AUBF 15: MICROSCOPIC EXAM OF URINE

Thomas Addis developed a procedure to quantitate formed elements in a 12-hour overnight urine collection. What was the purpose of this test, the Addis count? What indicated disease progression?

to follow the progress of renal disease, particularly acute glomerulonephritis

increased numbers of erythrocytes, white blood cells, or casts in the urine

disease indicated if the number of RBCs exceeded 500,000; the number of WBCs exceeded 2 million; or the number of casts exceeded 5000

Factors that require standardization in the microscopic examination of urine

urine volume used (e.g., 10 ml, 12 ml, 15 ml) speed of centrifugation (400 or 450 x g) time of centrifugation (5 minutes) concentration of sediment prepared (e.g., 10:1, 12:1, 15:1, 30:1) volume of sediment examined--determined by commercial slides used and microscope optical properties (i.e., ocular field number) result reporting--format, terminology, reference intervals, magnification used for assessment

To achieve consistency, several commercial urinalysis systems are available. What does each system seek to consistently do?

(1) produce the same concentration of urine or sediment volume (2) present the same volume of sediment for microscopic examination (3) control microscopic variables such as the volume of sediment viewed and the optical properties of the slides

Benefits of commercial slides (acrylic)

cost competitive easy to adapt to necessary to ensure reproducible and accurate results

What volume of urine is recommended for urinalysis? Pediatric patients?

12 ml, but volumes ranging from 10 ml to 15 ml can be used

for pediatric patients, or other patients, where you may not be able to get 12 ml, the volume of urine can be reduced to 6 ml, and all the numeric counts from the sediment examination must be doubled

After well-mixed urine is poured into a centrifuge tube, how long and at what speed is it centrifuged?

400 to 450 g for 5 min

Why is well-mixed urine centrifuged at a certain speed?

to allow for optimal sediment concentration without damaging fragile formed elements such as cellular casts

How do you calculate what RPM (revolutions per minute) is needed to generate a certain RCF,g (centrifugal force)

RCF (g) = 1.118 x 10^-5 x radius (cm) x RPM^2

Why must the centrifuge brake not be used?

because this will cause the sediment to resuspend, resulting in erroneously decreased numbers of formed elements in the concentrated sediment

Why must you check all centrifuge settings before use?

if other lab personnel use the centrifuge for other tests besides urinalysis, the settings will be different and the resultant urine sediments can show dramatic variations in their formed elements because of processing differences in speed, time, or braking

How is sediment concentrated?

supernatant urine is removed by decanting or using a disposable pipette until 1 ml of urine is retained. Then, a pipette is used to gently resuspend the sediment. Too vigorous agitation of sediment can cause fragile and brittle formed elements, sch as RBC casts and waxy casts, to break into pieces

Why are glass microscope slides and coverslips not recommended for viewing urine sediment?

they do not yield standardized, reproducible results

In a manual microscopic examination, how are urine components assessed or enumerated?

using at least 10 low-power (lpf) or 10 high-power fields (hpf)

How are some components (e.g., mucus, crystals, bacteria) assessed?

qualitatively assessed per field of view (FOV)

How are other components (RBCs, WBCs, casts) assessed?

they are enumerated as a range of formed elements present (e.g., 0 to 2, 2 to 5, 5 to 10)

When a microscopic examination is performed, what is the volume of sediment in each microscopic FOV determined by?

the optical lenses of the microscope and the standardized slide system used

FOV = ocular field number and objective lens; the larger the FOV, the greater is the number of components that may be visible

Qualitative Terms and Descriptions for Fields of View (FOVs)

rare--1+--present, but hard to find few--1+--one (or more) present in almost every field of view moderate--2+--easy to find; number present in FOV varies; "more than few, less than many" many--3+--prominent; large number present in all FOVs packed--4+--FOV is crowded by or overwhelmed with the elements

How do you convert the number of formed elements observed per low- or high-power field to the number present per mL of urine tested?

1. calculate the areas of the low-power and high-power fields of view for your microscope using the formula: Area = (pie)(r)^2

2. calculate the maximum number of low-power and high-power fields possible using your microscope and the standardized microscope slides in use: (total coverslip area for viewing) / (area per high power field (or low-power field)) = # of view fields possible

3. calculate the field conversion factor, which is the number of microscope fields per milliliter or urine tested: (# of view fields possible) / [(volume of sediment viewed (ml) x (concentration factor)] = (number of view fields) / (1 ml of urine tested)

4. convert the number of formed elements observed per high-power field (or low-power field) to the number present per milliliter of urine by multiplying the number observed per view field by the appropriate field conversion factor

What is the purpose of staining?

changes the refractive index of formed elements and increases their visibility

Which elements of urine are hard to see using brightfield microscopy?

hyaline casts, mucous threads, bacteria

Why can visualization of urine sediment components be difficult when brightfield microscopy is used? How is this remedied?

because the refractive index of urine and some sediment components are similar, lacking sufficient contrast for optimal viewing

staining changes the refractive index of formed elements and increases their visibility another approach is to change they type of microscopy, which can also facilitate visualization of low-refractibility components or can be used to confirm the identity of suspected substances such as fat

Sternheimer-Malbin

a supravital stain that characteristically stains cellular structures and other formed elements enables detailed viewing an differentiation of cells, cast inclusions, and low refractile elements (e.g., hyaline casts, mucus) most commonly used stain enhances formed element identification by enabling more detailed viewing of internal structures, particularly WBCs, epithelial cells, and casts

What is one disadvantage of the use of the Sternheimer-Malbin stain?

in strongly alkaline urines, this stain can precipitate, which obstructs the visualization of sediment components

0.5% toluidine blue

another supravital stain a metachromatic stain that enhances the nuclear detail of cells aids in differentiating WBCs and renal tubular epithelial cells stains various cell components differently, hence the differentiation between the nucleus and cytoplasm becomes more apparent aids in distinguishing cells of similar size, such as leukocytes from renal collecting duct cells

2% acetic acid

accentuates the nuclei of leukocytes (WBCs) and epithelial cells lyses RBCs

Fat stains: Sudan III, Oil Red O

stains triglyceride (neutral fat) globules a characteristic orange (Sudan III) or red (Oil Red O) color used to confirm the presence of fat in urine

Where can the lipids (triglyceride/neutral fat) be found after staining?

(1) free floating as droplets or globules (2) within renal cells or macrophages, aptly termed oval fat bodies (3) within the matrix of casts as globules or oval fat bodies

What types of fat stains? Which ones do not?

only neutral fats (e.g., triglycerides) stain

cholesterol and cholesterol esters do not stain and must be confirmed by polarizing microscopy

Are fats normally found in urine?

no

it implicates renal disease because changes have occurred in the glomeruli such that triglycerides and cholesterol from the bloodstream are now passing the glomerular filtration barriers with the plasma ultrafiltrate

Gram stain

identifies and classifies bacteria as gram-negative or gram-positive aids in the identification of bacterial and fungal casts gram-positive = pink gram-negative = dark purple

How do you perform a gram stain?

a dry preparation of the urine sediment is made on a microscope slide by smearing and air drying or by cytocentrifugation; the slide is heat-fixed and stained

Prussian blue reaction stain

identifies hemosiderin, which can be free-floating, in epithelial cells, or in casts stains the iron of hemosiderin granules a characteristic blue

Hansel stain

aids in the identification of eosinophils Wright's stain or Giemsa stain also distinguishes eosinophils, but Hansel stain is preferred

Patients with acute interstitial nephritis caused by hypersensitivity to a medication such as a penicillin derivative can have increased numbers of eosinophils in the urine sediment. Identification of this renal disease is important because it is one of the few renal diseases for which quick and effective treatment is available: cessation of drug administration

Phase-contrast microscopy

enhances the imaging of translucent or low-refractile formed elements and living cells allows identification of traditionally difficult to view formed elements: hyaline casts and mucous threads

Polarizing microscopy

used to confirm the presence of cholesterol globules by their characteristic Maltese cross pattern aids in identification of crystals assists in differentiating "look-alike" component: RBCs vs monohydrate calcium oxalate crystals--polarize light for monohydrate calcium oxalate crystal but NOT for RBCs

Casts, mucus vs fibers (clothing, diapers), plastic fragments--do NOT polarize light for casts, mucus, but DO for the other

Bacteria vs. amorphous crystals (urates: strongly; phosphates: very weakly)--do NOT polarize light for bacteria but DO for the other

Cells, cellular debris (membrane phospholipids) vs cholesterol globules, starch granules--do NOT polarize light for cells, cellular debris but DO for the other

Maltese cross pattern

in polarizing microscopy, cholesterol droplets appear as orbs against a black background divided into four quadrants by a bright Maltese-style cross starch granules also do this, but they can be distinguished from cholesterol by using brightfield microscopy

Interference contrast microscopy

enhances imaging of formed elements by producing three-dimensional images of high contrast and resolution 2 types: differential interference contrast (Nomarski) and modulation contrast (Hoffman) suited ideally for formed elements

Cytocentrifugation

a technique used to produce permanent microscopic slides of urine sediment and body fluids

How is cytocentrifugation performed?

Because a monolayer of sediment components is desired, an initial microscopic examination is required to determine the amount or volume of urine sediment to use when preparing the slide.

The appropriate amount of concentrated urine sediment is added to a specially designed cartridge fitted with a microscopic slide that is placed in a cytocentrifuge.

After cytocentrifugation, a dry circular monolayer of sediment components is fixed permanently using an appropriate fixative and is stained (Papanicolaou's stain or Wright's stain)

The end result is a monolayer of the urine sediment components with their structural details greatly enhanced by staining; this enables the quantitation and differentiation of WBCs and epithelial cells in the urine sediment

cytodiagnostic urinalysis

can play an important role in early detection of renal allograft rejection and in the differential diagnosis of renal disease

involves making a 10:1 concentration of first morning urine specimen, followed by cytocentrifugation of the urine sediment and Papanlaou's staining

uniquely valuable in identification of blood cell types, cellular fragments, epithelial cells (atypical and neoplastic), cellular inclusions (viral and nonviral), and cellular casts

Red Blood Cells (Erythrocytes): Microscopic Appearance

typical form--smooth, biconcave disks, 6-8 um in diameter; no nucleus crenated forms--in concentrated urine (high SG) ghost cells--in dilute urine (low SG) dysmorphic forms and cell fragments

Red Blood Cells (Erythrocytes): Look-alike elements

monohydrate calcium oxalate crystals yeast cells

Red Blood Cells (Erythrocytes): Correlation with physical and chemical examinations

urine color--note that a normal appearing urine can still have increased RBCs present blood reaction--can be negative owing to ascorbic acid interference; degree of interference varies with reagent strip brand

RBCs in hypertonic urine--crenated forms

RBCs become smaller as intracellular water is lost from the cell by osmosis, which causes them to become crenated = appear rough microscopically compared with normal erythrocytes

RBCs in hypotonic urine--"ghost" cells

erythrocytes swell and release their hemoglobin to become "ghost" cells, which are cells with intact cell membranes but no hemoglobin

difficult to see using brightfield microscopy; readily visible with phase-contrast or interference contrast microscopy

RBCs in alkaline urine

alkaline urine promotes red blood cell lysis and disintegration, resulting in ghost cells and erythrocyte remnants

Dismorphic or distorted erythrocytes

occasionally present with normal erythrocytes in the urine of healthy individuals

increased #'s of particularly acanthocytes are associated with glomerular disorders

sickle cells = sickle cell disease

RBC counts

normal = 0-3 per high-power field or 3-12 per microliter of urine sediment

Renal bleeding: either glomerular or tubular

increased numbers of RBCs along with red blood cell casts

Renal bleeding: below the kidney or due to contamination

increased # of RBCs without casts or proteinuria

Red urine

red blood cells present

positive chemical test for blood but microscopic exam reveals not RBCs

RBCs readily lyse and disintegrate in hypotonic or alkaline urine; such lysis can also occur within the urinary tract before urine collection; as a result, urine specimens can be encountered that contain only hemoglobin from RBCs that are no longer intact or microscopically visible

other substances, such as myoblobin, microbial peroxidases, and strong oxidizing agents can cause a positive blood chemical test

false-positives because RBCs or blood is not present

RBCs present microscopically but a chemical screen for blood is negative

ascorbic acid interference should be suspected

if ascorbic acid is ruled out, it is possible that the formed elements observed are not RBCs but a "look-alike" component such as yeast or monohydrate calcium oxalate crystals; in this case the identity should be confirmed by an alternative technique such as staining or using polarizing microscopy

How much hemoglobin must be present in urine to be detected by routine protein reagent test strips?

an amount exceeding 10 mg/dl

Even though hemoglobin is a protein, in most cases of hematuria, it does not contribute to the protein result obtained by the chemical reagent test strip. What results are obtained if it is contributing?

when the chemical reagent strip test for blood reads less than large (3+), hemoglobin is NOT causing or contributing to the protein result; when blood result is greater than or equal to large (3+), hemoglobin may be contributing to the protein reagent strip test results

crenated RBCs vs WBCs

use acetic acid or toluidine blue stain = easier to see the nuclei of WBCs

RBCs vs yeast and calcium oxalate crystals

a Sternheimer-Malbin stain characteristically colors RBCs, whereas neither yeast nor calcium oxalate crystals stain

polar microscopy can identify calcium oxalate crystals

2% acetic acid can be added, which lyses RBCs but does not eliminate yeast or calcium oxalate crystals

yeast varies in size and tends to be spherical or ovoid rather than biconcave, and often exhibits budding

RBCs vs small bubbles or droplets of oil contaminating the urine sediment

distinguished from RBCs by their variation in size, uniformity in appearance, and high refractility

RBCs--Clinical significance: Hematuria

numerous conditions can result in hematuria

smoking as well as exercise can cause hematuria

anticoagulant drugs and drugs that induce toxic reaction, such as sulfonamides, can also cause increase RBCs numbers in urine sediment

any condition that results in inflammation or that compromises the integrity of the vascular system throughout the urinary tract can result in hematuria

specimens contaminated with blood from vaginal secretions or hemorrhoidal blood can falsely imply hematuria

Reference Intervals: RBCs

number = 0 to 3 magnification = per hpf

Reference Intervals: WBCs

number = 0-8 magnification = per hpf

Reference Intervals: Casts

number = 0 to 2 hyaline (or finely granular) magnification = per lpf

Reference Intervals: Epithelial cells--Squamous

number = few magnification = per lpf

Reference Intervals: Epithelial cells--Transitional

number = few magnification = per hpf

Reference Intervals: Epithelial cells--Renal

number = few magnification = per hpf

Reference Intervals: Bacteria and yeast

number = negative magnification = per hpf

Reference Intervals: Abnormal crystals

number = none magnification = per lpf

Which urine components are viewed using high power?

red blood cells white blood cells transitional epithelial cells renal epithelial cells bacteria and yeast

Which urine components are viewed using low power?

casts squamous epithelial cells abnormal crystals

WBCs: Neutrophils: Microscopic features

spherical cells, 12-14 um in diameter granular cytoplasm lobed nuclei glitter cells--dilute urine (low SG)

WBCs: Look-alike elements

renal tubular epithelial cells (collecting ducts) dead trichomonads crenated red blood cells

WBCs: correlation with physical and chemical examinations

leukocyte esterase reaction--can be negative despite increased WBCs owing to excess hydration or when the WBCs are lymphocytes negative nitrite reaction: suggestive of inflammation or nonbacterial infection positive nitrite reaction: suggests bacterial infections

WBC distribution in urine in healthy individuals

mirrors that of peripheral blood

List the 5 types of WBCs that can be found in urine.

neutrophils basophils eosinophils lymphocytes monocytes (macrophages)

What WBC is the one that predominates in urine and peripheral blood? When are the other WBCs the predominant ones found?

neutrophils

with some renal conditions, other leukocytes predominate in urine; for example, in acute interstitial nephritis caused by drug hypersensitivity, the predominate leukocytes observed are eosinophils, whereas in renal allograft rejection, lymphocytes predominate

Which leukocyte predominates in urine during acute interstitial nephritis caused by drug hypersensitivity?

eosinophils

Which leukocyte predominates in urine during a renal allograft rejection?

lymphocytes

Neutrophils: Microscopic appearance

approx. 14 um in diameter, but range from 10 to 20 um, depending on the tonicity of the urine larger than erythrocytes similar in size to epithelial cells characteristic cytoplasmic granules and lobed or segmented nuclei unstained = grayish hue and appear grainy may occur singly or aggregated in clumps; clumping, which often occurs in acute inflammatory conditions, makes their enumeration difficult fresh urine specimens = features readily apparent by brightfield microscopy

What makes neutrophils hard to distinguish from renal tubular cells?

as neutrophils age they begin to disintegrate, their lobed nuclei fuse, and they can resemble a mononuclear cells; these changes make it difficult to distinguish them from renal tubular collecting duct cells

neutrophils in hypotonic urine--"glitter cells"

causes them to swell and become spherical balls that lyse as rapidly as 50% in 2 to 3 hours at room temperature

in these large swollen cells, brownian movement of the refractile cytoplasmic granules is often evident = "glitter cells"

neutrophils in hypertonic urine

become small as water is lost osmotically from the cells, but they do not crenate

neutrophils--formation of blebs (aging/disintegrating neutrophil)

vacuoles develop within the cell periphery or on their outer membranes; they appear to be empty or may contain a few small granules

as these changes continue, the blebs or vacuoles can detach and become free floating in the urine; they may also develop and remain within the cell, pushing the cytoplasm to one side and giving rise to large pale areas intracellularly

neutrophils--myelin forms (aging/disintegrating neutrophil)

numerous finger-like projections protruding from the cell surfaces

result from breakdown of the cell membrane

as these cells die, additional vacuolization, rupturing, or pseudopod formation may be observed

How is semiquantitation performed during microscopic examination of leukocytes?

by observing 10 representative high-power fields and determining the average number of WBCs present in each field

normal = 0 to 8 WBCs per high-power field or approx. 10 WBCs per microliter of urine sediment

Upper urinary tract infections

WBC casts in the urine

cellular casts (i.e., cell identity cannot be determined)

granular casts (result from cell degradation)

positive protein reagent strip

Lower urinary tract infections (below kidney)

increased WBCs but without cellular casts; if protein is present, it is usually at a low level

WBCs: physical findings

cloudy = WBCs in increased amounts strong, foul odor = extent of infection is great macroscopic exam of sediment button = large amount of gray-white material = the concentrated leukocytes

positive LE test despite few or no WBCs present microscopically

can occur because of WBC lysis and disintegration

different populations of WBCs have varying quantities of cytoplasmic granules and therefore differing amounts of leukocyte esterase; in fact, lymphocytes have no leukocyte esterase

negative LE test with increased #'s of WBCs present in urine

must ensure that the cells are granulocytic leukocytes, and that reagent strips are functioning properly

although the LE screening test usually detects 10 to 25 WBCs per microliter, the amount of esterase present may be insufficient to produce a positive response

owing to hydration, hypotonic urine could cause the leukocyte esterase to be diluted such that it is below the detection limit of the LE reaction

Leukocytes vs RBCs and renal tubular epithelial cells

use 2% acetic acid solution or a 0.5% toluidine blue stain to reveal the nuclear details present to determine proper cell identification

Leukocytes with cytoplasmic granulation vs collecting duct cells

collecting duct cells = large, dense nuclei and polygonal shape

staining with Sternheimer-Malbin stain or toluidine blue

Eosinophils vs neutrophils

are slightly larger than neutrophils and have bilobed nuceli

when specifically requested, eosinophils should be centrifuged and stained using Hansel stain

eosinophiluria

acute interstitial nephritis (AIN) occasionally, chronic urinary tract infections (UTIs)

What is the presence of eosinophil casts diagnostic of?

good predictor of AIN associated with drug hypersensitivity, particularly hypersensitivity to penicillin and its derivatives

eosinophils and acute allograft rejection

presence of large #'s of eosinophils in a kidney biopsy specimen is considered a poor prognostic indicator

WBCs: Lymphocytes: Microscopic features

spherical cells, 6-9 um in diameter mononuclear--single round to slightly oval nucleus and scant clear cytoplasm that usually extends out from one side of the cell

Lymphocytes are usually not recognized because of their small numbers. What should you do to make them more readily apparent and identifiable?

use a supravital stain or do a cytodiagnostic urinalysis using Wright's or Papanicolaou's stain

When are lymphocytes usually present in urine?

they are normally present in urine in small amounts but are present in increasing #s during inflammatory conditions such as acute pyelonephritis

lymphocytes predominant over neutrophils from patients experiencing renal transplant rejection

will NOT provide a positive LE test because they do NOT contain leukocyte esterases

WBCs: Monocytes and Macrophages: Microscopic features

spherical cells, 20-25 um in diameter granular cytoplasm mononuclear--round to oval cytoplasm often vacuolated with ingested debris azurophilic granules

What are the primary functions of monocytes/macrophages?

defend against microorganisms removed dead or dying cells and cellular debris interact immunologically with lymphoid cells