CHEM 110 - Clinical Chemistry 1 - LO3 PDF

Learning Outcome 3: Interpret microscopic urinalysis results and their correlation to clinical pathology. Learning Step 3.1: Describe preparation of urine sediment for microscopic urinalysis. 3.1.1 Reading Assignment Read the introductory paragraphs, Sediment Preparation and Microscopic Observation...

Learning Outcome 3: Interpret microscopic urinalysis results and their correlation to clinical pathology. Learning Step 3.1: Describe preparation of urine sediment for microscopic urinalysis. 3.1.1 Reading Assignment Read the introductory paragraphs, Sediment Preparation and Microscopic Observation and Enumeration pages 112-113 from the textbook, Mundt and Shanahan, Graff’s Textbook of Urinalysis and Body Fluids. 3rd Ed. Formatting 3.1.2 Instruction Sheet: Preparation of Urine Sediment Microscopic urinalysis may be done as requested by the physician or the laboratory may have a protocol to do microscopic analysis only on specimens that are cloudy and/or contain one of the following on chemical urinalysis: blood leukocytes nitrites protein Because consistency and standardization of microscopic examinations are important, there are various commercial systems available to laboratories to assist in the preparation of a urine sediment. Regardless of the system used, care must be taken that each step is followed consistently by all lab personnel to provide accurate results. Urine Volume Most systems use a 10-12 ml conical urine sample tube. If the specimen of less than the optimum volume is processed, according to general protocol a comment should be added to the microscopic report indicating that the evaluation was done on a smaller than normal volume. Some laboratories choose to do the microscopic urinalysis on an uncentrifuged specimen if the specimen volume is less than optimal. A comment would be added to the report that the microscopic analysis was done on an uncentrifuged specimen. Page 54 CHEM 110 – Clinical Chemistry 1 – LO3 Centrifugation When the chemical analyses have been completed, the conical tube is centrifuged at 400-450g (Relative Centrifugal Force) for 5 minutes. RCF(g) = 1.118 x 10-5 x radius in cm x (rpm)2 Centrifugation at 400-450g for 5 minutes will allow an adequate sediment to form without the disruption of the fragile formed elements. The supernatant is then removed from the sediment so that all sediment volumes are consistent. The sediment may or may not be stained before microscopic examination. Viewing the Sediment The urine sediment must be gently mixed to resuspend the particles and transferred to a standardized slide. Subdued light must be used to provide adequate contrast. This is obtained by closing the iris diaphragm located in the substage condenser. If too much light is used, some structures will be missed. The fine focus should be continuously adjusted while performing the microscopic examination. This ensures that objects on a different focal plane are counted and identified. Casts are counted on 10x but identified on 40x. Other microscopic elements are counted or observed on 40x. Scan a minimum of 10 fields. Final counts for casts and cells are an average of those fields. CHEM 110 - Clinical Chemistry 1 – LO3 Page 55 Learning Step 3.2: Describe the elements seen in microscopic urinalysis and correlate to clinical pathology. 3.2.1 Reading Assignment Read pages 113-136 from the textbook, Mundt and Shanahan, Graff’s Textbook of Urinalysis and Body Fluids. 3rd Ed. 3.2.2 Instruction Sheet: Microscopic Urinalysis Cells Three types of cells can be seen in urine sediment: red blood cells, leukocytes, and epithelial cells. Red Blood Cells In normal urine, the upper limit of normal is 2-5/HPF (high power field). An increased number of RBCs in urine is called hematuria. When increased red blood cells are accompanied by red blood cell casts, the source of the hematuria is probably renal. Increased red blood cells in the absence of red blood cell casts suggests the source of the hematuria is somewhere in the urinary tract other than the kidney. Page 56 CHEM 110 – Clinical Chemistry 1 – LO3 Depending on the osmotic pressure (OP) and the pH of the urine, the appearance of red cells can be altered. If the osmotic pressure is high, the RBCs will appear crenated; if the osmotic pressure is low or the pH very alkaline, the RBCs will be swollen or lysed. When lysing occurs, the microscopic examination may show the empty red blood cell membranes which are often referred to as “ghost cells” and are very difficult to see. Ghost red blood cells are still counted when performing microscopic urinalysis. At times, RBCs may be confused with WBCs, yeast, urate crystals, or atypical calcium oxalate. To help confirm the presence of RBCs, check the blood portion of the reagent strip: it should be positive if there are at least 5-10 RBC/HPF. Red blood cells may also be lysed with acetic acid to help with differentiation. Leukocytes In normal urine the upper limit is 5-8/HPF. Leukocytes probably enter the urine of healthy individuals by diapedesis through the glomerular membrane or the tubular cells. An increased number of leukocytes in urine is called pyuria. The most common type of leukocyte seen is the neutrophil which can sometimes be identified by the multilobed nucleus. Slight pyuria along with proteinuria, will occur in a healthy individual following strenuous exercise or stress. Hyaline or granular casts may also be observed. Pyuria is associated with an inflammatory process in or adjacent to the urinary tract. Leukocytes are attracted to any area of inflammation, and, because of their ameboid properties, they can penetrate the areas adjacent to the inflammatory site. Sometimes pyuria is seen in conditions such as appendicitis and pancreatitis. It is also found in non-infectious conditions such as glomerulonephritis, dehydration, fever, or irritation to the ureter, bladder, or urethra. Acute infection of the urinary tract is suggested when 50 or more leukocytes per HPF are seen in microscopic examination of urine sediment, especially when they occur in clumps. If this degree of pyuria is accompanied by minimal proteinuria and leukocyte casts, the probable cause is pyelonephritis. If accompanied by minimal proteinuria and no casts, it is likely an infection of the lower urinary tract. CHEM 110 - Clinical Chemistry 1 – LO3 Page 57 Leukocytes shrink if the urine is concentrated and swell up or are rapidly lysed in the urine is dilute or alkaline. Studies have shown the number of leukocytes decreases by 60% within two hours after collection if the urine is alkaline and dilute, and not refrigerated. When leukocytes swell in dilute urine, their granules may show Brownian movement. Cells which develop this characteristic are called “glitter cells”. Glitter cells are not reported. Leukocytes in urine sediment may be confused with RBCs or renal tubular epithelial cells (RTEs). leukocytes “lumpy nucleus” RTEs round central nucleus RBCs smaller, no nucleus To confirm the presence of Leukocytes, check the leukocyte portion of the reagent strip: it should be positive if there at least 10/HPF. Page 58 CHEM 110 – Clinical Chemistry 1 – LO3 Epithelial Cells Three types of epithelial cells can be found in urine: 1. Renal tubular: originate from the tubules of the nephrons in the kidney 2. Transitional: originate from the renal pelvis, ureters, bladder, and proximal two-thirds of the urethra 3. Squamous: originate from the terminal end of the urethra, and from the vagina. CHEM 110 - Clinical Chemistry 1 – LO3 Page 59 Renal Tubular Epithelial Cells Occur singly or in pairs Often round by may have a flat side or cuboidal or columnar shape Transitional Epithelial Cells Slightly larger than RTE’s; may have a teardrop, pear or round shape Squamous Epithelial Cells Large, flat cells; may be rolled up to give a cigar shape or folded Abundant cytoplasm In normal urine, very few renal tubular epithelial cells and transitional epithelial cells may be found due to sloughing of aged cells. Increased numbers of these cells indicate inflammation within the kidney or lower urinary tract. An increased number of renal tubular epithelial cells suggest tubular damage. This damage can occur due to nephrotoxic agents such as heavy metals, organic solvents, or ethylene glycol, or renal infections such as pyelonephritis, or diminished blood supply to the kidney. Any condition that is causing the RTEs to die, acute tubular necrosis. An increased number of transitional epithelial cells can be due to renal calculi (stones), a bladder infection, or trauma due to catheterization. Squamous epithelial cells are not generally considered to be significant. A large number in female urine is most often indicative of vaginal contamination due to improper specimen collection. In summary: Increased RTEs Page 60 Acute tubular necrosis Pyelonephritis Kidney Transplant rejection Increased Transitional Cystitis Renal Calculi Catheterization Increased Squamous Insignificant (usually means vaginal contamination) CHEM 110 – Clinical Chemistry 1 – LO3 Crystals Crystals are found in urine when chemicals are present in excess of their solubility. They frequently occur in normal urine, especially if the specimen has been refrigerated. Most crystals are diagnostically insignificant, and some laboratories do not bother reporting normal crystals. Only a few crystals are abnormal and must be reported. A good atlas of urine sediment is the best aid in identifying crystals to determine if they are normal or abnormal. The chart on the following pages shows some of the more common normal crystals. Normal Crystals Crystal Uric Acid Amorphous Urates Urine pH acid acid Description These crystals come in a variety of shapes: diamonds, hexagons, prisms. They are often yellow to red-brown in colour. Although considered normal, a large number may indicate gout. These appear microscopically as granular sediment. In large numbers they may obscure other elements in the sediment. Macroscopically they appear as pink precipitate which is sometimes referred to as “brick dust”. pink precipitate They can be dissolved with hot tap water. CHEM 110 - Clinical Chemistry 1 – LO3 Page 61 Calcium Oxalate acid (may also be found in neutral or alkaline) Sodium Urates acid Hippuric Acid acid Triple Phosphate Page 62 alkaline These crystals are 3 dimensional and are described as colourless, refractile envelopes; occasionally they appear as dumbbells. Although they are considered normal, they are found in cases of ethylene glycol (antifreeze) poisoning where they can cause considerable obstruction in the kidney. These appear as colourless or yellow needles or prisms occurring in sheaves or clusters These crystals are 2 dimensional and appear microscopically as colourless “picket fences” with both ends pointed. These 3-dimensional crystals are described as “coffin lids”. Sometimes they appear as feathery fern leaves. They may be associated with chronic urinary tract infections. CHEM 110 – Clinical Chemistry 1 – LO3 Amorphous Phosphates alkaline These appear microscopically as granular sediment which looks like amorphous urates. They may also obscure other urine sediment elements. Macroscopically they appear as white precipitate when present in large numbers. They cannot be dissolved with warm tap water. white precipitate Calcium Carbonate alkaline Colourless dumbbells or colourless granules. Calcium Phosphate alkaline These appear as colourless prisms which may form a rosette. They can also appear as a thin irregular shaped plate/sheet. Ammonium Biurate alkaline These appear microscopically as yellowbrown spheres with striations on the surface. Irregular projections may appear on the crystal giving it a “thorny apple” look. Care must be taken not to confuse with leucine crystals. Solubility tests may be required. CHEM 110 - Clinical Chemistry 1 – LO3 Page 63 Abnormal Crystals Abnormal crystals are rare and clinically significant. The chart below shows some of the abnormal crystals that can be found in urine sediment. All are found at an acid pH. Type Description Cystine Colourless hexagonal plates These crystals are diagnostic of cystinuria. It is an inherited metabolic disorder where cystine is not reabsorbed by the renal tubules. Formation of renal calculi composed of cystine may also occur. Cystine crystals should be confirmed by chemical solubility testing. Leucine Yellow, oily looking spheres with radial striations. Tyrosine Fine needles or sheaves Cholesterol Colourless transparent plates with notched corners. These crystals are found in the urine of patients with maple syrup disease which is an error in leucine metabolism. The urine excreted has a characteristic maple syrup odour. Leucine crystals can also be found in severe liver disease. Leucine crystals should be confirmed by chemical solubility testing. These crystals are seen in tyrosinosis, a disease of tyrosine metabolism. They can be found together with leucine crystals in patients with severe liver disease such as terminal cirrhosis or severe viral hepatitis. Tyrosine crystals should be confirmed by chemical solubility testing. Found in patients with the nephrotic syndrome or severe urinary tract infection. Cholesterol is normally too large to pass through the glomerular barrier. Therefore, they should be accompanied by proteinuria and lipiduria. Bilirubin Delicate needles: Found in patients with liver disease where red brown in bilirubin is present in the urine. colour, may form balls Page 64 CHEM 110 – Clinical Chemistry 1 – LO3 Drug Crystals Administration of drugs can occasionally result in the formation of crystals that can be found in the urine. If unusual crystals are found, you should obtain a list of medications the patient is taking. Antibiotics such as sulfonamides and ampicillin are known for their likelihood of crystal formation. Intravenous administration of radiographic dyes may also result in crystal formation. Ampicillin crystals appear as long, colourless thin prisms or needles. Sulfonamides appear in various forms: sheaves, rosettes, spheres. Radiographic dyes may appear as colourless, long pointed needles, singly or in sheaves. They may also appear as flat, elongated rectangular plates. They are usually highly refractile. The presence of needle crystals in urine with grossly elevated urine specific gravity, >1.050, by refractometer, is suspicious of radiographic dye. Solubility Properties of Urine Crystals: Reference Chart Differentiating crystals can be difficult and sometimes chemical solubility testing must be performed. Solubility testing should also be performed to confirm the presence of the abnormal crystals. Refer to Table 9-1 on page 119 in Mundt and Shanahan, Graff’s Textbook of Urinalysis and Body Fluids, 3rd ed. Solubility properties do not need to be memorized as they can be found in most urinalysis reference books. Casts Casts are cylindrical structures that form in the lumen of the distal convoluted tubule (DCT) or collecting duct (CD); they represent an actual cast or mold of the tubular lumen. The matrix of all casts is composed primarily of Tamm-Horsfall protein (THP) also called uromodulin. This is a protein which is synthesized by the renal tubular epithelial cells in the DCT. It is not found in plasma. When there is urinary stasis, the filtrate is concentrated and the pH is acid, uromodulin precipitates and adheres to the tubule wall forming a hollow cylindrical structure, which is filled in as more protein precipitates. When the uromodulin precipitates, extraneous material present in the filtrate may adhere to the surface. This includes red blood cells, leukocytes, renal tubular epithelial cells, and fat. When urine passes through the nephron, casts are washed loose and become part of the microscopic elements of the urine. Cast have an even and defined outline with parallel sides. They should have a uniform diameter and are several times longer than wide. CHEM 110 - Clinical Chemistry 1 – LO3 Page 65 Clinical Significance of Casts Some types of casts are not clinically significant and can be seen in healthy individuals. Because casts are formed in the tubules of the kidney, many are important indicators of renal disease (as opposed to lower urinary tract disease). In the late stages of many renal diseases, the renal tubules often become dilated. Casts developing in these tubules have large diameters (2-6 times wider than ordinary casts) and are called broad casts, or chronic renal failure casts. The prognosis is grave when these casts are found in significant numbers. At times it is difficult to distinguish between various types of casts because of degeneration or because the cast may contain a variety of structures. As cellular casts degenerate, they form granular casts which in turn further degenerate to fine granular and finally, to waxy casts. Page 66 CHEM 110 – Clinical Chemistry 1 – LO3 Classification of Casts Hyaline Casts The main constituent of hyaline casts is uromodulin. They are colourless and transparent and therefore difficult to see unless the urine sediment is stained or examined with reduced illumination. They can contain an individual cell or some debris which became trapped in the matrix during cast formation. A few hyaline casts per low power microscopic field may be seen in a healthy individual, especially after strenuous exercise or stress, and in elderly patients. Cylindroids Cylindroids resemble casts but have one end that tapers similar to mucus. Cylindroids are most frequently hyaline but may incorporate other material. Cylindroids are formed in the renal tubules and have the same significance as casts. Therefore, they are counted as casts. CHEM 110 - Clinical Chemistry 1 – LO3 Page 67 Red Blood Cell Casts A red blood cell cast is a hyaline cast with red blood cells embedded in its matrix. The cast may contain only a few RBC’s or there may be many cells packed into the matrix. If the red blood cells are still intact and their outlines are detectable then the cast is termed a red blood cell cast. Unstained, these casts may appear yellow orange in colour. With time, the red blood cells may disintegrate and lyse within the cast. This disintegration results in a cast where the hemoglobin discolours the cast to a gold brown colour and the cytoplasm of the cells becomes granular. As the cells within the cast disintegrate and their outlines are no longer visible, the cast is called a coarse granular or granular cast. The reddish-brown colour of the course granular cast is a clue that the cast contained red blood cell. Red blood cell casts usually occur in diseases where the basement membrane of the glomerulus is damaged, allowing RBCs and protein to be filtered. They are most commonly associated with glomerulonephritis. The most common route of red blood cell cast formation is RBCs passing through the glomerulus. However, these casts can also occur when there is bleeding into the renal tubule. Page 68 CHEM 110 – Clinical Chemistry 1 – LO3 Leukocyte Casts A leukocyte cast is also called a white blood cell cast and has leukocytes embedded in its matrix. The leukocytes may be difficult to distinguish from renal tubular epithelial cells (RTE’s); staining may help by emphasizing the multilobed nuclei of neutrophils, which are the most common type of leukocyte present in casts. The cast may contain a few leukocytes or may be packed with leukocytes. If the cells are intact, the cast is called a leukocyte cast. As the cast sits in the tubule, the neutrophils may disintegrate into granular debris. When this occurs, the cast would be reported as a granular cast. The most common cause of leukocyte casts is pyelonephritis, a bacterial infection that affects the interstitium of the kidney. The bacterial invasion most commonly ascends from the lower urinary tract (i.e., starts off as a bladder infection). Neutrophils accumulate at the site of the infection in the kidney and pass into the renal tubules where they become incorporated into casts. Pyelonephritis is usually manifested by many leukocyte casts in the urine sediment, accompanied by bacteriuria and proteinuria. Leukocyte casts may also be found in glomerulonephritis, wherein leukocytes appear at the site of glomerular injury and are filtered. CHEM 110 - Clinical Chemistry 1 – LO3 Page 69 Epithelial Cell Casts Epithelial cell casts are renal tubular epithelial cells surrounded by protein. RTE’s are small round cells (about the same size as a neutrophil) with a large, round, central nucleus. Epithelial cell casts indicate tubular necrosis. This means the renal tubular epithelial cells have been injured and are dying. Injured RTE’s are sloughed into the lumen of the nephron and form casts. The presence of these casts indicates damage to the renal tubules by: 1. interrupted blood supply (the RTEs are very oxygen-sensitive and die easily if the blood supply is interrupted, as can happen in post-surgical hemorrhage or following a myocardial infarction). 2. some nephrotoxic agents such as a heavy metal or ethylene glycol (antifreeze). 3. organ rejection following kidney transplant (their presence after the third post-operative day of a renal transplant is one of the most reliable indicators of rejection). Mixed Cell Casts A variety of cells may be found in the urinary filtrate. Therefore, it is not uncommon to find casts which have more than one type of cell. These casts are called mixed cell casts and can be any combination of leukocytes, erythrocytes, or renal tubular epithelial cells. Page 70 CHEM 110 – Clinical Chemistry 1 – LO3 Granular Casts Granular casts are former cellular casts in which the cells have disintegrated into granular debris. Urinary stasis allows the cells within cellular casts to disintegrate while remaining in the tubules. Coarse granular casts which have resulted from cellular casts where the cells have disintegrated and are no longer clearly visible within the matrix of the cast. The granules are larger and usually quite dark. Fine granular casts occur when a granular or coarse granular cast has been in the tubule for prolonged periods of time. Fine granular casts are often similar in texture to waxy casts and their clinical significance is similar to waxy casts. A fine granular cast is almost a waxy cast. Hyaline casts can sometimes take on a granular texture due to the entrapment of lysosomal material and debris which can become trapped inside the matrix of the cast. Debris also sticks to the outside of the cast which can give it a slightly granular appearance. These casts should not be called granular or fine granular casts because their significance is very different from a true granular or fine granular cast which results from the breakdown of cellular casts. Waxy Casts When urinary stasis is prolonged, a granular cast can further disintegrate, and the cast becomes waxy. Waxy casts represent an advanced stage of other casts. Waxy casts are brittle and highly refractile. They have an opaque, paraffin-like appearance, and typically have cracked or notched outlines. Numerous waxy casts indicate a long renal transit time and always indicate renal disease. CHEM 110 - Clinical Chemistry 1 – LO3 Page 71 Fatty Casts Fat can occur in urine in 3 forms: as free fat, oval fat bodies and fatty casts. Free fat is globular, yellow brown in colour, and tends to glisten due to its high refractive index. Oval fat bodies are RTE’s in which fat has accumulated. The presence of oval fat bodies in urine sediment suggests damage to the renal tubules. Both free fat and oval fat bodies may be incorporated into fatty casts. These casts may contain a few fat droplets in a hyaline matrix or may be filled with fat globules of different sizes. Fat is best recognized by using polarized microscopy which gives lipid globules a distinctive Maltese cross appearance. Chemical testing for lipids or staining with Sudan III or Oil Red O can be used to confirm chyluria, although not all fat will take up the stain. Chyluria (in the form of free fat, oval fat bodies, or fatty casts) accompanied by massive proteinuria is most commonly associated with the nephrotic syndrome. Page 72 CHEM 110 – Clinical Chemistry 1 – LO3 Miscellaneous Elements 1. Bacteria are often seen in urine sediment and cannot be considered significant unless proper sterile collection procedure is used. Many bacteria in the absence of pyuria suggests contamination, or a urine which has been allowed to stand at room temperature for longer than two hours. At Saskatchewan Polytechnic, if bacteria is observed during the microscopic, it is reported as present. 2. Mucus threads are faint, long ribbon-like strands with tapered ends. They are not clinically significant. At Saskatchewan Polytechnic, if mucus is observed during the microscopic, it is reported as present. 3. Trichomonas vaginalis is a parasite that causes a sexually transmitted disease called trichomoniasis. In females, the parasite is harboured in the vagina where it can produce itching, burning, and vaginal discharge. It can be seen in urine specimens due to vaginal contamination as the urine is voided. Although it can be seen in male urines, it is more commonly observed in female urine. The parasite is about the same size as a leukocyte. It must be recognized based on its jerky motility to report as present. It is usually accompanied by many squamous epithelial cells and leukocytes. 4. Spermatozoa may be present in the urine of men after seizures, nocturnal emissions, diseases of the genital organs and in spermatorrhea. It may be found in both male and female urine after intercourse. Sperm should be reported as present. CHEM 110 - Clinical Chemistry 1 – LO3 Page 73 5. Yeast may be normal flora or a significant pathogen, particularly in a diabetic or patients whose immune system is depressed. Most often the yeast is a vaginal contaminant accompanied by numerous squamous epithelial cells. Yeast cells are similar in size to red blood cells, oval, and are most easily recognized by characteristic “buds”. In severe infections yeast may appear as branched pseudohyphae. Yeast are reported as present. 6. Artifacts: a variety of foreign objects can find their way into the urine specimen during collection, transportation, or during testing. These extraneous structures should be recognized as being foreign, but they are not reported. Some artifacts that may be found are: Starch can vary in shape but is highly retractile. The most common type of starch found in urine is corn starch as it is used in body powders. Fibres are commonly found in urine from clothing, diapers, and toilet paper. Fibres from food can also be found in the urine because of fecal contamination. Page 74 CHEM 110 – Clinical Chemistry 1 – LO3