Urinalysis Microscopic Techniques in Clinical Microscopy

Renal Physiology

• Kidneys are the major organ of the excretory system, responsible for disposing waste products of metabolism and regulating plasma-water volume.
• Each kidney has around 1-1.5 million nephrons, capable of urine formation, with cortical and juxtamedullary nephrons.
• The main function of cortical nephrons is to remove waste products and reabsorb nutrients.
• Juxtamedullary nephrons extend into the medulla and concentrate in the urine.

Urine Formation

• Urine formation involves three steps: glomerular filtration, tubular reabsorption, and tubular secretion.
• Renal blood flow is approximately 1200 mL/min, with half of it being plasma.
• Normal plasma flow is around 600-700 mL/min.
• The glomerular tuft is the filtering apparatus, featuring eight (8) lobes of capillaries responsible for filtration.
• Glomerular filtration is non-selective, allowing substances with a molecular weight less than 70,000 kD to pass through.

Glomerular Filtration

• Glomerular filtration rate (GFR) is approximately 120 mL/min.
• The product of filtration is ultrafiltrate, with a specific gravity of 1.010.
• Non-selective filtration of plasma substances with MWs less than 70,000 kD occurs.
• Albumin is not allowed to pass through despite its MW being less than 70,000 kD due to its net electric charge and the membrane's electric charge.

Tubular Reabsorption

• Occurs mostly in the proximal tubule (PCT), transporting essential substances out of the tubular fluid back into the blood.
• Reabsorption of glucose, amino acids, electrolytes, and water occurs in the PCT.
• Substances reabsorbed in the PCT include 100% of glucose, 80-85% of nephrons, and essential nutrients.

Renal Function Tests

• Urinalysis is the first step/initial test to assess kidney function.
• Glomerular filtration rate (GFR) can be measured using creatinine clearance test (usually 24 hours).
• Sex-specific creatinine values differ between males and females.
• Urea test is the first glomerular filtration test, but not commonly used due to its flaws.

Urine Characteristics

• Urine volume is around 180 L every 24 hours.
• Urine composition is close to tissue fluid, with a specific gravity of 1.010.
• Healthy urine is negative for protein and glucose.
• Urine should always be negative for albumin or protein.### Renal Function Test
• 65% of filtrate is reabsorbed in the renal tubules, including reabsorption of sodium and potassium
• Urea is reabsorbed by the body, making it unreliable for determining GFR; creatinine is not reabsorbed and is a better marker
• Inulin clearance test is the gold standard for GFR, but it is not practical for everyday use
• Cystatin C is a marker for GFR that is produced by all nucleated cells at a constant rate

Tubular Reabsorption and Secretion

• Tubular reabsorption occurs in the ascending and descending loops of Henle and involves the reabsorption of sodium, potassium, and urea
• Tubular secretion occurs in the peritubular capillaries and involves the removal of non-filtered waste products from the blood to the filtrate
• Substances such as medication are bound to plasma carrier proteins and are too large to be filtered, but they can dissociate from the carrier proteins and be secreted into the filtrate
• Small hydrogen molecules are easily filtered and must be returned to the blood, where they combine with phosphate ions or ammonia secreted by the renal tubular cells

Regulation of Blood Pressure

• Renin is secreted in response to low blood pressure, which activates angiotensinogen to angiotensin 1
• Angiotensin 1 is converted to angiotensin 2 in the lungs, which causes vasodilation of afferent arterioles and constriction of efferent arterioles, increasing blood pressure
• Aldosterone promotes reabsorption of salt, specifically sodium, making blood pressure high

Urinalysis

• Routine urinalysis includes physical examination, chemical examination, and microscopic examination
• It is important for diagnosis of disease, monitoring progress of disease, and effectiveness of therapy
• It can screen for asymptomatic patients and detect undetected disorders

Types of Urine Specimens

• Random: collected at any time of day without specific instruction

• First Morning: collected immediately on arising

• Clean-Catch: requires patient to cleanse the genital area, void first into the toilet, then collect specimen

• Catheterized: collected via catheterization

• 24-Hour (Timed): collects all urine during a designated period, often used to measure hCG titer

• Drug Screening: requires strict chain-of-custody form requirements, and the sample must be safe and secured from collection to testing### Urine Collection Methods

• Voiding into the toilet: a process where the patient is asked to clean their genitalia with soap and water, and then urinate into a toilet

• 3-Glass Collection: a method used to diagnose prostatic infection and differentiate it from genitourinary tract infection

  • 1st vial: collects the first fraction of urine
  • 2nd vial: collects the midstream part of urine
  • 3rd vial: collects prostatic fluid with remaining urine volume (after digital prostate massage)

• Catheterized: collected from a catheter passed into the bladder

• SPA (Suprapubic Aspiration): direct puncturing of the bladder for urine collection

• Pediatric Sample: collected using a urine bag (“Wee Bag”) or pediatric bag attached to the infant's genitalia

Urine Preservation and Changes

• Preservatives: used to prevent bacterial growth and preserve the specimen
  • Chemical preservatives: e.g., thymol, boric acid, formalin, and sodium fluoride
  • Refrigeration: preserves the specimen for 2 hours
• Changes in Unpreserved Urine:
  • False increase: specific gravity, pH, urobilin, nitrite, bacteria, and odor
  • False decrease: glucose, clarity, urobilinogen, bilirubin, ketone bodies, and casts

Physical Examination of Urine

• Urine Volume:
  • Normal: 600-2000 mL/day
  • Oliguria: decreased output, less than 400 mL/day
  • Anuria: no urine output
• Nocturia: increased urine output at night (exceeding 500mL/night)
• Polyuria: increased urine output greater than 2.5L/day

Abnormalities and Conditions

• Diabetes Mellitus:
  • Polydipsia (excessive thirst)
  • Polyuria (excessive urination)
  • Polyphagia (excessive eating)
  • Low insulin/No insulin
  • Hyperglycemia
  • Glucosuria
• Diabetes Insipidus:
  • Polydipsia
  • Polyuria
  • Low sp. Gravity (as low as 1.003)
  • Decreased/absent ADH
  • No hyperglycemia
  • No glucosuria

Examination of Urine

• Urinalysis is a commonly performed laboratory test in clinical practice.
• Composition of normal urine is:
• Volume: 600-2000 ml
• Specific gravity: 1.003-1.030
• Osmolality: 300-900 mOsm/kg
• pH: 4.6-8.0

Indications for Urinalysis

• Suspected renal diseases (e.g. glomerulonephritis, nephrotic syndrome, pyelonephritis, renal failure)
• Detection of urinary tract infection
• Detection and management of metabolic disorders (e.g. diabetes mellitus)
• Differential diagnosis of jaundice
• Detection and management of plasma cell dyscrasias
• Diagnosis of pregnancy

Collection of Urine

• Methods of collection: single specimen, random specimen, post-prandial specimen
• First morning voided specimen is preferred for routine examination

Proteinuria

• Detection of microalbuminuria or early diabetic nephropathy
• Grading of albuminuria:
  • Normal: <30 mg/24 hours
  • Microalbuminuria: 30-300 mg/24 hours
  • Macroalbuminuria: >300 mg/24 hours
• Methods of quantitation:
  • Estimation of proteins in a 24-hour urine sample
  • Estimation of protein/creatinine ratio in a random urine sample

Microalbuminuria

• Significance:
  • Earliest sign of renal damage in diabetes mellitus (diabetic nephropathy)
  • Independent risk factor for cardiovascular disease in diabetes mellitus
• Methods of detection:
  • Quantitative estimation of proteins in a 24-hour urine sample
  • Estimation of protein/creatinine ratio in a random urine sample
  • Test strips for microalbuminuria

Bence Jones Proteinuria

• Definition: urinary excretion of monoclonal immunoglobulin light chains (κ or λ)
• Causes: plasma cell dyscrasias (e.g. multiple myeloma, primary amyloidosis)
• Characteristics: thermal instability, precipitation at 40-60°C, re-dissolution at 85-100°C

Glucosuria

• Definition: excretion of glucose in urine
• Causes:
  • Diabetes mellitus
  • Endocrine diseases (e.g. acromegaly, Cushing's syndrome, hyperthyroidism)
  • Non-endocrine diseases (e.g. central nervous system diseases, liver disorders)
  • Drugs (e.g. adrenocorticotrophic hormone, corticosteroids, thiazides)
  • Alimentary glycosuria (Lag-storage glycosuria)
• Tests:
  • Benedict's qualitative test
  • Clinitest tablet method
  • Reagent strip method

Ketones

• Definition: breakdown products of fatty acids
• Causes:
  • Decreased utilization of carbohydrates
  • Increased metabolic needs
  • Decreased availability of carbohydrates in the diet
• Tests:
  • Rothera's test
  • Acetest tablet method
  • Ferric chloride test
  • Reagent strip test

Bilirubin

• Definition: yellow pigment produced during hemoglobin breakdown
• Urine test for bilirubin:
  • Present in obstructive and hepatocellular jaundice
  • Absent in hemolytic jaundice
• Table 1.6: Urine bilirubin and urobilinogen in jaundice### Bilirubin Tests
• Bilirubin is a breakdown product of hemoglobin and is present in two forms: conjugated and unconjugated
• Unconjugated bilirubin is bound to albumin, is not water-soluble, and cannot pass through glomeruli, therefore it does not appear in urine
• Conjugated bilirubin is water-soluble, is filtered by glomeruli, and appears in urine
• Detection of bilirubin in urine is helpful in the differential diagnosis of jaundice

Ferric Chloride Test (Gerhardt's)

• Addition of 10% ferric chloride solution to urine causes a reddish or purplish color if acetoacetic acid is present
• The test is not specific and certain drugs can give a similar reaction
• Sensitivity of the test is 25-50 mg/dl

Reagent Strip Test

• Modifications of nitroprusside test
• Sensitivity is 5-10 mg/dl of acetoacetate
• Reagent strips are vulnerable to improper storage and can give false-negative results

Bilirubin Tests in Urine

• Foam test: development of yellowish foam indicates presence of bilirubin
• Gmelin's test: play of colors indicates positive test
• Lugol iodine test: development of green color indicates positive test
• Fouchet's test: development of blue-green color around the drop indicates presence of bilirubin

Bile Salts

• Salts of four different types of bile acids: cholic, deoxycholic, chenodeoxycholic, and lithocholic
• Bile salts enter the small intestine through bile and act as detergents to emulsify fat and reduce surface tension
• In the terminal ileum, bile salts are absorbed and enter the blood stream, then taken up by the liver and re-excreted in bile (enterohepatic circulation)

Urobilinogen

• Formed in the intestine through bacterial action on bilirubin
• A portion of urobilinogen is absorbed in blood, undergoes recycling (enterohepatic circulation), and a small amount is excreted in urine
• Normally about 0.5-4 mg of urobilinogen is excreted in urine in 24 hours
• Urinary excretion of urobilinogen shows diurnal variation with highest levels in the afternoon

Tests for Urobilinogen

• Ehrlich's aldehyde test: reaction with urobilinogen produces a pink color
• Reagent strip method: specific for urobilinogen
• Watson-Schwartz test: used to distinguish between urobilinogen and porphobilinogen

Hematuria

• Presence of abnormal number of intact red blood cells in urine
• Implies presence of a bleeding lesion in the urinary tract
• Causes: diseases of urinary tract, hematological conditions, and hemoglobinuria

Tests for Blood in Urine

• Microscopic examination of urinary sediment
• Chemical tests: benzidine test, orthotoluidine test, and reagent strip test
• Reagent strip test: detects both intracellular and extracellular hemoglobin

Hemosiderin

• Presence of free hemoglobin in plasma

• Appears as blue granules when urine sediment is stained with Prussian blue stain

• Indicates intravascular hemolysis or hemoglobinuria### Screening Tests for Myoglobinuria

• Myoglobin is soluble in 80% saturated solution of ammonium sulfate, while hemoglobin is insoluble and precipitates.

• A positive chemical test for blood done on supernatant indicates myoglobinuria.

Chemical Tests for Significant Bacteriuria

• Leucocyte esterase test detects esterase enzyme released in urine from granules of leucocytes.
• A positive test indicates pyuria, and urine culture should be done.

Microscopic Examination of Urine

• Microscopic examination of urine is also known as the "liquid biopsy of the urinary tract".
• Urine consists of various microscopic, insoluble, solid elements in suspension.
• These elements are classified as organized and unorganized substances.

Urinary Sediments

• Organized substances include red blood cells, white blood cells, epithelial cells, casts, bacteria, and parasites.
• Unorganized substances are crystalline and amorphous materials.
• Examination of urinary deposit is helpful in diagnosis of urinary tract diseases.

Differentiation between Hematuria, Hemoglobinuria, and Myoglobinuria

• Distinction between hematuria, hemoglobinuria, and myoglobinuria is based on urine color, plasma color, urine test, urine microscopy, serum haptoglobin, and serum creatine kinase.

Microscopic Examination of Urine Sediment

• Specimen: A mid-stream, freshly voided, first morning specimen is preferred.
• Method: A well-mixed sample of urine is centrifuged, and the supernatant is poured off.
• The tube is tapped at the bottom to resuspend the sediment, and a drop is placed on a glass slide and covered with a cover slip.

Urinary Findings in Renal Diseases

• Different conditions such as acute glomerulonephritis, nephrotic syndrome, and acute pyelonephritis have distinct urinary findings.

Red Blood Cells

• Normally, there are no or an occasional red blood cell in urine.
• Red cells appear as small, smooth, yellowish, anucleate biconcave disks about 7 μ in diameter.
• In glomerulonephritis, red cells are typically described as being dysmorphic.

White Blood Cells (Pus Cells)

• White blood cells are spherical, 10-15 μ in size, granular in appearance, with a visible nucleus.
• Presence of many white cells in urine is called pyuria.
• Clumps of pus cells or pus cells >10/HPF are suggestive of urinary tract infection.

Casts

• Urinary casts are cylindrical, cigar-shaped microscopic structures that form in distal renal tubules and collecting ducts.
• Casts are composed of a precipitate of a protein that is secreted by tubules (Tamm-Horsfall protein).
• Different types of casts include hyaline, granular, waxy, and fatty casts.
• Presence of casts is indicative of disease of the renal parenchyma.

Bacteria

• Bacteria in urine can be detected by microscopic examination, reagent strip tests, and culture.
• Significant bacteriuria exists when there are >105 bacterial colony forming units/ml of urine in a clean-catch midstream sample.

Examination of Urine

• Urinalysis is a commonly performed laboratory test in clinical practice.
• Composition of normal urine is:
• Volume: 600-2000 ml
• Specific gravity: 1.003-1.030
• Osmolality: 300-900 mOsm/kg
• pH: 4.6-8.0

Indications for Urinalysis

• Suspected renal diseases (e.g. glomerulonephritis, nephrotic syndrome, pyelonephritis, renal failure)
• Detection of urinary tract infection
• Detection and management of metabolic disorders (e.g. diabetes mellitus)
• Differential diagnosis of jaundice
• Detection and management of plasma cell dyscrasias
• Diagnosis of pregnancy

Collection of Urine

• Methods of collection: single specimen, random specimen, post-prandial specimen
• First morning voided specimen is preferred for routine examination

Proteinuria

• Detection of microalbuminuria or early diabetic nephropathy
• Grading of albuminuria:
  • Normal: <30 mg/24 hours
  • Microalbuminuria: 30-300 mg/24 hours
  • Macroalbuminuria: >300 mg/24 hours
• Methods of quantitation:
  • Estimation of proteins in a 24-hour urine sample
  • Estimation of protein/creatinine ratio in a random urine sample

Microalbuminuria

• Significance:
  • Earliest sign of renal damage in diabetes mellitus (diabetic nephropathy)
  • Independent risk factor for cardiovascular disease in diabetes mellitus
• Methods of detection:
  • Quantitative estimation of proteins in a 24-hour urine sample
  • Estimation of protein/creatinine ratio in a random urine sample
  • Test strips for microalbuminuria

Bence Jones Proteinuria

• Definition: urinary excretion of monoclonal immunoglobulin light chains (κ or λ)
• Causes: plasma cell dyscrasias (e.g. multiple myeloma, primary amyloidosis)
• Characteristics: thermal instability, precipitation at 40-60°C, re-dissolution at 85-100°C

Glucosuria

• Definition: excretion of glucose in urine
• Causes:
  • Diabetes mellitus
  • Endocrine diseases (e.g. acromegaly, Cushing's syndrome, hyperthyroidism)
  • Non-endocrine diseases (e.g. central nervous system diseases, liver disorders)
  • Drugs (e.g. adrenocorticotrophic hormone, corticosteroids, thiazides)
  • Alimentary glycosuria (Lag-storage glycosuria)
• Tests:
  • Benedict's qualitative test
  • Clinitest tablet method
  • Reagent strip method

Ketones

• Definition: breakdown products of fatty acids
• Causes:
  • Decreased utilization of carbohydrates
  • Increased metabolic needs
  • Decreased availability of carbohydrates in the diet
• Tests:
  • Rothera's test
  • Acetest tablet method
  • Ferric chloride test
  • Reagent strip test

Bilirubin

• Definition: yellow pigment produced during hemoglobin breakdown
• Urine test for bilirubin:
  • Present in obstructive and hepatocellular jaundice
  • Absent in hemolytic jaundice
• Table 1.6: Urine bilirubin and urobilinogen in jaundice### Bilirubin Tests
• Bilirubin is a breakdown product of hemoglobin and is present in two forms: conjugated and unconjugated
• Unconjugated bilirubin is bound to albumin, is not water-soluble, and cannot pass through glomeruli, therefore it does not appear in urine
• Conjugated bilirubin is water-soluble, is filtered by glomeruli, and appears in urine
• Detection of bilirubin in urine is helpful in the differential diagnosis of jaundice

Ferric Chloride Test (Gerhardt's)

• Addition of 10% ferric chloride solution to urine causes a reddish or purplish color if acetoacetic acid is present
• The test is not specific and certain drugs can give a similar reaction
• Sensitivity of the test is 25-50 mg/dl

Reagent Strip Test

• Modifications of nitroprusside test
• Sensitivity is 5-10 mg/dl of acetoacetate
• Reagent strips are vulnerable to improper storage and can give false-negative results

Bilirubin Tests in Urine

• Foam test: development of yellowish foam indicates presence of bilirubin
• Gmelin's test: play of colors indicates positive test
• Lugol iodine test: development of green color indicates positive test
• Fouchet's test: development of blue-green color around the drop indicates presence of bilirubin

Bile Salts

• Salts of four different types of bile acids: cholic, deoxycholic, chenodeoxycholic, and lithocholic
• Bile salts enter the small intestine through bile and act as detergents to emulsify fat and reduce surface tension
• In the terminal ileum, bile salts are absorbed and enter the blood stream, then taken up by the liver and re-excreted in bile (enterohepatic circulation)

Urobilinogen

• Formed in the intestine through bacterial action on bilirubin
• A portion of urobilinogen is absorbed in blood, undergoes recycling (enterohepatic circulation), and a small amount is excreted in urine
• Normally about 0.5-4 mg of urobilinogen is excreted in urine in 24 hours
• Urinary excretion of urobilinogen shows diurnal variation with highest levels in the afternoon

Tests for Urobilinogen

• Ehrlich's aldehyde test: reaction with urobilinogen produces a pink color
• Reagent strip method: specific for urobilinogen
• Watson-Schwartz test: used to distinguish between urobilinogen and porphobilinogen

Hematuria

• Presence of abnormal number of intact red blood cells in urine
• Implies presence of a bleeding lesion in the urinary tract
• Causes: diseases of urinary tract, hematological conditions, and hemoglobinuria

Tests for Blood in Urine

• Microscopic examination of urinary sediment
• Chemical tests: benzidine test, orthotoluidine test, and reagent strip test
• Reagent strip test: detects both intracellular and extracellular hemoglobin

Hemosiderin

• Presence of free hemoglobin in plasma

• Appears as blue granules when urine sediment is stained with Prussian blue stain

• Indicates intravascular hemolysis or hemoglobinuria### Screening Tests for Myoglobinuria

• Myoglobin is soluble in 80% saturated solution of ammonium sulfate, while hemoglobin is insoluble and precipitates.

• A positive chemical test for blood done on supernatant indicates myoglobinuria.

Chemical Tests for Significant Bacteriuria

• Leucocyte esterase test detects esterase enzyme released in urine from granules of leucocytes.
• A positive test indicates pyuria, and urine culture should be done.

Microscopic Examination of Urine

• Microscopic examination of urine is also known as the "liquid biopsy of the urinary tract".
• Urine consists of various microscopic, insoluble, solid elements in suspension.
• These elements are classified as organized and unorganized substances.

Urinary Sediments

• Organized substances include red blood cells, white blood cells, epithelial cells, casts, bacteria, and parasites.
• Unorganized substances are crystalline and amorphous materials.
• Examination of urinary deposit is helpful in diagnosis of urinary tract diseases.

Differentiation between Hematuria, Hemoglobinuria, and Myoglobinuria

• Distinction between hematuria, hemoglobinuria, and myoglobinuria is based on urine color, plasma color, urine test, urine microscopy, serum haptoglobin, and serum creatine kinase.

Microscopic Examination of Urine Sediment

• Specimen: A mid-stream, freshly voided, first morning specimen is preferred.
• Method: A well-mixed sample of urine is centrifuged, and the supernatant is poured off.
• The tube is tapped at the bottom to resuspend the sediment, and a drop is placed on a glass slide and covered with a cover slip.

Urinary Findings in Renal Diseases

• Different conditions such as acute glomerulonephritis, nephrotic syndrome, and acute pyelonephritis have distinct urinary findings.

Red Blood Cells

• Normally, there are no or an occasional red blood cell in urine.
• Red cells appear as small, smooth, yellowish, anucleate biconcave disks about 7 μ in diameter.
• In glomerulonephritis, red cells are typically described as being dysmorphic.

White Blood Cells (Pus Cells)

• White blood cells are spherical, 10-15 μ in size, granular in appearance, with a visible nucleus.
• Presence of many white cells in urine is called pyuria.
• Clumps of pus cells or pus cells >10/HPF are suggestive of urinary tract infection.

Casts

• Urinary casts are cylindrical, cigar-shaped microscopic structures that form in distal renal tubules and collecting ducts.
• Casts are composed of a precipitate of a protein that is secreted by tubules (Tamm-Horsfall protein).
• Different types of casts include hyaline, granular, waxy, and fatty casts.
• Presence of casts is indicative of disease of the renal parenchyma.

Bacteria

• Bacteria in urine can be detected by microscopic examination, reagent strip tests, and culture.
• Significant bacteriuria exists when there are >105 bacterial colony forming units/ml of urine in a clean-catch midstream sample.