SCBM343- Urinalysis Associate Professor Dr. Wannee Jiraungkoorskul Department of Pathobiology, Faculty of Science, Mahidol University Tel: 02-201-5563, E-mail: wannee.jir@mahidol.ac.th 1
OBJECTIVES 1. Discuss the properly collected urine specimen 2. Discuss the physical, chemical and microscopic urine analysis 3. List the cause of abnormal results 4. Identify and discuss the formation and clinical significance of casts and crystals 5. Discuss the reference values in normal urine 2
COMPOSITION OF URINE 95% water and 5% dissolved substances Organic substances Urea Uric acid Creatinine Inorganic substances Sodium Ammonium Potassium 3
URINE CONTAINER 4
METHODS OF URINE COLLECTION Random collection taken at any time of day with no precautions regarding contamination. The sample may be dilute, isotonic, or hypertonic and may contain white cells, bacteria, and squamous epithelium as contaminants. In female, the specimen may contain vaginal contaminants such as trichomonads, yeast, and red cells during menses. 5
METHODS OF URINE COLLECTION Early morning collection before ingestion of any fluid This is usually hypertonic and reflects the ability of the kidney to concentrate urine during dehydration which occurs overnight. If all fluid ingestion has been avoided since 6 p.m. the previous day, the specific gravity usually exceeds 1.022 in healthy individuals. 6
METHODS OF URINE COLLECTION 24 hour collection save all the urine pass in a 24-hour period and measure the amount of certain chemicals that kidneys clean from the body. Some chemicals are cleaned in different amounts during the day and night. With the 24-hour urine the chemical can be measured correctly. Another reason to collect urine for 24 hours may be to see if too little or too much urine is produced. 7
THE ROUTINE URINALYSIS 1. Physical properties 2. Chemical properties 3. Microscopic examination 8
1. Volume 2. Color 3. Transparency 4. Odor 5. Specific gravity VOLUME Normal = 1200-1500 ml in adult Polyuria = over 2000 ml/24hr. Oliguria = less than 500 ml/24hr. Anuria = complete absence of urine formation Nocturia = excretion of urine at night 9
COLOR Normal color of urine is yellow caused by a pigment (Urochrome) Intensity of color varies with the concentration Other colors may be due to variations of diet, medication or disease 10
COLOR Abnormal color: Dark yellow / amber Very concentrated and low volume, fever, dehydration White Pus Brown Bilirubin or yellow foam, may become green Black Melanin, homogentisic acid Bright orange certain azo dyes Clear red Hemoglobinuria Cloudy red Hematuria Dark red brown Myoglobin Blue / green Bacterial infections Other colors Caused by drugs 11
TRANSPARENCY Fresh urine is usually clear Cloudiness may be caused by several factors Amorphous urate or phosphate crystals Mucous threads White blood cells Red blood cells Epithelial cells Bacteria Crystals Casts 12
ODOR Normal faintly aromatic odor Characteristic odor caused by volatile acids Acetone fruity odor, diabetes Ammonia bacteria Amino acid inborn errors of metabolism Asparagus Stink bean (Parkia speciosa Hassk) 13
SPECIFIC GRAVITY Normal 1.005-1.030 (Indicates the weight of the urine as compared to the weight of an equal volume of water) First morning urine or dehydration will be the most concentrated urine Measured by 1. Reagent strip 2. Refractometer 14
CHEMICAL EXAMINATION A plastic strip with pads containing chemical reactants that will be discarded after one use. Test within one hour of collection or refrigerate urine. Mix urine well and compare to color chart after the exact time required for each test. Color charts provided by the manufacturer. Read manually or by automated instruments. 15
http://loudoun.nvcc.edu/vetonline/vet131/ Pictures/Strips.jpg
CHEMICAL EXAMINATION Reference range: Glucose: negative Bilirubin: negative Specific Gravity: ph: Infant: 1.002-1.006 newborn/neonate: 5-7 Adult: 1.001-1.030 Adult: 5-8 Ketone: negative Blood: negative Protein: negative Urobilinogen: 0.2 1 E.U. Nitrite: negative Leukocytes: negative 17
Methodology MICROSCOPIC EXAMINATION 10-15 ml urine is centrifuged 2,000-3,000 rpm for 5-10 minutes. Supernatant is decanted and a volume of 0.2 to 0.5 ml is left inside the tube. Sediment is resuspended in the remaining supernatant by flicking the bottom of the tube several times. Drop of resuspended sediment is poured onto a glass slide and cover-slipped. 18
MICROSCOPIC EXAMINATION Examination Low power - identify crystals, casts, squamous cells, and other large objects reported number per LPF. Since the number of elements found in each field may vary considerably from one field to another, several fields are averaged. High power - identify crystals, cells, and bacteria reported number per HPF 19
MICROSCOPIC EXAMINATION Red Blood Cells Hematuria is the presence of abnormal numbers of red cells in urine due to: glomerular damage, tumors, renal infarcts, acute tubular necrosis, upper and lower urinary tract infections. May contaminate from the vagina in menstruating women or from trauma produced by bladder catherization. 20
MICROSCOPIC EXAMINATION Note the irregular outlines of many of these abnormal RBC's are dysmorphic RBC's. 21
RBC Fat droplets RBC are more uniform and red-tinged Fat droplets are more variable in shape, slightly greenish-tinged (or refractile), have a darker edge, are more globular shape (this can be visualized when you focus up and down) and usually float to the top of the coverslip (thus when fat droplets are in focus, the other urine constituents are out of focus) http://ahdc.vet.cornell.edu/clinpath/modules/ua-sed/cells.htm 22
MICROSCOPIC EXAMINATION White Blood Cells 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, usually, are granulocytes. Wbc from the vagina, or the external urethral meatus in men and women may contaminate the urine. 23
Pyuria = presence of WBC Infection in either the upper or lower urinary tract or with acute glomerulonephritis Wbc from the vagina, or the external urethral meatus in men and women may contaminate the urine. 24
Epithelial Cells MICROSCOPIC EXAMINATION Squamous occur principally in the urethra & vagina and are of little diagnostic value Transitional (bladder) usually 2-4 x larger than a WBC. May be round or pear-shaped. Arise in the renal pelvis, ureters, bladder or urethra. Renal tubular slightly larger than a WBC. Appear flat, cuboidal, or columnar. Presence suggests tubular damage (tubular necrosis, transplant rejection, pyelonephritis) 25
Large polygonal squamous epithelial cells with small nuclei Transitional epithelial cells : Variable size and shape, round or polygonal, pear-shaped, tailed, spindle, may develop refractile, fatty inclusions with storage Renal epithelial cells : oval shape, single large nucleus that is eccentrically located 26
URINARY CAST MORPHOLOGY A urinary cast is congealed protein or cellular debris, that forms within a renal tubule. The material or cells that form a cast may be Damaged glomerulus Part of an interstitial inflammatory infiltrate Dead tubular cells 27
URINARY CAST MORPHOLOGY The cast is expelled into the urine, and maintains the shape of the tubule in which it formed. Urinary casts are formed only in the distal convoluted tubule or the collecting duct. 28
URINARY CAST MORPHOLOGY 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, later a finely granular cast, and ultimately, a waxy cast. http://ahdc.vet.cornell.edu/clinpath/modules/uarout/images/cast%20formation.gif 29
URINARY CAST MORPHOLOGY The factors which favor protein cast formation are low flow rate, high salt concentration, low ph, and all of which favor protein denaturation and precipitation Protein casts with long, thin tails formed at the junction of Henle's loop and the distal convoluted tubule are called cylindroids. Hyaline casts can be seen even in healthy patients. 30
Hyaline cast - notice the parallel sides, tubular shape with rounded edge, and colorless appearance http://www.udel.edu/mls/mclane/cast.jpg 31
Red blood cell casts are indicative of glomerulonephritis, with leakage of RBC's from glomeruli, or severe tubular damage. http://www.tabers.com/tabersonline/ub?cmd=repview&t ype=tabers_21&name=c190p0 White blood cell casts are most typical for acute pyelonephritis, glomerulonephritis. http://www.meddean.luc.edu/lumen/meded/pathology/images/renal23.jpg 32
Granular casts, with a roughly rectangular shape. Broad, waxy cast. Note that the edges are sharp and there are "cracks" in the cast. Renal tubular cell cast suggests injury to the tubular epithelium. http://www.labdd.com/uploads/allimg/100601/13131m4l-5.jpg 33
http://ahdc.vet.cornell.edu/clinpath/modules/ua-sed/images/cast_compilation1.jpg 34
Bacteria 1. Normal microbial flora of the vagina or external urethral meatus 2. Ability to rapidly multiply in urine standing at room temperature http://ahdc.vet.cornell.edu/clinpath/modules/ua-sed/images/wbc_bacilli_labeled.jpg 35
Yeast Yeast cells may be contaminants or a true yeast infection. They are often difficult to distinguish from red cells and amorphous crystals but are distinguished by their tendency to bud. Most often they are Candida, which may colonize bladder, urethra, or vagina. 36
MICROSCOPIC EXAMINATION Acid urine crystals (usually non-pathologic) Uric acid crystals may be seen normally 16% of patients with gout Calcium oxalate crystals oxalate high in dark green vegetables, tomatoes carbonated drinks, tea, chocolate Amorphous urates Hippuric acid crystals after eating fruits & vegetables that contain benzoic acid 37
http://www.marvistavet.com/assets/images/oxalate_crystals.gif Oxalate crystals, which look like little envelopes Uric acid crystals, hexagonal plate Amorphous urates 38
Alkaline urine crystals (usually non-pathologic): Triple phosphate MICROSCOPIC EXAMINATION Calcium carbonate Calcium phosphate Ammonium biurate Amorphous phosphates "triple phosphate" crystals look like rectangles, or coffin lids. http://www.utmb.edu/poc/images/ppmimages/ppm_2008/ua/ua_2008_03.jpg 39
Calcium phosphate crystals are long prism with one sharped end. They are found with triple phosphates. Amorphous phosphates
Pathologic crystals Cystine crystals: congenital metabolic disorder Cholesterol crystals: renal disease, nephrotic syndrome, and other conditions that form or deposit lipids in the kidney Sulfonamides (Sulfa): may form calculi and cause renal tubule damage Sulfa crystals Amoxycillin crystals 41
http://meded.ucsd.edu/isp/1994/im-quiz/images/cystine.jpg Pathologic crystals Cystine crystals are shaped like stop signs. Cholesterol crystals are flat, rectangular plates with notched corners 42
Miscellaneous Spermatozoa can sometimes be seen. Rarely, pinworm ova may contaminate the urine. In Egypt, ova from bladder infestations with schistosomiasis may be seen. Mucous 43
Fat droplets Trichomonas Talcum powder or Granular starch in gloves Cotton fiber 44
MICROSCOPIC EXAMINATION Microscopic reference ranges Red cells: 0-3/HPF (high power field) White cells: 0-5/HPF Squamous epithelial cells: present Bacteria for a routine specimen: < 10/HPF Bacteria for a catheterized specimen: absent Hyaline casts: 0-3/LPF Crystals: small of non-pathogenic crystals 45
References Textbook of Urinalysis and Body Fluids: A Clinical Approach. by Landy McBride 1998 Graff's Textbook of Urinalysis and Body Fluids. 2 nd ed. by Lillian Mundt Kristy Shanahan 2010 Urinalysis and Body Fluids by Susan King Strasinger, Marjorie Schaub Di Lorenzo 2008
SCBM343 Urinalysis 47