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Chemical examination of feces is usually carried out for the following tests (Figure 845.1):

  • Occult blood
  • Excess fat excretion (malabsorption)
  • Urobilinogen
  • Reducing sugars
  • Fecal osmotic gap
  • Fecal pH
Figure 845.17 Chemical examinations done on fecal sample
Figure 845.1 Chemical examinations done on fecal sample

Test for Occult Blood in Stools

Presence of blood in feces which is not apparent on gross inspection and which can be detected only by chemical tests is called as occult blood. Causes of occult blood in stools are:

  1. Intestinal diseases: hookworms, amebiasis, typhoid fever, ulcerative colitis, intussusception, adenoma, cancer of colon or rectum.
  2. Gastric and esophageal diseases: peptic ulcer, gastritis, esophageal varices, hiatus hernia.
  3. Systemic disorders: bleeding diathesis, uremia.
  4. Long distance runners.

Occult blood test is recommended as a screening procedure for detection of asymptomatic colorectal cancer. Yearly examinations should be carried out after the age of 50 years. If the test is positive, endoscopy and barium enema are indicated.

Tests for detection of occult blood in feces: Many tests are available which differ in their specificity and sensitivity. These tests include tests based on peroxidase-like activity of hemoglobin (benzidine, orthotolidine, aminophenazone, guaiac), immunochemical tests, and radioisotope tests.

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Tests Based on Peroxidase-like Activity of Hemoglobin

Principle: Hemoglobin has peroxidase-like activity and releases oxygen from hydrogen peroxide. Oxygen molecule then oxidizes the chemical reagent (benzidine, orthotolidine, aminophenazone, or guaiac) to produce a colored reaction product.

Benzidine and orthotolidine are carcinogenic and are no longer used. Benzidine test is also highly sensitive and false-positive reactions are common. Since bleeding from the lesion may be intermittent, repeated testing may be required.

Causes of False-positive Tests

  1. Ingestion of peroxidase-containing foods like red meat, fish, poultry, turnips, horseradish, cauliflower, spinach, or cucumber. Diet should be free from peroxidase-containing foods for at least 3 days prior to testing.
  2. Drugs like aspirin and other anti-inflammatory drugs, which increase blood loss from gastrointestinal tract in normal persons.

Causes of False-negative Tests

  1. Foods containing large amounts of vitamin C.
  2. Conversion of all hemoglobin to acid hematin (which has no peroxidase-like activity) during passage through the gastrointestinal tract.

Immunochemical Tests

These tests specifically detect human hemoglobin. Therefore there is no interference from animal hemoglobin or myoglobin (e.g. meat) or peroxidase-containing vegetables in the diet.

The test consists of mixing the sample with latex particles coated with anti-human haemoglobin antibody, and if agglutination occurs, test is positive. This test can detect 0.6 ml of blood per 100 grams of feces.

Radioisotope Test Using 51Cr

In this test, 10 ml of patient’s blood is withdrawn, labeled with 51Cr, and re-infused intravenously. Radioactivity is measured in fecal sample and in simultaneously collected blood specimen. Radioactivity in feces indicates gastrointestinal bleeding. Amount of blood loss can be calculated. Although the test is sensitive, it is not suitable for routine screening.

Apt test: This test is done to decide whether blood in the vomitus or in the feces of a neonate represents swallowed maternal blood or is the result of bleeding in the gastrointestinal tract. The test was devised by Dr. Apt and hence the name. The baby swallows blood during delivery or during breastfeeding if nipples are cracked. Apt test is based on the principle that if blood is of neonatal origin it will contain high proportion of hemoglobin F (Hb F) that is resistant to alkali denaturation. On the other hand, maternal blood mostly contains adult hemoglobin or Hb A that is less resistant.

Test for Malabsorption of Fat

Dietary fat is absorbed in the small intestine with the help of bile salts and pancreatic lipase. Fecal fat mainly consists of neutral fats (unsplit fats), fatty acids, and soaps (fatty acid salts). Normally very little fat is excreted in feces (<7 grams/day in adults). Excess excretion of fecal fat indicates malabsorption and is known as steatorrhea. It manifests as bulky, frothy, and foul-smelling stools, which float on the surface of water.

Causes of Malabsorption of Fat

  1. Deficiency of pancreatic lipase (insufficient lipolysis): chronic pancreatitis, cystic fibrosis.
  2. Deficiency of bile salts (insufficient emulsification of fat): biliary obstruction, severe liver disease, bile salt deconjugation due to bacterial overgrowth in the small intestine.
  3. Diseases of small intestine: tropical sprue, celiac disease, Whipple’s disease.

Tests for fecal fat are qualitative (i.e. direct microscopic examination after fat staining), and quantitative (i.e. estimation of fat by gravimetric or titrimetric analysis).

  1. Microscopic stool examination after staining for fat: A random specimen of stool is collected after putting the patient on a diet of >80 gm fat per day. Stool sample is stained with a fat stain (oil red O, Sudan III, or Sudan IV) and observed under the microscope for fat globules (Figure 845.2). Presence of ≥60 fat droplets/HPF indicates steatorrhea. Ingestion of mineral or castor oil and use of rectal suppositories can cause problems in interpretation.
  2. Quantitative estimation of fecal fat: The definitive test for diagnosis of fat malabsorption is quantitation of fecal fat. Patient should be on a diet of 70-100 gm of fat per day for 6 days before the test. Feces are collected over 72 hours and stored in a refrigerator during the collection period. Specimen should not be contaminated with urine. Fat quantitation can be done by gravimetric or titrimetric method. In gravimetric method, an accurately weighed sample of feces is emulsified, acidified, and fat is extracted in a solvent; after evaporation of solvent, fat is weighed as a pure compound. Titrimetric analysis is the most widely used method. An accurately weighed stool sample is treated with alcoholic potassium hydroxide to convert fat into soaps. Soaps are then converted to fatty acids by the addition of hydrochloric acid. Fatty acids are extracted in a solvent and the solvent is evaporated. The solution of fat made in neutral alcohol is then titrated against sodium hydroxide. Fatty acids comprise about 80% of fecal fat. Values >7 grams/day are usually abnormal. Values >14 grams/day are specific for diseases causing fat malabsorption.
Figure 845.2 Sudan stain on fecal sample
Figure 845.2 Sudan stain on fecal sample: (A) Negative; (B) Positive

Test for Urobilinogen in Feces

Fecal urobilinogen is determined by Ehrlich’s aldehyde test (see  Article “Test for Detection of Urobilinogen in Urine). Specimen should be fresh and kept protected from light. Normal amount of urobilinogen excreted in feces is 50-300 mg per day. Increased fecal excretion of urobilinogen is seen in hemolytic anemia. Urobilinogen is deceased in biliary tract obstruction, severe liver disease, oral antibiotic therapy (disturbance of intestinal bacterial flora), and aplastic anemia (low hemoglobin turnover). Stools become pale or clay-colored if urobilinogen is reduced or absent.

Test for Reducing Sugars

Deficiency of intestinal enzyme lactase is a common cause of malabsorption. Lactase converts lactose (in milk) to glucose and galactose. If lactase is deficient, lactose is converted to lactic acid with production of gas. In infants this leads to diarrhea, vomiting, and failure to thrive. Benedict’s test or Clinitest™ tablet test for reducing sugars is used to test freshly collected stool sample for lactose. In addition, oral lactose tolerance test is abnormal (after oral lactose, blood glucose fails to rise above 20 mg/dl of basal value) in lactase deficiency. Rise in blood glucose indicates that lactose has been hydrolysed and absorbed by the mucosa. Lactose tolerance test is now replaced by lactose breath hydrogen testing. In lactase deficiency, accumulated lactose in the colon is rapidly fermented to organic acids and gases like hydrogen. Hydrogen is absorbed and then excreted through the lungs into the breath. Amount of hydrogen is then measured in breath; breath hydrogen more than 20 ppm above baseline within 4 hours indicates positive test.

Fecal Osmotic Gap

Fecal osmotic gap is calculated from concentration of electrolytes in stool water by formula 290-2([Na+] + [K+]). (290 is the assumed plasma osmolality). In osmotic diarrheas, osmotic gap is >150 mOsm/kg, while in secretory diarrhea, it is typically below 50 mOsm/kg. Evaluation of chronic diarrhea is shown in Figure 845.3.

Figure 845.3 Evaluation of chronic diarrhea
Figure 845.3 Evaluation of chronic diarrhea

Fecal pH

Stool pH below 5.6 is characteristic of carbohydrate malabsorption.

Tests to Assess Proximal Tubular Function
 
Renal tubules efficiently reabsorb 99% of the glomerular filtrate to conserve the essential substances like glucose, amino acids, and water.
 
1. Glycosuria: In renal glycosuria, glucose is excreted in urine, while blood glucose level is normal. This is because of a specific tubular lesion which leads to impairment of glucose reabsorption. Renal glycosuria is a benign condition. Glycosuria can also occur in Fanconi syndrome.
 
2. Generalized aminoaciduria: In proximal renal tubular dysfunction, many amino acids are excreted in urine due to defective tubular reabsorption.
 
3. Tubular proteinuria (Low molecular weight proteinuria): Normally, low molecular weight proteins2 –microglobulin, retinol-binding protein, lysozyme, and α1 –microglobulin) are freely filtered by glomeruli and are completely reabsorbed by proximal renal tubules. With tubular damage, these low molecular weight proteins are excreted in urine and can be detected by urine protein electrophoresis. Increased amounts of these proteins in urine are indicative of renal tubular damage.
 
4. Urinary concentration of sodium: If both BUN and serum creatinine are acutely increased, it is necessary to distinguish between prerenal azotemia (renal underperfusion) and acute tubular necrosis. In prerenal azotemia, renal tubules are functioning normally and reabsorb sodium, while in acute tubular necrosis, tubular function is impaired and sodium absorption is decreased. Therefore, in prerenal azotemia, urinay sodium concentration is < 20 mEq/L while in acute tubular necrosis, it is > 20 mEq/L.
 
5. Fractional excretion of sodium (FENa): Measurement of urinary sodium concentration is affected by urine volume and can produce misleading results. Therefore, to avoid this, fractional excretion of sodium is calculated. This refers to the percentage of filtered sodium that has been absorbed and percentage that has been excreted. Measurement of fractional sodium excretion is a better indicator of tubular absorption of sodium than quantitation of urine sodium alone.
 
This test is indicated in acute renal failure. In oliguric patients, this is the most reliable means of early distinction between pre-renal failure and renal failure due to acute tubular necrosis. It is calculated from the following formula:
 
 
(Urine sodium × Plasma creatinine) × 100%
(Plasma sodium × Urine creatinine)
 
 
In pre-renal failure this ratio is less than 1%, and in acute tubular necrosis it is more than 1%. In pre-renal failure (due to reduced renal perfusion), aldosterone secretion is stimulated which causes maximal sodium conservation by the tubules and the ratio is less than 1%. In acute tubular necrosis, maximum sodium reabsorption is not possible due to tubular cell injury and consequently the ratio will be more than 1%. Values above 3% are strongly suggestive of acute tubular necrosis.
 
Tests to Assess Distal Tubular Function
 
1. Urine specific gravity: Normal specific gravity is 1.003 to 1.030. It depends on state of hydration and fluid intake.
 
  1. Causes of increased specific gravity:
    a. Reduced renal perfusion (with preservation of concentrating ability of tubules),
    b. Proteinuria,
    c. Glycosuria,
    d. Glomerulonephritis.
    e. Urinary tract obstruction.
  2. Causes of reduced specific gravity:
    a. Diabetes insipidus
    b. Chronic renal failure
    c. Impaired concentrating ability due to diseases of tubules.
 
As a test of renal function, it gives information about the ability of renal tubules to concentrate the glomerular filtrate. This concentrating ability is lost in diseases of renal tubules.
 
Fixed specific gravity of 1.010, which cannot be lowered or increased by increasing or decreasing the fluid intake respectively, is an indication of chronic renal failure.
 
2. Urine osmolality: The most commonly employed test to evaluate tubular function is measurement of urine/plasma osmolality. This is the most sensitive method for determination of ability of concentration. Osmolality measures number of dissolved particles in a solution. Specific gravity, on the other hand, is the ratio of mass of a solution to the mass of water i.e. it measures total mass of solute. Specific gravity depends on both the number and the nature of dissolved particles while osmolality is exact number of solute particles in a solution. Specific gravity measurement can be affected by the presence of solutes of large molecular weight like proteins and glucose, while osmolality is not. Therefore measurement of osmolality is preferred.
 
When solutes are dissolved in a solvent, certain changes take place like lowering of freezing point, increase in boiling point, decrease in vapor pressure, or increase of osmotic pressure of the solvent. These properties are made use of in measuring osmolality by an instrument called as osmometer.
 
Osmolality is expressed as milliOsmol/kg of water.
 
Urine/plasma osmolality ratio is helpful in distinguishing pre-renal azotemia (in which ratio is higher) from acute renal failure due to acute tubular necrosis (in which ratio is lower). If urine and plasma osmolality are almost similar, then there is defective tubular reabsorption of water.
 
3. Water deprivation test: If the value of baseline osmolality of urine is inconclusive, then water deprivation test is performed. In this test, water intake is restricted for a specified period of time followed by measurement of specific gravity or osmolality. Normally, urine osmolality should rise in response to water deprivation. If it fails to rise, then desmopressin is administered to differentiate between central diabetes insipidus and nephrogenic diabetes insipidus. Urinary concentration ability is corrected after administration of desmopressin in central diabetes insipidus, but not in nephrogenic diabetes insipidus.
 
If urine osmolality is > 800 mOsm/kg of water or specific gravity is ≥1.025 following dehydration, concentrating ability of renal tubules is normal. However, normal result does not rule out presence of renal disease.
False result will be obtained if the patient is on low-salt, low-protein diet or is suffering from major electrolyte and water disturbance.
 
4. Water loading antidiuretic hormone suppression test: This test assesses the capacity of the kidney to make urine dilute after water loading.
 
After overnight fast, patient empties the bladder and drinks 20 ml/kg of water in 15-30 minutes. The urine is collected at hourly intervals for the next 4 hours for measurements of urine volume, specific gravity, and osmolality. Plasma levels of antidiuretic hormone and serum osmolality should be measured at hourly intervals.
 
Normally, more than 90% of water should be excreted in 4 hours. The specific gravity should fall to 1.003 and osmolality should fall to < 100 mOsm/kg. Plasma level of antidiuretic hormone should be appropriate for serum osmolality. In renal function impairment, urine volume is reduced (<80% of fluid intake is excreted) and specific gravity and osmolality fail to decrease. The test is also impaired in adrenocortical insufficiency, malabsorption, obesity, ascites, congestive heart failure, cirrhosis, and dehydration.
 
This test is not advisable in patients with cardiac failure or kidney disease. If there is failure to excrete water load, fatal hyponatremia can occur.
 
5. Ammonium chloride loading test (Acid load test): Diagnosis of renal tubular acidosis is usually considered after excluding other causes of metabolic acidosis. This test is considered as a ‘gold standard’ for the diagnosis of distal or type 1 renal tubular acidosis. Urine pH and plasma bicarbonate are measured after overnight fasting. If pH is less than 5.4, acidifying ability of renal tubules is normal. If pH is greater than 5.4 and plasma bicarbonate is low, diagnosis of renal tubular acidosis is confirmed. In both the above cases, further testing need not be performed. In all other cases in which neither of above results is obtained, further testing is carried out. Patient is given ammonium chloride orally (0.1 gm/kg) over 1 hour after overnight fast and urine samples are collected hourly for next 6-8 hours. Ammonium ion dissociates into H+ and NH3. Ammonium chloride makes blood acidic. If pH is less than 5.4 in any one of the samples, acidifying ability of the distal tubules is normal.
Normally, a very small amount of albumin is excreted in urine. The earliest evidence of glomerular damage in diabetes mellitus is occurrence of microalbuminuria (albuminuria in the range of 30 to 300 mg/24 hours). An albuminuria > 300-mg/24 hour is termed clinical or overt and indicates significant glomerular damage. (See “Proteinuria” under Article “Chemical Examination of Urine”).
Two biochemical parameters are commonly used to assess renal function: blood urea nitrogen (BUN) and serum creatinine. Although convenient, they are insensitive markers of glomerular function.
 
Blood Urea Nitrogen (BUN)
 
Urea is produced in the liver from amino acids (ingested or tissue-derived). Amino acids are utilized to produce energy, synthesize proteins, and are catabolized to ammonia. Urea is produced in the liver from ammonia in the Krebs urea cycle. Ammonia is toxic and hence is converted to urea, which is then excreted in urine (Figure 842.1).
 
Figure 842.1 Formation of urea from protein breakdown
Figure 842.1 Formation of urea from protein breakdown 
 
The concentration of blood urea is usually expressed as blood urea nitrogen. This is because older methods estimated only the nitrogen in urea. Molecular weight of urea is 60, and 28 grams of nitrogen are present in a gm mole of urea. As the relationship between urea and BUN is 60/28, BUN can be converted to urea by multiplying BUN by 2.14, i.e. the real concentration of urea is BUN × (60/28).
 
Urea is completely filtered by the glomeruli, and about 30-40% of the filtered amount is reabsorbed in the renal tubules depending on the person’s state of hydration.
 
Blood level of urea is affected by a number of non-renal factors (e.g. high protein diet, upper gastrointestinal hemorrhage, liver function, etc.) and therefore utility of BUN as an indicator of renal function is limited. Also considerable destruction of renal parenchyma is required before elevation of blood urea can occur.
 
The term azotemia refers to the increase in the blood level of urea; uremia is the clinical syndrome resulting from this increase. If renal function is absent, BUN rises by 10-20 mg/dl/day.
 
Causes of increased BUN:
 
  1. Pre-renal azotemia: shock, congestive heart failure, salt and water depletion
  2. Renal azotemia: impairment of renal function
  3. Post-renal azotemia: obstruction of urinary tract
  4. Increased rate of production of urea:
    • High protein diet
    • Increased protein catabolism (trauma, burns, fever)
    • Absorption of amino acids and peptides from a large gastrointestinal hemorrhage or tissue hematoma
 
Methods for estimation of BUN:
 
Two methods are commonly used.
 
  1. Diacetyl monoxime urea method: This is a direct method. Urea reacts with diacetyl monoxime at high temperature in the presence of a strong acid and an oxidizing agent. Reaction of urea and diacetyl monoxime produces a yellow diazine derivative. The intensity of color is measured in a colorimeter or spectrophotometer.
  2. Urease- Berthelot reaction: This is an indirect method. Enzyme urease splits off ammonia from the urea molecule at 37°C. Ammonia generated is then reacted with alkaline hypochlorite and phenol with a catalyst to produce a stable color (indophenol). Intensity of color produced is then measured in a spectrophotometer at 570 nm.
 
Reference range for BUN in adults is 7-18 mg/dl. In adults > 60 years, level is 8-21 mg/dl.
 
Serum Creatinine
 
Creatinine is a nitrogenous waste product formed in muscle from creatine phosphate. Endogenous production of creatinine is proportional to muscle mass and body weight. Exogenous creatinine (from ingestion of meat) has little effect on daily creatinine excretion.
 
Serum creatinine is a more specific and more sensitive indicator of renal function as compared to BUN because:
 
  • It is produced from muscles at a constant rate and its level in blood is not affected by diet, protein catabolism, or other exogenous factors;
  • It is not reabsorbed, and very little is secreted by tubules.
 
With muscle mass remaining constant, increased creatinine level reflects reduction of glomerular filtration rate. However, because of significant kidney reserve, increase of serum creatinine level (from 1.0 mg/dl to 2.0 mg/dl) in blood does not occur until about 50% of kidney function is lost. Therefore, serum creatinine is not a sensitive indicator of early renal impairment. Also, laboratory report showing serum creatinine “within normal range” does not necessarily mean that the level is normal; the level should be correlated with body weight, age, and sex of the individual. If renal function is absent, serum creatinine rises by 1.0 to 1.5 mg/dl/day (Figure 842.2).
 
Figure 842.2 Relationship between glomerular filtration rate and serum creatinine
Figure 842.2 Relationship between glomerular filtration rate and serum creatinine. Significant increase of serum creatinine does not occur till a considerable fall in GFR

Causes of Increased Serum Creatinine Level
 
  1. Pre-renal, renal, and post-renal azotemia
  2. Large amount of dietary meat
  3. Active acromegaly and gigantism
 
Causes of Decreased Serum Creatinine Level
 
  1. Pregnancy
  2. Increasing age (reduction in muscle mass)
 
Methods for Estimation of Serum Creatinine
 
The test for serum creatinine is cheap, readily available, and simple to perform. There are two methods that are commonly used:
 
  1. Jaffe’s reaction (Alkaline picrate reaction): This is the most widely used method. Creatinine reacts with picrate in an alkaline solution to produce spectrophotometer at 485 nm. Certain substances in plasma (such as glucose, protein, fructose, ascorbic acid, acetoacetate, acetone, and cephalosporins) react with picrate in a similar manner; these are called as non-creatinine chromogens (and can cause false elevation of serum creatinine level). Thus ‘true’ creatinine is less by 0.2 to 0.4 mg/dl when estimated by Jaffe’s reaction.
  2. Enzymatic methods: These methods use enzymes that cleave creatinine; hydrogen peroxide produced then reacts with phenol and a dye to produce a colored product, which is measured in a spectrophotometer.
 
Reference range:
 
Adult males: 0.7-1.3 mg/dl.
Adult females: 0.6-1.1 mg/dl.
 
Serum creatinine alone should not be used to assess renal function. This is because serum creatinine concentration depends on age, sex, muscle mass, glomerular filtration and amount of tubular secretion. Thus, normal serum creatinine range is wide. Serum creatinine begins to rise when GFR falls below 50% of normal. Minor rise of serum creatinine is associated with significant reduction of GFR (Figure 842.2). Therefore early stage of chronic renal impairment cannot be detected by measurement of serum creatinine alone.
 
BUN/Serum Creatinine Ratio
 
Clinicians commonly calculate BUN/creatinine ratio to discriminate pre-renal and post-renal azotemia from renal azotemia. Normal ratio is 12:1 to 20:1.
 
Causes of Increased BUN/Creatinine Ratio (>20:1):
 
  1. Increased BUN with normal serum creatinine:
    • Pre-renal azotemia (reduced renal perfusion)
    • High protein diet
    • Increased protein catabolism
    • Gastrointestinal hemorrhage
  2. Increase of both BUN and serum creatinine with disproportionately greater increase of BUN:
    • Post-renal azotemia (Obstruction to the outflow of urine)
    Obstruction to the urine outflow causes diffusion of urinary urea back into the blood from tubules because of backpressure.

Causes of Decreased BUN/Creatinine Ratio (<10:1)
 
  • Acute tubular necrosis
  • Low protein diet, starvation
  • Severe liver disease
One can estimate GFR from age, sex, body weight, and serum creatinine value of a person from the following formula (Cockcroft and Gault):
 
 
Creatinine clearance in ml/min = (140 - Age in years) × (Body weight in kg)
                                                              (72 × Serum creatinine in mg/dl)
 
 
In females, the value obtained from above equation is multiplied by 0.85 to get the result.
 
It is recommended by National Kidney Foundation (USA) to calculate creatinine clearance by Cockcroft and Gault or other equation from serum creatinine value rather than estimating creatinine clearance from a 24-hour urine sample. This is because the latter test is inconvenient, time-consuming, and often inaccurate.
Glomerular filtration rate refers to the rate in ml/min at which a substance is cleared from the circulation by the glomeruli. The ability of the glomeruli to filter a substance from the blood is assessed by clearance studies. If a substance is not bound to protein in plasma, is completely filtered by the glomeruli, and is neither secreted nor reabsorbed by the tubules, then its clearance rate is equal to the glomerular filtration rate (GFR). Clearance of a substance refers to the volume of plasma, which is completely cleared of that substance per minute; it is calculated from the following formula:
 
Clearance = UV
                      P
 
where, U = concentration of a substance in urine in mg/dl; V = volume of urine excreted in ml/min; and P = concentration of the substance in plasma in mg/dl. Since U and P are in the same units, they cancel each other and the clearance value is expressed in the same unit as V i.e. ml/min. All clearance values are adjusted to a standard body surface area i.e. 1.73 m2.

The agents used for measurement of GFR are:
 
  • Exogenous: Inulin, Radiolabelled ethylenediamine tetraacetic acid (51Cr- EDTA), 125I-iothalamate
  • Endogenous: Creatinine, Urea, Cystatin C
 
The agent used for measurement of GFR should have following properties: (1) It should be physiologically inert and preferably endogenous, (2) It should be freely filtered by glomeruli and should be neither reabsorbed nor secreted by renal tubules, (3) It should not bind to plasma proteins and should not be metabolized by kidneys, and (4) It should be excreted only by the kidneys. However, there is no such ideal endogenous agent.
 
Clearance tests are cumbersome to perform, expensive, and not readily available. One major problem with clearance studies is incomplete urine collection.
 
Abnormal clearance occurs in: (i) pre-renal factors: reduced blood flow due to shock, dehydration, and congestive cardiac failure; (ii) renal diseases; and (iii) obstruction to urinary outflow.
 
Inulin Clearance
 
Inulin, an inert plant polysaccharide (a fructose polymer), is filtered by the glomeruli and is neither reabsorbed nor secreted by the tubules; therefore it is an ideal agent for measuring GFR. A bolus dose of inulin (25 ml of 10% solution IV) is administered followed by constant intravenous infusion (500 ml of 1.5% solution at the rate of 4 ml/min). Timed urine samples are collected and blood samples are obtained at the midpoint of timed urine collection. This test is considered as the ‘gold standard’ (or reference method) for estimation of GFR. However, this test is rarely used because it is time consuming, expensive, constant intravenous infusion of inulin is needed to maintain steady plasma level, and difficulties in laboratory analysis. Average inulin clearance for males is 125 ml/min/1.73 m2 and for females is 110 ml/min/1.73 m2. In children less than 2 years and in older adults, clearance is low. This test is largely limited to clinical research.
 
Clearance of Radiolabeled Agents
 
Urinary clearance of radiolabeled iothalamate (125Iiothalamate) correlates closely with inulin clearance. However, this method is expensive with risk of exposure to radioactive substances. Other radiolabelled substances used are 51Cr-EDTA and 99Tc-DTPA.
 
Cystatin C Clearance
 
This is a cysteine protease inhibitor of MW 13,000, which is produced at a constant rate by all the nucleated cells. It is not bound to protein, is freely filtered by glomeruli and is not returned to circulation after filtration. It is a more sensitive and specific marker of impaired renal function than plasma creatinine. Its level is not affected by sex, diet, or muscle mass. It is thought that cystatin C is a superior marker for estimation of GFR than creatinine clearance. It is measured by immunoassay.
 
Creatinine Clearance
 
This is the most commonly used test for measuring GFR.
 
Creatinine is being produced constantly from creatine in muscle. It is completely filtered by glomeruli and is not reabsorbed by tubules; however, a small amount is secreted by tubules.
 
A 24-hour urine sample is preferred to overcome the problem of diurnal variation of creatinine excretion and to reduce the inaccuracy in urine collection. 
 
After getting up in the morning, the first voided urine is discarded. Subsequently all the urine passed is collected in the container provided. After getting up in the next morning, the first voided urine is also collected and the container is sent to the laboratory. A blood sample for estimation of plasma creatinine is obtained at midpoint of urine collection. Creatinine clearance is calculated from (1) concentration of creatinine in urine in mg/ml (U), (2) volume of urine excreted in ml/min (V) (this is calculated by the formula: volume of urine collected/collection time in minutes e.g. volume of urine collected in 24 hours ÷ 1440), and (3) concentration of creatinine in plasma in mg/dl (P). Creatinine clearance in ml/min per 1.73 m2 is then derived from the formula UV/P.
 
Because of secretion of creatinine by renal tubules, the above formula overestimates GFR by about 10%. In advanced renal failure, secretion of creatinine by tubules is increased and thus overestimation of GFR is even more.
 
Jaffe’s reaction (see serum creatinine) used for estimation of creatinine measures creatinine as well as some other substances (non-creatinine chromogens) in blood and thus gives slightly higher result. Thus effect of tubular secretion of creatinine is somewhat balanced by slight overestimation of serum creatinine by Jaffe’s reaction.
 
To provide values closer to the actual GFR, cimetidine (which blocks secretion by renal tubules) can be administered before commencing urine collection (cimetidine-enhanced creatinine clearance).
 
Creatinine clearance is not an ideal test for estimation of GFR because of following reasons:
 
  1. A small amount of creatinine is secreted by renal tubules that increase even further in advanced renal failure.
  2. Collection of urine is often incomplete.
  3. Creatinine level is affected by intake of meat and muscle mass.
  4. Creatinine level is affected by certain drugs like cimetidine, probenecid, and trimethoprim (which block tubular secretion of creatinine).
 
Urea Clearance
 
Urea is filtered by the glomeruli, but about 40% of the filtered amount is reabsorbed by the tubules. The reabsorption depends on the rate of urine flow. Thus it underestimates GFR, depends on the urine flow rate, and is not a sensitive indicator of GFR.
 
BUN and serum creatinine, by themselves, are not sensitive indicators of early renal impairment since values may be normal e.g. if baseline values of serum creatinine is 0.5 mg/dl, then 50% reduction in kidney function would increase it to 1.0 mg/dl. Thus clearance tests are more helpful in early cases. If biochemical tests are normal and renal function impairment is suspected, then creatinine clearance test should be carried out. If biochemical tests are abnormal, then clearance tests need not be done.
 
Further Reading:
 
Renal biopsy refers to obtaining a small piece of kidney tissue for microscopic examination. Percutaneous renal biopsy was first performed by Alwall in 1944. In renal disease, renal biopsy is helpful to:
 
  • Establish the diagnosis
  • Assess severity and activity of disease
  • Assess prognosis by noting the amount of scarring
  • To plan treatment and monitor response to therapy
 
Renal biopsy is associated with the risk of procedure-related morbidity and rarely mortality. Therefore, before performing renal biopsy, risks of the procedure and benefits of histologic examination should be evaluated in each patient.
 
Indications for Renal Biopsy
 
  1. Nephrotic syndrome in adults (most common indication)
  2. Nephrotic syndrome not responding to corticosteroids in children.
  3. Acute nephritic syndrome for differential diagnosis
  4. Unexplained renal insufficiency with near-normal kidney dimensions on ultrasonography
  5. Asymptomatic hematuria, when other diagnostic tests fail to identify the source of bleeding
  6. Isolated non-nephrotic range proteinuria (1-3 gm/24 hours) with renal impairment
  7. Impaired function of renal graft
  8. Involvement of kidney in systemic disease like systemic lupus erythematosus or amyloidosis
 
Contraindications
 
  1. Uncontrolled severe hypertension
  2. Hemorrhagic diathesis
  3. Solitary kidney
  4. Renal neoplasm (to avoid spread of malignant cells along the needle track)
  5. Large and multiple renal cysts
  6. Small, shrunken kidneys
  7. Acute urinary tract infection like pyelonephritis
  8. Urinary tract obstruction
 
Complications
 
  1. Hemorrhage: As renal cortex is highly vascular, major risk is bleeding in the form of hematuria or perinephric hematoma. Severe bleeding may occasionally necessitate blood transfusion and rarely removal of kidney.
  2. Arteriovenous fistula
  3. Infection
  4. Accidental biopsy of another organ or perforation of viscus (liver, spleen, pancreas, adrenals, intestine, or gallbladder)
  5. Death (rare).
 
Procedure
 
  1. Patient’s informed consent is obtained.
  2. Ultrasound/CT scan is done to document the location and size of kidneys.
  3. Blood pressure should be less than 160/90 mm of Hg. Bleeding time, platelet count, prothrombin time, and activated partial thromboplastin time should be normal. Blood sample should be drawn for blood grouping and cross matching, as blood transfusion may be needed.
  4. Patient is sedated before the procedure.
  5. Patient lies in prone position and kidney is identified with ultrasound.
  6. The skin over the selected site is disinfected and a local anesthetic is infiltrated.
  7. A small skin incision is given with a scalpel (to insert the biopsy needle). Localization of kidney is done with a fine bore 21 G lumbar puncture needle. A local anesthetic is infiltrated down to the renal capsule.
  8. A tru-cut biopsy needle or spring loaded biopsy gun is inserted under ultrasound guidance and advanced down to the lower pole. Biopsy is usually obtained from lateral border of lower pole. Patient should hold his/her breath in full inspiration during biopsy. After obtaining the biopsy and removal of needle, patient is allowed to breath normally.
  9. The biopsy should be placed in a drop of saline and examined under a dissecting microscope for adequacy.
  10. Patient is turned to supine position. Vital signs and appearance of urine should be monitored at regular intervals. Patient is usually kept in the hospital for 24 hours.
 
Kidney biopsy can be divided into three parts for light microscopy, immunofluorescence, and electron microscopy. For light microscopy, renal biopsy is routinely fixed in neutral buffered formaldehyde. Sections are stained by:
 
  • Hematoxylin and eosin (for general architecture of kidney and cellularity)
  • Periodic acid Schiff: To highlight basement membrane and connective tissue matrix.
  • Congo red: For amyloid.
 
For electron microscopy, tissue is fixed in glutaraldeyde. In immunohistochemistry, tissue deposits of IgG, IgA, IgM, C3, fibrin, and κ and λ light chains can be detected by using appropriate antibodies. Many kidney diseases are immune-complex mediated.

In DM, applications of laboratory tests are as follows:

  • Diagnosis of DM
  • Screening of DM
  • Assessment of glycemic control
  • Assessment of associated long-term risks
  • Management of acute metabolic complications.

LABORATORY TESTS FOR DIAGNOSIS OF DIABETES MELLITUS

Diagnosis of DM is based exclusively on demonstration of raised blood glucose level (hyperglycemia).

The current criteria (American Diabetes Association, 2004) for diagnosis of DM are as follows:

Typical symptoms of DM (polyuria, polydipsia, weight loss) and random plasma glucose ≥ 200 mg/dl (≥ 11.1 mmol/L)

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Or

Fasting plasma glucose ≥ 126 mg/dl (≥ 7.0 mmol/L)

Or

2-hour post glucose load (75 g) value during oral glucose tolerance test ≥ 200 mg/dl (≥ 11.1 mmol/L).

If any one of the above three criteria is present, confirmation by repeat testing on a subsequent day is necessary for establishing the diagnosis of DM. However, such confirmation by repeat testing is not required if patient presents with (a) hyperglycemia and ketoacidosis, or (b) hyperosmolar hyperglycemia.

The tests used for laboratory diagnosis of DM are (1) estimation of blood glucose and (2) oral glucose tolerance test.

Estimation of Blood Glucose

Measurement of blood glucose level is a simple test to assess carbohydrate metabolism in DM (Figure 837.1). Since glucose is rapidly metabolized in the body, measurement of blood glucose is indicative of current state of carbohydrate metabolism.

Figure 837.1 Blood glucose values in normal individuals
Figure 837.1 Blood glucose values in normal individuals, prediabetes, and diabetes mellitus

Glucose concentration can be estimated in whole blood (capillary or venous blood), plasma or serum. However, concentration of blood glucose differs according to nature of the blood specimen. Plasma glucose is about 15% higher than whole blood glucose (the figure is variable with hematocrit). During fasting state, glucose levels in both capillary and venous blood are about the same. However, postprandial or post glucose load values are higher by 20-70 mg/dl in capillary blood than venous blood. This is because venous blood is on a return trip after delivering blood to the tissues.

When whole blood is left at room temperature after collection, glycolysis reduces glucose level at the rate of about 7 mg/dl/hour. Glycolysis is further increased in the presence of bacterial contamination or leucocytosis. Addition of sodium fluoride (2.5 mg/ml of blood) maintains stable glucose level by inhibiting glycolysis. Sodium fluoride is commonly used along with an anticoagulant such as potassium oxalate or EDTA. Addition of sodium fluoride is not necessary if plasma is separated from whole blood within 1 hour of blood collection.

Plasma is preferred for estimation of glucose since whole blood glucose is affected also by concentration of proteins (especially hemoglobin).

There are various methods for estimation of blood glucose:

  • Chemical methods:
    – Orthotoluidine method
    – Blood glucose reduction methods using neocuproine, ferricyanide, or copper.

Chemical methods are less specific but are cheaper as compared to enzymatic methods.

  • Enzymatic methods: These are specific for glucose.
    – Glucose oxidase-peroxidase
    – Hexokinase
    – Glucose dehydrogenase

Chemical methods have now been replaced by enzymatic methods.

Terms used for blood glucose specimens: Depending on the time of collection, different terms are used for blood glucose specimens.

  • Fasting blood glucose: Sample for blood glucose is withdrawn after an overnight fast (no caloric intake for at least 8 hours).
  • Post meal or postprandial blood glucose: Blood sample for glucose estimation is collected 2 hours after the subject has taken a normal meal.
  • Random blood glucose: Blood sample is collected at any time of the day, without attention to the time of last food intake.

Oral Glucose Tolerance Test (OGTT)

Glucose tolerance refers to the ability of the body to metabolize glucose. In DM, this ability is impaired or lost and glucose intolerance represents the fundamental pathophysiological defect in DM. OGTT is a provocative test to assess response to glucose challenge in an individual (Figure 837.2).

Figure 837.2 Oral glucose tolerance curve
Figure 837.2 Oral glucose tolerance curve

American Diabetes Association does not recommend OGTT for routine diagnosis of type 1 or type 2 DM. This is because fasting plasma glucose cutoff value of 126 mg/dl identifies the same prevalence of abnormal glucose metabolism in the population as OGTT. World Health Organization (WHO) recommends OGTT in those cases in which fasting plasma glucose is in the range of impaired fasting glucose (i.e. 100-125 mg/dl). Both ADA and WHO recommend OGTT for diagnosis of gestational diabetes mellitus.

Preparation of the Patient

  • Patient should be put on a carbohydrate-rich, unrestricted diet for 3 days. This is because carbohydrate-restricted diet reduces glucose tolerance.
  • Patient should be ambulatory with normal physical activity. Absolute bed rest for a few days impairs glucose tolerance.
  • Medications should be discontinued on the day of testing.
  • Exercise, smoking, and tea or coffee are not allowed during the test period. Patient should remain seated.
  • OGTT is carried out in the morning after patient has fasted overnight for 8-14 hours.

Test

  1. A fasting venous blood sample is collected in the morning.
  2. Patient ingests 75 g of anhydrous glucose in 250-300 ml of water over 5 minutes. (For children, the dose is 1.75 g of glucose per kg of body weight up to maximum 75 g of glucose). Time of starting glucose drink is taken as 0 hour.
  3. A single venous blood sample is collected 2 hours after the glucose load. (Previously, blood samples were collected at ½, 1, 1½, and 2 hours, which is no longer recommended).
  4. Plasma glucose is estimated in fasting and 2-hour venous blood samples.

Interpretation of blood glucose levels is given in Table 837.1.

Table 837.1 Interpretation of oral glucose tolerance test
Parameter Normal Impaired fasting glucose Impaired glucose tolerance Diabetes mellitus
(1) Fasting (8 hr) < 100 100-125 ≥ 126
(2) 2 hr OGTT < 140 < 140 140-199 ≥ 200

OGTT in gestational diabetes mellitus: Impairment of glucose tolerance develops normally during pregnancy, particularly in 2nd and 3rd trimesters. Following are the recent guidelines of ADA for laboratory diagnosis of GDM:

  • Low-risk pregnant women need not be tested if all of the following criteria are met, i.e. age below 25 years, normal body weight (before pregnancy), absence of diabetes in first-degree relatives, member of an ethnic group with low prevalence of DM, no history of poor obstetric outcome, and no history of abnormal glucose tolerance.
  • Average-risk pregnant women (i.e. who are in between low and high risk) should be tested at 24-28 weeks of gestation.
  • High-risk pregnant women i.e. those who meet any one of the following criteria should be tested immediately: marked obesity, strong family history of DM, glycosuria, or personal history of GDM.

Initially, fasting plasma glucose or random plasma glucose should be obtained. If fasting plasma glucose is ≥ 126 mg/dl or random plasma glucose is ≥ 200 mg/dl, repeat testing should be carried out on a subsequent day for confirmation of DM. If both the tests are normal, then OGTT is indicated in average-risk and high-risk pregnant women.

There are two approaches for laboratory diagnosis of GDM

  • One step approach
  • Two step approach

In one step approach, 100 gm of glucose is administered to the patient and a 3-hour OGTT is performed. This test may be cost-effective in high-risk pregnant women.

In two-step approach, an initial screening test is done in which patient drinks a 50 g glucose drink irrespective of time of last meal and a venous blood sample is collected 1 hour later (O’Sullivan’s test). GDM is excluded if glucose level in venous plasma sample is below 140 mg/dl. If level exceeds 140 mg/dl, then the complete 100 g, 3-hour OGTT is carried out.

In the 3-hour OGTT, blood samples are collected in the morning (after 8-10 hours of overnight fasting), and after drinking 100 g glucose, at 1, 2, and 3 hours. For diagnosis of GDM, glucose concentration should be above the following cut-off values in 2 or more of the venous plasma samples:

  • Fasting: 95 mg/dl
  • 1 hour: 180 mg/dl
  • 2 hour: 155 mg/dl
  • 3 hour: 140 mg/dl

LABORATORY TESTS FOR SCREENING OF DIABETES MELLITUS

Aim of screening is to identify asymptomatic individuals who are likely to have DM. Since early detection and prompt institution of treatment can reduce subsequent complications of DM, screening may be an appropriate step in some situations.

Screening for type 2 DM: Type 2 DM is the most common type of DM and is usually asymptomatic in its initial stages. Its onset occurs about 5-7 years before clinical diagnosis. Evidence indicates that complications of type 2 DM begin many years before clinical diagnosis. American Diabetes Association recommends screening for type 2 DM in all asymptomatic individuals ≥ 45 years of age using fasting plasma glucose. If fasting plasma glucose is normal (i.e. < 100 mg/dl), screening test should be repeated every three years.

Another approach is selective screening i.e. screening individuals at high risk of developing type 2 DM i.e. if one or more of the following risk factors are presentobesity (body mass index ≥ 25.0 kg/m2), family history of DM (first degree relative with DM), high-risk ethnic group, hypertension, dyslipidemia, impaired fasting glucose, impaired glucose tolerance, or history of GDM. In such cases, screening is performed at an earlier age (30 years) and repeated more frequently.

Recommended screening test for type 2 DM is fasting plasma glucose. If ≥126 mg/dl, it should be repeated on a subsequent day for confirmation of diagnosis. If <126 mg/dl, OGTT is indicated if clinical suspicion is strong. A 2-hour post-glucose load value in OGTT ≥200 mg/dl is indicative of DM and should be repeated on a different day for confirmation.

Screening for type 1 DM: Type 1 DM is detected early after its onset since it has an acute presentation with characteristic clinical features. Therefore, it is not necessary to screen for type 1 DM by estimation of blood glucose. Detection of immunologic markers (mentioned earlier) has not been recommended to identify persons at risk.

Screening for GDM: Given earlier under OGTT in gestational diabetes mellitus.

LABORATORY TESTS TO ASSESS GLYCEMIC CONTROL

There is a direct correlation between the degree of blood glucose control in DM (both type 1 and type 2) and the development of microangiopathic complications i.e. nephropathy, retinopathy, and neuropathy. Maintenance of blood glucose level as close to normal as possible (referred to as tight glycemic control) reduces the risk of microvascular complications. There is also association between persistently high blood glucose values in DM with increased cardiovascular mortality.

Following methods can monitor degree of glycemic control:

  • Periodic measurement of glycated hemoglobin (to assess long-term control).
  • Daily self-assessment of blood glucose (to assess day-to- day or immediate control).

Glycated Hemoglobin (Glycosylated Hemoglobin, HbA1C)

Glycated hemoglobin refers to hemoglobin to which glucose is attached nonenzymatically and irreversibly; its amount depends upon blood glucose level and lifespan of red cells.

Hemoglobin + Glucose ↔ Aldimine → Glycated hemoglobin

Plasma glucose readily moves across the red cell membranes and is being continuously combined with hemoglobin during the lifespan of the red cells (120 days). Therefore, some hemoglobin in red cells is present normally in glycated form. Amount of glycated hemoglobin in blood depends on blood glucose concentration and lifespan of red cells. If blood glucose concentration is high, more hemoglobin is glycated. Once formed, glycated hemoglobin is irreversible. Level of glycated hemoglobin is proportional to the average glucose level over preceding 6-8 weeks (about 2 months). Glycated hemoglobin is expressed as a percentage of total hemoglobin. Normally, less than 5% of hemoglobin is glycated.

Numerous prospective studies have demonstrated that a good control of blood glucose reduces the development and progression of microvascular complications (retinopathy, nephropathy, and peripheral neuropathy) of diabetes mellitus. Mean glycated hemoglobin level correlates with the risk of these complications.

The terms glycated hemoglobin, glycosylated hemoglobin, glycohemoglobin, HbA1, and HbA1c are often used interchangeably in practice. Although these terms refer to hemoglobins that contain nonenzymatically added glucose residues, hemoglobins thus modified differ. Most of the studies have been carried out with HbA1c.

Glycated hemoglobin should be routinely measured in all diabetic patients (both type 1 and type 2) at regular intervals to assess degree of long-term glycemic control. Apart from mean glycemia (over preceding 120 days), glycated hemoglobin level also correlates with the risk of the development of chronic complications of DM. In DM, it is recommended to maintain glycated hemoglobin level to less than 7%.

Box 837.1 Glycated hemoglobin 
  • Hemoglobin A1C of 6% corresponds to mean serum glucose level of 135 mg/dl. With every rise of 1%, serum glucose increases by 35 mg/dl. Approximations are as follows:
    – Hb A1C 7%: 170 mg/dl
    – Hb A1C 8%: 205 mg/dl
    – Hb A1C 9%: 240 mg/dl
    – Hb A1C 10%: 275 mg/dl
    – Hb A1C 11%: 310 mg/dl
    – Hb A1C 12%: 345 mg/dl
  • Assesses long-term control of DM (thus indirectly confirming plasma glucose results or self-testing results).
  • Assesses whether treatment plan is working
  • Measurement of glycated hemoglobin does not replace measurement of day-to-day control by glucometer devices.

Spurious results of glycated hemoglobin are seen in reduced red cell survival (hemolysis), blood loss, and hemoglobinopathies.

In DM, if glycated hemoglobin is less than 7%, it should be measured every 6 months. If >8%, then more frequent measurements (every 3 months) along with change in treatment are advocated.

There are various methods for measurement of glycated hemoglobin such as chromatography, immunoassay, and agar gel electrophoresis.

Role of glycated hemoglobin in management of DM is highlighted in Box 837.1.

Self-Monitoring of Blood Glucose (SMBG)

Diabetic patients are taught how to regularly monitor their own blood glucose levels. Regular use of SMBG devices (portable glucose meters) by diabetic patients has improved the management of DM. With SMBG devices, blood glucose level can be monitored on day-to-day basis and kept as close to normal as possible by adjusting insulin dosage. SMBG devices measure capillary whole blood glucose obtained by fingerprick and use test strips that incorporate glucose oxidase or hexokinase. In some strips, a layer is incorporated to exclude blood cells so that glucose in plasma is measured. Aim of achieving tight glycemic control introduces the risk of severe hypoglycemia. Daily use of SMBG devices can avoid major hypoglycemic episodes.

SMBG devices yield unreliable results at very high and very low glucose levels. It is necessary to periodically check the performance of the glucometer by measuring parallel venous plasma glucose in the laboratory.

Portable glucose meters are used by patients for day-to-day self-monitoring, by physicians in their OPD clinics, and by health care workers for monitoring admitted patients at the bedside. These devices should not be used for diagnosis and population screening of DM as they lack precision and there is variability of results between different meters.

Goal of tight glycemic control in type 1 DM patients on insulin can be achieved through self-monitoring of blood glucose by portable blood glucose meters.

Glycosuria

Semiquantitative urine glucose testing for monitoring of diabetes mellitus in home setting is not recommended. This is because (1) even if glucose is absent in urine, no information about blood glucose concentration below the renal threshold (which itself is variable) is obtained (Normally, renal threshold is around 180 mg/dl; it tends to be lower in pregnancy (140 mg/dl) and higher in old age and in long-standing diabetics; in some normal persons it is low), (2) urinary glucose testing cannot detect hypoglycemia, and (3) concentration of glucose in urine is affected by urinary concentration. Semiquantitative urine glucose testing for monitoring has now been replaced by self-testing by portable glucose meters.

LABORATORY TESTS TO ASSESS LONG-TERM RISKS

Urinary Albumin Excretion
 
Diabetes mellitus is one of the leading causes of renal failure. Diabetic nephropathy develops in around 20-30% of patients with type 1 or type 2 DM. Diabetic nephropathy progresses through different stages as shown in Figure 837.3. Hypertension also develops along the course of nephropathy with increasing albumin excretion. Evidence indicates that if diabetic nephropathy is detected early and specific treatment is instituted, further progression of nephropathy can be significantly ameliorated. Early detection of diabetic nephropathy is based on estimation of urinary albumin excretion. In all adult patients with DM, usual reagent strip test for proteinuria should be carried out periodically. Positive test means presence of overt proteinuria or clinical proteinuria and may be indicative of overt nephropathy. In all such patients quantitation of albuminuria should be carried out to plan appropriate therapy. If the routine dipstick test for proteinuria is negative, test for microalbuminuria should be carried out.
 
Figure 837.3 Evolution of diabetic nephropathy
Figure 837.3 Evolution of diabetic nephropathy. In 80% of patients with type 1 DM, microalbuminuria progresses in 10-15 years to overt nephropathy that is then followed in majority of cases by progressive fall in GFR and ultimately end-stage renal disease. Amongst patients with type 2 DM and microalbuminuria, 20-40% of patients progress to overt nephropathy, and about 20% of patients with overt nephropathy develop end-stage renal disease. Abbreviation: GFR: Glomerular filtration rate
 
The term ‘microalbuminuria’ refers to the urinary excretion of albumin below the level of detection by routine dipstick testing but above normal (30-300 mg/ 24 hrs, 20-200 μg/min, or 30-300 μg/mg of creatinine). Albumin excretion rate is intermediate between normal (normal albumin excretion in urine is < 30 mg/24 hours) and overt albuminuria (> 300 mg/24 hours). Significance of microalbuminuria in DM is as follows:
 
  • It is the earliest marker of diabetic nephropathy. Early diabetic nephropathy is reversible.
  • It is a risk factor for cardiovascular disease in both type 1 and type 2 patients.
  • It is associated with higher blood pressure and poor glycemic control.
 
Specific therapeutic interventions such as tight glycemic control, administration of ACE (angiotensinconverting enzyme) inhibitors, and aggressive treatment of hypertension significantly slow down the progression of diabetic nephropathy.
 
In type 2 DM, screening for microalbuminuria should begin at the time of diagnosis, whereas in type 1 DM, it should begin 5 years after diagnosis. At this time, a routine reagent strip test for proteinuria is carried out; if negative, testing for microalbuminuria is done. Thereafter, in all patients who test negative, screening for microalbuminuria should be repeated every year.
 
Screening tests for microalbuminuria include:
 
 
Reagent strip tests to detect microalbuminuria are available. Positive results should be confirmed by more specific quantitative tests like radioimmunoassay and enzyme immunoassay. For diagnosis of microalbuminuria, tests should be positive in at least two out of three different samples over a 3 to 6 month period.
 
Lipid Profile
 
Abnormalities of lipids are associated with increased risk of coronary artery disease (CAD) in patients with DM. This risk can be reduced by intensive treatment of lipid abnormalities. Lipid parameters which should be measured include:
 
  • Total cholesterol
  • Triglycerides
  • Low-density lipoprotein (LDL) cholesterol
  • High-density lipoprotein (HDL) cholesterol
 
The usual pattern of lipid abnormalities in type 2 DM is elevated triglycerides, decreased HDL cholesterol and higher proportion of small, dense LDL particles. Patients with DM are categorized into high, intermediate and low-risk categories depending on lipid levels in blood (Table 837.2).
 
Table 837.2 Categorization of cardiovascular risk in diabetes mellitus according to lipid levels (American Diabetes Association)
Category Low density lipoproteins High density lipoproteins Triglycerides
High-risk ≥130 < 35 (men) ≥ 400
    < 45 (women)  
Intermediate risk 100-129 35-45 200-399
Low-risk < 100 > 45 (men) < 200
    > 55 (women)  
 
Annual lipid profile is indicated in all adult patients with DM.
 
LABORATORY TESTS IN THE MANAGEMENT OF ACUTE METABOLIC COMPLICATIONS OF DIABETES MELLITUS
 
The three most serious acute metabolic complications of DM are:
 
  • Diabetic ketoacidosis (DKA)
  • Hyperosmolar hyperglycemic state (HHS)
  • Hypoglycemia
 
The typical features of DKA are hyperglycemia, ketosis, and acidosis. The common causes of DKA are infection, noncompliance with insulin therapy, alcohol abuse and myocardial infarction. Patients with DKA present with rapid onset of polyuria, polydipsia, polyphagia, weakness, vomiting, and sometimes abdominal pain. Signs include Kussmaul’s respiration, odour of acetone on breath (fruity), mental clouding, and dehydration. Classically, DKA occurs in type 1, while HHS is more typical of type 2 DM. However, both complications can occur in either types. If untreated, both events can lead to coma and death.
 
Hyperosmolar hyperglycemic state is characterized by very high blood glucose level (> 600 mg/dl), hyperosmolality (>320 mOsmol/kg of water), dehydration, lack of ketoacidosis, and altered mental status. It usually occurs in elderly type 2 diabetics. Insulin secretion is adequate to prevent ketosis but not hyperglycemia. Causes of HHS are illness, dehydration, surgery, and glucocorticoid therapy.
 
Differences between DKA and HHS are presented in Table 837.3.
 
Table 837.3 Comparison of diabetic ketoacidosis and hyperosmolar hyperglycemic state
Parameter Diabetic ketoacidosis Hyperosmolar hyperglycemic state
1. Type of DM in which more common  Type 1 Type 2 
2. Age  Younger age  Older age
3. Prodromal clinical features  < 24 hrs  Several days
4. Abdominal pain, Kussmaul’s respiration  Yes  No
5. Acidosis  Moderate/Severe  Absent
6. Plasma glucose  > 250 mg/dl  Very high (>600 mg/dl)
7. Serum bicarbonate  <15 mEq/L  >15 mEq/L
8. Blood/urine ketones  ++++  ±
9. β-hydroxybutyrate  High  Normal or raised
10. Arterial blood pH  Low (<7.30)  Normal (>7.30)
11. Effective serum osmolality*  Variable  Increased (>320)
12. Anion gap**  >12  Variable
Osmolality: Number of dissolved (solute) particles in solution; normal: 275-295 mOsmol/kg
** Anion gap: Difference between sodium and sum of chloride and bicarbonate in plasma; normal average value is 12
 
Laboratory evaluation consists of following investigations:
 
  • Blood and urine glucose
  • Blood and urine ketone
  • Arterial pH, Blood gases
  • Serum electrolytes (sodium, potassium, chloride, bicarbonate)
  • Blood osmolality
  • Serum creatinine and blood urea.
 
Testing for ketone bodies: Ketone bodies are formed from metabolism of free fatty acids and include acetoacetic acid, acetone and β-hydroxybutyric acid.
 
Indications for testing for ketone bodies in DM include:
 
  • At diagnosis of diabetes mellitus
  • At regular intervals in all known cases of diabetes, during pregnancy with pre-existing diabetes, and in gestational diabetes
  • In known diabetic patients: during acute illness, persistent hyperglycemia (> 300 mgs/dl), pregnancy, and clinical evidence of diabetic acidosis (nausea, vomiting, abdominal pain).
 
An increased amount of ketone bodies in patients with DM indicate impending or established diabetic ketoacidosis and is a medical emergency. Method based on colorimetric reaction between ketone bodies and nitroprusside (by dipstick or tablet) is used for detection of both blood and urine ketones.
 
Test for urine ketones alone should not be used for diagnosis and monitoring of diabetic ketoacidosis. It is recommended to measure β-hydroxybutyric acid (which accounts for 75% of all ketones in ketoacidosis) for diagnosis and monitoring DKA.
 
REFERENCE RANGES
 
  • Venous plasma glucose:
    Fasting: 60-100 mg/dl
    At 2 hours in OGTT (75 gm glucose): <140 mg/dl
  • Glycated hemoglobin: 4-6% of total hemoglobin
  • Lipid profile:
    – Serum cholesterol: Desirable level: <200 mg/dl
    – Serum triglycerides: Desirable level: <150 mg/dl
    – HDL cholesterol: ≥60 mg/dl
    – LDL cholesterol: <130 mg/dl
    – LDL/HDL ratio: 0.5-3.0
  • C-peptide: 0.78-1.89 ng/ml
  • Arterial pH: 7.35-7.45
  • Serum or plasma osmolality: 275-295 mOsm/kg of water.

Serum Osmolality can also be calculated by the following formula recommended by American Diabetes Association:
 
Effective serum osmolality (mOsm/kg) = (2 × sodium mEq/L) + Plasma glucose (mg/dl)
                                                                                                            18
 
  • Anion gap:
    – Na+ – (Cl + HCO3): 8-16 mmol/L (Average 12)
    – (Na+ + K+) – (Cl + HCO3): 10-20 mmol/L (Average 16)
  • Serum sodium: 135-145 mEq/L
  • Serum potassium: 3.5-5.0 mEq/L
  • Serum chloride: 100-108 mEq/L
  • Serum bicarbonate: 24-30 mEq/L
 
CRITICAL VALUES
 
  • Venous plasma glucose: > 450 mg/dl
  • Strongly positive test for glucose and ketones in urine
  • Arterial pH: < 7.2 or > 7.6
  • Serum sodium: < 120 mEq/L or > 160 mEq/L
  • Serum potassium: < 2.8 mEq/L or > 6.2 mEq/L
  • Serum bicarbonate: < 10 mEq/L or > 40 mEq/L
  • Serum chloride: < 80 mEq/L or > 115 mEq/L

PREGNANCY TESTS

  • 16 Aug 2017

Pregnancy tests detect human chorionic gonadotropin (hCG) in serum or urine. Although pregnancy is the most common reason for ordering the test for hCG, measurement of hCG is also indicated in other conditions as shown in Box 836.1.

Human chorionic gonadotropin is a glycoprotein hormone produced by placenta that circulates in maternal blood and excreted intact by the kidneys. It consists of two polypeptide subunits: α (92 amino acids) and β (145 amino acids) which are non-covalently bound to each other. Structurally, hCG is closely related to three other glycoprotein hormones, namely, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). The α subunits of hCG, LH, FSH, and TSH are similar, while β subunits differ and confer specific biologic and immunologic properties. Immunological tests use antibodies directed against β-subunit of hCG to avoid cross-reactivity against LH, FSH, and TSH.

Box 836.1 Indications for measurement of β human chorionic gonadotropin

• Early diagnosis of pregnancy
• Diagnosis and management of gestational trophoblastic disease
• As a part of maternal triple test screen
• Follow-up of malignant tumors that produce β human chorionic gonadotropin.

Syncytiotrophoblastic cells of conceptus and later of placenta synthesize hCG. Human chorionic gonadotropin supports the corpus luteum of ovary during early pregnancy. Progesterone, produced by corpus luteum, prevents ovulation and thus maintains pregnancy. After 7-10 weeks of gestation, sufficient amounts of progesterone are synthesized by placenta, and hCG is no longer needed and its level declines.

CLINICAL APPLICATIONS OF TESTS FOR HUMAN CHORIONIC GONADOTROPIN

  1. Early diagnosis of pregnancy: Qualitative serum hCG test becomes positive 3 weeks after last menstrual period (LMP), while urine hCG test becomes positive 5 weeks after LMP.
  2. Exclusion of pregnancy before prescribing certain medications (like oral contraceptives, steroids, some antibiotics), and before ordering radiological studies, radiotherapy, or chemotherapy. This is necessary to prevent any teratogenic effect on the fetus.
  3. Early diagnosis of ectopic pregnancy: Trans-vaginal ultrasonography (USG) and quantitative estimation of hCG are helpful in early diagnosis of ectopic pregnancy (before rupture).
  4. Evaluation of threatened abortion: Serial quantitative estimation of hCG is helpful in following the course of threatened abortion.
  5. Diagnosis and follow-up of gestational trophoblastic disease (GTD).
  6. Maternal triple test screen: This consists of measurement of hCG, α-fetoprotein, and unconjugated estriol in maternal serum at 14-19 weeks of gestation. The maternal triple screen identifies pregnant women with increased risk of Down syndrome and major congenital anomalies like neural tube defects.
  7. Follow-up of ovarian or testicular germ cell tumors, which produce hCG.

Normal Pregnancy

In women with normal menstrual cycle, conception (fertilization of ovum to form a zygote) occurs on day 14 in the fallopian tube. Zygote travels down the fallopian tube into the uterus. Division of zygote produces a morula. At 50-60-cell stage, morula develops a primitive yolk sac and is then called as a blastocyst. About 5 days after fertilization, implantation of blastocyst occurs in the uterine wall. Trophoblastic cells (on the outer surface of the blastocyst) penetrate the endometrium and develop into chorionic villi. There are two main forms of trophoblasts—syncytiotrophoblast and cytotrophoblast. Placental development occurs from chorionic villi. After formation of placenta, the conceptus is called as an embryo. When embryo develops most major organs, it is called as fetus (after 10 weeks of gestation).

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Figure 836.1 Level of human chorionic gonadotropin during pregnancy
Figure 836.1Level of human chorionic gonadotropin during pregnancy

Box 836.2 Diagnosis of early pregnancy

• Positive serum hCG test: 8 days after conception or 3 weeks after last menstrual period (LMP)
• Positive urine hCG test: 21 days after conception or 5 weeks after LMP
• Ultrasonography for visualization of gestational sac:
– Transvaginal: 21 days after conception or 5 weeks after LMP
– Transabdominal: 28 days after conception or 6 weeks after LMP

Human chorionic gonadotropin is synthesized by syncytiotrophoblasts (of placenta) and detectable amounts (~5 mIU/ml) appear in maternal serum about 8 days after conception (3 weeks after LMP). In the first trimester (first 12 weeks, calculated from day 1 of LMP) of pregnancy, hCG levels rapidly rise with a doubling time of about 2 days. Highest or peak level is reached at 8-10 weeks (about 100,000 mIU/ml). This is followed by a gradual fall, and from 15-16 weeks onwards, a steady level of 10,000-20,000 mIU/ml is maintained for the rest of the pregnancy (Figure 836.1). After delivery, hCG becomes non-detectable by about 2 weeks.

Box 836.2 shows minimum time required for the earliest diagnosis of pregnancy by hCG test and ultrasonography (USG).

Two types of pregnancy tests are available:

  • Qualitative tests: These are positive/negative result types that are done on urine sample.
  • Quantitative tests: These give numerical result and are done on serum or urine. They are also used for evaluation of ectopic pregnancy, failing pregnancy, and for follow-up of gestational trophoblastic disease.

Ectopic Pregnancy

Ectopic pregnancy refers to the implantation of blastocyst at a site other than the cavity of uterus. The most common of such sites (>95% cases) is fallopian tube. Early diagnosis and treatment of tubal ectopic pregnancy is essential since it can lead to maternal mortality (from rupture and hemorrhage) and future infertility. Ectopic pregnancy is a leading cause of maternal death during first trimester. Diagnosis of ectopic pregnancy can be readily made in most cases by ultrasonography and estimation of β-subunit of human chorionic gonadotropin.

Early diagnosis of unruptured tubal pregnancy can be made by quantitative estimation of serum hCG and ultrasonography. In normal intrauterine pregnancy, hCG titer doubles every 2 days until first 40 days of gestation. If hCG rise is abnormally slow, then an unviable pregnancy (either ectopic or abnormal intrauterine pregnancy) should be suspected.

Transabdominal USG can detect gestational sac in intrauterine pregnancy 6 weeks after LMP. The level of hCG in serum at this stage is >6500 mIU/ml. If gestational sac is not visualized at this level of hCG, then there is a possibility of ectopic pregnancy. Transvaginal ultrasonography can detect ectopic pregnancy average 1 week earlier than abdominal ultrasonography; it can detect gestational sac if β-hCG level is 1000-1500 mIU/ml. Therefore, if gestational sac is not visualized in the presence of >1500 mIU/ml of β-hCG level, an ectopic pregnancy can be suspected.

Early diagnosis of ectopic pregnancy provides the option of administration of intramuscular methotrexate (rather than surgery), which causes dissolution of conceptus. This improves the chances of patient’s future fertility. Serial measurements of hCG after surgical removal of ectopic pregnancy can help in detecting persistence of trophoblastic tissue.

Abortion

Termination of pregnancy before fetus becomes viable (i.e. before 20 weeks) is called as abortion.

In threatened abortion, vaginal bleeding is present but internal os is closed and process of abortion, though started, is still reversible. It is possible that pregnancy will continue.

Serial quantitative titers of hCG showing lack of expected doubling of hCG level and USG are helpful in diagnosis and management of abortion.

Gestational Trophoblastic Disease (GTD)

It is characterized by proliferation of pregnancyassociated trophoblastic tissue. The two main forms of GTD are hydatidiform (vesicular) mole (benign) and choriocarcinoma (malignant). Clinical features of GTD are as follows:

  • Short history of amenorrhea followed by vaginal bleeding.
  • Size of uterus larger than gestational age; uterus is soft and doughy on palpation with no fetal parts and no fetal heart sounds.
  • Excessive nausea and vomiting due to high hCG.
  • Characteristic snowstorm appearance on pelvic USG.

Quantitative estimation of hCG is helpful in diagnosis and management of GTD.

Trophoblastic cells of GTD produce more hCG as compared to the trophoblasts of normal pregnancy for the same gestational age. Concentration of hCG parallels tumor load. Also, hCG continues to rise beyond 10 weeks of gestation without reaching plateau (as expected at the end of first trimester).

After evacuation of uterus, weekly estimation of hCG is advised till subsequent three (weekly) results are negative; following evacuation of vesicular mole, hCG becomes undetectable (after 2-3 months) on follow-up in 80% of cases. Plateau or rising hCG indicates persistent GTD. In such cases, chemotherapy is indicated.

Negative results for hCG after therapy should be regularly followed up every 3 months for 1-2 years.

LABORATORY TESTS FOR HUMAN CHORIONIC GONADOTROPIN

These are classified into two main groups:

  • Biological assays or bioassays
  • Immunological assays

Bioassays

In bioassay, effect of hCG is tested on laboratory animals under standardized conditions. There are several limitations of bioassays like need for animal facilities, need for standardization of animals, long time required for the test results, low sensitivity, and high cost. Therefore, bioassays have been replaced by immunological assays.

In Ascheim-Zondek test, urine from pregnant woman is injected into immature female mice. Formation of hemorrhagic corpora lutea in ovaries (after 4 days) is a positive test. Friedman test is similar except that urine is injected into female rabbit. In rapid rat test, injection of urine containing hCG into female rats is followed by hyperaemia and hemorrhage in ovaries. Yet another test measures release of spermatozoa from male frog after injection of urine containing hCG.

Immunological Assays

These are rapid and sensitive tests for detection and quantitation of hCG. Variable results are obtained by different immunological tests with the same serum sample; this is due to differences in specificity of different immunoassays to complete hCG, β-subunit, and β-core fragment. A number of immunological tests are commercially available based on different principles like agglutination inhibition assay, enzyme immunoassay including enzyme linked immunosorbent assay or ELISA, radioimmunoassay (RIA), and immunoradiometric assay.

A commonly used qualitative urine test is agglutination inhibition assay. Early morning urine specimen is preferred because it contains the highest concentration of hCG. Causes of false-positive test include red cells, leukocytes, bacteria, some drugs, proteins, and excess luteinizing hormone (menopause, midcycle LH surge) in urine. Some patients have anti-mouse antibodies (that are used in the test), while others have hCG-like material in circulation, producing false-positive test. Anti-mouse antibodies also interfere with other antibody-based tests and are known as ‘heterophil’ antibodies. Fetal death, abortion, dilute urine, and low sensitivity of a particular test are causes of false-negative test. Renal failure leads to accumulation of interfering substances causing incorrect results.

Figure 836.2 Principle of agglutination inhibition test for diagnosis of pregnancy
Figure 836.2 Principle of agglutination inhibition test for diagnosis of pregnancy

In latex particle agglutination inhibition test (Figure 836.2), anti-hCG antibodies are incubated with patient’s urine. This is followed by addition of hCGcoated latex particles. If hCG is present in urine, anti-hCG serum is neutralized, and no agglutination of latex particles occurs (positive test). If there is no hCG in urine, there is agglutination of latex particles (negative test). This is commonly used as a slide test and requires only a few minutes.

Sensitivity of agglutination inhibition test is >200 units/liter of hCG.

Radioimmunoassay, enzyme immunoassay, and radioimmunometric assay are more sensitive and reliable than agglutination inhibition assay.

Quantitative tests are employed for detection of very early pregnancy, estimation of gestational age, diagnosis of ectopic pregnancy, evaluation of threatened abortion, and management of GTD.

REFERENCE RANGES

  • Serum human chorionic gonadotropin:
    – Non-pregnant females: <5.0 mIU/ml
    – Pregnancy: 4 weeks after LMP: 5-100 mIU/ml
    – 5 weeks after LMP: 200-3000 mIU/ml
    – 6 weeks after LMP: 10,000-80,000 mIU/ml
    – 7-14 weeks: 90,000-500,000 mIU/ml
    – 15-26 weeks: 5000-80000 mIU/ml
    – 27-40 weks: 3000-15000 mIU/ml

Further Reading: SEMEN ANALYSIS FOR INVESTIGATION OF INFERTILITY

 Box 835.1 Contributions to semen volume
 
• Testes and epididymis: 10%
• Seminal vesicles: 50%
• Prostate: 40%
• Cowper’s glands: Small volume
Semen (or seminal fluid) is a fluid that is emitted from the male genital tract and contains sperms that are capable of fertilizing female ova. Structures involved in production of semen are (Box 835.1):
 
  • Testes: Male gametes or spermatozoa (sperms) are produced by testes; constitute 2-5% of semen volume.
  • Epididymis: After emerging from the testes, sperms are stored in the epididymis where they mature; potassium, sodium, and glycerylphosphorylcholine (an energy source for sperms) are secreted by epididymis.
  • Vas deferens: Sperms travel through the vas deferens to the ampulla which is another storage area. Ampulla secretes ergothioneine (a yellowish fluid that reduces chemicals) and fructose (source of nutrition for sperms).
  • Seminal vesicles: During ejaculation, nutritive and lubricating fluids secreted by seminal vesicles and prostate are added. Fluid secreted by seminal vesicles consists of fructose (energy source for sperms), amino acids, citric acid, phosphorous, potassium, and prostaglandins. Seminal vesicles contribute 50% to semen volume.
  • Prostate: Prostatic secretions comprise about 40% of semen volume and consist of citric acid, acid phosphatase, calcium, sodium, zinc, potassium, proteolytic enzymes, and fibrolysin.
  • Bulbourethral glands of Cowper secrete mucus.
 
Normal values for semen analysis are shown in Tables 835.1 and 835.2.
 
Table 835.1 Normal values of semen analysis (World Health Organization, 1999)
Test Result
1. Volume ≥2 ml
2. pH 7.2 to 8.0
3. Sperm concentration ≥20 million/ml
4. Total sperm count per ejaculate ≥40 million
5. Morphology ≥30% sperms with normal morphology
6. Vitality ≥75% live
7. White blood cells <1 million/ml
8. Motility within 1 hour of ejaculation  
    • Class A ≥25% rapidly progressive
    • Class A and B ≥50% progressive
9. Mixed antiglobuiln reaction (MAR) test <50% motile sperms with adherent particles
10. Immunobead test <50% motile sperms with adherent particles
 
Table 835.2 Biochemical variables of semen analysis (World Helath Organization, 1992)
 1. Total fructose (seminal vesicle marker) ≥13 μmol/ejaculate 
 2. Total zinc (Prostate marker)  ≥2.4 μmol/ejaculate
 3. Total acid phosphatase (Prostate marker)  ≥200U/ejaculate
 4. Total citric acid (Prostate marker)  ≥52 μmol/ejaculate
 5. α-glucosidase (Epididymis marker)  ≥20 mU/ejaculate
 6. Carnitine (Epididymis marker)  0.8-2.9 μmol/ejaculate
 
INDICATIONS FOR SEMEN ANALYSIS
 
Box 835.2 Tests done on seminal fluid
 
• Physical examination: Time to liquefaction, viscosity, volume, pH, color
• Microscopic examination: Sperm count, vitality, motility, morphology, and proportion of white cells
• Immunologic analysis: Antisperm antibodies (SpermMAR test, Immunobead test)
• Bacteriologic analysis: Detection of infection
• Biochemical analysis: Fructose, zinc, acid phosphatase, carnitine.
• Sperm function tests: Postcoital test, cervical mucus penetration test, Hamster egg penetration assay, hypoosmotic swelling of flagella, and computer-assisted semen analysis
Availability of semen for examination allows direct examination of male germ cells that is not possible with female germ cells. Semen analysis requires skill and should preferably be done in a specialized andrology laboratory.
 
  1. Investigation of infertility: Semen analysis is the first step in the investigation of infertility. About 30% cases of infertility are due to problem with males.
  2. To check the effectiveness of vasectomy by confirming absence of sperm.
  3. To support or disprove a denial of paternity on the grounds of sterility.
  4. To examine vaginal secretions or clothing stains for the presence of semen in medicolegal cases.
  5. For selection of donors for artificial insemination.
  6. For selection of assisted reproductive technology, e.g. in vitro fertilization, gamete intrafallopian transfer technique.
 
COLLECTION OF SEMEN FOR INVESTIGATION OF INFERTILITY
 
Semen specimen is collected after about 3 days of sexual abstinence. Longer period of abstinence reduces motility of sperms. If the period of abstinence is shorter than 3 days, sperm count is lower. The sample is obtained by masturbation, collected in a clean, dry, sterile, and leakproof wide-mouthed plastic container, and brought to the laboratory within 1 hour of collection. The entire ejaculate is collected, as the first portion is the most concentrated and contains the highest number of sperms. During transport to the laboratory, the specimen should be kept as close to body temperature as possible (i.e. by carrying it in an inside pocket). Ideally, the specimen should be obtained near the testing site in an adjoining room. Condom collection is not recommended as it contains spermicidal agent. Ejaculation after coitus interruptus leads to the loss of the first portion of the ejaculate that is most concentrated; therefore this method should not be used for collection. Two semen specimens should be examined that are collected 2-3 weeks apart; if results are significantly different additional samples are required.
 
Box 835.3 Semen analysis for initial investigation of infertility
 
• Volume
• pH
• Microscopic examination for (i) percentage of motile spermatozoa, (ii) sperm count, and (iii) sperm morphology
EXAMINATION OF SEMINAL FLUID
 
The tests that can be done on seminal fluid are shown in Box 835.2. Tests commonly done in infertility are shown in Box 835.3. The usual analysis consists of measurement of semen volume, sperm count, sperm motility, and sperm morphology.
 
Terminology in semen analysis is shown in Box 835.4.
 
EXAMINATION OF SEMEN TO CHECK THEEFFECTIVENESS OF VASECTOMY
 
 Box 835.4 Terminology in semen analysis

• Normozoospermia: All semen parameters normal
• Oligozoospermia: Sperm concentration <20 million/ml (mild to moderate: 5-20 million/ml; severe: <5 million/ml)
• Azoospermia: Absence of sperms in seminal fluid
• Aspermia: Absence of ejaculate
• Asthenozoospermia: Reduced sperm motility; <50% of sperms showing class (a) and class (b) type of motility OR <25% sperms showing class (a) type of motility.
• Teratozoospermia: Spermatozoa with reduced proportion of normal morphology (or increased proportion of abnormal forms)
• Leukocytospermia: >1 million white blood cells/ml of semen
• Oligoasthenoteratozoospermia: All sperm variables are abnormal
• Necrozoospermia: All sperms are non-motile or non-viable
The aim of post-vasectomy semen analysis is to detect the presence or absence of spermatozoa. The routine follow-up consists of semen analysis starting 12 weeks (or 15 ejaculations) after surgery. If two successive semen samples are negative for sperms, the semen is considered as free of sperm. A follow-up semen examination at 6 months is advocated by some to rule out spontaneous reconnection.
 
Further Reading:
 
These tests are available only in specialized andrology laboratories. The tests are not standardized thus making interpretation difficult. If used singly, a sperm function test may not be helpful in fertility assessment. They are more predictive if used in combination.
 
Postcoital (Sims-Huhner) Test
 
This is the examination of the cervical mucus after coitus and assesses the ability of the sperm to penetrate the cervical mucus. The quality of the cervical mucus varies during the menstrual cycle, becoming more abundant and fluid at the time of ovulation (due to effect of estrogen); this facilitates penetration of the mucus by the spermatozoa. Progesterone in the secretory phase increases viscosity of the mucus. Therefore cervical mucus testing is scheduled just before ovulation (determined by basal body temperature records or follicular sizing by ultrasonography). Postcoital test is the traditional method to detect the cervical factor in infertility. Cervical mucus is aspirated with a syringe shortly before the expected time of ovulation and 2-12 hours after intercourse. Gross and microscopic examinations are carried out to assess the quality of cervical mucus (elasticity and drying pattern) and to evaluate the number and motility of sperms (Box 834.1). If ≥ 10 motile sperms are observed the test is considered as normal. An abnormal test may result from: (a) poor quality of cervical mucus due to wrong judgment of ovulation, cervicitis or treatment with antioestrogens (e.g. Clomid), and (b) absence of motile sperms due to ineffective technique of coitus, lack of ejaculation, poor semen quality, use of coital lubricants that damage the sperm, or presence of antisperm antibodies. Antisperm antibodies cause immotile sperms, or agglutination or clumping of sperms; they may be present in either partner. If cervical factor is present, intrauterine insemination is the popular treatment. The value of the postcoital test is disputed in the medical literature.
 
Box 834.1 Interpretation of postcoital test
  • Normal: Sperms are normal in amount and moving forward in the mucus; mucus stretches atleast 2 inches (5 cm) and dries in a fern-like manner.
  • Abnormal: Absence of sperms or large number of sperms are dead or sperms are clumped; cervical mucus cannot stretch 2 inches (5 cm) or does not dry in a fern-like manner.
 
This test can be carried out if semen analysis is normal, and the female partner is ovulating and fallopian tubes are not blocked. It is also done if antisperm antibodies are suspected and male partner refuses semen analysis.
 
Cervical Mucus Penetration Test
 
In this test, greatest distance traveled by the sperm in seminal fluid placed and incubated in a capillary tube containing bovine mucus is measured. Majority of fertile men show score >30 mm, while most infertile men show scores <20 mm.
 
Hamster Egg Penetration Assay
 
Hamster oocytes are enzymatically treated to remove the outer layers (that inhibit cross-species fertilization). They are then incubated with sperms and observed for penetration rate. It can be reported as (a) Number of eggs penetrated (penetration rate <15% indicates low fertility), or as (b) Number of sperm penetrations per egg (Normal >5). This test detects sperm motility, binding to oocyte, and penetration of oocyte. There is a high incidence of false-negative results.
 
Hypo-osmotic Swelling of Flagella
 
This test assesses the functional integrity of the plasma membrane of the sperm by observing curling of flagella in hypo-osmotic conditions.
 
Computer-assisted Semen Analysis
 
Computer software measures various characteristics of the spermatozoa; however, its role in predicting fertility potential is not confirmed.
ANTISPERM ANTIBODIES
 
The role of antisperm antibodies in causation of male infertility is controversial. The immunological tests done on seminal fluid include mixed antiglobulin reaction (MAR test) and immunobead test.
 
The antibodies against sperms immobilize or kill them, thus preventing their passage through the cervix to the ovum. The antibodies can be tested in the serum, seminal fluid, or cervical mucus. If the antibodies are present bound to the head of the sperm, they will prevent the penetration of the egg by the sperm. If antibodies are bound to the tail of the sperm, they will retard motility.
 
a. SpermMAR™ test: This test can detect IgG and IgA antibodies against sperm surface in semen sample. In direct SpermMAR™ IgG test, a drop each of semen (fresh and unwashed), IgG-coated latex particles, and anti-human immunoglobulin are mixed together on a glass slide. At least 200 motile spermatozoa are examined. If the spermatozoa have antibodies on their surface, antihuman immunoglobulin will bind IgG-coated latex particles to IgG on the surface of the spermatozoa; this will cause attachment of latex particles to spermatozoa, and motile, swimming sperms with attached particles will be seen. If the spermatozoa do not have antibodies on their surface, they will be seen swimming without attached particles; the latex particles will show clumping due to binding of their IgG to antihuman immunoglobulin.
 
In direct SpermMAR™ IgA test, a drop each of fresh unwashed semen and of IgA-coated latex particles, are mixed on a glass slide. The latex particles will bind to spermatozoa if spermatozoa are coated with IgA antibodies.
 
In indirect SpermMAR™ tests, fluid without spermatozoa (e.g. serum) is tested for the presence of antisperm antibodies. First, antibodies are bound to donor spermatozoa which are then mixed with the fluid to be analyzed. These antibodies are then detected as described above for direct tests.

Atleast 200 motile spermatozoa should be counted. If >50% of spermatozoa show attached latex particles, immunological problem is likely.
 
b. Immunobead test: Antibodies bound to the surface of the spermatozoa can be detected by antibodies attached to immunobeads (plastic particles with attached anti-human immunoglobulin that may be either IgG, IgA, or IgM). Percentage of motile spermatozoa with attached two or more immunobeads are counted amongst 200 motile spermatozoa. Finding of >50% spermatozoa with attached beads is abnormal.
The most important test in semen analysis for infertility is microscopic examination of the semen.
 
SPERM MOTILITY
 
The first laboratory assessment of sperm function in a wet preparation is sperm motility (ability of the sperms to move). Sperm motility is essential for penetration of cervical mucus, traveling through the fallopian tube, and penetrating the ovum. Only those sperms having rapidly progressive motility are capable of penetrating ovum and fertilizing it.
 
Principle: All motile and non-motile sperms are counted in randomly chosen fields in a wet preparation under 40× objective. Result is expressed as a percentage of motile spermatozoa observed.
 
Method: A drop of semen is placed on a glass slide, covered with a coverslip that is then ringed with petroleum jelly to prevent dehydration, and examined under 40× objective. Atleast 200 spermatozoa are counted in several different microscopic fields. Result is expressed as a percentage of (a) rapidly progressive spermatozoa (moving fast forward in a straight line), (b) slowly progressive spermatozoa (slow linear or non-linear, i.e. crooked or curved movement), (c) non-progressive spermatozoa (movement of tails, but with no forward progress), and (d) immotile spermatozoa (no movement at all) (WHO critera). Sperms of grades (c) and (d) are considered to be poorly motile (asthenospermia). Normally, ≥ 25% of sperms show rapid progressive motility, or ≥ 50% of sperms show rapid progressive and slow progressive motility.
 
If the proportion of motile spermatozoa is < 50%, then proportion of viable sperms should be determined by examining an eosin preparation.
 
SPERM VIABILITY OR VITALITY
 
Principle: A cell with intact cell membrane (a vital or viable cell) will not take up the eosin Y and will not be stained, while a non-viable or dead cell will have damaged cell membrane, will take up the dye, and will be stained pink-red (Figure 832.1). Another stain (e.g. nigrosin) may  be used to stain the background material. The test is performed if motility is abnormal.
 
Figure 832.1 Eosin nigrosin stain
Figure 832.1 Eosin-nigrosin stain. Dead sperms are stained pink-red, while live sperms are stained white
 
Method
 
  1. Mix one drop of semen with 1 drop of eosin-nigrosin solution and incubate for 30 seconds.
  2. A smear is made from a drop placed on a glass slide.
  3. The smear is air-dried and examined under oilimmersion objective. White sperms are classified as live or viable, and red sperms are classified as dead or non-viable. At least 200 spermatozoa are examined.
  4. The result is expressed as a proportion of viable sperms against non-viable as an integer percentage.
 
Seventy-five percent or more of sperms are normally live or viable.
 
SPERM COUNT
 
Principle: The sperm count is done after liquefaction in a counting chamber following dilution and the total number of spermatozoa is reported in millions/ml (106/ml).
 
Method
 
  1. Semen is diluted 1:20 with sodium bicarbonateformalin diluting fluid (Take 1 ml liquefied semen in a graduated tube and fill with diluting fluid to 20 ml mark. Mix well).
  2. A coverslip is placed over the improved Neubauer counting chamber and the counting chamber is filled with the well-mixed diluted semen sample using a Pasteur pipette. The chamber is then placed in a humid box for 10-15 minutes for spermatozoa to settle.
  3. The chamber is placed on the microscope stage. Using the 20× or 40× objective and iris diaphragm lowered sufficiently to give sufficient contrast, number of spermatozoa is counted in 4 large corner squares. Spermatozoa whose heads are touching left and upper lines of the square should be considered as ‘belonging’ to that square.
  4. Sperm count per ml is calculated as follows:

    Sperm count =                Sperms counted × correction factor             × 1000
                              Number of squares counted × Volume of 1 square
                           = Sperms counted × 20 1000
                                        4 × 0.1
                           = Sperms counted × 50, 000

  5. Normal sperm count is ≥ 20 million/ml (i.e. ≥ 20 × 106/ml). Sperm count < 20 million/ml may be associated with infertility in males.
 
SPERM MORPHOLOGY
 
A smear is prepared by spreading a drop of seminal fluid on a glass slide, stained, and percentages of normal and abnormal forms of spermatozoa are counted. The staining techniques used are Papanicolaou, eosinnigrosin, hematoxylin-eosin, and Rose Bengal-toluidine blue stain. Atleast 200 spermatozoa should be counted under oil immersion. Percentages of normal and abnormal spermatozoa should be recorded.
 
Normal morphology: A spermatozoon consists of three main components: head, neck, and tail. Tail is further subdivided into midpiece, main (principle) piece, and end piece (Figure 832.2 and Box 832.1).
 
Figure 832.2 Morphology of spermatozoa
Figure 832.2 Morphology of spermatozoa
 
Head is pear-shaped. Most of the head is occupied by the nucleus which has condensed chromatin and few areas of dispersed chromatin (called nuclear vacuoles). The anterior 2/3rds of the nucleus is surrounded by acrosomal cap. Acrosomal cap is a flattened membranebound vesicle containing glycoproteins and enzymes. These enzymes are required for separation of cells of corona radiata and dissolution of zona pellucida of ovum during fertilization.
 
Neck is a very short segment that connects the head and the tail. Centriole in the neck gives rise to axoneme of the flagellum. Axoneme consists of 20 microtubules (arranged as a central pair surrounded by 9 peripheral doublets) and is surrounded by condensed fibrous rings.
 
Middle piece is the first part of the tail and consists of central axoneme surrounded by coarse longitudinal fibers. These are surrounded by elongated mitochondria that provide energy for movement of tail.
 
Principle or main piece constitutes most of the tail and is composed of axoneme that is surrounded by 9 coarse fibers. This central core is surrounded by many circularly arranged fibrous ribs.
 
Endpiece is the short tapering part composed of only axoneme.
 
Normally, > 30% of spermatozoa should show normal morphology (WHO, 1999). The defects in morphology that are associated with infertility in males include defective mid-piece (causes reduced motility), an incomplete or absent acrosome (causes inability to penetrate the ovum), and giant head (defective DNA condensation).
 
Box 832.1 Normal sperm morphology
• Total length of sperm: About 60 μ
• Total length of sperm: About 60 μ
• Head:
   – Length: 3-5 μ
   – Width: 2-3 μ
   – Thickness: 1.5 μ
• Neck: Length: 0.3 μ
• Middle piece:
   – Length: 3-5 μ
   – Width: 1.0 μ
• Principal piece:
   – Length: 40-50 μ
   – Width: 0.5 μ
• End piece: 4-6 μ
 
Abnormal morphology (Figure 832.3): WHO morphological classification of human spermatozoa (1999) is given below:
 
  1. Normal sperm
  2. Defects in head:
    • Large heads
    • Small heads
    • Tapered heads
    • Pyriform heads
    • Round heads
    • Amorphous heads
    • Vacuolated heads (> 20% of the head area occupied by vacuoles)
    • Small acrosomes (occupying < 40% of head area)
    • Double heads
  3. Defects in neck:
    • Bent neck and tail forming an angle >90° to the long axis of head
  4. Defects in middle piece:
    • Asymmetric insertion of midpiece into head
    • Thick or irregular midpiece
    • Abnormally thin midpiece
  5. Defects in tail:
    • Bent tails
    • Short tails
    • Coiled tails
    • Irregular tails
    • Multiple tails
    • Tails with irregular width
  6. Pin heads: Not to be counted
  7. Cytoplasmic droplets
    • > 1/3rd the size of the sperm head
  8. Precursor cells: Considered abnormal
 
Figure 832.3 Abnormal morphological sperm forms
Figure 832.3 Abnormal morphological sperm forms: (1) Normal sperm, (2) Large head, (3) Small head, (4) Tapered head, (5) Pyriform head, (6) Round head, (7) Amorphous head, (8) Vacuoles in head, (9) Round head without acrosome, (10) Double head, (11) Pin head, (12) Round head without acrosome and thick midpiece, (13) Coiled tail, and (14) Double tail

ROUND CELLS
 
Round cells on microscopic examination may be white blood cells or immature sperm cells. Special stain (peroxidase or Papanicolaou) is required to differentiate between them. White blood cells >1 million/ml indicate presence of infection. Presence of large number of immature sperm cells indicates spermatogenesis dysfunction at the testicular level.
Biochemical markers (Table 831.1) can be measured in semen to test the secretions of accessory structures. These include fructose (seminal vesicles), zinc, citric acid or acid phosphatase (prostate), and α-glucosidase or carnitine (epididymis).
 
Table 831.1 Biochemical variables of semen analysis (World Helath Organization, 1992)
1. Total fructose (seminal vesicle marker) ≥13 μmol/ejaculate
2. Total zinc (Prostate marker) ≥2.4 μmol/ejaculate
3. Total acid phosphatase (Prostate marker) ≥200U/ejaculate
4. Total citric acid (Prostate marker) ≥52 μmol/ejaculate
5. α-glucosidase (Epididymis marker) ≥20 mU/ejaculate
6. Carnitine (Epididymis marker) 0.8-2.9 μmol/ejaculate
 
TEST FOR FRUCTOSE
 
Resorcinol method is used for detection of fructose. In this test, 5 ml of resorcinol reagent (50 mg resorcinol dissolved in 33 ml concentrated hydrochloric acid; dilute up to 100 ml with distilled water) is added to 0.5 ml of seminal fluid. The mixture is heated and brought to boil. If fructose is present, a red-colored precipitate is formed within 30 seconds.
 
Absence of fructose indicates obstruction proximal to seminal vesicles (obstructed or absent vas deferens) or a lack of seminal vesicles. In a case of azoospermia, if fructose is absent, it is due to the obstruction of ejaculatory ducts or absence of vas deferens, and if present, azoospermia is due to failure of testes to produce sperm.
Examination is carried out after liquefaction of semen that occurs usually within 20-30 minutes of ejaculation.
 
1. VISUAL APPEARANCE
 
Normal semen is viscous and opaque gray-white in appearance. After prolonged abstinence, it appears slightly yellow.
 
 
Immediately following ejaculation, normal semen is thick and viscous. It becomes liquefied within 30 minutes by the action of proteolytic enzymes secreted by prostate. If liquefaction does not occur within 60 minutes, it is abnormal. The viscosity of the sample is assessed by filling a pipette with semen and allowing it to flow back into the container. Normal semen will fall drop by drop. If droplets form ‘threads’ more than 2 cm long, then viscosity is increased. Increased semen viscosity affects sperm motility and leads to poor invasion of cervical mucus; it results from infection of seminal vesicles or prostate.
 
3. VOLUME
 
Volume of ejaculated semen sample should normally be > 2 ml. It is measured after the sample has liquefied. Volume < 2.0 ml is abnormal, and is associated with low sperm count.
 
4. pH
 
A drop of liquefied semen is spread on pH paper (of pH range 6.4-8.0) and pH is recorded after 30 seconds. Normal pH is 7.2 to 8.0 after 1 hour of ejaculation. The portion of semen contributed by seminal vesicles is basic, while portion from prostate is acidic. Low pH (< 7.0) with absence of sperms (azoospermia) suggests obstruction of ejaculatory ducts or absence of vas deferens. Low pH is usually associated with low semen volume (as most of the volume is supplied by seminal vesicles).

This includes examination of material obtained from vagina, stains from clothing, skin, hair, or other body parts for semen. This is carried out in cases of alleged rape or sexual assault.

Collection of Sample

  • Vagina: Direct aspiration or saline lavage
  • Clothing: When scanned with ultraviolet light, semen produces green white fluorescence. A small piece (1 m2) of clothing from stained portion is soaked in 1-2 ml of physiologic saline for 1 hour. A similar piece of clothing distant from the stain is also soaked in saline as a control.

LABORATORY PROCEDURES

1. MICROSCOPIC EXAMINATION FOR SPERMS

Presence of motile sperms in vaginal fluid indicates interval of < 8 hours. Smears prepared from collected samples are stained and examined for the presence of sperms.

2. ACID PHOSPHATASE

Acid phosphatase is determined on vaginal or clothing samples. Due to the high level of acid phosphatase in semen, its presence indicates recent sexual intercourse. Level of ≥50 U/sample is considered as positive evidence of semen.

3. DETERMINATION OF BLOOD GROUP SUBSTANCES

When semen is positively identified in vaginal fluid or other sample, test can be carried out for the presence of blood group substances in the same sample. The ‘secretor’ individuals (80% individuals are secretors) will secrete the blood group substances in body fluids, including semen.

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4. FLORENCE TEST

This test detects the presence of choline found in high concentration in semen. To several drops of sample, add equal volume of reagent (iodine 2.54 g, potassium iodide 1.65 g, distilled water 30 ml); in positive test rhombic or needle-like crystals of periodide of choline form. False-positive tests can occur due to high choline content of some other body fluids.

1. MICROSCOPIC EXAMINATION OF URINARY SEDIMENT

Definition of microscopic hematuria is presence of 3 or more number of red blood cells per high power field on microscopic examination of urinary sediment in two out of three properly collected samples. A small number of red blood cells in urine of low specific gravity may undergo lysis, and therefore hematuria may be missed if only microscopic examination is done. Therefore, microscopic examination of urine should be combined with a chemical test.

2. CHEMICAL TESTS

These detect both intracellular and extracellular hemoglobin (i.e. intact and lysed red cells) as well as myoglobin. Heme proteins in hemoglobin act as peroxidase, which reduces hydrogen peroxide to water. This process needs a hydrogen donor (benzidine, orthotoluidine, or guaiac). Oxidation of hydrogen donor leads to development of a color (Figure 828.1). Intensity of color produced is proportional to the amount of hemoglobin present.

Chemical tests are positive in hematuria, hemoglobinuria, and myoglobinuria.

Figure 828.1 Principle of chemical test for red cells
Figure 828.1 Principle of chemical test for red cells, hemoglobin, or myoglobin in urine

Benzidine Test

Make saturated solution of benzidine in glacial acetic acid. Mix 1 ml of this solution with 1 ml of hydrogen peroxide in a test tube. Add 2 ml of urine. If green or blue color develops within 5 minutes, the test is positive.

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Orthotoluidine Test

In this test, instead of benzidine, orthotoluidine is used. It is more sensitive than benzidine test.

Reagent Strip Test

Various reagent strips are commercially available which use different chromogens (o-toluidine, tetramethylbenzidine).

Causes of false-positive tests:

  • Contamination of urine by menstrual blood in females
  • Contamination of urine by oxidizing agent (e.g. hypochlorite or bleach used to clean urine containers), or microbial peroxidase in urinary tract infection.

Causes of false-negative tests:

  • Presence of a reducing agent like ascorbic acid in high concentration: Microscopic examination for red cells is positive but chemical test is negative.
  • Use of formalin as a preservative for urine

Evaluation of positive chemical test for blood is shown in Figure 828.2.

Figure 828.2 Evaluation of positive chemical test for blood in urine
Figure 828.2 Evaluation of positive chemical test for blood in urine
Have you ever wondered, what is your physiological age? Is it more or less than your chronological age? Physiological age determines a person’s health condition. Are we able to determine physiological age? You would think the answer is NO. but it can be done by determining telomere’s length. “Telomere is a repetitive nucleotide sequence (having no meaningful information) at each end of chromosome to protect DNA from deterioration and or from fusion with other chromosomes.” This sequence is about 3000-15000 base pairs in length. In vertebrates this repeated sequence is TTAGGG.
 
Significance of Telomeres
 
Cells divide and increase their number, DNA duplication also occurs. Enzymes involved in this duplication process, can’t continue duplication all the way to the end so some part of DNA is lost and chromosome is shortened. This lost part is some base pairs of telomere. Somatic cells lose about 50-100 nucleotides on each cell division. In this way, telomeres, having no meaningful information, act as CAPS preventing the important information (DNA) from deterioration and preserve the critical information. Telomeres are never tied to each other which allows chromosomes to remain segregate. Without telomeres, chromosomes would fuse with each other. Telomere Shortening Telomeres shorten because of the two major factors:
 
  1. End replication problem in eukaryotes accounts for loss of 20 base pairs per cell division.
  2. Oxidative stress accounts for loss of 50-100 base pairs per cell division.
 
Figure 827.1
 
Oxidative stress in the body depends on lifestyle factors. Smoking, poor diet and stress can cause increase in oxidative stress. With each cell division telomeres shorten, so there are limited number of divisions that a cell can undergo, this limit is called Hayflick Limit. This is to prevent the loss of vital DNA information and to prevent production of abnormal cells. When a cell reaches this limit it undergoes apoptosis that is a programmed cell death. Telomere Lengthening to reverse telomere shortening, there is an enzyme named Telomerase that adds telomere sequence nucleotides and replenish the lost telomere nucleotides. Telomerase activity is not present in all cells. It is almost absent in somatic cells including; lung, liver, kidney cells, adult tissues, cardiac and skeletal muscles etc. In the presence of telomerase enzyme, a cell can divide to unlimited extent without ageing giving rise to tumors. That’s why it is found only in some cells in considerable concentration including germline cells and stem cells. These cells don’t show signs of ageing.
 
Figure 827.3
 
Relation between Telomere’s Shortening and Ageing
 
Figure 827.2It is still controversial that whether telomere shortening is a reason of ageing or is a sign of ageing just like grey hair. Whatever it is, the thing is, it determines your physiological age because ageing cells mean an ageing body. Telomere shortening is related with poor lifestyle. People who are active and have a healthy lifestyle have the same telomere length as someone 10 years younger than them has. Depression causes increase in oxidative stress in the body so the higher the stress, the shorter the telomere is Link between Telomeres and Cancer “Cancer in general is defined as an uncontrollable rapid growth of cells.”
 
What causes these cells to grow uncontrollably?
 
These cells have active telomerase enzyme, which doesn’t let the telomere to shorten, so no Hayflick limit reaches and cell continues to divide. This is the reason why telomerase is not used as an anti-ageing medicine because it has potential to turn normal body cells into cancerous cells. Without telomerase activity cancer cells activity would stop, which is an under research treatment for cancer. However, drugs inhibiting telomerase activity, can interfere with normal functioning of cells that require telomerase. In healthy female breast there is a portion of cells named, luminal progenitors, with critically short telomere length. In these cells telomerase becomes active causing these cells to turn into cancer cells on higher activity. To tackle breast cancer, use of telomerase inhibiting drugs should be practiced. Telomere biology is very important for understanding cancer biology and scientists are working hard on it.
 
 
Reviewed by Dr. Nida Hayat Khan
Editor @ BioScience.pk 

The chemical examination is carried out for substances in urine are listed below:

  • Proteins
  • Glucose
  • Ketones
  • Bilirubin
  • Bile salts
  • Urobilinogen
  • Blood
  • Hemoglobin
  • Myoglobin
  • Nitrite or leukocyte esterase

PROTEINS

Normally, kidneys excrete scant amount of protein in urine (up to 150 mg/24 hours). These proteins include proteins from plasma (albumin) and proteins derived from urinary tract (Tamm-Horsfall protein, secretory IgA, and proteins from tubular epithelial cells, leucocytes, and other desquamated cells); this amount of proteinuria cannot be detected by routine tests.

(Tamm-Horsfall protein is a normal mucoprotein secreted by ascending limb of the loop of Henle).

Proteinuria refers to protein excretion in urine greater than 150 mg/24 hours in adults.

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Causes of Proteinuria

Box 826.1: Causes of proteinuria
  • Glomerular proteinuria
  • Tubular proteinuria
  • Overflow proteinuria
  • Hemodynamic (functional) proteinuria
  • Post-renal proteinuria

Causes of proteinuria can be grouped as shown in Box 826.1.

  • Glomerular proteinuria: Proteinuria due to increased permeability of glomerular capillary wall is called as glomerular proteinuria.

    There are two types of glomerular proteinuria: selective and nonselective. In early stages of glomerular disease, there is increased excretion of lower molecular weight proteins like albumin and transferrin. When glomeruli can retain larger molecular weight proteins but allow passage of comparatively lower molecular weight proteins, the proteinuria is called as selective. With further glomerular damage, this selectivity is lost and larger molecular weight proteins (γ globulins) are also excreted along with albumin; this is called as nonselective proteinuria.

    Selective and nonselective proteinuria can be distinguished by urine protein electrophoresis. In selective proteinuria, albumin and transferrin bands are seen, while in nonselective type, the pattern resembles that of serum (Figure 826.1).

    • Massive proteinuria (>3.5 gm/24 hr)
    • Hypoalbuminemia (<3.0 gm/dl)
    • Generalised edema
    • Hyperlipidemia (serum cholesterol >350 mg/dl)
    • Lipiduria
    Causes of glomerular proteinuria are glomerular diseases that cause increased permeability of glomerular basement membrane. The degree of glomerular proteinuria correlates with severity of disease and prognosis. Serial estimations of urinary protein are also helpful in monitoring response to treatment. Most severe degree of proteinuria occurs in nephrotic syndrome (Box 826.2).

  • Tubular proteinuria: Normally, glomerular membrane, although impermeable to high molecular weight proteins, allows ready passage to low molecular weight proteins like β2-microglobulin, retinol-binding protein, lysozyme, α1-microglobulin, and free immunoglobulin light chains. These low molecular weight proteins are actively reabsorbed by proximal renal tubules. In diseases involving mainly tubules, these proteins are excreted in urine while albumin excretion is minimal.

    Urine electrophoresis shows prominent α- and β-bands (where low molecular weight proteins migrate) and a faint albumin band (Figure 826.1).

    Tubular type of proteinuria is commonly seen in acute and chronic pyelonephritis, heavy metal poisoning, tuberculosis of kidney, interstitial nephritis, cystinosis, Fanconi syndrome and rejection of kidney transplant.

    Purely tubular proteinuria cannot be detected by reagent strip test (which is sensitive to albumin), but heat and acetic acid test and sulphosalicylic acid test are positive.

  • Overflow proteinuria: When concentration of a low molecular weight protein rises in plasma, it “overflows” from plasma into the urine. Such proteins are immunoglobulin light chains or Bence Jones proteins (plasma cell dyscrasias), hemoglobin (intravascular hemolysis), myoglobin (skeletal muscle trauma), and lysozyme (acute myeloid leukemia type M4 or M5).

  • Hemodynamic proteinuria: Alteration of blood flow through the glomeruli causes increased filtration of proteins. Protein excretion, however, is transient. It is seen in high fever, hypertension, heavy exercise, congestive cardiac failure, seizures, and exposure to cold.

    Postural (orthostatic) proteinuria occurs when the subject is standing or ambulatory, but is absent in recumbent position. It is common in adolescents (3-5%) and is probably due to lordotic posture that causes inferior venacaval compression between the liver and vertebral column. The condition disappears in adulthood. Amount of proteinuria is <1000 mg/day. First-morning urine after rising is negative for proteins, while another urine sample collected after patient performs normal activities is positive for proteins. In such patients, periodic testing for proteinuria should be done to rule out renal disease.

  • Post-renal proteinuria: This is caused by inflammatory or neoplastic conditions in renal pelvis, ureter, bladder, prostate, or urethra.

Further reading: Tests for Detection of Proteinuria

Figure 826.1 Glomerular and tubular proteinuria
Figure 826.1 Glomerular and tubular proteinuria. Upper figure shows normal serum protein electrophoresis pattern. Lower part shows comparison of serum and urine electrophoresis in (1) selective proteinuria, (2) non-selective proteinuria, and (3) tubular proteinuria

GLUCOSE

The main indication for testing for glucose in urine is detection of unsuspected diabetes mellitus or follow-up of known diabetic patients.

Box 826.3: Urine glucose
  • Urine should be tested for glucose within 2 hours of collection (due to lowering of glucose by glycolysis and by contaminating bacteria which degrade glucose rapidly)
  • Reagent strip test is a rapid, inexpensive, and semi-quantitative test
  • In the past this test was used for home-monitoring of glucose; the test is replaced by glucometers.
  • Urine glucose cannot be used to monitor control of diabetes since renal threshold is variable amongst individuals, no information about level of blood glucose below renal threshold is obtained, and urinary glucose value is affected by concentration of urine.

Practically all of the glucose filtered by the glomeruli is reabsorbed by the proximal renal tubules and returned to circulation. Normally a very small amount of glucose is excreted in urine (< 500 mg/24 hours or <15 mg/dl) that cannot be detected by the routine tests. Presence of detectable amounts of glucose in urine is called as glucosuria or glycosuria (Box 826.3). Glycosuria results if the filtered glucose load exceeds the capacity of renal tubular reabsorption. Most common cause is hyperglycemia from diabetes mellitus.

Causes of Glycosuria

1. Glycosuria with hyperglycemia:

  • Endocrine diseases: diabetes mellitus, acromegaly, Cushing’s syndrome, hyperthyroidism, pancreatic disease
  • Non-endocrine diseases: central nervous system diseases, liver disorders
  • Drugs: adrenocorticotrophic hormone, corticosteroids, thiazides
  • Alimentary glycosuria (Lag-storage glycosuria): After a meal, there is rapid intestinal absorption of glucose leading to transient elevation of blood glucose above renal threshold. This can occur in persons with gastrectomy or gastrojejunostomy and in hyperthyroidism. Glucose tolerance test reveals a peak at 1 hour above renal threshold (which causes glycosuria); the fasting and 2-hour glucose values are normal.

2. Glycosuria without hyperglycemia

  • Renal glycosuria: This accounts for 5% of cases of glycosuria in general population. Renal threshold is the highest glucose level in blood at which glucose appears in urine and which is detectable by routine laboratory tests. The normal renal threshold for glucose is 180 mg/dl. Threshold substances need a carrier to transport them from tubular lumen to blood. When the carrier is saturated, the threshold is reached and the substance is excreted. Up to this level glucose filtered by the glomeruli is efficiently reabsorbed by tubules. Renal glycosuria is a benign condition in which renal threshold is set below 180 mgs/dl but glucose tolerance is normal; the disorder is transmitted as autosomal dominant. Other conditions in which glycosuria can occur with blood glucose level remaining below 180 mgs/dl are renal tubular diseases in which there is decreased glucose reabsorption like Fanconi’s syndrome, and toxic renal tubular damage. During pregnancy, renal threshold for glucose is decreased. Therefore it is necessary to estimate blood glucose when glucose is first detected in urine.

Further reading: Tests for Detection of Glucose in Urine

KETONES

Excretion of ketone bodies (acetoacetic acid, β-hydroxybutyric acid, and acetone) in urine is called as ketonuria. Ketones are breakdown products of fatty acids and their presence in urine is indicative of excessive fatty acid metabolism to provide energy.

Causes of Ketonuria

Box 826.4: Urine ketones in diabetes
Indications for testing
  • At diagnosis of diabetes mellitus
  • At regular intervals in all known cases of diabetes, and in gestational diabetes
  • In known diabetic patients during acute illness, persistent hyperglycemia (>300 mg/dl), pregnancy, clinical evidence of diabetic acidosis (nausea, vomiting, abdominal pain)

Normally ketone bodies are not detectable in the urine of healthy persons. If energy requirements cannot be met by metabolism of glucose (due to defective carbohydrate metabolism, low carbohydrate intake, or increased metabolic needs), then energy is derived from breakdown of fats. This leads to the formation of ketone bodies (Figure 826.2).

  1. Decreased utilization of carbohydrates:
    a. Uncontrolled diabetes mellitus with ketoacidosis: In diabetes, because of poor glucose utilization, there is compensatory increased lipolysis. This causes increase in the level of free fatty acids in plasma. Degradation of free fatty acids in the liver leads to the formation of acetoacetyl CoA which then forms ketone bodies. Ketone bodies are strong acids and produce H+ ions, which are neutralized by bicarbonate ions; fall in bicarbonate (i.e. alkali) level produces ketoacidosis. Ketone bodies also increase the plasma osmolality and cause cellular dehydration. Children and young adults with type 1 diabetes are especially prone to ketoacidosis during acute illness and stress. If glycosuria is present, then test for ketone bodies must be done. If both glucose and ketone bodies are present in urine, then it indicates presence of diabetes mellitus with ketoacidosis (Box 826.4).
    In some cases of diabetes, ketone bodies are increased in blood but do not appear in urine.
    Presence of ketone bodies in urine may be a warning of impending ketoacidotic coma.
    b. Glycogen storage disease (von Gierke’s disease)
  2. Decreased availability of carbohydrates in the diet:
    a. Starvation
    b. Persistent vomiting in children
    c. Weight reduction program (severe carbohydrate restriction with normal fat intake)
  3. Increased metabolic needs:
    a. Fever in children
    b. Severe thyrotoxicosis
    c. Pregnancy
    d. Protein calorie malnutrition

Further reading: Tests for Detection of Ketones in Urine

Figure 826.2 Formation of ketone bodies
Figure 826.2 Formation of ketone bodies. A small part of acetoacetate is spontaneously and irreversibly converted to acetone. Most is converted reversibly to β-hydroxybutyrate

BILE PIGMENT (BILIRUNIN)

Bilirubin (a breakdown product of hemoglobin) is undetectable in the urine of normal persons. Presence of bilirubin in urine is called as bilirubinuria.

There are two forms of bilirubin: conjugated and unconjugated. After its formation from hemoglobin in reticuloendothelial system, bilirubin circulates in blood bound to albumin. This is called as unconjugated bilirubin. Unconjugated bilirubin is not water-soluble, is bound to albumin, and cannot pass through the glomeruli; therefore it does not appear in urine. The liver takes up unconjugated bilirubin where it combines with glucuronic acid to form bilirubin diglucuronide (conjugated bilirubiun). Conjugated bilirubin is watersoluble, is filtered by the glomeruli, and therefore appears in urine.

Detection of bilirubin in urine (along with urobilinogen) is helpful in the differential diagnosis of jaundice (Table 826.1).

Table 826.1 Urine bilirubin and urobilinogen in jaundice
Urine test Hemolytic jaundice Hepatocellular jaundice Obstructive jaundice
1. Bilirubin Absent Present Present
2. Urobilinogen Increased Increased Absent

In acute viral hepatitis, bilirubin appears in urine even before jaundice is clinically apparent. In a fever of unknown origin bilirubinuria suggests hepatitis.

Presence of bilirubin in urine indicates conjugated hyperbilirubinemia (obstructive or hepatocellular jaundice). This is because only conjugated bilirubin is water-soluble. Bilirubin in urine is absent in hemolytic jaundice; this is because unconjugated bilirubin is water-insoluble.

Further reading: Tests for Detection of Bilirubin in Urine

BILE SALTS

Bile salts are salts of four different types of bile acids: cholic, deoxycholic, chenodeoxycholic, and lithocholic. These bile acids combine with glycine or taurine to form complex salts or acids. Bile salts enter the small intestine through the bile and act as detergents to emulsify fat and reduce the surface tension on fat droplets so that enzymes (lipases) can breakdown the fat. In the terminal ileum, bile salts are absorbed and enter in the blood stream from where they are taken up by the liver and re-excreted in bile (enterohepatic circulation).

Further reading: Test for Detection of Bile Salts in Urine

UROBILINOGEN

Conjugated bilirubin excreted into the duodenum through bile is converted by bacterial action to urobilinogen in the intestine. Major part is eliminated in the feces. A portion of urobilinogen is absorbed in blood, which undergoes recycling (enterohepatic circulation); a small amount, which is not taken up by the liver, is excreted in urine. Urobilinogen is colorless; upon oxidation it is converted to urobilin, which is orange-yellow in color. Normally about 0.5-4 mg of urobilinogen is excreted in urine in 24 hours. Therefore, a small amount of urobilinogen is normally detectable in urine.

Urinary excretion of urobilinogen shows diurnal variation with highest levels in afternoon. Therefore, a 2-hour post-meal sample is preferred.

Causes of Increased Urobilinogen in Urine

  1. Hemolysis: Excessive destruction of red cells leads to hyperbilirubinemia and therefore increased formation of urobilinogen in the gut. Bilirubin, being of unconjugated type, does not appear in urine. Increased urobilinogen in urine without bilirubin is typical of hemolytic anemia. This also occurs in megaloblastic anemia due to premature destruction of erythroid precursors in bone marrow (ineffective erythropoiesis).
  2. Hemorrhage in tissues: There is increased formation of bilirubin from destruction of red cells.

Causes of Reduced Urobilinogen in Urine

  1. Obstructive jaundice: In biliary tract obstruction, delivery of bilirubin to the intestine is restricted and very little or no urobilinogen is formed. This causes stools to become clay-colored.
  2. Reduction of intestinal bacterial flora: This prevents conversion of bilirubin to urobilinogen in the intestine. It is observed in neonates and following antibiotic treatment.

Testing of urine for both bilirubin and urobilinogen can provide helpful information in a case of jaundice (Table 826.1).

Further reading: Tests for Detection of Urobilinogen in Urine

BLOOD

The presence of abnormal number of intact red blood cells in urine is called as hematuria. It implies presence of a bleeding lesion in the urinary tract. Bleeding in urine may be noted macroscopically or with naked eye (gross hematuria). If bleeding is noted only by microscopic examination or by chemical tests, then it is called as occult, microscopic or hidden hematuria.

Causes of Hematuria

1. Diseases of urinary tract:

  • Glomerular diseases: Glomerulonephritis, Berger’s disease, lupus nephritis, Henoch-Schonlein purpura
  • Nonglomerular diseases: Calculus, tumor, infection, tuberculosis, pyelonephritis, hydronephrosis, polycystic kidney disease, trauma, after strenuous physical exercise, diseases of prostate (benign hyperplasia of prostate, carcinoma of prostate).

2. Hematological conditions:

Coagulation disorders, sickle cell disease Presence of red cell casts and proteinuria along with hematuria suggests glomerular cause of hematuria.

Further reading: Tests for Detection of Blood in Urine

HEMOGLOBIN

Presence of free hemoglobin in urine is called as hemoglobinuria.

Causes of Hemoglobinuria

  1. Hematuria with subsequent lysis of red blood cells in urine of low specific gravity.
  2. Intravascular hemolysis: Hemoglobin will appear in urine when haptoglobin (to which hemoglobin binds in plasma) is completely saturated with hemoglobin. Intravascular hemolysis occurs in infections (severe falciparum malaria, clostridial infection, E. coli septicemia), trauma to red cells (march hemoglobinuria, extensive burns, prosthetic heart valves), glucose-6-phosphate dehydrogenase deficiency following exposure to oxidant drugs, immune hemolysis (mismatched blood transfusion, paroxysmal cold hemoglobinuria), paroxysmal nocturnal hemoglobinuria, hemolytic uremic syndrome, and disseminated intravascular coagulation.

Tests for Detection of Hemoglobinuria

Tests for detection of hemoglobinuria are benzidine test, orthotoluidine test, and reagent strip test.

HEMOSIDERIN

Hemosiderin in urine (hemosiderinuria) indicates presence of free hemoglobin in plasma. Hemosiderin appears as blue granules when urine sediment is stained with Prussian blue stain (Figure 826.3). Granules are located inside tubular epithelial cells or may be free if cells have disintegrated. Hemosiderinuria is seen in intravascular hemolysis.

Figure 826.3 Staining of urine sediment with Prussian blue stain
Figure 826.3 Staining of urine sediment with Prussian blue stain to demonstrate hemosiderin granules (blue)

MYOGLOBIN

Myoglobin is a protein present in striated muscle (skeletal and cardiac) which binds oxygen. Causes of myoglobinuria include injury to skeletal or cardiac muscle, e.g. crush injury, myocardial infarction, dermatomyositis, severe electric shock, and thermal burns.

Chemical tests used for detection of blood or hemoglobin also give positive reaction with myoglobin (as both hemoglobin and myoglobin have peroxidase activity). Ammonium sulfate solubility test is used as a screening test for myoglobinuria (Myoglobin is soluble in 80% saturated solution of ammonium sulfate, while hemoglobin is insoluble and is precipitated. A positive chemical test for blood done on supernatant indicates myoglobinuria).

Distinction between hematuria, hemoglobinuria, and myoglobinuria is shown in Table 826.2

Table 826.2 Differentiation between hematuria, hemoglobinuria, and myoglobinuria
Parameter Hematuria Hemoglobinuria Myoglobinuria
1. Urine color Normal, smoky, red, or brown Pink, red, or brown Red or brown
2. Plasma color Normal Pink Normal
3. Urine test based on peroxidase activity Positive Positive Positive
4. Urine microscopy Many red cells Occasional red cell Occasional red cell
5. Serum haptoglobin Normal Low Normal
6. Serum creatine kinase Normal Normal Markedly increased

Chemical Tests for Significant Bacteriuria (Indirect Tests for Urinary Tract Infection)

In addition to direct microscopic examination of urine sample, chemical tests are commercially available in a reagent strip format that can detect significant bacteriuria: nitrite test and leucocyte esterase test. These tests are helpful at places where urine microscopy is not available. If these tests are positive, urine culture is indicated.

1. Nitrite test: Nitrites are not present in normal urine; ingested nitrites are converted to nitrate and excreted in urine. If gram-negative bacteria (e.g. E.coli, Salmonella, Proteus, Klebsiella, etc.) are present in urine, they will reduce the nitrates to nitrites through the action of bacterial enzyme nitrate reductase. Nitrites are then detected in urine by reagent strip tests. As E. coli is the commonest organism causing urinary tract infection, this test is helpful as a screening test for urinary tract infection.

Some organisms like Staphylococci or Pseudomonas do not reduce nitrate to nitrite and therefore in such infections nitrite test is negative. Also, urine must be retained in the bladder for minimum of 4 hours for conversion of nitrate to nitrite to occur; therefore, fresh early morning specimen is preferred. Sufficient dietary intake of nitrate is necessary. Therefore a negative nitrite test does not necessarily indicate absence of urinary tract infection. The test detects about 70% cases of urinary tract infections.

2. Leucocyte esterase test: It detects esterase enzyme released in urine from granules of leucocytes. Thus the test is positive in pyuria. If this test is positive, urine culture should be done. The test is not sensitive to leucocytes < 5/HPF.

Microscopic examination of urine is also called as the “liquid biopsy of the urinary tract”.

Urine consists of various microscopic, insoluble, solid elements in suspension. These elements are classified as organized or unorganized. Organized substances include red blood cells, white blood cells, epithelial cells, casts, bacteria, and parasites. The unorganized substances are crystalline and amorphous material. These elements are suspended in urine and on standing they settle down and sediment at the bottom of the container; therefore they are known as urinary deposits or urinary sediments. Examination of urinary deposit is helpful in diagnosis of urinary tract diseases as shown in Table 825.1.

Table 825.1 Urinary findings in renal diseases
Condition Albumin RBCs/HPF WBCs/HPF Casts/LPF Others
1. Normal 0-trace 0-2 0-2 Occasional (Hyaline)
2. Acute glomerulonephritis 1-2+ Numerous;dysmorphic 0-few Red cell, granular Smoky urine or hematuria
3. Nephrotic syndrome > 4+ 0-few 0-few Fatty, hyaline, Waxy, epithelial Oval fat bodies, lipiduria
4. Acute pyelonephritis 0-1+ 0-few Numerous WBC, granular WBC clumps, bacteria, nitrite test
HPF: High power field; LPF: Low power field; RBCs: Red blood cells; WBCs: White blood cells.

Different types of urinary sediments are shown in Figure 825.1. The major aim of microscopic examination of urine is to identify different types of cellular elements and casts. Most crystals have little clinical significance.

Figure 825.1 Different types of urinary sediment
Figure 825.1 Different types of urinary sediment

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Specimen

The cellular elements are best preserved in acid, hypertonic urine; they deteriorate rapidly in alkaline, hypotonic solution. A mid-stream, freshly voided, first morning specimen is preferred since it is the most concentrated. The specimen should be examined within 2 hours of voiding because cells and casts degenerate upon standing at room temperature. If preservative is required, then 1 crystal of thymol or 1 drop of formalin (40%) is added to about 10 ml of urine.

Method

A well-mixed sample of urine (12 ml) is centrifuged in a centrifuge tube for 5 minutes at 1500 rpm and supernatant is poured off. The tube is tapped at the bottom to resuspend the sediment (in 0.5 ml of urine). A drop of this is placed on a glass slide and covered with a cover slip (Figure 825.2). The slide is examined immediately under the microscope using first the low power and then the high power objective. The condenser should be lowered to better visualize the elements by reducing the illumination.

Figure 825.2 Preparation of urine sediment for microscopic examination
Figure 825.2 Preparation of urine sediment for microscopic examination

CELLS

Cellular elements in urine are shown in Figure 825.3.

Figure 825.3 Cells in urine
Figure 825.3 Cells in urine (1) Isomorphic red blood cells, (2) Crenated red cells, (3) Swollen red cells, (4) Dysmorphic red cells, (5) White blood cells (pus cells), (6) Squamous epithelial cell, (7) Transitional epithelial cells, (8) Renal tubular epithelial cells, (9) Oval fat bodies, (10) Maltese cross pattern of oval fat bodies, and (11) spermatozoa

Red Blood Cells

Normally there are no or an occasional red blood cell in urine. In a fresh urine sample, red cells appear as small, smooth, yellowish, anucleate biconcave disks about 7 μ in diameter (called as isomorphic red cells). However, red cells may appear swollen (thin discs of greater diameter, 9-10 μ) in dilute or hypotonic urine, or may appear crenated (smaller diameter with spikey surface) in hypertonic urine. In glomerulonephritis, red cells are typically described as being dysmorphic (i.e. markedly variable in size and shape). They result from passage of red cells through the damaged glomeruli. Presence of > 80% of dysmorphic red cells is strongly suggestive of glomerular pathology.

The quantity of red cells can be reported as number of red cells per high power field.

Causes of hematuria have been listed earlier.

White Blood Cells (Pus Cells)

White blood cells are spherical, 10-15 μ in size, granular in appearance in which nuclei may be visible. Degenerated white cells are distorted, smaller, and have fewer granules. Clumps of numerous white cells are seen in infections. Presence of many white cells in urine is called as pyuria. In hypotonic urine white cells are swollen and the granules are highly refractile and show Brownian movement; such cells are called as glitter cells; large numbers are indicative of injury to urinary tract.

Normally 0-2 white cells may be seen per high power field. Pus cells greater than 10/HPF or presence of clumps is suggestive of urinary tract infection.

Increased numbers of white cells occur in fever, pyelonephritis, lower urinary tract infection, tubulointerstitial nephritis, and renal transplant rejection.

In urinary tract infection, following are usually seen in combination:

  • Clumps of pus cells or pus cells >10/HPF
  • Bacteria
  • Albuminuria
  • Positive nitrite test

Simultaneous presence of white cells and white cell casts indicates presence of renal infection (pyelonephritis).

Eosinophils (>1% of urinary leucocytes) are a characteristic feature of acute interstitial nephritis due to drug reaction (better appreciated with a Wright’s stain).

Renal Tubular Epithelial Cells

Presence of renal tubular epithelial cells is a significant finding. Increased numbers are found in conditions causing tubular damage like acute tubular necrosis, pyelonephritis, viral infection of kidney, allograft rejection, and salicylate or heavy metal poisoning.

These cells are small (about the same size or slightly larger than white blood cell), polyhedral, columnar, or oval, and have granular cytoplasm. A single, large, refractile, eccentric nucleus is often seen.

Renal tubular epithelial cells are difficult to distinguish from pus cells in unstained preparations.

Squamous Epithelial Cells

Squamous epithelial cells line the lower urethra and vagina. They are best seen under low power objective (×10). Presence of large numbers of squamous cells in urine indicates contamination of urine with vaginal fluid. These are large cells, rectangular in shape, flat with abundant cytoplasm and a small, central nucleus.

Transitional Epithelial Cells

Transitional cells line renal pelvis, ureters, urinary bladder, and upper urethra. These cells are large, and diamond- or pear-shaped (caudate cells). Large numbers or sheets of these cells in urine occur after catheterization and in transitional cell carcinoma.

Oval Fat Bodies

These are degenerated renal tubular epithelial cells filled with highly refractile lipid (cholesterol) droplets. Under polarized light, they show a characteristic “Maltese cross” pattern. They can be stained with a fat stain such as Sudan III or Oil Red O. They are seen in nephrotic syndrome in which there is lipiduria.

Spermatozoa

They may sometimes be seen in urine of men.

Telescoped urinary sediment: This refers to urinary sediment consisting of red blood cells, white blood cells, oval fat bodies, and all types of casts in roughly equal proportion. It occurs in lupus nephritis, malignant hypertension, rapidly proliferative glomerulonephritis, and diabetic glomerulosclerosis.

ORGANISMS

Organisms detectable in urine are shown in Figure 825.4.

Figure 825.4 Organisms in urine
Figure 825.4 Organisms in urine: (A) Bacteria, (B) Yeasts, (C) Trichomonas, and (D) Egg of Schistosoma haematobium

Bacteria

Bacteria in urine can be detected by microscopic examination, reagent strip tests for significant bacteriuria (nitrite test, leucocyte esterase test), and culture.

Significant bacteriuria exists when there are >105 bacterial colony forming units/ml of urine in a cleancatch midstream sample, >104 colony forming units/ml of urine in catheterized sample, and >103 colonyforming units/ml of urine in a suprapubic aspiration sample.

  1. Microscopic examination: In a wet preparation, presence of bacteria should be reported only when urine is fresh. Bacteria occur in combination with pus cells. Gram’s-stained smear of uncentrifuged urine showing 1 or more bacteria per oil-immersion field suggests presence of > 105 bacterial colony forming units/ml of urine. If many squamous cells are present, then urine is probably contaminated with vaginal flora. Also, presence of only bacteria without pus cells indicates contamination with vaginal or skin flora.
  2. Chemical or reagent strip tests for significant bacteriuria: These are given earlier.
  3. Culture: On culture, a colony count of >105/ml is strongly suggestive of urinary tract infection, even in asymptomatic females. Positive culture is followed by sensitivity test. Most infections are due to Gram-negative enteric bacteria, particularly Escherichia coli.

If three or more species of bacteria are identified on culture, it almost always indicates contamination by vaginal flora.

Negative culture in the presence of pyuria (‘sterile’ pyuria) occurs with prior antibiotic therapy, renal tuberculosis, prostatitis, renal calculi, catheterization, fever in children (irrespective of cause), female genital tract infection, and non-specific urethritis in males.

Yeast Cells (Candida)

These are round or oval structures of approximately the same size as red blood cells. In contrast to red cells, they show budding, are oval and more refractile, and are not soluble in 2% acetic acid.

Presence of Candida in urine may suggest immunocompromised state, vaginal candidiasis, or diabetes mellitus. Usually pyuria is present if there is infection by Candida. Candida may also be a contaminant in the sample and therefore urine sample must be examined in a fresh state.

Trichomonas vaginalis

These are motile organisms with pear shape, undulating membrane on one side, and four flagellae. They cause vaginitis in females and are thus contaminants in urine. They are easily detected in fresh urine due to their motility.

Eggs of Schistosoma haematobium

Infection by this organism is prevalent in Egypt.

Microfilariae

They may be seen in urine in chyluria due to rupture of a urogenital lymphatic vessel.

CASTS

Urinary casts are cylindrical, cigar-shaped microscopic structures that form in distal renal tubules and collecting ducts. They take the shape and diameter of the lumina (molds or ‘casts’) of the renal tubules. They have parallel sides and rounded ends. Their length and width may be variable. Casts are basically composed of a precipitate of a protein that is secreted by tubules (Tamm-Horsfall protein). Since casts form only in renal tubules their presence is indicative of disease of the renal parenchyma. Although there are several types of casts, all urine casts are basically hyaline; various types of casts are formed when different elements get deposited on the hyaline material (Figure 825.5). Casts are best seen under low power objective (×10) with condenser lowered down to reduce the illumination.

Figure 825.5 Genesis of casts in urine
 Figure 825.5 Genesis of casts in urine. All cellular casts degenerate to granular and waxy casts

Casts are the only elements in the urinary sediment that are specifically of renal origin.

Casts (Figure 825.6) are of two main types:

  1. Noncellular: Hyaline, granular, waxy, fatty
  2. Cellular: Red blood cell, white blood cell, renal tubular epithelial cell.

Hyaline and granular casts may appear in normal or diseased states. All other casts are found in kidney diseases.

Figure 825.6 Urinary casts
Figure 825.6 Urinary casts: (A) Hyaline cast, (B) Granular cast, (C) Waxy cast, (D) Fatty cast, (E) Red cell cast, (F) White cell cast, and (G) Epithelial cast

Non-cellular Casts

Hyaline casts: These are the most common type of casts in urine and are homogenous, colorless, transparent, and refractile. They are cylindrical with parallel sides and blunt, rounded ends and low refractive index. Presence of occasional hyaline cast is considered as normal. Their presence in increased numbers (“cylinduria”) is abnormal. They are composed primarily of Tamm-Horsfall protein. They occur transiently after strenuous muscle exercise in healthy persons and during fever. Increased numbers are found in conditions causing glomerular proteinuria.

Granular casts: Presence of degenerated cellular debris in a cast makes it granular in appearance. These are cylindrical structures with coarse or fine granules (which represent degenerated renal tubular epithelial cells) embedded in Tamm-Horsfall protein matrix. They are seen after strenuous muscle exercise and in fever, acute glomerulonephritis, and pyelonephritis.

Waxy cast: These are the most easily recognized of all casts. They form when hyaline casts remain in renal tubules for long time (prolonged stasis). They have homogenous, smooth glassy appearance, cracked or serrated margins and irregular broken-off ends. The ends are straight and sharp and not rounded as in other casts. They are light yellow in color. They are most commonly seen in end-stage renal failure.

Fatty casts: These are cylindrical structures filled with highly refractile fat globules (triglycerides and cholesterol esters) in Tamm-Horsfall protein matrix. They are seen in nephrotic syndrome.

Broad casts: Broad casts form in dilated distal tubules and are seen in chronic renal failure and severe renal tubular obstruction. Both waxy and broad casts are associated with poor prognosis.

Cellular Casts

To be called as cellular, casts should contain at least three cells in the matrix. Cellular casts are named according to the type of cells entrapped in the matrix.

Red cell casts: These are cylindrical structures with red cells in Tamm-Horsfall protein matrix. They may appear brown in color due to hemoglobin pigmentation. These have greater diagnostic importance than any other cast. If present, they help to differentiate hematuria due to glomerular disease from hematuria due to other causes. RBC casts usually denote glomerular pathology e.g. acute glomerulonephritis.

White cell casts: These are cylindrical structures with white blood cells embedded in Tamm-Horsfall protein matrix. Leucocytes usually enter into tubules from the interstitium and therefore presence of leucocyte casts indicates tubulointerstitial disease like pyelonephritis.

Renal tubular epithelial cell casts: These are composed of renal tubular epithelial cells that have been sloughed off. They are seen in acute tubular necrosis, viral renal disease, heavy metal poisoning, and acute allograft rejection. Even an occasional renal tubular cast is a significant finding.

CRYSTALS

Crystals are refractile structures with a definite geometric shape due to orderly 3-dimensional arrangement of its atoms and molecules. Amorphous material (or deposit) has no definite shape and is commonly seen in the form of granular aggregates or clumps.

Crystals in urine (Figure 825.7) can be divided into two main types: (1) Normal (seen in normal urinary sediment), and (2) Abnormal (seen in diseased states).

Figure 825.7 Crystals in urine
Figure 825.7 Crystals in urine. (A) Normal crystals: (1) Calcium oxalate, (2) Triple phosphates, (3) Uric acid, (4) Amorphous phosphates, (5) Amorphous urates, (6) Ammonium urate. (B) Abnormal crystals: (1) Cysteine, (2) Cholesterol, (3) Bilirubin, (4) Tyrosine, (5) Sulfonamide, and (6) Leucine

However, crystals found in normal urine can also be seen in some diseases in increased numbers.

Most crystals have no clinical importance (particularly phosphates, urates, and oxalates). Crystals can be identified in urine by their morphology. However, before reporting presence of any abnormal crystals, it is necessary to confirm them by chemical tests.

Normal Crystals

Crystals present in acid urine:

  1. Uric acid crystals: These are variable in shape (diamond, rosette, plates), and yellow or red-brown in color (due to urinary pigment). They are soluble in alkali, and insoluble in acid. Increased numbers are found in gout and leukemia. Flat hexagonal uric acid crystals may be mistaken for cysteine crystals that also form in acid urine.
  2. Calcium oxalate crystals: These are colorless, refractile, and envelope-shaped. Sometimes dumbbell-shaped or peanut-like forms are seen. They are soluble in dilute hydrochloric acid. Ingestion of certain foods like tomatoes, spinach, cabbage, asparagus, and rhubarb causes increase in their numbers. Their increased number in fresh urine (oxaluria) may also suggest oxalate stones. A large number are seen in ethylene glycol poisoning.
  3. Amorphous urates: These are urate salts of potassium, magnesium, or calcium in acid urine. They are usually yellow, fine granules in compact masses. They are soluble in alkali or saline at 60°C.

Crystals present in alkaline urine:

  1. Calcium carbonate crystals: These are small, colorless, and grouped in pairs. They are soluble in acetic acid and give off bubbles of gas when they dissolve.
  2. Phosphates: Phosphates may occur as crystals (triple phosphates, calcium hydrogen phosphate), or as amorphous deposits.
    Phosphate crystals
    Triple phosphates (ammonium magnesium phosphate): They are colorless, shiny, 3-6 sided prisms with oblique surfaces at the ends (“coffinlids”), or may have a feathery fern-like appearance.
    Calcium hydrogen phosphate (stellar phosphate): These are colorless, and of variable shape (starshaped, plates or prisms).
    Amorphous phosphates: These occur as colorless small granules, often dispersed.
    All phosphates are soluble in dilute acetic acid.
  3. Ammonium urate crystals: These occur as cactus-like (covered with spines) and called as ‘thornapple’ crystals. They are yellow-brown and soluble in acetic acid at 60°C.

Abnormal Crystals

They are rare, but result from a pathological process.

These occur in acid pH, often in large amounts. Abnormal crystals should not be reported on microscopy alone; additional chemical tests are done for confirmation.

  1. Cysteine crystals: These are colorless, clear, hexagonal (having 6 sides), very refractile plates in acid urine. They often occur in layers. They are soluble in 30% hydrochloric acid. They are seen in cysteinuria, an inborn error of metabolism. Cysteine crystals are often associated with formation of cysteine stones.
  2. Cholesterol crystals: These are colorless, refractile, flat rectangular plates with notched (missing) corners, and appear stacked in a stair-step arrangement. They are soluble in ether, chloroform, or alcohol. They are seen in lipiduria e.g. nephrotic syndrome and hypercholesterolemia. They can be positively identified by polarizing microscope.
  3. Bilirubin crystals: These are small (5 μ), brown crystals of variable shape (square, bead-like, or fine needles). Their presence can be confirmed by doing reagent strip or chemical test for bilirubin. These crystals are soluble in strong acid or alkali. They are seen in severe obstructive liver disease.
  4. Leucine crystals: These are refractile, yellow or brown, spheres with radial or concentric striations. They are soluble in alkali. They are usually found in urine along with tyrosine in severe liver disease (cirrhosis).
  5. Tyrosine crystals: They appear as clusters of fine, delicate, colorless or yellow needles and are seen in liver disease and tyrosinemia (an inborn error of metabolism). They dissolve in alkali.
  6. Sulfonamide crystals: They are variably shaped crystals, but usually appear as sheaves of needles. They occur following sulfonamide therapy. They are soluble in acetone.

Bile salts are salts of four different types of bile acids: cholic, deoxycholic, chenodeoxycholic, and lithocholic. These bile acids combine with glycine or taurine to form complex salts or acids. Bile salts enter the small intestine through the bile and act as detergents to emulsify fat and reduce the surface tension on fat droplets so that enzymes (lipases) can breakdown the fat. In the terminal ileum, bile salts are absorbed and enter in the blood stream from where they are taken up by the liver and re-excreted in bile (enterohepatic circulation).

Bile salts along with bilirubin can be detected in urine in cases of obstructive jaundice. In obstructive jaundice, bile salts and conjugated bilirubin regurgitate into blood from biliary canaliculi (due to increased intrabiliary pressure) and are excreted in urine. The test used for their detection is Hay’s surface tension test. The property of bile salts to lower the surface tension is utilized in this test.

Take some fresh urine in a conical glass tube. Urine should be at the room temperature. Sprinkle on the surface particles of sulphur. If bile salts are present, sulphur particles sink to the bottom because of lowering of surface tension by bile salts. If sulphur particles remain on the surface of urine, bile salts are absent.

Thymol (used as a preservative) gives false positive test.

Bilirubin is converted to non-reactive biliverdin on exposure to light (daylight or fluorescent light) and on standing at room temperature. Biliverdin cannot be detected by tests that detect bilirubin. Therefore fresh sample that is kept protected from light is required. Findings associated with bilirubinuria are listed below.

Methods for detection of bilirubin in urine are foam test, Gmelin’s test, Lugol iodine test, Fouchet’s test, Ictotest tablet test, and reagent strip test.

  1. Foam test: About 5 ml of urine in a test tube is shaken and observed for development of yellowish foam. Similar result is also obtained with proteins and highly concentrated urine. In normal urine, foam is white.
  2. Gmelin’s test: Take 3 ml of concentrated nitric acid in a test tube and slowly place equal quantity of urine over it. The tube is shaken gently; play of colors (yellow, red, violet, blue, and green) indicates positive test (Figure 823.1).
  3. Lugol iodine test: Take 4 ml of Lugol iodine solution (Iodine 1 gm, potassium iodide 2 gm, and distilled water to make 100 ml) in a test tube and add 4 drops of urine. Mix by shaking. Development of green color indicates positive test.
  4. Fouchet’s test: This is a simple and sensitive test.
    i. Take 5 ml of fresh urine in a test tube, add 2.5 ml of 10% of barium chloride, and mix well. A precipitate of sulphates appears to which bilirubin is bound (barium sulphate-bilirubin complex).
    ii. Filter to obtain the precipitate on a filter paper.
    iii. To the precipitate on the filter paper, add 1 drop of Fouchet’s reagent. (Fouchet’s reagent consists of 25 grams of trichloroacetic acid, 10 ml of 10% ferric chloride, and distilled water 100 ml).
    iv. Immediate development of blue-green color around the drop indicates presence of bilirubin (Figure 823.2).
  5. Reagent strips or tablets impregnated with diazo reagent: These tests are based on reaction of bilirubin with diazo reagent; color change is proportional to the concentration of bilirubin. Tablets (Ictotest) detect 0.05-0.1 mg of bilirubin/dl of urine; reagent strip tests are less sensitive (0.5 mg/dl).
Figure 823.1 Positive Gmelins test for bilirubin showing play of colors
Figure 823.1 Positive Gmelin’s test for bilirubin showing play of colors

Figure 823.2 Positive Fouchets test for bilirubin in urine
Figure 823.2 Positive Fouchet’s test for bilirubin in urine

The proportion of ketone bodies in urine in ketosis is variable: β-hydroxybutyric acid 78%, acetoacetic acid 20%, and acetone 2%.

No method for detection of ketonuria reacts with all the three ketone bodies. Rothera’s nitroprusside method and methods based on it detect acetoacetic acid and acetone (the test is 10-20 times more sensitive to acetoacetic acid than acetone). Ferric chloride test detects acetoacetic acid only. β-hydroxybutyric acid is not detected by any of the screening tests.

Methods for detection of ketone bodies in urine are Rothera’s test, Acetest tablet method, ferric chloride test, and reagent strip test.

1. ROTHERA’S’ TEST (Classic Nitroprusside Reaction)

Acetoacetic acid or acetone reacts with nitroprusside in alkaline solution to form a purple-colored complex (Figure 822.1). Rothera’s test is sensitive to 1-5 mg/dl of acetoacetate and to 10-25 mg/dl of acetone.

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Figure 822.1 Principles of Rothera Test in Urine
Figure 822.1 Principles of Rothera’s test and reagent strip test for ketone bodies in urine. Ketones are detected as acetoacetic acid and acetone but not β-hydroxybutyric acid

Method

  1. Take 5 ml of urine in a test tube and saturate it with ammonium sulphate.
  2. Add a small crystal of sodium nitroprusside. Mix well.
  3. Slowly run along the side of the test tube liquor ammonia to form a layer.
  4. Immediate formation of a purple permanganate colored ring at the junction of the two fluids indicates a positive test (Figure 822.2).

False-positive test can occur in the presence of L-dopa in urine and in phenylketonuria.

Figure 822.2 Rotheras tube test and reagent strip test for ketone bodies in urine
Figure 822.2 Rothera’s tube test and reagent strip test for ketone bodies in urine

2. ACETEST TABLET TEST

This is Rothera’s test in the form of a tablet. The Acetest tablet consists of sodium nitroprusside, glycine, and an alkaline buffer. A purplelavender discoloration of the tablet indicates the presence of acetoacetate or acetone (≥ 5 mg/dl). A rough estimate of the amount of ketone bodies can be obtained by comparison with the color chart provided by the manufacturer.

The test is more sensitive than reagent strip test for ketones.

3. FERRIC CHLORIDE TEST (Gerhardt’s)

Addition of 10% ferric chloride solution to urine causes solution to become reddish or purplish if acetoacetic acid is present. The test is not specific since certain drugs (salicylate and L-dopa) give similar reaction. Sensitivity of the test is 25-50 mg/dl.

4. REAGENT STRIP TEST

Reagent strips tests are modifications of nitroprusside test (Figures 822.1 and 822.2). Their sensitivity is 5-10 mg/dl of acetoacetate. If exposed to moisture, reagent strips often give false-negative result. Ketone pad on the strip test is especially vulnerable to improper storage and easily gets damaged. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.

1. HEAT AND ACETIC ACID TEST (BOILING TEST)
 
This test is based on the principle that proteins get precipitated when boiled in an acidic solution.
 
Method
 
Urine should be clear; if not, filter or use supernatant from a centrifuged sample.
 
Urine should be just acidic (check with litmus paper); if not, add 10% acetic acid drop by drop until blue litmus paper turns red.
 
A test tube is filled 2/3rds with urine. The tube is inclined at an angle and the upper portion is boiled over the flame. (Only the upper portion is heated so that convection currents generated by heat do not disturb the precipitate and the upper portion can be compared with the lower clear portion). Compare the heated part with the lower part. Cloudiness or turbidity indicates presence of either phosphates or proteins (Figure 821.1). A few drops of 10% acetic acid are added and the upper portion is boiled again. Turbidity due to phosphates disappears while that due to proteins does not.
 
Figure 821.1 Principle of heat test for proteins
Figure 821.1 Principle of heat test for proteins
 
False-positive test occurs with tolbutamide and large doses of penicillins.
 
2. REAGENT STRIP TEST
 
The reagent area of the strip is coated with an indicator and buffered to an acid pH which changes color in the presence of proteins (Figures 821.2 and 821.3). The principle is known as “protein error of indicators”.
 
Figure 821.2 Principle of reagent strip test for proteins
Figure 821.2 Principle of reagent strip test for proteins. The principle is called as ‘protein error of indicators’ meaning that one color appears if protein is present and another color if protein is absent. Sensitivity is 5-10 mg/dl. The test does not detect Bence Jones proteins, hemoglobin, and myoglobin
 
The reagent area is impregnated with bromophenol blue indicator buffered to pH 3.0 with citrate. When the dye gets adsorbed to protein, there is change in ionization (and hence pH) of the indicator that leads to change in color of the indicator. The intensity of the color produced is proportional to the concentration of protein. The test is semi-quantitative.
 
Figure 821.3 Grading of proteinuria with reagent strip test
Figure 821.3 Grading of proteinuria with reagent strip test (above) and sulphosalicylic acid test (below)
 
Reagent strip test is mainly reactive to albumin. It is false-negative in the presence of Bence Jones proteins, myoglobin, and hemoglobin. Overload (Bence Jones) proteinuria and tubular proteinuria may be missed entirely if only reagent strip method is used. This test should be followed by sulphosalicylic acid test, which is a confirmatory test. Highly alkaline urine, gross hematuria, and contamination with vaginal secretions can give false-positive reactions. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.
 
3. SULPHOSALICYLIC ACID TEST
 
Addition of sulphosalicylic acid to the urine causes formation of a white precipitate if proteins are present (Proteins are denatured by organic acids and precipitate out of solution).
 
Take 2 ml of clear urine in a test tube. If reaction of urine is neutral or alkaline, a drop of glacial acetic acid is added. Add 2-3 drops of sulphosalicylic acid (3 to 5%), and examine for turbidity against a dark background (Figure 821.3).
 
This test is more sensitive and reliable than boiling test.
 
False-positive test may occur due to gross hematuria, highly concentrated urine, radiographic contrast media, excess uric acid, tolbutamide, sulphonamides, salicylates, and penicillins.
 
False-negative test can occur with very dilute urine.
 
The test can detect albumin, hemoglobin, myoglobin, and Bence Jones proteins.
 
Comparison of reagent strip test and sulphosalicylic acid test is shown in Table 821.1.
 
Table 821.1 Comparison of two tests for proteinuria
Parameter Reagent strip test Sulphosalicylic acid test
1. Principle Colorimetric Acid precipitation
2. Proteins detected Albumin All (albumin, Bence Jones proteins, hemoglobin, myoglobin)
3. Sensitivity 5-10 mg/dl 20 mg/dl
4. Indicator Color change Turbidity
5. Type of test Screening Confirmatory
 
QUANTITATIVE ESTIMATION OF PROTEINS
 
Indications for quantitative estimation of proteins in urine are:
 
  • Diagnosis of nephrotic syndrome
  • Detection of microalbuminuria or early diabetic nephropathy
  • To follow response to therapy in renal disease
 
Proteinuria >1500 mg/ 24 hours indicates glomerular disease; proteinuria >3500 mg/24 hours is called as nephrotic range proteinuria; in tubular, hemodynamic and post renal diseases, proteinuria is usually < 1500 mg/24 hours.
 
Grading of albuminuria is shown in Table 821.2. There are two methods for quantitation of proteins:
 
  1. Estimation of proteins in a 24-hour urine sample, and
  2. Estimation of protein/creatinine ratio in a random urine sample.
 
Table 821.2 Grading of albuminuria
Condition mg/24 hr mg/L mg/g creatinine μg/min μg/mg creatinine g/mol creatinine
Normal < 30 < 20 < 20 < 20 < 30 < 2.5
Microalbuminuria 30-300 20-200 20-300 20-200 30-300 2.5-25
Overt albuminuria > 300 > 200 > 300 > 200 > 300 > 25
 
1. Quantitative estimation of proteins in a 24-hour urine sample: Collection of a 24-hour sample is given earlier. Adequacy of sample is confirmed by calculating expected 24-hour urine creatinine excretion. Daily urinary creatinine excretion depends on muscle mass and remains relatively constant in an individual patient. In adult males creatinine excretion is 14-26 mg/kg/24 hours, while in women it is 11-20 mg/kg/24 hours. Various methods are available for quantitative estimation of proteins: Esbach’s albuminometer method, turbidimetric methods, biuret reaction, and immunologic methods.
 
2. Estimation of protein/creatinine ratio in a random urine sample: Because of the problem of incomplete collection of a 24-hour urine sample, many laboratories measure protein/creatinine ratio in a random urine sample. Normal protein/creatinine ratio is < 0.2. In low-grade proteinuria it is 0.2-1.0; in moderate, it is 1.0-3.5; and in nephrotic- range proteinuria it is > 3.5.
 
MICROALBUMINURIA
 
This is defined as urinary excretion of 30 to 300 mg/24 hours (or 2-20 mg/dl) of albumin in urine.
 
Significance of Microalbuminuria
 
  1. Microalbuminuria is considered as the earliest sign of renal damage in diabetes mellitus (diabetic nephropathy). It indicates increase in capillary permeability to albumin and denotes microvascular disease. Microalbuminuria precedes the development of diabetic nephropathy by a few years. If blood glucose level and hypertension are tightly controlled at this stage by aggressive treatment then progression to irreversible renal disease and subsequent renal failure can be delayed or prevented.
  2. Microalbuminuria is an independent risk factor for cardiovascular disease in diabetes mellitus.
 
Detection of Microalbuminuria: Microalbuminuria cannot be detected by routine tests for proteinuria. Methods for detection include:
 
  • Measurement of albumin-creatinine ratio in a random urine sample
  • Measurement of albumin in an early morning or random urine sample
  • Measurement of albumin in a 24 hr sample
 
Test strips that screen for microalbuminuria are available commercially. Exact quantitation can be done by immunologic assays like radioimmunoassay or enzyme linked immunosorbent assay.
 
BENCE JONES PROTEINURIA
 
Bence Jones proteins are monoclonal immunoglobulin light chains (either κ or λ) that are synthesized by neoplastic plasma cells. Excess production of these light chains occurs in plasma cell dyscrasias like multiple myeloma and primary amyloidosis. Because of their low molecular weight and high concentration they are excreted in urine (overflow proteinuria).
 
Bence Jones proteins have a characteristic thermal behaviour. When heated, Bence Jones proteins precipitate at temperatures between 40°C to 60°C (other proteins precipitate between 60-70°C), and precipitate disappears on further heating at 85-100°C (while precipitate of other proteins does not). When cooled (60-85°C), there is reappearance of precipitate of Bence Jones proteins. This test, however, is not specific for Bence Jones proteins and both false-positive and -negative results can occur. This test has been replaced by protein electrophoresis of concentrated urine sample (Figure 821.4).
 
Figure 821.4 Urine protein electrophoresis showing heavy Bence Jones proteinuria
Figure 821.4 Urine protein electrophoresis showing heavy Bence Jones proteinuria (red arrow) along with loss of albumin and other low molecular weight proteins in urine
 
Further evaluation of persistent overt proteinuria is shown in Figure 821.5.
 
Figure 821.5 Evaluation of proteinuria
Figure 821.5 Evaluation of proteinuria.
Note: Quantitation of proteins and creatinine clearance are done in all patients with persistent proteinuria

1. COPPER REDUCTION METHODS

A. Benedict’s qualitative test:

When urine is boiled in Benedict’s qualitative solution, blue alkaline copper sulphate is reduced to red-brown cuprous oxide if a reducing agent is present (Figure 820.1). The extent of reduction depends on the concentration of the reducing substance. This test, however, is not specific for glucose.

Figure 820.1 Principle of Benedict’s qualitative test for sugar in urine
Figure 820.1 Principle of Benedict’s qualitative test for sugar in urine. Sensitivity is 200 mg of glucose/dl

Other carbohydrates (like lactose, fructose, galactose, pentoses), certain metabolites (glucuronic acid, homogentisic acid, uric acid, creatinine), and drugs (ascorbic acid, salicylates, cephalosporins, penicillins, streptomycin, isoniazid, para-aminosalicylic acid, nalidixic acid, etc.) also reduce alkaline copper sulphate solution.

Method

  1. Take 5 ml of Benedict’s qualitative reagent in a test tube (composition of Benedict’s qualitative reagent: copper sulphate 17.3 gram, sodium carbonate 100 gram, sodium citrate 173 gram, distilled water 1000 ml).
  2. Add 0.5 ml (or 8 drops) of urine. Mix well.
  3. Boil over a flame for 2 minutes.
  4. Allow to cool at room temperature.
  5. Note the color change, if any.

Sensitivity of the test is about 200 mg reducing substance per dl of urine. Since Benedict’s test gives positive reaction with carbohydrates other than glucose, it is also used as a screening test (for detection of galactose, lactose, fructose, maltose, and pentoses in urine) for inborn errors of carbohydrate metabolism in infants and children.

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For testing urine only for glucose, reagent strips are preferred (see below).

The result is reported in grades as follows (Figure 820.2):

  • Nil: no change from blue color
  • Trace: Green without precipitate
  • 1+ (approx. 0.5 grams/dl): Green with precipitate
  • 2+ (approx. 1.0 grams/dl): Brown precipitate
  • 3+ (approx. 1.5 grams/dl: Yellow-orange precipitate
  • 4+ (> 2.0 grams/dl): Brick- red precipitate.
Figure 820.2 Grading of Benedicts test
Figure 820.2 Grading of Benedict’s test (above) and reagent strip test (below) for glucose

B. Clinitest tablet method (Copper reduction tablet test):

This is a modified form of Benedict’s test in which the reagents are present in a tablet form (copper sulphate, citric acid, sodium carbonate, and anhydrous sodium hydroxide). Sensitivity is 200 mgs/dl of glucose.

2. REAGENT STRIP METHOD

This test is specific for glucose and is therefore preferred over Benedict’s and Clinitest methods. It is based on glucose oxidase-peroxidase reaction. Reagent area of the strips is impregnated with two enzymes (glucose oxidase and peroxidase) and a chromogen. Glucose is oxidized by glucose oxidase with the resultant formation of hydrogen peroxide and gluconic acid. Oxidation of chromogen occurs in the presence of hydrogen peroxide and the enzyme peroxidase with resultant color change (Figure 820.3). Nature of chromogen and buffer system differ in different strips. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.

The strip is dipped into the urine sample and color is observed after a specified time and compared with the color chart provided (Figure 820.2).

Figure 820.3 Principle of reagent strip test for glucose in urine
Figure 820.3 Principle of reagent strip test for glucose in urine. Each mole of glucose produces one mole of peroxide, and each mole of peroxide reduces one mole of oxygen. Sensitivity is 100 mg glucose/100 ml

This test is more sensitive than Benedict’s qualitative test and specific only for glucose. Other reducing agents give negative reaction.

Sensitivity of the test is about 100 mg glucose/dl of urine.

False-positive test occurs in the presence of oxidizing agent (bleach or hypochlorite used to clean urine containers), which oxidizes the chromogen directly.

False-negative test occurs in the presence of large amounts of ketones, salicylates, ascorbic acid, and severe Escherichia coli infection (catalase produced by organisms in urine inactivates hydrogen peroxide).

The parameters to be examined on physical examination of urine are listed below.

  • Volume
  • Color
  • Appearance
  • Odor
  • Specific Gravity
  • pH

VOLUME

Volume of only the 24-hr specimen of urine needs to be measured and reported. The average 24-hr urinary output in adults is 600-2000 ml. The volume varies according to fluid intake, diet, and climate. Abnormalities of urinary volume are as follows:

  • Polyuria means urinary volume > 2000 ml/24 hours. This is seen in diabetes mellitus (osmotic diuresis), diabetes insipidus (failure of secretion of antidiuretic hormone), chronic renal failure (loss of concentrating ability of kidneys) or diuretic therapy.
  • Oliguria means urinary volume < 400 ml/24 hours. Causes include febrile states, acute glomerulonephritis (decreased glomerular filtration), congestive cardiac failure or dehydration (decreased renal blood flow).
  • Anuria means urinary output < 100 ml/24 hours or complete cessation of urine output. It occurs in acute tubular necrosis (e.g. in shock, hemolytic transfusion reaction), acute glomerulonephritis, and complete urinary tract obstruction.

COLOR

Normal urine color in a fresh state is pale yellow or amber and is due to the presence of various pigments collectively called urochrome. Depending on the state of hydration urine may normally be colorless (over hydration) or dark yellow (dehydration). Some of the abnormal colors with associated conditions are listed in Table 819.1.

Table 819.1 Different colors of urine
Colors Conditions
Colorless Dilute urine (diabetes mellitus, diabetes insipidus, overhydration)
Red Hematuria, Hemoglobinuria, Porphyria, Myoglobinuria
Dark brown or black Alkaptonuria, Melanoma
Brown Hemoglobinuria
Yellow Concentrated urine
Yellow-green or green Biliverdin
Deep yellow with yellow foam Bilirubin
Orange or orange-brown Urobilinogen/Porphobilinogen
Milky-white Chyluria
Red or orange fluorescence with UV light Porphyria
Note: Many drugs cause changes in urine color; drug history should be obtained if there is abnormal coloration of urine

APPEARANCE

Normal, freshly voided urine is clear in appearance. Causes of cloudy or turbid urine are listed in Table 819.2. Foamy urine occurs in the presence of excess proteins or bilirubin.

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Table 819.2 Causes of cloudy or turbid urine
Cause Appearance Diagnosis
1. Amorphous phosphates White and cloudy on standing in alkaline urine Disappear on addition of a drop of dilute acetic acid
2. Amorphous urates Pink and cloudy in acid urine Dissolve on warming
3. Pus cells Varying grades of turbidity Microscopy
4. Bacteria Uniformly cloudy; do not settle at the bottom following centrifugation Microscopy, Nitrite test

ODOR

Freshly voided urine has a typical aromatic odor due to volatile organic acids. After standing, urine develops ammoniacal odor (formation of ammonia occurs when urea is decomposed by bacteria). Some abnormal odors with associated conditions are:

  • Fruity: Ketoacidosis, starvation
  • Mousy or musty: Phenylketonuria
  • Fishy: Urinary tract infection with Proteus, tyrosinaemia.
  • Ammoniacal: Urinary tract infection with Escherichia coli, old standing urine.
  • Foul: Urinary tract infection
  • Sulfurous: Cystinuria.

SPECIFIC GRAVITY (SG)

This is also called as relative mass density. It depends on amount of solutes in solution. It is basically a comparison of density of urine against the density of distilled water at a particular temperature. Specific gravity of distilled water is 1.000. Normal SG of urine is 1.003 to 1.030 and depends on the state of hydration. SG of normal urine is mainly related to urea and sodium. SG increases as solute concentration increases and decreases when temperature rises (since volume expands with rise in temperature).

SG of urine is a measure of concentrating ability of kidneys and is determined to get information about this tubular function. SG, however, is affected by proteinuria and glycosuria.

Causes of increase in SG of urine are diabetes mellitus (glycosuria), nephrotic syndrome (proteinuria), fever, and dehydration.

Causes of decrease in SG of urine are diabetes insipidus (SG consistently between 1.002-1.003), chronic renal failure (low and fixed SG at 1.010 due to loss of concentrating ability of tubules) and compulsive water drinking.

Methods for measuring SG are urinometer method, refractometer method, and reagent strip method.

1. Urinometer method:

This method is based on the principle of buoyancy (i.e. the ability of a fluid to exert an upward thrust on a body placed in it). Urinometer (a hydrometer) is placed in a container filled with urine (Figure 819.1A). When solute concentration is high, upthrust of solution increases and urinometer is pushed up (high SG). If solute concentration is low, urinometer sinks further into the urine (low SG).

Figure 819.1 A. Urinometer method and B. Reagent strip method for measuring specific gravity of urine
Figure 819.1 (A) Urinometer method and (B) Reagent strip method for measuring specific gravity of urine

Accuracy of a urinometer needs to be checked with distilled water. In distilled water, urinometer should show SG of 1.000 at the temperature of calibration. If not, then the difference needs to be adjusted in test readings taken subsequently.

The method is as follows:

  1. Fill a measuring cylinder with 50 ml of urine.
  2. Lower urinometer gently into the urine and let it float freely.
  3. Let urinometer settle; it should not touch the sides or bottom of the cylinder.
  4. Take the reading of SG on the scale (lowest point of meniscus) at the surface of the urine.
  5. Take out the urinometer and immediately note the temperature of urine with a thermometer.

Correction for temperature: Density of urine increases at low temperature and decreases at higher temperature. This causes false reading of SG. Therefore, SG is corrected for difference between urine temperature and calibration temperature. Check the temperature of calibration of the urinometer To get the corrected SG, add 0.001 to the reading for every 3°C that the urine temperature is above the temperature of calibration. Similarly subtract 0.001 from the reading for every 3°C below the calibration temperature.

Correction for dilution: If quantity of urine is not sufficient for measurement of SG, urine can be appropriately diluted and the last two figures of SG are multiplied by the dilution factor.

Correction for abnormal solute concentration: High SG in the presence of glycosuria or proteinuria will not reflect true kidney function (concentrating ability). Therefore it is necessary to nullify the effect of glucose or proteins. For this, 0.003 is subtracted from temperature-corrected SG for each 1 gm of protein/dl urine and 0.004 for every 1 gm of glucose/dl urine.

2. Refractometer method:

SG can be precisely determined by a refractometer, which measures the refractive index of the total soluble solids. Higher the concentration of total dissolved solids, higher the refractive index. Extent of refraction of a beam of light passed through urine is a measure of solute concentration, and thus of SG. The method is simple and requires only 1-2 drops of urine. Result is read from a scale or from digital display.

3. Reagent strip method:

Reagent strip (Figure 819.1B) measures the concentration of ions in urine, which correlates with SG. Depending on the ionic strength of urine, a polyelectrolyte will ionize in proportion. This causes a change in color of pH indicator (bromothymol blue). Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.

REACTION AND pH

The pH is the scale for measuring acidity or alkalinity (acid if pH is < 7.0; alkaline if pH is > 7.0; neutral if pH is 7.0). On standing, urine becomes alkaline because of loss of carbon dioxide and production of ammonia from urea. Therefore, for correct estimation of pH, fresh urine should be examined.

There are various methods for determination of reaction of urine: litmus paper, pH indicator paper, pH meter, and reagent strip tests.

  1. Litmus paper test: A small strip of litmus paper is dipped in urine and any color change is noted. If blue litmus paper turns red, it indicates acid urine. If red paper turns blue, it indicates alkaline urine (Figure 819.2A).
  2. pH indicator paper: Reagent area (which is impregnated with bromothymol blue and methyl red) of indicator paper strip is dipped in urine sample and the color change is compared with the color guide provided. Approximate pH is obtained.
  3. pH meter: An electrode of pH meter is dipped in urine sample and pH is read off directly from the digital display. It is used if exact pH is required.
  4. Reagent strip test: The test area (Figure 819.2B) contains polyionic polymer bound to H+; on reaction with cations in urine, H+ is released causing change in color of the pH-sensitive dye. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.
Figure 819.2 A. Testing pH of urine with litmus paper and B. with reagent strip test
Figure 819.2 Testing pH of urine with litmus paper (A) and with reagent strip test (B)

Normal pH range is 4.6 to 8.0 (average 6.0 or slightly acidic). Urine pH depends on diet, acid base balance, water balance, and renal tubular function.

Acidic urine is found in ketosis (diabetes mellitus, starvation, fever), urinary tract infection by Escherichia coli, and high protein diet. Alkaline urine may result from urinary tract infection by bacteria that split urea to ammonia (Proteus or Pseudomonas), severe vomiting, vegetarian diet, old ammoniacal urine sample and chronic renal failure.

Determining pH of urine helps in identifying various crystals in urine. Altering pH of urine may be useful in treatment of renal calculi (i.e. some stones form only in acid urine e.g. uric acid calculi; in such cases urine is kept alkaline); urinary tract infection (urine should be kept acid); and treatment with certain drugs (e.g. streptomycin is effective in urinary tract infection if urine is kept alkaline). In unexplained metabolic acidosis, measurement of urine pH is helpful in diagnosing renal tubular acidosis; in renal tubular acidosis, urine pH is consistently alkaline despite metabolic acidosis.

Fresh urine sample should be used because on standing urobilinogen is converted to urobilin, which cannot be detected by routine tests. A timed (2-hour postprandial) sample can also be used for testing urobilinogen.

Methods for detection of increased amounts of urobilinogen in urine are Ehrlich’s aldehyde test and reagent strip test.

1. EHRLICH’S ALDEHYDE TEST

Ehrlich’s reagent (pdimethylaminobenzaldehyde) reacts with urobilinogen in urine to produce a pink color. Intensity of color developed depends on the amount of urobilinogen present. Presence of bilirubin interferes with the reaction, and therefore if present, should be removed. For this, equal volumes of urine and 10% barium chloride are mixed and then filtered. Test for urobilinogen is carried out on the filtrate. However, similar reaction is produced by porphobilinogen (a substance excreted in urine in patients of porphyria).

Fig. 818.1 Ehrlichs aldehyde test for urobilinogen
Figure 818.1 Ehrlich’s aldehyde test for urobilinogen

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Method

Take 5 ml of fresh urine in a test tube. Add 0.5 ml of Ehrlich’s aldehyde reagent (which consists of hydrochloric acid 20 ml, distilled water 80 ml, and paradimethylaminobenzaldehyde 2 gm). Allow to stand at room temperature for 5 minutes. Development of pink color indicates normal amount of urobilinogen. Darkred color means increased amount of urobilinogen (Figure 818.1).

Since both urobilinogen and porphobilinogen produce similar reaction, further testing is required to distinguish between the two. For this, Watson-Schwartz test is used. Add 1-2 ml of chloroform, shake for 2 minutes and allow to stand. Pink color in the chloroform layer indicates presence of urobilinogen, while pink coloration of aqueous portion indicates presence of porphobilinogen. Pink layer is then decanted and shaken with butanol. A pink color in the aqueous layer indicates porphobilinogen (Figure 818.2).

Figure 818.2 Interpretation of Watson Schwartz test
Figure 818.2 Interpretation of Watson-Schwartz test

False-negative reaction can occur in the presence of (i) urinary tract infection (nitrites oxidize urobilinogen to urobilin), and (ii) antibiotic therapy (gut bacteria which produce urobilinogen are destroyed).

2. REAGENT STRIP METHOD

This method is specific for urobilinogen. Test area is impregnated with either p-dimethylaminobenzaldehyde or 4-methoxybenzene diazonium tetrafluoroborate. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.

Porphyrias (from Greek porphura meaning purple pigment; the name is probably derived from purple discoloration of some body fluids during the attack) are a heterogeneous group of rare disorders resulting from disturbance in the heme biosynthetic pathway leading to the abnormal accumulations of red and purple pigments called as porphyrins in the body. Heme, a component of hemoglobin, is synthesized through various steps as shown in Figure 817.1. Each of the steps is catalyzed by a separate enzyme; if any of these steps fails (due to hereditary or acquired cause), precursors of heme (porphyrin intermediates) accumulate in blood, get deposited in skin and other organs, and excreted in urine and feces. Depending on the site of defect, different types of porphyrias are described with varying clinical features, severity, and the nature of accumulated porphyrin.
 
Porphyria has been offered as a possible explanation for the medieval tales of vampires and werewolves; this is because of the number of similarities between the behavior of persons suffering from porphyria and the folklore (avoiding sunlight, mutilation of skin on exposure to sunlight, red teeth, psychiatric disturbance, and drinking of blood to obtain heme).
 
Porphyrias are often missed or wrongly diagnosed as many of them are not associated with definite physical findings, screening tests may yield false-negative results, diagnostic criteria are poorly defined and mild disorders produce an enzyme assay result within ‘normal’ range.
 
Heme is mainly required in bone marrow (for hemoglobin synthesis) and in liver (for cytochromes). Therefore, porphyrias are divided into erythropoietic and hepatic types, depending on the site of expression of disease. Hepatic porphyrias mainly affect the nervous system, while erythropoietic porphyrias primarily affect the skin. Porphyrias are also classified into acute and nonacute (or cutaneous) types depending on clinical presentation (Table 817.1).
 
Table 817.1 Various classification schemes for porphyrias
Classification based on predominant clinical manifestations
Classification based on site of expression of disease
Classification based on mode of clinical presentation
Neuropsychiatric
Hepatic
Acute
1. Acute intermittent porphyria
1. ALA-dehydratase porphyria
1. ALA-dehydratase porphyria (Plumboporphyria)
2. ALA-dehydratase porphyria (Plumboporphyria)
2. Acute intermittent porphyria
2. Acute intermittent porphyria
Cutaneous (Photosensitivity)
3. Hereditary coproporphyria
3. Hereditary coproporphyria
1. Congenital erythropoietic porphyria
4. Variegate porphyria
4. Variegate porphyria
2. Porphyria cutanea tarda
Erythropoietic porphyria
Non-acute (cutaneous)
3. Erythropoietic protoporphyria
1. Congenital erythropoietic porphyria
1. Porphyria cutanea tarda
Mixed (Neuropsychiatric and cutaneous)
2. Erythropoietic protoporphyria
2. Congenital erythropoietic porphyria
1. Hereditary coproporphyria
Hepatic/Erythropoietic
3. Erythropoietic protoporphyria
2. Variegate porphyria
1. Porphyria cutanea tarda
 
 
Inheritance of porphyrias may be autosomal dominant or recessive. Most acute porphyrias are inherited in an autosomal dominant manner (i.e. inheritance of one abnormal copy of gene). Therefore, the activity of the deficient enzyme is 50%. When the level of heme falls in the liver due to some cause, activity of ALA synthase is stimulated leading to increase in the levels of heme precursors up to the point of enzyme defect. Increased levels of heme precursors cause symptoms of acute porphyria. When the heme level returns back to normal, symptoms subside.
 
Accumulation of porphyrin precursors can occur in lead poisoning due to inhibition of enzyme aminolevulinic acid dehydratase in heme biosynthetic pathway. This can mimick acute intermittent porphyria.
 
CLINICAL FEATURES
 
Clinical features of porphyrias are variable and depend on type. Acute porphyrias present with symptoms like acute and severe abdominal pain/vomiting/constipation, chest pain, emotional and mental disorders, seizures, hypertension, tachycardia, sensory loss, and muscle weakness. Cutaneous porphyrias present with photosensitivity (redness and blistering of skin on exposure to sunlight), itching, necrosis of skin and gums, and increased hair growth over the temples (Table 817.2).
 
Table 817.2 Clinical characteristics of porphyrias
Porphyria Deficient enzyme Clinical features Inheritance Initial test
1. Acute intermittent porphyria (AIP)* PBG deaminase Acute neurovisceral attacks; triggering factors+ (e.g. drugs, diet restriction) Autosomal dominant Urinary PBG; urine becomes brown, red, or black on standing
2. Variegate porphyria Protoporphyrinogen oxidase Acute neurovisceral attacks + skin fragility, bullae Autosomal dominant Urinary PBG
3. Hereditary coproporphyria Coproporphyrinogen oxidase Acute neurovisceral attacks + skin fragility, bullae Autosomal dominant Urinary PBG
4. Congenital erythropoietic porphyria Uroporphyrinogen cosynthase Onset in infancy; skin fragility, bullae; extreme photosensitivity with mutilation; red teeth and urine (pink red urinestaining of diapers) Autosomal recessive Urinary/fecal total porphyrins; ultraviolet fluorescence of urine, feces, and bones
5. Porphyria cutanea tarda* Uroporphyrinogen decarboxylase Skin fragility, bullae Autosomal dominant (some cases) Urinary/fecal total porphyrins
6. Erythropoietic protoporphyria* Ferrochelatase Acute photosensitivity Autosomal dominant Free erythrocyte protoporphyrin
Disorders marked with * are the three most common porphyrias. PBG: Porphobilinogen
  
Symptoms can be triggered by drugs (barbiturates, oral contraceptives, diazepam, phenytoin, carbamazepine, methyldopa, sulfonamides, chloramphenicol, and antihistamines), emotional or physical stress, infection, dieting, fasting, substance abuse, premenstrual period, smoking, and alcohol. Autosomal dominant porphyrias include acute intermittent porphyria, variegate porphyria, porphyria cutanea tarda, erythropoietic protoporphyria (most cases), and hereditary coproporphyria. Autosomal recessive porphyrias include: congenital erythropoietic porphyria, erythropoietic protoporphyria (few cases), and ALAdehydratase porphyria (plumboporphyria).
 
LABORATORY DIAGNOSIS
 
Porphyria can be diagnosed through tests done on blood, urine, and feces during symptomatic period. Timely and accurate diagnosis is required for effective management of porphyrias. Due to the variability and a broad range of clinical features, porphyrias are included under differential diagnosis of many conditions. All routine hospital laboratories usually have facilities for initial investigations in suspected cases of porphyrias; laboratory tests for identification of specific type of porphyrias are available in specialized laboratories.
 
INITIAL STUDIES
 
In suspected acute porphyrias (acute neurovisceral attack), a fresh randomly collected urine sample (10-20 ml) should be submitted for detection of excessive urinary excretion of porphobilinogen (PBG) (see Figure 817.2). In AIP, urine becomes red or brown on standing (see Figure 817.3). In suspected cases of cutaneous porphyrias (acute photosensitivity without skin fragility), free erythrocyte protporphyrin or FEP in EDTA blood (for diagnosis of erythrocytic protoporphyria) and for all other cutaneous porphyrias (skin fragility and bullae), examination of fresh, random urine (10-20 ml) and either feces (5-10 g) or plasma for excess porphyrins are necessary (see Figure 817.4 and Table 817.2).
 
Figure 817.2 Evaluation of acute neurovisceral porphyria
 Figure 817.2 Evaluation of acute neurovisceral porphyria
 
Figure 817.3 Red coloration of urine on standing in acute intermittent porphyria
Figure 817.3 Red coloration of urine on standing in acute intermittent porphyria
 
Figure 817.4 Evaluation of cutaneous porphyrias
Figure 817.4 Evaluation of cutaneous porphyrias
 
Apart from diagnosis, the detection of excretion of a particular heme intermediate in urine or feces can help in detecting site of defect in porphyria. Heme precursors up to coproporphyrinogen III are water-soluble and thus can be detected in urine. Protoporphyrinogen and Protoporphyrin are insoluble in water and are excreted in bile and can be detected in feces. All samples should be protected from light.
 
Samples required are
 
  1. 10-20 ml of fresh random urine sample without any preservative;
  2. 5-10 g wet weight of fecal sample, and
  3. blood anticoagulated with EDTA.
 
Test for Porphobilinogen in Urine
 
Ehrlich’s aldehyde test is done for detection of PBG. Ehrlich’s reagent (p-dimethylaminobenzaldehyde) reacts with PBG in urine to produce a red color. The red product has an absorption spectrum with a peak at 553 nm and a shoulder at 540 nm. Since both urobilinogen and porphobilinogen produce similar reaction, further testing is required to distinguish between the two. Urobilinogen can be removed by solvent extraction. (See Watson-Schwartz test). Levels of PBG may be normal or near normal in between attacks. Therefore, samples should be tested during an attack to avoid false-negative results.
 
Test for Total Porphyrins in Urine
 
Total porphyrins can be detected in acidified urine sample by spectrophotometry (Porphyrins have an intense absorbance peak around 400 nm). Semiquantitative estimation of porphyrins is possible.
 
Test for Total Porphyrins in Feces
 
Total porphyrins in feces can be determined in acidic extract of fecal sample by spectrophotometry; it is necessary to first remove dietary chlorophyll (that also absorbs light around 400 nm) by diethyl ether extraction.
 
Tests for Porphyrins in Erythrocytes and Plasma
 
Visual examination for porphyrin fluorescence, and solvent fractionation and spectrophotometry have now been replaced by fluorometric methods.
 
Further Testing
 
If the initial testing for porphyria is positive, then concentrations of porphyrins should be estimated in urine, feces, and blood to arrive at specific diagnosis (Tables 817.3 and 817.4).
 
Table 817.3 Diagnostic patterns of concentrations of heme precursors in acute porphyrias
Porphyria Urine Feces
Acute intermittent porphyria PBG, Copro III
Variegate porphyria PBG, Copro III Proto IX
Hereditary coproporphyria PBG, Copro III Copro III
PBG: Porphobilinogen; Copro III: Coproporphyrinogen III; Proto IX: Protoporphyrin IX
 
Table 817.4 Diagnostic patterns of concentrations of heme precursors in cutaneous porphyrias
Porphyria Urine Feces Erythrocytes
Congenital erythropoietic porphyria Uro I, Copro I Copro I
Porphyria cutanea tarda Uroporphyrin Isocopro
Erythropoietic protoporphyria Protoporphyrin
Uro I: Uroporphyrinogen I; Copro I: Coproporphyrinogen I; Isocopro: Isocoproporphyrinogen
 
In latent porphyrias and in patients during remission, porphyrin levels may be normal; in such cases, enzymatic and DNA testing is necessary for diagnosis.
 
If porphyria is diagnosed, then it is necessary to investigate close family members for the disorder. Positive family members should be counseled regarding triggering factors.
This is done by flow cytometric analysis for detection of lack of GpIb/IX in Bernanrd Soulier syndrome (deficiency of CD42), and lack of GpIIb/IIIa in Glanzmann’s thrombasthenia (deficiency of CD41, CD61).
 
What is the best protocol for platelet glycoprotein (GPIIb/IIIa) analysis using flow cytometry?
 
Fresh platelets should always be used. Storing platelets dramatically changes the level of transmembrane proteins. The best way is to follow one of standardized protocols defined in: Immunophenotypic analysis of platelets. Krueger LA, Barnard MR, Frelinger AL 3rd, Furman MI, Michelson AD.Curr Protoc Cytom. 2002 Feb;Chapter 6:Unit 6.10

TEST FOR D-DIMER

  • 03 Aug 2017
D-dimer is derived from the breakdown of fibrin by plasmin and D-dimer test is used to evaluate fibrin degradation. Blood sample can be either serum or plasma. Latex or polystyrene microparticles coated with monoclonal antibody to D-dimer are mixed with patient’s sample and observed for microparticle agglutination. As the particle is small, turbidometric endpoint can be determined in automated instruments. D-dimer and FDPs are raised in disseminated intravascular coagulation, intravascular thrombosis (myocardial infarction, stroke, venous thrombosis, pulmonary embolism), and during postoperative period or following trauma. D-dimer test is commonly used for exclusion of thrombosis and thrombotic tendencies.
 
Further Reading:
 
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