- 05 Sep 2017
Box 855.1 Determination of basal acid output, maximum acid output, and peak acid output
- Volume: Normal total volume is 20-100 ml (usually < 50 ml). Causes of increased volume of gastric juice are—
• Delayed emptying of stomach: pyloric stenosis
• Increased gastric secretion: duodenal ulcer, Zollinger-Ellison syndrome.
- Color: Normal gastric secretion is colorless, with a faintly pungent odor. Fresh blood (due to trauma, or recent bleeding from ulcer or cancer) is red in color. Old hemorrhage produces a brown, coffee-ground like appearance (due to formation of acid hematin). Bile regurgitation produces a yellow or green color.
- pH: Normal pH is 1.5 to 3.5. In pernicious anemia, pH is greater than 7.0 due to absence of HCl.
- Basal acid output:
• Normal: Up to 5 mEq/hour.
• Duodenal ulcer: 5-15 mEq/hour.
• Zollinger-Ellison syndrome: >20 mEq/hour.
Normal BAO is seen in gastric ulcer and in some patients with duodenal ulcer.
- Peak acid output:
• Normal: 1-20 mEq/hour.
• Duodenal ulcer: 20-60 mEq/hour.
• Zollinger-Ellison syndrome: > 60 mEq/hour.
• Achlorhydria: 0 mEq/hour.
Normal PAO is seen in gastric ulcer and gastric carcinoma. Values up to 60 mEq/hour can occur in some normal individuals and in some patients with Zollinger-Ellison syndrome.
In pernicious anemia, there is no acid output due to gastric mucosal atrophy. Achlorhydria should be diagnosed only if there is no free HCl even after maximum stimulation.
- Ratio of basal acid output to peak acid output (BAO/PAO):
• Normal: < 0.20 (or < 20%).
• Gastric or duodenal ulcer: 0.20-0.40 (20-40%).
• Duodenal ulcer: 0.40-0.60 (40-60%).
• Zollinger-Ellison syndrome: > 0.60 (> 60%).
Normal values occur in gastric ulcer or gastric carcinoma.
|Increased gastric acid output||Decreased gastric acid output|
|• Duodenal ulcer||• Chronic atrophic gastritis|
|• Zollinger-Ellison syndrome||1. Pernicious anemia|
|• Hyperplasia of antral G cells||2. Rheumatoid arthritis|
|• Systemic mastocytosis||3. Thyrotoxicosis|
|• Basophilic leukemia||• Gastric ulcer|
|• Gastric carcinoma|
|• Chronic renal failure|
- 04 Sep 2017
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- 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.
- 04 Sep 2017
- 30 Aug 2017
Microscopic examinations done on fecal sample are shown in Figure 846.1.
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.
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.
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).
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 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.
Classification of Intestinal Parasites of Humans
Intestinal parasites of humans are classified into two main kingdoms: protozoa and metazoa (helminths) (Figure 846.5).
- 30 Aug 2017
Chemical examination of feces is usually carried out for the following tests (Figure 845.1):
- Occult blood
- Excess fat excretion (malabsorption)
- Reducing sugars
- Fecal osmotic gap
- Fecal pH
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:
- Intestinal diseases: hookworms, amebiasis, typhoid fever, ulcerative colitis, intussusception, adenoma, cancer of colon or rectum.
- Gastric and esophageal diseases: peptic ulcer, gastritis, esophageal varices, hiatus hernia.
- Systemic disorders: bleeding diathesis, uremia.
- 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
- 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.
- Drugs like aspirin and other anti-inflammatory drugs, which increase blood loss from gastrointestinal tract in normal persons.
Causes of False-negative Tests
- Foods containing large amounts of vitamin C.
- Conversion of all hemoglobin to acid hematin (which has no peroxidase-like activity) during passage through the gastrointestinal tract.
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
- Deficiency of pancreatic lipase (insufficient lipolysis): chronic pancreatitis, cystic fibrosis.
- Deficiency of bile salts (insufficient emulsification of fat): biliary obstruction, severe liver disease, bile salt deconjugation due to bacterial overgrowth in the small intestine.
- 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).
- 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.
- 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.
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.
Stool pH below 5.6 is characteristic of carbohydrate malabsorption.
- 28 Aug 2017
(Plasma sodium × Urine creatinine)
- Causes of increased specific gravity:
a. Reduced renal perfusion (with preservation of concentrating ability of tubules),
e. Urinary tract obstruction.
- Causes of reduced specific gravity:
a. Diabetes insipidus
b. Chronic renal failure
c. Impaired concentrating ability due to diseases of tubules.
- 27 Aug 2017
- 27 Aug 2017
- Pre-renal azotemia: shock, congestive heart failure, salt and water depletion
- Renal azotemia: impairment of renal function
- Post-renal azotemia: obstruction of urinary tract
- 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
- 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.
- 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.
- 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.
Causes of Increased Serum Creatinine Level
- Pre-renal, renal, and post-renal azotemia
- Large amount of dietary meat
- Active acromegaly and gigantism
- Increasing age (reduction in muscle mass)
- 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.
- 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.
- Increased BUN with normal serum creatinine:
• Pre-renal azotemia (reduced renal perfusion)
• High protein diet
• Increased protein catabolism
• Gastrointestinal hemorrhage
- 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
(72 × Serum creatinine in mg/dl)
The agents used for measurement of GFR are:
- Exogenous: Inulin, Radiolabelled ethylenediamine tetraacetic acid (51Cr- EDTA), 125I-iothalamate
- Endogenous: Creatinine, Urea, Cystatin C
- A small amount of creatinine is secreted by renal tubules that increase even further in advanced renal failure.
- Collection of urine is often incomplete.
- Creatinine level is affected by intake of meat and muscle mass.
- Creatinine level is affected by certain drugs like cimetidine, probenecid, and trimethoprim (which block tubular secretion of creatinine).
- 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
- Nephrotic syndrome in adults (most common indication)
- Nephrotic syndrome not responding to corticosteroids in children.
- Acute nephritic syndrome for differential diagnosis
- Unexplained renal insufficiency with near-normal kidney dimensions on ultrasonography
- Asymptomatic hematuria, when other diagnostic tests fail to identify the source of bleeding
- Isolated non-nephrotic range proteinuria (1-3 gm/24 hours) with renal impairment
- Impaired function of renal graft
- Involvement of kidney in systemic disease like systemic lupus erythematosus or amyloidosis
- Uncontrolled severe hypertension
- Hemorrhagic diathesis
- Solitary kidney
- Renal neoplasm (to avoid spread of malignant cells along the needle track)
- Large and multiple renal cysts
- Small, shrunken kidneys
- Acute urinary tract infection like pyelonephritis
- Urinary tract obstruction
- 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.
- Arteriovenous fistula
- Accidental biopsy of another organ or perforation of viscus (liver, spleen, pancreas, adrenals, intestine, or gallbladder)
- Death (rare).
- Patient’s informed consent is obtained.
- Ultrasound/CT scan is done to document the location and size of kidneys.
- 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.
- Patient is sedated before the procedure.
- Patient lies in prone position and kidney is identified with ultrasound.
- The skin over the selected site is disinfected and a local anesthetic is infiltrated.
- 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.
- 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.
- The biopsy should be placed in a drop of saline and examined under a dissecting microscope for adequacy.
- 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.
- 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.
- 17 Aug 2017
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:
Fasting plasma glucose ≥ 126 mg/dl (≥ 7.0 mmol/L)
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.
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
– 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).
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.
- A fasting venous blood sample is collected in the morning.
- 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.
- 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).
- Plasma glucose is estimated in fasting and 2-hour venous blood samples.
Interpretation of blood glucose levels is given in Table 837.1.
|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
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.
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
- 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.
- Albumin to creatinine ratio in a random urine sample
- Urinary albumin excretion in a 24-hour urine sample.
- Total cholesterol
- Low-density lipoprotein (LDL) cholesterol
- High-density lipoprotein (HDL) cholesterol
|Category||Low density lipoproteins||High density lipoproteins||Triglycerides|
|High-risk||≥130||< 35 (men)||≥ 400|
|< 45 (women)|
|Low-risk||< 100||> 45 (men)||< 200|
|> 55 (women)|
- Diabetic ketoacidosis (DKA)
- Hyperosmolar hyperglycemic state (HHS)
|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|
|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
- 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.
- 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).
- 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:
- 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
- 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
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
- 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.
- 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.
- Early diagnosis of ectopic pregnancy: Trans-vaginal ultrasonography (USG) and quantitative estimation of hCG are helpful in early diagnosis of ectopic pregnancy (before rupture).
- Evaluation of threatened abortion: Serial quantitative estimation of hCG is helpful in following the course of threatened abortion.
- Diagnosis and follow-up of gestational trophoblastic disease (GTD).
- 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.
- Follow-up of ovarian or testicular germ cell tumors, which produce hCG.
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).
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 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.
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
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.
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.
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.
- 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
- 15 Aug 2017
Box 835.1 Contributions to semen volume
• Testes and epididymis: 10%
• Seminal vesicles: 50%
• Prostate: 40%
• Cowper’s glands: Small volume
- 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.
|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|
|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|
|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
- 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.
- To check the effectiveness of vasectomy by confirming absence of sperm.
- To support or disprove a denial of paternity on the grounds of sterility.
- To examine vaginal secretions or clothing stains for the presence of semen in medicolegal cases.
- For selection of donors for artificial insemination.
- For selection of assisted reproductive technology, e.g. in vitro fertilization, gamete intrafallopian transfer technique.
|Box 835.3 Semen analysis for initial investigation of infertility
• Microscopic examination for (i) percentage of motile spermatozoa, (ii) sperm count, and (iii) sperm morphology
| 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
- PHYSICAL EXAMINATION OF SEMEN FOR INVESTIGATION OF INFERTILITY
- BIOCHEMICAL ANALYSIS OF SEMEN FOR INVESTIGATION OF INFERTILITY
- MICROSCOPIC EXAMINATION OF SEMEN FOR INVESTIGATION OF INFERTILITY
- IMMUNOLOGIC ANALYSIS OF SEMEN FOR INVESTIGATION OF INFERTILITY
- SPERM FUNCTION TESTS OR FUNCTIONAL ASSAYS
- EXAMINATION FOR THE PRESENCE OF SEMEN IN MEDICOLEGAL CASES
- 15 Aug 2017
Atleast 200 motile spermatozoa should be counted. If >50% of spermatozoa show attached latex particles, immunological problem is likely.
- Mix one drop of semen with 1 drop of eosin-nigrosin solution and incubate for 30 seconds.
- A smear is made from a drop placed on a glass slide.
- 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.
- The result is expressed as a proportion of viable sperms against non-viable as an integer percentage.
- 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).
- 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.
- 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.
- 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
- Normal sperm count is ≥ 20 million/ml (i.e. ≥ 20 × 106/ml). Sperm count < 20 million/ml may be associated with infertility in males.
• Total length of sperm: About 60 μ
• Total length of sperm: About 60 μ
– 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
- Normal sperm
- 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
- Defects in neck:
• Bent neck and tail forming an angle >90° to the long axis of head
- Defects in middle piece:
• Asymmetric insertion of midpiece into head
• Thick or irregular midpiece
• Abnormally thin midpiece
- Defects in tail:
• Bent tails
• Short tails
• Coiled tails
• Irregular tails
• Multiple tails
• Tails with irregular width
- Pin heads: Not to be counted
- Cytoplasmic droplets
• > 1/3rd the size of the sperm head
- Precursor cells: Considered abnormal
|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|
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.
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.
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.
- 13 Aug 2017
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.
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.
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.
- 12 Aug 2017
- End replication problem in eukaryotes accounts for loss of 20 base pairs per cell division.
- Oxidative stress accounts for loss of 50-100 base pairs per cell division.
- 10 Aug 2017
The chemical examination is carried out for substances in urine are listed below:
- Bile salts
- Nitrite or leukocyte esterase
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.
Causes of Proteinuria
Box 826.1: Causes of 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).
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).Box 826.2: Nephrotic syndrome
- 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
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
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
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
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).
- 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)
- Decreased availability of carbohydrates in the diet:
b. Persistent vomiting in children
c. Weight reduction program (severe carbohydrate restriction with normal fat intake)
- Increased metabolic needs:
a. Fever in children
b. Severe thyrotoxicosis
d. Protein calorie malnutrition
Further reading: Tests for Detection of Ketones in Urine
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).
|Urine test||Hemolytic jaundice||Hepatocellular jaundice||Obstructive jaundice|
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 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
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
- 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).
- Hemorrhage in tissues: There is increased formation of bilirubin from destruction of red cells.
Causes of Reduced Urobilinogen in Urine
- 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.
- 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
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
Presence of free hemoglobin in urine is called as hemoglobinuria.
Causes of Hemoglobinuria
- Hematuria with subsequent lysis of red blood cells in urine of low specific gravity.
- 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 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.
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
|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.
- 10 Aug 2017
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.
|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|
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.
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.
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.
Cellular elements in urine are shown in Figure 825.3.
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
- 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.
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 detectable in urine are shown in Figure 825.4.
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.
- 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.
- Chemical or reagent strip tests for significant bacteriuria: These are given earlier.
- 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.
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.
They may be seen in urine in chyluria due to rupture of a urogenital lymphatic vessel.
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.
Casts are the only elements in the urinary sediment that are specifically of renal origin.
Casts (Figure 825.6) are of two main types:
- Noncellular: Hyaline, granular, waxy, fatty
- 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.
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.
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 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).
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.
Crystals present in acid urine:
- 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.
- 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.
- 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:
- 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.
- 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.
- 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.
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.
- 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.
- 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.
- 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.
- 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).
- 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.
- Sulfonamide crystals: They are variably shaped crystals, but usually appear as sheaves of needles. They occur following sulfonamide therapy. They are soluble in acetone.