Food Science

Published in Food Science
Monday, 04 September 2017 17:26
Food science is the applied science devoted to the study of food. The Institute of Food Technologists defines food science as "the discipline in which the engineering, biological, and physical sciences are used to study the nature of foods, the causes of deterioration, the principles underlying food processing, and the improvement of foods for the consuming public". The textbook Food Science defines food science in simpler terms as "the application of basic sciences and engineering to study the physical, chemical, and biochemical nature of foods and the principles of food processing".


Published in Ecology
Monday, 04 September 2017 17:01
Ecology (from Greek: οἶκος, "house", or "environment"; -λογία, "study of") is the scientific analysis and study of interactions among organisms and their environment. It is an interdisciplinary field that includes biology, geography, and Earth science. Ecology includes the study of interactions that organisms have with each other, other organisms, and with abiotic components of their environment. Topics of interest to ecologists include the diversity, distribution, amount (biomass), and number (population) of particular organisms, as well as cooperation and competition between organisms, both within and among ecosystems. Ecosystems are composed of dynamically interacting parts including organisms, the communities they make up, and the non-living components of their environment. Ecosystem processes, such as primary production, pedogenesis, nutrient cycling, and various niche construction activities, regulate the flux of energy and matter through an environment. These processes are sustained by organisms with specific life history traits, and the variety of organisms is called biodiversity. Biodiversity, which refers to the varieties of species, genes, and ecosystems, enhances certain ecosystem services.
Ecology is not synonymous with environment, environmentalism, natural history, or environmental science. It is closely related to evolutionary biology, genetics, and ethology. An important focus for ecologists is to improve the understanding of how biodiversity affects ecological function. Ecologists seek to explain:
  • Life processes, interactions, and adaptations
  • The movement of materials and energy through living communities
  • The successional development of ecosystems
  • The abundance and distribution of organisms and biodiversity in the context of the environment.
There are many practical applications of ecology in conservation biology, wetland management, natural resource management(agroecology, agriculture, forestry, agroforestry, fisheries), city planning (urban ecology), community health, economics, basic and applied science, and human social interaction (human ecology). For example, the Circles of Sustainability approach treats ecology as more than the environment 'out there'. It is not treated as separate from humans. Organisms (including humans) and resources compose ecosystems which, in turn, maintain biophysical feedback mechanisms that moderate processes acting on living (biotic) and non-living (abiotic) components of the planet. Ecosystems sustain life-supporting functions and produce natural capital like biomass production (food, fuel, fiber, and medicine), the regulation of climate, global biogeochemical cycles, water filtration, soil formation, erosion control, flood protection, and many other natural features of scientific, historical, economic, or intrinsic value.
The word "ecology" ("Ökologie") was coined in 1866 by the German scientist Ernst Haeckel (1834–1919). Ecological thought is derivative of established currents in philosophy, particularly from ethics and politics. Ancient Greek philosophers such as Hippocrates and Aristotle laid the foundations of ecology in their studies on natural history. Modern ecology became a much more rigorous science in the late 19th century. Evolutionary concepts relating to adaptation and natural selection became the cornerstones of modern ecological theory.

Forensic Science

Published in Forensic Science
Monday, 04 September 2017 16:36
Forensic science is the application of science to criminal and civil laws, mainly—on the criminal side—during criminal investigation, as governed by the legal standards of admissible evidence and criminal procedure.
Forensic scientists collect, preserve, and analyze scientific evidence during the course of an investigation. While some forensic scientists travel to the scene of the crime to collect the evidence themselves, others occupy a laboratory role, performing analysis on objects brought to them by other individuals.
In addition to their laboratory role, forensic scientists testify as expert witnesses in both criminal and civil cases and can work for either the prosecution or the defense. While any field could technically be forensic, certain sections have developed over time to encompass the majority of forensically related cases.


Published in Clinical Pathology
Wednesday, 30 August 2017 23:26
Microscopic examinations done on fecal sample are shown in Figure 846.1.
Figure 846.1 Microscopic examinations carried out on fecal sample
Figure 846.1 Microscopic examinations carried out on fecal sample
Collection of Specimen for Parasites
A random specimen of stool (at least 4 ml or 4 cm³) is collected in a clean, dry, container with a tightly fitting lid (a tin box, plastic box, glass jar, or waxed cardboard box) and transported immediately to the laboratory (this is because trophozoites of Entameba histolytica rapidly degenerate and alter in morphology). About 20-40 grams of formed stool or 5-6 tablespoons of watery stool should be collected. Stool should not be contaminated with urine, water, soil, or menstrual blood. Urine and water destroy trophozoites; soil will introduce extraneous organisms and also hinder proper examination. Parasites are best detected in warm, freshly passed stools and therefore stools should be examined as early as possible after receipt in the laboratory (preferably within 1 hour of collection). If delay in examination is anticipated, sample may be refrigerated. A fixative containing 10% formalin (for preservation of eggs, larvae, and cysts) or polyvinyl alcohol (for preservation of trophozoites and cysts, and for permanent staining) may be used if specimen is to be transported to a distant laboratory.
One negative report for ova and parasites does not exclude the possibility of infection. Three separate samples, collected at 3-day intervals, have been recommended to detect all parasite infections.
Patient should not be receiving oily laxatives, antidiarrheal medications, bismuth, antibiotics like tetracycline, or antacids for 7 days before stool examination. Patient should not have undergone a barium swallow examination.
In the laboratory, macroscopic examination is done for consistency (watery, loose, soft or formed) (Figure 846.2), color, odor, and presence of blood, mucus, adult worms or segments of tapeworms.
Figure 846.2 Consistency of feces
Figure 846.2 Consistency of feces
Trophozoites are most likely to be found in loose or watery stools or in stools containing blood and mucus, while cysts are likely to be found in formed stools. Trophozoites die soon after being passed and therefore such stools should be examined within 1 hour of passing. Examination of formed stools can be delayed but should be completed on the same day.
Color/Appearance of Fecal Specimens
  • Brown: Normal
  • Black: Bleeding in upper gastrointestinal tract (proximal to cecum), Drugs (iron salts, bismuth salts, charcoal)
  • Red: Bleeeding in large intestine, undigested tomatoes or beets
  • Clay-colored (gray-white): Biliary obstruction
  • Silvery: Carcinoma of ampulla of Vater
  • Watery: Certain strains of Escherichia coli, Rotavirus enteritis, cryptosporidiosis
  • Rice water: Cholera
  • Unformed with blood and mucus: Amebiasis, inflammatory bowel disease
  • Unformed with blood, mucus, and pus: Bacillary dysentery
  • Unformed, frothy, foul smelling, which float on water: Steatorrhea.
Preparation of Slides
After receipt in the laboratory, saline and iodine wet mounts of the sample are prepared (Figure 846.3).
Figure 846.3 Saline and iodine wet mounts of fecal sample
Figure 846.3 Saline and iodine wet mounts of fecal sample 
A drop of normal saline is placed near one end of a glass slide and a drop of Lugol iodine solution is placed near the other end. A small amount of feces (about the size of a match-head) is mixed with a drop each of saline and iodine using a wire loop, and a cover slip is placed over each preparation separately. If the specimen contains blood or mucus, that portion should be included for examination (trophozoites are more readily found in mucus). If the stools are liquid, select the portion from the surface for examination.
Saline wet mount is used for demonstration of eggs and larvae of helminths, and trophozoites and cysts of protozoa. It can also detect red cells and white cells. Iodine stains glycogen and nuclei of the cysts. The iodine wet mount is useful for identification of protozoal cysts. Trophozoites become non-motile in iodine mounts. A liquid, diarrheal stool can be examined directly without adding saline.
Concentration Procedure
Concentration of fecal specimen is useful if very small numbers of parasites are present. However, in concentrated specimens, amebic trophozoites can no longer be detected since they are destroyed. If wet mount examination is negative and there is clinical suspicion of parasitic infection, fecal concentration is indicated. It is used for detection of ova, cysts, and larvae of parasites.
Various concentration methods are available; the choice depends on the nature of parasites to be identified and the equipment/reagent available in a particular laboratory. Concentration techniques are of two main types:
  • Sedimentation techniques: Ova and cysts settle at the bottom. However, excessive fecal debris may make the detection of parasites difficult. Example: Formolethyl acetate sedimentation procedure.
  • Floatation techniques: Ova and cysts float on surface. However, some ova and cysts do not float at the top in this procedure. Examples: Saturated salt floatation technique and zinc sulphate concentration technique.
The most commonly used sedimentation method is formol-ethyl acetate concentration method since: (i) it can detect eggs and larvae of almost all helminths, and cysts of protozoa, (ii) it preserves their morphology well, (iii) it is rapid, and (iv) risk of infection to the laboratory worker is minimal because pathogens are killed by formalin.
In this method, fecal suspension is prepared in 10% formalin (10 ml formalin + 1 gram feces). This suspension is then passed through a gauze filter till 7 ml of filtered material is obtained. To this, ethyl acetate (3 ml) is added and the mixture is centrifuged for 1 minute. Eggs, larvae, and cysts sediment at the bottom of the centrifuge tube (Figure 846.4). Above this deposit, there are layers of formalin, fecal debris, and ether. Fecal debris is loosened with an applicator stick and the supernatant is poured off. One drop of sediment is placed on one end of a glass slide and one drop is placed at the other end. One of the drops is stained with iodine, cover slips are placed, and the preparation is examined under the microscope.
Figure 846.4 Formol ethyl acetate concentration technique
Figure 846.4 Formol-ethyl acetate concentration technique
Classification of Intestinal Parasites of Humans
Intestinal parasites of humans are classified into two main kingdoms: protozoa and metazoa (helminths) (Figure 846.5).
Figure 846.5 Classification of intestinal parasites of humans
Figure 846.5 Classification of intestinal parasites of humans


Published in Clinical Pathology
Wednesday, 30 August 2017 01:21
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.
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.


Published in Clinical Pathology
Monday, 28 August 2017 01:46
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.


Published in Clinical Pathology
Sunday, 27 August 2017 13:15
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”).


Published in Clinical Pathology
Sunday, 27 August 2017 01:43
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

Estimation of Creatinine Clearance from Serum Creatinine by Prediction Equations

Published in Clinical Pathology
Saturday, 26 August 2017 22:35
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.


Published in Clinical Pathology
Saturday, 26 August 2017 12:44
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
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:




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