Microscopic Examination of Urine - Introduction PDF

Microscopic Examination of Urine - Introduction URINALYSIS & BODY FLUIDS by STRASINGER, S. & DI LORENZO, M. 7TH ED. - CHAPTER 7  Microscopic Examination After a chemical examination of the urine has been completed, you will determine whether a microscopic examination is needed. M...

Microscopic Examination of Urine - Introduction URINALYSIS & BODY FLUIDS by STRASINGER, S. & DI LORENZO, M. 7TH ED. - CHAPTER 7  Microscopic Examination After a chemical examination of the urine has been completed, you will determine whether a microscopic examination is needed. Most labs will not do a microscopic on every urine. Typically, a microscopic examination is done if one or more of the following tests were positive: Blood (RBCs) Leukocyte esterase (WBCs) Protein Nitrite (Bacteria)  Microscopic Examination 1) Pour urine in conical tube, cover with cap 2) Centrifuge at speed noted in your lab manual 3) Decant over dirty sink: Uncap, flip urine tube upside down over sink, and allow excess fluid to run off sop only important sediment(s) remain 4) Use a plastic piette to apply sediment to blank glass slide 5) Coverslip sediment 6) Scan entire preparation for at least 10 lpfs & 10 hpfs Low power 10X High power magnification 40X  Stains Microscopic examinations can be done with or without stain The cellular detail can be seen better with stain Common stains: Sternheimer-Malbin Sedi-stain Sudan III or IV Oil Red O We will not be using stains here at Conestoga College :) Most labs also do not use stains due to expensive nature  Microscope Technique/Filters Bright-Field Microscopy Used for routine analysis Objects appear dark against a light background Phase contrast Microscopy A phase condenser ring can be added to a bright-field microscope Easier to see elements with low refractive indexes Hyaline casts, mixed cellular casts, mucous threads, Trichomonas Polarizing Microscopy Polarizing filters can be added to a bright-field microscope Identifies cholesterol Maltese cross Allows better identification of lipids and crystals Lipids exhibit a birefringent maltese cross Brings out cast details  Normal Sediment Normal sediment may contain a variety of formed elements in small amounts: RBCs WBCs Epithelial cells Amorphous urates / amorphous phosphates  Red Blood Cells Smooth, non-nucleated, biconcave discs (7 μm) Identified using high power objective (40X) Average of 10 high-power fields (hpfs) reported Normal range: 0 - 3/hpf  RBCs and Urine Concentration In hypersthenuric (concentrated) urine, RBCs shrink due to loss of water and appear crenated (irregulary shaped). SG > 1.010 In hyposthenuric (dilute) urine, RBCs swell and lyse rapidly. SG < 1.010 Hemoglobin is released and only the cell membrane remains. (Ghost Cells) Figure 6-13 Dysmorphic RBCs (x400) Dysmporphic cells vary in size, have cellular protrusions or are fragmented. Seen with glomerular bleeding. Also seen with: - Strenuous exercise - Nonglomerular hematuria  RBCs - Identification Difficulties RBCs can be confused with yeast cells, oil droplets, and air bubbles. Crenated RBCs may look like they have granules and can be confused with WBCs Acetic acid can be added to help differentiate RBCs will lyse Yeast, oil droplets, air bubbles and WBCs will remain intact  RBCs vs. Yeast Yeast will have a more oval shape than RBCs. Yeast can be in its budding or mycelial forms. Budding yeast Mycelial Form of Yeast RBCs vs. Oil Droplets and Air Bubbles Oil Air droplets Notice that no Bubble formed elements are in focus  RBCs - Clinical Significance Non-pathologic causes: Menstrual contamination in females Strenuous exercise Also increase in hyaline, granular, and RBC casts Traumatic catheterization Pathological causes: Damage to the glomerular membrane Pyelonephritis, glomerulonephritis Renal tumor The number of cells is an indicator of the extent of the damage/injury. Remember: Red/pink, cloudy urine = intact red cells = hematuria Red, clear urine = hemoglobin = hemoglobinuria  White Blood Cells Larger than RBCs, smaller than epithelial cells (12 μm) Identified using high power objective (40X) Average of 10 high-power fields (hpfs) reported Normal range: 0 - 5/hpf Capable of amoeboid migration through tissues to sites of inflammation or infection  WBCs - Neutrophils Predominant WBC found in urine sediment Contain granules and multilobed nuclei which makes them easy to identify Lyse rapidly in dilute alkaline urine Absorb water and swell in hypotonic urine Brownian movement of the granules produces a sparkling appearance Called Glitter Cells Stain light blue with Sternheimer-Malbin No pathologic significance in low numbers In high numbers = Bacterial infection  WBCs - Eosinophils Normally not seen in urine Urine sediment must be concentrated and stained Preferred stain is Hansel Wright’s stain can also be used. A finding of > 1% after counting 100 (or 500) cells is considered significant. Increased in: Primarily drug-induced interstitial nephritis Small numbers in UTIs and renal transplant rejection  WBCs - Mononuclear Cells May be present in small numbers Include lymphocytes, monocytes, macrophages, and histiocytes Lymphocytes Smallest WBCs May be mistaken for RBCs Increased in Renal Transplant Rejection  WBC - Identification Difficulties Mononuclear cells may be mistaken for renal tubular epithelial cells (RTEs) Lymphocytes may be mistaken for RBCs Adding acetic acid or a supravital stain can enhance nuclear detail, if necessary.  WBCs - Clinical Significance An increase in urinary leukocytes is called pyuria. Pyuria can be caused by: Bacterial infections Non-bacterial disorders Pyelonephritis (inflammation of the Glomerulonephritis kidney) (inflammation of the kidney as a result of an immune response) Cystitis (inflammation of the bladder) Lupus erythematosus (autoimmune disease) Prostatitis Interstitial nephritis (swelling in between the kidney tubules) Urethritis Tumors >50/hpf is strongly suggestive of acute inflammation (pyelonephritis, cystitis) Clumps of WBCs strongly suggest a kidney origin (i.e. not bladder) Will also see WBC casts and WBC/epithelial casts = pyelonephritis  Epithelial Cells Small amounts are not unusual and represent the normal sloughing of old cells. Three types are classified based on their origin within the genitourinary system 1) Squamous Vagina, male and female urethra 2) Transitional (urothelial) Bladder, renal pelvis, calyces, ureters, upper male urethra 3) Renal tubular Renal tubules from kidneys Epithelial cells are reported using the high-power magnification  Squamous Epithelial Cells Largest cell found in urine sediment Abundant, irregular cytoplasm Prominent nucleus about the size of an RBC Fried egg appearance Originate from the linings of the female urethra and vagina and the lower portion of the male urethra A large number of squamous cells may indicate that the sample is not mid-stream. Why? Squamous cells are shed in the first part of the void, if this is collected in the container you will see a large number which can interfere with viewing other elements.  Squamous Epithelial Cells - Clue Cells Usually squamous epithelial cells have no pathologic significance. One variation of the squamous cell that is pathologic is the clue cell. Squamous cell covered with the Gardnerella vaginalis coccobacillus The squamous cell will have a grainy, irregular appearance Testing is performed by examining a vaginal wet preparation, but small numbers may be seen in urinary sediment.  Transitional Epithelial Cells Line the renal pelvis, calyces, ureters, bladder and upper male urethra Smaller than squamous cells Centrally located nucleus Poached egg appearance  Alternate Forms of Transitional Epithelial Cells Caudate form Syncytia - clumped transitional epithelials - occurs during invasive urological procedures - no clinical significance  Transitional Epithelial Cells - Clinical Significance Small numbers are normal Increased numbers may indicate inflammation/infection in the areas where they originate: renal pelvis, calyces, ureters, bladder, upper male urethra Cells containing vacuoles or irregular nuclei could indicate a malignancy or viral infection.  Renal Tubular Epithelial Cells Vary in size and shape depending on what part of the renal tubule they originate from Those from proximal convoluted tubule are larger and columnar Those from the distal convoluted tubule are smaller and oval Those from the collecting ducts are cuboidal (at least one straight edge) Can appear in large sheets called renal fragments Coarsely granular cytoplasm Eccentric nuclei  Renal Tubular Epithelial Cells RTE columnar proximal RTE oval distal convoluted RTE cuboidal collecting convoluted tubule cell. tubule cells duct cells Fat globules attached. Note the eccentrically placed nuclei.  Renal Tubular Epithelial Cells - Clinical Significance The most clinically significant of the epithelial cells. More than 2 RTE cells/hpf indicates tubular injury and further testing should be done. Increased RTEs are indicative of renal tubular necrosis. This can be caused by: Exposure to heavy metals Drug-induced toxicity Viral infections (hepatitis B) Pyelonephritis Malignant infiltrations Salicylate poisoning Renal fragments indicate severe tubular injury  Oval Fat Bodies Fat can be found as free droplets or within disintegrating cells Oval fat bodies - RTE cells containing lipids Confirmation Stain with Sudan III or Oil Red O Triglycerides and neutral fats will stain orange Polarized light Cholesterol will demonstrate a Maltese cross Found in patients with nephrotic syndrome Also seen with severe tubular necrosis, diabetes mellitus and trauma Figure 6-19 p. 114  Bacteria Urine of healthy individuals should not contain bacteria A few bacteria may be present from contamination during the collection of the specimen. These contaminant bacteria can multiply rapidly at room temperature but have no clinical significance. Can be cocci or bacilli Reported as few, moderate or many/hpf If a UTI is suspected, bacteria should be accompanied by WBCs Enterobacteriaceae (gram-negative rods) are most associated with UTIs  Bacte Bacteria are observed under theria microscope but the reagent strip was negative for nitrite. Why would this occur? So much bacteria are present that nitrite was further reduced to nitrogen. Nitrogen would not be identified by the reagent strip. The bacteria present lack the enzyme needed to reduce nitrate to nitrite. Only gram-negative bacteria have this enzyme. The urine was not incubated in the bladder long enough for the bacteria to reduce the nitrate The patient has not been getting nitrate from their diet (no green vegetables). The patient is taking antibiotics (inhibits bacterial metabolism)  Yeast Small, refractile oval structures May or may not be budding Can be mistaken for RBCs Look for budding RBCs lyse in acetic acid while yeast does not Common in diabetics Acidic urine containing glucose is ideal for the growth of yeast. Primarily Candida albicans Also seen in immunocompromised patients Yeast infection: Nitrite negative; presence  Spermatozoa Oval, tapered heads with long, flagella-like tails Motility is rarely seen as urine is toxic to spermatozoa Rarely of clinical significance except in cases of male infertility Retrograde ejaculation may occur where sperm is expelled into the bladder instead of the urethra Laboratories may or may not report spermatozoa. Refer to your laboratories operating procedures. Often not reported due to the lack of clinical significance and the possible legal consequences. Positive protein on reagent strip when large amounts of semen are present  Mucus Thread-like structures with a low refractive index Uromodulin is the major constituent Glycoprotein excreted by RTE cells of the distal convoluted tubules and upper collecting ducts Can be confused with hyaline casts Mucus has an irregular appearance whereas casts are more uniform. Found more frequently in specimens from females  Parasites - Trichomonas vaginalis Most frequently seen parasite Pear-shaped with an undulating membrane When motile, has a rapid darting movement Only report if you see motility Non-motile Trichomonas can be mistaken with WBCs and RTEs Is a sexually transmitted pathogen Correlations: LE positive, presence of WBCs Trichomonas vaginalis under the microscope  Parasites - Schistosoma haematobium Bladder parasite Ova may be found in the urine (eggs ova) Not often seen in North America Mainly Africa and the Middle East Schistosoma haematobium under the micros cope  Parasites - Enterobius vermicularis Intestinal parasite Pinworm ova may be found in urine following fecal contamination. They have a characteristic oval shape with one flat side.  Artifacts Starch Granules Highly refractive spheres with a dimpled center Contamination from the use of powered gloves Will produce a Maltese cross formation when polarized. Hair and Fibers Long and refractile with dark edges Can be mistaken for casts Contaminant from clothing and diapers