CLS 161 UNIT 2 MICROSCOPIC EXAMINATION

what is the purpose for microscopically examining urine?

to detect and identify insoluble materials present in urine

what are the procedural variations for microscopic analysis?

• method of how sediment is prepared

• volume of sediment actually examined

• method and equipment used to obtain visualization

• manner in which results are reported

describe the protocol of initiation of microscopic examination based on macroscopic examination

a microscopic examination is only triggered when the specimen meets specific criteria which would be based on physical and chemical results

• color, clarity (cloudy urines trigger microscopic examinations), and odor even though it is not reported

• presence of blood, protein, nitrite, LE, and possibly glucose

what are the patient populations that must be considered when developing protocols for when a microscopic examination is triggered?

• pregnant women

• old people

• pediatric

• diabetics

• immunocompromised

what does CLSI recommend when a microscopic examination should be performed?

• physical requests

• lab-specified population is being tested

• abnormal physical or chemical results are obtained

describe the microscopic examination of urine specimens

• microscopic examination is used to detect and identify insoluble materials that are present in the urine

• blood, kidney, lower genitourinary tract, and external contamination all contribute formed elements

  • RBCs, WBCs

  • epithelial cells

  • casts

  • bacteria, yeast, parasites, mucus, spermatozoa

  • crystals

  • artifacts

• some formed elements have no clinical significance and are actually considered normal until they are present in increased amounts

• examination of urinary sediment must include both ID and quantitation of elements present

describe the recommended methods for standardizing specimen prep and volume, centrifugation, sediment prep, volume, and examination and reporting of results

• regarding specimen prep, specimens should be examined while the urine is fresh or properly preserved as formed elements will disintegrate quickly, especially in dilute alkaline urine.

  • a clean-catch midstream specimen will have less contamination regarding epithelial cells.

• regarding specimen volume, the recommended volume is between 10-15 mL although 12 mL is usually used since reagent strips and capped centrifuged tubes is calibrated to 12 mL.

  • if 12 mL isnt possible then it needs to be noted onto the report so a physician can make corrections to the results

• regarding centrifugation, specimens are centrifuged so the sediment falls to the bottom.

  • the speed of the centrifuge and the length of time that the specimen is spinning for needs to be consistent.

  • specimens should be capped to avoid aerosols

  • centrifuging for 5 mins at 400 RCF or 1000 RPM is optimum for amount of sediment with the least chance of damaging the sediment

• regarding sediment prep, a uniform amount of urine and sediment should be present after decantation.

  • sediment should be gently resuspended through gentle agitation with a pipetted or by tapping the tip of the tube.

  • there should be a consistent amount of sediment placed on the slide and if commercial systems are used then it must be filled completely

• regarding examining the sediment, it should be performed in a consistent manner by observing a minimum of 10 fields under low and high power fields

  • first, slides are examined under low power to detect casts and to overall determine the general composition of sediment

  • if the sediment is unstained, the sediments should be viewed under reduced light when using bright field microscopy

• regarding reporting the microscopic examination, casts are reported as average number per low power field (lpf), RBCs and WBCs are reported as average number of high power field (hpf), and epithelial cells, crystals, and other elements are reported semiquantitatively.

  • semiquantitative = rare, few, moderate, and many or +1, +2, +3, +4 followed by per lpf or hpf

• MICROSCOPIC RESULTS SHOULD CORRELATE WITH PHYSICAL AND CHEMICAL RESULTS

what formed elements will disintegrate quickly in dilute alkaline urine?

• RBC

• WBC

• hyaline casts

why should you refrain from using the centrifuges braking mechanism to slow down the centrifuge?

disrupts the sediment before decantation

what amount is common after decanting the sediment?

0.5 and 1.0 mL

what is the concentration factor? what does it relate to?

• volume of urine centrifuged divided by sediment volume

• related to the probability of detecting elements present in low quantities

  • also used when quantitating the number of elements per mL

what is necessary to quantitate cellular elements per mL of urine?

chamber volume, size of viewing area, and approx. number of low power and high power viewing areas (commercial systems do this) + sediment concentration factor

what does the CLSI recommend regarding commercial systems?

the use of commercial systems and standardization of all phases of the methodology which includes the conventional method

how should the physical and chemical tests correlate when RBCs are seen microscopically? what are the exceptions?

• physical

  • turbidity

  • red color

• chemical

  • + blood

  • + protein

• exceptions

  • number of them

  • hemolysis

how should the physical and chemical tests correlate when WBCs are seen microscopically? what are the exceptions?

• physical

  • turbidity

• chemical

  • + protein

  • + nitrite

  • + LE

• exception

  • number

  • lysis

how should the physical and chemical tests correlate when epithelial cells are seen microscopically? what are the exceptions?

• physical

  • turbidity

• chemical

  • none

• exceptions

  • number

how should the physical and chemical tests correlate when casts are seen microscopically? what are the exceptions?

• physical

  • none

• chemical

  • + protein

• exceptions

  • number

how should the physical and chemical tests correlate when bacteria’s are seen microscopically? what are the exceptions?

• physical

  • turbidity

• chemical

  • pH

  • + nitrite

  • + leukocyte

• exceptions

  • number

  • type

how should the physical and chemical tests correlate when crystals are seen microscopically? what are the exceptions

• physical

  • turbidity

  • color

• chemical

  • pH

  • + bilirubin

• exceptions

  • number

  • type

what are the 4 factors that could affect the appearance of a sediment?

1. cells and casts in various stages of development and degeneration

2. distortion of cells and crystals (will reflect light) by the chemical content of the specimen

3. presence of inclusions in cells and casts

4. contamination of artifacts

   • how perfect is it? (nature is not perfect)

   • how is light being reflected? 

when someone is talking about counting the elements found in a sediment, what are they talking about?

talking about the field of view meaning the microscope needs to be moved in 10 unique spots

what does staining do?

increases the overall visibility when using bright field microscopy (use subdued light) by changing the refractive index

what does staining show?

shows identifying characteristics to cellular structures like nuclei, cytoplasm, and inclusions

what are the two stains for supravital stains? explain why each stain is used

• Sternheimer-Malbin

  • WBCs, epithelial cells, and casts absorb this dye well which allows a clear description of its structure and contrasting colors of their nuclei and cytoplasm

• 0.5% toluidine blue (metachromatic stain)

  • used to differentiate between WBCs and renal tubular epithelial cells

  • also used to examine other cells from other body fluids

is the sediment fixed on the slide when using supravital stains? why or why not?

not fixed onto the slide because the cells need to be alive when examining them

what is acetic acid used for? why should it not be used for initial analysis?

• enhances the nuclear detail of WBCs and epithelial cells

• should not be used for initial analysis as RBCs will lyse

  • use if you need to see other things that may be present like WBCs, yeast, oil droplets, and crystals

what does the passage of lipids across the glomerular membrane result in?

appearance of free fat droplets and lipid-containing cells and casts in the sediment

what are the two lipid stains? explain their usage. what else is used in conjunction with lipid stains?

• two lipid stains → Oil red O and Sudan III

  • stains triglycerides and neutral fats an orange-red

• polarizing microscopy is used in conjunction with lipid stains to confirm the presence of cholesterol

what are gram stains used for?

identification of bacterial casts but can be easily confused with granular casts

what is almost always associated with microbial infections?

polynuclear WBC, neutrophil

what is the hansel stain used for? what is in it? how is it performed?

• used to stain eosinophils (preferred)

  • can use Wright stain and Giemsa stain

• contains methylene blue + eosin Y in methanol

• performed on a dried smear of centrifuged specimen or a cytocentrifuged prep of sediment

what are the other causes of eosinophils to be present?

• renal transplant rejection

• pyelonephritis

• prostatitis

• cystitis

what is the prussian blue stain?

stains structures containing iron which will stain hemosiderin in cases of hemoglobinuria blue

what is cytodiagnostic urine testing? what is it used in combination with?

• performed independently but is used for detection of malignancies of lower urinary tract

• used with the Papanicolaou stain to detect:

  • renal tubular changes associated with transplant rejection

  • viral, fungal, and parasitic infections

  • cellular inclusions

  • pathological casts

  • inflammatory conditions

when is microscopy best performed?

when the laboratorian is knowledgeable about the types of microscopes, primary characteristics of them, and the properly use and maintenance of them

what does the type of microscopy depend on?

• specimen type

• refractive index of object

• ability to image unstained living cells

what is bright field microscopy?

objects appear dark against light background and is the most common type of microscopy used but can present issues if the light is too bright

what is phase difference influenced by?

• thickness of object

• refractive index

• other light-absorbance properties

what is phase-contrast microscopy useful for?

ID of low refractive index hyaline casts or mixed cellular casts and mucous threads by forming a halo of light around the specimen

what does polarizing microscopy used for? (two things)

• used to confirm the identification of fat droplets, oval fat bodies, and fatty casts that produce the maltese cross pattern characteristic

• can also help differentiate birefringent uric acid crystals from:

  • cystine crystals and monohydrate calcium oxalate crystals from nonpolarizing RBCs

  • calcium phosphate crystals from nonpolarizing bacteria

what is interference-contrast microscopy?

provides 3D image that shows very fine structure detail by splitting the light ray so the beam passes through different areas of the specimen

what is dark field microscopy used for?

to enhance the visualization of specimen (usually unstained) that cannot be easily with a bright-field microscopy

what does fluorescence microscopy detect?

bacteria and viruses within cells and tissues through immunofluorescence

what can cause cellular elements to be easily distorted?

• concentration that can vary

• pH

• presence of metabolities in urine

what would normal red blood cells look like in urine?

appear smooth, nonnucleated, biconcave disk that is ~7 μm in diameter

what power should RBCs be ID’d with? what are they reported as?

• ID using high power

• reported as average number seen in 10 hpfs

what happens to RBCs in concentrated (hypersthenuric, high SG) urine?

cells will shrink due to loss of water and may appear crenated or irregularly shaped

what happens to RBCs in dilute (hyposthenuric, low SG) urine?

cells will absorb water and swell which will lyse rapidly causing them to release their hemoglobin and leaving their cell membrane behind → ghost cell is the result

what are the reasons for RBCs being difficult to identify?

• lack of characteristics structure

• variations in size

• close resemblance to other constituents

what are the five things that can easily be confused with RBCs?

• yeasts (usually exhibits budding and should expect to see glucose)

• oil droplets (highly refractile)

• air bubbles (highly refractile)

• starch (highly refractile)

• may resemble granules seen in WBCs if RBCs are roughly crenated but smaller

what should be done if identification is still doubtful with RBCs?

add acetic acid to lyse the RBCs which would leave the yeasts, oil droplets, and WBCs intact

what does the morphology of urinary RBCs help with?

determining the site of renal bleeding

what can cause dysmorphic RBCs?

• primarily associated with glomerular bleeding

• strenuous exercise → indicates glomerular origin

what would a dysmorphic RBC that is related to glomerular bleeding look like?

acanthocytic (abnormal RBCs with thorn like protrusions) with multiple protrusions and is not as uniform

what would the dysmorphic cells look like if a wright stain is used to further analyze them?

shows the cells to be hypochromatic and would show a better description of the presence of cellular blebs and protrustions

what is the clinical significance of RBCs in urine sediment? what do the number of cells present indicate?

• associated with damage to glomerular membrane or vascular injury within genitourinary tract

• the number of cells present indicate the extend of the damage or injury

what would the macroscopic hematuria determine with RBCs in urinary sediment?

• cloudy red or brown urine

• associated w/ advanced glomerular damage

• also seen with damage to vascular integrity of urinary tract caused by trauma, coagulant disorders, and acute infection or inflammation

what would the microscopic hematuria determine with RBCs in urinary sediment?

• helps with the early diagnosis of glomerular disorders and malignancies of the urinary tract

• confirms the presence of renal calculi

• can be transient if pt participated in strenuous exercise which is nonpathological

what would cause a positive for blood with a chemical test but would be absent in microscopic hematuria?

hemoglobin that has been filtered by glomerulus

how big are WBCs? what power are they identified under?

• 12 μm

• reported as average number seen in 10 hpfs

what would neutrophils look like under a microscope?

is the predominant WBCs and contains granules and multilobed nuclei

what happens to neutrophils when they are exposed to dilute alkaline urine?

lyses rapidly and begins to lose nuclear detail

what happens to neutrophils when exposed to hypotonic urine?

absorbs water and begins to swell which causes brownian movement and forms a glitter cell (NONPATHOLOGICAL)

when would you see eosinophils in urinary sediment?

• drug induced interstitial nephritis

• small number would be seen in UTI and renal transplant rejection

finding more than 1% is significant

how is an eosinophil test performed? what is determined for the percentage?

• performed on a concentrated, stained (Hansel) urine sediment

  • can concentrate by centrifugation or cytocentrifugation

• percentage per 100 to 500 cells

what would the mononuclear cells look like under a microscope?

• lymphocytes are the smallest WBC and may look like RBCs

  • seen in increased numbers in early stages of renal transplant rejection

• monocytes, macrophages, and histiocytes are larger and may appear vacuolated or contain inclusions

mononuclear cells are rare

what is the primary concern with the ID of WBCs?

differentiating between WBCs and disintegrated neutrophils from round renal tubular epithelial cells (RTE)

what is considered normal when seeing WBCs in urinary sediment?

<5 leukocytes per hpf but more in females

how can leukocytes enter urine?

through glomerular or capillary trauma but also through ameboid migration (can be hard to differentiate from epithelial cells) through the tissue to the site of infection or inflammation

what bacterial infections would be associated with an increased of WBCs in the urine (pyuria)?

• pyelonephritis

• cystitis

• prostatitis

• urethritis

what nonbacterial infections would be associated with pyuria?

• glomerulonephritis

• lupus erythematosus

• interstitial nephritis

• tumors

what are the three types of epithelial cells?

1. squamous

2. transitional (urothelial)

3. renal tubular

what would squamous epithelial cells look like under a microscope? what are they good for?

• is the largest cell and contains abundant, irregular cytoplasm and has a prominent nucleus as big as RBC (~7 μm)

• often the first structure observed and is a good reference point

what do squamous epithelial cells originate from? what do they represent? what are they reported as?

• originate from linings of vagina, female urethra, and lower portions of male urethra

• represents normal sloughing of cells and have no pathological significance

  • increased amounts are seen in female patients

• reported as rare, few, moderate, or many (semiquantitative) and lpf or hpf depending on the lab

why would someone have difficulty identifying an epithelial cell on the rare occasion?

• may appear folded which would resemble a cast

• begins to disintegrate in unfresh urine

• urine with large amounts of epithelial cells can clump which can make it difficult to see smaller pathological elements like RBCs and WBCs

what is a clue cell? what determines a clue cell? what is used to examine a clue cell?

• a pathologically significant squamous epithelial cell and is indicative of a vaginal infection by Gardnerella vaginalis

• bacteria covers 70% of cell surface and extends beyond the edges of the squamous cell which gives it a granular, irregular appearance

• a vagina wet prep (saline) is used to examine a clue cell

what are transitional epithelial (urothelial) cells? where do they originate from? what are they reported as?

• smaller than squamous cell with a centrally located nucleus and has three forms

  • spherical = absorb water in bladder and become large and round

    • can be difficult to differentiate from RTE cells

  • polyhedral = multiple sides

  • caudate = has a tail

• originates from lining of renal pelvis, calyces, ureters, bladder, and upper portion of male urethra

  • appears in small numbers and represent normal cellular sloughing 

• ID’d in high power and reported as rare, few, moderate (semiquantitative) 

what can increased numbers of transitional epithelial cells come in? what would an increase of transitional epithelial cells with abnormal morphology indicate?

• singles, pairs, clumps (syncytia) which is present after invasive urological procedures and have no clinical significance 

• indicative of malignancy or viral infections which should be referred to a pathologist

why do renal tubular epithelial cells vary in size and shape? what areas would they come from and describe them

depends on the area of renal tubules from where they originated from

• proximal convoluted tubule (PCT) = larger, rectangular, cytoplasm is coarsely granular

  • referred to as columnar or convoluted (coiled) cells

• distal convoluted tube (DCT) = smaller than PCT and are round or oval with an eccentrically placed nuclei

  • can be mistaken for WBCs and spherical transitional epithelial cells

• collecting duct = cuboidal, never round, has at least one straight edge with an eccentrically placed nucleus

  • appears in groups of three or more called renal fragments which is seen as large sheets of cells (not seen with PCT and DCT)

  • indication of severe tubular injury w/ basement membrane distruption

how are RTE’s ID’d and reported? what does their presence indicate?

• reported either semiquantitatively (rare, few, moderate, or many) or quantitatively (actual number per hpf)

• presence of two or more per hpf → indicative of tubular injury

what is the clinical significance of RTEs?

presence of increased amounts of RTEs is indicative of necrosis of renal tubules which could possible overall affect renal function

what are the 8 conditions that could produce tubular necrosis?

1. exposure to heavy metals

2. drug-induced toxicity

3. hemoglobin and myoglobin toxicity

4. viral infections (hep b)

   • seen with bilirubin

5. pyelonephritis

6. allergic rxns

7. malignant infiltrations

8. salicylate poisoning

   • single cuboidal cells are noticeable with salicylate poisoning

9. acute allogenic (donor to host) transplant rejection

what is one of the functions of RTE? based on one of its functions, why can it be clinically significant?

• reabsorption of glomerular filtrate so seeing substance from the filtrate is not unusual

• may be clinically significant because hemosiderin can be seen in episodes of hemoglobinuria

  • use prussian blue to stain hemosiderin blue

• RTE cells also absorb bilirubin present in filtrate → due to liver damage

  • often occurs in viral hepatitis and appear deep yellow color

what are oval fat bodies? what are oval fat bodies reported as? how would you know oval fat bodies are present?

• RTE cells absorb lipids that are present in the glomerular filtrate and appears highly refractile

  • seen in conjunction with free-floating fat droplets

    • may float on top of the specimen

• reported quantitative as average number per hpf

• confirmation = droplets containing cholesterol would have maltese cross formation with polarized light 

  • droplets would stain orange red with Oil Red O and Sudan III with triglycerides and neutral fats

what should be considered when observing free-floating fat droplets?

specimens that are contaminated due to vaginal preparations and lubricants

what is lipiduria? what are they most associated with? what can they also be seen with?

• presence of free lipids in urine

• associated most often with damage to glomerulus caused by nephrotic syndrome

• also seen with severe tubular necrosis, diabetes mellitus, and trauma cases that cause the release of bone marrow fat from long bones

what would be present in lipid-storage disease?

large-fat laden histiocytes which can be differentiated from oval fat bodies due to their large size

what could also be seen in cases of acute tubular necrosis? what are they referred to?

• RTE cells that have large, nonlipid-filled vacuoles can be seen along with normal renal tubule cells and oval fat bodies 

• referred to as “bubble cells”

  • represents injured cells with the ER has dilated before apoptosis

what are the nonpathological reasons for bacteria to be in urine sediment?

• specimen is contaminated from urethra, vagina, external genitalia, collection container

• specimen was left unpreserved at room temp for longer than 2 hours

  • increases pH to 8

  • may cause positive nitrite

  • cause amount of glucose and ketone to decrease

  • not actually representative of urine

what is bacteria reported as and what will it look like under a microscope?

• reported as few, moderate, or many per hpf

• small and may be present in cocci or bacilli

what is needed for bacteria to be considered significant?

must be accompanied by WBCs and some labs only report bacteria when it is observed in fresh specimens with WBCs

what would correlate well with a positive urine culture?

• the presence of motile organisms in a drop of fresh urine that was collected under sterile conditions

  • motility is also useful in differentiating them from amorphous phosphates and urates which look similar

what does the presence of bacteria indicate? what cause a follow up with a urine culture? what bacteria is associated with UTIs?

• indicative of upper or lower UTI

• specimens with increased bacteria and leukocytes are followed up with specimen urine culture

• bacteria that is associated with UTI would be Enterobacteriaceae (gram-negative)

  • Staphylococcus and Enterococcus can also cause UTIs but are gram-positive which would be negative for nitrites but positive for LE

what would yeast look like under a microscope?

• appears as small, refractile oval structures that may or may not contain a bud

• may also appear branched, mycelial forms in severe infections

what are yeasts reported as?

rare, few, moderate, or many per hpf

where would yeast cells be seen in?

• diabetics

  • acidic, glucose containing urine is an ideal medium for yeast to grow

• immunocompromised

• women with vaginal moniliasis

what should be accompanied with a true yeast infection?

presence of WBCs

what is the most common parasite encountered and describe it

Trichomonas vaginalis

• pear-shaped flagellate with an undulating membrane

• easily ID’d in a wet prep due to its rapid, darting movement but when it isn’t moving it could look like a WBC, transitional, or RTE cell

• sexually transmitted and primarily associated with vaginal inflammation

  • males are asymptomatic → infections of male urethra and prostate are asymptomatic

• reported as rare, few, moderate, or many per hpf

related to parasites, what could also appear in the urine sediment other than Trichomonas vaginalis?

• ova (eggs) of the pinworm Enterobius vermicularis is commonly seen

• ova (eggs) of the bladder parasite, Schistosoma haematobium is seldom seen

• fecal contamination in the urine specimen can result in the presence of ova from intestinal parasites

what would spermatozoa look like under a microscope?

oval, slightly taped heads, long flagella like tails and will rarely exhibit motility