Chapter-6-Microscopic-examination PDF

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Chapter 6: MICROSCOPIC EXAMINATION OF URINE Purpose: to detect and identify insoluble materials present in the urine A. Macroscopic Screening  Abnormalities in the physical and chemical portions of urinalysis play a primary role in the decision to perform a microscopic analysis.  Protocols of consideration on population under macroscopic screening: Pregnant women, pediatric, geriatric, diabetic, immunocompromised and renal patients. *(Refer to Table 6-1 “Macroscopic Screening and Microscopic Correlations”)* Specimen Preparation  Specimen should be examined while fresh or adequately preserved  Formed elements- RBCs, WBCs and hyaline castsdisintegrate rapidly, particularly in dilute alkaline urine  Refrigeration- may cause precipitation of amorphous urates and phosphates and other non-pathologic crystals that can obscure other elements in the urine sediments  Warming the specimen to 37oC- can dissolve some of the crystals  Midstream clean-catch specimen- minimizes external contamination of the sediment  Dilute random specimen- cause false negative readings Specimen Volume  Standard amount of urine: between 10 and 15 mL, is centrifuged in conical tube-provides an adequate volume of the elements present in the specimen  12-mL volume-frequently used because multiparameter reagent strips are easily immersed ad capped centrifuge tubes often calibrated to this volume  If obtaining a 12 mL is not possible- for pediatric patients, it should be noted on the report form Centrifugation  Centrifugation time: 5 minutes Centrifugal force: 400 RCF Effect: optimum amount of sediment with least chance of damaging elements  Use of braking mechanism to slow centrifuge: cause disruption of the sediment prior to decantation and should not be used.  To prevent biohazardous aerosols: all specimens must be centrifuged in capped bottles Sediment Preparation  Uniform amount of sediment and urine should remain in the tube after decantation  Frequently used volume: 0.5 and 1.0 mL  Concentration Factor- volume of urine centrifuged divided by the sediment volume  To maintain a uniform sediment concentration factor, urine should be aspirated off rather than poured off, unless otherwise specified by the commercial system in use  The sediment must be thoroughly resuspended by gentle agitation.  Vigorous agitation should be avoided  Thorough resuspension is essential to provide equal distribution of elements in the microscopic fields Volumes of Sediment Examined  Volume of sediment place in the microscope slide should be consistent for each specimen  Conventional glass slide method: recommended volume is 20 uL covered by a 22x22 mm glass cover slip- which will allow the specimen to flow outside the cover slip- result in the loss of heavier elements such as casts  Commercial Systems: control the volume of sediment examined by providing slides with chambers capable of containing specified volume

Commercial Systems  System provide a variety of options including capped, calibrated centrifuge tubes; decanting pipettes to control the sediment volume; and slides that control the amount of sediment examined; produce a consistent monolayer of sediment for examination, and provide calibrated grids for more consistent quantitation.  Cen-Slide and R/s Workstations do not require manual loading of the centrifuged specimen onto a slide and are considered closed systems that minimize the exposure to the specimen  Cen-Slide provides a specially designed tube that permits direct reading of the urine sediment  R/S Workstations consist of glass flow cell into which urine sediment is pumped, microscopically examined, and then flushed from the system Examining the Sediment  Microscopic examination must be performed in a consistent manner and include observation of a minimum 10 fields under both low (10x) and high (40x)  The slide is first examined in low power to detect cast then if it is identified changed to high power  Bright filed microscopy: essential for unstained sediment Reporting the Microscopic Examination  Casts: reported as the average number per lpf following the examination f 10 fields  RBCs and WBCs: as the average number per 10 hpf  Epithelial cells, crystals and other elements: reported in semiquantitative terms such as rare, few, moderate, and many, or as 1+, 2+, 3+, and 4+ following lpf or hpf formatwas use Correlating Results  Microscopic results should be correlated with the physical and chemical finings to ensure the accuracy of the report  Specimens in which the results do not correlate must be rechecked for both technical and clerical errors *(Refer to table 6-2 “Routine Urinalysis Correlations”)* B. Sediment Examination Techniques  Many factors can influence the appearance of urinary sediments including cells and cats, distortion of cells and crystals by the chemical content of specimen and contamination of artifacts.  Identification can be enhanced through the use of sediment stains and different types of microscopy 

*(Refer to table 6-3 “Urine Sediment Stain Characteristics”)*

Sediment Stains  Staining increases the overall visibility of sediment elements being examined in bright field microscopy by changing their refractive index  Sternheimer-Malbin Stain: most frequentlyused stain which is consisting of crystal violet and safranin O. -The stain is available commercially under a variety of names, including Sedi-Stain and KOVA stain  0.5% solution of toluidine blue a metachromatic stain that provides enhancement of a nuclear detail. -It can be useful in the differentiation between WBCs and renal tubular epithelial cells and is also used in examination of cells from other body fluids  0.2 % acetic acid: alsoenhance the nuclear detail -This method is cannot be used for initial sediment because RBCs lyses on acetic acid *(Refer to table 6-4 “Expected Staining Reactions of Urine Sediment Constituents”)* Lipid Stains  Passage of lipids across he glomerular membrane results in the appearance of free droplets and lipid containing cells and casts in the urinary sediment  Sudan III and Oil Red O are lipid stains and Polarizing microscopy can be used to detect the presence of the lipids.  Triglycerides and neutral fats: stains orange-red

 Cholesterol: do not stain but capable of polarization Gram Stain  Primarily used in microbiology section for differentiation between gram positive (blue) and gram negative (red) bacteria.  In routine analysis it is limited in determining bacterial casts, which can easily be confused with granular casts  To perform gram staining, a dried, heat fixed preparation is used Hansel Stain  The preferred stain for urinary eosinophils is Hansel stain, consisting of Methylene Blue and eosin Y.  Wright’s stains: it can also be used on a dried smear of the centrifuged specimen or a centrifuged preparation of sediments Prussian Blue Stain  Due to episodes of hemoglobinuria, yellow brown granules may be seen in renal tubular epithelial cells and casts or free floating in the urine sediment.  Confirmation of Hemosideran in granules is used and the resulting color is blue

TYPES OF MICROSCOPY 

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Cytodiagnostic Urine Testing  Frequently performed independently of routine urinalysis for detecting the malignancies of the lower urinary tract.  A voided first morning urine specimen is recommended for testing  Provides more definitive information about real tubular changes associated with transplant rejection; viral, fungal and parasitic infections; cellular inclusions; pathologic casts; and inflammatory conditions Microscopy  Bright field microscopy: most common type of microscopy performed in urinalysis laboratory.  The type of microscopy used depends on the specimen type, the refractive index of the object, and the ability to image unstained living cells  All microscopes are designed to magnify small objects to such a degree that the details of their structure can be analyzed *(Refer to table 6-5 “Urinalysis Microscopic Technique”)* The Microscope  Lens system: primary components are the oculars, objective and the coarse and fine adjustment knobs.  Illuminationsystem: contains the light source, condenser and field and iris diaphragm  Mechanical stage: platform where objects to be examined are placed  Compound bright field microscope: used primarily in urinalysis laboratory and consist of two lens system combined with a light source First lens system: located in theobjective and is adjusted near the specimen Second lens system: the ocular lens is located in the eyepiece  Ocular or Eyepiece: located at the top of the body; designed to further magnify the object that has been enhanced by the objectives for viewing  Objectives:contained in the revolving nose piece located above the mechanical stage  Resolution:ability to visualize fine details; ability of the lens to distinguish two small objects that are specific distance apart -Objectives used in urinalysis are 10x and 40x  Final Magnification: product of the objective magnification times the ocular magnification  Numerical aperture number: represents the refractive index of the material between the slide and the outer lens and the angle of light passing through it  Coarse and fine focusing knob: the distance between the slide and the objective  Coarse knob- initial focusing is performed  Fine focusing knob- to sharpen the image  Condenser- located below the stage then focuses the light on the specimen and controls the light for uniform illumination  Aperture diaphragm- controls the amount of light and the angle of light rays that passes the specimen

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Bright-Field Microscopy o Most frequently used in the clinical laboratory o Objects appear dark against a light background o Has a light source emitting light in the visible wavelength range o For use in examination of urine sediments:  Must be examined using decreased light(adjust the rheostat, not the condenser) *Sediment with low refractive index – overlooked when subjected to high light intensity  Staining of sediment – increases the visualization of the elements Phase-Contrast Microscopy o Phase difference – light rays that pass through an object are slowed in comparison to the rays passing through the air, producing increased light intensity and contrast Affected by:  thickness of the object  refractive index  other light absorbance properties o Provide best contrast by:  shifting one quarter of a wavelength, of the light that does not pass through the specimen, and compare it with the phase difference of the specimen o Adaptation of a bright-field microscope with a phasecontrast objective lens and a matching condenser *Image has best contrast when the background isdarkest o Advantageous in identifying:  Low refractive hyaline casts or mixed cellular casts  Mucous threads Polarizing Microscopy o Aids in identification of crystals and lipids  Crystals and lipids – have the ability to rotate the path of the unidirectional polarized light beam to produce:  Characteristic colors – crystals  Maltese cross formation – lipids – Birefringent (a property indicating that the element can refract light in two dimensions at 90 degrees to each other) o Halogen quartz lamp – produces light rays of many different waves. [Each wave has its (1) Distinct direction and (2) Vibration perpendicular to its direction]  Normal or unpolarized light - vibrates in equal intensity in all directions  Polarized light - vibrates in the same plane or direction o Birefringent substance–substance from which light passes through as it splits into two beams o Isotropic substances– (ex. Blood cells) light passes through this substance unchanged; does not have refractive property o A substance that rotates the plane of polarized light 90 degrees clockwise direction have positive birefringence o A substance that rotates the plane in a counterclockwisedirection has negative birefringence o Polarized light – obtained by using two polarizing filters  The light emerging from one filter vibrates in one plane  Second filter – placed at 90-degree angle and blocks all incoming light *The filters are in opposite directions called “crossed configuration” o Bright-field microscopes can be adapted for polarizing microscopy o Used in urinalysis to confirm identification of:  Fat droplets  Oval fat bodies  Fatty casts – produce Maltese cross pattern o For distinctionbetween the following by their polarizing characteristics:

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Birefringent uric acid crystals -- cystine crystals Monohydrate calcium oxalate crystals -- nonpolarizing RBCs  Calcium phosphate crystals-- nonpolarizing bacteria Interference-Contrast Microscopy o Provides a three-dimensional image showing very fine structural detail by splitting the light ray so that beams pass through different areas of the specimen o Object appears bright against a dark background w/o the diffraction halo associated with phase-contrast microscopy o Not routinely used in the urinalysis laboratory o Two types: [provide (1) Layer-by-layer imaging of the specimen and (2) enhance detail for specimens w/ either low or high refractive index]  Modulation contrast (Hoffman) Polarized light rays pass through a split aperture to the various areas of the specimen and to the modulator where they are converted to variations of light intensity producing a 3-D image 3 zones of light transmission of the modulator:  Dark zone – transmits 1% of light  Gray zone – transmits 15% of light  Clear zone – transmits 100%of light  Differential-interference contrast (Nomarski) Uses prism Two-layered Nomarski-modified Wollaston prism (required to separate individual rays of light into pairs Dark-Field Micriscopy o Enhance visualization of specimens hat cannot be seen easily viewed with a bright-field microscope o For unstained specimens o Identify the spirochete Treponema pallidum o Specimen appear light against black back-ground or dark-field o Bright-Field microscope is easily adapted for dark-field microscopy by:  Replacing the condenser w/ a dark-field condenser that contains an opaque disk Fluorescence Microscopy o Detect bacteria and viruses within cells and tissues through immunofluorescence o Visualization of naturally fluorescent substances or those that are stained with fluorochrome or fluorophore o Fluorescence –property by which some atoms absorb light at a particular wavelength and subsequently emit fluorescence lifetime o Fluorescence lifetime – light of a longer wavelength o Fluorescent substances – absorb energy and emit a longer wavelength of light and is visualized with the use of special filters:  Excitation filter –selects the excitation wavelength of light from a light source  Emission filter – selects a specific wavelength of emitted light from the specimen to become visible o Dichroic mirror – reflects the excitation light to the specimen and transmits the emitted light to the emission filter o Object observed as bright against a dark background with high contrast when ultraviolet light source is used o Powerful light sources: Mercury or xenon arc lamps URINE SEDIMENT CONSTITUENTS o

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Normal urine sediment may contain variety of formed elements:  Pathologically significant RBCs, WBCS and casts  Rare epithelial cell or mucous strand Urine sediment preparation methods – determine the actual (1) concentration of the sediment and (2) number of elements that may be present in a microscopic field Commonly listed values:  0-2 or 3 RBCs/hpf  0-5-8 WBCs/hpf  0-2 hyaline casts/Ipf

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RED BLOOD CELLS o Smooth, non-nucleated and biconcave disks o Identified in high-power (40x) objective o Reported as average number seen in 10 hpfs o Concentrated urine (hypersthenuric) – Cells shrink (loss of water) and may appear crenated or irregularly shaped o Dilute urine (hyposthenuric) – Cells swell (absorb water) and lyse rapidly,, releasing their hemoglobin and leaving only cell membrane – Ghost cells–large empty cells o Most difficult for students to recongnize due to RBCs’:  Lack of characteristic structures  Variations in size  Close resemblance to other urine sediment constituents o Confused with: (sources of error)  yeast cells  oil droplets  air bubbles o Dysmorphic RBCs – RBCs that vary in size, have cellular protrusions or are fragmented – Primarily associated with glomerular bleeding – found with nonglomerular hematuria – ↑after strenuous exercise – use of Wright’s stained prep – shows cells to be hypochromic and better delineates the presence of cellular blebs and protrusions * Acanthocyte with multiple protrusions – the dysmorphic cell most closely associated with glomerular bleeding o Clinical Significance Presence of RBCs in urine is associated with:  Glomerular membrane or vascular injury within genitourinary tract No. of cells present  Indicative of the extent of damage or injury Hematuria  Macroscopic hematuria:Urine appears cloudy with a red to brown color  Microscopic: reported in terms of greater than 100 per hpf or as specified by laboratory protocol Refer to SUMMARY 6-1 (Microscopic RBCs) Observation of microscopic hematuria  Critical in the early diagnosis of glomerular disorders and malignancy of the urinary tract to confirm the presence of renal calculi RBCs, hyaline, granular and RBC casts in urine  Seen after strenuous exercise  Nonpathologic and disappear after rest *Presence or absence of RBCs in the urine cannot always be correlated with specimen color or a positive chemicaltest result for clood (Ex. Hemoglobin in urine – Red urine – (+) chemical test in absence of hematuria) WHITE BLOOD CELLS o Larger than RBCs o Neutrophil – predominant WBC found in urine  Much easier to identify than RBCs  Contain granules with multilobed nuclei  Reported as average number seen in 10 hpfs  Lyse rapidly in dilute alkaline urine  Brownian mov’t of granules within this cell produces a sparkling appearance referred to as “glitter cells”(no pathologic significance)  Glitter cells – large cells that stain light blue with Sternheinmer-Malbin stain as opposed to the violet color usually seen with neutrophils o Eosinophils  Primarily associated with drug-induced interstitial nephritis  Small numbers in (1) Urinary tract infections (UTI) and (2) Renal transplant rejection  Concentrated and stained urine sediment – required for urinary eosinophil test  Centrifugation/cytocentrifugation –for concentration of urine sediment  Hansel – preferred eosinophil stain

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*Wright’s stain can also be used *Percentage of eosinophils in 100 to 500 cells is determined  Not normally seen in urine, finding >1% is significant o Mononuclear cells – usually not identifiedin the wet preparation urine microscopic analysis  Lymphocytes – the smallest WBCs and may resemble RBCs – ↑in early stages of renal transplant rejection  Monocytes, macrophages and histiocytes – large cells that appear vacuolated or contain inclusions * Cytodiagnostic urine testing – referred to if specimens containing an increased amt of mononuclear cells cannot be identified as epithelial cells  Source of error:  Renal tubular epithelial (RTE) cells – larger than WBCs with an eccentrically located nucleus – may be difficult to distinguish from WBCs in the process of ameboid motion because of their irregular shape * Supravital stainingor addition of acetic acid – can be used to enhance nuclear detail if necessary  ↓than 5 leukocytes per hpf are found in normal urine, but ↑numbers in females  Pyuria - ↑in urinary WBCs – Indicates the presence of an infection or inflammation in the genitourinary system – Can cause bacterial infections (pyelonephritis, cystitis, prostatitis and urethritis) * Refer to SUMMARY 6-2 (Microscopic WBCs) EPITHELIAL CELLS o Represent normal sloughing of old cells o Derived from the linings of the genitourinary system o Not unusual to find in the urine unless found in large or abnormal numbers 3 TYPES OF EPITHELIAL CELLS o Squamous Epithelial Cells  Largest cells found in the urine sediment  Contain abundant, irregular cytoplasm and a prominent nucleus about a size of an RBC  First structures observed under low-power magnification  Good ...