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in the distal and collector tubules (renal NGAL pool); at the
same time, AKI induces an increase in NGAL synthesis also
in organs such as the liver and lungs (systemic NGAL pool)
and, for this reason, in the early stages of AKI, NGAL increases
both in the circulation and in the urine. Both clinical and
experimental studies in animal models and in human cells
invitro have unequivocally demonstrated that, just 3–6h after
the onset of ischemia or anoxia, tubule cells express amounts
of NGAL increased by approximately 10-fold from the baseline. Unfortunately, NGAL is affected by some issues that
have so far limited its use in clinical practice. First, NGAL is a
protein expressed by various tissues and organs, as highlighted
above, and, therefore, can increase in circulation even in many
extrarenal pathologies. Clinical pictures such as systemic
infections and sepsis are associated with increased NGAL due
to hyperactivity of neutrophil granulocytes. Moreover,
“noxae” of the tubule, such as ischemia, anoxia, oxidative
stress, etc., cause not only the increased synthesis of NGAL by
tubular cells but also the recall of inammatory cells and,
more specically, of neutrophil polymorphonucleates, which,
in turn, express NGAL. Therefore, the amount of NGAL
detectable in the tubular interstitium, and, thus, also in the
urine, derives from both the tubule and the neutrophils. There
are three molecular isoforms of NGAL: monomers
(MW≅25kDa), dimers formed by the binding of two monomers (MW≅45kDa), and heterodimers formed by the binding of a monomer to neutrophil matrix metalloproteinase-9 or
gelatinase (NGAL-MMP-9, MW≅135kDa). Monomers and
dimers are expressed and released by neutrophils, whereas
monomers and heterodimers are isoforms expressed and
released by renal tubule cells. It follows that a heterodimer can
be considered tubule-specic, and its presence in the urine or
in the circulation identies release by tubular cells, excluding
the share of NGAL from neutrophils or other cell types or tissues. Since 2010, automated methods for the determination of
NGAL also in urine have been developed, which allow the test
to be introduced in clinical laboratories on an urgent/emergency basis. Unfortunately, these methods use mixtures of
antibodies that recognize the epitopes common to the three
isoforms, and this prevents the differentiation of the isoform
corresponding to the heterodimer. In a restricted population of
healthy adult subjects (n= 174), the 95th percentile urinary
NGAL concentration was 107μg/L; distinguishing the population into men (n=100) and women (n=74), the 95th percentile was 91 and 129μg/L, respectively. To reduce intra- and
interindividual variability, it is recommended to express
NGAL in urine in relation to creatininuria.
Proteinuria
An organic nephropathy is almost always characterized by
the presence of proteinuria, which, in many cases, is the earliest sign of renal impairment. Most of the proteins present in
the urine are of plasma origin; however, if proteinuria originates from renal parenchymal lesions, secondary to ischemic, hypoxic, or toxic insults, proteinuria may also be
represented by tissue proteins of the kidney and urinary tract.
Of particular interest is the Tamm–Horsfall protein (THP), or
uromodulin, a glycoprotein with immunosuppressive action
secreted by the ascending tract of Henle’s loop: its average
urinary excretion value is around 20–200mg/day. THP has a
MW of 85 kDa and is found in the urine in polymerized
form, in aggregated subunits; it has an important role in the
formation of urinary cylinders and shows antiviral activities,
but, due to the high content in carbohydrates, it escapes the
common methods used for the measurement of total urinary
proteins. Recently, the role of THP in CKD and hypertension
has been reevaluated, especially as a risk factor. It has also
been shown that high THP concentrations reect good renal
reserve and are inversely correlated with the risk of GFR
decay and AKI.Other urinary proteins of renal origin are
protein 1, or Clara cell protein (CC16; PM=15.8kDa), and
tissue enzymes, such as urokinase, an enzyme with antibrinolytic activity, as well as other enzymes, such as alanine
aminopeptidase (AAP; E.C. 3.4.11.2) and N-acetyl-β-D-
glucosaminidase (NAG; E.C. 3.2.1.30), whose increase in
urine is of particular interest in the diagnosis of toxic
nephropathy and, more generally, of tubulointerstitial nephritis. From a general point of view, proteinuria can be classied into physiological, transitory (intermittent or functional),
and associated with nephropathies. There is a general agreement in dening “physiological” a content of urinary proteins in healthy adults not higher than 150–200 mg/
die/1.73m
2
of body surface (about 100–150mg/L). Transient
proteinuria is the nonconstant presence of protein in the
urine. This type of proteinuria is also dened as functional,
and it is essentially related to transient hemodynamic alterations (renal blood ow), which affect the reversible increase
in glomerular permeability both in certain physiological situations, such as prolonged physical activity or heavy exertion,
pregnancy, and intense cold, and in pathologies involving
extrarenal organs and apparatuses, such as fever and hyperthermia, emotional stress, noradrenaline infusion, heart failure, and prolonged hyperlordotic posture, typically
characteristic of orthostatic proteinuria. When the concentration of proteinuria stably exceed (at least three successive
determinations) the threshold of physiological proteinuria,
we talk about pathological or overt proteinuria. In the presence of proteinuria, it is absolutely necessary to establish its
entity and composition. According to its composition, proteinuria can be classied into glomerular, tubular, mixed, and
overow proteinuria.
Glomerular proteinuria consists mainly of plasma proteins of PM >60kDa. The term “selective proteinuria” indicates a glomerular proteinuria consisting almost exclusively
of proteins with a MW between 60 and 150kDa. The selectivity of glomerular proteinuria positively correlates with the
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ability of the glomerular lter to retain proteins with high
and very high MWs (>150kDa), distinguishing them from
those with lower MWs. “Non- nephrotic glomerular proteinuria” refers to the excretion that does not exceed 3.5g/day;
above this limit, we talk about “nephrotic proteinuria,” which
is usually accompanied by the clinical and humoral manifestations of the nephrotic syndrome: edemas, hypoalbuminemia, and increased globulins with the simultaneous presence
of hypogammaglobulinemia. In these cases, protein loss can
be marked (5–10g/day), even reaching massive levels (more
than 40g/day). Tubular proteinuria consists of plasma proteins of low MW or microglobulins (MW ≤50kDa). Mixed
proteinuria results from the presence of high- and low-MW
plasma proteins and proteins and enzymes of renal origin. It
is predominantly found in conditions of impaired glomerular
and tubular function. Finally, proteinuria due to overload,
also called extrarenal proteinuria, is constituted by low-MW
plasma proteins that are not normally present in the circulation or at most are present in low concentrations. In this case,
the presence of extrarenal pathologies characterized by specic pathological mechanisms affecting organs and tissues
(proliferation, cytolysis, etc.) causes an abnormal increase of
proteins in the plasma and in the ultraltrate; this increase
quickly saturates the tubular reabsorption mechanisms, causing their appearance in the urine. Typical examples include
myoglobin, and immunoglobulin free light chains.
The approach to the study of proteinuria is developed
through three phases: research, measurement, and characterization of the composition of proteinuria. The presence of
proteinuria must be ascertained in all cases of suspected
nephropathy or, in the absence of signs and/or symptoms, in
all cases of hematuria, urinary tract infection, etc. If the presence of proteinuria is ascertained, it should always be measured to ascertain its extent. Finally, the characterization of
the composition of proteinuria is an important evaluation
criterion for the diagnosis and clinical classication of renal
pathology. For these reasons, the methods can be schematically divided into qualitative and quantitative methods.
Qualitative methods, such as dry chemistry methods (dipstick, see the section “Standard Urine Test”) and electrophoretic separative methods (solid support or liquid phase,
namely capillary), allow the detection and characterization
of proteinuria. Separative electrophoretic methods are the
only ones able to highlight the morphology of the free light
chains, allowing the diagnosis of Bence Jones proteinuria in
the case of monoclonality. Quantitative methods allow determining the concentration of total proteins and of the main
urinary specic proteins. In particular, the determination of
total proteins in urine has several critical issues that have not
yet been resolved, mainly due to the poor standardization of
methods and numerous analytical interferences. The direct
colorimetric methods constantly show, even if in a pronounced form, a limitation given by the different afnity of
the dye for the different protein structures. It follows that in
a urine sample, the prevalence of a certain type of protein
structure rather than another (globulins rather than albumin,
or vice versa, etc.) inevitably leads to overestimates or underestimates of the real protein content, with poor accuracy of
the determination. To overcome this serious problem, the use
of sodium dodecyl sulfate (SDS) was introduced, to be added
in a dened amount to the reaction mixture containing the
dye. The use of SDS increases the linearity range of the colorimetric method. To date, there is no denitive consensus on
the choice of reference method for the determination of total
urinary proteins: one of the few recommended methods for
urinary proteins is the biuret colorimetric method after gel
ltration, identied as the selected method. However, other
recommended methods are affected, to a greater or lesser
extent, by pitfalls related to interferences, protein pattern
composition, etc. However, it is desirable that a good method
can achieve wide ranges of linearity, allowing accurate measurements of protein concentrations between about 0.025
and 4.0g/L.
Albuminuria
Plasma albumin is normally ltered in very low amount by
the glomerulus, and then more than 99% of it is reabsorbed
by the cells of the proximal convoluted tubule. The term
albuminuria refers to the loss of albumin by urine, regardless
of the extent of the loss. Under stable physiological conditions and at rest, albuminuria is present only in trace amounts,
i.e., in concentrations ≤10mg/L.The most important physiological factors that can cause signicant changes in albuminuria are sex, age, body mass index (BMI), and a
high-protein diet. Other factors that cause albuminuria, independently of kidney function, are physical exercise over the
24h prior to urine collection, fever, congestive heart failure,
marked hyperglycemia, urinary tract infections, severe
hypertension, menstrual period, and certain medications.
Albuminuria is a prognostic index of progression of diabetic
nephropathy in both type 1 and type 2 diabetes, according to
the scheme shown in Table 18.10, broken down by various
modes of expression of the outcome. In 80% of patients with
type 1 diabetes and albuminuria, urinary albumin excretion
increases at an annual rate of 10–20%, with development of
clinical proteinuria within 10–15 years. After the onset of
clinical proteinuria, most of these patients (>80%) manifest
reductions in GFR until the development, in a relatively short
time, of ESKD.20–40% of patients with type 2 diabetes and
albuminuria evolve to clinical proteinuria, but, in the 20years
following the onset of clinical proteinuria, only 20% of these
patients evolve to ESKD.Finally, in patients with type 1 or 2
diabetes and albuminuria between 30 and 300 mg/g creatinine, the risk of cardiovascular disease is signicantly
increased: meta-analysis studies have shown that in type 2
diabetes, albuminuria doubles cardiovascular morbidity and
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Table 18.10 Levels of excretion of albuminuria referred to as impromptu and timed collection
Extemporaneous collection (sample spot)
mg/mmol
Category
Normoalbuminuria Men <2.5 <25 <30 <20
Women <3.5 <35
Microalbuminuria Men 2.5–30 <25–299 30–299 20–199
Women 3.5–30 <35–299
Clinical albuminuria (overt nephropathy)
Modied from Karalliedde and Viberti (2004)
(creatinine)
≥30 ≥300 ≥300 ≥200
μg/mg
(creatinine) mg/24h μg/min
Timed collection
M. Mussap
mortality (OR 2; 95% CI 1.4–2.7) and more than doubles all
other causes of death (OR 2.4; 95% CI 95%). According to
the new recommendations of the American Diabetes
Association (ADA) on the standards of care for diabetic
patients, a signicant albuminuria value must be found in at
least two out of three samples over 3–6 months before a
patient can be considered albuminuric. The threshold values
dened by the scheme reported in Table18.10 are very useful for dening the onset and the extent of progression of
diabetic nephropathy; regarding CKD, it is dened by a constant value of albuminuria >30mg/24h or >30mg/g of urinary creatinine in the extemporaneous sample, for more than
3months. The choice of this threshold value is based, among
other things, on the evidence that 30mg corresponds to a
value three times higher than the average value found in a
healthy young adult (10 mg). Recent studies have shown
that, in healthy adult populations, changes in urinary albumin excretion expressed in relation to urinary creatinine
(albumin-to--creatinine ratio (ACR)) are signicantly associated with the risk of ESKD and death. However, nowadays,
albuminuria has assumed a more extensive clinical signicance as a marker of cardiac and extracardiac organ damage
and as an independent cardiovascular risk factor, and values
<30mg/24h or <30mg/g urinary creatinine should be clinically evaluated. Albuminuria is a marker of endothelial damage: factors such as hyperglycemia and hypertension, as
mentioned above, and dyslipidemia induce an increase in
endothelial permeability, both in the renal and systemic compartments, mediated by different metabolic pathways, with
passage of molecules from the vascular to the interstitial
compartment, including albumin. This passage triggers the
acute phase, and the consequent recall of immunocompetent
cells and accumulation of cytokines and acute-phase proteins, and this process maintains and increases endothelial
damage. The prevalence of albuminuria in essential hypertension varies between 10% and 30% and increases with age
and disease duration. Albuminuria is associated with signs of
extracardiac vascular damage, such as increased carotid
intima-media thickness, an expression of atherosclerosis,
and increased pulse wave propagation velocity, an expression of arterial stiffness. In addition, albuminuria is associated with left ventricular hypertrophy and increased
intrarenal vascular resistance. For these reasons, concentrations below 30mg/day are signicant, since they dene the
risk, which is a continuous variable and, therefore, changes
considerably, for example, between an albuminuria value of
15 mg and one of 25 mg. As described above, in a CKD
patient, albuminuria and eGFR dene the cumulative risk of
adverse outcome and progression to ESKD, as reported in
Table18.4. The determination of albuminuria requires great
attention from the clinical laboratory, which is called to
choose analytical methods with excellent quality specications, especially those able to guarantee reliable measurements even in the range of concentrations below 30mg. The
choice of the type of sample is very important and is closely
related to the expression of the results. Spot urine sample
allows expressing albuminuria as albumin-to-creatinine ratio
(ACR) (mg/g or mg/mmol of urinary creatinine); the timed
collection (albumin excretion rate (AER)) allows expressing
albuminuria as a share of daily excretion or in any case
referred to as a time frame (mg for 24, 12, 6, or 3h). There is
a wide consensus on the recommendation to avoid expressing the results in relation to the urinary volume (mg/L)
because too many variables contribute in this case to make
the results not very reproducible. The “normalization” of
results for urinary creatinine decreases the variability of
albuminuria. In a 1989 study, it was shown that the intraindividual biological variability (coefcients of variation (CVs))
is lower in the rst morning sample (36%) and further
decreases (31%) when the result is expressed as an albuminuria/creatinuria ratio. In a more recent study, these results
have been conrmed: the total variability of albuminuria is
about 26% on the second morning urination when related to
volume (mg/L) and drops to about 13% when related to urinary creatinine (mg/g creatinine). Several studies have conrmed that the best accuracy and reproducibility in the
measurement of albuminuria is achieved by using the spot
urine sample and expressing the result in relation to urinary
creatinine, and, therefore, it is recommended that all laboratories follow this recommendation. Some recommendations
for the determination and reporting of albuminuria are summarized in Table18.11. However, factors related to reduced
muscle mass, such as older age, female sex, and low body
weight, result in reduced urinary excretion of creatinine,
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Table 18.11 Recommendations for the laboratory evaluation of
albuminuria
The use of the terms “microalbuminuria” and “microalbumin” is not
recommended because they are misleading. The use of the term
“albuminuria” is recommended to indicate the presence of albumin
in urine, regardless of concentration.
For screening and outpatient monitoring, the sample of choice is the
midstream urine collected upon awakening or, alternatively, the
midstream urine from a sample spot or random.
The determination of albuminuria on timed samples (24h, 12h, etc.)
is foreseen in particular cases (follow-up kidney transplant, nephrotic
syndrome, highly developed muscle mass as in competitive athletes,
malnutrition, etc.); in these cases, the result refers to diuresis in 24h
(die).
Sample collection must be performed in a sterile container. Since
albumin is relatively stable in urine at pH >5.0, the sample can be
stored for approximately 8h after urination at 6–20°C.
Samples collected during a feverish episode or after even modest
physical exertion (walking, etc.) or in the presence of bacteriuria
give rise to poorly reliable results from the clinical point of view and
must be conrmed in subsequent checks.
To reduce the variability of the result, it is necessary to “normalize”
the albuminuria concentration values (mg/L) in relation to the
creatininuria concentration and report the albuminuria/creatininuria
ratio expressed in mg/g creatinine (ACR) in the laboratory report.
Therefore, each request for albuminuria involves the determination
of two parameters (albumin and creatinine) and the calculation of the
ratio.
The method of choice for the determination of albuminuria must be
endowed with high sensitivity and good analytical performance even
for ranges of concentration<10mg/g of creatinuria.
The diagnosis of albuminuria requires at least two determinations of
albuminuria over a period of at least 2–3months.
which, in turn, affects the ACR calculation at least partially,
thus resulting in falsely increased results. Various remedies
have been proposed to overcome this problem, including the
development of equations to estimate urinary creatinine and
their use as a correction factor to be multiplied by
ACR. Finally, about the time of sample collection, it has
been found that ACR measured in the extemporaneous random sample leads to an overestimation of the prevalence of
albuminuria compared with ACR measured in the rst morning urination. The clinical importance of albuminuria determination requires an effort to standardize analytical methods
commonly used in routine practice. Although a candidate
method has been developed as a reference method, all the
elements of a “reference system” to standardize the determination of albuminuria, such as a reference material with a
“target” value assigned by a reference method, are not yet
available. Analytical issues related to the determination of
albuminuria involve conformational differences of the molecule, which include the amount and types of bound
substances and the degree of glycation. In urine, in addition,
factors such as pH, ionic strength, and high concentrations of
urea, glucose, and ascorbic acid contribute to albumin
changes. Most of the routine methods for the determination
of albuminuria are represented by immunoturbidimetric
ones, but a further factor of variability among methods is just
the type or mixture of antibodies used (polyclonal, monoclonal, mixed). This variability makes the methods nonhomogeneous, assigning different specications and performance to
each. Based on the data of biological variability, the analytical goal of inaccuracy, expressed as a coefcient of variation
(CV, in percentage), should be <15%, a result conrmed by
the external quality control schemes managed by various
international bodies. There is also sufcient evidence to
dene stable albumin in urine samples up to 7days between
+2 and +8°C, even if it is preferable to measure it on fresh
samples or stored at −80°C.Ultimately, the clinical importance of microalbuminuria requires the utmost commitment
of the clinical laboratory in providing precise and accurate
results, in the choice of sample type and mode of expression
of results, and in the constant monitoring of analytical quality. These requirements are essential not only in the screening but also in the follow-up of patients with CKD,
cardiovascular diseases, diabetes, and patients under therapeutic treatment, to improve the analytical reliability of a test
so important in the denition of cardiovascular risk and
mortality.
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Panteghini M (2008) Enzymatic assays for creatinine: time for action.
Clin Chem Lab Med 46:567–572
Rampoldi L, Scolari F, Amoroso A etal (2011) The redisco-very of uro-
modulin (Tamm-Horsfall protein): from tubulointerstitial nephropathy to chronic kidney disease. Kidney Int 80:338–347
Schwartz GJ, Brlon LP, Spitzer A (1987) The use of plasma creatinine
concentration for estimating glomerular ltration rate in infants,
children and adolescents. Pediatr Clin N Am 34:571–590
Stevens PE, Levin A, Kidney Disease: Improving Global Outcomes
Chronic Kidney Disease Guideline Development Work Group
Members (2013) Evaluation and management of chronic kidney
disease: synopsis of the kidney disease: improving global outcomes
2012 clinical practice guideline. Ann Intern Med 158:825–830
Physical, Chemical, andMorphological
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Urine Examination
FabioManoni
19
Introduction
The physical, chemical, and morphological urine examination (ECMU) is a widely used test because of the wealth of
information it can provide, the ease of sample collection, the
possibility of performing it in any laboratory in a practical,
accurate, and safe way, and the advantageous costeffectiveness ratio. ECMU is a prole of tests aimed at evaluating different aspects of the urinary system, including
indicators of injury (proteins, erythrocytes) and indicators of
function (urinary concentration expressed as relative density,
conductivity, or osmolality). Since ECMU, together with
creatinine and estimated glomerular ltration rate (eGFR), is
the rst approach to diagnose lesions and/or dysfunction of
the kidney and urinary system, it is particularly important
that it is appropriately performed. Specically, it can be
divided into three phases: preanalytical, analytical, and
postanalytical.
Urine is a uid with very labile physical, chemical, and
corpuscular characteristics. Thus, a sample collected or stored
inappropriately should be eliminated. The attending physician
should instruct the patient with the indications on how to collect and deliver the urine sample to the clinical laboratory.
The ECMU typically includes some or all the following
investigations:
• Visual inspection: Color and appearance
• Physical analysis: Concentration (relative density/con-
ductivity/osmolality)
• Chemical analysis: Protein, albumin, creatinine, hemo-
globin, esterase, nitrite, pH, ascorbic acid, glucose,
ketones, bilirubin, urobilinogen
• Counting and morphology of the corpuscular component:
On automatic analyzers and/or by microscopy for eryth-
rocytes, leukocytes, epithelial cells, cylinders, crystals,
lipids, bacteria, mycetes, protozoa, parasites, contaminants, atypical cells.
Each laboratory denes the procedures to use and, in
agreement with the clinicians, the test to perform considering different aspects, including guidelines, published studies,
the prevalence of diseases in the population, and the type of
patients studied. Indeed, the pretest probability in relation to
the prevalence of renal or urological diseases should condition the use of highly specic procedures or, on the contrary,
of highly sensitive tests. Specically, if mainly nephrological/urological patients are evaluated, high-specicity methods should be preferred, while in mainly normal subjects, for
example, in sports medicine, highly sensitive methods should
be preferred.
Preanalytical Phase
The request for ECMU should be made based on a clinical
question. However, ECMU is often requested to exclude the
presence of pathology or as part of an overall holistic
approach to frame a patient. The request for chemical and
morphological examination of urine nds its rationale in the
following conditions:
• Suspected urinary tract infection
• Suspicion or follow-up of kidney disease
• Suspicion or follow-up of noninfectious urinary tract dis-
ease, either primary or secondary to systemic diseases,
such as rheumatic disease, hypertension, toxemia of preg-
nancy, or drug side effects
• Recurrent formation of urinary stones
F. Manoni (*)
Department of Diagnostics and Care Service, Ospedali Riuniti
Padova Sud “Madre Teresa di Calcutta”, Padova, Italy
e-mail: fabio.manoni@aulss6.veneto.it
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. Ciaccio (ed.), Clinical and Laboratory Medicine Textbook, https://doi.org/10.1007/978-3-031-24958-7_19
The patient should be instructed to avoid strenuous exertion prior to urine specimen collection, as well as to avoid
dietary overload. Women of childbearing age should avoid
collection during the menstrual period. Sexual relations
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F. Manoni
should be avoided for at least 12hours prior to collection.
Urine obtained from the rst urination in the morning should
normally be collected on an empty stomach and before
undertaking any physical activity.
Sample Collection
Urine from the rst-morning urination or, alternatively, a
urine specimen that has been in the bladder for at least 4
hours under resting conditions is recommended. The patient
should be properly informed about how to prepare for the
collection of a urine specimen. Specically, the patient
should be advised to: wash hands; precede urine collection
by thoroughly cleansing the genitals using a nonantiseptic
soap and plenty of water for rinsing, and then dry; discard
the rst urination, collecting the intermediate urination in a
wide-mouthed, sterile container, avoiding contact between
container and genitals; collect at least 15–30mL of urine;
close the container hermetically and send it to the laboratory
as soon as possible; always report any pathologies or major
urological interventions; in the presence of urine with abnormal pigments, report the intake of particular drugs or foods.
The sample collected from intermediate urination is the most
suitable for microbiological examination and for chemicalphysical and morphological evaluation. Indeed, it is only
minimally inuenced by the presence of urethral secretions
and mucus. The latter represents an important disturbing element in the chemical-morphological evaluation of urine as it
can induce false positivity in the evaluation of proteins and,
on automatic instrumentation, of cylindruria.
be requested. In both cases, the reasons for this choice must
be adequately explained in a note in the report.
Analytical Phase
Freshly emitted urine usually has the following
characteristics:
• Relative density between 1007 and 1035 (individuals who
have recently received iodinated contrast media may have
a urinary relative density greater than 1035)
• pH between 4.5 and 7.5
• Temperature between 32.5 and 37.5°C
• Creatinine, with a concentration 50–100 times higher
than in other body uids.
The concentration of urea, Na+, K+, and Cl− in urine is
signicantly higher than in other body uids. Negligible
amounts of glucose and protein are usually found in the urine
of healthy subjects, whereas they are present in high concentrations in plasma, amniotic uid, and exudates (but not necessarily in exudates).
Visual Inspection
Volume
The volume of urine normally produced in 24 hours is
between 600 and 1500mL.This is important in the case of
timed collections and in the calculation of clearance.
Sample Storage
The ECMU should be performed as soon as possible. If it is
anticipated that the urine sample cannot be examined within
4 hours, refrigerated storage at +4/8 °C is recommended.
Alternatively, and only if the alterations they induce are
known, preserving agents may be used limited to the examination of certain parameters. The borate has a bacteriostatic
action (it prevents bacterial replication during storage);
higher alcohols are instead able to preserve the morphology
of corpuscular elements of urine to allow the microscopic
examination even after a few hours from sample collection.
Arrival at theLaboratory
The laboratory must always express an opinion on the suitability and acceptability of the sample received; depending
on the type of nonconformity (NC) found, only analyses not
affected by the NC may be carried out, or a new sample must
Color
Urine usually has its own color, which is more or less markedly yellow according to the different concentrations of urochromes, but it can assume different colors in case of
systemic, renal, or urological diseases: dark red or colacolored in case of hemoglobinuria, myoglobinuria, porphyria; brown in case of jaundice and alkaptonuria; blue in
blue diaper syndrome due to the presence of indole in urine;
esh-colored in case of macroscopic hematuria. Different
colors related to the intake of foods containing pigments and
of drugs are not pathologically relevant (Table 19.1). In
ECMU, the color is always evaluated, but it is expressed in
the report only in case of abnormal coloration, with the obligation to comment.
Turbidity
Normal urine appears clear. Various degrees of turbidity
are related to an increase in suspended corpuscles. This
feature may not be stated in the report because the corpuscular elements that originate it are the subject of a
specific evaluation.
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Table 19.1 Main changes in urine color and their possible causes
Color Pathology Medicines and foods
Red Hematuria,
Orange Dehydration Fluorescin, rifampicin,
Greenblue
Brown Alkaptonuria, tyrosinosis
Black Black water fever
Violet Porphyrinuria
Modied from Manoni etal. (2016)
hemoglobinuria,
myoglobinuria,
porphyrinuria
Jaundice, Pseudomonas
urinary infections, blue
diaper syndrome
Porphyrinuria, jaundice
(hemoglobinuric fever in
course of some infectious
diseases such as malaria,
dengue, acute HBV+HDV
coinfection) Malignant
melanoma
Catheter syndrome bladder
(purple urine bag
syndrome)
Cascara, desferroxamine,
doxorubicin, epirubucin,
phenothiazines, phenytoin,
ibuprofen, levodopa,
rifampicin, senna (alkaline
urine), sulfamethoxazole
beets, blackberries, rhubarb
sulfasalanzina (alkaline
urine), warfarin
chilli, rhubarb
Amitriptyline, methylene
blue, indomethacin,
triamterene
Cascara, quinine,
phenothiazines, phenytoin,
iron, levodopa, metronidazole,
methyldopa, nitrofurantoin,
senna (alkaline
urine)
Cascara, quinine, iron,
methyldopa
Seine
Foam
The presence of abundant whitish foam is often linked to the
presence of protein. Also, in this case, it is not necessary to
evaluate and report this parameter because the protein constituents, which are the relevant element from a diagnostic
point of view, are analyzed and reported.
Chemical-Physical Examination
Usually, in clinical laboratories, ECMU is performed using
multipad test strips (dipsticks), whose sensitivity and specicity have been evaluated by several authors. Table 19.2
summarizes the characteristics of the main products on the
market. However, dipsticks, if not stored correctly (humidity), can give false-positive results (Table19.3).
Although to date no commercial methods are available in
kits applicable on clinical chemistry analyzers for evaluation
of the entire ECMU prole, an “upgrade” can be made by
providing: (1) the reporting of only the clinically useful
parameters (Table19.4); (2) the liquid chemistry measurement of urinary proteins with more sensitive and specic
methods (in medium-large laboratories and/or territorial ref-
erence laboratories); (3) the on-demand analysis by traditional chemistry methods of parameters useful only in
particular clinical conditions (glucose, ketones, etc.).
Albumin/Protein
When proteinuria exceeds physiological limits, it almost
always indicates the presence of impaired kidney function
and integrity or systemic disease. The Kidney Disease:
Improving Global Outcomes (KDIGO) guidelines state that
the presence of protein in the urine is a negative prognostic
factor for the development of chronic renal failure and for
cardiovascular risk even at concentrations considered
“physiological” (between 100–300 mg/L of albumin; or,
using the albumin/creatinine ratio, between 10 and 30mg/
mmol), also when the eGFR is still normal. Consequently,
early detection of albuminuria (and/or proteinuria) with sensitive and accurate methods can be the most effective
weapon for an early diagnosis of renal disease and for cardiovascular prevention.
Classically, it is possible to distinguish three types of proteinuria: prerenal, renal, postrenal. In the prerenal form, we
distinguish the functional form and the overload form. In the
functional form, proteinuria, usually modest (always <1.5g/
day), appears after the subject has maintained an upright
posture for some time (orthostatic proteinuria) or during
febrile states, after physical activity, acute heart failure,
while it is absent in the rst morning urine. The overload
form is due to an increase in ultraltrated plasma proteins.
This increased load of low-molecular-weight proteins can
occur, for example, in renal pathologies due to monoclonal
gammopathies associated with light chain elimination, in
severe septicemia, in acute (hemoglobin) or chronic (hemosiderin) hemolysis, in massive muscle traumas (myoglobin).
Proteinuria of renal origin can be distinguished into tubular
and glomerular. Glomerular proteinuria is the most severe
and common form of proteinuria, often marked (>3.5g/day,
so-called proteinuria in the nephrotic range), which can be
associated with hematuria in the case of glomerulonephritis
or with lipiduria together with hypoalbuminemia and hyperlipemia in nephrotic syndrome. Usually, the protein most
represented in the urine is albumin, but as the disease evolves,
other protein species of higher molecular weight may appear.
The presence of modest amounts of albumin in the urine
(erroneously called microalbuminuria) has assumed an
important prognostic value in some common diseases, such
as diabetes and hypertension. Based on this, KDIGO considers albuminuria a marker of chronic kidney disease (CKD),
if reconrmed after at least 3months.
Tubular proteinuria occurs when the normal tubular function of protein reabsorption fails; it is typically relatively
modest proteinuria (<1.5 g/day), characterized by lowmolecular- weight proteins (<35kDa), such as lysozyme, retinol-binding globulin, α1-microglobulin. Mixed glomerular
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F. Manoni
Table 19.2
globin, esterases
AimStick 15mg/dL 50mg/dL
AutionSticks 15mg/dL 50mg/dL
Chemistrip 6mg/dL 40mg/dL
CombiScreen plus 15mg/dL 40mg/dL
DiaScreen 5mg/dL 50mg/dL
Dirui H Series 15mg/dL 50mg/dL
MediTest C9 30mg/dL 50mg/dL
Mission 18mg/dL 25mg/dL –
Multistix 15mg/dL 75mg/dL
Sismex Meditape UC-11A 10mg/dL 50mg/dL
Uriet S2 5mg/dL 10mg/dL
Uriscan 10mg/dL 50mg/dL
Uritest 13G 10mg/dL 40mg/dL –
Uro-Dip 10C – 100mg/dL –
Uropaper alfa 3-9L 15mg/dL 50mg/dL
URS 15mg/dL 100mg/dL
vChem 15mg/dL 45mg/dL
Modied from Manoni etal. (2016)
RBC Red Blood Cells, WBC White Blood Cells, leukocytes
Comparative analytical sensitivity evaluation of some commercially available test strips for albumin, glucose, red blood cells/hemo-
Albumin Glucose RBCs/hemoglobin Esterase
5 RBC/μL
0.3mg/dL
20 RBC/μL
0.06mg / dL
5 RBC/μL
–
5 RBC/μL
–
5 RBC/μL
0.02mg/dL
5 RBC/μL
–
10 RBC/μL
–
0.018mg/dL
5 RBC/μL
0.015mg/dL
10 RBC/μL
0.03
10 RBC/μL
0.03mg/dL
5 RBC/μL
0.015mg/dL
0.3mg/dL
0.05mg/dL
10 RBC/μL
0.3mg/dL
5 RBC/μL
0.3mg/dL
5 RBC/μL
0.2mg/dL
5 WBC/μL
5 WBC/μL
20 WBC/μL
10 WBC/μL
20 WBC/μL
5 WBC/μL
–
9 WBC/μL
5 WBC/μL
25 WBC/μL
20 WBC/μL
2 WBC/μL
15 WBC/μL
–
25 WBC/μL
10 WBC/μL
20 WBC/μL
Table 19.3
Parameter Specicity/interferences
Relative density Only ionic solutes
pH Interference in reduction: Formaldehyde
Blood/hemoglobin False positives: Bacterial peroxidase, oxidizing agents, hydrochloric acid
Leukocyte esterase Only in granulocytes
Nitrites False positives: Hyperchromic urine, drugs, poor sample storage
Proteins False positives: Strongly alkaline urine, hyperchromic urine, drugs, quaternary ammonium, plasma expander
Glucose Specic method for glucose but interference from low temperature and/or high relative density
Main interferers with dipstick
Interference in reduction: Alkaline pH, glucose, and urea >1g/L
Increasing interference: Protein 500mg/dL, ketoacidosis
False negatives: Ascorbate, high relative density, reducing agents, formalin, nitrites, drugs
False positives: Hyperchromic urine, formalin, drugs, sodium azide detergents
False negatives: Ascorbate, borate, glucose >3g/dL, gg proteins >0.5g/dL High relative density, oxidizing agents, soaps
and detergents, drugs
False negatives: Non-nitrite-forming bacteria, diet low in nitrates, urine that has not stayed in the bladder, ascorbate
False negatives: Presence of globulins, hyperchromic urine
False positives: Oxidizing agents, peroxides, hydrochloric acid
False negatives: Ascorbate, poorly stored
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Table 19.3 (continued)
Parameter Specicity/interferences
Ketones It does not show hydroxybutyric acid.
False positives: Free sulfhydryl groups (N-acetyl cysteine), hyperchromic urine, metabolites of levodopa,
phenolphthalein
False negatives: Incorrect conservation
Bilirubin False positives: Hyperchromic urine, chlorpromazine
False negatives: Ascorbate, nitrites, poor storage, direct sunlight
Urobilinogen False positives: Hyperchromic urine, sulfonamides, para-aminosalyl acid
False negatives: Formalin, oxidizing agents, poor storage
Ascorbate False positives: Free sulfhydryl groups (N-acetyl cysteine), reducing agents
Creatinine False positives: Hemoglobin, myoglobin
False negatives: EDTA
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Table 19.4
available strips for albumin, glucose, red blood cells/hemoglobin,
esterase
Clinical utility Parameter
High Albumin/protein
High in specic
clinical conditions
Good Leukocyte esterase
Poor Biliary pigments
Comparative analytical sensitivity of some commercially
Urinary concentration
Albumin/protein
Urinary concentration
Nitrites
Ascorbate
and tubular proteinuria occurs when glomerular and tubular
damage is associated; it is nonselective proteinuria characterized by proteins with different molecular weights.
Urinary protein determination should be expressed as a
ratio to urinary creatinine as an indicator of urinary concentration: PCR (protein creatinine ratio) or ACR (albumin creatinine ratio).
The postrenal form is characteristic of all phlogistic or
erosive pathologies of the urothelial mucosae.
The determination of urinary proteins must be performed
on the intermediate urination of the rst-morning sample by
sensitive methods in liquid chemistry, and the result must be
expressed in relation to the concentration of urine. ACR (rst
choice test in adults) and PCR (rst choice test in children)
are suggested. The lower sensitivity of ACR in pediatric subjects is determined by the higher frequency, in this age group,
of tubular pathologies than the glomerular ones.
The analytical method used in the ECMU is very relevant.
Indeed, immunoturbidimetry on automatic analyzers is the
most reliable but also the least used at present; this is followed by dry chemistry methods on dipsticks by specic
dyes for albumin and expression in relation to urinary creatinine; methods that assess protein concentration (almost
exclusively albumin) on the basis of the variation of a pH-
sensitive indicator with marked interferences in case of alkaline pH are not recommended. The accuracy (and
harmonization) of urinary albumin and creatinine measurement is still an open issue. The International Federation of
Clinical Chemistry (IFCC) is currently working to overcome
it. In any case, the choice of the method must ensure the
detection, at an adequate sensitivity, of both albumin and
globulins. The term microalbuminuria, coined to identify the
measurement of albumin at low concentrations, is wrong and
should be avoided. When performing the test strip, we should
not refer to protein but only to albumin. Indeed, the method
used is sensitive to detect almost exclusively the presence of
albumin and transferrin. Some multireactive strips use,
besides the pad based on the above principle, a reactive area
for the determination of albumin at low concentration (about
100mg/L). Test strips capable of detecting the albumin/creatinine ratio have recently been marketed. Since it is a simple, inexpensive, and rapid method, dry chemistry on dipstick
has spread to all laboratories and is currently still the most
widely used method for protein determination.
Urinary Concentration
Urine is composed of 97–99% water and the remaining
1–3% of a mixture of solutes. The concentration of solutes in
urine is an important index of the ability to concentrate urine
by the kidney, as well as the state of hydration of the subject.
It turns out, therefore, to be an indicator of considerable clinical value. It also has relevance in the analysis of urine sediment as the concentration of urine affects the preservation of
cellular elements, which may undergo lysis, especially in
low-concentrated urine, and morphological alterations, especially in strongly hypertonic urine. The expression of solutes
in urine can be evaluated using different parameters, which
differ in terms of meaning and type of solutes that will be
detected: relative density, osmolality, and conductivity. Each
of these parameters can be determined using different
methods.
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