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King Saud University College of Science Department of Biochemistry Disclaimer • The texts, tables, figures and images contained in this course presentation (BCH 376) are not my own, they can be found on: • References supplied • Atlases or • The web Chapter 6 Urinalysis-2 Professor A. S. Alhomida 1 Examination of Urine Sediment Macroscopic Screening • Abnormalities in the physical and chemical portion of urinalysis play a particular role in the decision to perform a microscopic examination • Parameters considered significant vary among Lab but include macroscopic screening 2 Examination of Urine Sediment, Cont’d Correlation of Results • Microscopic results should be correlated with the physical and chemical findings • Results of Specimen don’t correclate must be rechecked for both technical and clerical errors 3 Examination of Urine Sediment, Cont’d General Aspects 1. Preservation • Cells and casts begin to disintegrate in 1 - 3 hr at room temperature • Refrigeration for up to 48 h (little loss of cells) 2. Specimen Concentration • Ten to twenty-fold concentration by centrifugation 4 Examination of Urine Sediment, Cont’d General Aspects 3. Types of Microscopy • • • Phase contrast microscopy Polarized microscopy Bright field microscopy with special staining (e.g., Sternheimer-Malbin stain) 5 Examination of Urine Sediment, Cont’d Microscopic Techniques 6 Examination of Urine Sediment, Cont’d Procedure 1. A sample of well-mixed urine (usually 10-15 mL) is centrifuged in a test tube at relatively low speed (about 2-3,000 rpm) for 5-10 minutes until a moderately cohesive button is produced at the bottom of the tube 2. The supernate is decanted and a volume of 0.5 to 1.0 mL is left inside the tube 3. The sediment is resuspended in the remaining supernate by flicking the bottom of the tube several times 4. A drop of resuspended sediment is poured onto a glass slide and coverslipped 7 Preparing the Sediment for Analysis • Tubes, pipets and slides used for standardizing urine sediments 8 Preparing the Sediment for Analysis • Mix the urine well and pour 12 milliliters of urine in a clean centrifuge tube • Cap the tube and centrifuge the urine for five minutes at 450 g. RPMs • At this speed the sediment will be optimally concentrated without destroying any of the fragile elements in the urine 9 Preparing the Sediment for Analysis, Cont’d • After centrifugation, remove the supernatant fluid with a polyethylene, transfer pipet leaving 0.5 to 1.0 mL urine on the sediment • Resuspend the sediment in the remaining urine by flicking the bottom of the tube or by running the tube across a test tube rack 10 Preparing the Sediment for Analysis, Cont’d 11 Preparing the Sediment for Analysis, Cont’d 12 Preparing the Sediment for Analysis, Cont’d • Using a plastic or polypropylene transfer pipet, mount a drop of the urine in the counting chamber of the prepared commercial plastic slides • Avoid using glass pipets when mounting the urine sediment as elements like casts tend to cling to the glass surface 13 Preparing the Sediment for Analysis, Cont’d • Exam the sediment using the microscope • After counting the elements in the sediment, you may calculate the number of elements per mL instead of lpf (low- or hight power field or hpf), to provid standardization among the various techniques 14 Preparing the Sediment for Analysis, Cont’d 1. Calculate the area of an lpf or hpf using the diameter: 2 2. Area = pr 3. For example: 4. r for hpf 0.35 m= 0.175 m 2 2 5. Area = 3.14 X (0.175) = 0.096 mm 15 Preparing the Sediment for Analysis, Cont’d 6. Calculate the maximum number of lpf or hpf in the viewing area under area 22 X 22 mm for 2 cover slip = 484 m 7. hpf = 484 / 0.096 = 5040 8. Convert hpf into per mL 9. 5040/ 0.02 mL X 12 = 21,000 hpf/mL • where is 0.02 the volume of sediment and 12 is concentration factor 16 Preparing the Sediment for Analysis, Cont’d • Special stains can be used to enhance the elements in the sediment • Sternheimer-Malbin stain, tolui Papanicolau • Wright’s • Immunoperoxidase • Immunofluorescence dine blue • They will enhance the internal structures of the cells making them easier to identify 17 Preparing the Sediment for Analysis, Cont’d 18 Preparing the Sediment for Analysis, Cont’d 19 Examination of Urine Sediment, Cont’d At Low Power 1. To identify most crystals, casts, squamous cells, and other large objects 2. The numbers of casts seen are usually reported as number of each type found per low power field (LPF) 3. Example: 5-10 hyaline casts/L casts/LPF 4. Since the number of elements found in each field may vary considerably from one field to another, several fields are averaged 20 Examination of Urine Sediment, Cont’d At High Power 1. To identify crystals, cells, and bacteria 2. The various types of cells are usually described as the number of each type found per average high power field (HPF). Example: 1-5 WBC/HPF 21 Examination of Urine Sediment, Cont’d Abnormal Findings 1. Per High Power Field (HPF) (400x) • • • • > > > > 3 erythrocytes 5 leukocytes 2 renal tubular cells 10 bacteria 22 Examination of Urine Sediment, Cont’d Abnormal Findings 2. Per Low Power Field (LPF) (200x) • • • > 3 hyaline casts or > 1 granular cast > 10 squamous cells (indicative of contaminated specimen) Any other cast (RBCs, WBCs) 23 Examination of Urine Sediment, Cont’d Abnormal Findings 3. Presence of: • • • Fungal hyphae or yeast, parasite, viral inclusions Pathological crystals (leucine, tyrosine, cystine) Large number of uric acid or calcium oxalate crystals 24 Components of Urine Sediment Organized Components 1. 2. 3. 4. 5. 6. 7. 8. Red blood cells White blood cells Epithelial cells Casts Bacteria Yeast Parasites Spermatozoa 25 Components of Urine Sediment, Cont’d Unorganized Components 1. Crystals • Mostly insignificant • Exceptions • Cystine • Uric acid • Sulfa drug crystals 2. Chemical elements 26 Examination of Urine Sediment, Cont’d Cells 1. Erythrocytes • 2. 3. • • • • • • “Dysmorphic” vs. “normal” Leukocytes Neutrophils (glitter cells) Eosinophils Epithelial Cells Renal tubular epithelial cells Oval fat bodies Transitional epithelial cells Squamous cells (> 10 per HPF) More than 1 per 3 HPF Hansel test (special stain) Few are normal Abnormal, indicate Nephrosis Few are normal Indicate level of contamination 27 Examination of Urine Sediment, Cont’d Cells 1. Erythrocytes (GN, stone, tumor, exogenous) 2. Renal tubular epithelial cells (many renal diseases) • Oval Fat bodies (Fat means nephrotic syndrome) 3. PMNs (Infection or sterile inflammation) 4. Eosinophils (Some allergic interstitial nephritis) 5. Squamous cells (CONTAMINATION) 28 Examination of Urine Sediment, Cont’d Red Blood Cells 1. Hematuria is the presence of abnormal numbers of red cells in urine due to: glomerular damage, tumors which erode the urinary tract anywhere along its length, kidney trauma, urinary tract stones, renal infarcts, acute tubular necrosis, upper and lower uri urinary tract infections, nephrotoxins, and physical stress 2. Red cells may also contaminate the urine from the vagina in menstruating women or from trauma produced by bladder catherization 3. Theoretically, no red cells should be found, but some find their way into the urine even in very healthy individuals 29 Examination of Urine Sediment, Cont’d Red Blood Cells 4. 5. 6. If one or more red cells can be found in every high power field, and if contamination can be ruled out, the specimen is probably abnormal RBC's may appear normally shaped, swollen by dilute urine (in fact, only cell ghosts and free hemoglobin may remain), or crenated by concentrated urine Both swollen, partly hemolyzed RBC's and crenated RBC's are sometimes difficult to distinguish from WBC's in the urine 30 Examination of Urine Sediment, Cont’d Red Blood Cells, Cont’d 7. In addition, red cell ghosts may simulate yeast. The presence of dysmorphic RBC's in urine suggests a glomerular disease such as a glomerulonephritis 8. Dysmorphic RBC's have odd shapes as a consequence of being distorted via passage through the abnormal glomerular structure 9. Dysmorphic RBCs Irregular shape, size, density Indicate glomerular bleeding 31 Examination of Urine Sediment, Cont’d RBCs • There are many RBCs in this field • Note the center pallor of most of the cells especially the one labeled with a "B" • Can you picture the donut shape or biconcave disc form of the cell? • “A" points to a red cell on its side 32 Examination of Urine Sediment, Cont’d RBCs • This is another field of RBCs • Note that some of them look granular • That is because they are crenated or puckered • The spicules make the cell look granular 33 Examination of Urine Sediment, Cont’d RBCs • Note the irregular outlines of many of these RBC's, compared to two relatively normal RBC's at the center left of the right panel. These abnormal RBC's are dysmorphic RBC's. • Dysmorphic RBC's have odd shapes as a consequence of being distorted via passage through the abnormal glomerular structure 34 Examination of Urine Sediment, Cont’d Summary of RBC 35 Examination of Urine Sediment, Cont’d WBCs 1. Pyuria refers to the presence of abnormal numbers of leukocytes that may appear with infection in either the upper or lower urinary tract or with acute glomerulonephritis 2. Usually, the WBC's are granulocytes 3. White cells from the vagina, especially in the presence of vaginal and cervical infections, or the external urethral meatus in men and women may contaminate the urine 36 Examination of Urine Sediment, Cont’d WBCs 4. If two or more leukocytes per each high power field appear in non-contaminated urine, the specimen is probably abnormal 5. Leukocytes have lobed nuclei and granular cytoplasm 37 Examination of Urine Sediment, Cont’d WBCs • WBC in urine are most neutrophils • Like erythrocytes, WBC may lyse in very dilute or highly alkaline urine; WBC cytoplasmic granules released into the urine often resemble cocci bacteria 38 Examination of Urine Sediment, Cont’d WBCs • In this field, "C" points to a white cell and "B" points to two transitional epithelial cells • Note that you can distinguish a nucleus in the white cell if you look very closely • Also note the granular appearance 39 Examination of Urine Sediment, Cont’d Summary of WBCs 40 Examination of Urine Sediment, Cont’d Renal Tubular Cells 1. Usually larger than granulocytes, contain a large round or oval nucleus and normally slough into the urine in small numbers 2. In nephrotic syndrome and in conditions leading to tubular degeneration, the number sloughed is increased 3. When lipiduria occurs, these cells contain endogenous fats. When filled with numerous fat droplets, such cells are called oval fat bodies 4. Oval fat bodies exhibit a "Maltese cross" configuration by polarized light microscopy 41 Examination of Urine Sediment, Cont’d Squamous Cells • This image provides a good example of two unstained squamous epithelial cells • Note the size of the cell, its nucleus and its irregular shape • These two cells look somewhat granular which is an artifact of the urine preservation 42 Examination of Urine Sediment, Cont’d Renal Tubular Cells • This frame compares a WBC ("B") and the renal tubular cells ("A") • Note the size comparison • Also note how prominent the nucleus is in the renal tubular cells • It is much easier to see than the nucleus of the WBC. 43 Examination of Urine Sediment, Cont’d Transitional Cells • They originate from the renal pelvis, ureter, or bladder • It shows a transitional epithelial cell at "B" • The three cells just above this one are also transitional epithelials • "A" points to a squamous epithelial cell 44 Examination of Urine Sediment, Cont’d Oval Fat Body • Oval fat bodies consist of degenerated tubular cells containing abundant lipid, which appears refractile • Indicate heavy proteinuria 45 Examination of Urine Sediment, Cont’d Oval Fat Body • Under polarized light, oval fat bodies demonstrate the "Maltese cross" appearance 46 Examination of Urine Sediment, Cont’d LE Cell • Positive leukocyte esterase test results from the presence of white blood cells either as whole cells or as lysed cells. • Pyuria can be detected even if the urine sample contains damaged or lysed WBC's. • A negative leukocyte esterase test means that an infection is unlikely and that, without additional evidence of urinary tract infection, microscopic exam and/or urine culture need not be done to rule out significant bacteriuria 47 Examination of Urine Sediment, Summary of Epithelial Cells Cont’d 48 Examination of Urine Sediment, Cont’d Bacteria and Yeasts 1. Bacteria • Bacteriuria 2. Yeasts • Candidiasis 3. Viruses • CMV inclusions More than 10 per HPF Most likely a contaminant but should correlate with clinical picture Probable viral cystitis. 49 Examination of Urine Sediment, Cont’d Bacteria 1. They are common in urine specimens because of the abundant normal microbial flora of the vagina or external urethral meatus and because of their ability to rapidly multiply in urine standing at room temperature 2. Diagnosis of bacteriuria in a case of suspected urinary tract infection requires culture 3. A colony count may also be done to see if significant numbers of bacteria are present 50 Examination of Urine Sediment, Cont’d Bacteria 4. More than 100,000/ml of one organism reflects significant bacteriuria 5. Multiple organisms reflect contamination. However, the presence of any organism in catheterized or suprapubic tap specimens should be considered significant 51 Examination of Urine Sediment, Cont’d Bacteria • This is an excellent example of a mixed field of red blood cells and bacteria • "C" is pointing to the bacteria • Note how small they are compared to the RBC 52 Examination of Urine Sediment, Cont’d Yeasts 1. Yeast cells may be contaminants or represent a true yeast infection 2. They are often difficult to distinguish from red cells and amorphous crystals but are distinguished by their tendency to bud 3. Most often they are Candida, which may colonize bladder, urethra, or vagina 53 Examination of Urine Sediment, Cont’d Yeasts • This frame shows an excellent example of yeast ("B") • Note the budding and the more oval appearance of each cell • "A" is a crenated red blood cell • "C" is also a red cell 54 Examination of Urine Sediment, Trichomonas Parasite Cont’d • Trichomonas vaginalis is the most common parasite in urine in USA and is found in approximately 25% of women in casual vaginal cervical examination. • The presence of this organism in urine specimens of females is due to the contamination of the urine with vaginal secretions 55 Examination of Urine Sediment, Cont’d Cytomegalovirus • CMV is a common virus that infects people of all ages and once CMV is in a person’s body, it stays there for life • Most infections with CMV are “silent,” no signs or symptoms • It is rarely causes serious consequences except in people with suppressed or impaired immune systems • It is found in saliva, urine, and other bodily fluids • Because it is often found in semen as well as in cervical secretions, the virus can be spread by sexual contact 56 Examination of Urine Sediment, Cont’d 57 Significance of Cellular Casts Erythrocyte Casts Leukocyte Casts Bacterial Casts Individual Erythrocytes Individual Leukocytes Individual Bacteria 58 Renal Tubular Cast Formation 59 Renal Tubular Cast Formation, Cont’d 60 Examination of Urine Sediment, Cont’d Casts, Cont’d 1. 2. 3. 4. Casts are cylinders of Tamm-Horsfall protein that solidified in distal tubules Cellular casts most commonly result when disease processes such as ischemia, infarction or nephrotoxicity cause degeneration and necrosis of tubular epithelial cells A common scenario is the patient with decreased renal perfusion and oliguria secondary to severe dehydration Ischemic injury results in degeneration and sloughing of the epithelial cells 61 Examination of Urine Sediment, Cont’d Casts, Cont’d 5. 6. 7. 8. The resulting casts often are prominent in urine produced following rehydration with fluid therapy The restoration of urine flow flushes numerous casts out of the tubules Leukocytes can also be incorporated into casts in cases of tubulo-interstitial inflammation (eg., pyelonephritis) Red Cell - Proliferative GN or GBM defect 62 Examination of Urine Sediment, Cont’d Casts, Cont’d 9. Urinary casts are formed only in the distal convoluted tubule (DCT) or the collecting duct (distal nephron) 10. WBC - Intrarenal infection or inflammation 11. The proximal convoluted tubule (PCT) and loop of Henle are not locations for cast formation 12. Hyaline casts are composed primarily of a mucoprotein (Tamm-Horsfall protein) secreted by tubule cells 63 Examination of Urine Sediment, Cont’d Casts, Cont’d 13. Even with glomerular injury causing increased glomerular permeability to plasma proteins with resulting proteinuria, most matrix or "glue" that cements urinary casts together is Tamm-Horsfall mucoprotein 14. The Tamm-Horsfall protein secretion (green dots) is illustrated in the diagram below, forming a hyaline cast in the collecting duct 15. Granular - Many types of renal disease 64 Examination of Urine Sediment, Cont’d Casts, Cont’d 16. Although albumin and some globulins are also incorporated 17. An example of glomerular inflammation with leakage of RBC's to produce a red blood cell cast is shown in the diagram below 18. Hyaline - may be normal unless numerous 19. Waxy - Chronic renal disease 65 Factors Favoring Protein Cast Formation 1. 2. 3. 4. Low flow rate High salt concentration Low pH All of which favor protein denaturation and precipitation, particularly that of the Tamm-Horsfall protein 5. Protein casts with long, thin tails formed at the junction of Henle's loop and the distal convoluted tubule are called cylindroids 6. Hyaline casts can be seen even in healthy patients 66 Examination of Urine Sediment, Cont’d Casts, Cont’d Tamm-Horsfall protein Red blood cell cast 67 Examination of Urine Sediment, RBCs Cast - Histology Cont’d • The presence of this red blood cell cast on urine microscopic analysis suggests a glomerular or renal tubular injury 68 Examination of Urine Sediment, RBCs Cast - Histology Cont’d • This histologic section at medium power with trichrome stain highlights red blood cells grouping together in tubules to form casts • The tubular epithelium is also damaged, with a foamy appearance, and is the basis for the appearance of oval fat bodies in urine in this case 69 Examination of Urine Sediment, RBCs Cast - Histology Cont’d • Red blood cells may stick together and form red blood cell casts • Such casts are indicative of glomerulonephritis, with leakage of RBC's from glomeruli, or severe tubular damage 70 Examination of Urine Sediment, Cont’d WBCs Cast • White blood cell casts are most typical for acute pyelonephritis, but they may also be present with glomerulonephritis • Their presence indicates inflammation of the kidney, because such casts will not form except in the kidney 71 Examination of Urine Sediment, Cont’d Tubular Epith. Cast • Renal Tubular Cells are originally cubic in shape; but once exfoliated, they adopt a rounded shape • These cells are slightly larger than leukocytes (10-14 um) with lightly granular cytoplasm • The nucleus is round, well defined and usually centric. • The cytoplasm often shows a perinuclear halo when stained. Note the "glitter" cell (fatty degenerated WBC) in the lower-left corner 72 Examination of Urine Sediment, Tubular Epith. Cast Cont’d • This renal tubular cell cast suggests injury to the tubular epithelium 73 Examination of Urine Sediment, Cont’d Granular Cast • When cellular casts remain in the nephron for some time before they are flushed into the bladder urine, the cells may degenerate to become a coarsely granular cast • Casts which persist may break down, so that the cells forming it are degenerated into granular debris 74 Examination of Urine Sediment, Cont’d Granular and waxy Cast • This slide shows waxy cast and granular casts • Granular casts have a textured appearance which ranges from fine to coarse • Since they usually form as a stage in the degeneration of cellular casts, the interpretation is similar to that for cellular casts 75 Examination of Urine Sediment, Cont’d Waxy Cast • They have a smooth consistency but are more refractile and easier to see compared to hyaline casts • They commonly have squared off ends, as if brittle and easily broken • They are found especially in chronic renal diseases • Diabetic nephropathy • Malignant hypertension • Glomerulonephritis 76 Examination of Urine Sediment, Cont’d Hyaline Cast • Hyaline casts, which appear very pale and slightly refractile, are common findings in urine • Greater numbers of hyaline casts may be seen associated with proteinuria of renal glomerular disease or extrarenal (overflow proteinuria as in myeloma) 77 Examination of Urine Sediment, Cont’d Fatty Cast • They are identified by the presence of refractile lipid droplets • The background matrix of the cast may be hyaline or granular • Interpretation of the significance of fatty casts should be based on the character of the cast matrix, rather than on the lipid content 78 Examination of Urine Sediment, Cont’d Summary of Urine Casts 79 Examination of Urine Sediment, Cont’d Summary of Urine Casts 80 Examination of Urine Sediment, Cont’d Crystals 1. Urate • • Ammonium biurate Uric acid 2. Triple Phosphate 3. Calcium Oxalate 4. Amino Acids • • • Leucine Cystine Tyrosine 5. Sulfonamide 81 Examination of Urine Sediment, Cont’d Dihydrated Calcium Oxalate Crystals • These are oxalate crystals • They look like little envelopes (or tetrahedrons, depending upon your point of view) • Oxalate crystals are common 82 Examination of Urine Sediment, Cont’d Monohydrated Calcium Oxalate Crystals • Rarely found, vary in size and may have a spindle, oval, or dumbbell shape • Appear as flat, elongated, six-sided crystals ("fence pickets") • The arrow indicates "daughter" crystal forming on the face of a larger underlying crystal 83 Examination of Urine Sediment, Cont’d Triple Phosphate Crystals • Usually appear as colorless, prism-like "coffin lids" • Often seen in urine from normal individuals. • Can be found in urine of any pH, their formation is favored in neutral to alkaline urine • Urinary tract infection with urease producing bacteria can promote it (and urolithiasis) by raising urine pH and increasing free ammonia 84 Examination of Urine Sediment, Cont’d Urate Crystals • Uric acid crystals may appear as yellow to brown rhombic or hexagonal plates, needles or rosettes • With rare exceptions, the finding of uric acid crystals in urine is of little clinical value 85 Examination of Urine Sediment, Cont’d Leucine Crystals • Leucine crystals are seen as yellow spheres with concentric and radial strias • These crystals can sometimes be mistaken for cells, with the center resembling a nucleus 86 Examination of Urine Sediment, Cont’d Cystine Crystals • These cystine crystals are shaped like stop signs • Cystine crystals are seen as flat colorless hexagonal plates • They often aggregate in layers, and their formation is favored in acidic urine 87 Examination of Urine Sediment, Cont’d Ammonium Biurate Crystals • These crystals generally appear as yellow-brown, radially-striated spheres with irregular "thornapple" or "ox-horn" projections • They may be seen in acid urine, their formation is favored in neutral to alkaline urine 88 Examination of Urine Sediment, Cont’d Cholesterol Crystals • The cholesterol crystals appear to be needleshaped • They are actually rectangles in crosssection • The surrounding tissue is all necrotic (dead) debris 89 Examination of Urine Sediment, Cont’d Sulfonamide Crystals • Sulfonamide crystals are typically yellow in color and often resemble uric acid crystals. However, sulfa crystals are easily distinguished from uric acid by confirmatory tests • Sulfa crystals are readily soluble in acetone and exhibit a positive dextrine/sulfuric acid test ("old yellow newspaper" test) 90 Examination of Urine Sediment, Cont’d 91 Examination of Urine Sediment, Cont’d 92 THE END Any questions? 93