Assistant Professor of Biochemistry Examination of Urine Dr/Mohamed Mahmoud Nour Eldein PhD Biochemistry Assistant Professor of Biochemistry Faculty of Medicine Umm-ALOura University

Urinary System

Introduction Urine is formed in the kidneys, is a product of ultrafiltration of plasma by the renal glomeruli which is a network of arteriolar capillaries, each glomeruli is surrounded by Bowman’s capsule (a double epithelial sac) like a rounded funnel which leads to the tube.

Purpose Urine contains important metabolic information Urine is cheap, simple, & readily available General evaluation of health Diagnosis of disease or disorders of the kidneys or urinary tract Diagnosis of other systemic disease that affect kidney function Monitoring of patients with diabetes Screening for drug abuse (eg. Sulfonamide or aminoglycosides)

Collection of urine specimens The first voided morning urine (most concentrated) - qualitative Random urine (routine) 24hrs sample- quantitative Mid-stream clean catch (MSCC) (for urine culture)- UTI Post prandial sample-D.M Attention Need to be examined within 1 hour

Clean Catch

24 hour urine sample For quantitative estimation of proteins For estimation of vanillyl mandelic acid, 5-hydroxyindole acetic acid, metanephrines For detection of AFB in urine For detection of microalbuminuria

Types of Analysis 1- Macroscopic Examination Physical characteristics : color, odor, turbidity, volume, & specific gravity Chemical Analysis (Urine Dipstick) : pH, glucose, protein, ketones, pus (WBC’s & bacteria), RBC’s, hemoglobin, bile 2- Microscopic Examination of urine sediment: crystals, cells, etc.

Physical examination Volume Color Odour Reaction or urinary pH Specific gravity

Urinary volume The average daily urine output 1200 - 1500 mL (1.2 - 1.5 L) The normal daily range of urine output 600 - 2000 mL (0.6 - 2.0 L) Polyuria- >2000ml Oliguria- <400ml Anuria-complete cessation of urine(<200ml) Nocturia-excretion of urine by an adult of >500ml with a specific gravity of <1.018 at night (characteristic of chronic glomerulonephritis)

Causes of polyuria > 2000ml Diabetes mellitus Diabetes insipidus Polycystic kidney Chronic renal failure Diuretics Intravenous saline/glucose

Oliguria <400ml Dehydration-vomiting, diarrhea, excessive sweating Renal ischemia Acute tubular necrosis Obstruction to the urinary tract Acute renal failure

Urine Color and Clarity Urine color and clarity can indicate what substances may be present in urine. Confirmation of suspected substances is obtained during the chemical and microsopic examination.

Urine Color Normal urine color ranges from pale yellow to deep amber — the result of a pigment called urochrome Most changes in urine color are harmless and temporary and may be due to: Certain foods – beets may turn urine red Dyes in foods/drinks Supplements – vitamins Prescription drugs

Abnormal colors of urine & possible causes Unusual urine color can indicate an infection or serious illness . Colourless- dilution, diabetes mellitus, diabetes insipidus, diuretics Milky- genitourinary tract infection Orange-fever, excessive sweating, bilirubin Red-beetroot ingestion,haematuria Brown/ black- alkaptunuria, melanin Green - bile, Pseudomonas bacteria

Examples of Urine Color

Urine Clarity Urine clarity refers to how clear the urine is. Terms used: clear, transparent, slightly cloudy, cloudy, or turbid. “Normal” urine can be clear or cloudy. The clarity of the urine is not as important as the substance that is causing the urine to be cloudy. Turbidity - “cloudiness” due to particulate matter suspended in urine

Urine Clarity Substances that cause cloudiness but that are not considered unhealthy include: mucous, sperm and prostatic fluid, cells from the skin, normal urine crystals, and contaminants (like body lotions and powders). Other substances that can make urine cloudy (such as red blood cells, white blood cells, or bacteria) indicate a condition that requires attention.

Examples of Urine Clarity

Odour Normal= aromatic due to the volatile fatty acids Standing (“old”) urine takes on an ammonia odor due to urea-splitting bacterial Foul, offensive: Old specimen, pus or inflammation Sweet: Glucose Fruity: Ketones

Chemical Analysis

Urine dipsticks (Reagent Strips) Urine dipstick are plastic strips on which are attached to a series of chemically impregnated absorbent pads, each pad contain certain chemicals that react with substance in the urine producing a color change in pad, this color change is compared with a series of known standards.

Chemical Analysis Urine Dipstick Glucose Bilirubin Ketones Specific Gravity Blood pH Protein Urobilinogen Nitrite Leukocyte Esterase

Reagent Strips

Procedure Testing Reagent strips are used only once and discarded. Perform within 1 hour after collection Allow refrigerated specimens to return to room temperature. Dip strip briefly, but completely into well mixed, room temperature urine sample. Withdraw strip. Blot briefly on its side. Keep the strip flat, read results at the appropriate times by comparing the color to the appropriate color on the chart provided.

Procedure Instruments are available which detect color changes electronically and prints out results

Handling and Storage of Strips Keep strips in original container Do not touch reagent pad areas Reagents and strips must be stored properly to retain activity Protect from moisture and volatile fumes Stored at room temperature Use before expiration date

Sources of Error Timing - Failure to observe color changes at appropriate time intervals may cause inaccurate results. Lighting - Observe color changes and color charts under good lighting. QC - Reagent strips should be tested with positive controls on each day of use to ensure proper reactivity. Sample - Proper collection and storage of urine is necessary to insure preservation of chemical.

Sources of Error Testing cold specimens - would result in a slowing down of reactions; test specimens when fresh or bring them to RT before testing Inadequate mixing of specimen - could result in false reduced or negative reactions to blood and leukocyte tests; mix specimens well before dipping Over-dipping of reagent strip - will result in leaching of reagents out of pads; briefly, but completely dip the reagent strip into the urine

Horseradish Peroxidase The Urine Dipstick: Glucose Chemical Principle Negative Trace (100 mg/dL) + (250 mg/dL) ++ (500 mg/dL) +++ (1000 mg/dL) ++++ (2000+ mg/dL) Glucose Oxidase Glucose + 2 H2O + O2 ---> Gluconic Acid + 2 H2O2 Horseradish Peroxidase 3 H2O2 + KI ---> KIO3 + 3 H2O Read at 30 seconds RR: Negative

Uses and Limitations of Urine Glucose Detection Significance Diabetes mellitus. Renal glycosuria. Limitations Interference: reducing agents, ketones. Only measures glucose and not other sugars. Renal threshold must be passed in order for glucose to spill into the urine. Other Tests CuSO4 test for reducing sugars.

Urinalysis Glucose Result Urine versus Blood Glucose ++ + Urinalysis Glucose Result trace Negative 200 400 600 800 1000 Blood Glucose (mg/dL)

The Urine Dipstick: Bilirrubin Negative Chemical Principle + (weak) ++ (moderate) +++ (strong) Chemical Principle Acidic Bilirubin + Diazo salt ---------> Azobilirubin Read at 30 seconds RR: Negative

Bilirubin Bilirubin is a byproduct of the breakdown of hemoglobin. Normally contains no bilirubin. Presence may be an indication of liver disease, bile duct obstruction or hepatitis. Since the bilirubin in samples is sensitive to light, exposure of the urine samples to light for a long period of time may result in a false negative test result.

Ketones Ketones are excreted when the body metabolizes fats incompletely (ketonuria)

Acetoacetic Acid + Nitroprusside ------> Colored Complex The Urine Dipstick: Ketones Negative Trace (5 mg/dL) + (15 mg/dL) ++ (40 mg/dL) +++ (80 mg/dL) ++++ (160+ mg/dL) Chemical Principle Acetoacetic Acid + Nitroprusside ------> Colored Complex Read at 40 seconds RR: Negative

Uses and Limitations of Urine Ketone Detection Significance - Diabetic ketoacidosis - Prolonged fasting Limitations - Interference: expired reagents (degradation with exposure to moisture in air) - Only measures acetoacetate not other ketone bodies (such as in rebound ketosis). Other Tests - Ketostix (more sensitive tablet version of same assay) - Serum glucose measurement to confirm DKA

Specific gravity Depends on the concentration of various solutes in the urine. Measured by-urinometer - refractometer - dipsticks Specific gravity reflects kidney's ability to concentrate. Want concentrated urine for accurate testing, best is first morning sample. Low – specimen not concentrated, kidney disease. High – first morning, certain drugs

Urinometer Take 2/3 of urinometer container with urine Allow the urinometer to float into the urine Read the graduation at the lowest level of urinary meniscus Correction of temperature & albumin is a must. Urinometer is calibrated at 15or 200c So for every 3oc increase/decrease add/subtract 0.001 For 1gm/dl of albumin add0.001

The Urine Dipstick: Specific Gravity 1.000 Chemical Principle 1.005 1.010 1.015 1.020 1.025 1.030 Chemical Principle X+ + Polymethyl vinyl ether / maleic anhydride ---------------> X+-Polymethyl vinyl ether / maleic anhydride + H+ H+ interacts with a Bromthymol Blue indicator to form a colored complex. Read up to 2 minutes RR: 1.003-1.035

Uses and Limitations of Urine Specific Gravity Significance - Diabetes insipidus Limitations - Interference: alkaline urine - Does not measure non-ionized solutes (e.g. glucose) Other Tests - Refractometry - Hydrometer - Osmolality measurement (typically used with water deprivation test)

High specific gravity(hyperosthenuria) Normal-1.016-1.022 Causes All causes of oliguria Glycosuria

Low specific gravity(hyposthenuria) All causes of polyuria except glycosuria Fixed specific gravity (isosthenuria)=1.010 Seen in chronic renal disease when kidney has lost the ability to concentrate or dilute

Blood Presence of blood may indicate infection, trauma to the urinary tract or bleeding in the kidneys. False positive readings most often due to contamination with menstrual blood.

The Urine Dipstick: Blood Negative Trace (non-hemolyzed) Chemical Principle Trace (non-hemolyzed) Lysing agent to lyse red blood cells Moderate (non-hemolyzed) Diisopropylbenzene dihydroperoxide + Tetramethylbenzidine ------------> Colored Complex Trace (hemolyzed) Heme + (weak) ++ (moderate) Read at 60 seconds RR: Negative Analytic Sensitivity: 10 RBCs +++ (strong)

Uses and Limitations of Urine Blood Detection Significance - Hematuria (nephritis, trauma, etc) - Hemoglobinuria (hemolysis, etc) - Myoglobinuria (rhabdomyolysis, etc) Limitations - Interference: reducing agents, microbial peroxidases - Cannot distinguish between the above disease processes Other Tests - Urine microscopic examination - Urine cytology

Urinary pH/ reaction Reaction reflects ability of kidney to maintain normal hydrogen ion concentration in plasma & ECF Normal= 4.6-8 Tested by- 1.litmus paper 2. pH paper 3. dipsticks

Methyl Red (at high concentration; low pH) and The Urine Dipstick: pH 5.0 6.0 6.5 7.0 7.5 8.0 8.5 Chemical Principle H+ interacts with: Methyl Red (at high concentration; low pH) and Bromthymol Blue (at low concentration; high pH), to form a colored complexes (dual indicator system) Read up to 2 minutes R.R.: 4.5-8.0

Acidic urine Ketosis-diabetes, starvation, fever Systemic acidosis UTI- E.coli Acidification therapy

Alkaline urine Strict vegetarian Systemic alkalosis UTI- Proteus Alkalization therapy

Uses and Limitations of Urine pH Detection Significance - Acidic (less than 4.5): metabolic acidosis, high-protein diet - Alkaline (greater than 8.0): renal tubular acidosis (>5.5) Limitations - Interference: bacterial overgrowth (alkaline or acidic), “run over effect” effect of protein pad on pH indicator pad Other Tests - Titrable acidity - Blood gases to determine acid-base status

pH Run Over Effect Glucose Bilirubin Ketones Specific Gravity Blood pH Protein Urobilinogen Nitrite Leukocyte Esterase Buffers from the protein area of the strip (pH 3.0) spill over to the pH area of the strip and make the pH of the sample appear more acidic than it really is.

Protein Presence of protein (proteinuria) is an important indicator of renal disease. False negatives can occur in alkaline or dilute urine or when primary protein is not albumin.

The Urine Dipstick: Protein Chemical Principle “Protein Error of Indicators Method” Negative Trace + (30 mg/dL) ++ (100 mg/dL) +++ (300 mg/dL) ++++ (2000 mg/dL) Pr H Tetrabromphenol Blue (buffered to pH 3.0) H+ Pr Pr Pr Pr Pr Pr Read at 60 seconds RR: Negative

Causes of Proteinuria Functional Renal Pre-Renal Post-Renal - Severe muscular exertion - Glomerulonephritis - Pregnancy - Nephrotic syndrome - Orthostatic proteinuria - Renal tumor or infection Pre-Renal Post-Renal - Fever - Cystitis - Renal hypoxia - Urethritis or prostatitis - Hypertension - Contamination with vaginal secretions

Uses and Limitations of Urine Protein Detection Significance - Proteinuria and the nephrotic syndrome. Limitations - Interference: highly alkaline urine. - Much more sensitive to albumin than other proteins (e.g., immunoglobulin light chains). Other Tests - Sulfosalicylic acid (SSA) turbidity test. - Urine protein electrophoresis (UPEP) - Bence Jones protein

Urobilinogen Urobilinogen is a degradation product of bilirubin formed by intestinal bacteria. It may be increased in hepatic disease or hemolytic disease

Urobilinogen + Diethylaminobenzaldehyde -------> Colored Complex The Urine Dipstick: Urobilinogen 0.2 mg/dL 1 mg/dL 2 mg/dL 4 mg/dL 8 mg/dL Chemical Principle Urobilinogen + Diethylaminobenzaldehyde -------> Colored Complex (Ehrlich’s Reagent) Read at 60 seconds RR: 0.02-1.0 mg/dL

Uses and Limitations of Urobilinogen Detection Significance - High: increased hepatic processing of bilirubin - Low: bile obstruction Limitations - Interference: prolonged exposure of specimen to oxygen (urobilinogen ---> urobilin) - Cannot detect low levels of urobilinogen Other Tests - Serum total and direct bilirubin

Nitrite Nitrite formed by gram negative bacteria converting urinary nitrate to nitrite

The Urine Dipstick: Nitrite Chemical Principle Negative Positive Acidic Negative Positive Nitrite + p-arsenilic acid -------> Diazo compound Diazo compound + Tetrahydrobenzoquinolinol ----------> Colored Complex Read at 60 seconds RR: Negative

Uses and Limitations of Nitrite Detection Significance - Gram negative bacteriuria Limitations - Interference: bacterial overgrowth - Only able to detect bacteria that reduce nitrate to nitrite Other Tests - Correlate with leukocyte esterase and - Urine microscopic examination (bacteria) - Urine culture

Leukocytes Leukocytes (white blood cells) usually indicate infection. Leucocyte esterase activity is due to presence of WBCs in urine while nitrites strongly suggest bacteriuria.

The Urine Dipstick: Leukocyte Esterase Chemical Principle Derivatized pyrrole amino acid ester ------------> 3-hydroxy-5-phenyl pyrrole Negative Trace + (weak) ++ (moderate) +++ (strong) Esterases 3-hydroxy-5-phenyl pyrrole + diazo salt -------------> Colored Complex Read at 2 minutes RR: Negative Analytic Sensitivity: 3-5 WBCs

Significance Limitations Other Tests Uses and Limitations of Leukocyte Esterase Detection Significance - Pyuria - Acute inflammation - Renal calculus Limitations - Interference: oxidizing agents, menstrual contamination Other Tests - Urine microscopic examination (WBCs and bacteria) - Urine culture

Normal Values Negative results for glucose, ketones, bilirubin, nitrites, leukocyte esterase and blood. Protein negative or trace. pH 5.5-8.0 Urobilinogen 0.2-1.0 Ehrlich units

Microscopic examination Microscopic urinalysis is done simply pouring the urine sample into a test tube and centrifuging it (spinning it down in a machine) for a few minutes. The top liquid part (the supernatant) is discarded. The solid part left in the bottom of the test tube (the urine sediment) is mixed with the remaining drop of urine in the test tube and one drop is analyzed under a microscope

Microscopic Examination Abnormal Findings Per High Power Field (HPF) (400x) > 3 erythrocytes > 5 leukocytes > 2 renal tubular cells > 10 bacteria Per Low Power Field (LPF) (200x) > 3 hyaline casts or > 1 granular cast > 10 squamous cells (indicative of contaminated specimen) Any other cast (RBCs, WBCs) Presence of: Fungal hyphae or yeast, parasite, viral inclusions Pathological crystals (cystine, leucine, tyrosine) Large number of uric acid or calcium oxalate crystals

Microscopic Examination Cells Erythrocytes - “Dysmorphic” vs. “normal” (> 10 per HPF) Leukocytes - Neutrophils (glitter cells) More than 1 per 3 HPF - Eosinophils Hansel test (special stain) Epithelial Cells - Squamous cells Indicate level of contamination - Renal tubular epithelial cells Few are normal - Transitional epithelial cells Few are normal - Oval fat bodies Abnormal, indicate Nephrosis

Microscopic Examination RBCs

Microscopic Examination RBCs

Microscopic Examination WBCs

Microscopic Examination Squamous Cells

Tubular Epithelial Cells Microscopic Examination Tubular Epithelial Cells

Microscopic Examination Transitional Cells

Microscopic Examination Transitional Cells

Microscopic Examination Oval Fat Body

Microscopic Examination LE Cell

Microscopic Examination Bacteria & Yeasts Bacteria - Bacteriuria More than 10 per HPF Yeasts - Candidiasis Most likely a contaminant but should correlate with clinical picture. Viruses - CMV inclusions Probable viral cystitis.

Microscopic Examination Bacteria

Microscopic Examination Yeasts

Microscopic Examination Yeasts

Microscopic Examination Cytomegalovirus

casts Urinary casts are cylindrical aggregations of particles that form in the distal nephron, dislodge, and pass into the urine. In urinalysis they indicate kidney disease. They form via precipitation of Tamm-Horsfall mucoprotein which is secreted by renal tubule cells.

Microscopic Examination Casts

Types of casts Acellular casts Hyaline casts Granular casts Waxy casts Fatty casts Pigment casts Crystal casts Cellular casts Red cell casts White cell casts Epithelial cell cast

Microscopic Examination Casts Erythrocyte Casts: Glomerular diseases Leukocyte Casts: Pyuria, glomerular disease Degenerating Casts: - Granular casts Nonspecific (Tamm-Horsfall protein) - Hyaline casts Nonspecific (Tamm-Horsfall protein) - Waxy casts Nonspecific - Fatty casts Nephrotic syndrome (oval fat body casts)

Red cell casts The presence of red blood cells within the cast is always pathologic, and is strongly indicative of glomerular damage. They are usually associated with nephritic syndromes.

Microscopic Examination RBCs Cast - Histology

Microscopic Examination RBCs Cast

Microscopic Examination RBCs Cast - Histology

White blood cell casts Indicative of inflammation or infection, pyelonephritis acute allergic interstitial nephritis, nephrotic syndrome, or post-streptococcal acute glomerulonephritis

Microscopic Examination WBCs Cast

Epithelial casts This cast is formed by inclusion or adhesion of desquamated epithelial cells of the tubule lining. These can be seen in acute tubular necrosis and toxic ingestion, such as from mercury, diethylene glycol, or salicylate.

Microscopic Examination Tubular Epith. Cast

Microscopic Examination Tubular Epith. Cast

Granular casts Granular casts can result either from the breakdown of cellular casts or the inclusion of aggregates of plasma proteins (e.g., albumin) or immunoglobulin light chains indicative of chronic renal disease

Microscopic Examination Granular Cast

Hyaline casts The most common type of cast, hyaline casts are solidified Tamm-Horsfall mucoprotein secreted from the tubular epithelial cells Seen in fever, strenuous exercise, damage to the glomerular capillary

Microscopic Examination Hyaline Cast

Waxy casts waxy casts suggest severe, longstanding kidney disease such as renal failure(end stage renal disease).

Waxy casts

Microscopic Examination Waxy Cast

Fatty casts Formed by the breakdown of lipid-rich epithelial cells, these are hyaline casts with fat globule inclusions They can be present in various disorders, including nephrotic syndrome, diabetic or lupus nephropathy, Acute tubular necrosis

Fatty casts

Microscopic Examination Fatty Cast

Crystal casts Though crystallized urinary solutes, such as oxalates, urates, or sulfonamides, may become enmeshed within a hyaline cast during its formation. The clinical significance of this occurrence is not felt to be great.

Contents of normal urine m/s Contains few epithelial cells, occasional RBC’s, few crystals.

Crystals in urine Crystals in acidic urine Uric acid Calcium oxalate Cystine Leucine Crystals in alkaline urine Ammonium magnesium phosphates(triple phosphate crystals) Calcium carbonate

crystals

Calcium Oxalate Crystals Microscopic Examination Calcium Oxalate Crystals

Calcium Oxalate Crystals Microscopic Examination Calcium Oxalate Crystals Dumbbell Shape

Triple Phosphate Crystals Microscopic Examination Triple Phosphate Crystals

Microscopic Examination Urate Crystals

Microscopic Examination Cystine Crystals