide quantitative albumin also measure creatinine to allow for the calculation of the albumin-creatinine ratio. If the POC methodology is of high quality and provides accurate quantitative results, it is unlikely that there would be a need to refer positive tests to a laboratory.

4. Currently, there is a considerable interest in developing immunosensors, which detect microalbuminuria without sample dilution. Carboxyl-enriched porous screen-printed carbon electrode is used in an immunosensor-based method to measure the microalbuminuria. It is based on the principle that the voltage gradient of the electrode decreases when antialbumin antibody immobilized on detector surface interacts with albumin in the sample. The quantitative detection is done with chronoamperometry. Its detection limit is 9.7 mg/mL. This method is rapid, sensitive, and highly reproducible [13].

5. Newer systems use a dye-binding-based albumin test strip assay in combination with a complementary metal-oxide semiconductor (CMOS) sensor technology (Sysmex UFC 3500 reader + CMOS). It showed a strong correlation (r = 0.92) compared with immunonephelometric assay with a limit of detection limit being as low as 5 mg/L. The effect of urinary pH on test results was negligible. Carbamylated, glycated, and partially hydrolyzed isoforms of albumin were detected [10].

Unlike immunoassay-based methods, high-pressure liquid chromatography (HPLC) can detect both the fragmented and immunounreactive albumin present in the urine sample, which produces higher results particularly in samples with low concentrations leading to higher rates of patients with microalbuminuria. The transition to use of tandem mass spectrometry and combined with HPLC provides ability to detect very low concentrations of albumin as it takes into account all its physicochemical properties. This provides the ability to establish standards, which will significantly improve the traceability and standardization of methods. Although they are more precise and accurate, however, instrumentation is specialized and costly, which limits their use for routine use. The human serum albumin standard ERM-DA470, which is serum matrix, is the most frequently used standard by manufacturers.

3.2 Total urine protein

There is no recognized reference method for measuring total urine proteins. The mix of different proteins in urine makes it impossible to optimize the current methodologies to provide consistent comparative response to the concentrations of different proteins. The two methods that are equally used are either pyrogallol red molybdate (colourimetric method) or benzethonium chloride (turbidimetric method). Both of these methods are easily adoptable on high-throughput general chemistry analyzers and have similar performance characteristics. The variability in protein composition and other compounds and concentration plus choice of standards has been found to be the factor in comparability between the two methods. Additionally, the concentration of protein in urine taken from patients can vary widely depending on dieting, exercising, and time of the day a patient urinated. In healthy individuals, Tamm-Horsfall (TH) protein (also known as uromodulin) is the most abundant protein in urine (50%), followed by albumin (20%) and immunoglobulin (5%) [14]. Tamm-Horsfall (TH) protein is apparently not significantly detected by these automated assays. Either human or bovine albumin, e.g., NIST SRM 927, is used as standard by manufacturers.

In the pyrogallol method, proteins form colored complexes, and these are spectrophotometrically measured. The presence of Bence-Jones protein can cause negative interference. Positive interference has been reported in patient receiving colloidal-based fillers, but this can be minimized if the reagent has added sodium dodecyl sulfate.

The benzethonium chloride method causes the precipitation of the proteins; turbidity and absorbance are directly related to the concentration of the urine proteins. Positive interferences have been observed in patients taking drugs, e.g., levodopa and methyldopa, while negative interference has been reported with gelatin-based plasma substitutes and one method in patients receiving contrast agent, povidone-iodine (PVP-iodine) solution.

POCT methods use a dye that changes in color. It is recommended that strips should be used to measure albumin not total proteins due to the increased accuracy. Alkaline urine (e.g., infection) can lead to false-positive results. The presence of highly colored urine (e.g., high bilirubin, other colored compounds) can make the reading of the strip more difficult. These strips may not be reactive to Bence-Jones proteins. The use of urine dipstick protein may help in CKD screening, staging, and prognosis.

Routinely, in laboratories, only turbid urines (e.g., urinary infection) are centrifugated to remove insoluble material before sample analysis. Centrifugation is recommended with all the samples for protein analysis [15]. The effect is greater at low protein concentration and the benzethonium (turbidimetric) methods being more affected due to turbidity being removed during centrifugation; hence, protein values near the cutoff ~140 mg/L without centrifugation can lead to false-positive results and subsequently impact on diagnosis [16].

Results must be interpreted against reference or cutoff limits provided in the report. The differences in results among urine protein and albumin methods can be significant and alter risk classification and treatment decisions for individuals with kidney disease.

4. Cystatin-C (serum)

Cystatin C is an alternative to the widely available creatinine in determining renal function with the eGFR. It is a small protein produced by all the nucleated cells, which is freely filtered at the glomerulus. The production rate is relatively constant from 4 months to 70 years and is proportional to the GFR. Its clearance is predominantly renal, and since it is not reabsorbed from the renal tubular lumen, its level is directly proportional to the glomerular infiltrate. The concentration levels are not greatly influenced by height, weight, sex, race, and changes in muscle mass or nutrition [17].

Since creatinine is so widely available, has a lot of guidelines and data reinforcing its use, and is very cheap to run in laboratories, cystatin C has not been widely implemented in most commercial laboratory test catalogs. Its use is mostly confined to confirming renal function in situations where creatinined use is contraindicated, such as reduced skeletal muscle mass or to confirm the presence of a creatinine interferent as an alternative test. In the context of CKD, no clinical advantage has been found in using cystatin C over creatinine except in very exceptional situations [18]. However, some studies do note that cystatin C may be more sensitive in early CKD, where the eGFR is between 60 and 90 mL/min/1.73 m² [19]. In cases where there is suspected renal dysfunction, the cystatin-C-based eGFR should be calculated, since it gives more accurate and less biased estimates than the creatinine-based eGFR, and should be confirmed by the mGFR (iohexol) to reduce systematic or random variation.

Analytically, cystatin C levels are determined with immunoassays, so they are liable to all the same theoretical interferences, such as heterophilic antibodies. The immunoassay technique utilizes turbidimetric or nephelometric principles, and the analytical error can also be caused by analytes causing turbidity, such as hypertriglyceridemia. In vivo cystatin C levels can be increased in corticosteroid use, hyperthyroidism, increased cell turnover, and malignancy; therefore, unexpectedly high results may require these conditions to be ruled out [20, 21]. Finally, results must be interpreted based on the reference ranges provided by the specific method.

5. Urea

Urea (blood urea nitrogen) is a marker of renal function. It is the body’s primary marker of nitrogenous excretion. It is produced by the liver during protein catabolism through the urea cycle, and > 90% is excreted via the kidneys. It is less utilized than creatinine though as a marker of direct renal function, because it cannot be used to accurately estimate the glomerular filtration rate. To be a marker of glomerular filtration, the analyte has to be completely filtered at the glomerulus and not reabsorbed or secreted by the tubules. While urea is completely filtered at the glomerulus, it is able to be excreted, and 40–50% is reabsorbed through the tubules by passive diffusion, limiting its utility as a specific glomerular filtration marker [22]. Furthermore, its excretion in urine can be affected by the urine flow rate, which explains why plasma levels are increased in dehydration, since the reduced urine flow rate allows for more urea to be reabsorbed from the tubular lumen to the interstitial space and ultimately back into the blood. A number of other factors influence urea levels, including protein loading, gut bleeds, catabolism, malignancy (increase urea), starvation, and urea cycle defects (decrease urea) [23].

Urea is analyzed in the laboratory using either a chemical method or an enzymatic method. The chemical method involves the reaction of the urea present in the specimen with diacetyl to form diazine, which absorbs light at 540 nm, which is detected using a spectrophotometer. Chemical methods are no longer in use. The enzymatic method utilizes the specificity of urease for urea, which produces ammonia. The ammonia, then, reacts in an indicator reaction with α-ketoglutarate and the enzyme glutamate dehydrogenase (GLDH), with this reaction oxidizing NADH to NAD⁺, which produces a change in light absorbance at 340 nm [24]. The enzymatic method can be affected by endogenous ammonia, which can be increased in urine specimens with aged specimens and patients with metabolic diseases, including urea cycle defects. CKD is associated with significant elevations in urea; however, its utility in determining renal function has been largely superseded by creatinine and the eGFR.

6. Fibroblast growth factor 23 (FGF23)

FGF23 is a phosphatonin that reduces the level of phosphate in the medium-to-long term. Hyperphosphatemia is a significant problem in chronic kidney disease, and it can be very resistant to treatment. It is postulated that FGF23 is an excellent marker in predicting deterioration in CKD [25]. It acts by binding to α-Klotho, a transmembrane FGF receptor predominantly expressed by the proximal tubules. The effect of FGF23 binding to the α-Klotho transmembrane complex is reduction in the insertion in sodium proximal tubular transporter 2a into the renal tubular membrane, which is involved in renal phosphate reabsorption. In the kidneys, it also reduces calcitriol (1, 25-dihydroxy vitamin D) production by suppressing 1a-hydroxylase and stimulating 24 hydroxylase. This results in less active vitamin D, which limits phosphate absorption from the gut and phosphate reabsorption from the kidney [26]. This would result in a reduced phosphate level in normal physiology.

CKD results in an elevation in FGF23. FGF23 production is stimulated by calcitriol, phosphate, and parathyroid hormone (PTH) [27], the latter two of which are already raised in CKD. Elevations in FGF23 are not specific for CKD, with elevations also occurring in genetic forms of rickets (X-linked, autosomal dominant, and autosomal recessive rickets), oncogenic osteomalacia, and fibrous dysplasia, but these tend to be readily differentiated clinically from CKD and are associated with hypophosphatemia. FGF23 looks to be a particularly useful marker in predicting refractory hyperparathyroidism, with its predictive ability superior to that of the PTH [28]. CKD begins to increase very early in kidney dysfunction, with end-stage renal failure associated with 1000fold increases. In CKD, FGF23 is correlated with endothelial dysfunction, CKD progression, and mortality [29].

FGF23 levels are typically done using immunoassay techniques and, as such, can be affected by the various causes of analytical issues of immunoassays, such as heterophile antibodies and high-molecular-weight interferents that can cause nonspecific binding to a molecularly similar epitope, as well as the theoretical risk of hook effects at very high levels. An FGF23 specific analytical issue is related to the availability of different assays. Some detect the C-terminal peptide (cFGF23), while others detect the whole or intact FGF23 molecule. The difference between these molecules means that results between different assays are not interchangeable and must be interpreted in the context of the quoted reference interval. This difference in molecular FGF23 can be important to consider in renal failure, as iron deficiency, which is also common in relatively common in CKD, can falsely elevate cFGF23, while iFGF23 is unaffected [26].

7. Parathyroid hormone (PTH)

Parathyroid hormone is a principal mediator in calcium and phosphate biochemistry. It is a hormone that is secreted by the parathyroid glands on the back of the thyroid and responds to ionized calcium through the calcium sensing receptor. Hence, clinical units frequently request the estimation of the ionized calcium. The PTH performs a number of actions, including increased bone resorption of calcium and phosphate, increased renal calcium reabsorption, and decreased phosphate renal reabsorption and indirectly increases calcium and phosphate absorption from the small intestine by inducing active vitamin D production (calcitriol, 1, 25-dihydroxyvitamin D) through the induction of 1alpha hydroxylase in the kidneys [26].

In CKD, the regulation of calcium and phosphate is deranged due to the central role of the kidney in the homeostasis of these compounds. The kidneys play a pivotal role in the maintenance of calcium levels by eliminating and regulating solutes that can precipitate calcium out of solution, such as oxalate and phosphate, as well as contributing to the control of the acid–base status. This can lead to calcification throughout the body termed CKD-mineral and bone disorder, renal osteodystrophy, and adynamic bone disease [30]. It is in this setting that the PTH can prove useful in CKD. As the GFR deteriorates, the PTH levels also increase exponentially [31]. This is to compensate for the declining ionized calcium that results because of the reduced loss of oxalate and phosphate. The more the PTH goes up and the longer it does, the more likely of developing tertiary hyperparathyroidism, which is essentially a secondary hyperparathyroidism that has transitioned to autonomous PTH production. However, if the PTH is kept too low, patients are then at risk of developing adynamic bone disease [32]. Thus, CKD patients who are not on dialysis should have their PTH regularly monitored, and vitamin D status assessed and replaced if deficient. If the patient is on dialysis, the PTH should be maintained within two to nine times the upper limit of normal for the assay [33].

PTH is performed in the laboratory using immunoassays. Historically, many PTH immunoassays were susceptible to cross interference with the inactive PTH C-terminal metabolite, which would accumulate in CKD. Current assays are more specific for the intact, biologically active PTH molecule, with the immunoassay antibodies recognizing the biologically active N-terminus [26]. Being an immunoassay, it is liable to all the same interference types common to all immunoassays, such as heterophile antibody interference. Hook effect is rare in modern day immunoassays, but warrants theoretical consideration if a much lower PTH result than expected is produced. Care must be taken to ensure that the specimen is promptly separated from cells and analyzed to avoid in vitro lowering of levels [34].

8. Conclusion

Biochemical analysis is essential to the diagnosis, management, and monitoring of CKD. This chapter has served as a primer to such testing, looking at the analytes and laboratory techniques that commonly make up renal function testing. It has not gone into depth in the hematological testing, endocrine testing, or dynamic function testing commonly employed in CKD management. However, even routine renal function testing warrants vigilance in interpretation. The laboratory is a suitable place to assist in these matters.