The role of laboratory testing in detection and classification of chronic kidney disease: national recommendations

Chronic kidney disease (CKD) is a common clinical condition with significant adverse consequences for the patient and it is recognized as a significant public health problem. The role of laboratory medicine in diagnosis and management of CKD is of great importance: the diagnosis and staging are based on estimation of glomerular filtration rate (eGFR) and assessment of albuminuria (or proteinuria). Therefore, the joint working group of the Croatian society of medical biochemistry and laboratory medicine and Croatian chamber of medical biochemists for laboratory diagnostics in CKD issued this national recommendation regarding laboratory diagnostics of CKD. Key factors for laboratories implementing the national guidelines for the diagnosis and management of CKD are: 1. Ensure good communication between laboratory professionals and clinicians, such as nephrologists or specialists in general/family medicine, 2. Ensure all patients are provided with the same availability of laboratory diagnostics, 3. Ensure creatinine assays are traceable to isotope dilution mass spectrometry (IDMS) method and have minimal bias and acceptable imprecision, 4. Select the appropriate GFR estimating formula. Recommended equation is the 2009 Chronic Kidney Disease Epidemiology Collaboration (CKD – EPI) equation, 5. In reporting the key laboratory tests (creatinine, eGFR, urine albumin-to-creatinine ratio, urine protein-to-creatinine ratio) use the appropriate reporting units, 6. Provide adequate information on limitations of creatinine measurement. The manuscript has been organized to identify critical points in laboratory tests used in basic laboratory diagnostics of CKD and is based on the Kidney Disease: Improving Global Outcomes (KDIGO) 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease.


Introduction
Chronic kidney disease (CKD) is a common clinical condition with significant adverse consequences for the patient. It is recognized as a significant public health problem throughout the world (1).
Many publications report the prevalence of CKD in the general population, however there are considerable variation in methods for sampling general population and assessment of kidney function across studies (2). This makes comparison of studies rather difficult, however worldwide prevalence of adult CKD is about 10%, reaching up to 50% in high-risk population (3). Late recognition and diagnosis of disease inevitably leads to kidney failure (1). In this case the only possible therapeutic measure is dialysis or transplantation in health care systems where such treatment is available. In those countries, where access to dialysis and transplantation service may be limited or unavailable, the final consequence of progressive CKD is death. Earlier stages of kidney disease are often asymptomatic and are usually discovered through various comorbid conditions, and may be reversible. It is of great importance, due to right time treatment and improving the quality of life of patients with CKD, but also because of the significant financial savings, to identify disease at an early stage where it is still possible to stop or slow down progression (1). Although, to this point, there had not been official complete epidemiological studies conducted regarding CKD (prof. Mirjana Sabljar Matovinović, personal communication), the availability of treatment in Croatia includes both, dialysis and transplantation (4).
In 2002 the US Kidney Disease Outcomes Quality Initiative (KDOQI) group published the Clinical Practice Guidelines for Chronic Kidney Disease: Evaluation, Classification, and Stratification. Update of these guidelines and recommendations: Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease was released in 2012 under the direction of Kidney Disease: Improving Global Outcomes (KDIGO). Our national recommendations for laboratory diagnosis of chronic kidney disease are mainly based on the 2012 KDIGO guidelines, and written permission was obtained to reproduce the parts of the KDIGO guidelines. KDIGO guidelines are the product of cooperation of a large number of international experts who created recommendations, among other things, to be used for good laboratory practice in the diagnosis and management of CKD.
Chronic kidney disease is defined as an abnormality of kidney structure or function with implications on the health of an individual, and it is present for more than three months. Chronic kidney disease is a general term for heterogeneous disorders affecting kidney structure and function with variable clinical presentation (1). Rational approach to the diagnosis and evaluation of CKD involves simultaneous assessment and monitoring of renal function (through estimated glomerular filtration rate (eGFR), serum creatinine) and kidney damage (albuminuria and/or proteinuria) ( Table 1).
One of the prominent criteria for the diagnosis of CKD is decreased GFR value (< 60 mL/min/1.73m 2 ). GFR is widely accepted as the best index of kidney function. The normal value in young adult men and woman is approximately 125 mL/min/1.73m 2 . Values below 15 mL/min/1.73m 2 indicate kidney failure and the person can be identified as a candidate for dialysis or renal replacement therapy/kidney transplantation (1). The role of laboratory medicine in diagnosis and management of CKD is of great importance because a very simple test can identify people who are at risk of developing CKD. All that is required is measuring the concentration of serum creatinine and reporting of eGFR, using the available predictive equations.
For an initial assessment of proteinuria the following measurements are recommended (in descending order of preference): 1. urine albumin-to-creatinine ratio (ACR), or 2. urine protein-to-creatinine ratio (PCR). In all cases an early morning urine sample is preferred, but does not exclude spot urine samples. Measuring the concentration of urine albumin is preferred to measuring the concentration of urine total protein. Use of urine albumin measurement as the preferred test for proteinuria detection will improve the sensitivity, quality, and consistency of the approach to early detection and management of kidney disease. Albumin (or total protein) concentration in the urine sample should be reported in proportion to the concentration of creatinine (ACR or PCR) in the same sample to minimize the influence of the patient's hydration and the concentration of the urine sample. The reporting of the results is the same for both first morning and spot urine samples, respectively. A positive finding of albuminuria in a random sample of urine needs to be confirmed in the next morning void urine. If a more accurate assessment of albuminuria (or total proteinuria) is required, it is recommended to measure albumin excretion rate (AER) or total protein excretion rate in a timed urine sample (1,5,6). The choice of a suitable timed sample type should be of a laboratory manager. Adequate sample type, time of collection and instructions for patients should be provided by institution.

Markers
Some of the other laboratory criteria for diagnosing CKD include urine sediment abnormalities as markers of kidney damage (Table 1). This may include some formed elements, such as renal tubular cells, red blood cells (RBC) casts, white blood cell (WBC) casts, coarse granular casts, wide casts and large numbers of dysmorphic RBCs. Abnormalities of electrolytes (Table 1) may result from disorders of renal tubular reabsorption and secretion. These syndromes are uncommon but pathognomonic of kidney disease (1).
Grading of CKD is based almost exclusively on two laboratory parameters: eGFR (GFR categories 1 to 5 (G1 -G5)) and albuminuria (albuminuria categories 1 to 3 (A1 -A3)) ( Table 2). It is also used for the prognosis of progression of the disease (1).
Depending on the category, patients are classified as low-risk patients, highlighted in green, moderate risk (yellow), high risk (orange) and very high risk patients (highlighted in red).
CKD testing using eGFR and ACR should be offered to people with any of the following risk factors: • diabetes • hypertension • acute kidney injury • cardiovascular disease (ischaemic heart disease, chronic heart failure, peripheral vascular disease or cerebral vascular disease)

Background
Recently, it was shown that laboratory diagnostics of chronic kidney disease in Croatia is not standardized (8). There is a large heterogeneity among Croatian medical biochemistry laboratories regarding creatinine methods and used reference intervals and types of preferred samples for urine albumin (or protein). The most important issue that occured is the fact that laboratories still use non-standardized methods for creatinine results and do not report eGFR values. Also, the majority of laboratories do not measure urine albumin, especially in primary care health setting (8). These facts set the background for the process of standardization and harmonization in this area of laboratory medicine. These national guidelines, based on the relevant 2012 KDIGO Guideline (1), represent the first step in accomplishing this goal.
Key factors for laboratories implementing the national guidelines for the diagnosis and management of CKD are:  5. In reporting the key laboratory tests (creatinine, eGFR, urine albumin-to-creatinine ratio (ACR), urine protein-to-creatinine ratio (PCR)) use the appropriate reporting units, 6. Provide adequate information on limitations to creatinine measurement (9).

Recommedations
The national recommendations are mainly based on the KDIGO 2012 guidelines, however, novel literature findings are also incorporated. Our main goal was to provide recommendations that can be easily applied in every medical biochemistry laboratory in Croatia. The draft of the recommendations was sent to numerous national and international experts for their comments. The manuscript was also made available for public consultation. All comments were carefully considered and incorporated into the final version of the recommendations.
The document consists of four main parts with corresponding subheadings: The manuscript is organized to identify critical points in four major laboratory tests used in basic laboratory diagnostics of CKD. It is rather difficult to give unique and uniform recommendations, regarding a large heterogeneity amongst methods and populations. Our intention was to point out to some weak points in pre-and analytical phase, but every laboratory must set their own specifications for method performance and handling the specimens, according to their possibilities and conditions.
An easy-to-follow step-by-step approach in implementation of the recommendations is shown in Appendix 1.
To ensure the better flow of information in implementing national guidelines laboratories can use the provided template (Appendix 2) (10).

. Creatinine
An important limiting factor in the use of predictive equations for GFR estimation is an accurate method for determining serum creatinine concentration.

.. Preanalytical phase
There are numerous well known preanalytical variations that affect serum creatinine concentration which are listed in the Table 3.
The majority of listed variations are non-controllable, however both laboratory professionals and clinicians should be aware of those limitations. The laboratory professionals are referred to previously

Non-controllable variations
Non-steady state AKI Laboratory professionals and clinicians should be aware of listed limitations.
For a better flow of information please consult the Appendix 2.  Table 3. Sources of errors in GFR estimation using creatinine mentioned Appendix 2 which will be of assistance in communication with patients, as well as with the ordering physicians. For a minor part of variations that can be controlled regarding sample quality, laboratory professionals are referred to the published national recommendations for venous and capillary blood sampling (11,12).

Stability
Creatinine, in a non-separated serum sample, which is in contact with a blood clot, is stable for 24 hours. In a separate serum sample creatinine is stable for 7 days at room temperature (20-25 °C), or stored in a refrigerator (2-8 °C). Serum samples stored at -20 °C are stable for 3 months with 10 freeze-thaw cycles (13).

.2 . Analytical phase
To universally implement eGFR based on serum creatinine measurements, standardization of routine serum creatinine measurements is required. For the measurement of serum creatinine, laboratories should use the recommended method with calibration traceable to international standard reference material and minimal bias compared to the IDMS method (1).
Desirable and optimal specification for imprecision, bias and total error according to biological variation database (14,15) are shown in Table 4.
Recommended methods for serum creatinine measurements in Croatia are: photometric compensated Jaffé method traceable to IDMS method and enzymatic method traceable to IDMS method and The National Institute of Standards and Technology Standard Reference Material (NIST SRM) 967 for creatinine in serum (16). However, there is emerging evidence that enzymatic creatinine assays lead to less variability in measurements of serum creatinine and are preferably used in clinical practice in order to generate more reliable GFR estimates (17)(18)(19).
Standardization (compensation) of photometric Jaffé method involves changing the values of calibrators in terms of traceability to the IDMS method and change of corresponding intercept (or factor B, depending on the analyzer). Considering the fact that there are many creatinine assays available that may not be IDMS traceable, and that for assays which may be IDMS traceable, the information supplied does not make this clear to the user (20), for new values of calibrators, controls and intercept, laboratory professionals should contact the person in charge of applications from companies providing reagents, controls and calibrators or whose analyzer is on which creatinine is measured. The list of available creatinine assays, as well as traceability information, is given in the Appendix 3. However, laboratory professionals should be aware that this list is susceptible to changes and they should always be correctly informed by the latest Information for use (IFU) leaflet.
Standardization of calibration does not correct for analytical interferences. The enzymatic assays may be less influenced by non-creatinine chromogens  compared to the Jaffé assays (21,22), but no procedure was unaffected. The most common analytical interferences are caused by endogenous substances: high bilirubin concentration, glucose, proteins, pyruvate, β-hydroxybutyric acid, low albumin, as well as many drugs (cephalosporins, dobutamine, lidocaine) (23). High bilirubin concentrations may interfere with the Jaffé method, where the assay absorbance is near the bilirubin absorbance peak of ~456 nm. Jaffé reaction may also be affected by lipemia and/or haemolysis. Haemolysed samples that contain fetal haemoglobin (HbF) interfere with the Jaffé reaction, and it is possible to obtain negative creatinine results (24). Management of lipemic samples was extensively explained in the review by Nikolac et al. (25).
The influence of interfering substances is greater at creatinine concentrations within reference range than at higher concentrations. Magnitude and direction of bias in creatinine concentration depends on the details of implementation of the method principle (26). The influence of interfering substances is less frequent with the enzymatic procedures, however no procedure is unaffected and is method and analyzer dependent (21,27). Interference (endogenous or exogenous) if unrecognized lead to false laboratory result and consequently to incorrect diagnosis. To systematize corrective actions, as a part of the total quality system, when interference appears first step must be manufacturer´s method specification in which are listed interference studies conducted by the manufacturer (28). However, it was shown that there are serious discrepancies between manufacturer's declarations and measured data (29). Each laboratory should verify the data declared by the manufacturer and define its own acceptability criteria (30).
Because urine contains relatively little or no protein, both enzymatic and Jaffé method are suitable for urine creatinine measurement (1).

.3 . Postanalytical phase
When reporting serum and urine creatinine concentrations obtained by a standardized assay, laboratories should use revised reference intervals published by Croatian chamber of medical biochemists (CCMB) in 2010 (31,32) which are shown in Table 5.
The applicability of the recommended "common" reference intervals in all Croatian laboratories measuring serum creatinine concentrations were confirmed in the study conducted by Flegar-Meštrić et al. (33). This fulfils the prerequisite for implementation of international guidelines for early diagnosis and prediction of progression of chronic kidney disease using glomerular filtration rate CKD-EPI estimating equation (34,35).

.4 . Pediatric considerations
Children show lower reference ranges for total protein, thus the protein error in Jaffé method is considerably smaller, which, in consequence, with restandardized Jaffé-type assays, could lead to negative values in children with a decreased muscle mass (36). Therefore, the only recommended method for the measurement of serum creatinine in pediatric patients (individuals younger than 18 years) is enzymatic assay (37).
The persisting problem of pediatric reference intervals has been substantially reduced with the establishment of a new comprehensive database of pediatric reference intervals as a part of the Canadian Laboratory Initiative in Pediatric Reference Intervals (CALIPER) study (38,39). It should assist laboratorians and pediatricians in interpreting test results more accurately. There are already some transference studies with other analytical platforms and local populations, as recommended by the CLSI (40,41). Laboratory professionals should also be aware of the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) Pediatric Reference Range Initiatives with many useful information on this delicate topic (42).

.1 . Equations
The recommended equation for GFR estimation in adult population (≥ 18 years) is CKD-EPI equation (1,43,44). The equation includes four variables: serum creatinine concentration, age, sex and race ( Table 6) (34). Although all equations for GFR estimation are essentially mathematical abstractions that relate patients to the populations from which the equations were derived (45), there is growing body of evidence that CKD-EPI equation is superior in general population (5,46), as well as in diabetic patients (47)(48)(49).
In situations where GFR estimating equations are limited including extremes of body size and age, conditions after limb amputation, pregnancy, severe malnutrition or obesity, muscle wasting diseases, paraplegia and quadriplegia, vegetarian diet, rapidly changing kidney function, when determining eligibility for kidney donation or adjusting dosage of toxic drugs that are excreted by the kidneys and in research projects in which estimating glomerular filtration rate is a primary goal, GFR should be measured using standardized creatinine clearance measurement (1,50).

.. Preanalytical phase
eGFR may be falsely decreased after a meal of high meat content, as blood creatinine concentration increases after meal intake (51).
Blood creatinine is predominantly derived from muscle, eg. a muscular young man may have increased serum creatinine concentration and a falsely lowered eGFR. eGFR increases by 2.3 mL/ min/kg of lean mass in healthy men (52).
Low eGFR finding should be confirmed with a repeated sample taken after avoidance of meat for at least 12 hours. Spurious causes of low eGFR, such as high muscle mass, should be considered.
Additionally, a low eGFR should prompt a check for proteinuria assessment (51).

.3 . Postanalytical phase
Diagnosis, prognosis prediction and progression of CKD using eGFR is not based on comparing the values to population based reference intervals but on diagnostic values defined as categories in classification system shown in Table 2. Implementation of equations listed in  Based on the biological and analytical variation of serum creatinine, the reference change value (RCV) for eGFR is about 14%.

.4 . Pediatric considerations
For estimation of GFR in pediatric population we recommend Schwartz equation with obligatory use of enzymatic assay for the measurement of serum creatinine concentration (54,55). The equation is applicable for children from 1 to 18 years old.
Routine calculation of eGFR is not recommended in children and youth (5). Every eGFR result calculated by Schwartz equation above than 75 mL/ min/1.73 m 2 should not be reported as a whole number but as "> 75 mL/min/1.73 m 2 ".
In children younger than 2 years of age with CKD, the GFR categories as per the adult in Table 2

.1 . Preanalytical phase
Albumin intra-and inter-individual biological variation are important factors for selecting appropriate urine sample, for the interpretation of the confirmation results and for assessing clinically signifi-cant difference in albumin concentration. ACR in the first morning urine has a significantly lower intra-individual variation, compared to the albumin concentration in 24-hour urine. This represents an important fact considering the pitfalls in collecting 24-hour urine samples (56). Table 7 presents the factors affecting urinary ACR (1,57).
The majority of the variations listed are non-controllable, however both laboratory professionals and clinicians should be aware of those limitations. Controllable variations include obtaining an adequate urine sample. The patient should be adequately prepared and told why a urine specimen requires to be examined. Instructions on how it should be collected should be given, ideally both orally and in written form, following the national recommendations issued by CCMB about standards of good laboratory practice in obtaining adequate urine samples (58). Biological (in vivo) factors, changing the true concentration of a measured component, cause problems in the interpretation of laboratory results, although the measurement process itself is correct. They are called influence factors and patients should be adequately explained about possible interferences (59).

Stability
Albumin is stable in urine samples without preservatives at least one week when stored at 2-8 °C.
For an extended period it is recommended to freeze the sample at -80 °C, without centrifugation or filtration. After sample thawing, possible precipitates can be easily removed by dissolving the sample at 37 °C. Blurry urine samples should be centrifuged (60).

.2 . Analytical phase
Recommended methods for the measurement of urine albumin are immunochemistry assays, specific and precise at low albumin concentrations, that produce quantitative results in clinically relevant range. Albumin is mainly measured using turbidimetric assays. Currently there is no reference measurement procedure or standardized reference material recommended by the Joint Committee on Traceability in Laboratory Medicine (JCTLM). At the moment the LC-IDMS method developed at the Mayo Clinic Renal Reference Laboratory is under validation at the National Institute for Standards and technology (NIST) before being submitted to the JCTLM for listing (61 (1), however the NIST SRM 2925 containing pure albumin intended for calibration of LC-IDMS is now available. The commutability assessment of the NIST SRM 3666 containing albumin in frozen human urine intended for calibration of routine measurements procedures is at the moment under investigation (61).
Since the results are expressed in proportion to the concentration of creatinine, urine creatinine should be measured in the same urine sample.
Desirable and optimal specifications for imprecision, bias and total error according to biological variation database (14) are shown in Table 4.
Samples with very high albumin concentration may give falsely low or normal results due to the prozone effect. In this case it is necessary to repeat the analysis after dilution of the sample.
The main causes of variation in urine albumin measurement are outside the analytical process, in preanalytical (as described in the previous subheading) and postanalytical phases (different units, different cut-off values, different ways of reporting the results). Other causes of variation are different forms of albumin in urine, which are significantly different from each other even between healthy individuals. Urine albumin is exposed to modifying factors such as wide range of pH and ionic strength, high concentrations of urea, glucose and ascorbate, and cleavage by peptidases (62).

.. Postanalytical phase
The presence of albumin in urine should be expressed as categories of classification system shown in Table 2. The term microalbuminuria is no longer recommended.
The results should be reported as ACR expressed as mg/mmol. If the presence of albumin in urine is measured as AER results should be reported using the units mg/24 hours with reference interval < 30 mg/24hours, independently of sex and age.

.4 . Pediatric considerations
There is no set standard encompassing all children with respect to the normal range of urinary protein (or albumin) excretion. Values vary across age, sex, For a better flow of information please consult the Appendix 2.
Non-renal causes of variability in creatinine excretion Age (lower in children and elderly) Race (lower in Caucasians than black people) Muscle mass (lower in people with amputations, paraplegia, muscular dystrophy) Gender (lower in women)

Preanalytical storage conditions
Degradation of albumin before analysis Albumin is stable in urine samples without pre ser vatives at least one week when stored at 2-8 °C.
For an extended period it is recommended to freeze the sample at -80 °C, without centrifugation or filtration. After thawing of the sample, possible precipitates can be easily removed by dissolving the sample at 37 °C. Blurry urine samples should be centrifuged.

Degradation of total protein before analysis
The proteins are susceptible to bacterial degradation at room temperature. Analysis should be performed as quickly as possible. Samples may be stored for up to 1 week at + 4 °C, for longer storage frozen at -20 °C or at -80 °C. Samples should be dissolved at 37 °C to prevent degradation of proteins and after homogenizing, samples should be centrifuged prior to analysis ACR -albumin-to-creatinine ratio. PCR -protein-to-creatinine ratio. AKI -acute kidney injury. CCMB -Croatian Chamber of Medical Biochemists.  Table 7. Factors affecting urinary ACR and PCR puberty, the presence of obesity (high BMI) and may be modified by exercise, fever and posture (1).
In children with CKD any expression of abnormal urinary protein excretion may utilize proteinuria in place of albuminuria. Children older than 24 months of age are expected to achieve normal (adult) protein values (1).

.1 . Preanalytical phase
Filtered serum proteins, proteins derived from the kidney and urinary tract make normal urine protein content (63). Their appearance is influenced by renal, pre-and postrenal conditions (64). Urine as a body fluid for clinical analysis is relatively stable, probably due to the fact that it is "stored" for hours in the bladder; hence, proteolytic degradation by endogenous proteases may be essentially complete by the time of voiding (65).
Total protein in urine may be increased after heavy exercise, dehydration, very high protein intake and emotional stress (66). Vaginal and urethral secretions can produce false positive, and diluted urine specimens can give false negative protein results (67). Because urine albumin is predominant protein in most proteinuric kidney diseases, all factors affecting urinary ACR also affect PCR (Table 7).

Stability
Proteins are susceptible to bacterial degradation at room temperature. Analysis should be performed as quickly as possible. Samples may be stored for up to 1 week at + 4 °C, for longer storage frozen at -20 °C or at -80 °C. Samples should be dissolved at 37 °C to prevent degradation of proteins and after homogenizing, samples should be centrifuged prior to analysis (68).

.2 . Analytical phase
There is no recommended method for measuring of total protein in the urine. The majority of laboratories use turbidimetric or colorimetric assays. These methods do not have the same analytical specificity and sensitivity for all proteins. Most methods reacts more strongly with albumin than with globulins and other non-albumin proteins.
There is no reference method and no standardized reference material for urine protein recommended by JCTLM. Different methods and calibrators lead to significant between-laboratory variation. It is difficult to define a standardized reference material since a variable mixture of different proteins is measured (1).
Desirable and optimal specifications for imprecision, bias and total error according to biological variation database (14) are shown in Table 4.

.3 . Postanalytical phase
Results of total urine protein measurement should be reported as PCR using the units mg/mmol (Table 8).

Recommendations
1. Measure albumin preferably in a morning urine specimen.
2. Measure urine creatinine in the same urine sample.
3. Express the results as albumin-to-creatinine ratio (ACR) in recommended units (mg/mmol). 4. A positive finding of albuminuria in a random sample of urine needs to be confirmed in the next morning void urine.
5. If a more accurate estimate of albuminuria is required, it is recommended to measure albumin excretion rate (AER), with reference interval < 30 mg/24hours, independently of sex and age.
6. In children with CKD proteinuria should be preferred over albuminuria, especially in children < 2 years of age.
7. Adult values for AER and ACR apply for children older than 24 months of age.   2. Measure urine creatinine in the same urine sample.
3. Express the results as protein-to-creatinine ratio (PCR) in recommended units (mg/mmol).

4.
A positive finding of proteinuria in a random sample of urine should be confirmed in the next morning void urine.
5. In adults, for a measure of protein excretion rate (PER) apply reference interval < 150 mg/24hours, independently of sex and age. 6. In children, the quantification of total protein, as compared to the albumin only fraction, may be preferred method.
Measurement of PCR to total protein concentration, in initial assessment of proteinuria, is to overcome variation in urine concentration and dilution (63).

.. Pediatric considerations
Neonates and young infants/children are both expected and allowed to have higher urinary losses of both glomerular and tubular proteinuria due to lack of maturation in the proximal tubular reabsorption of proteins. In children the quantification of total protein, as compared to the albumin only fraction, may be preferred method (1).
The normal ranges for albuminuria and proteinuria in children are shown in Table 9.

Conclusion
There are many issues that need to be resolved in the laboratory diagnostics of CKD in Croatia (8). Although there were many potential biomarkers suggested for the early diagnosis of CKD (69,70), considering the issues that were raised via the conducted survey (8), we need to approach the Croatian medical biochemistry laboratories at the very basic level.
The principal clinical purpose of assessing a patient's renal function is to anticipate complications, enabling better screening and treatment decisions. Determining with great accuracy a certain physiologic parameter -actual GFR -is a less important goal (71) and inexpensive, easy and accurate measurement of serum creatinine could lead to reduction in the global burden of CKD (3). In connection to this, the very first goal is to introduce standardized assays for creatinine measurement and eGFR reporting in all medical biochemistry laboratories. The second goal is to harmonize the choice of the sample for ACR/PCR measurement and the reporting units, consequently.
The future perspectives include education in implementing the recommendations and conducting tho follow-up survey to observe the completeness and identifying "weak spots" of the recommendations implementation process. The obtained data will be a starting point for the second edition of the recommendations.
In conclusion, reporting the results of laboratory tests for the diagnosis of CKD should be aligned with the adopted general recommendations with the applicable reference intervals, diagnostic value, and the source is acknowledged criteria. An example of the recommended reporting in laboratory diagnostics of CKD is shown in Table 10.

Question Answer
What is a standardized creatinine?
Standardized creatinine is a result of the determination of serum creatinine by method (enzymatic or compensated Jaffé) calibrated with standard NIST SRM 967th Why we introduce a standardized creatinine?
Generally: By standardization of creatinine we compensated systematic analytical error in current non-specific methods, which allowed standardization and enabled global comparability of results of serum creatinine and application of uniform reference intervals. Specifically: Standardized creatinine is a prerequisite for the implementation of international guidelines for the early diagnosis and monitoring progression of chronic kidney disease in risk populations (hypertension, diabetes, etc.).