Urine complete Analysis

Sample

1. The urine may be random or morning samples.
2. Urine must be tested within 1 to 2 hours of collection.

Precautions

1. Urine can be refrigerated for 6 to 8 hours.
   1. At low temperatures, there will be precipitation of amorphous urates or phosphates.
2. Bring the refrigerated urine to room temperature before testing.
3. Urine collected for more than 2 hours may be rejected.
4. The decomposition of urine begins in 30 minutes at room temperature and 4 hours in refrigeration.
5. For longer periods, boric acid can be used as a preservative.

Complete Urine Analysis

The volume of urine:

1. It is 600 to 2500 ml /24 hours, and the average volume is 1200 ml/24 hours.
2. Polyuria when urine amount exceeds 2000 mL/24 hours.
   1. This is seen in diabetes mellitus.
   2. In diabetes Insipidus.
   3. Excessive fluid intake.
   4. Drugs like diuretic therapy.
3. Oliguria when urine volume is less than 500 mL/ 24 hours. This is seen in the following conditions.
   1. Renal tubular dysfunction.
   2. Edema.
   3. End-stage kidneys.
   4. Obstructive uropathy.
   5. Dehydration.
   6. Vomiting.
   7. Shock.
   8. Diarrhea.
4. Anuria when there is no urine output. This is seen in the following conditions:
   1. Acute renal failure.
   2. Heart failure.
   3. Acute Glomerulonephritis.
   4. Obstructive uropathy.
   5. Shock.

1. urine output can be calculated as follows:
   1. Adult = >0.5 mL/Kg/hour.
   2. Child = >1 mL/Kg/hour .
   3. Neonate  (< 1-year-old) = >2 mL/Kg/hour.
2. Urine volume varies according to age.
   1. Urine volume is 600 to 2500 mL/day, and the average is 1200 mL/day.
   2. At night urine volume is usually <700 mL and specific gravity more than 1.018.
   3. The ratio of the night to day urine volume is 1:2 to 1:4.
   4. The range of 600 to 2000 mL/day is considered normal.
3. Table showing the variation of the urine volume in various age groups:
   

   Various groups	Various age groups	Normal volume per day
   Newborn	1 to 4 days	30 to 60 mL
   	3 to 10 days	100 to 300 mL
   Infants	60 days to one year	400 to 500 mL
   Children	1 to 3 years	500 to 600 mL
   	8 to 14 days	800 to 1400 mL
   Middle-aged	Male	800 to 1800 mL
   	Female	600 to 1600 mL
   Old people		250 to 2400 mL
   Urine volume

Color of urine:

1. The normal color is Pale to dark yellow.
2. Abnormal Color may be due to some of the foods and medicines.
   1. Urine with no color may be caused by long-term kidney disease or uncontrolled diabetes.
   2. Dark yellow urine can be caused by dehydration.
   3. Red urine can be caused by blood in the urine.
   4. The Orange color is due to bilirubin.
   5. The dark yellow color may be seen due to bilirubin and urobilin.
   6. Brown and black color may be seen due to melanin, homogentisic acid, and biliary pigments.
   7. Green color urine may be seen due to Biliverdin, pseudomonas infection, and oxidized bilirubin.
3. Cloudy urine can be caused by :
   1. pus (white blood cells).
   2. Blood (red blood cells).
   3. Sperm, Bacteria, and yeast.
   4. Crystals.
   5. Mucus.
   6. Parasite infection, such as trichomoniasis.
4. Red color urine may be:
   1. When clear, this will indicate:
      1. Hemoglobinuria.
      2. Myoglobinuria.
   2. When red cloudy urine indicates:
      1. 1. Hematuria.
            

            Urine colors in various conditions

            Various colors of urine in different conditions:

            Color of the urine	Effect of the foods	Color change due to drugs	Medical causes for the color
            Orange	Carrots Vitamin C rich foods	Phenazopyridine Rifampicin Isoniazid	Liver diseases
            Green	Asparagus	Vitamin B12	Pseudomonas infection
            Brown	Fava beans	Metronidazole Chloroquine Levodopa Nitrofurantoin	Liver diseases Tyrosinemia Gilbert’s syndrome
            Purple	Beets	Rifampicin	Infection in  patients with an indwelling catheter
            White		Propofol	Pyuria Phosphate crystalluria Chyluria
            Black		Methyldopa Phenol derivatives Metronidazole	Alkaptonuria Malignant melanoma
            Brown-black	Iron use
            Red (pink)	Blackberries Beets	Chlorpromazine Thioridazine Rifampin	Hemoglobinuria Porphyrias Urinary tract infection Nephrolithiasis
            Blue		Indomethacin Amitriptyline Methylene blue	Malabsorption Tryptophan
            Blue-green	Asparagus	Vitamin B 12 Propofol
            Bright yellow	Riboflavin

Clarity of urine:

1. Normally it is Clear. No visible particles are seen.
   1. Hazy urine where there are few particulates, and through urine, print can be seen easily.
   2. Cloudy when there are many particulates, and through urine, the print is blurred.
   3. Turbid urine when through urine print cannot be seen.
   4. Milky where there are many particulates, and it may be clotted.
2. Urine turbidity is seen in nonpathological conditions like:
   1. With the presence of squamous epithelial cells.
   2. The presence of mucus.
   3. The presence of spermatozoa.
   4. Amorphous phosphates, urates, and carbonates.
   5. The case of contrast media.
   6. The case of fecal contamination.
3. Urine turbidity is seen in pathological conditions like:
   1. White blood cells.
   2. Red blood cells.
   3. Presence of bacteria.
   4. Presence of yeast.
   5. Abnormal crystals.
   6. Lymph fluids and lipids.
      

      Urine analysis showing turbidity

Turbid urine solubility in various conditions:

Solubility  in various conditions	Substances/ chemicals solubilized
In alkaline urine	Amorphous phosphates and carbonates
In acidic urine	Amorphous urates and radiographic contrast media
With heat	Amorphous urates and uric acid crystals
Soluble in dilute acetic acid	RBCs, amorphous urates, and carbonates
Insoluble in dilute acetic acid	Spermatozoa, bacteria, yeast, and WBCs
Soluble in ether	lipids, chyle, and lymphatic fluid

1. In Alkaline urine, amorphous phosphate and carbonates are seen.
2. In acidic urine, amorphous urates, and radiographic contrast media.
3. Soluble by heat are amorphous urates and uric acid crystals.

The odor of urine:

1. The odor is not clinically significant.  This is not part of the urine analysis.
2. Normally odor is a slightly “nutty” odor (faint, aromatic odor).
   1. On keeping the urine, the ammonia odor is more prominent. This is because of the breakdown of the urea.
      

      Urine giving an ammonia smell

3. Abnormal Odor may be due to:
   1. Some foods, such as asparagus, onions, garlic, cause a pungent odor.
   2. Vitamins and antibiotics (such as penicillin) can cause urine to have a different odor.
   3. A sweet, fruity odor may be caused by uncontrolled diabetes mellitus.
   4. A urinary tract infection (UTI) can cause a bad, unpleasant odor.
   5. Urine that smells like maple syrup means maple syrup urine disease when the body cannot break down certain amino acids.
   6. Phenylketonuria gives a mousy smell.
   7. Bleach like odor is due to contamination.
      

      Various types of odor (smell)	Etiology of the odor
      Aromatic	Normal
      Fruity,  sweet	Ketone (in diabetes mellitus) Starvation Vomiting
      Mousy (musty odor)	Phenylketonuria
      Foul, ammonia-like	Bacterial contamination Urinary tract infection
      Rancid (unpleasant smell)	Tyrosinemia
      Maple syrup	Maple syrup urine disease
      Bleech	Contamination
      Cabbage like	Methionine malabsorption
      Sweety feet	Isovaleric acidemia
      Fetid odor	Infection and may be due to E.Coli.
      Bleach

The specific gravity of urine:

1. The kidneys’ ability to selectively reabsorb essential chemicals and water from the glomerular filtrate is one of the most important functions.
2. If the urine is refrigerated, then bring it to room temperature.
3. Specific gravity is the density of a substance relative to the density of distle water.
4. The normal range of urine specific gravity = 1.005 to 1.030
   1. Normal sample ranges between 1.010 to 1.025.
   2. Specific gravity is higher in the first-morning specimen (>1.020).
      

      Urine specific gravity formula

5. The normal range of urine specific gravity = 1.005 – 1.030.
   1. Neonates = 1.012.
   2. Infants = 1.002 to 1.006.
   3. Adults = 1.003 to 1.030.
6. The urinometer measures specific gravity.
   1. To check the urinometer’s reliability, put this into the water, and it should read 1.000.
   2. It can check by using the know specific gravity solution.

7. A urine refractometer measures the refractive index of the urine.
   7. Its principle is that the refractive index compares the velocity of the light in air with the velocity of light in a solution.
   8. The dissolved particle concentration in the solution will determine the velocity and angle at which the light will pass through the solution.
   9. The refractometer’s advantage is that it uses only 1 to 2 drops of the urine when the urinometer needs more.

 

Abnormal Specific Gravity classification:

Types of specific gravity	Etiology or causes
Consistent low specific gravity  = 1.010  (Isosthenuria)	Chronic renal diseases Impairment of the renal function The specific gravity of urine is like plasma filtrate.
Very low specific gravity (hyposthenuria) = <1.010 , urine is diluted	Diabetes insipidus (Lake of antidiuretic hormone) Drinking too much fluids Severe kidney diseases due to tubular damage Glomerulonephritis Pyelonephritis Use of diuretics
A very high specific gravity  (Hypersthenuria) = >1.010, Urine is is concentrated	Lake of during enough amount of the water Loss of too much fluid like excessive vomiting, sweating, or diarrhea Substances (such as sugar or protein) in the urine (diabetes mellitus) Adrenal insufficiency. Hepatic disease. Congestive cardiac failure

The pH of the urine:

1. Normal pH = 4.6  to  8.0.
2. The average person has a pH of 5 to 6.
   

   Urine analysis pH normal range

3. pH is helpful for the identification of crystals.
4. pH is helpful for the acid-base status.
   

   Control of Urine pH by the kidneys and the lungs

    

   

   Urine pH role in acid-base balance

   

   Urine role in pH control

5. Factors affecting the urine pH:
   1. Acidic urine is produced in:
      1. High protein diet.
      2. Lung diseases when CO 2 is retained in the body.
      3. Diabetes mellitus in uncontrolled diabetics.
   2. Conditions producing alkaline urine :
      1. Urinary tract infection.
      2. Bacterial contamination of the urine.
      3. Diet high in vegetables, citrus fruits,  or dairy products makes more alkaline urine.
      4. Hyperventilation and loss of CO 2.
      5. When treated by NaHCO3 and K citrate along with antibiotics in treating the urinary tract infection.
6. Abnormal pH.
   1. Some foods (such as citrus fruit and dairy products) and medicines (such as antacids) can affect urine pH.
      

      The pH of the urine	Etiology (causes)
      A highly alkaline urine	Severe vomiting Kidneys disease Urinary tract infection Asthma
      A low acidic urine	Severe lung disease  like emphysema Uncontrolled diabetes mellitus Aspirin overdose High protein diet severe diarrhea dehydration Starvation Excessive use of alcohol drinking antifreeze (ethylene glycol)

7. Significance of pH:
   1. Maintenance of acidic urine can be good to treat urinary tract infections caused by urea splitting microbes. Because these bacteria can not grow in an acid medium.
   2. People on a high protein diet produce acidic urine, people on a vegetarian diet produce alkaline urine.
   3. The exception is cranberry produces acidic urine, and it can be used as a home remedy for urinary tract infection.
   4. Alkaline urine is seen in patients with renal calculi made of calcium phosphate or magnesium-ammonium phosphate.
8. Measurement of urine pH:
   1. There are various commercially available urine reagent strips to measure pH—these measure pH between 5 to 9.
   2. Indicators are methyl red and bromthymol blue.
   3. Methyl red changes color from red to yellow with a pH change of 4 to 6.
   4. Bromthymol blue changes from yellow to blue with the change in pH from 6 to 9.
      

      Urine pH reagent strip

Proteins in the urine:

1. Normally urine is negative.
   1. It is <10 mg/dL or
   2. Around 100 mg/24 hours.
2. The protein consists of primarily low molecular weight serum proteins that are filtered by the glomeruli.
   1. Albumin is the major protein found in normal urine.
   2. The tubules reabsorb the majority of the albumin.
   3. Proteinuria may be seen after vigorous exercise, dehydration, and fever.
   4. Benign proteinuria is postural and is intermittent; this accounts for 5% of the people.
3. Proteinuria may be classified based on the origin of the protein into:
   1. Prerenal proteinuria:
      1. This is not indicative of kidney disease.
      2. This is due to the increased level of low-molecular-weight plasma proteins such as hemoglobin, myoglobin, and acute-phase protein.
      3. This is caused by  nonrenal diseases and is transient; it is seen in:
         1. Hemoglobinuria.
         2. Myoglobinuria.
         3. Acute phase proteinuria.
         4. This is usually not detected by the routine urine reagent strips.
   2. Renal proteinuria. This is due to renal diseases involving glomeruli or tubules.
      1. 1. In glomerular damage, selective filtration is impaired, and an increased amount of albumin and red blood cells, and white cells are excreted in the urine.
         2. It is seen in:
            1. SLE.
            2. Streptococcal glomerulonephritis.
            3. Strenuous exercise  (reversible condition).
            4. Pre-eclampsia and hypertension. (reversible condition).
            5. Toxic heavy metals.
            6. Severe viral infection.
            7. Microalbuminuria is seen in diabetic nephropathy indicating kidney damage in diabetes mellitus.
   3. Postrenal proteinuria. Proteins can be added as the urine passes through the ureter, urinary bladder, urethra, prostate, and vagina.
      1. 1. Bacterial and fungal infection of the lower urinary tract,
         2. Menstrual contamination also contains proteins.
         3. Prostatic fluid and spermatozoa.
         4.  Bacterial and fungal infections and inflammation produce exudate containing protein from the interstitial fluids.
         5. The presence of blood resulting from injury or menstrual contamination contributes to protein, as does the presence of prostatic fluid and a large number of spermatozoa.
   4. Orthostatic or postural proteinuria. This is a persistent benign condition frequently seen in young patients.
      1. 1. It appears when the person is upright and disappears when the patient lies down.
         2. Procedure to confirm the diagnosis:
            1. These patients are advised to empty their bladder before going to bed.
            2. Take the first urine sample when patients get up.
            3. Take another sample when patients upright for several hours.
               1. The first sample will be negative, and the second sample will be positive in orthostatic proteinuria.
   5. Microalbuminuria. This is seen in diabetic nephro[athy.
      
      1. 1. This microalbuminuria indicates diabetic complications.
         2. The further complication of diabetes can be prevented by controlling diabetes.
         3. This is also associated with an increased risk of cardiovascular diseases.
         4. Microalbuminuria is reported as albumin excreted as µg/min or in mg/24 hours.
            1. The level is significant from 20 to 200 µg/min Or 30 to 300 mg/24 hours.
            2. In these cases, the albumin/creatinine ratio is >3.4 mg/mmol.
            3. The first-morning specimen is recommended.
   6. Bence Jones proteinuria is seen in multiple myeloma patients.
      1. 1. These are monoclonal light chain immunoglobulins.
         2. This is a low molecular weight protein and is excreted in the urine.
         3. Procedure to detect BJ protein:
            1. Heat the urine, and these proteins coagulate at 40 to 60 °C.
            2. Dissolve when the temperature reaches 100 °C.
               1. Reading the result: Urine turbid at 40 to 60 °C and clear at 100 °C is considered positive.

Summary of the proteinuria:

Type of the proteinuria	Etiology of the proteinuria
Prerenal proteinuria	Multiple myeloma Severe infection and inflammation Intravascular hemolysis Muscle injury
Renal Proteinuria	Glomerular disorders: Amyloidosis Immune complex disorders Diabetic nephropathy Toxic agents Pre-eclampsia Hypertension Dehydration Orthostatic (postural) proteinuria Tubular diseases Severe viral infections Fanconi’s syndrome Heavy metal poisoning Toxic agents injury
Postrenal proteinuria	Injury and trauma Lower urinary tract infections and inflammation vaginal secretion menstrual contamination prostatic fluid Contamination by the spermatozoa

 

1. Method to detect proteinuria:
   1. The urine dipstick reagents detect urine protein. This is a semiquantitative test.
      1. Trace positive reaction = 15 to 30 mg/dL.
      2. When 4+ positive reaction = 2.0g/dL.
      3. Trace proteinuria = 10 to 30 mg/dL.
         1. 1+ = 30 mg/dL.
         2. 2+ = 100 mg/dL
         3. 3+ = 300 mg/dL
         4. 4+ = >1000 mg/dL
   2. Sulfosalicylic acid is a precipitation method.
      1. Transfer 3 mL of centrifuged urine into a test tube.
      2. Add 3 drops of sulfosalicylic acid (250 g/L) along the side of the test tube.
      3. The acid will form a layer underneath the urine.
      4. Do not mix, and let it remain for one minute.
      5. Reading:
         1. There is barely perceptible turbidity = reported as traces or ± (roughly 5 mg/dL).
         2. The heavier turbidity graded = 1+ to 4+.
            1. No turbidity = 0 mg/dL (<6 mg/dL)
            2. Slightly turbid = Traces, 20 mg/dL (6 to 30 mg/dL)
            3. 1+ = 50 mg/dL (Print visible through specimen, 30 to 100 mg/dL).
            4. 2+ = 200 mg/dL  (Print invisible, 100 to 200 mg/dL).
            5. 3+ = 500 mg/dL (Flocculation, 200 to 400 mg/dL).
            6. 4+ = >1000 mg/dL (Dense precipitate, >400 mg/dL).
               

               Urine protein reagent strip

   3. Trichloroacetic acids (TCA) is also a precipitation method. This method is used in automation.
      1. TCA precipitate urine proteins as a fine suspension. The turbidity of the urine suspension is proportional to the amount of proteins in the urine.
      2. Procedure:
         1. Take TCA 12.5 g/dL.
         2. Dissolve it in the distle water and makeup to 100 mL.
         3. Prepare the solution fresh every month.
         4. Make the standard protein solution in the sodium chloride.
      3. Take the urine clear of the turbidity; if needed, can centrifuge the urine.
         1. Take 4 mL of urine, 4 mL of the standard, and 4 ml of urine as blank.
         2. Add TCA 1.0 mL.
         3. Add 1.0 mL of NaCl (9 g/L) to all three test tubes.
         4. After 5 to 10 minutes, read all three tubes at 420 nm.
         5. Calculation formula =
            

            Urine TCA protein calculation

            

            Urine TCA procedure

1. 4. Heat coagulation test:
      1. Centrifuge the urine, and make urine clear.
      2. Take 7.5 mL of urine in heat resistant test tube.
      3. Add 3  drops of 5M acetate buffer (pH 4.0 ). Can make this buffer by mixing 24 ml of glacial acetic acid and 7.5 g of anhydrous sodium acetate in 100 ml of distle water, and mix well.
      4. Reading the result:
         1. If precipitate form at this stage will indicate mucin (a mucoprotein).
         2. Centrifuge and remove this precipitate.
         3. Now heat the upper half of the urine, and bring to boiling.
            

            Urine heat test for the protein

         4. Now examine for turbidity, comparing the upper half to the lower half.
            1. Hardly visible turbidity = 1+ (10 to 30mg/dL) or  ±.
            2. Moderate turbidity = 2+ (40 to 100 mg/dL).
            3. Heavy turbidity = 3+ (200 to 500 mg/dL).
         5. A flocculant precipitate = 4+ (>500 mg’dL).
   5. Postural (Orthostatic) proteinuria test:
      4. Ask the patient to empty the bladder before going into bed.
      5. Collect the urine immediately after getting up in the morning.
      6. Get a second sample after the patient is in the vertical position for several hours.
      7. Check the protein in both samples.
         4. Postural (Orthostatic) proteinuria positive case=
            4. First-morning sample =   negative for urine proteins.
            5. Second sample             = Positive for urine protein.

 Table showing proteinuria grading: 

1. The degree of proteinuria	Protein excreted per 24 hours
   Mild	<1.0 g /24 hours
   Moderate	1.0 to 3 or 4 g /24 hours
   Severe	> 3 g / 24 hours

Various urine test for proteins and their characteristics:

Characteristics	Heat and acid	Reagent strip	Salfosalicylic acid
Protein detected Minimum level	Albumin = 5 to 10 mg/dL Bence-Jones clears with boiling. Globulins	Albumin = 5 to 10 mg/dL	Albumin = 0.25 mg/dL Bence-Jones Glycoproteins Globulins
Proteins not detected		Bence-Jones Globulins
False-negative result	Highly alkaline urine	Very dilute urine High salt concentration	Highly alkaline urine
False-positive result	PO4 and urates may confuse Drugs metabolites like: Sulfonamides Tolbutamide Penicillin high doses	Skin disinfectants	PO4 and urates may confuse Drugs metabolites like: Sulfonamides Tolbutamide Penicillin Chlorpromazine Cephalothin

 

Classification of the proteinuria:

Type of proteinuria	Amount of protein excreted in urine	Characteristic features
Minimal proteinuria	<0.5 g/24 hours of urine	Protein in the urine is<500 mg/day This is due to: Chronic pyelonephritis Polycystic kidney disease Renal tubular disease Diseases of the lower urinary tract
Moderate proteinuria	).5 to 4 g/24 hours of urine	Protein in the urine is 0.4 to 4 g/day This is due to: Nephrotic syndrome Acute and chronic glomerulonephritis chronic pyelonephritis Nephrosclerosis Amyloidosis Multiple myeloma Pre-eclampsia of pregnancy Toxic nephropathy Venous congestion of the kidney Inflammatory condition of the lower urinary tract, urinary bladder. Urinary bladder stones
Marked proteinuria	>4 g/24 hours of the urine	protein in the urine is >4 g/day. This is due to: Nephrotic syndrome Acute and chronic glomerulonephritis Nephrosclersis Amyloidosis Severe venous congestion of the kidney Systemic lupus erythematosus
Postural proteinuria (Orthostatic)	<1.0 g/24 hours of the urine	This is persistent benign proteinuria Frequently seen in young adults It appears when the person in a vertical position and disappears in a horizontal position
Functional proteinuria	Benign and transient	This is usually transient This is a benign condition This is due to: Associated with fever Emotional stress Exposure to cold Excessive exercise

1. Protein in the urine may be due to the following conditions:
   1. Kidney damage.
   2. Infection.
   3. Cancer.
   4. High blood pressure.
   5. Diabetes.
   6. The systemic lupus erythematosus (SLE).
   7. Glomerulonephritis.
   8. Heart failure.
   9. Leukemia.
   10. Poison (lead or mercury poisoning).
   11. High blood pressure during pregnancy (preeclampsia).
   12. Protein in the urine may mean :
   13. Kidney damage.
   14. Infection.
   15. Cancer.
   16. High blood pressure.
   17. Diabetes.
   18. The systemic lupus erythematosus (SLE).
   19. Glomerulonephritis.
   20. Heart failure.
   21. Leukemia.
   22. Poison (lead or mercury poisoning).
   23. High blood pressure during pregnancy (preeclampsia).
   24. Proteinuria can be classified into different grades according to the amount of protein in the urine.

Beta-2-Microglobulin:

1. It is structurally related to Immunoglobulin. It is used to differentiate Glomerular from tubular dysfunction.

    Glucose in urine:

1. the healthy individual has a blood glucose of 100 mg/dL or less.
2. This condition may be benign due to emotional stress or the ingestion of a large meal.
3. When due to pathological condition then is seen in the diabetes mellitus.
4. Normally urine is negative.
5. Urine glucose is detected by:
   1. The dipstick.
   2. The copper sulfate method (Clinitest).
6. Under normal circumstances, almost all glucose filtered by the glomerulus is reabsorbed in the proximal convoluted tubules.
   1. The renal threshold of glucose: The level at which the tubular reabsorption is stopped for glucose is 160 to 180 mg/dL, called the renal threshold. But mostly,
      it is considered around 180 mg/dL.
      

      Urine glucose appears after the renal threshold

   2. The main function of insulin is to convert glucose to glycogen for storage (glycogenesis).
   3. The opposing hormone causes glycogen’s breakdown to glucose (glycogenolysis), leading to an increased circulating glucose level in the blood.
   4. Epinephrine is a strong inhibitor of insulin secretion.
      1. Epinephrine is secreted in response to severe stress, cerebrovascular trauma, and myocardial infarction.
   5. Renal glycosuria may be seen when the renal tubule absorption of glucose  is compromised; this may be seen in:
      1. End-stage renal disease.
      2. Osteomalacia.
      3. Fanconi’s syndrome.
   6. Method to detect glucose in the urine:
      1. Copper reduction method:
         1. This test was used in the early days.
         2. This test is based on the reduction of copper sulfate (CuSO4) to cuprous oxide in the presence of alkali and heat.
         3. Reading: The color changes from blue (negative = CuSO4) to green, yellow, orange/red (CuO).
         4. Urine glucose benedict reaction:
            

            Benedicts reaction principle

1. 1. 2. Glucose-oxidase reagent strips:
         1. Glucose-oxidase detects specific glucose in the urine only, while copper reduction detects other reducing substances as well.
         2. The result is reported as 1+, 2+, 3+, 4+ and these colors also provide quantitative measurements ranging from 100 mg/dL to 2000 mg/dL or 0.1% to 2%.
            

            Urine glucose oxidase method principle

Comparison of Benedict reaction and Oxidase method:

Characteristics	Benedict reaction (CuSO4)	Glucose oxidase
Minimum level detected	Glucose 50 to 250 mg/dL	Glucose 50 mg/dL
Other sugars detected	Galactose Lactose Fructose Pentose Maltose
False-negative		Ascorbic acid (Vitamin C) A large number of salicylates Homogentisic acid
False-positive	Vitamin C (Ascorbic acid) Salicylates Many antibiotics Levodopa X-ray contrast media Homogentisic acid

1.  Glucose in urine is seen in:
   1. Intravenous (IV) fluids.
   2. Uncontrolled diabetes.
   3. Adrenal gland diseases.
   4. Liver damage.
   5. Brain Injury.
   6. Poisoning.
   7. Kidney diseases.
   8. Pregnant women can have glucose in their urine, which is normal during pregnancy.
2. Hyperglycemia may be seen in the nondiabetic cases where hormones act against the insulin, seen in conditions like:
   1. Pancreatitis.
   2. Pancreatic cancers.
   3. Cushing’s syndrome.
   4. Acromegaly.
   5. Hyperthyroidism.
   6. Pheochromocytoma.

Ketones in urine:

1. Ketones bodies are mainly three intermediate products from the lipolysis, and these are excreted in the urine:
   1. Acetone 2%.
   2. Acetoacetate  20%.
   3. β-hydroxybutyric acid 78%.
      

      Ketone bodies in diabetic patients

2. Normally the measurable amount of ketones don’t appear in the urine. All metabolized fat is broken down into carbon dioxide (CO2) and water (H2O).
   1. When there is no carbohydrate for the body’s energy, then stores of the fat are metabolized to supply energy, and ketone bodies appear in the urine.
   2. The reason for the fat metabolism is the inability to metabolized carbohydrates, as seen in the:
      1. Diabetes Mellitus.
      2. Vomiting.
      3. Inadequate intake of carbohydrates as in starvation.
   3. Ketonuria indicates a deficiency of insulin and can regulate the dose of insulin.
   4. Ketones are excreted in combination with basic ions (Na⁺, K⁺. and Ca⁺⁺), so ketonuria produces ketoacidosis.
   5. These patients have a fruity odor in their breath because acetone is very volatile blown off from the lungs.
   6. Normally urine is negative.
   7. It has importance in Diabetes Mellitus and particularly in uncontrolled diabetes mellitus patients.
      

      Ketone bodies formation

      

      Ketone bodies formation

3. Procedure to detect ketones bodies:
   1. Ketone test strips or tablets are used based on sodium nitroprusside (nitroferricyanide) reaction to measure ketone bodies.
      1. This will detect acetoacetic acid or acetone in alkaline conditions.
      2. β-hydroxybutyric acid does not react with this reagent.
      3. Reporting of the result:
      4. Color changes from lavender to maroon (purple).
      5. Acetoacetate+acetone is expressed as :
      6. Negative = 0 mg/dL
      7. Small amount =  10 mg/dL
      8. Moderate amount = 30 mg/dL
      9. Large amount = 80 mg/dL

1. 1. Rothera’s test:
      1. Grind fine powder of :
         1. 10 g Na2CO3 (anhydrous).
         2.  20 g (NH4)2SO4.
         3. 0.5 g sodium nitroprusside.
         4. Mix the powder completely and stopper it tightly.
      2. Procedure:  Take about 0.5 g of the above powder on a late surface.
         1. Add 2 drops of the urine to the powder.
         2. Ketone positive urine will change the color from lavender to purple in 30 seconds.
            

            Rothera’s test for ketones in the urine

2. Abnormal Ketonuria
   1. uncontrolled diabetes.
   2. Very low-carbohydrate diet.
   3. Starvation.
   4. Eating disorders, e.g., anorexia nervosa or bulimia.
   5. Alcoholism.
   6. Poisoning from drinking rubbing alcohol (isopropanol).
   7. Ketones are often found in the urine during fasting for 18 hours or longer.
   8. The sick person cannot eat or vomits for several days.
   9. Low levels of ketones are sometimes found in the urine of healthy pregnant women.

Bilirubin in urine:

1. Bilirubin is a yellow-orange bile pigment compound formed when RBCs are hemolysed or ruptured.
2. The appearance of the bilirubin in the urine indicates liver disease and is called bilirubinuria.
3. Bilirubin is a pigmented yellow compound, which is a degradation product of hemoglobin.
   1. It is detected long before jaundice clinically appears.
4. It is negative in normal urine.
   1. Negative in hemolytic jaundice.
5. Increased bilirubinuria is seen in the hepatic and obstructive jaundice.
   

   Bilirubin metabolism and excretion in the urine

   

   Bilirubin metabolism and excretion in the urine

   6. Conjugated bilirubin appears in the urine when the normal degradation cycle is disrupted by obstruction of the bile duct-like gall stones or cancer.
      1. Another possibility is when the liver cells are damaged and allowing the leakage of bilirubin directly into the circulation.
      2. Hepatitis and cirrhosis are common causes of liver cell damage and resulting in bilirubinuria.
      3. Increased hemolysis does not produce bilirubinuria.
   7. Bilirubin in the urine is detected only if the urine sample is fresh because bilirubin decomposes rapidly in bright light.
   8. If the urine is left in the lab, then bilirubin is converted into biliverdin, a green compound by the light not detected by the bilirubin strips.
   9. Clinical importance of the bilirubin in the urine (bilirubinuria):
      1. Hepatitis (infectious).
      2. Liver diseases.
      3. Cirrhosis.
      4. Gall bladder stones  (cholelithiasis) or cancer-causing obstruction.
   10. Normal:
       1. Urine bilirubin is negative (0 to 0.2 mg/dL (0 to 0.34 µmol/L).
       2. Bilirubin can be detected in urine by the Foam test.
   11. Procedure for the foam test:
       1. Place a small volume of the urine in the test tube, cap it, and shake it vigorously.
       2. Result:
          1. The foam at the top is white means bilirubin is negative.
          2. If the foam is orange, color means bilirubin is present.
   12. Other methods to detect the bilirubin in the urine are:
       1. Diazo reaction in the form of tablets or Dipstick.
   13. Fouchet’s test (Ferric chloride test.): This is basically an oxidizing reaction.

1. 14. Another modification of Fouchet’s test for urine bilirubin:
       
       1. Composition of the Fouchet’s reagent:
          1. Trichloracetic acid = 25 g
          2. Ferric chloride = 10% aquos solution.
          3. Distle water = 100 mL
       2. procedure: Take 10 mL of urine.
          1. Add 1 g barium chloride.
          2. Mix and filter.
          3. Leave the precipitate on filter paper and let it dry.
          4. Add Fouchet’s reagent or yellow nitric acid to the precipitate.
             

             Fouchet’s modified reaction

Comparison of Fouchet’s, Ferric chloride, and Diazo reaction for Bilirubin:

1. 1. 1. Characteristic feature	Fouchet’s test (Ferric chloride test)	Diazo reaction (tablet or dipstick)
         Sensitivity	Many compounds give different colors to FeCl3.	Tablet = 0.2 to 0.4 mg/dL Dipstick = 0.0 to 0.1 mg/dL
         False-negative result	If delayed, then bilirubin is oxidized.	High level of nitrite Vitamin C (Ascorbic acid) If the test is delayed >4 hours, then bilirubin is oxidized.
         False-positive result	Urobilin Urobilinogen Aspirin metabolites	Chlorpromazine

Urobilinogen in urine:

1. Urobilinogen is like bilirubin bile pigment, which is the result of the hemoglobin degradation product.
2. Urobilinogen is a colorless compound derived from bilirubin., it is excreted into the bile and partially hydrogenated by bacteria in the intestine.
   1. It is produced in the intestine by bacterial action on the bilirubin and shown in the above diagram.
      1. The intestine reabsorbs approximately 50% of urobilinogen.
      2. Rest excreted in the feces.
      3. A small amount in enterohepatic circulation passed from the kidney and excreted in the urine as urobilin.
      4. This is why urobilinogen is positive normally in the urine.
3. Urobilirubin excreted from 0.5 to 4 mg /24 hours.
   1. <1 mg/dL is normally found in the urine. Atleast 0.1 mg/dL is needed for the detection.
4. How to check in the urine:
   1. The reagent strip for urobilinogen involves the Ehrlich aldehyde reaction. This will form the red azo dye.
   2. This test must be done on the fresh sample because urobilinogen is an unstable compound and breakdown into urobilin on standing.
5. Significance:
   1. It helps in the early detection of liver diseases.
   2. It is an increase in hemolytic disorder.
   3. In hepatic jaundice, liver disorder, hepatitis, cirrhosis, and cancers.
   4. It is absent in obstructive jaundice.
      

      Urobilinogen metabolism and excretion into stool and urine

1. The normal :
   1. Normal value = 0.2 to 1.0 mg/dL.
   2. The level of 2 mg/dL is the cut-off point for the result to be abnormal.
   3. There are 0 to 4 mg /24 hours.
   1. 1. Random urine = 0.1 to 1 Ehrlich U/dL or <1 mg/dL.
         1. 2 hours urine = 0.1 to 1.0 Ehrlich U/2 hours or <1 mg/2 hours.
         2. 24 hours urine = 0.5 to 4.0 Ehrlich U/24 hours or 0.5 to 4.0 mg/24 hours.
2. Increased level of urobilinogen is seen in:
   1. Hemolytic anemia.
   2. Pernicious (megaloblstic) anemia.
   3. Malarial attack.
   4. Excessive bruising.
   5. Pulmonary infarction.
   6. Cirrhosis.
   7. Acute hepatitis.
   8. Cholangitis.
3. A decreased level of urobilinogen is seen in:
   1. Complete or partial obstruction of the biliary tract.
   2. Cholelithiasis.
   3. Biliary duct inflammation.
   4. Cancer of the head of the pancreas.
   5. Antibiotic therapy will suppress intestinal bacterial flora.

The difference between Bilirubin and urobilinogen in the urine:

Chemical	In normal  condition	Hepatic diseases	Biliary obstruction	Hemolytic  disease
Urine bilirubin	Negative	positive or negative	Positive (+++)	Negative
Urine urobilinogen	Normal	Increased (++)	Absent or low (normal)	Increased (+++)

Microscopic analysis:

1. Normal  microscopic findings:
   1. Normally Very few or no red or white blood cells or casts are seen.
   2. No bacteria, yeast cells, or parasites are present.
   3. A few crystals are normally seen.

Abnormal Microscopic findings

(Please see more detail in part 6 urine analysis)

RBCs in urine:

1. The positive blood test indicates the presence of RBCs.
2. Hematuria is a disorder of renal or genitourinary origin where the bleeding is due to trauma or damage to the organs.
   1. The main causes are:
      1. Renal calculi.
      2. Glomerular diseases.
      3. Malignancies.
      4. Pyelonephritis.
      5. Trauma.
3. Hemoglobinuria may result from the lysis of RBCs. There will be mixed hemoglobinuria and hematuria.
   1. In alkaline urine, there may be lysis of RBCs in the urinary tract.
   2. Intravascular hemolysis, no RBCs are seen in the urine.
4. Myoglobinuria is a heme-containing protein found in the muscles. This is seen in Rhabdomyolysis like :
   1. Trauma.
   2. Crush syndrome.
   3. Convulsions.
   4. Muscle-wasting disease.
   5. Heroin abuse.
   6. Prolonged come.
   7. Extensive exertion.
5. Red blood cells in the urine may be caused by:
   1. Kidney or bladder injury.
   2. Kidney stones.
   3. Urinary tract infection (UTI).
   4. Inflammation of the kidneys (glomerulonephritis).
   5. Kidney or bladder tumor.
   6. An autoimmune disease like systemic lupus erythematosus (SLE).

Lab test	Myoglobinuria	Hemoglobinuria
Serum Creatinine kinase	Increased	Normal
Serum LDH	Increased	Normal
Urine Precipitation test supernatant	Positive (Red color)	Negative

White blood cells in urine:

1. Pus cells in the urine may be caused by :
   1. Urinary tract infection.
   2. Urinary bladder tumor.
   3. Inflammation of the kidneys.
   4. The systemic lupus erythematosus (SLE).
   5. Inflammation in the vagina or under the foreskin of the penis.
      

      urine microscopy, bacteria, and polys

CAST in urine:

1. Casts mean inflammation or damage to :
   1. The tiny tubes in the kidneys.
   2. Poor blood supply to the kidneys.
   3. Poisoning (such as lead or mercury).
   4. Heart failure.
   5. Bacterial infection.
2. Depending on the types. There are the following types.
   1. Granular.
   2. Hyline.
   3. Cellular.
   4. Epithelial cast.
      

      Types of Urine casts

   5. Waxy cast.

Crystals in urine:

1. The presence of large amounts of crystals or certain types of crystals indicates :
   1. Kidney stones.
   2. Damaged kidneys.
   3. Problems with metabolism.
   4. Medicines.
   5. Some types of urinary tract infections can also increase the number of crystals in the urine.

Types of crystals	Acidic urine	Alkaline urine	Soluble with heat
Amorphous urates	present	absent	yes
Uric acid crystals	present	absent	yes
Contrast media	present
Amorphous phosphates	absent	present
Amorphous carbonates	absent	present

Bacteria in urine:

1. In the urine mean a urinary tract infection (UTI).

Yeast cells or parasites

1. Such as the parasite that causes trichomoniasis, which means an infection of the urinary tract.

 

Normal urine picture:

Physical features	Chemical features	Microscopic findings
Color = Pale yellow or amber Appearance = Clear to slightly hazy pH = 4.5 to 8.0 Specific gravity = 1.015 to 1.025	Blood = Negative Glucose = Negative Ketones= Negative Protein = Negative Bilirubin = Negative Urobilinogen = Negative (±) Leucocyte esterase = Negative Nitrite for bacteria = Negative	RBCs = Rare or Negative WBC = Rare or Negative Epithelial cells = Few Cast = Negative (Occasional hyaline) Crystal = Negative (Depends upon the pH of the urine) Bacteria = Negative

Note: The details of urine analysis see in parts 4, 5, and 6.

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