Nima Naimi and Seth Goldberg
Urinalysis
Urinalysis is an integral part of the evaluation of kidney and urinary tract disease. It consists of two components:
· Macroscopic examination. This includes dipstick evaluation, which yields information regarding the physical and chemical properties of a urine sample, whereas microscopy provides for evaluation of formed elements in the urine.
· Microscopic evaluation. A microscopic evaluation of urine sediment should be performed in the presence of abnormal renal function, hematuria or proteinuria on macroscopy, or clinical concern for urinary tract infection. This analysis should be done personally instead of relying on the laboratory technologist report.
MACROSCOPIC EXAMINATION OF THE URINE
Specimen Collection and Testing Procedure
· Urine samples should be examined within 2 hours of collection, as urea breakdown into ammonia results in alkaline urine, which promotes cell lysis and cast degradation.
· Ideally, samples should be collected from midstream catch of an early-morning specimen. Alternatively, bladder catheterization, either transurethral or suprapubic, may be used, although it may cause hematuria. There is no proven benefit of cleaning the genitalia prior to the collection.
· First perform dipstick testing to interpret colorimetric reaction results. It is important to assess the color and clarity of urine as well.
· For microscopic evaluation, collect 10 to 15 mL of a freshly voided specimen and centrifuge at 1,500 to 3,000 rpm for 5 minutes. Most of the supernatant is then discarded and the sediment resuspended in the remaining fluid. Remove approximately 0.5 mL of supernatant using a pipette and apply one drop of this solution onto a clean glass slide and cover with a cover slip. View the sample with phase-contrast microscopy at 100× and 400× magnification to examine the formed elements in the urine.
Physical and Chemical Properties of Urine
Color
· Normal urine should appear pale yellow in color. Dilute specimens are lighter in color, and concentrated urine has an amber appearance.
· Red urine is seen with hematuria, hemoglobinuria, myoglobinuria, and porphyrinuria. The presence of blood on dipstick testing without red blood cells (RBCs) on microscopy is suggestive of myoglobinuria. Consumption of certain foods (beets, rhubarb, and blackberries) and drugs (phenytoin and rifampin) may also color the urine red.1
· Brown urine may be seen with fava beans, chloroquine, nitrofurantoin, levodopa, metronidazole or with jaundice.1
Clarity
· Normal urine is clear but turns turbid with any urine particles.
· Turbid urine is seen with organisms, cells, and casts in the urine. Urinary tract infections usually produce turbid urine.
· Other causes include hematuria, lipiduria, and metabolic disease (oxaluria, uricosuria).
Odor
· Normal urine does not have a strong odor.
· Foul-smelling urine may be encountered with urinary tract infections due to ammonia production.1
· Diabetic ketoacidosis is associated with fruity odor, and gastrointestinal-vesical fistulas may result in a fecal odor to urine.1
Specific Gravity
· Specific gravity refers to the relative density of urine with respect to water.
· Normal values range from 1.005 to 1.020.
· Specific gravity ≥1.020 is consistent with concentrated urine in the setting of volume depletion; higher values may suggest glycosuria or other osmotically active substances in the urine such as contrast material.1
· Values ≤1.005 suggest dilute urine, which may be seen in water intoxication and diabetes insipidus.
· A fixed specific gravity of 1.010 is often seen in intrinsic renal disease where the kidneys can neither concentrate nor dilute the urine. As a result, the urine is isosthenuric (i.e., it has the same osmolality as serum).
· Proteinuria (>7 g/dL) may cause falsely elevated specific gravity, and falsely decreased values may be seen with urine pH <6.5.
Urine pH
· The normal urine pH ranges from 4.5 to 8.0.
· Acidic urine is associated with metabolic acidosis (e.g., starvation ketosis, diabetic ketoacidosis), dehydration, and large protein loads. Respiratory acidosis can lead to compensatory metabolic alkalosis and decreased urine pH. Extrarenal bicarbonate losses (e.g., diarrhea) may promote urinary acidification.
· Alkaline urine is typically seen in distal renal tubular acidosis. Urea-splitting organisms (e.g., Proteus spp.) can raise urinary pH. Similarly, prolonged storage of urine results in conversion of urea to ammonia, and urinary pH increases.
Glucose
· In patients with preserved renal function and serum glucose concentrations <180 mg/dL, urine glucose is typically absent as it is almost completely reabsorbed in the proximal tubule.
· Dipstick analysis can provide qualitative information regarding the presence or absence of glycosuria.
· Urine glucose may be seen in diabetes mellitus, liver disease, pancreatic disease, Fanconi syndrome, and Cushing syndrome.
Protein
· Urine dipstick is the main screening test for proteinuria.
· The test is a pH-based assay, and albumin is the primary protein identified.
· False-positive results may be seen with highly concentrated specimens, alkaline urine, phenazopyridine, or quaternary ammonia compounds. False-negative tests are associated with dilute urine and nonalbumin proteins, including immunoglobulins and tubular proteins.
· A sulfosalicylic acid test may be used to identify nonalbumin proteins.
· If the dipstick test is positive, the protein should be measured using either a 24-hour protein excretion or a random protein-to-creatinine ratio (PCR) if the creatinine is at a stable baseline.
Hemoglobin
· The dipstick can detect as few as four RBCs per high-power microscopic field (HPF).
· Free hemoglobin or myoglobin in urine, as can be seen with hemolysis or rhabdomyolysis, catalyzes the same dipstick reaction. These conditions should be suspected when the urine dipstick is positive for occult blood in the absence of RBCs on microscopic examination of the urine sediment.
· False-negative tests result from the presence of substances such as ascorbic acid (ingestion of >200 mg/day vitamin C) that diminish the oxidizing potential of the reagent strip.
· Detection of hematuria by dipstick should always be confirmed by microscopic examination of the urine. The presence of dysmorphic RBCs or RBC casts (a so-called active urine sediment) or the coexistence of proteinuria suggests that the hematuria is of glomerular origin.
Leukocyte Esterase
· The detection of leukocyte esterase relies on the release of esterases from lysed neutrophils.
· False-positive results are seen when significant delay occurs between sampling and testing and when the sample is contaminated by vaginal cells.
· False-negative results occur with inhibition of granulocyte function, including glycosuria, proteinuria, high specific gravity, and high urinary concentrations of certain antibiotics (tetracycline, cephalexin, gentamicin).
Urine Nitrite
· Urine dipstick testing depends on bacterial conversion of urinary nitrates into nitrite.1
· While many gram-negative bacteria can reduce nitrates to nitrite, certain organisms, including Enterococcus spp., Neisseria gonorrhoeae, Pseudomonas, and mycobacteria, do not; this may result in negative results.
· False-negative results are also seen with insufficient bladder incubation time, low consumption of nitrates (found in vegetables), and reduction of nitrates to nitrogen by bacteria.
· The test has low sensitivity but high specificity.
· Both leukocyte esterase and nitrite testing must be combined with microscopic examination and clinical context to accurately diagnose urinary tract infections.
MICROSCOPIC EXAMINATION OF URINE
Red Blood Cells
· Presence of three or more RBCs/HPF on two of three urine specimens is diagnostic of microscopic hematuria.1
· Dysmorphic RBCs and RBC casts are suggestive of glomerular disease, whereas normal RBCs suggest nonglomerular bleeding in the urinary tract.
· Hematuria is discussed in further detail in Chapter 25.
White Blood Cells
· Urinary white blood cells (WBCs) are associated with either infection or inflammation.
· Pyuria is defined as more than five WBCs/HPF.
· Extraurinary inflammation, as seen in appendicitis, can also result in pyuria.
· Urine eosinophils may be seen in allergic interstitial nephritis, but this finding is neither sensitive nor specific. Urine eosinophils are also present with parasitic infection of the urinary tract (e.g., schistosomiasis), cholesterol emboli, chronic pyelonephritis, and prostatitis. Eosinophiluria is identified with Hansel staining of the urine.
Epithelial Cells
· Various types of epithelial cells are commonly encountered on urine microscopy.
· Squamous epithelial cells are large, flat cells with central nuclei; they are found in the distal urinary tract and are almost always contaminants.
· Transitional epithelial cells are small, pear-shaped cells lining the bladder and ureters. They may be seen after bladder catheterization but are also found in genitourinary malignancy.
· Renal tubular epithelial cells are notable for large nuclei; their presence suggests renal tubular injury.
· Oval fat bodies are lipid-laden renal epithelial cells. They are identified by the presence of a Maltese cross appearance on polarized light microscopy. Their presence is suggestive of nephrotic syndrome.
Casts
· Casts form when cells or other intraluminal debris (e.g., lipids, bacteria) are trapped within the tubular protein matrix (Tamm-Horsfall protein). They often take the shape of the tubular lumen and represent a snapshot of the intraluminal environment.
· Hyaline casts are formed by Tamm-Horsfall proteins secreted by tubular epithelium into the tubular lumen. Tamm-Horsfall proteins accumulate into casts under states of volume depletion or acidic urine but may also occur in normal urine.
· Granular casts are formed by Tamm-Horsfall proteins and products of cellular breakdown. Muddy brown granular casts may be seen in any renal disease, but in the right clinical context, they suggest a diagnosis of acute tubular necrosis (debris from tubular epithelial cell destruction).
· RBC casts are easily identified as tubular, orange-red structures on light microscopy. Their presence indicates glomerular hematuria.
· WBC casts consist of WBCs and tubular proteins trapped in the tubular lumen. These are seen with interstitial inflammation, including pyelonephritis, acute interstitial nephritis, and occasionally with glomerulonephritis.
· Waxy casts are formed from the degradation of other casts. They are smooth, well-defined structures visible without polarized light. Waxy casts are mainly seen in chronic kidney disease (CKD).
· Fatty casts are characterized by Maltese crosses visible under polarized light. Fatty casts are seen with ethylene glycol poisoning and nephrotic syndrome.
Organisms
· Bacteriuria is frequently encountered, as many urine specimens are not collected under sterile conditions. The clinical context of this finding aids in determining the presence of infection.
· Common bacterial causes of urinary tract infection include E. coli, Staphylococcus saprophyticus (especially in menstruating females), Proteus mirabilis, Enterococcus spp., group B Streptococcus, and other gram-negative bacillus infections (e.g., Klebsiella spp., Citrobacter spp., and Pseudomonas spp.).
· Candida spp. are frequently seen as contaminants from genital secretions or indwelling Foley catheter colonization.
Crystals
· A discussion of pathologic urinary crystals and nephrolithiasis may be found in Chapter 25.
· Acidic urine can result in precipitation of calcium oxalate or uric acid.
· Alkaline urine can result in precipitation of calcium phosphate or struvite (triple phosphate).
· Crystals may form from precipitation of drugs and drug metabolites (e.g., sulfadiazine, amoxicillin, ciprofloxacin, acyclovir, indinavir).
Assessment of Renal Function
ESTIMATION OF GFR
· Kidney function is best assessed by the glomerular filtration rate (GFR). The gold standard to measure GFR is infusion of an exogenous marker such as inulin; however, this technique is too cumbersome for clinical practice and thus only used for research purposes.
· Alternatively, the GFR can be estimated using creatinine-based equations or a 24-hour creatinine clearance. This estimation of GFR is accurate only if the serum creatinine (SCr) is stable and in steady state.
CREATININE AS A MARKER OF RENAL FUNCTION
· Creatinine is a metabolite of skeletal muscle and dietary meat that is freely filtered at the glomerulus and undergoes no significant tubular reabsorption. It is secreted to a small degree by the renal tubules into the urine. Its production is relative to an individual’s muscle mass.
· Aging and weight loss contribute to reduced creatinine production rates.
· In patients with normal renal function, most of the creatinine elimination occurs by glomerular filtration. As renal function declines, creatinine elimination by tubular secretion may exceed filtration. Subsequently, estimates of renal function based on creatinine overestimate actual GFR with worsening renal function.
· Several medications are known to increase serum creatinine (SCr) by either inhibiting the secretion (trimethoprim, cimetidine) or increasing the production (fenofibrate).
· SCr varies inversely with GFR. Any elevation of SCr above the normal range (0.6 to 1.2 mg/dL) should alert the physician to the presence of reduced GFR and renal insufficiency.
· Changes in plasma creatinine are not linearly related to renal function. For example, an increase in creatinine from 1.0 to 1.5 signifies a greater decline in renal function than an increase from 2.0 to 2.5.
· Because SCr is a function of muscle mass and is dependent on age, gender, race, and size, it can often be difficult to assess the degree of renal impairment from SCr alone. Therefore, the following equations should be used to estimate the GFR.
THE COCKCROFT-GAULT CRCL FORMULA
· CrCl (mL/minute) = [(140 - age) × lean body weight (kg) × (0.85 if female)]/(72 × SCr).
· The Cockcroft-Gault formula was derived from studies on adult inpatient male patients. Problems with this formula include estimation of lean body weight and overestimation of true GFR with worsening renal function.2
· It takes age, ideal body weight, and gender into account to estimate GFR from a measurement of SCr.
· This formula estimates CrCl in mL/minute (normal, 100 to 125 mL/minute for males and 85 to 100 mL/minute for females).
· This rapid estimation of GFR is useful in adjustment of drug dosages for decreased renal function based on SCr.
THE MODIFICATION OF DIET IN RENAL DISEASE EQUATIONS
· GFR = 186 × SCr−1.154 × age−0.203 × (1.210 if black) × (0.742 if female).
· The modification of diet in renal disease equation (MDRD) was developed in a mostly white outpatient CKD population. In patients with GFR <60 mL/minute/1.73 m2, the equation accurately estimates renal function.3However, the MDRD is less reliable in patients with normal renal function as it underestimates the GFR.
· The MDRD cannot be generalized in patients with diabetes, patients >70 years of age, and nonwhite patients.
· The abbreviated equation uses age, serum creatinine, gender, and race.
CHRONIC KIDNEY DISEASE EPIDEMIOLOGY COLLABORATION EQUATION
· GFR = 141 × min(SCr/κ,1)α × max(SCr/κ,1)−1.209 × 0.993Age × (1.018 if female) × (1.159 if black), where κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min indicates the minimum of SCr/κor 1, and max indicates the maximum of SCr/κ or 1.4
· The Chronic Kidney Disease Epidemiology Collaboration Equation (CKD-EPI) was developed in an effort to create a more generalizable equation than the MDRD.
· It has been studied in a much larger and diverse population than the other two equations.
· In comparison studies with the MDRD, the CKD-EPI has been shown to be more generalizable, more accurate in estimating GFR, and more accurate in assessing prognosis.
· Current Kidney Disease Improving Global Outcomes (KDIGO) guidelines recommend the CKD-EPI equation for evaluation of CKD.
TWENTY-FOUR–HOUR URINE COLLECTION FOR CrCl
· A 24-hour urine CrCl is preferable to the above equations in extremes of age and weight, pregnancy, individuals with normal GFR, and malnutrition.
· Patients should be instructed to discard their first morning urine and then begin the 24-hour urine collection, ending the collection with inclusion of the following morning’s first void.
· The amount of total creatinine in the collection can be used to assess the adequacy of the collection; an adequate 24-hour urine collection contains 15 to 20 mg/kg creatinine for females and 20 to 25 mg/kg for males.
· Accuracy may be altered by incorrect collection of the urinary specimen or hypersecretion of creatinine in advanced kidney disease.
CYSTATIN C
· Cystatin C is a low molecular weight protein that is part of the cysteine protease inhibitor family. It is produced at a constant rate by all nucleated cells and freely filtered by the glomerulus. Once in the proximal tubules, it is catabolized by the epithelial cells. Therefore, it cannot be used to measure clearance.
· Because it is not secreted in the tubules and is less affected by muscle mass and diet, cystatin C was thought to be a better marker to estimate GFR than creatinine. Many factors, however, have been found to affect its level, such as adiposity, diabetes, and inflammation.
· Equations that use cystatin C are not more accurate than creatinine-based estimates of GFR.
· The most accurate estimation of GFR is obtained when using an equation that combines both cystatin C and creatinine, especially around a GFR of 60 mL/minute/1.73 m2.5
Proteinuria
GENERAL PRINCIPLES
· Patients with normal renal function excrete <150 mg of total protein or <30 mg of albumin in 24 hours.
· Proteinuria is defined as protein excretion of >150 mg/day, while nephrotic syndrome is defined as protein excretion >3.5 g/day associated with hypoalbuminemia, edema, lipiduria, and hyperlipidemia. Nephrotic-range proteinuria refers to urine protein excretion >3.5 g/day without the other findings.
· Albuminuria is now used to further classify the level of CKD as it is a marker of severity and is associated with progression of kidney disease.6
· Treatment of proteinuria may slow progression of kidney disease and reduce the risk of cardiovascular disease.
· If proteinuria is detected on a dipstick test, the test should be repeated after at least a week to rule out transient proteinuria. If the repeat test is negative, no further workup is needed.
· Transient proteinuria (usually <1 g/day) can occur with fever, emotional stress, heavy physical exercise, urinary tract infection, or acute medical illness.
· Orthostatic proteinuria should also be ruled out. It is defined by an elevated protein excretion in the upright position, which normalizes while the patient is supine. The diagnosis is usually made in patients younger than 30 and carries no increased risk of kidney disease. A normal first-void PCR may effectively rule out the diagnosis.
· Once proteinuria is confirmed, it should be quantified.
· Patients with persistent albuminuria (>300 mg/day) or proteinuria (>500 mg/day) should be referred to a nephrologist. In cases of unexplained proteinuria or hematuria, a biopsy is usually required to make a definitive diagnosis and to initiate disease-specific therapy.
DIAGNOSIS
Clinical Presentation
· Focus on signs and symptoms of conditions associated with proteinuria, such as hypertension, diabetes mellitus, or connective tissue diseases.
· All medications should be reviewed (prescription, illicit, over the counter, herbal). Nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with a variety of renal diseases and should be discontinued in the presence of proteinuria pending further evaluation.
· Clinical features of malignancies, especially multiple myeloma and lymphoma, should be sought. Furthermore, solid organ malignancies are associated with glomerulonephritis.
· Findings consistent with infections, such as HIV, viral hepatitis, and bacterial endocarditis, should be evaluated, as all these can cause glomerular disease.
· A complete family history should be obtained.
Diagnostic Testing
Urine Reagent Strip
· The urine reagent dipstick is frequently used as the initial screening test for proteinuria as it is quick and inexpensive. It detects albumin at concentrations >10 to 20 mg/dL, which equates to approximately 300 to 500 mg of protein in 24 hours.
· The test is only semiquantitative, has low sensitivity for nonalbumin proteins (e.g., light chains of multiple myeloma), has variability among different manufacturers, and is dependent on hydration.6
· False positives can occur within 24 hours of iodinated radiocontrast infusion or with high urine pH.
· Any patient with persistent proteinuria should undergo a quantification study.
Twenty-Four–Hour Urine Collection for Protein
· The 24-hour urine collection, when obtained correctly, remains the reference point to accurately quantify the amount of daily protein excretion in the urine.
· Concurrent collection of urine creatinine should also be done to ensure the completeness of the collection.
· Although the gold standard, this test is cumbersome for patients and frequently leads to an inaccurate/incomplete urine collection.
Spot Urine Albumin-to-Creatinine or Protein-to-Creatinine Ratio
· The latest KDIGO guidelines recommend using the albumin to creatinine ratio (ACR) as the initial testing for proteinuria in CKD.7 ACR is preferred over PCR as it is more sensitive for kidney disease in CKD. PCR should still be used in certain cases, such as inpatients with monoclonal gammopathies.
· These tests use a random spot urine sample for protein or albumin (mg/dL) and creatinine (mg/dL) concentrations to calculate a unitless ratio. The creatinine must be at a stable baseline. For best results, an early-morning urine sample should be used.
· ACR of 30 to 300 mg/g is referred to as moderately increased (formerly microalbuminuria) and ACR >300 mg/g is referred to as severely increased (formerly macroalbuminuria).
· The results can be unreliable in patients at either extremes of muscle mass. Also, the amount of proteinuria varies throughout the day, being higher during the day than at night.
Other Laboratories
· Urine sediment should be carefully examined for other signs of glomerular disease, such as dysmorphic RBCs, RBC casts, and oval fat bodies.
· Obtain a complete blood count (CBC), basic metabolic profile (BMP) with GFR estimation, albumin, and quantification of the protein with either a spot urine or 24-hour collection.
· If multiple myeloma is considered, a urine and serum protein electrophoresis with immunofixation and serum free light chains should be ordered.
· If a connective tissue disease or vasculitis is suspected, a complete autoimmune workup is required (Chapter 24).
Imaging
A renal ultrasound should also be obtained to assess the renal and urinary tract architecture. The finding of symmetrically small kidneys (<10 cm) suggests CKD. Notable exceptions include the kidneys of diabetic nephropathy, HIV-associated nephropathy, polycystic kidney disease, and deposition disorders, in which the kidneys may be enlarged.
REFERENCES
1.Simerville JA, Maxted WC, Pahira JJ. Urinalysis: a comprehensive review. Am Fam Physician 2005;71:1153–1162.
2.Poggio ED, Wang X, Greene T, et al. Performance of the modification of diet in renal disease and Cockcroft-Gault equations in the estimation of GFR in health and in chronic kidney disease. J Am Soc Nephrol 2005;16:459–466.
3.Levey AS, Bosch JP, Lewis JB, et al. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med1999;130:461–470.
4.Madero M, Sarnak MJ. Creatinine-based formulae for estimating glomerular filtration rate: is it time to change to chronic kidney disease epidemiology collaboration equation? Curr Opin Nephrol Hypertens2011;20:622–630.
5.Inker LA, Schmid CH, Tighiouart H, et al. Estimating glomerular filtration rate from serum creatinine and cystatin C. N Engl J Med 2012;367:20–29.
6.Lamb EJ, MacKenzie F, Stevens PE. How should proteinuria be detected and measured? Ann Clin Biochem 2009;46:205–217.
7.Kidney Disease: Improving Global Outcomes (KDIGO) CKD Work Group. KDIGO 2012 clinical practice guideline for the evaluation and management of chronic kidney disease. Kidney Int Suppl2013;3:1–150.