Urinalysis: Urine Formation

Urinalysis Overview

• Urinalysis involves urine formation, composition, and analysis.

Urine Formation: Nephron Structure

• The nephron includes the proximal convoluted tubule, distal convoluted tubule, Bowman's capsule, the renal corpuscles, loop of Henle, cortex and medulla.
• Urine passes through the collecting duct.

Urine Formation: Filtration

• Step 1 of urine formation is filtration.
• Plasma is filtered at the renal corpuscles in the kidneys.
• Molecules larger than 15 kDa, like proteins and cells, are not filtered.
• Filtered substances pass through endothelial pores and filtration slits.
• About 180 L/day of filtrate is produced.

Urine Formation: Reabsorption and Secretion

• Step 2 is reabsorption and secretion.
• Substances move from the filtrate back into the blood during reabsorption.
• The body selectively adds molecules from the blood to the filtrate during secretion.
• Reabsorption and secretion occur in the proximal tubule, loop of Henle, and distal tubule.
• Over 99% of plasma volume entering the kidney returns to systemic circulation.

Urine Formation: Excretion

• Step 3 is excretion.
• Filtrate remaining in the lumen at the end of the nephron is excreted as urine.
• Less than 1% of plasma volume is excreted.

Normal Urine Composition

• Urine is a liquid byproduct of metabolism.
• Urine removes toxins and wastes.
• Normal urine is composed of organic and inorganic solutes in water, with water comprising over 95%.
• Urea makes up about 50% of the total dissolved solids.
• Additional components include creatinine, uric acid, chloride, phosphate, nitrate, sodium, potassium, calcium, ammonium, hormones, vitamins, drugs, and metabolites.

Urine Composition: Urea

• Urea is a product of the metabolic breakdown of proteins and amino acids.
• Urea is synthesized in the liver by enzymes of the urea cycle.
• Urea is created in the process of eliminating toxic ammonia and is less toxic than ammonia.

Urine Composition: Creatinine

• Creatinine is a breakdown product of phosphocreatine, especially in muscles.
• Phosphocreatine can rapidly regenerate ATP during heavy activity.
• Creatinine production rate is consistent, depending on muscle mass and diet.
• The body degrades a few percent of its total creatine into creatinine daily.

Urine Composition: Uric Acid

• Uric acid is a breakdown product of purine nucleotides.
• High uric acid levels lead to gout or kidney stones.
• Gout involves painful, arthritic inflammation due to deposition of sodium urate crystals.

Abnormal Urine Composition

• Abnormal urine samples contain protein, glucose, blood, cells, crystals, casts, bacteria, or mucus.
• Abnormal urine is indicative of diseases.

Introduction to Urinalysis

• A urine specimen is easily collected and non-invasive.
• Urinalysis involves inexpensive laboratory tests.
• Urinalysis provides information about the body's metabolic functions.

Urine Sample Collection

• Types of urine specimens include morning, random, and timed collections.
• Morning urine is preferred for assessing kidney concentration and trace substance detection.
• Urine should be examined within 1-2 hours at room temperature to avoid changes in unpreserved urine.

Introduction to Urinalysis: Potential Changes in Unpreserved Urine

• Physical changes include darkened color and decreased clarity.
• Urine becomes ammoniacal with a foul smell.
• Chemical changes include increased pH and decreased glucose and ketones.

Urine Sample Preservation

• The choice of preservative depends on tests to be performed and the testing delay.
• Refrigeration at 4°C preserves urine for up to 24 hours but can induce precipitation.
• Commercial tubes with preservatives can be used.
• BD Vacutainer tubes, with chlorhexidine, ethyl paraben, and sodium propionate, can preserve samples for up to 72 hours unrefrigerated.
• Boric acid tubes with sodium formate, d-Sorbitol, and sodium acetate can stabilize samples for up to 48 hours unrefrigerated.

Abnormal Urine Types

• Oliguria is an abnormally small amount of urine from shock or kidney damage.
• Polyuria is an abnormally large amount of urine caused by diabetes.
• Dysuria is painful urination, often from urinary tract infections.
• Proteinuria is protein content from leaky or damaged glomeruli.
• Hematuria is red blood cells from infection or injury.
• Glycosuria is glucose due to excess plasma glucose or diabetes.

Types of Urinalysis

• Urinalysis includes physical examination, chemical examination, and microscopic examination.

Physical Examination of Urine

• Physical examination assesses color, odor, clarity, and specific gravity.
• The examination provides preliminary information about potential disorders.
• Physical examinations confirm or explain findings in chemical/microscopic tests.

Urine Colour

• Urine color ranges from colorless to dark yellow, indicating urine concentration.
• Color reflects hydration, ingested material, metabolic functions, and pathological conditions.
• Urobilin causes the yellow color, derived from hemoglobin breakdown.

Urine Colour Abnormalities

• Red, pink, or brown urine indicates blood.
• Blood can be due to red blood cells (cloudy), hemoglobin (clear), or myoglobin (clear).
• Relevant causes include: menstrual contamination, urinary system diseases, intravascular hemolysis, and muscle damage.
• Rifampin turns urine reddish-orange, and B-complex vitamins turn urine bright yellow.

Clarity

• Clarity is described as clear, hazy, cloudy, turbid, or milky.
• Nonpathological turbidity may come from mucus, semen, or improper preservation.
• Pathological turbidity may come from red blood cells, white blood cells, or crystals.
• Clear urine has no visible particulates.
• Hazy urine has few particulates.
• Cloudy urine has many particulates, making the print blurred through the urine.
• Turbid urine means the print cannot be seen through the urine.
• Milky urine may precipitate or be clotted.

Specific Gravity

• Specific gravity is the density of urine relative to distilled water.
• Specific gravity rapidly estimates urine concentration and molecular size of solutes.
• Specific gravity can indicate hydration, kidney function, diabetes mellitus, or disorders.

Methods for Measuring Specific Gravity

• Methods include urine dipstick tests and refractometry.
• Refractometry measures refractive index.
• As the number of solutes increases, the velocity of light decreases and the angle of light refraction decreases.
• Specific gravity of 1.000 indicates the same as pure water and adulteration.
• 1.001-1.009 points to dilute urine from increased water intake or diuresis.
• 1.010-1.025 indicates average solute and water intake.
• 1.025-1.035 indicates concentrated urine from dehydration.
• Above 1.040 suggests an iatrogenic substance like radiographic contrast media.

Chemical Examination of Urine

• Chemical examination uses urine dipstick tests.
• Dipsticks have chemically-impregnated absorbent pads attached to a plastic strip.
• A color-producing chemical reaction takes place when the strip comes in contact with urine.
• The color is compared with a chart to determine the concentration of substances.

Chemical Examination: Specific Gravity

• The dipstick test measures only ionic solutes.
• Ionic solutes cause protons to be released from the cation exchange polymer, decreasing pH.
• The test relies on color change of the bromthymol blue pH indicator.

Chemical Examination: pH

• The normal pH range of urine is 4.5 to 8.0.
• Acidic urine (pH < 4.5) can indicate respiratory acidosis, lactic acid accumulation, high-protein diets, or diabetes.
• Alkaline urine (pH > 8.0) can indicate respiratory alkalosis, vegetarian diets, contamination, or vomiting.

Chemical Examination: Glucose

• Urine normally contains minute amounts of glucose, less than 20 mg/dL.
• Almost all glucose that passes through the glomerular filtration is reabsorbed.
• The maximum reabsorptive capacity is about 350 mg/min.
• Hyperglycemia, such as that seen in diabetes mellitus or pregnancy, can cause glucose in the urine.
• The presence of glucose can be from related drugs or renal diseases.

Chemical Examination: Chemical Principle - Glucose

• Glucose in urine is detected with glucose oxidase.
• Glucose + O2 convert to gluconic acid + H2O2.
• H2O2 + chromogen (KI) convert to oxidized colored chromogen + H2O

Chemical Examination: Ketones

• Body stores of fat must be metabolized to supply energy when glucose metabolism is compromised.
• Ketones include acetone (2%), acetoacetic acid (20%), and β-hydroxybutyrate (78%).
• Ketones get released into the bloodstream.
• Ketones are excreted in urine (ketonuria) when blood concentration is above 70 mg/dL.
• Causes include diabetes mellitus, pancreatic disorders, starvation, strenuous exercise, or vomiting.

Chemical Examination: Chemical Principle - Ketones

• Key reaction is acetoacetate/acetone + sodium nitroprusside
• In alkaline conditions, this reaction produces a purple color.

Chemical Examination: Protein

• Normal urine contains very little protein because high molecular weight proteins can't to penetrate a healthy glomerular filtration barrier.
• Low-MW proteins pass through the barrier but are reabsorbed.
• Causes can be renal disorders or secondary renal disease.
• The increase in plasma can be due to muscle injury, inflammation, and hemolysis.
• Can also be due to urinary tract caused by inflammation and trauma.

Chemical Examination: Chemical Principle - Protein

• The test primarily detects albumin.
• An indicator + protein react at pH 3.0 to form protein + H+ indicator, visualized by a color change from yellow to blue-green.

Chemical Examination: Blood

• Key test is H2O2 + chromogen reacting with hemaglobin/myoglobin to produce oxidized chromogen + H2O with a green-blue color.
• Free hemoglobin/myoglobin presents with uniform color.
• RBCs present with a speckled pattern.

Chemical Examination: Significance of Blood

• Hematuria (renal and urinary malfunction) is caused by renal calculi, trauma, tumors, anticoagulants, or menstruation.
• Myoglobinuria (muscle destruction) is caused by trauma, prolonged coma, alcoholism, or cholesterol-lowering meds.
• Hemoglobinuria is derived from lysis of RBCs or from intravascular hemolysis caused by transfusion reactions, hemolytic anemias, severe burns, or infections.

Chemical Examination: Nitrite

• The test is the primary way to diagnose and monitor UTIs.
• UTIs are caused by gram-negative bacteria like E. coli.
• Nitrate converts to nitrite, a gram negative bacteria.
• Key reactions are aromatic amine + nitrite forming a diazonium salt under acidic conditions.
• The diazonium salt + tetrahydrobenzoquinolin gives rise to an azo dye that creates a pink color.

Chemical Examination: Leukocyte Esterase

• Increased numbers of leukocytes is an indication of inflammation of the urinary system from bacteria, yeast, viruses, or Trichomonas vaginalis.
• Leukocyte esterases are granules in the cytoplasm of leukocytes such as neutrophils, eosinophils, and basophils.

Chemical Examination: Leukocyte Esterase Principles

• Ester on the test pad reacts with leukocyte esterases to form aromatic compounds.
• Then, diazonium salt reacts with aromatic compound under acidic conditions to form an Azo dye.

Chemical Examination: Bilirubin

• Bilirubin is a degradation product of hemoglobin.
• Bilirubin in urine indicates early liver disease.
• The presence of bilirubin and bilirubinuria is associated with hepatitis, cirrhosis and carcinoma.
• Bilirubin glucuronide reacts with diazonium salt in acidic conditions to produce an azo dye.

Case Study - Urinalysis

• The study cites frequent urination, temperature and blood pressure, and positive tests for WBCs, nitrites and bacteria.
• Proper interpretation is key to the best initial therapy.