Practical Laboratories Physiology III

URINALYSIS
Urinalysis is a screening test used for detection of certain disorders, but it is not sufficient for establishing a diagnosis. Disorders inducing changes in urinalysis are: water balance and acid-base balance disorders renal diseases: glomerular nephropathies (glomerulonephritis) and tubule-interstitial nephropathies (pyelonephritis), renal stones altered renal function: ARF (acute renal failure), CRF (chronic renal failure) post-renal disorders: infections of urinary tract (urethritis, cystitis) endocrine disorders: diabetes mellitus, diabetes insipidus (ADH deficiency) liver diseases: jaundice syndromes Urinalysis is performed in the morning, from the first urine eliminated a jeun and comprises: macroscopic examination (volume, aspect, color, smell) physical exam (density and pH) and biochemical exam (nitrite, proteins, glucose, ketone bodies, pus, bilirubin, urobilinogen), which are performed using dipstick screening method, fast, semi-quantitative, based on color reactions that can be compared with a standard scale after 60 120 seconds from the initial contact of urine with the dipstick microscopic examination of urine sediment (cells, casts, bacterial flora, crystals) 1. MACROSCOPIC URINE EXAMINATION 1.1. URINE VOLUME (diuresis) represents the urine volume eliminated/24 hours. It presents variations depending on water balance status and ratio between fluid volume intake and water volume eliminated, both on renal and extra-renal pathway (digestive, skin) and renal function, meaning the kidney capacity to secrete and reabsorb water (altered in renal failure). Normal value: 1200 - 2000 ml/24 h (mean value of 1500 ml/day) volume < 500 ml/24 h = oliguria functional ARF (pre-renal azotemia) volume < 150 ml/24 h = anuria intrinsic ARF (acute tubular necrosis) volume > 2500 ml/24 h = polyuria CRF (compensatory diuresis), osmotic diuresis due to glucose presence in urine (diabetes mellitus) or proteins presence in urine (nephrotic syndrome), hyperhydration (diabetes insipidus), diuretics therapy Urine elimination is changing in urinary tract infections: inversed urine elimination between day / night = nocturia frequent and reduced amount micturitions = polakiuria difficult micturition = dysuria 1.2. URINE ASPECT - normal urine is clear and transparent. In urine left to rest at room temperature, a thin cloud will appear, formed of epithelial cells and mucus (nebule). turbulent urine can be found in presence of excess salts (phosphates, urates, carbonates, oxalates), pus (piuria), lipids (lipiduria), and lymph. 1.3. URINE COLOR normal urine is light yellow due to pigments content (urochrome, urobilin, porphyrin). Color changes depending on density, composition, time from collection, urine pH (diluted or alkaline urine is lighter in color, while concentrated or acid urine is darker), nutrition, and medication. changes in urine color consist of: colorless urine (renal failure, diabetes insipidus, hyperhydration, diuretics use), dark-yellow (dehydration), yellow-brown (jaundice 1

Practical Laboratories Physiology III syndrome), brown-black (methemoglobinuria), red (hematuria, hemoglobinuria, porphyrinuria, myoglobinuria), yellow-green (urinary infection with Pseudomonas) 1.4. URINE SMELL fresh urine has a characteristic smell due to urinoid substances (volatile acids). urine smell can be changed as follows: ammonium smell (urinary infections with bacteria decomposing urea into ammonium), fermented apples (presence of ketone bodies), fetid (severe infections of urinary bladder, recto-vesical fistulae), putrid (urinary bladder cancer) 2. PHYSICAL EXAMINATION OF URINE 2.1. URINE DENSITY (D) expresses kidney capacity to concentrate and dilute urine. Density has inversely proportional variations with hydration status and diuresis, and varies proportional with amount of solutes excreted by kidneys, excepting the following circumstances: metabolic uncompensated diabetes mellitus increased diuresis + increased density arterial hypertension normal diuresis + decreased density acute renal failure decreased diuresis + decreased density chronic renal failure increased diuresis + decreased density Normal value: 1.015-1.025 g/cm3 Interpretation: density > 1.025 g/cm3 = hyperstenuria presence of glucose (diabetes mellitus) or proteins (nephritic syndrome) in urine, dehydration (fever, vomiting, diarrhea) density < 1.015 g/cm3 = hypostenuria renal failure (loss of urine concentration capacity), hyperhydration, diabetes insipidus, diuretics use density = 1.010 1.011 g/cm3 constant (fixed) in repeated determinations/24h = isostenuria terminal stage renal failure (loss of urine concentration and dilution capacity) 2.2. URINE REACTION (pH) reflects kidney capacity to acidify urine Normal value: pH 6 (limits 5.5 7.5). Physiological variations can be found in the following circumstances: nutrition based on meat consumption induces a pH = 5 5.5 (more acid) strictly vegetarian alimentation induces a pH = 7 7.5 (more alkaline) in contact with air, ammonium fermentation is produced, and urine becomes alkaline Interpretation: acid pH < 5.5 presence of ketone bodies in urine (ketoacidosis in diabetes mellitus metabolic uncompensated, starvation), urinary infection with Escherichia Coli neutral or slightly acid pH (5.5 7) renal failure (decreased renal capacity for acidification of urine) alkaline pH > 7.5 in systemic alkalosis (vomiting, prolonged fever), renal tubular acidosis (decreased capacity for urine acidification), urinary infection with Proteus Urinary pH control is important in: renal stones for reducing salts precipitation calcium and magnesium phosphates, as well as calcium carbonate precipitate in alkaline urine, so that urine should be maintained acid, while uric acid (urates) and calcium oxalates precipitate in acid urine, so that urine should be maintained alkaline

Practical Laboratories Physiology III urinary infection in order to reduce bacterial flora proliferation in Escherichia Coli urinary infection, urine should be maintained alkaline, while in Proteus urinary infection, urine should be maintained acid medication antibiotics treatment (streptomycin, neomycin, kanamycin) in case of urinary infection is effective if urine is alkaline, while in salycilates intoxication urine should be maintained acid in order to increase salycilates excretion 3. CHEMICAL EXAMINATION OF URINE 3.1. NITRITE every nitrate present in urine is transformed by bacterial reduction into nitrite, which can be identified by a color reaction in acid medium. Color intensity (pink - red) is directly proportional to nitrite concentration, but is not correlated with urinary infection severity. Interpretation: normal urine DOES NOT contain nitrites negative test (-) in urinary infection positive test (+) or intense positive (++) nitrites absence DOES NOT exclude bacterial infection in urine sample collected, bacteria did not have time (< 4 hours) to transform nitrate into nitrite, or urinary infection is induced by germs which are not reducing the nitrate (e.g: enteroccocus, gonococcus, mycobacterium tuberculosis). 3.2. PROTEINS are glomerulary filtered and totally reabsorbed at the PCT level Interpretation: normal urine contains SMALL amount of albumins (< 30 mg/day) negative test (-) amount of 30 300 mg/day = MICROALBUMINURIA characteristic for diabetic nephropathy, but it cannot be revealed during urinalysis quantity > 300 mg/day = PROTEINURIA (ALBUMINURIA) (altered glomerular filtration and/or tubular reabsorption) positive test from 1 (+) to 4(+) (15 500 mg/dl). Proteinuria can be: - physiologic (transient) after strong emotions, strenuous physical exercise, cold exposure, prolonged orthostatic position, fever - pathologic (persistent) renal cause (nephropathies), post-renal (urinary tract inflammation) and extra-renal (acute infectious diseases and acute clinical disorders, such as pains, epilepsy, myocardial infarction, stroke, trauma, after surgery) (a) SULPHOSALICYLIC ACID method allows identification of albumins and globulins from urine. Adding 10 drops of 20% sulfosalicylic acid on 5 ml of freshly collected urine, will induce proteins precipitation in acid medium, which can be interpreted when compared to control sample (5 ml of urine), using ESBACH method: clear urine = absent proteins (-), opalescent urine = traces of proteins (+), turbulent urine without precipitate = decreased amount of proteins (++), turbulent urine with precipitate = quantifiable proteins in urine (+++). The results obtained using the dipstick method and sulfosalicylic method must be correlated as follows: - in presence of albumins similar results are obtained using both dipstick method and sulfosalicylic method - in presence of albumins and globulins - proteinuria identified using dipstick method is lower when compared to the one determined using sulfosalicylic method (b) Method for determination of proteinuria ESBACH METHOD - urinary proteins precipitate in presence of Esbach reagent and sediment within the Esbach albuminometer. This method allows evaluation of proteinuria severity: mild < 1 g/day (physiologic, urinary tract infections, kidney stones), moderate 1-3 g/day (glomerular and tubule-interstitial nephropathies), and severe > 3.5 g/day (nephrotic syndrome) 3

Practical Laboratories Physiology III 3.3. GLUCOSE is filtered at the glomerular level and totally reabsorbed at the level of PCT Interpretation: normal urine DOES NOT contain glucose negative test (-) glucose presence in urine = GLYCOSURIA positive test from 1 (+) to 4 (+) (50 - 1000 mg/dl). Glycosuria can be: - physiologic pregnancy, ingestion of large amounts of carbohydrates, emotional stress - pathologic exceeding renal glucose threshold of 180 mg/dl (metabolic uncompensated diabetes mellitus) 3.4. KETONE BODIES - acetone, beta-hydroxybutyric acid, acetoacetic acid Interpretation: normal urine contains LOW AMOUNTS (15 - 30 mg/day) negative test (-) increased amount of urinary ketone bodies = KETONURIA positive test from 1 (+) to 3 (+) - ketonuria is the most important parameter for monitoring blood glucose level in diabetic subjects, because ketone bodies are detected in urine before they can significantly increase in plasma - ketonuria occurs in increased vomiting (pregnancy, in children), starvation, lipids-rich diet and low carbohydrates intake dipstick method is not able to detect beta-hydroxybutyric acid (increased in alcoholic persons) 3.5. PUS mixture of dead bacteria and leukocytes Interpretation: normal urine DOES NOT CONTAIN pus negative test (-) presence of pus in urine = PIURIA urinary infection Piuria can be: - macroscopic turbulent urine + positive test (+) + uroculture (+) - microscopic clear urine + positive test (+) + uroculture (+) Leukocytic esterase (LE) qualitative test for identification in urine of lysosomal enzymes released from leukocytes hydrolyzing aromatic and aliphatic esters. Reveals sterile piuria, when LE (+) is associated with uroculture (+) urinary infection with Chlamydia 3.6. BILIARY PIGMENTS form in which hemoglobin degradation products are eliminated BILIRUBIN normal urine contains REDUCED amounts of direct bilirubin (< 0.02 mg/dl) negative test (-) positive test from 1(+) to 3(+) in jaundice syndrome UROBILINOGEN normal urine contains REDUCED amounts of urobilinogen (0.1 1 mg/dl) positive test (+) intense positive test (increased urobilinogen) from 2(+) to 4(+) (2 12 mg/dl) in hemolytic jaundice (pre-hepatic) and parenchymatous (hepatic) negative test (-) (absent urobilinogen) in obstructive jaundice (post-hepatic) 3.7. BLOOD normal urine does not contain blood negative test (-) positive test from 1 (+) to 4(+) in the following circumstances: 4

Practical Laboratories Physiology III hematuria renal trauma, kidney stones hemoglobinuria intravascular hemolytic anemia myoglobinuria lesions of skeletal muscle fiber (rhabdomyolysis) because the dipsticks are not distinguishing between hemoglobin and myoglobin, hematuria must be confirmed by red blood cells presence in urinary sediment 4. MICROSCOPIC EXAMINATION OF URINARY SEDIMENT Principle by centrifugation of 10 ml of urine for 10 minutes at 2000 rpm, a sediment is obtained, from which a native preparation is collected (1 drop between 2 glass slides), further to be examined using optic microscopy, magnification 400x (HPF). Depending on solubility, both at hot temperature, HCl, and acetic acid, the following can be described: (a) organized sediment, insoluble, comprising physiologic elements (cells, casts) and pathologic elements (bacterial flora, parasites, neoplastic cells), and (b) unorganized sediment, soluble, which is formed of crystals. Leukocytes (neutrophilic granulocytes) in normal urine < 5 leukocytes/HPF. Increased leukocytes count = LEUKOCYTURIA pyelonephritis, urinary tract infections Red blood cells in normal urine < 5 RBC/HPF. Increased RBC count = HEMATURIA glomerular nephropathy, kidney stones, renal trauma, anti-coagulant therapy. Hematuria can be macroscopic (red urine) and microscopic (clear urine). Epithelial cells absent or rare in normal urine (1-3 epithelial cells/HPF). Frequent epithelial cells tubule-interstitial nephropathy, urinary tract infection Casts represent moulds of renal tubules lumen, formed of proteins (Tamm-Horsfall protein), cellular elements (RBC, WBC, epithelial cells), and tissue detritus. Within normal urine, 1 hyaline cast/HPF is admitted. Increased number of casts = CYLINDRURIA, having pathologic significance only if associated with proteinuria and changes of organized sediment. Urinary casts can be: hyaline (non-cellular) acid urine, after physical exercise, hematic glomerular nephropathy, leukocytic tubule-interstitial nephropathy, fat (contain cholesterol) nephrotic syndrome, epithelial acute tubular necrosis ( intrinsic ARF) Microbial flora in normal urine THERE IS NO bacterial flora. Presence of bacterial flora, estimated as rare, moderate or abundant = BACTERIURIA. This is associated with uroculture (+) tubule-interstitial nephropathies and common urinary tract infections or uroculture (-) urinary infection with Chlamydia Crystals in normal urine, crystals are rare. Increased number of crystals = CRYSTALURIA kidney stones and consequent nephropathy. Acid urine can contain uric acid (gout), calcium oxalate, while alkaline urine can contain crystals of calcium and magnesium phosphates, and calcium carbonates, respectively. Addis-Hamburger method (minute urine sediment) is performed on morning-collected urine, during 3 hours, when suspicion of renal disorder exists, but urinalysis revealed normal urine sediment. Normal values: RBC: 0-100/min/ml, leukocytes: 0-500/min/ml, casts: 0-7/min/ml

Practical Laboratories Physiology III INTERPRETATION OF URINALYSIS BULLETINS Table I. Renal cause changes of urinalysis
Urinalysis Glomerular Nephrotic Urinary tract infection nephropathy syndrome Volume (ml/24 h) 1200 - 2000 N N, , Density (g/cm3) 1.015 1.025 N N, , pH 5.5 7.5 N, (RF) N,(RF) N, , Nitrite ++ Proteins +++ ++++ Glucose Ketone bodies Pus / LE (-) + or LE (+) Bilirubin Urobilinogen + + + + Sediment N LC H, Ch L, H , EC, MF + Normal sediment (N): < 5L/HPF, < 5H/HPF, 1HC/HPF, rare EC, absent MF (-) Legend: RF = renal failure, LE = leukocytic esterase, L = leukocytes, H = red blood cells, EC = epithelial cells, Ch = hyaline casts, HC = hematic casts, LC = lipid casts, MF = presence of microbial flora Normal

Table 2. Extra-renal cause changes of urinalysis Urinalysis Normal

Jaundice syndrome
Hemolytic (pre-hepatic) Parenchymatous (hepatic) Obstructive (post-hepatic)

Volume (ml/24 h) 1200 - 2000 3 Density (g/cm ) 1.015 1.025 pH 5.5 7.5 Nitrite Proteins Glucose + Ketone bodies Pus /LE (-) Bilirubin + ++ Urobilinogen + + ++ +++ Sediment N N N Normal sediment (N): < 5L/HPF, < 5H/HPF, 1 Ch/HPF, rare CE, MF absent (-)

+++ -

Practical Laboratories Physiology III INTERPRETATION BULLETINS 1. Diuresis = 100 ml/day Density = 1.005 g/cm3 pH = 6 Nitrite (-) Proteins (+++) Glucose (-) Ketone bodies (-) Pus (-) Bilirubin (-) Urobilinogen (+) Sediment: > 10 RBC/HPF > 10 HC/HPF 2. Diuresis = 3000 ml/day Density = 1.035 g/cm3 pH = 6.5 Nitrite (-) Proteins (++++) Glucose (-) Ketone bodies (-) Pus (-) Bilirubin (-) Urobilinogen (+) Sediment: > 10 FC/HPF 3. Diuresis = 150 ml/zi Density = 1.011 g/cm3 pH = 5 Nitrite (++) Proteins () Glucose (-) Ketone bodies (-) Pus (++) Bilirubin (-) Urobilinogen (+) Sediment: > 10 WBC/HPF > 10 RBC/HPF, frequent uric acid crystals, abundant MF 6. Diuresis = 1600 ml/day Density = 1.020 g/cm3 pH = 7 Nitrite (-) Proteins (-) Glucose (-) Ketone bodies (-) Pus (-) Bilirubin (+++) Urobilinogen (-) Sediment: 2WBC/HPF, 4RBC/HPF

4. Diuresis = 3000 ml/day Density = 1.035 g/cm3 pH = 4.5 Nitrite (-) Proteins (-) Glucose (++) Ketone bodies (+) Pus (-) Bilirubin (-) Urobilinogen (+) Sediment: 2 WBC/HPF 1 RBC/HPF

5. Diuresis = 2000 ml/day Density = 1.025 g/cm3 pH = 6.2 Nitrite (-) Proteins (-) Glucose (-) Ketone bodies (-) Pus (-) Bilirubin (++) Urobilinogen (+++) Sediment: 2 WBC/HPF 4 RBC/HPF

A: 1. Acute glomerular nephropathy. Anuria; 2. Nephrotic syndrome; 3. Uric nephropathy and kidney stones. Urinary tract infection. Anuria; 4. Uncompensated diabetes mellitus with ketoacidosis; 5. Hepatic jaundice; 6. Obstructive jaundice (post-hepatic)

Practical Laboratories Physiology III Case study 1. Evaluate functional status of kidneys for a 65 year-old female, diabetic for 15 years, complaining of edema at the inferior limbs, nocturia and hematuria. The following parameters are determined: BP = 180/100 mm Hg, urea = 80 mg/dl (normal 15 45 mg/dl), creatinine = 2.5 mg/dl (normal 0.6 1 mg/dl), glycemia = 240 mg/dl. Urinalysis revealed: diuresis = 3000 ml/day, density = 1.012 g/cm3, pH = 5, nitrite (+), proteins (+++), glucose (++), ketone bodies (+), pus (-), LE (-), bilirubin (-), urobilinogen (+). Urinary sediment: < 10 leukocytes /HPF, > 10 RBC/HPF, > 10 hematic casts / HPF. Based on the presented data, establish the correct answer to the following questions. 1. The following parameters of urinalysis reflect kidneys function: A. Diuresis for renal reabsorption and excretion of water B. Density for renal concentration and dilution of urine C. Proteinuria for selective permeability of glomerular filtrating barrier D. All statements are true 2. In the above case, diuresis reflects: A. Occurrence of acute renal failure B. Dehydration status of the body C. Presence of glycosuria (osmotic diuresis) D. All statements are true 3. In the above case, urine density reflects: A. Kidneys inability to concentrate urine B. Kidneys inability to dilute urine C. Total amount of solutes the kidneys can excrete D. All statements are true 4. Urinary pH reveals: A. Decreased capacity of urine acidification in context of acute renal failure B. Decreased capacity of urine acidification in the context of chronic renal failure C. Presence of ketone bodies due to unbalanced glucose plasma level D. Presence of nitrate in urine 5. The following urinalysis parameters show renal glomerular disorder: A. Proteinuria B. Hematuria C. Presence of hematic casts in urinary sediment D. All statements are true 6. Results of urinalysis could be correlated with other data, as follows: A. Proteinuria with edema B. Diuresis and urine density with arterial hypertension C. Urine density with hematuria D. Urinary pH with nitrogen retention (increased urea and creatinine) 7. In conclusion, the patient presents: A. Glomerular nephropathy B. Diabetic nephropathy C. Chronic renal failure D. All statements are true A: 1D, 2C, 3A, 4C, 5D, 6A, 7D. 8

Practical Laboratories Physiology III Clinical case 2. 40 year-old male subject presents for 2 days intense abdominal pain in right hypochondrium, nausea, vomiting, prolonged fever. The following parameters are determined: BP = 90/55 mm Hg, urea = 45 mg/dl, creatinine = 1 mg/dl, glycemia = 62 mg/dl, plasma pH = 7.59, PCO2 = 32 mm Hg, HCO3- = 30 mEq/l. Urinalysis showed: diuresis = 800 ml/day, density = 1.030 g/cm3, pH = 7.6, nitrite (+), proteins (-), glucose (-), ketone bodies (-), pus (-), LE (+), bilirubin (++), urobilinogen (-). Urinary sediment: 1-2 RBC/HPF, 1-2 WBC/HPF, rare epithelial cells, rare calcium and magnesium phosphates crystals, rare microbial flora. Based on the presented data, establish the correct answer to the following questions. 1. Urinalysis shows disorders of: A. Hydration status of the body B. Acid-base balance C. Metabolism of biliary pigments D. All statements are true 2. Based on clinical data and urinalysis possible diagnosis is: A. Pre-hepatic jaundice B. Hepatic jaundice C. Obstructive jaundice D. Acute intrinsic renal failure 3. Possible diagnosis previously stated is based on: A. Absence of urobilinogen from urine B. Presence of bilirubin in urine C. Normal urinary sediment D. All statements are true 4. Physical examination of urine revealed: A. Dehydration status, as a result of vomiting B. Dehydration status, as a result of prolonged fever C. Systemic alkalosis, as a result of vomiting and prolonged fever D. All statements are true 5. In case of identified water balance disorder, functional kidney response: A. Is normal because diuresis is decreased and urine density is increased B. Is normal because diuresis and urine density are within normal limits C. Is decreased, due to oliguria and hyperstenuria D. Cannot be evaluated based on urinalysis 6. Within the identified acid-base disorder, plasma pH shows that kidneys: A. Compensated metabolic alkalosis induced by vomiting B. Compensated respiratory alkalosis induced by fever C. Eliminates alkaline urine by increased excretion of HCO3 D. All statements are true 7. In the above case, alkaline urinary pH: A. Induced precipitation of calcium and magnesium phosphates crystals B. Favored urinary infection revealed by presence of nitrites and LE (+) C. Could favor development of a urinary infection with Proteus D. All statements are true A: 1D, 2C, 3D, 4D, 5A, 6C, 7D. 9