URINALYSIS
Unit One
1.0. INTRODUCTION
Ross D.L. (1983) defines urinalysis as a laboratory test in urine used to evaluate the function of the kidneys and the quality of urine produced. A urinalysis usually consists of three parts: examining the physical sample, a dipstick analysis to evaluate the presence of substances and microscopic examination of the sediment.
1.1. The Importance of Urinalysis
In 1637 Thomas Brian wrote “ there is no judgement of diseases to be given by the urine alone; that the physician ought not to give judgement of the urine, before he have strictly examined how the sick partie is affected.” Prior to Brian admonition, greater emphasis was placed on the crude examination of the urine than on obtaining a proper history and performing a thorough examination of the patient. Brian put the examination of urine- urinalysis in the context by which it is still viewed today: an important adjunct to the workup of a patient’s problem. Nearly two centuries later in 1827, Richard Bright introduced urinalysis as part of the routine examination of a patient. Bright emphasized using both macroscopic and microscopic examination of urine to diagnose for renal disease, a view later reinforced by the American physician Thomas Addis who refined the microscopic examination technique, whereas the early investigations advocated using urinalysis to screen for diseases of other organ systems. With the method of testing being a single, simple dipstick, urinalysis may now be performed well by relatively less skilled individuals. In fact in many clinical practices only dipstick tests are performed. Nevertheless, the microscopic examination can be critically important in the examination of diseases of the urogenital tract. In many situations the macroscopic examination enables discrimination of the diseases of the lower urinary tract from renal diseases, and often enables determination of the type and activity of the renal diseases.
1.2. Urinalysis Today
In today’s cost-focused environment, when should urinalysis-and what type- be performed? For the periodic physical examination dipstick testing should be routinely performed to pick up asymptomatic diseases. Macroscopic examinations should be performed if there is a history suggesting renal disease or if any of the dipstick tests are abnormal, for the patient admitted in the hospital, a dipstick urinalysis can be readily justified since it is inexpensive and may detect unsuspected asymptomatic disease the finding of which may spare the hospital and medical staff considerable embarrassment. When there is any suggestion of renal disease, a thorough microscopic examination is always indicated to help localize the site of the problem and to facilitate the diagnosis of the disorder.
1.3. Statement of The Problem
The Biomedical Science programme (BMS) has for a long time, not been able to purchase up to date teaching and learning resources in most taught courses including urinalysis, which forms the basis for routine examination of clients. Consequently it has not been easy to work effectively without reliable text materials available. This then means that the programme has been running with inadequate stocks of books. The BMS programme is going to review its curriculum shortly in late 2005 or early 2006 as such the programme requires to develop teaching and learning materials for instance manuals which can be reproduced to conform to the demands of the reviewed curriculum. Development of this manual is significant because the BMS textbooks are expensive to purchase and inadequate supply in the college library. Most textbooks offer wide ranging content, none of which is presented in depth (Shutes and Peterson 1994). Students have no access to the Internet service.
1.4. Justification
An updated manual on urinalysis was developed during an independent study. The manual complements the books available in the MCHS library. Trainees, qualified laboratory technicians and lecturers at the college can use this manual. This manual I believe provides an alternative source of information to the trainees, qualified laboratory technicians and lecturers and to the BMS programme.
1.5. Methodology
The study was a literature based using the Internet service and the few books available at Mzuzu University Library.
Unit Two
2.0. AIM
To provide the student with the knowledge, skills and attitudes necessary to understand, the principles and the significance of the tests performed in urinalysis.
2.1. OBJECTIVES
1. Discuss the physiology of the genitourinary system and the formation of urine.
2. Relate the physical and chemical properties of normal and abnormal urine specimens.
3. Perform a complete urinalysis, including all biochemical confirmatory tests, with accuracy.
4. Describe other tests that are used to detect renal function.
Unit Three
3.0. Anatomy of the human kidney
3.1. Objectives of Unit Two
§ Describe the anatomical structure of the kidney.
3.1.2. Structure of the kidney
3.1.3. Gross anatomic features of the kidney:
Palastanga et al (1994) describes the kidneys as bean shaped reddish-brown organs that are located at the back of the abdomen, below the diaphragm, one on each side of the spine. It is approximately 11.0 cm in length, 5.0 to 7.0 cm in diameter and 2.5 cm in thickness. The right kidney is slightly lower than the left because the liver is located above it (See figure 1). Surrounding the kidneys is a layer of adipose tissue that helps protect and support them. A fibrous layer of connective tissue called (renal fascia) encapsulates and anchors the kidneys in place in the abdomen. Each kidney is enclosed in a fibrous capsule and is composed of an outer cortex and an inner medulla located externally at the concave portion of the kidney is a notch called the hilum. Structures at the hilum of each kidney are the renal artery and vein lymphatics, nerves and renal pelvis (See figure 2). The renal pelvis is a funnel shaped extension of the upper ureter that provides a passageway for urine to the bladder.
Each kidney is enclosed in a fibrous capsule and is composed of an outer cortex and an inner medulla. The cortex, the outer portion lies under the renal fascia. Substantial portions of the inner medullary layer are separated by cortical substances that are called renal columns (columns of bertis) also originating in the cortex and extending into the medulla are the uriniferous tubules which are made up of the paranchymal functioning units of the kidneys, the nephrons. The medulla the inner portion of the kidney contain an estimated 8-18 pyramids so called because they are triangular (See figure 2). They are striated due to the collecting ducts, nephrons and blood vessels of which they are composed. The apex of each pyramid is called the papilla. Below the papilla is a large cavity called the renal pelvis, which is interrupted by cuplike extensions.
The active units of the kidney, nephrons are located within the cortex and medulla.
Figure 1 showing the Urogenital System
Figure 2 showing the human kidney
3.2. The Nephron:
Structures of the Nephron:
· Glomerulus
· Proximal Convoluted Tubules
· Loop of Henle
· Distal Convoluted Tubules
· Collecting Duct
Bullock (1999) describes the nephron as the functional unit of the kidney and the structure in which urine forms (See figure 3). Each kidney contains over a million nephrons. A nephron consists of small vessels carrying blood and a set of tubules carrying fluid derived from the blood. The fluid is a result from capillary filtration. It enters the tubules and is then modified by a number of different processes as the blood reabsorbs materials of value to the body. The fluid left over after this modification is the urine.
Arterial blood enters the kidney through the renal artery (See figure 2). This artery then divides into smaller arteries passing into the renal medulla. The arteries give rise to still smaller arteries that enter the kidney's cortex. Finally, the small arteries subdivide into numerous microscopic afferent arterioles. The microscopic afferent arterioles deliver blood into a tuft of capillaries called a glomerulus. Each nephron has one glomerulus. In the glomerulus, blood plasma passes into the hollow walls of a surrounding capsule. The remaining blood then flows into an efferent arteriole. The efferent arteriole then forms a capillary network called the peritubular capillaries. These surround the nephron tubules. Later, the peritubular capillaries then drain into small veins, which then unite to form larger veins. The larger veins eventually form the renal vein, which conducts blood out of the kidney.
The tubular portion of a nephron consists of several structures: the hollow capsule surrounding the glomerulus, the proximal convoluted tubule, the descending limb of the loop of Henle, the loop of Henle, the ascending limb of the loop of Henle, and the distal convoluted tubule.
The capsule surrounding the glomerulus is called Bowman's capsule, also known as the glomerular capsule. It is somewhat analogous to a long balloon pushed in at one end by a fist so the balloon surrounds the fist. The fist represents the glomerulus; the balloon represents Bowman's capsule. In order for filtration to occur, the pressure of the glomerulus must exceed the pressure of Bowman's capsule. Changes in the oncotic pressure of the glomerular capillaries, hydrostatic pressure in Bowman's capsule, or hydrostatic pressure in the glomerular capillaries can change the glomerular filtration rate.
Figure 3 showing the structure of nephron
3.3. Glomerulus (Kidney)
Tortora and Grabowski (2000) describe the glomerulus as a capillary bed found surrounded by the Bowman's capsule of the nephron in the vertebrate kidney (See figure 4). Glomerular endothelial cells are perforated by thousands of small 'cracks' known as "fenestrae". These 'cracks' allow water and small solutes to pass through, but not proteins and cells. Blood is fed to the glomerulus through the afferent arteriole, and empties into the efferent arteriole. The difference in pressure in the arterioles results in the process of ultrafiltration where fluids and soluble materials in the blood are forced out of the capillaries and into the Bowman’s capsule. Fluids collected in the Bowman's capsule is known as glomerular filtrate, which eventually becomes urine after further processing along the nephron.
3.4. Bowman’s capsule (bo’manz)
Cheesebrough (2000) describes a double-walled, cup-shaped structure around the glomerulus of each nephron of the vertebrate kidney as the Bowman’s capsule (see figure 4). It serves as a filter to remove organic wastes, excess inorganic salts, and water. Also called Malpighian capsule.
Figure 4 showing the glomerulus and Bowman’s capsule (courtesy of ADAM)
3.5. Renal Corpuscle
Palstanga (1994) describes a tuft of glomerular capillaries with the Bowman's capsule that encloses it as the renal corpuscle (See figure 3). It is also known as Malpighian corpuscle, named after Marcello Malpighi, (1628-1694 AD), an Italian physician and biologist. This name is not used widely anymore, probably to avoid confusion with a Malpighian corpuscle in the spleen. The renal corpuscle is the initial filtering component of a nephron in the kidney. It consists of a glomerulus, a small network of capillaries, enclosed in a Bowman's capsule, a sac-like structure. Fluids from blood in the glomerulus is collected in the Bowman's capsule to form "glomerulus filtrate", which is then further processed along the nephron to form urine.
3.6. Proximal Convoluted Tubule
In the biology of the kidney, the proximal convoluted tubule is the segment of the renal tubule that drains Bowman's capsule. Tortora (2000) explains that the proximal convoluted tubule is some 15 mm long and is located within the cortex or outer segment of the kidney (See figure 5). Virtually all filtered glucose, amino acids, bicarbonate and potassium are absorbed here, together with two-thirds of filtered sodium and water “salty fluid”. Some substances are secreted into this area, including penicillin, creatinine, and steroid glucuronides.
Figure 5 showing the structure of proximal convoluted tubule
3.7. Loop of Henle (hĕn'lē)
Tortora and Grabowski (2000) explain that the segment of the nephron of a vertebrate kidney, which is situated between the proximal and distal convoluted tubules, is named after its discoverer a German pathologist. a German pathologist, Friedrich Gustav Jacob Henle. (1809–1885 AD), The loop of Henle is a section of the nephron that leads from the proximal convoluted tubule to the distal convoluted tubule in the kidney. The loop has a hairpin bend in the medulla. Its primary function uses a countercurrent mechanism in the medulla to reabsorb water and ions from the urine. The loop of Henlé has a descending and ascending limb (See figure 6). Some 15 percent of the loops are relatively long and extend into the inner portion (medulla) of the kidney, with the remainder having a short loop, which are in the cortex only. The thinner descending limb loops back on itself and then forms the thick ascending limb which then passes close to its own glomerulus to form the juxta-glomerulus apparatus where renin is produced.
Figure 6 showing the structure of loop of Henle
It can be divided into four parts:
Descending limb
Thin ascending limb
Medullary thick ascending limb
Cortical thick ascending limb
3.8. Juxtaglomerular apparatus
Tortora and Grabowski (2000) describe the juxtaglomerular apparatus as a renal structure consisting of the macula densa and juxtaglomerular cells. Juxtaglomerular cells (JG cells) are the sites of renin secretion (See figure 7).
The JG cells are found in the afferent arterioles of the glomerulus act as an intra-renal pressure sensor. Lowered pressure leads to secretion of renin, which acts to increase systemic blood pressure via the rennin-angiotensin system.
The macula densa senses fluid flow rate in the distal tubule of the kidney and secretes a locally active vasopressor, which acts on the adjacent afferent arteriole to decrease GFR.
Example: A patient having a 0.1 mg/ml blood concentration of inulin can have his GFR shown by 0.1 mg/ml x 100 ml/min = 10 mg/min. From this, the patient has a clearance rate of 10mg of inulin in one minute so a 20 mg injection would be cleared in 2 minutes.
Figure 7 showing a structure of Juxtaglomerular apparatus 3.9. Distal Convoluted Tubule
Tortora and Grabowski (2000) describe the distal convoluted tubule (DCT) as a portion of kidney nephron between the loop of Henle and the collecting duct system. It is partly responsible for the regulation of potassium, sodium, and calcium and pH.
3.10. Collecting Duct System
The collecting duct system of the kidney consists of:
The connecting tubule
The cortical collecting duct
The medullary collecting duct
Unit Four
4.0. Physiology of the Human Kidney
Tortora (2000) explains that renal physiology is the study of the functions of the kidneys.
4.1. Functions of the Kidney
4.2. Objective
· Explain the physiology of the genitourinary system
4.3. Filtering Wastes from the Bloodstream
Wastes are filtered out from the blood in the glomeruli via the process of ultrafiltration.
The ultrafiltrate is passed through, in turn; the proximal convoluted tubules, the loop of Henle, and the distal convoluted tubules and is then collected by the collecting ducts to form urine.
The renal collecting ducts open into the renal pelvis and drain into the ureters, which pass on the urine to the bladder.
4.4 Secretion of Hormones
Tortora (2000) describes secretion as the process by which hormones move from a producing or storing organ to a target organ or peripheral site.
§ Secretion of erythropoietin, which regulates red blood cell production in the bone marrow.
§ Secretion of rennin
§ Secretion of the active form of vitamin D- 1,25-dihydroxycholecalciferol (calcitriol) and prostaglandins
4.4.1. Secretion of Erythropoietin
Mayne (2000) states that erythropoietin (EPO) is a hormone produced especially by the kidney and stimulates the formation of red blood cells by the bone marrow.The kidney cells that make EPO are specialized so that they are sensitive to low oxygen levels in the blood coming into the kidney. These cells make and release EPO when the oxygen level is too low.As the prime regulator of red cell production, EPO’s major functions are to:
1. Promote the development of red blood cells.
2. Initiate the synthesis of hemoglobin, the molecule within red cells that transports oxygen.
4.4.2. Secretion of Rennin
Bishop et al (1992) describes the rennin-angiotensin system (RAS) or the rennin-angiotensin aldosterone system as a hormone system that assists to regulate long-term blood pressure and blood volume in the body. The system can be activated when there is a loss of blood volume or a drop in blood pressure (such as in hemorrhage). If the perfusion of the juxtaglomerular apparatus in the kidneys decreases, then the juxtaglomerular cells release the enzymatic hormone renin. Renin cleaves an inactive peptide called angiotensinogen converting it into Angiotensin I is then converted to angiotensin II by angiotensin converting enzyme (ACE), which is found mainly in lung capillaries. Angiotensin I may have some minor activity, but angiotensin II is more potent.
Angiotensin II has a variety of effects on the body:
It is a potent vasoconstrictor.
In the kidneys, it constricts glomerular afferent arterioles. This decreases the glomerular filtration rate (GFR), which in turn raises systemic arterial blood pressure.
It also acts on the adrenal cortex causing the release of aldosterone. Aldosterone acts on the tubules (i.e. the distal convoluted tubules and the cortical collecting ducts) in the kidneys, causing them to reabsorb more sodium and water from the urine. Aldosterone also acts on the central nervous system to increase a person's appetite for salt, and to make them feel thirsty.
These effects directly act to increase the amount of fluid in the blood, making up for a loss in volume, and to increase blood pressure.
The renin-angiotensin system is often manipulated clinically to treat high blood pressure. Inhibitors of angiotensin-converting enzyme are often used to reduce the formation of the more potent angiotensin II. Alternatively, angiotensin receptor blockers (ARBs) can be used to prevent angiotensin II from acting on angiotensin receptors.
4.4.4. Secretion of the Active Form of Vitamin D 1,25-Dihydroxycholecalciferol (Calcitriol) and Prostaglandins.
The active metabolite of vitamin D is synthesized and released by the kidney following hepatic hydroxylation of 25-hydroxyvitamin D. 1,25 (OH)2 vitamin D increases calcium absorption by intestinal mucosal cells. In conjunction with PTH it stimulates osteoclastic activity releasing calcium from bone.
4.5. Maintaining Body Sodium and Water Balance
There is a stable balance of sodium and water in the body. The major homeostatic control point for maintaining this stable balance is renal excretion.
The kidney is directed to excrete or retain sodium via the action of aldosterone, ADH (anti-diuretic hormone or vasopressin), ANP (atrial natriuretic peptide) and other hormones.
Cl- always follows (Na+)
4.6. Acid-Base Homeostasis
Metabolic reactions are very sensitive to the pH level H+ or hydronium ion concentration) of the fluid in which they occur. This is because hydronium ions can influence enzyme function.
The kidneys maintain blood plasma acid-base homeostasis by hydronium regulation. Gain and loss of hydronium must be balanced.
4.6.1. Sources of Hydrogen-Ion Gain:
Carbon dioxide
Production of nonvolatile acids from the metabolism of proteins and other organic molecules.
Gain in (H+) hydrogen ions due to loss of bicarbonate in diarrhea or other nongastric (gastro intestinal, (GI) fluids.
Gain in hydrogen ions due to loss of bicarbonate in the urine.
Hypoventilation
4.6.2. Sources of Hydrogen Ion Loss:
Use of hydrogen ions in the metabolism of various organic anions.
Loss of hydrogen ions in vomitus.
Loss of hydrogen ions in the urine.
Hyperventilation.
When hydrogen ion loss exceeds gain, alkalosis occurs. When gain exceeds loss acidosis occurs. There are various renal responses to acidosis and alkalosis:
4.6.3. Responses to acidosis:
Bicarbonate is added to the blood plasma by tubular cells.
This is caused by sufficient hydrogen ion secretion from the tubular epithelial cells.
Extra (H+) hydrogen ion secretion will bind to non-bicarbonate urinary buffers and this will lead to more new bicarbonate in the blood plasma.
This is also caused by increased glutamine metabolism and ammonia excretion.
4.6.4. Responses to alkalosis:
Excretion of bicarbonate in urine.
This is caused by lowered rate of hydrogen ion secretion from the tubular epithelial cells.
This is also caused by lowered rates of glutamine metabolism and ammonia excretion.
4.6.5. Hydronium Ions and Carbon Dioxide
Carbon dioxide + water + carbonic anhydrase (catalyst) will produce carbonic acid + bicarbonate + hydronium ion
4.6.6. Buffering of hydrogen ions
Any substance that reversibly binds hydrogen ions is called a buffer. Extracellular and intracellular buffers buffer hydrogen ions.
4.6.6. Homeostatic controls
Hydrogen-ion (H+) gain and loss must be balanced to maintain a relatively stable concentration.
Unit five
5.0. Formation of Urine
5.1. Objective
Describe the formation of urine
5.2. Filtration, Reabsorption and Secretion
Every one of us depends on the process of urination for the removal of certain waste products in the body. The production of urine is vital to the health of the body. Most of us have probably never thought of urine as valuable, but we could not survive if we did not produce it and eliminate it. Urine is composed of water, certain electrolytes, and various waste products that are filtered out of the blood system. Remember, as the blood flows through the body, wastes resulting from the metabolism of foodstuffs in the body cells are deposited into the bloodstream, and this waste must be disposed of in some way. A major part of this "cleaning" of the blood takes place in the kidneys and, in particular, in the nephrons, where the blood is filtered to produce the urine. Both kidneys in the body carry out this essential blood cleansing function. Normally, about 20% of the total blood pumped by the heart each minute will enter the kidneys to undergo filtration. This is called the filtration fraction. The rest of the blood (about 80%) does not go through the filtering portion of the kidney, but flows through the rest of the body to service the various nutritional, respiratory, and other needs that are always present.
For the production of urine, the kidneys do not simply pick waste products out of the bloodstream and send them along for final disposal. The kidneys' 2 million or more nephrons (about a million in each kidney) form urine by three precisely regulated processes: filtration, reabsorption, and secretion (See figure 8).
Figure 8 showing processes of filtration, reabsorption and secretion
of nephron
5.3. Filtration
Urine Formation begins with the process of filtration, which goes on continually in the renal corpuscles (See figure 8). As blood courses through the glomeruli, much of its fluid, containing both useful chemicals and dissolved waste materials, soaks out of the blood through the membranes (by osmosis and diffusion) where it is filtered and then flows into the Bowman's capsule. This process is called glomerular filtration. The water, waste products, salt, glucose, and other chemicals that have been filtered out of the blood are known collectively as glomerular filtrate. The glomerular filtrate consists primarily of water, excess salts (primarily Na+ and K+), glucose, and a waste product of the body called urea. Urea is formed in the body to eliminate the very toxic ammonia products that are formed in the liver from amino acids. Since humans cannot excrete ammonia, it is converted to the less dangerous urea and then filtered out of the blood. Urea is the most abundant of the waste products that must be excreted by the kidneys. The total rate of glomerular filtration (glomerular filtration rate or GFR) for the whole body (i.e., for all of the nephrons in both kidneys) is normally about 125 ml per minute. That is, about 125 ml of water and dissolved substances are filtered out of the blood per minute. The following calculations may help you visualize how enormous this volume is. The GFR per hour is:
125 ml/min X 60min/hr= 7500 ml/hr.
The GFR per day is:7500 ml/hr X 24 hr/day = 180,000 ml/day or 180 liters/day.
Now, see if you can calculate how many gallons of water we are talking about. Here are some conversion factors for you to consider: 1 quart = 960 ml, 1 liter = 1000 ml, 4 quarts. = 1 gallon. Remember to cancel units and you will have no problem.
Now, what we have just calculated is the amount of water that is removed from the blood each day - about 180 liters per day. (Actually it also includes other chemicals, but the vast majority of this glomerular filtrate is water.) Imagine the size of a 2-liter bottle of soda pop. About 90 of those bottles equal 180 liters! Obviously no one ever excretes anywhere near 180 liters of urine per day! Why? Because almost all of the estimated 43 gallons of water (which is about the same as 180 liters - did you get the right answer?) that leaves the blood by glomerular filtration, the first process in urine formation, returns to the blood by the second process - reabsorption.
5.3.1. Glomerular Filtration Rate (GFR)
Tortora and Grabowski (2000) define glomerular filtration rate as the volume of fluid filtered from the renal glomerular capillaries into Bowman's capsule per unit time. Clinically, this is often measured to determine renal function.
The creatinine clearance was originally determined by injecting inulin (not insulin) into the plasma. Since inulin is not absorbed into any body systems, it is 100% filtered by the glomerulus and was represented with (volume of plasma per unit time). However, today we use creatinine clearance in order to know the filtration rate. Reasons for this are because it is like inulin and not reabsorbed from the renal tubules and is also synthesized by the body, making it a much cheaper yet still effective way to obtain clearance rates. The rate is typically recorded in 100 ml/min.
5.3.2. Reabsorption
Reabsorption, by definition Mayne (2000), is the movement of substances out of the renal tubules back into the blood capillaries located around the tubules (called the peritubular capillaries). Substances reabsorbed are water, glucose and other nutrients, and sodium (Na+) and other ions. Reabsorption begins in the proximal convoluted tubules and continues in the loop of Henle, distal convoluted tubules, and collecting tubules. Let's discuss for a moment the three main substances that are reabsorbed back into the bloodstream.
Large amounts of water - more than 178 liters per day - are reabsorbed back into the bloodstream from the proximal tubules because the physical forces acting on the water in these tubules actually push most of the water back into the blood capillaries. In other words, about 99% of the 180 liters of water that leaves the blood each day by glomerular filtration returns to the blood from the proximal tubule through the process of passive reabsorption.
The nutrient glucose (blood sugar) is entirely reabsorbed back into the blood from the proximal tubules. In fact, it is actively transported out of the tubules and into the peritubular capillary blood. Being lost in the urine wastes none of this valuable nutrient. However, even when the kidneys are operating at peak efficiency, the nephrons can reabsorb only so much sugar and water. Their limitations are dramatically illustrated in cases of diabetes mellitus, a disease that causes the amount of sugar in the blood to rise far above normal. As already mentioned, in ordinary cases all the glucose that seeps out through the glomeruli into the tubules is reabsorbed into the blood. But if too much is present, the tubules reach the limit of their ability to pass the sugar back into the bloodstream, and the tubules retain some of it. It is then carried along in the urine, often providing a doctor with her first clue that a patient has diabetes mellitus. The value of urine as a diagnostic aid has been known to the world of medicine since as far back as the time of Hippocrates. Since then, examination of the urine has become a regular procedure for physicians as well as scientists.
Sodium ions (Na+) and other ions are only partially reabsorbed from the renal tubules back into the blood. For the most part, however, sodium ions are actively transported back into blood from the tubular fluid. The amount of sodium reabsorbed varies from time to time; it depends largely on how much salt we take in from the foods that we eat. (As stated earlier, sodium is a major component of table salt, known chemically as sodium chloride.) As a person increases the amount of salt taken into the body, that person's kidneys decrease the amount of sodium reabsorption back into the blood. That is, more sodium is retained in the tubules. Therefore, the amount of salt excreted in the urine increases. The process works the other way as well. The less the salt intake, the greater the amount of sodium reabsorbed back into the blood, and the amount of salt excreted in the urine decreases.
5.4. Secretion
Now, let's describe the third important process in the formation of urine. Mayne (2000) defines secretion as the process by which substances move into the distal and collecting tubules from blood in the capillaries around these tubules. In this respect, secretion is reabsorption in reverse. Whereas reabsorption moves substances out of the tubules and into the blood, secretion moves substances out of the blood and into the tubules where they mix with the water and other wastes and is converted into urine. These substances are secreted through either an active transport mechanism or as a result of diffusion across the membrane. Substances secreted are hydrogen ions (H+), potassium ions (K+), ammonia (NH3) and certain drugs. Kidney tubule secretion plays a crucial role in maintaining the body's acid-base balance, another example of an important body function that the kidney participates in.
Summary
In summary, three processes occurring in successive portions of the nephron accomplish the function of urine formation:
Filtration of water and dissolved substances out of the blood in the glomeruli and into Bowman's capsule.
Reabsorption of water and dissolved substances out of the kidney tubules back into the blood (note that this process prevents substances needed by the body from being lost in the urine).
Secretion of hydrogen ions (H+), potassium ions (K+), ammonia (NH3), and certain drugs out of the blood and into the kidney tubules, where they are eventually eliminated in the urine.
Unit Six
6.0. Composition and Appearance of Urine Changes During Disease Conditions
6.1. Objective
· Relate the physical and chemical properties of normal and abnormal urine.
6.1. Appearance
Surprisingly little information can be gained from looking at the urine. Cheesebrough (2000) explains that the colour depends very much upon the volume of the urine, concentrated being darker (stronger). She further explains that the turbidity of the urine sample is gauged subjectively and reported as: clear, slightly cloudy, cloudy, opaque, or flocculent.
Normally, fresh urine is clear to very slightly cloudy. Excess turbidity results from the presence of suspended particles in the urine. The cause can usually be determined based on the results of the microscopic urine sediment examination. The common causes of abnormal turbidity include:
v Increased cells (RBC, WBC)
v Numerous crystals
v Bacteria
v Lipiduria (lipids often rise to the surface)
v Mucus (especially in horses)
v Semen
v Fecal contamination
6.1.1. Turbidity
Anderson and Cockayne (1999) define turbidity as the cloudiness of a urine sample depends on both its pH and it’s dissolved solids composition. Causes of turbidity include gross Bacteriuria.
6.1.2. Normal Urine
Urine is normally transparent, has thick viscous urine that is cloudy on examination. Turbidity suggests the presence of cells, casts, or crystals (See figure 9 A and B). Often refrigeration will propagate the sedimentation of crystals in the urine, producing a cloudy appearance. This is usually of no significance.
6.1.3. Cloudy Urine
Urine usually has an unpleasant smell and contains WBC’s.
Possible cause is bacterial urinary infection.
6.1.4. Red and Cloudy Urine
This type of urine is probable due to red cells. The possible cause is urinary schistosomiasis.
6.1.5. Brown and Cloudy urine
This is normally due to haemoglobin. The possible causes are; Blackwater fever and other conditions hat cause intravascular haemolysis.
6.1.6. Yellow-Brown or Green-Brown Urine
Is probable due to bilirubin. The possible causes include acute viral hepatitis and obstructive jaundice.
6.1.7. Yellow-Orange
This is due to urobilin i.e. oxidized urobilinogen. The possible causes include haemolysis and hepatocellular jaundice.
6.1.8. Milky-White Urine
Is probably due to chyle. The possible cause is Bancroftian filariasis.
6.1.9. Smoky Appearance
Smoky appearance is seen in haematuria. Threadlike cloudiness is observed when the specimen is full of mucus. In alkaline urine amorphous phosphates and carbonates may be responsible for the turbidity. While in acidic urine amorphous urates may be the cause.
Note: Other changes in the colour of urine can be caused by the ingestion of certain foods, herbs and drugs especially vitamins. Normal, freshly passé urine is clear and pale yellow-to-yellow depending on concentration. Note that when left to stand, cloudiness may develop due to the precipitation of urates in acid urine or phosphates and carbonates in alkaline urine. Urates may give the urine a pink-orange colour.
A B
Figure 9 A and B showing turbidity of urine
The turbidity in the above sample was due to numerous crystals, which precipitated upon cooling of the specimen to room temperature.
6.2. Colour
Tilton (2000) states that urine colour vary between normally some shades of yellow depending on the concentration. Colour intensity of urine correlates with concentration. The darler the colour, the more concentrated is the specimen. The various colours observed in urine are due to different excreted pigments. Yellow and amber are generally due to urochromes (derivatives of urobilin, the end product of bilirubin degradation), while yellowish brown to green are due to bile pigment oxidation. Red and brown after standing are due to porphyrins, while reddish brown in fresh specimens comes from haemoglobin or red cells. Brownish black after standing is seen in alkaptonuria (due to excreted homogentisic acid) and in malignant melanoma (in which the precursor melanogen oxidizes in the air to melanin). Drugs also may affect urine colour methylene blue can turn urine blue, while the analgesic dye pyridium stains urine orange. Some foods such as beets also may alter urine colour. The Abnormal color changes in the urine could be due to drugs, increased urinary pigments or red blood cells. Red to reddish-brown could be due to either, haematuria (See figure 10), hemoglobinuria, or myoglobinuria. Yellow-green to yellow-brown is associated with bilirubinuria. Occasionally, unusual colors may be caused by dyes associated with food or drugs. The urine colour may vary from watery to lager-like, depending on when excreted. Early-morning urine is more concentrated and will be darker than that excreted following breakfast or recent fluid intake.
Figure 10 showing a urine sample exhibiting haematuria
The color of the urine sample is assessed subjectively and reported as red, brown, yellow, etc, or combination thereof, along with a modifier for the depth of color, for instance, light yellow, dark red or brown, etc (See figure 11). Some examples of various urine colors and corresponding common causes are shown below:
Light to medium ------------------ yellow normal
Colorless ------------------ very dilute urine
Very dark yellow ------------------ extremely concentrated; bilirubinuria
Red to brownish red ----------------- hematuria, hemoglobinuria, myoglobinuria
Reddish brown-to-brown ------------ myoglobinuria, hemoglobinuria, methemoglobin
Greenish tint ------------------ bilirubinuria
.
Figure 11 showing some shades of urine colours
6.3. Odor
Bishop et al (1992) explains that urine has a characteristic smell due to the concentration of the sample. Odor ordinarily has little diagnostic significance. The characteristic pungent odor of fresh urine is due to volatile aromatic acids, in contrast to urine that has been allowed to stand. A particularly foul odor may occur in the presence of bacteria for instance urinary tract infections impart a noxious, faecal smell to urine, while the urine of diabetics often smells fruity. Thus, strong smelling urine is common in cases of cystitis. Ketonuria produces a very sweet smell, as does glucosuria. Sweet smelling urine is commonly associated with acetonemia, pregnancy toxemia, and diabetes mellitus. Other distinctive odors are present in the urine of patients with maple syrup urine disease or phenylketonuria (PKU).
6.4. Composition
Tortora and Grabowski (2000) describe urine as a transparent solution that is light yellow to amber in colour. It is the byproduct or waste fluid secreted by the kidneys, transported by the ureters to the urinary bladder where it is stored until it is voided through the urethra. Urine is made up of a watery solution of metabolic wastes (such as urea), dissolved salts and organic materials. Fluid and materials being filtered by the kidneys -destined to become urine- comes from the blood or interstitial fluid. The composition of urine is adjusted in the process of reabsorption when essential molecules needed by the body, such as glucose, are reabsorbed back into the blood stream via carrier molecules. The remaining fluid contains high concentrations of urea and other excess or potentially toxic substances that will be released from the body via urination. Urine flows through the following structures: the kidney, ureter, bladder, and finally the urethra, Urine is produced by a process of filtration, reabsorption, and tubular secretion.
Urine contains large amounts of urea, an excellent source of nitrogen for plants. As such it is a useful accelerator for compost. Urea is 10,000 times less toxic than ammonia and is a byproduct of deamination (2 NH3 molecules) and cellular respiration's (1 CO2 molecule) products combining together. Other components include various inorganic salts such as sodium chloride. Liquid solution of metabolic wastes and other, often toxic, substances filtered from plasma. The fluid in the Bowman capsule at the start of each nephron is essentially plasma without the large molecules (e.g., proteins). The concentrated fluid (final urine) that exits the kidney consists of water, urea, inorganic salts, uric acid, creatinine, ammonia, and broken-down blood pigments, including urochrome, which makes urine yellow, plus any unusual substances not reabsorbed into the blood.
6.5. Volume and Frequency of Urine.
Most people have very little idea of how much urine they are passing, although they can usually make a guess at how many times they pass it. In health, the minimum volume of urine an adult can pass and remain healthy is about a pint a day and the maximum on an average fluid intake in temperate climates is about 4 - 5 pints. Most people pass less urine when they have been sweating a lot, especially in hot and above all, humid climates, and fluid intake needs to be adjusted accordingly if you take exercise. However, most normal individuals pass urine four to eight times a day, occasionally at night. The ability to hold urine for 12-24 hours may be useful but (except when dehydrated) is probably abnormal. In children, ability to hold urine for these very long periods is one of the signs of a large, slack bladder.
Passing urine regularly at night, unless it is merely a habit or the result of taking fluids just before going to bed, is a valuable sign that something is wrong. It indicates that the usual fall in urine volume during the night is not happening, and probably the volume of urine to be passed has increased. These are signs that the concentrating power of the kidney is impaired. Some causes of this are malfunction of the medulla, where the concentrating mechanism is found; overall renal failure; or much more rarely, an absence of the hormone ADH; or resistance of an otherwise normal kidney to ADH. It is often caused merely from a distended sigmoid colon pressing on the bladder, reducing its capacity. This will give a full feeling much sooner than if the bladder was not being impinged. Colon cleansing will eliminate symptoms if involved. Passing urine very frequently, especially if accompanied by pain in the urethra and bladder and a feeling that the bladder has not been emptied, is the classical symptom of what is called a ‘urinary tract infection’. This is often caused by a pH imbalance. When the urine pH is too alkaline, the condition exists for a urinary tract ‘inflammation’ to happen.
Sometimes, in older men, this may be the main result of obstruction of the urinary outflow from the bladder by an enlarged prostate, but will usually be accompanied by a poor stream, an inability to start or stop passing urine as promptly as before, and perhaps dribbling between times. Again, this is usually caused by an enlarged sigmoid colon that wraps around the prostate gland and the uterus. Not only is there mechanical pressure applied to these organs, but, in the presence of leaky gut syndrome, bowel toxins leak through the wall of the colon from bowel herniations or diverticuli, into the bladder, prostate gland and uterus, causing urinary tract infections, prostatitis (enlarged prostate), endometriosis, fibroid uterus, prostate cancer and uterus cancer. Incontinence of urine, in an adult individual previously continent, is obviously abnormal. In older people it may be the only sign of a urinary tract infection, and will disappear when corrected. Again though, incontinence is usually caused by a distended sigmoid colon, that presses on the bladder and in women also the uterus and causes it to prolapse onto the bladder, causing constant pressure than the dilute urine passed if a good deal of water is drunk. The appearance of fresh blood, or dark-brown altered blood, indicates a problem. There are many causes of blood in the urine (hematuria), many of them benign, but the appearance of fresh blood is always alarming to the individual concerned. Renal bleeding requires investigation. In jaundice, the urine will often be dark, or even yellow from the pigment retained because the liver is obstructed or unable to get rid of this substance. Normally, fresh urine has very little smell, but on standing, the urea breaks down to ammonia, which gives the characteristic odor, most associate with it. In infection, urine may smell foul even when first passed, and this is a valuable sign. Some foods or drugs give unusual odors to the urine.
Frothy Urine
Normal urine froths a little, but persistent frothing which will not flush away is abnormal. This may indicate that there is an excess of protein (albumin) in the urine, which when beaten up by the passage of the urine stream, forms a stable foam–just as egg white will when beaten. Frothy urine may also be seen in jaundice, because of the bile salts in the urine. The volume of urine is indicative of the balance and water lost from ingestion and water lost from the lungs, sweat and intestines.
6.6. Cells in Urine Sediment
Cheesebrough (2000) states that to identify urinary sediment cells is a difficult task. Some cells have characteristic features, therefore easily identifiable. On the other hand, some cells, even with sophisticated stains, remain a challenge. The diversity of cells that one can meet is notable. Cells found in urine can belong to the reticuloendothelial system (leukocyte, macrophage) or to the epithelial system. Urinary cells can originate from the kidney or from the lower urinary tract, from the superficial lining or from a deeper source. In an adult male, cells can also originate from the prostate and the urethra.
Urine is not a favorable media for maintaining cell structures. Most cells undergo rapid changes (degeneration, rupture, vacuolization, granulation of the cytoplasm) that profoundly affect the visual aspect of the cell. These changes are more pronounced if the cell comes from the higher urinary tract because aggression due to osmotic variation adds to the energy lack and other causes of morphological change. The best-preserved cells are usually bladder cells, while a well preserved proximal renal tubular cell is exceptional. Some pathologies, like inflammation, metaplasia, neoplasia, are known to affect the cellular aspect.
All experienced urinary sediment microscopists have seen some cases of cells, so special, that identification was a best guess based on the sediments context. This situation, while being frustrating, should not be considered as a failure. Identification of all the figures seen in the urinary sediment is quite impossible so, one must focus his effort on element of clinical value.
Urine is a hostile environment for cells since they encounter abnormal osmotic pressures, pH changes, and exposure to toxic metabolites. For these reasons, post-collection delay of examination should be minimized. If delay is unavoidable, refrigeration will slow degeneration of cells
For routine purposes, cells are examined as unstained wet-mounts of sedimented urine. Under some circumstances, air-dried smears are prepared and stained with hematologic stains.
Red blood cells and leukocytes are quantified as cells/HPF (High Power Field-40 X objective). Other cell types are usually subjectively listed as "few, moderate, or many".
6.6.1. White Blood Cells
White blood cells (WBC’s) in unstained urine sediments typically appear as round, granular cells which are1.5-2.0 times the diameter of RBC(See figure 12 A). The details of nuclear shape often are difficult to discern, especially if the specimen is not fresh. WBC in urine are most commonly neutrophils. Staining of air-dried sediment smears with a hematologic stain sometimes is useful for more specific identification. Like erythrocytes, WBC may lyse in very dilute or highly alkaline urine. WBC up to 5/HPF are commonly accepted as normal. Greater numbers (pyuria) generally indicate the presence of an inflammatory process somewhere along the course of the urinary tract (or urogenital tract in voided specimens).
Pyuria often is caused by urinary tract infections (See figure 12 B), and many times bacteria can be seen on sediment preparations. Depending on clinical signs, pyuria may be an indication for culture of urine even if no bacteria are seen. Non-septic causes of inflammation, such as uroliths and tumors, also must be considered.
Pyuria is usually regarded as significant when moderate or many pus cells are present i.e. more than 10 WBC/microlitre. Bacteriuria without Pyuria may occur in diabetes enteric fever, bacterial endocarditis or when the urine contains many contaminating organisms. Pyuria with a sterile routine culture may be with renal tuberculosis gonococcal urethritis, C trachomatis infection, leptospirosis or when a patient with urinary infection has been treated with antimicrobials.
WBC in urine:
WBC are reported semi quantitatively as number seen per high power field (HPF)
None seen, <5,>100 WBC HPF.
WBC are normal in urine in low numbers. Up to 5 WBC/HPF generally are considered acceptable
Figure 12 A showing WBC in urine
Figure 12 B showing WBC in urine Figure 13 showing RBC in urine
6.6.2. Red Blood Cells
The appearance of red blood cells (RBC’s) in urine depends largely on the concentration of the specimen and the length of time the red cells have been exposed. These are smaller and more refractile than white blood cells (See plate 2) they have a definite outline and contain no granules. Fresh red cells tend to have a red or yellow color (lower panel). Prolonged exposure results in a pale or colorless appearance as hemoglobin may be lost from the cells (upper panel). In fresh samples with S.G. of 1.010-1.020, RBC may retain the normal disc shape (upper panel). In more concentrated urines (>1.025), red cells tend to shrink and appear as small, crenated cells (lower panel). In more dilute samples, they tend to swell. At urine S.G. <1.008 and/or highly alkaline pH, red cell lysis is likely. Lysed red cells appear as very faint "ghosts", or may be virtually invisible. Red blood cells up to 5/HPF are commonly accepted as normal. An increased red cell in urine is termed hematuria, which can be due to hemorrhage, inflammation, necrosis, trauma, or neoplasia somewhere along the urinary tract (or urogenital tract in voided specimens). The method of collection must be considered in interpreting hematuria to aid in localizing the source, and because catheterization, cystocentesis, and manual compression can induce hemorrhage
RBC's are reported semi quantitatively as number seen per high power field (HPF):
None seen, <5,>100.
RBC are normal in urine in low numbers. Up to 5 RBC/HPF generally are considered acceptable.
Red blood cells in the urine can be due to vigorous exercise or exposure to toxic chemicals. Bloody urine can also be a sign of bleeding in the genitourinary tract as a result of systemic bleeding disorders, various kidney diseases, bacterial infections, parasitic infections including malaria, obstructions in the urinary tract, scurvy, subacute bacterial endocarditis, traumatic injuries, and tumors
Note; when haematuria is due glomerulonephritis the red cells often vary in size and shape dysmorphic. In sickle cell disease, sickled red cells can sometimes be seen in the urine see figure. Haematuria red cells in urine may be found in urinary schistosomiasis usually with proteinuria bacterial infections, acute glomeruli of the kidneys sickle cell disease leptospirosis in the urinary tract malignancy of the urinary tract and hemorrhagic conditions. Note the finding of red cells in the urine of women may be due to menstruation.
6.6.3. Epithelial Cells
Epithelial cells in urine are generally of little specific diagnostic utility. Cells lining the urinary tract at any level may slough into the urine. In the case of voided samples, even cells from the genital tract can appear in the sample.
Figure 14 showing epithelial cell Figure 15 showing bacteria in urine
Most commonly seen are squamous epithelial cells from the distal urethra and/or vulva (upper panel at right), and transitional epithelial cells from the bladder and urethra (lower panel). These are easily seen with 10X objective (See Figure 14). They are nucleated and vary in size and shape. They are usually reported as few, moderate, or many in number per low power (10 X objective) field. It is normal to find a few epithelial cells in urine. When seen in large numbers, however hey usually indicate inflammation of the urinary tract or vaginal contamination of the specimen. A large number of cells from tissue lining (epithelial cells) can indicatedamage to the small tubes that carry material into and out of the kidneys.
16 A 16 B
Figure 16 A & B showing granular and waxy casts
6.6.4. Yeast Cells
These can be differentiated from red cells by their oval shape and some of the yeasts usually show single budding. If in doubt run a drop of dilute acetic acid under the cover glass. Red cells will be haemolysed by the acid but not yeast cells.
Note: Glove powder in urine also resembles yeasts. It can be distinguished by adding a drop of iodine (as used in Gram’s stain). Glove powder starch turns blue black.
Yeast cells are usually reported as few, moderate, or many per high power field (HPF). They can be seen in urine of women with vaginal candidiasis and occasionally in specimens from diabetes and those with immuno suppression.
6.6.5. Bacteria
Bacteria can be identified in unstained sediments when present in sufficient numbers. Rod shaped bacteria and chains of cocci are often identifiable. If there is doubt about the presence of bacteria, a Gram stained smear of urine sediment should be examined. Urine in the bladder is sterile. Though bacteria from the distal urethra or genital tract may contaminate voided samples, they are usually too few to see if a good mid stream collection was obtained. Bacteriuria of clinical significance for example bacterial cystitis is usually accompanied by increased numbers of white blood cells (Pyuria) as shown in figure 15.
6.7.
6.7. Casts in Urine
6.7.1. Casts
Casts are small fibrous objects that are formed when protein and other materials settle in the kidney tubules and collecting ducts. Casts are dislodged by normal urine flow. A large number of them in a urine specimen are a sign of kidney disease. These can usually be seen with the 10 X objective provided the condenser iris is closed sufficiently to give good contrast. They consist of solidified protein and are cylindrical in shape because they are formed in the kidney tubules. The following casts can be found in urine.
6.7.2. Hyaline Casts
Hyaline casts, which are colourless and empty (See plate 6). They are associated with damage to the glomerular filter membrane. A few may be seen following strenuous exercise or during fever.
6.7.3. Waxy Casts
Waxy casts are hyaline casts that have remained in the kidney tubules a long time (see figure 16 A). They are thicker and denser than hyaline casts, often appear indented or twisted, and may be yellow in colour. They usually indicate tubular damage and can sometimes be seen in renal failure.
6.7.4. Cellular Casts
Cellular casts may contain white blood cells or red blood cells (See plate 2 and 3). Red cell casts appear orange red. They indicate haemorrhage into the renal or glomerular bleeding. White cell casts are found when there is inflammation of the kidney pelvis or tubules. Yellow brown-pigmented casts may be seen in the urine of jaundiced patients.
Plate 1 transitional epithelial cell Plate 2 RBC cast
Plate 3 WBC cast Plate 4 spermatozoa
Plate 5 squamous epithelial cell Plate 6 hyaline cast
6.7.5. Granular Casts
Granular casts which contain irregular sized granules originating from degenerated cells and protein (See Figure 16 B). They are also associated with renal damage.
6.8. Crystals in Urine
These have a characteristic refractile appearance. Normal urine contains many chemicals from which crystals can form, and therefore the finding of most crystals has little importance. Crystals should be looked for in fresh urine when calculi (stones) in the urinary tract are suspected. Crystals which may be found in rare disorders include:
6.8.1. Cystine Crystals
Cystine crystals, are identified by their six sides (See plate 13). They are soluble in 30 % v/v hydrochloric acid (unlike uric acid crystals which they may resemble). They can be found in cystinuria a rare congenital metabolic disorder in which cystine is excreted in the urine.
6.8.2. Cholesterol Crystals
Cholesterol crystals, look like rectangles with, cutout corners (See plate 14). They are insoluble in acids and alkalis but soluble in ether, ethanol, and chloroform. They are rarely found except in severe kidney disease or when a lymphatic vessel has ruptured into the renal pelvis.
6.8.3. Tyrosine Crystals
Tyrosine crystals, which are yellow or dark coloured and look like needles massed together (See plate 16). They are insoluble in ethanol, ether and acetone, they are occasionally found in severe liver disease.
6.8.4. Sulphonamide Crystals
Occasionally sulphonamide crystals are found in the urine of patients being treated with sulphonamide (See plate 17). When deposited in the urinary tract they can cause haematuria and other complications.
6.8.5. Triple Phosphate Crystals
Triple phosphate crystals are occasionally found in alkaline urine (See plate 10).
6.8.6. Calcium Oxalate Crystals
Calcium oxalate crystals are frequently seen (See plate 9). When found in freshly voided urine they may indicate calculi in the urinary tract.
6.8.7. Uric Acid Crystals
Uric acid crystals are yellow or pink-brown (See plate 7). They can sometimes be found with calculi.
6.8.8. Amorphous Phosphate Crystals
Amorphous crystals appear as aggregates of finely granular material without any defining shape at the light microscopic level (See plate 8). Amorphous urates tend to form in acidic urine, and may have a yellow or yellow-brown appearance.
Amorphous phosphates are similar in general appearance, but tend to form in alkaline urine and lack colour.
Plate 7 uric acid crystals Plate 8 amorphous phosphates
Plate 9 calcium oxalate crystals Plate 10 triple phosphate crystals
Plate 11 sodium urate crystals Plate 12 calcium phosphate crystals
Plate 13 cystine crystals Plate 14 cholesterol crystals
Plate 15 leucine crystals Plate 16 tyrosine crystals
Plate 17 sulphonamide crystals Plate 18 fat
6.9. Parasites in urine
6.9.1. Trichomonas vaginalis
Found in the urine of women with acute vaginitis (occassionally seen in the urine of men). The trichomonads are a little larger than white cells and are usually detected in fresh urine because they are motile. They move by flagella and an undulating membrane (see figure 17).
Figure 17 showing Trichomonas vaginalis
6.9.2. Egg of Schistosoma haematobium
The ova of Schistosoma haematobium (SH) are pale yellow- brown and oval in shape contains a fully developed miracidium. Recognized by their large size approximately about 145 x 55 micro metre and spine at one end (terminal spine, see Figure 18) the urine will contain red cells and protein. Note that sometimes the miracidium hatch from the eggs and can be seen swimming in the urine. In urinary Schistosomiasis there is haematuria, proteinuria and eosinophilia. SH egg excretion in urine is bright between 100.00 hours and 14.00 hours with a peak around midday. Note: Even when persons are heavily infected eggs may not be present in the urine all the time. It may be necessary to examine several specimens collected on different days due to the irregular pattern of the egg excretion.
1. Routine examination of urine for SH eggs
· Collect 10-15 ml of urine between 10.00 hours and 14.00 hours in a clean dry container.
Note: Neither exercising before passing urine nor collecting terminal urine (last few drops) increase the number of eggs present in the specimen (as once was thought). To avoid the miracidia hatching from the eggs, keep the specimen in the dark if unable to examine it within 30 minutes.
· Report the appearance of the urine. In moderate to heavy infections, the urine will usually contain blood and appear red or red-brown and cloudy. When visible blood is present, add 2 drops of saponin solution to lyze red cells. This will make it easier to detect the eggs. If blood is not seen, test the specimen chemically for blood and protein.
· Transfer 10 ml of well mixed urine to a conical tube and centrifuge at RCF 500-1000 g to sediment the schistosome eggs (avoid centrifuging at greater force because this can cause the eggs to hatch 0. if a centrifuge is not available, allow the eggs to sediment by gravity for one hour.
· Discard the supernatant fluid. Transfer all the sediment to a slide with a cover glass and examine the entire sediment microscopically using the 10x objective with the condenser iris closed sufficiently to give good contrast.
· Count the number of eggs in the preparation and report the number per 10 ml of urine. If more than 50 eggs are present, there is no need to continue counting. Report the count as more than 50 eggs per ml. Such counts indicate a heavy infection. In the early stages of urinary schistosomiasis the egg count is an indicator of the severity of disease.
Miracidia in urine: If the urine is dilute or has been left to stand for several hours in the light, the miracidia will hatch from the eggs. The ciliated miracidium are motile.
2. Filtration technique for detecting and quantifying SH eggs in urine.
Filtration is the most sensitive, rapid and reproducible technique for detecting and quantifying SH eggs in urine. Polycarbonate membrane filters are expensive, although with care they can be reused. Other types of filters include Nytrel woven filters.
3. Requirements
· 10 ml luer syringe
· Syringe filter holder 13 mm in diameter (swimmer type). This is suitable for holding filters of 12 mm or 13 mm.
4. Procedure
· Using blunt ended forceps, carefully place a polycarbonate filter on the filter support of the filter holder. Reassemble the filter holder and attach it to the end of a 10 ml luer syringe.
· Remove the plunger from the syringe.
· Fill the syringe to the 10 ml mark with well-mixed urine and replace the plunger. Holding the syringe over a beaker or other suitable container slowly pass the urine through the filter.
Note: filling the syringe is preferred to drawing up the urine into the syringe because it does not require tubing and the air which passes through the membrane after the urine help to stick the eggs to the filter.
· Remove the filter holder and unscrew it. Using blunt ended forceps carefully remove the filter and transfer it face upwards (eggs on surface) to a slide.
· Add a drop of physiological saline and cover with a cover glass.
· Using the 10X objective the condenser iris closed sufficiently to give good contrast, examine systemically the entire filter for SH eggs.
· Count the number per 10 ml of urine.
Figure 18 showing ova of Schistosoma haematobium
6.9.2. Other parasites that may be found in urine;
· Very occasionally the microfilariae of Wuchereria Bancrofti can be found in urine. This happens when a urogenital lymphatic vessel ruptures. The urine appears milky-white or reddish- pink (chyle mixed with blood). The microfilariae are large 225-20 X micormetre, motile and sheathed. No nuclei are present in the tail feature looked for in a Giemsa stained preparation. W.bancrofti microfilariae are shown in figure.
· Microfilariae of Onchocerca volvulusl may be found in the urine in Onchocercaiasis especially in heavy infections. The larvae are large 280-330X 7 micrometre, unsheathed, with a slightly enlarged head-end and a tail which is sharply pointed and contains no nuclei (See figure).
· Occasionally the eggs of Enterobius vermicularis are found in urine, especially from young girls when the eggs are washed off the external genitals when urine is being passed. See figure.
· Spermatozoa
Occasionally found in the urine of men, they can be easily recognized by their head and long thread-like tail, see figure. They may be motile in fresh urine.
Contaminants, which can be found in urine. These include cotton fibres; starch granules oil droplets pollen grains moulds, single celled plants (diatoms) and debris from dirty slides or containers.
A. Preparation and Examination of a Wet Preparation.
1. Aseptically transfer about 10 ml of well-mixed urine to a labeled conical tube.
2. Centrifuge at 500-1000 g for 5 minutes.
3. Pour supernatant fluid by completely inverting the tube into a second container not the original one. This can be used for biochemical tests to avoid contaminating the original urine which may need to be cultured depending on the findings of the microscopical examination.
4. Remix the sediment by tapping the bottom of the tube.
5. Transfer one drop of well-mixed sediment to a slide and cover with cover glass. Note; do not discard the remaining sediment because this may be needed to prepare a Gram smear if WBC’s and or bacteria are seen in the wet preparation.
6. Examine the preparation microscopically using 10x objective with the condenser iris closed sufficiently to give good contrast.
7. Report the following;
· WBC’s
· RBC’s
· Epithelial cells
· Casts
· Crystals
· Schistosoma heamatobium ova
· Bacteria
· Others
· Protein
· Glucose
· Others
B. Examination of a Gram Stain Smear
· Prepare and examine a gram stained smear of the urine when bacteria or white cells are seen in the wet preparation.
· Transfer a drop of the urine sediment to a slide and spread it to make a thin smear.
· Allow to air dry protected from insects and dust.
· Heat fix or methanol fix the smear and stain it by the Gram technique.
· Examine the smear first with a 40 X objective to see the distribution of material and then with the oil immersion objective.
· Look especially for bacteria associated with urinary infections especially gram-negative rods. Occasionally Gram-positive cocci and Streptococci may be seen.
· Neisseria gonorrhgoeae in urine
§ In male patients with acute urethriitis. It is often possible to make a presumptive diagnosis of gonorrhoeae by finding Gram negative intracellular diplococci in pus cells passed in urine see Color figure.
Finding intestinal parasites in urine indicates faecal contamination.
6.10. Specimen of Choice
Specimen of choice is the first-morning, midstream and clean-catch urine.
For routine urinalysis, the specimen of choice is the first-morning urine. This choice is a compromise. For the chemical testing, the first-morning urine is usually the preferred specimen because it is the most concentrated urine for the microscopical examination of cells: imagine cells incubated overnight in 37°C urine. Cytologists prefer the second-morning specimen but the aim of the tests is quite different.
A free-voided specimen is often used, this being unavoidable for the emergency patient. The main disadvantage comes from the dilution of the specimen. This dilution, in some cases, can be sufficiently high to cause some false-negative results, especially with the chemistry testing.
6.10.1. Types of Specimen
It is important to obtain the appropriate specimen, to optimize the information needed for diagnosis of a patient’s disease.
A. First Morning Specimen
This is the most concentrated is preferred for examination of both chemical and microscopic components. Although some microscopic elements may be destroyed with prolonged standing in the bladder.
B. To minimize the destruction of cells, a second voided sample may be examined. This is a specimen collected some time after the first morning specimen at a time when the urine is still relatively concentrated, but without standing in the bladder for an excessive period of time (Young D, 2004).
C. Postprandial Specimen
A variant of this type of specimen is collected after a meal with the primary objective of detecting glucose. However in the presence of gross abnormalities.
D. Random Urine Specimen
A random urine specimen will often suffice for examination. In outpatient practice this specimen is often the most convenient for both physician to patient.
E. A Two-Hour Specimen
A two-hour specimen collected in the afternoon is traditionally used for the semi quantitative measurement of urobilinogen, since urobilinogen is relatively unstable and undergoes considerable circadian variation. This is because of the circadian variation in which excretion of many components of urine are largely due to the influence of meals, posture and activity.
F. Twenty-Four Hour Urine
The collection of all urine over 24 hours is required for accurate quantification of the excretion of substance is urine. When such collections are made, appropriate preservatives should be added to the urine. Recommended preservatives for the different components of urine include.
E. Clean Catch Specimen
If a urine specimen is to be examined for bacteria or other microorganisms a clean catch specimen should be examined. After the external genitalia have been cleaned.
When testing formed elements or organisms the examination should done within 30 minutes of the urine specimen collection. Examinations should not be performed if they cannot be done within 2 hours of voiding the specimen.
6.10.2. Precautions of Voided Specimens
· Urinalysis should not be performed while a woman is menstruating or having a vaginal discharge. A woman who must have a urinalysis while she has a vaginal discharge or is having her period should insert a fresh tampon before beginning the test. She should also hold a piece of clean material over the entrance to her vagina to avoid contaminating the specimen.
· Patients do not have to fast or change their food intake before a urine test. They should, however, avoid intense athletic training or heavy physical work before the test because it may result in small amounts of blood in the urine.
The following drugs can affect urinalysis results. The patient may be asked to stop taking them until after the test:
Nitrofurantoin (Macrodantin, Furadantin). Nitrofurantoin is prescribed for infections of the urinary tract and other bacterial infections.
Phenazopyridine (Pyridium). This medication is used to relieve burning and pain caused by urinary-tract infections.
Rifampin (Rifadin). This medication is prescribed to treat tuberculosis, prevent the spread of meningitis, and treat other infections. Methods of Collection
6.10.3. Specimen Containers
The laboratory technician or hospital will supply a sterile container for a specimen being collected for a colony count. A colony count is a test that detects bacteria in urine that has been cultured for 24–48 hours. It is used instead of a routine urinalysis when a patient's symptoms suggest a urinary tract infection. Non-sterile containers can be used for routine specimens that will not be tested immediately after being collected. An ordinary open-necked jar may be used after it and its lid have been soaked in very hot water for 15–20 minutes and then air-dried.
6.10.4. Laboratory Procedures A. Storage
Urine specimens should not remain unrefrigerated for longer than two hours. A urine specimen that cannot be delivered to a laboratory within two hours should be stored in a refrigerator. The reason for this precaution is that urine samples undergo chemical changes at room temperature. Blood cells begin to dissolve and the urine loses its acidity.
B. Visual Examination
A clinician, nurse, or laboratory technician will look at the specimen to see if the urine is red, cloudy, or looks unusual in any way. He or she will also note any unusual odor.
C. Testing Techniques
Urine samples are tested with a variety of different instruments and techniques. Some tests use dipsticks, which are thin strips of plastic that change color in the presence of specific substances. Dipsticks can be used to measure the acidity of the urine (its pH) or the presence of blood, protein, sugar, or substances produced during the breakdown of fatty acids (ketones). A urinometer is used to compare the density of the urine specimen with the density of plain water. This measurement is called specific gravity. The testing techniques are dealt with in unit 7.
The urine specimen is also examined under a microscope to determine whether it contains blood cells, crystals, or small pieces of fibrous material (casts).
6.10.5. Urine Collection Methods
A. Preparation of Voided Specimens
The patient’s voiding into a suitable container collects most urine specimens from adults or older children. Soaps and disinfectants may contaminate urine specimens and should not be used. The clinician or laboratory may supply a special antiseptic solution that won't irritate the skin. The method for collection varies somewhat according to age and sex.
B. Women and girls
· Before collecting a urine sample, a woman or girl should use a clean cotton ball moistened with lukewarm water to cleanse the external genital area.
· Gently separating the folded skin (labia) on either side of her vagina, she should move the cotton ball from the front of the area to the back.
· After repeating this process several times, using a fresh piece of cotton each time, she should dry the area with a clean towel.
· To prevent menstrual blood, vaginal discharge, or germs from the external genitalia from contaminating the specimen, a woman or girl should release some urine before she begins to collect her sample. A urine specimen obtained this way is called a midstream clean catch.
C. Men and boys
· A man or boy should use a piece of clean cotton, moistened with antiseptic, to cleanse the head of his penis and the passage through which urine leaves his body (the urethral meatus).
· He should draw back his foreskin if he has not been circumcised. He should move the cotton in a circular motion away from the urinary opening, using a fresh piece of cotton each time.
· After repeating this process several times, he should use a fresh piece of cotton to remove the antiseptic.
· After the area has been thoroughly cleansed, he should begin urinating and collect a small sample in a container without interrupting the stream of urine.
D. Infants
· A parent, nurse, or doctor should cleanse the child's genitals and as much of the surrounding area as will fit into the sterile urine-collection bag provided by the hospital.
· When the area has been thoroughly cleansed, the bag should be attached to the child's genital area and left in place until the child has urinated.
· It is important to remember not to touch the inside of the bag and to remove it as soon as a specimen has been obtained.
6.10.5. Bladder Catheterization
Bladder catheterization is a hospital procedure used to collect uncontaminated urine when the patient cannot void. A catheter is a thin flexible tube that the clinician inserts through the urethra into the bladder to allow urine to flow out. To minimize the risk of infecting the patient's bladder with bacteria, many doctors use a so-called Robinson catheter, which is a plain rubber or latex tube that is removed as soon as the specimen is collected. It should not, however, be used to collect specimens from males with acute inflammation of the prostate or from a patient of either sex with a fractured pelvis.
6.10.6. Suprapubic Bladder Aspiration
Suprapubic bladder aspiration is a technique that is sometimes used to collect urine from infants younger than six months. The clinician withdraws urine from the bladder into a syringe through a needle inserted through the skin over the bladder. This technique is used only when the child cannot void because of an abnormal urethra or if he or she has a urinary tract infection that has not responded to treatment.
Aftercare
The patient may return to normal activities after collecting the sample and may start taking medications that were discontinued before the test.
Risks
There are no risks associated with voided specimens. The risk of bladder infection from catheterization with a Robinson catheter is about 3%.
6.10.7. Early Morning Urine
Urine specimens are usually collected early in the morning before breakfast. Urine collected eight hours after eating and at least six hours after the most recent urination is more likely to indicate abnormalities. Some people may be asked to void into a clean container before getting out of bed in the morning. Collecting a urine sample from emptying the bladder takes about two or three minutes. The sample can be collected at home as well as in a clinician’s office
6.11. Delay between collection and processing.
The ideal situation is when the specimen is analyzed shortly after collection. But let's stay realistic! There will always be a certain delay between collection and analysis. It is important to set some rules for an acceptable period and conditions of conservation.
6.11.1. What is an acceptable delay?
This is an easier question to ask than to answer. While it is reassuring to have clearly determined delay rules, strict obedience to rules is undesirable; it all depends on the specific situation. Some decisions must be left to common sense.
Before rejecting a specimen, we must consider the following:
Specimens are unique and represent a punctual situation in time. A second specimen is necessarily obtained later and perhaps in a changed clinical condition.
The patient that comes to a lab for analyses will have to come back in the case of a rejected specimen. We hope that the former does not return for only a urinalysis prescribed without conviction.
Some substances can disappear quite rapidly on standing, for example, glucose in the case of a urinary tract infection. But what was the first reason of the clinician request: to know if there's any sugar in his patient’s urine or to know if he or she has a urinary tract infection?
As for the serum, urinary substances and elements don't have the same stability. Cells degenerate rapidly while the characteristics of casts will be more easily preserved. Many crystals seen in urine are absent in the freshly voided specimen.
Specimens that no longer represent the patient at the time of collection and those that could lead to false interpretation should be rejected. It should be remembered that these are an extreme situation.
For several reasons, we think that all specimens should be analyzed. The specimen with a delay problem should have a mention as;” unacceptable delay: many results possibly invalid."
The quality of a specimen, which cannot be analyzed rapidly, depends on its conservation method. Several centers recommend that specimens be refrigerated if they cannot be processed shortly after collection. A major disadvantage with this practice is the precipitation of crystals. Amorphous urates are easily recognizable by the pink pellet they form upon centrifugation. In some cases, the precipitation is so abundant that it obscures all the elements rendering microscopical analysis quite difficult. A simple way to get rid of these urates is to place the complete uncentrifuged specimen at 37°C (once centrifuged and decanted, it is often impossible to dissolve the amount of precipitate in the small remaining volume). When the precipitate is formed of phosphate, it can be dissolved by incubation at 37°C or by the addition of 1 or 2 drops of 2% acetic acid to the sediment.
6.12. Analysis Volume
The widely accepted urine volume is 12 mL. In a majority of centers, specimens are received in a graduated conical tube. The excess volume can be aspirated through a special device before centrifugation.
In some cases, filling to the 12 ml mark is impossible. Some have suggested that the missing volume be completed with a suitable solution (normally saline). In some cases, a low volume should be used as is. Another problem with the unfilled container is whether to correct or not the results to a 12 ml volume. We prefer not to correct and add the mention to the report; "Results obtained with a.... mL specimen". The main reason for this choice is that it is not obvious that a count made from 6 ml of urine difficultly obtained from a urinary tract infected child would be doubled with a 12 ml specimen. This solution leaves the clinician with the opportunity to correct the values, if indicated.
6.13. Centrifugation
The recommended parameter for the urine centrifugation is 5 minutes at 400 RCF. The term RCF means « relative centrifugal force » and is dependent of the squared rotation speed (RPM) and the head's radius. In most centrifuge instruction manuals, a calculation nomogram is illustrated. This graphic is an easy way to calculate the correct RPM to obtain a 400 RCF. A speed of 1200 RPM is a representative example.
Specimens must not be over spun. With compacted pellets, resuspension is more difficult, leucocytes and some other elements form clumps. These factors will give unevenly distributed slides. Supernatant aspiration
The most efficient way to eliminate the supernatant is by aspiration
It is relatively easy to construct a constant volume aspiration device with a water siphon. The device shown here is efficient and clean. Some recommend keeping a residual volume of 1 ml, which gives a concentration factor of 12.
We think that a concentration factor of 20 (residual volume of 0,6 ml) is a good compromise. This choice increases the probability of finding some rare elements, like blood casts, which are usually present in a small number. If the slide is too loaded, it is always possible to dilute it with a proper dilution fluid.
Figure 19 showing aspiration of supernatant
6.14. Resuspension
It is frequent to have an unequal distribution of elements on the microscope slide. This is especially true for the leucocytes that have a tendency to aggregate. An inadequate resuspension can be the cause of an uneven distribution although, the presence of mucus, to which elements may adhere, can cause a significant variation in the different field counts. The resuspension procedure has to provide the better homogeneous distribution possible. The use of a vortex mixer at low speed is an efficient mixing method. Vortex mixing does not seem to disrupt the different urinary elements. With well-distributed sediment, examination of ten fields is often sufficient to obtain a representative average.
6.15. Examination Volume
It is highly recommended that the microscopical examination be done on a constant volume of sediment. Some companies (Kova...) propose special calibrated slides that always contain the same examination volume. The recent version of the Kova acrylic slide is: ready to use, has multiple wells, and allows examination under polarized light with the 40x objective.
For those that prefer the usual slide and cover slip, an examination volume of 20 uL, with a coverslip of 22 x 22 mm, is well suited. With this coverslip size, 20 uL is the ideal volume preparation being neither too thick nor too thin. The sediment volume is dispensed with a pipetting device. With an SMI™ type pipette that is used properly, carry over is not significant.
6.16. Report Format
The Microscopical analysis report for routine urinalysis should be concise and clear. A heavy listing of elements must be avoided. We prefer the use of a grid report form designed so that reporting is resumed to making a check in the correct square. Checklist-style reports are easier to fill and easier to read.
Cell count should be done at high power field (40 X objective) while the cast count is done at low power (10 X objective).
Statland recommends the use of a unique graduation for all the elements. The proposed scale (0-2, 3-5, 6-10, 11-20, 21-100, >100) is practical and has a good discriminating power.
Reference Values
It is not easy to establish the microscopical urinalysis reference values for a single voided specimen. Some values are reported in Ringsrud and Linné (Urinalysis and Body Fluid.
6.17. Structured Microscopical Analysis
One of the difficulties with the routine microscopical analysis is the number of samples to examine. Some labs use selection criteria, based on the aspect of the urine and the reaction of the dipstick (blood, protein, leukocytes), to eliminate some microscopical analysis. This operation allows an elimination of 40 to 60% of the microscopy depending on the center's clients and the activity of specialized clinics. Even after a first selection, there are still many specimens that can be classified as "ordinary". Real interesting specimens usually represent less than 4% of the workload. In such a context, it is difficult to forget the fifty undone specimens in front of a complex sediment. In this situation, it is normal to rush, and maybe not give enough time to the special sediment. the report is transmitted and the test ends, or the specimen is put aside for the phase II analysis.
7.0. Parameters
7.1. Glucose
Mayne (2001) defines glycosuria as a concentration of urinary glucose detectable using relatively insensitive, but specific screening tests. These tests depend on the action of the enzyme glucose oxidase, incorporated into a diagnostic strip. Usually the proximal tubular cells reabsorb most of the glucose in the glomerular filtrate. Although very low urinary glucose concentrations may be detectable by more sensitive methods even in normal subjects, glycosuria as defined above, occurs only when the plasma, and therefore glomerular filtrate concentrations, exceeds the tubular reabsorptive capacity. The causes of glycosuria include:
· A rise in the concentration of blood glucose with the kidney tubules being unable to reabsorb the increased amount of glucose in the glomerular filtrate for example in untreated Diabetes mellitus. Raised renal threshold for glucose.
· A reduced rate of reabsorption of glucose by the kidney tubules as occurs in serious tubular damage or an inherited defect of tubular absorption causing a lowering of the glucose renal threshold. Glucose appears in the urine when the blood glucose level is well below 10 mmol/l (180 mg/dl). This is often referred to as renal glycosuria for example, Fanconi Syndrome. When glucose, amino acids, phosphates and other substances are excreted due to impaired reabsorption this is termed the Fanconi syndrome.
· An increase in the rate of glomerular filtration as may sometimes occur during pregnancy.
Note: the blood glucose level should be measured whenever glucose is found in urine and the patient is not a known diabetic receiving glucose intravenously.
7.1.1. Urine Glucose
Figure 20 showing a clinistix
Glucose is measured on the clinistix by a glucose oxidase method. The reaction of glucose with glucose oxidase forms nascent oxygen (O), which converts potassium iodide in the dipstick pad to iodine, forming a brown color change. Normal urinary glucose is below the level of sensitivity of the commonly used detection techniques. Therefore, glucose is an abnormal finding in urine.7.1.2. False Positive Reactions
The presence of hydrogen peroxide, bacterial peroxidases (e.g. cystitis), hypochlorite and chlorine will produce false positive reactions.
Formaldehyde
Outdated reagents
7.1.3. False Negative Reactions
High concentrations of ascorbic acid inhibit the reaction.
Drugs: salicylates, tetracyclines.
7.1.4. Pathologic Glycosuria
HyperglycemiaIn nearly all cases, glycosuria is a result of prior (often, continuing) hyperglycemia to a level in excess of the renal threshold for reabsorption.
1. Persistent Hyperglycemia:
Glucosuria is seen in diabetes mellitus, hyperadrenocorticism, acromegaly, and phaeochromocytoma. Remember that a 4+ glucosuria will increase the USG by 0.010.2. Transient Hyperglycemia:
Stress-related hyperglycemia above the renal threshold will result in glucosuria. This is especially true in cats, which develop marked stress-related hyperglycemias, and where the finding of glucosuria is not diagnostic for diabetes mellitus. Other conditions that produce transient hyperglycemia, for example, pancreatitis, may induce a mild, transient glucosuria. Note also that a Transient glucosuria may be seen 1-2 hours after a heavy meal.
· Abnormal Proximal Renal Tubule FunctionGlucose is absorbed by a carrier-mediated process in the proximal renal tubules. Abnormal tubular function can result in glucosuria without hyperglycemia, but these conditions are quite rare. 1. Renal tubule damage: This could be due to drugs (aminoglycosides), hypoxia, and infections.2. Inherited renal diseases: Primary renal glycosuria and Fanconi syndrome.
7.2. Protein
7.2.1. Proteinuria
Mayne (2001) describes proteinuria as the presence of more than a trace amount of protein in urine. The condition is often referred to as albuminuria because when there is glomerular damage most of the protein which passé through the glomerular filter is albumin because this protein molecule is smaller than most of the globulins. Most proteins are too large to pass through the glomeruli of the kidney. The small amount is protein, which does filter through, is normally reabsorbed back into the blood by the kidney. Only trace amounts of protein (that is less than 50 mg per 24 hours) can therefore be found I normal urine. These amounts are insufficient for detection by routine laboratory tests.
7.2.2. Types of Proteinuria
7.2.2.1. Glomerular Proteinuria
According to Bishop et al (1992) describes glomerular proteinuria as a consequence of loss of glomerular membrane integrity, which normally keeps proteins from passing through to the urine because of their large molecular size leading to presence of protein urine.
7.2.2.2. Tubular Proteinuria
Mayne (2000) states that tubular proteinuria is due to renal tubular damage from any cause. This leads to the renal tubules being unable to perform their usual function of reabsorption because of dysfunction or because the amount of protein appearing in the tubular fluid exceeds the absorptive capability (capacity) of a normal functioning tubule. In tubular proteinuria small protein molecules of low molecular weight that normally pass through the glomerulus and are reabsorbed for example alpha and beta microglobulin and retinol binding protein appear in the urine.
7.2.2.3. Orthostatic Proteinuria (Postural)
Mayne (2000) explains that orthostatic proteinuria (postural) is usually more severe in the upright than in the prone position. The term orthostatic or postural has been applied to proteinuria, often severe, which disappears at night. It appears to be glomerular in origin and is common in adolescents and young adults. Although it is often harmless, evidence of renal disease may occur after some years.
7.2.2.4. Microalbuminuria
Bishop et al (1992) describes microalbuminuria in poorly controlled diabetic patients as the finding of albumin of less than 0.05 g in the urine often indicates the development of progressive renal disease (diabetic nephropathy) than those whose albumin excretion is normal. Only a very small amount of albumin is excreted usually below that which can be detected by routine reagent tests or chemical tests. Screening for microalbuminuria can be performed in specialist laboratories using sensitive immunological assays.
7.3. Causes of Proteinuria
7.3.1. The Cause of Proteinuria include:
· Glomerular or tubular urinary disease
· Proteinuria accompanies acute glomerulonephritis and is due to increased permeability of the glomerular basement membrane. The degree of the condition helps in assessing prognosis and responses to treatment.
· Pyogenic or tuberculous pyelonephritis.
· Severe lower urinary tract infection
· Nephrotic syndrome
· Eclampsia when there is moderate t o marked proteinuria.
· Hypertension
· Severe febrile illnesses including malaria.
· Accompanying haematuria
· Urinary schistosomiasis
7.3.2. Nephrotic Syndrome
Nephrotic syndrome is a condition characterized by heavy proteinuria, hypoalbuminaemia and oedema. The clinical condition is caused by increased glomerular permeability, resulting in a daily urinary protein loss of 5 g. The oedema is caused by a reduction in the colloid osmotic pressure due to a fall in the level of plasma albumin brought about by heavy proteinuria of up to 10 g/l per day.
The syndrome may be caused by:
7.3.3. Primary Renal Disease
· Most types of glomerulonephritis, usually due to deposition of circulating immune complexes in the glomeruli in about 80 percent of cases. In children minimal change glomerulonephritis is the most common cause.
7.3.4. Secondary Renal Disease associated with:
· Diabetes mellitus
· Amyloidosis
· Systemic lupus erythromatosus (SLE) due to depression of immune complexes.
· Malaria due to Plasmodium malariae due to immune complexes.
· Inferior vena cava or renal vein thrombosis.
7.3.5. Drugs and Toxins
· Gold
· Penicillamine
Note: Proteinuria should always be considered to indicate underlying disease until proved otherwise.
Important:
Whenever proteinuria is found the urine should be examined for bacteria, pus cells, red cells and casts.
7.4. Types of Proteinuria Tests
Bishop et al (1992) describes microalbuminuria in poorly controlled diabetic patients as the finding of albumin in the urine often indicates the development of renal disease (diabetic nephropathy). Only a very small amount of albumin is excreted usually below that which can be detected by routine reagent tests or chemical tests. Screening for microalbuminuria can be performed in specialist laboratories using sensitive immunological assays.
7.4.1. Bence Jones Protein in Urine
Anderson and Cockayne (1992) describe Bence Jones Protein (BJP) as an abnormal low molecular weight globulin consisting of monoclonal free light chains of immunoglobulins, which contain the amino acids, found in other proteins except methionine. It may be found in the urine of patients with multiple myeloma, which is a malignant disease of the plasma cells mainly affecting bone. If myeloma is suspected urine protein electrophoresis is required to demonstrate BJP and serum electrophoresis to detect the paraprotein ‘M’’(monoclonal immunoglobulin).
7.4.2. Heat Precipitation Test
This test is based on the demonstrating a protein in urine, which precipitates at forty degrees Celsius to sixty degrees Celsius. The test lacks sensitivity and specificity and should be used as a screening test for myeloma.
7.4.3. Urine Protein: SSA
Figure 21 showing a sulfosalicylic acid (SSA) reaction
PROT-SSA represents the reaction observed on the sulfosalicylic acid (SSA) precipitation test. The SSA reagent is added to a small volume of urine. Acidification causes precipitation of protein in the sample (seen as increasing turbidity), which is subjectively graded as trace, 1+, 2+, 3+ or 4+.
The SSA reaction will detect albumin and globulins (although it is more sensitive to albumin). In addition, the SSA detects Bence-Jones proteins, although it often underestimates them. In alkaline urine, the SSA reaction is a more accurate measure of urine protein content than the dipstick.The most accurate measurement of urine protein output is measurement of urine protein excretion over 24-hours. A good alternative to this test is the urine protein to creatinine ratio.
A. False Positives
Contrast media
Antibiotics in high concentration, e.g. penicillin and cephalosporin derivatives
Uncentrifuged turbid urines can look positive. Therefore, SSA should always be performed on urine supernatant.
B. False Negatives
Highly buffered alkaline urine. The urine may require acidification to a pH of 7.0 before performing the SSA test.
Dilute urine
Turbid urine - may mask a positive reaction
7.4.4. Urine Protein: Albustix
Figure 22 showing an albustix
The protein pad on the multireagent dipstick (Multistix®) or albustix is based on the "protein error of pH indicator dyes". Basically, the test is dependent on the ability of amino groups in proteins to bind to and alter the color of acid-base indicators, even though the pH is unchanged. The reaction is extremely sensitive to albumin (as it contains the most amino groups), but is much less sensitive to globulins. It is insensitive to Bence-Jones proteins. Generally this differential sensitivity is not a significant problem (nearly all cases of significant proteinuria involve albuminuria).
InterpretationThe urine protein results should always be interpreted in context with the urine specific gravity and pH. Normal urine contains little protein; negative to trace reactions are usual in concentrated urine. A trace to 1+ reaction in very dilute urine is suggestive of significant proteinuria. A dipstick protein reaction > 2+ in concentrated or dilute urine indicates significant proteinuria. There are numerous causes of proteinuria, the most common of which are urinary tract inflammation, hematuria, and glomerular disease.
A. False Positive Results
Alkaline Urine: False positives occur rarely in highly buffered or alkaline urine samples, as the citrate buffer is overcome, resulting in a shift in pH. Titration of the sample to a more neutral pH and retesting is indicated. (See discussion under "PROT-SSA").
Contact Time: Leaching of the citrate buffer occurs if the urine remains in contact with the pad for a long time.
Detergents: Quaternary ammonium compounds and chlorhexidine can result in false positives.
B. False Negative Results
Bence- Jones proteinuria: A positive SSA protein reaction, with a negative dipstick protein reaction, in a person with a high index of suspicion for multiple myeloma, is suggestive of the presence of free light chains in the urine.
C. Limitations of Protein Screening Tests
Rapid protein screening test were developed to detect presence of albumin and may be negative in the presence of other proteins such as BJP.
Because the test depends on protein concentrations very dilute urine may give negative results despite significant proteinuria.
It is essential that the sample should be fresh.
7.5. Ketones
7.5.1. Ketonuria
Cheesebrough (2000) defines ketonuria as the presence of ketones in urine and indicates deranged energy metabolism such that fat is used in excess of carbohydrate. This can result in production of the ketone bodies in amounts greater than can be metabolized by peripheral tissue; filtration into urine in excess of tubular reabsorption then results in ketonuria. Examples of ketones include acetone, acetoacetic acid and beta- hydroxybutyric acid.
Some specific causes include:
v Unregulated diabetes mellitus
v Pregnancy toxemia
7.5.2. Formation of Ketones
The metabolism of glucose normally provides the body with its energy requirements. If however the intake of glucose is insufficient as in the starvation, or glucose metabolism is defective due to a lack of insulin as occurs in untreated or uncontrolled diabetes the body obtains its energy by breaking down fats. It is this increase in fat metabolism that leads to a buildup of ketones in the body. An accumulation of ketones in the body I referred to as ketosis. Ketones are toxic to the brain and if present in sufficiently high concentration in the blood they can contribute to the coma found in diabetes ketoacidosis. They are strong acids and may overcome the buffering system in the blood and so cause ketoacidosis. With coma due to ketoacidosis, ketonuria is always present.
7.5.3. Causes of Ketonuria
· Untreated diabetes mellitus. The finding of ketonuria in a diabetic who is on treatment usually indicates an out of control state.
· Conditions of starvation when fat metabolism is increased.
· Eating a diet that is very low in carbohydrates.
· Severe dehydration following prolonged vomiting or diarrhoea.
· Glycogen storage disease.
· Toxemia of pregnancy
7.5.4. Urine Ketones
Figure 23 showing a ketostix
The ketone pad on the multi-reagent dipstick detects mainly acetoacetic acid and acetone; ß-hydroxybutyrate is not detected. The ketones react with sodium nitroprusside, which forms a purple color change. Because the color change on the dipstick can be quite subtle, positive reactions are confirmed with the Acetest. This comes in tablet form and contains lactulose to enhance the color change. The reaction with the Acetest is much more apparent than on the dipstick pad. The Acetest is useful for semi-quantitatively measuring ketones in other fluids, such as plasma and serum.
7.6. Urine Blood: Multistix
Figure 24 showing a urine blood Multistix
Blood-stix" represents the reaction observed on the hemoprotein pad of the multireagent urine dipstick. This test is based on detection of the "peroxidase-like" activity inherent in molecules of heme (present in hemoglobin or myoglobin). The reaction is very sensitive and will detect:
7.7. Urine pH
Figure 25 showing a urine pH Multistix
pH
Cheesebrough (2000) explains that the pH of urine varies with the acid or base status of a patient. The kidneys share with the lungs the primary responsibility for acid or base homeostasis. The renal tubules are involved in the reabsorption of bicarbonate and the excretion of nonvolatile organic acids and ammonium ions, but urinary ph rarely falls outside the range 4.7 to 7.8. Measurements of urine pH provide a guide to the overall acid (base state of a patient, but it is also a useful adjunct the proper interpretation of haematuria and haemoglobinuria.
A pH factor of greater than 7 (more alkaline) may result from Fanconi's syndrome, urinary tract infections, or metabolic or respiratory alkalosis. A pH factor below 7 (more acid) may be due to fever, phenylketonuria (PKU), the secretion of homogentisic acid in the urine (alkaptonuria), and acidosis.
The pH of a urine sample is affected by a variety of factors including:
Renal H+ Excretion and HCO3- Resumption
Pathologic abnormalities of systemic acid or base balance.
Pathologic abnormalities of tubular function: with failure to excrete an acid load or failure to absorb bicarbonate.
Dietary factors - due to differences in dietary "acid load", herbivores usually have alkaline urine, carnivores tend to have acidic urine. Urine in carnivores does become slightly less acidic after eating, associated with a post-prandial alkaline tide (due to increased secretion of HCl into the stomach).
2. Age of Specimen (loss of CO2 from the sample to the air raises the pH)
3. Presence of Contaminant or Pathogenic Bacteria (some convert
Urea to ammonia, raising pH)
Knowledge of the urine pH is important in interpreting urine sediment findings. Erythrocytes, leukocytes, and casts tend to disintegrate in alkaline urine (pH > 8.0). In addition, precipitation of urine crystals in supersaturated urine is highly dependent on urine pH (e.g. struvite will precipitate in alkaline not acidic urine).Note that although the kidneys play a central role in the control of acid/base metabolism, the pH of a random urine sample is not a reliable indicator of total body acid/base status. In some conditions, impaired renal tubular function in fact causes or perpetuates the underlying acid or base derangement
Meaningful evaluation of acid-base status generally requires blood gas analysis and consideration of clinical signs
7.8. Specific Gravity of Urine
7.8.1. Specific Gravity
A range of diseases and disorders can affect the specific gravity of urine. Low specific gravity (below 1.005) is associated with diabetes insipidus, nephrogenic diabetes insipidus, acute tubular necrosis, and inflammation of the upper urinary tract (pyelonephritis). In fixed specific gravity, the specific gravity of the urine remains at 1.010 no matter how much fluid the person drinks. This condition occurs in patients who have chronic inflammation of the small blood vessels in the kidneys (glomerulonephritis) and serious kidney damage. High specific gravity (above 1.035) occurs in patients who are in shock or who suffer from nephrotic syndrome, dehydration, acute glomerulonephritis, congestive heart failure, or liver failure.
Bishop et al (1992) explains that the urine specific gravity (USG) and osmolality are measures of the solute concentration in urine and are used to assess the ability of the renal tubules to concentrate or dilute the glomerular filtrate. The diagram and notes below detail how the kidney concentrates urine will increase the USG by 0.001 units.
Urine osmolality is directly related to the number of particles in solution and is unaffected by molecular weight and size. Osmolality can be measured by freezing point depression (technique used at Cornell University) and changes in vapor pressure. Urine osmolality can be approximated from the USG, by multiplying the last 2 digits of the USG by 36.
Knowledge of urinary solute concentration is essential for proper interpretation of BUN and serum creatinine, which are indicators of glomerular filtration rate. The USG is very useful for identifying the cause of azotemia. The interpretation of several urine chemical parameters also is influenced by the specific gravity of the specimen.
In addition, urinary constituents (erythrocytes, leukocytes and casts) can lyse in dilute urine (USG < 1.008), affecting interpretation of the urine sediment results.
7.8.2. Urine Specific Gravity Strip Test
The specific gravity reagent test area responds to the concentration of ions in the urine. It contains certain pretreated polyelectrocytes the pKa of which changes depending on the ionic concentration of the urine. The indicator bromothymol blue is used to detect the change. Colours range from deep blue –green when the urine is low ionic concentration, through green to yellow green when the specimen is of high ionic concentration.
The strip SG test does not indicate the amount of nonionic urinary constituents present such as urea, creatinine or glucose. The SG scale covers the range 1.000 to 1.030 in steps of 0.005. Adding 0.005 to the reading can obtain increased accuracy if the pH of the urine is 7.0 or more.
False Reactions.
· High readings may be obtained if more than 0.1 g/l of protein is present.
· Highly buffered alkaline urines may cause low readings.
· Large quantities of divalent cations for example calcium (Ca 2+)
7.8.3. Refractometer Method
Bishop et al (1992) explains that a refractometer measures refractive index. The measurement is based on the number of dissolved particles in urine. The higher the concentration of particles, the greater the increase in refractive index (refraction). From refractive index measurements a scale of relative mass density (SG) values can be prepared.
Procedure
· Check that the instrument scale is correctly adjusted by placing a few drops of distilled water on the face the prison.
· Gently close the cover plate. With the refractometer facing the light, bring the scale into focus turning the eyepiece. If the boundary line does not coincide with the wt (water) line, make the necessary adjustment by rotating the scale adjustment knob.
· To measure urine specific gravity, place one or two drops of urine on the prism surface and gently close the cover plate
· Rotate the eyepiece close until the scale becomes clearly visible.
· Observe the point on the urine SG scale where the dark part of the field meets the light area.
· Read off the urine SG value.
7.8.4. Urinometer
Cheesebrough (2000) explains that this technique uses a specially calibrated hydrometer. The lower the concentration of solute the further the urinometer will sink in the urine.
A new urinometer must be checked for accuracy by being floated in distilled water. The reading should be 1.000 at the temperature specified on the urinometer. If the reading is not 1.000, subtract the difference in value from each urine reading. For example if the density of the distilled water is 1.002 subtract 0.002 from each urine reading.
7.8.4.1. Procedure
· Obtain at least 50 ml of urine.
Note: The cylinder and urine must be detergent free because even a trace of detergent will lower the surface tension of the urine and give an incorrect reading.
· Transfer urine to a cylinder.
· Immerse the urinometer in the urine and make sure that it floats centrally and does not touch the bottom or sides of the cylinder.
· Take the reading at the lowest point of the meniscus. This must be done at eye level.
· The scale of the urinometer is calibrated from 1.000-1.060 with each division being equal to 0.001.
7.8.4.2. Adjustment for Temperature
Most urinometers are calibrated for use at 15 degrees Celsius or 20 degrees Celsius. For each 3degrees Celsius difference, 0.001 must be added if above or subtracted if below the calibration temperature.
Example: If the mass density of reading of the urine is 1.022 at 23 degrees Celsius and the urinometer has been calibrated at 20 degrees Celsius, the corrected reading is 1.022 plus 0.001 minus 1.023.
Figure 26 showing a refractometer
7.8.4.3. Interpretation of Urine Relative Mass Density Results
The reference range for urine relative mass density is 1.010- 1.030.
A. Low values
A consistently low value mass density usually indicates poor tubular reabsorption. Excessive fluid intake, however, will also result in a low mass density. In general, the greater the volume of urine excreted the lower is its density and the lighter its colour.
B. High values
A high urine mass density may be the result of heavy perspiration, dehydration, or to the presence of substance not normally found in the urine such as glucose or protein (if not corrected for in the test method).
C. Concentration of urine expressed as osmolality/kg
Bishop et al (1992) explains that this is the best way of expressing the concentration of urine because it depends on the number of osmolality active solute particles per unit of solvent whereas mass density depends on the type as well as the number of solutes particles. It is however, much simpler to measure the mass density of urine rather than is osmolality. A mass density of 1.002 is equivalent approximately to an osmolality of 100mosmol/kg and a mass density of 1.025 to about 1000 mosmol/kg.
7.9. Urine Bilirubin
7.9.1. Bilirubinuria
Cheesbrough (2001) defines bilirubinuria as the presence of bilirubin in urine. Bilirubin is not normally detected in urine. When it is found, the condition is termed bilirubinuria. Urine containing 8.4 micromoles per litre (0.5 mg /dl) or more of bilirubin has a characteristic yellow- brown colour (hepatocellular jaundice) or a yellow- green appearance (obstructive jaundice).
7.9.2. Formation of Bilirubin
Bilirubin is formed from the breakdown of erythrocytes and other haem containing proteins such as myoglobin and cytochromes. The haem (iron porphyrin) of the haemoglobin molecule is separated from the globin and the porphyrins is converted to Biliverdin by haem oxygenase. Biliverdin is reduced by Biliverdin reductase top bilirubin. This bilirubin is referred to as unconjugated bilirubin (indirect). It is insoluble in water and cannot be excreted in the urine. It is bound to albumin and transported in the blood to the liver. In the liver cells the enzyme glucuronyltransferase conjugates (joins) glucuronic acid to bilirubin forming bilirubin glucuronides mainly (diglucuronides). This bilirubin is called conjugated bilirubin. Conjugated bilirubin is water soluble and less toxic. It passes into the bile canaliculi through the bile duct and into the intestine. In the terminal ileum and colon, the bilirubin is deconjugated and reduced by bacteria to various pigments and colourless chromogens commonly termed urobilinogen. Most of the urobilinogen excreted in the faeces as stercobilinogen from the intestine is reabsorbed into the liver where it re-enters the intestine in the bile and is excreted in the faeces.
A small amount of reabsorbed urobilinogen is carried in the blood through the liver and transported to the kidneys where it is excreted in the urine. When exposed to air urobilinogen is rapidly oxidized to the brown coloured pigment urobilin (and stercobilinogen to stercobilin).
7.9.3. Causes of Bilirubinuria
· Partial or complete biliary obstruction.
· Hepatocellular damage associated with the hepatitis or cirrhosis when blood usually contains both conjugated and unconjugated bilirubin.
Note: a combination of measurements of urinary bilirubin and urobilirubin allows some differentiation as to whether jaundice is due to an obstructive, hepatocellular or hemolytic origin.
7.9.4. Urine Bilirubin: Ictotest
The main advantage is that observation of the true color reaction is less affected by the inherent color of the urine itself. It is, therefore, useful in confirming or refuting apparently positive reactions on the dipstick in cases where the urine sample is deeply colored. It is also more sensitive to bilirubin than the dipstick (detects 0.1 mg/dL of bilirubin).
The Ictotest® uses the same chemical reaction as the bilirubin pad on the multi-reagent dipstick, but presented in a tablet format
Purple color around the tablet is a positive result.
Figure 27 showing the reaction of Ictotest
7.9.5. Urine Bilirubin: Multistix
Figure 28 showing the bilirubin multistix
The bilirubin pad on the multireagent dipstick detects bilirubin using a specific diazotization reaction and is sensitive to 0.2-0.4 mg/dL of conjugated bilirubin. The color change indicating a positive reaction, however, is a rather subtle transition among shades of beige, and sometimes is obscured by color inherent in the urine itself (e.g., marked hemoglobinuria). In such instances, confirmation of a suspected positive reaction is attempted using the Ictotest® method.7.9.5.1. False Positive Reactions
Hepatobiliary Disease: Detection of bilirubin in urine is generally an abnormal finding. Bilirubinuria generally results when conjugated bilirubin levels in blood are elevated as a result of Hepatobiliary disease. In the dog, bilirubinuria may be seen prior to bilirubinemia due to the low renal threshold for bilirubin in this species.
Bilirubinuria indicates cholestasis.
In some cases of hemolytic anemia, bilirubinuria may be secondary to the hemolysis without any evidence of cholestasis. The renal tubular epithelium is capable of absorbing hemoglobin from the glomerular filtrate and converting it to conjugated bilirubin, which is then excreted in the urine. This will only occur with intravascular hemolysis, when free hemoglobin is filtered by the glomerulus.
7.9.5.2. False Negative Reactions
Aged Urine Samples: Conjugated bilirubin hydrolyzes to unconjugated bilirubin if left at room temperature.
Exposure to UV Light: UV light converts bilirubin to Biliverdin, resulting in false negative reactions.
Ascorbic Acid: High concentrations of vitamin C inhibit the reaction.
7.10. Fouchet’s Test
This text is recommended for detecting bilirubin in urine because it is sensitive, easy to perform, inexpensive and stable when compared with the strip tests and Ictotest tablet test. Barium chloride is used to precipitate the sulphates in the urine. Any bilirubin present becomes attached to the barium sulphates. When fouchet’s reagent is added to the precipitate, the iron III ferric chloride oxidizes the bilirubin to green-blue Biliverdin.
Reagents
· Barium chloride 0.48 mol/l
· Fouchet’s reagent
Procedure
· Dispense about 5 ml of fresh urine into a tube or small bottle.
· Add 2.5 ml of barium chloride reagent and mix well. The sample will become cloudy.
· Filter or centrifuge to obtain a precipitate, which will contain any bilirubin that is present. Unfold the filter paper and place it, precipitate upwards, on a piece of absorbent paper.
· Add 1 drop of Fouchet’s reagent to the precipitate on the filter paper or the sediment in the tube (after discarding the supernatant urine)
· Report as follows
Immediate blue green bilirubin positive
Colour around the drop
No blue green colour bilirubin negative
Note: any pink-mauve colour, which develops, is due to salicylates in the urine.
7.11. Urine Urobilinogen
7.11.1. Urobilinogen in Urine
Cheesebrough (2000) explains that it is normal to find small amounts of urobilinogen in the urine derived from that, which is reabsorbed from the intestine. The concentration of urobilinogen in the urine is therefore dependent on the amount of bilirubin being produced and entering the intestine and on the ability of the liver to excrete the urobilinogen coming to it from the intestine. Urine is often tested for urobilinogen when investigating haemolysis or liver disorders in which liver function is impaired.
7.11.2. Causes of Urobilinogen in Urine
· Haemolytic disease when the amount of bilirubin being produced is increased leading to greater amounts of urobilirubin being formed.
· Paralytic ileus or enterocolitis when there is an increase in the production of urobilinogen in the intestine.
· Hepatocellular damage or hepatic congestion resulting in loss of the absorbed urobilinogen being excreted by the liver. The urobilinogen then passes into the general circulation leading to more being excreted by the kidneys.
· Liver cirrhosis
· Viral hepatitis
At first it is increased but as liver cell damage increases the small biliary ducts become obstructed leading to a reduction or even an absence of urobilinogen in the urine.
7.12. Haemoglobinuria
· Bishop (1992) defines haemoglobinuria as the presence of haemoglobin in urine. It occurs with severe intravascular haemolysis when the amount of haemoglobin being released into the plasma is more than can be taken up by haptoglobin. The plasma protein that binds free haemoglobin to prevent it being lost from the body. The renal threshold for free haemoglobin is 1.0-1.4 g/l.
7.12.1. Causes of Haemoglobinuria
Conditions that cause haemoglobinuria include the following:
· Severe intravascular haemolysis. This may result from burns or crush injuries, transfusion reactions, malaria, and toxic effects of various chemical agents or drugs or from congenital or acquired haemolytic anaemias.
· Urinary schistosomiasis
· Malignant disease
· Renal damage
· An overdose of anticoagulants.
· Severs falciparum malaria
· Typhoid fever
· Glucose 6 phosphate dehydrogenase deficiency following the ingestion of certain drugs.
· Escherichia coli septicaemia
· Incompatible blood transfusion
· Snake bites that cause acute hemolysis sickle cell disease with severe viral haemorrhagic fever accompanied by intravascular haemolysis.
7.12.2. False Positive Reactions
False positive reactions can result from the presence of contaminating oxidizing detergents in the urine such as bleach. Heavy proteinuria may reduce the colour reaction (that is over 5 g/l).
7.13. Nitrite
Urine from a healthy person does not contain nitrite. The detection of nitrite in urine is a useful test in the investigation of urinary tract infection caused nitrite- reducing bacteria particularly when cultural facilities are not available. Most pathologic bacteria reduce nitrate normally found in urine to nitrate.
7.13.1. Causes of Nitrite in Urine
Bacteria that produce the enzyme nitrate reductase are able to reduce nitrate to nitrite. Examples of microorganisms that are able to do this include,
· Escherichia coli
· Proteus species
· Klebsiella species
· Citrobacter species
· Salmonella species
7.14. Leukocyte esterase.
Normal urine specimen generally yields negative results; positive results (+ or greater) are clinically significant. Individually observed trace results may be questionable clinical significance; however, trace results observed repeatedly may be clinically significant. Positive results may occasionally be found with random specimen from females due to contamination of the specimen by vaginal discharge. Elevated glucose concentration 160 micromoles per litre or high SG may cause decreased test results.
7.15. Urinalysis Procedure
7.15.1. Principle
Urine undergoes many changes during states of disease or body dysfunction before blood composition is altered to a significant extent. It is a useful procedure as an indicator of health or disease, especially in the areas of metabolic and renal disorders. Urine dipstick testing is screening test performed on fresh urine. A chemically- impregnated reagent strip is dipped in fresh urine to determine, glucose, bilirubin, ketones, specific gravity, blood, pH, protein, Urobilinogen, nitrites and leukocyte esterase.
7.15.2. Specimen Requirements
1. A random fresh urine sample.
2. Perform test as soon as possible.
3. Urine sample is stable at room temperature for one hour and refrigerated for four hours. If sample is refrigerated, allow it to come to room temperature before testing.
4. Minimum volume is one mL.
7.15.3. Interferences
Note: Abnormal urine colour may affect the readability of the reagent areas on the urinalysis reagent test strips.
7.15.4. Procedure
1. Observe and note the colour of urine.
2. Observe and note the appearance of urine.
3. Briefly dip a urine dipstick into a well- mixed urine sample. Remove excess urine by drawing the edge of the strip along the rim of the container or tip sideways on a paper towel.
4. If testing quality control (QC) solution, apply a drop of QC to each pad on the dipstick. Tip dipstick sideways on a paper towel to remove excess.
5. Never blot the test pads.
6. Read each colour pad at the designated time on the colour chart on the dipstick bottle. Reading the colour at the indicated time is critical for optimal results. Any colour change that occurs after two (2) minutes is of no diagnostic value.
7. If the test strip colour does not match a particular colour square on the colour cart, then match the test strip to a colour square of equal intensity.
8. If the colour development is slightly uneven, then match the average colour on the test pad.
7.15.5. Reporting of Results
1. Report the biochemical results using the following:
a. Urine colour as: colourless, straw, yellow, amber, red, brown, or green.
b. Urine appearance as: clear, slightly cloudy, cloudy, moderately cloudy, or turbid
c. Glucose as: 100, 250, 500, 1000, or >2000 mg/dl or alternatively as: trace, 1+, 2++, 3+++, or 4++++.
d. Bilirubin as: negative, small, moderate, or large.
e. Ketone as: negative, trace, small, moderate, or large.
f. Specific gravity as: 1.000,1.005, 1.010, 1.015.1.020, 1.025, or 1.030.
g. Blood as: negative, trace, small, moderate, or large.
h. PH as; 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5.
i. Protein as: negative, trace, 30, 100, 300, > 2000 mg/dl
j. Urobilinogen as: negative or positive
k. Nitrites as: negative or positive
l. Leukocytes as: negative, trace, small, moderate, or large.
2. Record results on patient’s request laboratory form, chart, health passport or
in computer.
7.15.6. Quality Control of Test Reagent Strips
Cheesebrough (2000) explains that the performance of test strips should be controlled by regularly checking the strip reactions against those obtained by standard chemical tests. Control urines of known negative or positive reactions should be prepared and tested with patients’ specimens.
7.15.7. Correct Storage of Urine Reagent Strips (Dipsticks)
The reliability of reagent strip test results depend on the correct urine sample being used, the correct storage, use, and control of the strips, and knowledge of the causes of false reactions. It is therefore essential to read carefully the literature supplied by the manufacturer, which will be found in each strip container.
Read carefully the expiry date and the storage instructions supplied with the strips. To prevent the deterioration of reagent strips the following are precautions important:
(i) Protect the strips from moisture and excessive heat and light, but do not refrigerate.
(ii) Remove strips only as required. After removing a strip replace the lid immediately and tightly close it to prevent against moisture. This is particularly important in humid climates. The reagents impregnating the test areas deteriorate rapidly if they become damp. This can occur after only fifteen minutes of exposure in humid conditions.
(iii) Do not touch test pads on the test strip.
(iv) Do not store the bottle in direct sunlight or near heat source.
(v) Store at room temperature until expiration date on the vial.
REFERENCES.
Bishop Michael L. et al, (1992), Clinical Chemistry, Principles, Procedures, and Correlations. J.B. Lippincort Company, Philadelphia.
Bullock N (1999), Anatomy and Physiology, 2nd edition, Mosby. New York.
Cheesebrough Monica (2000), District Laboratory Practice in Tropical Countries, Part I. Cambridge. Cambrigeshire.
Cheesebrough Monica (2000), District Laboratory Practice in Tropical Countries, Part I1. Cambridge. Cambridgeshire.
Young D. (2004), Urinalysis. Laboratory Science –Infectious Disease, Vol. no.2.
Mayne Phillip D (2000), Clinical Chemistry in Diagnosis and Treatment, 6th edition. Cambridge, Cambridgeshire.
Martin Rubin and Per Louis. (1992), Education and Training For Clinical Chemistry, Wheaton and Company. Exeter
Mesko Duncan (2003). Differential Diagnosis In Laboratory Medicine, Springer, New York.
http://www.oshu.edu/pathology/POC/procedure/urinalysis.html
Palstanga Nigel et al, (1994), Anatomy and Human Movement, Structure and Function, 2nd edition, Butterworth Heinemann.
Ross D.L. (1983), Textbook on Urinalysis and Body fluids, New York.
Richard Tilton C. (1992), Clinical Laboratory Medicine, Mosby, New York
.
Tortora G.J. and Grabowski S.R. (2000), Principles of Anatomy And Physiology, 9th edition. John Watery And Sons. New York.
About Me
- Henry Chigwalale
- Principled man. Married with 3 children. First wife passed away. Now with another one who has one child from her previous marriage. No child with me but I am keeping her child. Together now there are 4 children. Also Keeping 3 dependants. One from my late sister Rose Msiska. 2 from my on-and-off-sick brother at home Mackenzie. My children are: Chimwemwe (son) in form four, Hendrina in standard 8, Naomie in standard 6. Their ages are eighteen, fourteen and eleven, respectively. The other children are: Khwima from my late sister he is twenty but in form 1, Temwa (14)and Mbiko(11)in std 7 and 5 respectively (children of my brother). Joseph in std 5 is 14 and is the child to my new wife. I do not intend to have more children. In total I am keeping 4 extra children apart from my 3 children.
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