Point of Care
Vol. 2, No. 1, 20–32
© 2003 Lippincott Williams & Wilkins, Inc., Philadelphia
Provider-Performed Microscopy
A Review
Frederick L. Kiechle, MD, PhD, Isabel Gauss, MT(ASCP), and Barbara Robinson-Dunn, PhD
The examinations of labile specimens under the bright-field or phase contrast microscope are classified as provider-performed microscopy (PPM). With a PPM certificate
issued by the Centers for Medicare and Medicaid Services Administration, a physician,
nurse practitioner, nurse midwife, physician assistant, or dentist may perform PPM and
waived procedures. Provider-performed microscopy procedures use specimens, such as
body fluids or skin scrapings. Because quality control material is not available for PPM
procedures, quality assurance assessment is complicated. Policies and procedures must
be written for each test procedure. Provider-performed microscopy procedures include
wet mounts, KOH preparations, pinworm detection, fern test, postcoital test, microscopic urinalysis, fecal leukocytes, semen analysis for presence of sperm and motility,
and eosinophils in nasal smears. The quality of PPM is dependent on adequate training
and retraining to achieve optimal skills.
Key Words: Provider-performed microscopy—Waived tests—Bright-field microscopy—Phase-contrast microscopy—KOH preparation—Wet mounts—Pinworm detection—Postcoital test—Microscopic urinalysis—Semen analysis—Nasal eosinophils.
rovider-performed microscopy (PPM) refers
to a group of procedures that evaluate labile
specimens using bright-field or phase-contrast
microscopy, which were defined in the Federal Register
of April 25, 1995 and revised on October 1, 1998. A
summary of the criteria for these PPM procedures is
listed in Table 1. The evaluation of synovial fluid by
phase-contrast microscopy for monosodium urate
crystals (gout) or calcium pyrophosphate dihydrate
crystals (pseudogout)1,2 is excluded from this group
of PPM procedures. The microscopic evaluation of
P
From the Department of Clinical Pathology, William Beaumont Hospital,
Royal Oak, Michigan.
Address correspondence and reprint requests to Frederick L. Kiechle, MD,
PhD, Department of Clinical Pathology, William Beaumont Hospital, 3601
West 13 Mile Road, Royal Oak, MI 48073–6769
(e-mail: fkiechle@beaumont.edu).
20
crystals in synovial fluid may be performed initially
with an ordinary bright-field microscope (Fig 1);
however, the definitive identification of the crystals
requires a polarizing microscope. 2 Providerperformed microscopy procedures use specimens
such as body fluids or skin scrapings (Table 2).3 The
federal law described by Centers for Medicare and
Medicaid Services (CMS) Administration in the Clinical Laboratory Improvement Amendments of 1988
(CLIA’88) classify laboratory procedures based on
their complexity using well-defined criteria: waived,
moderately complex, provider-performed microscopy, and highly complex.4,5 Provider-performed
microscopy, as defined by CLIA’88, is a subset of tests
classified as moderately complex. Quality assurance
assessment of these PPM procedures is complicated
by the absence of a readily available source of quality
Table 1
Criteria for provider-performed microscopy
procedures
1. The procedure must be categorized as moderately complex.
2. The examination must be personally performed by a practitioner.
3. The primary instrument for performing the test is the microscope, limited
to bright-field or phase-contrast microscopy.
4. The specimen is labile, or delay in performing the test could compromise
the accuracy of the test result.
5. Limited specimen handling or processing is required.
6. Control materials are not available to monitor the entire testing process.
control testing material (Table 1). However, there is
the potential for developing home brew material to
simulate the fern test or others.6
The CMS issues certificates of registration, which
may be limited to PPM or waived testing (Table 3). A
certificate for performing waived testing is not sufficient to cover PPM testing. However, waived testing
may be performed under a PPM certificate. With a
PPM certificate, only physicians, nurse practitioners,
nurse midwifes, physician assistants, or dentists may
perform the testing at the time of the patient’s visit. A
PPM certificate costs $200 every 2 years and in a hospital, it is usually obtained by a physician designated as
responsible for PPM testing in a specific area.6
The Joint Commission on Accreditation of Health
Care Organizations (JCAHO) indicates that documentation of competence or training is not required
for physicians or dentists when the test is a logical part
of his or her specialty.7 However, if the test is performed by a midlevel practitioner, like a nurse practitioner, nurse midwife, or physician assistant, docu-
mentation of initial training and ongoing competency
assessment is required. The frequency of the determination of competency assessment is a decision left to
the health care organization.
Also, JCAHO states that there should be a system
in place to ensure the accuracy and reliability of the
test results.7 Although proficiency testing is not required, it could be used to meet this standard. Kodachrome or digital images could be used to organize
proficiency and competency testing. Intranet-enabled
competency challenges may be custom built using Microsoft (Redmond, WA) technologies, such as Active
Server Pages and Access and filing user details or using
Healthstream (Nashville, TN) (formerly “m3 Learning” (Dallas, TX).8 The latter uses a combination of
third-party authoring software (Macromedia Authorware, San Francisco, CA) and server scripting. Both
formats provide the user with a list of questions. The
answers may be scored and calculated as a percent of
correct responses. Electronic documentation of test
Table 2
Provider-performed microscopy tests and CPT
codes
CPT Code
Test Description
Q0111
Wet mounts, including preparations of vaginal, cervical or
skin specimens
Q0112
All potassium hydroxide (KOH) preparations
Q0113
Pinworm examinations
Q0114
Fern test
Q0115
Post-coital direct, qualitative examinations of vaginal or cervical mucous
81015
Urinalysis; microscopic only
81000
Urinalysis, by dipstick or tablet reagent for bilirubin, glucose,
hemoglobin, ketones, leukocytes, nitrite, pH, protein, specific gravity, urobilinogen, or any number of these constituents; non-automated, with microscopy
81001
Urinalysis, by dipstick or tablet reagent for bilirubin, glucose,
hemoglobin, ketones, leukocytes, nitrite, pH, protein, specific gravity, urobilinogen, or any number of these constituents; automated, with microscopy
NOTE: May only be used when the laboratory is using an
automated dipstick urinalysis instrument approved as
waived.
Fig 1
Colorless monosodium urate crystals (bright field,
original magnification x 10).
Point of Care, vol. 2, no. 1
81020
Urinalysis; two or three glass test
G0026
Fecal leukocyte examination
G0027
Semen analysis; presence and/or motility of sperm excluding
Huhner
89190
Nasal smears for eosinophils
Centers for Medicare and Medicaid Services, as of April 1, 2002.
CPT; Current procedural terminology.
Provider-Performed Microscopy
21
information permits filterable reports that may be
sorted by physician or date for documentation of
compliance activity. Alternatively, this goal may be
accomplished by purchasing the Excel program XL-G
from the College of American Pathologists (CAP). In
conclusion, competency indicators for PPM might include monitoring the number of proficiency-testing
or blind samples testing failures or failed procedural
observations.9 In this way, the staff and resident
physician performing these procedures may be
monitored.
Policies and procedures must be written for each
test procedure.9 The procedure manual should include a section on specimen collection, method, handling and disposal of infectious specimens, interpretation, and equipment maintenance. Maintenance includes both the microscope and the centrifuges,
which should be in good condition and routinely serviced. All PPM results should be filed in the patient’s
chart, along with the date and name of the person who
performed the test.6,9 Regulatory agencies evaluate
the performance of PPM testing using the same criteria as all other laboratory procedures with the exception of quality control records (Table 1). A recent
evaluation by CMS of laboratories performing PPM
revealed a variety of quality problems.10 The top four
quality issues included 38% of sites did not evaluate
test accuracy two times a year, 36% had no microscope and/or centrifuge maintenance, 28% had no director approved standard operating procedure
manual, and 25% did not document personnel
competency.10
PPM Procedures: Examples
KOH and wet mount preparations
Direct microscopic examination of many clinical
specimens with saline or with potassium hydroxide
(KOH) can reveal the presence of microorganisms
such as fungi and Trichomonas vaginalis. This test can
also be used to demonstrate the presence of clue cells
indicative of bacterial vaginosis.
Potassium hydroxide is used for direct examination
of hair, skin scrapings, nails, and fluids (e.g., vaginal
exudate). The fungal cell wall contains chitin making
the fungi relatively resistant to the lytic action of
KOH.11 Because of this property KOH can be used to
22
F. L. Kiechle et al
dissolve clinical material (either solid or liquid) leaving the fungal elements. Over time (2 to 3 days), the
KOH will also dissolve any fungi present in the
sample. To prevent this, a small amount of glycerol
added to the KOH solution will protect the fungi for
several days.12
Skin scrapings should be obtained from the leading
edges of a presumed “ringworm” lesion. Often, a
Wood’s lamp can be used to localize the area from
which the specimen should be obtained. Care must be
taken to ensure that clinical specimens are not obtained with cotton or fiber swabs because those fibers
may resemble fungal elements when viewed under
the microscope.
A 10% aqueous solution of KOH is used in this
test. The KOH must be stored in a nonglass vial because the extremely basic solution will leach minerals
from glass thus rendering the solution cloudy and unusable.13 A drop of KOH is mixed with the clinical
specimen on a clean glass slide and a cover slip is
placed over the specimen. The specimen is then gently warmed and should still be cool enough to be
touched. Do not allow the KOH to boil and evaporate. The slide preparation must “cure” at room temperature until the clinical material clears (dissolves).
The amount of time necessary for digestion of the
proteinaceous material varies with the type and size of
the specimen. For example, a piece of fingernail will
take much longer to dissolve than will a skin scraping.
A brightfield microscope is commonly used to examine KOH and wet mount preparations. Because
most microorganisms and human cells have a very low
refractive index (unless they are dematiaceous or
darkly colored), it is necessary to reduce the light
used for viewing the specimen. After the slide preparation is placed on the stage of the microscope, the
condenser, which serves to focus the beam of light,
should be lowered. (The condenser is the component
of the microscope that is between the stage and the
light source.) The specimen can be screened on low
power and then examined in more detail on high
power.
Saline is used to emulsify vaginal fluids for microscopic examination. A drop of saline is mixed with a
drop of the specimen on a clean glass slide and then
covered with a glass coverslip. It may be gently
Point of Care, vol. 2, no. 1
pressed down on the slide to provide for better viewing through the suspension. When examining the saline wet mount on low power, the microscopist
should look for clue cells and for movement due to
Trichomonas vaginalis. Clue cells are epithelial cells
whose edges are obscured by the presence of adherent
bacteria (Fig 2). T. vaginalis is a flagellated protozoan
parasite whose movement has been described as
“jerky.” Trichomonads are 7 to 23 µm long and 5 to
15 µm wide, with an obvious axostyle and an undulating membrane that stops halfway down the side of
the protozoan (Fig 3).14 Because they are often difficult to see in a wet mount, it may be necessary to
examine several specimens before finding T. vaginalis
in the specimen. As it slows down, the undulating
membrane may become visible. Specimens to be examined for the presence of T. vaginalis must not be
refrigerated because the protozoan will rapidly become nonviable.
If the clinical specimen has been digested by KOH,
it should be relatively easy to determine if fungal elements are present in the specimen. It is not necessary
to use an oil immersion lens to examine a KOH preparation of a clinical specimen and may actually provide
some degree of confusion and “over-reading” of the
specimen if it is used. The fungal elements will either
appear as small pieces of septate hyphae or as budding
yeasts (oval or elongated) (Fig 4).
Cellulose tape preparations for detection
of pinworms
Infection with pinworms (Enterobius vermicularis) is
one of the most common parasitic diseases and has
Fig 2
Clue cells in vaginal specimen. Epithelia cell edges are
obscured by bacteria (bright field, original magnification x 40).
Point of Care, vol. 2, no. 1
Fig 3
Wet mount of Trichomonas vaginalis (right) and two
epithelial cells (left) (bright field, original magnification x 40).
been recognized since ancient times. Pinworms are
acquired by fecal-oral contamination via unwashed
hands, soiled pajamas, contaminated toys, or other
objects. Eggs contaminating the environment can be
killed by sunlight or radiation from ultraviolet lights.
Following ingestion of infective eggs, the parasites
hatch and develop into adult worms. The female
worm matures and within approximately 1 month, is
capable of producing eggs. Following fertilization by
the male worm, the female migrates out of the anus
and deposits eggs on the anal and perianal surfaces (Fig
5). The eggs mature rapidly and are fully infective
within a few hours.
Persons infected with pinworms complain of intense itching, irritability, and insomnia. The itching is
due to the female worm migrating out of the anus to
deposit her eggs. Since the worms migrate and lay
eggs at night, specimens should be obtained after the
patient has been asleep for several hours or early in the
morning before the patient has used the bathroom.
The irritability and insomnia that are common complaints from a person suspected of pinworm infection
are also due to the itching, which interrupts normal
sleep patterns.
The specimen to be examined for pinworms consists of touch preparations of anal and perianal skin
and can be obtained by wrapping a piece of clear cellulose tape (sticky side against the glass slide) lengthwise around the end of a glass slide. To obtain the
Provider-Performed Microscopy
23
Fig 4
(A) KOH preparation of fungal elements (oval yeast and hyphae) in a skin scraping (bright field, original magnification x 40).
(B) KOH preparation of budding oval yeast in a vaginal exudate (bright field, original magnification x 40).
specimen, one end of the tape is released. A tongue
depressor is placed against the side of the tape that is
still secured to the slide and the free end of the tape is
bent back against it (sticky side out). The tape is then
pressed against several of the areas in which the itching is intense.
To examine the specimen microscopically, the side
of tape that has been used to collect the specimen is
bent back around the slide and pressed against it. A
drop of xylene or toluene may be added under the
edge of the tape to clear it. The glass slide/tape preparation is placed on the stage of the microscope with
the specimen on the top of the slide. The slide is examined under low power (10 × or 20 ×) with reduced
light intensity. If a pinworm has deposited eggs, they
will appear to be football-shaped with one side flattened and are 20 to 30 µm by 50 to 60 µm in size (Fig
Fig 5
Female Enterobius vermicularis warm releasing eggs
(bright field, original magnification x 10).
24
F. L. Kiechle et al
6). It is necessary to examine 4 to 6 consecutive negative tape preparations to rule out infection. Additionally, it is not necessary to submit fecal samples to the
laboratory for diagnosis of pinworms as they are only
occasionally recovered from these specimens.15
Fern test
The fern test is performed to determine if rupture of
the fetal membranes has occurred before the onset of
labor. This premature rupture of membranes occurs
in approximately 10% of pregnant patients. The test
is performed by placing a small amount of vaginal fluid
onto a glass slide, which is allowed to air dry. The
slide is then examined microscopically for fern formation (Fig 7). The fern pattern occurs when the relative
Fig 6
Scotch tape preparation demonstrating Enterobius vermicularis eggs (bright field, original magnification x 10).
Point of Care, vol. 2, no. 1
Fig 7
Dried amniotic fluid on a slide demonstrating a positive fern test with an arborization pattern (bright field, original
magnification x 10).
concentration of electrolytes, proteins, and carbohydrates, especially sodium chloride, is sufficient to support crystallization in an arborization pattern.16,17
The salt concentration is determined by the amount of
estrogen in the mucus. Amniotic fluid will crystallize
after 20 weeks gestation. If the specimen is amniotic
fluid, it will dry in the characteristic fern-like pattern.
Other body fluids that will display this slide arborization include CSF, serum, and saliva.18,19 Urine will
not dry in this unique pattern.6
Fern testing is considered to be as sensitive but less
specific than Nitrazine pH testing.18 Meconium and
blood at dilutions of less than 1:1 will not interfere
with ferning. The best sensitivity can be achieved by
using a combination of medial history, ferning, and
Nitrazine testing.18 Estrogen promotes crystallization
while progesterone inhibits it. Alpha fetoprotein, fetal fibronectin, and other cytologic stains may be useful in evaluating patients with possible premature rupture of membranes, but are more costly.18,19
Postcoital test
The postcoital test evaluates factors that affect fertility
and is performed during the middle of the ovulatory
cycle. After 3 days of sexual abstinence, the patient
should remain in bed 10 to 15 minutes following coitus to allow the semen to contact the cervical mucus.
A sterile speculum is used to aspirate an endocervical
mucus specimen, which should be collected within 24
hours of coitus.20 The complete postcoital test20–22
includes qualitative assessment (PPM procedure,
Point of Care, vol. 2, no. 1
Table 2) including color, viscosity, tenacity, and the
presence of ferning, as well as direct microscopic examination of sperm number and motility in cervical
mucus (Sims-Huhner test; not a PPM procedure,
Table 2). The cervical mucus should be clear with
minimal viscosity. Its elasticity or spinnbarkeit increases at ovulation. A positive cervical mucus test is
defined as a thread of mucus attached to a glass coverslip lifted from a glass slide on which cervical mucus
has been placed.20,21 The clinical utility of the qualitative postcoital cervical mucus tests are in dispute.22–25 For example, qualitative cervical mucus
characteristics were not predictive of a couple’s future pregnancy, whereas quantitative sperm characteristics were predictive.22 The reverse was found to
be true when inducing singleton pregnancies in anovulatory women treated with human menopausal
gonadotropin.24
Urinalysis: microscopic
A complete urinalysis includes visual, microscopic,
and chemical examination of urine.26 The kidneys and
urinary tract are not available for direct physical examination.27 Therefore, evaluation of a urine specimen represents a liquid tissue biopsy of the urinary
tract.3,26 Uroscopists or pisse-prophets used a urine
circle composed of 21 segments describing urine
color compared with known substances and associated clinical interpretation during the Byzantine era
(330 to 1453 AD).28–30 For example, green coloration at the top of the specimen was associated with
pleurisy.30 Today, green urine is associated with the
presence of a variety of substances including bile pigment, porphyrins, mitoxantrone, and food dye and
color blue number 1.28,31,32 The invention and introduction of scientific methods provided early reports
of urinary microscope crystals in the 17th and 18th
century, followed by more systematic evaluation
of urinary sediment in the 19th century.33,34 The
most accurate laboratory data is obtained from a
specimen that has been properly collected.26,28 A
contaminated specimen should be suspected if a large
number of squamous epithelial cells and bacteria are
observed.35
Formed elements in the urinary sediment including red cells (Fig 8), red cell casts (Fig 9), white cells
Provider-Performed Microscopy
25
Fig 8
Red cells in urine sediment (bright field, original magnification x 10).
Fig 10
Polymorphonuclear leukocytes in urine sediment (oil
immersion, original magnification x 50).
(Fig 10), white cell casts (Fig 11), tubular casts,
granular casts, and fatty casts are associated with glomerular, tubulointerstitial, or vascular kidney disease. Acute allergic interstitial nephritis is associated
with the presence of urinary eosinophils.36 These
formed elements are unstable in urine. Urinary microscopy should be performed within 1 hour using
storage at room temperature or within 4 hours using
storage in refrigeration. In the pediatric age group,
white cells are less stable and begin to degrade between 2 and 4 hours in the refrigerator.37 In a CAP
Q-Probe Study,38 11.2% of never-refrigerated urine
specimens from hospitalized patients were evaluated
after 2 hours. To prevent this degradation of formed
elements over time, a variety of preservatives have
been evaluated.39,40 Some cellular elements exhibit
improved survival in the presence of preservative after 3 days compared with refrigeration without a pre-
servative.39 However, statistical significance between
the differences was not achieved.
Variations also exist in specimen preparation. The
urinary elements may be examined under the microscope after centrifugation and resuspension of the pellet26,40,41 or in unspun urine39 using the Kova system
(ICL Scientific, Fountain Valley, CA)42 or a hemocytometer for leukocyte counts.43 For centrifuged urine
specimens, 12 mL of well-mixed, fresh urine should
be spun for 400 × g for 5 minutes. The supernatant
should be decanted and the sediment resuspended. A
drop of resuspended sediment should be placed on a
glass slide and a coverslip placed on top. Microscopic
examination by bright field or phase contrast may be
initiated after the cellular elements have been allowed
to settle for 30 to 60 seconds.3 The centrifugation
process may remove protein including casts and lipids, which will adhere to the glass surface of the tube41
and crystal formation may be altered.44
Fig 9
Red cell cast in urine sediment (bright field, original
magnification x 50).
26
F. L. Kiechle et al
Fig 11
Polymorphonuclear leukocyte cast in urine sediment
(bright field, original magnification x 50).
Point of Care, vol. 2, no. 1
The relationship between laboratory testing regulations on the availability of laboratory tests and quality practices in physician offices has been investigated.45–47 Generally, there is lower availability of
tests and a reduced number of tests performed in physician office laboratories in states with laboratory
regulations for the evaluation of urinary tract infections.45 For example, the American Academy of Pediatrics practice parameter48 recommends urinalysis
as a screening method for urinary tract infection in
young children. Results that are suggestive of a urinary tract infection in a young child or infant include a
positive leukocyte esterase or nitrite on a urine dipstick analysis (a waived procedure), greater than five
white cells per high power field on a spun urine sediment (a PPM procedure), or the presence of bacteria
in an unspun gram-stained specimen (a moderately
complex procedure). Forty-six percent of physician
office laboratories have a certificate to perform only
waived procedures and 32% have a PPM certificate,
which would allow them to perform the first two procedures (waived and PPM) only (Table 3). Therefore,
all three laboratory evaluations in the practice parameter cannot be adequately completed in most physician office laboratories.49
The three glass test (CPT code 81020, Table 2) of
Meares and Stamey is used to evaluate the pathogenic
site of urinary tract problems in males.50 The urine is
collected sequentially in three containers and each is
examined microscopically. Abnormal findings (white
cells, bacteria, etc) in the first collection container
Table 3
would represent a urethral problem; the second,
bladder or upper urinary tract; the third, prostate,
and this collection has the highest concentration of
prostatic secretions. A two glass test has been used to
screen for urethritis in males,51,52 as well as females.53 The scientific basis and clinical usefulness of
the two glass test has been questioned.51,52
Fecal leukocytes
The cause of loose, watery stools observed in patients
with diarrhea may be evaluated using bacterial culture, investigation for ova and parasites, and fecal leukocytes.54 To perform this PPM procedure, a fleck of
stool or mucus is placed on a glass slide and stained
with Wright’s or Löffler methylene blue.55 After
placing a coverslip over the mixture, a rough quantitative count of mononuclear and polymorphonuclear
cells are distinguished among 200 total cells or their
numbers are estimated per high power field. Greater
than five leukocytes per high power field is usually
considered positive.56,57 At five leukocytes per high
power field, the test’s sensitivity (63.2%) and specificity (84.3%) is similar to that reported for fecal lactoferrin,57 but not as good as myeloperoxidase.58 The
sensitivity of the fecal leukocyte count in the detection of shigellosis is dependent on the collection technique.59 The cup stool specimen (95% sensitive) had
a sensitivity greater than swab or diaper specimens
(44% sensitive).
The presence of fecal leukocytes indicates the presence of an inflammatory condition caused by a specific
CLIA registration and fees
Enrollment
No. of Labs
No. of
Physician
Office
Laboratories
Total laboratories in nonexempt states
167,744
96,922
—
—
—
Total laboratories in CLIA exempt
states (New York, Washington)
5,447
Biennial Fee
Application type
Compliance (CMS surveys)
22,056 (13%)
14,715 (15%)
—
Waiver
91,516 (55%)
44,111 (46%)
$150
Provider-performed microscopy
37,455 (22%)
31,376 (32%)
$200
Accreditation by CMS approved
organization
16,717 (10%)
6,720 (7%)
Volume dependent
$150–$6,220
Centers for Medicare and Medicaid Services (CMS), data from October, 2001.
CLIA; Clinical Laboratory Improvement Amendments of 1988.
Point of Care, vol. 2, no. 1
Provider-Performed Microscopy
27
organism like Salmonella, Shigella, invasive Escherichia
coli, Campylobacter jejuni, Vibrio cholerae, Yersinia or
Entamoeba histolytica, or a nonspecific etiology like ulcerative colitis, antibiotic-associated colitis, or collagenous colitis.54,55,59–64 Cases of diarrhea are associated with viruses, most parasites (excluding Entamoeba) or bacterial toxins are not associated with the
presence of fecal leukocytes.
Semen analysis
Semen or seminal fluid is composed of products contributed by various male reproductive organs including seminal vesicles, prostate, epididymis, vas deferens, bulbourethral glands and urethral glands.65,66
Spermatozoa are maintained in semen. For example,
seminal vesicular secretions are important for the
maintenance of normal semen coagulation, sperm
motility, and sperm chromatin stability.66 Semen
analysis may be performed to evaluate an infertile
couple,23,67 to detect sexual assault,68 or to investigate the effectiveness of bilateral vasectomy or the
sperm’s suitability for use in artificial insemination
procedures.67,69 Qualitative semen analysis, limited
to the presence or absence of sperm and the detection
of sperm viability, is classified as a PPM procedure
(CPT code G0027, Table 2). This CPT code does not
include the evaluation of the number of motile sperm
per high power field in cervical mucus during the
postcoital or Sims-Huhner test.21,23 Postvasectomy
patients should provide several semen specimens to
assure that no viable or nonviable sperm are present
(Fig 12). A wet mount preparation on a glass slide
with a coverslip is adequate. In the emergency center,
a saline wet mount of a swap of a specimen obtained
from a sexual assault victim may be used for a pointof-care search for motile or nonmotile sperm under a
microscope.68
Semen should be collected in a sterile container by
masturbation or with a special collection condom
during intercourse after a minimum of 2 days, but not
longer than 5 days, of sexual abstinence.23 The viscus
yellow-gray semen forms a coagulum in which the
sperm are trapped. Liquefaction begins within 10 to
20 minutes at room temperature and is complete
within 1 hour.23,65 Laboratory evaluation should be
performed with 1 to 2 hours after collection.
Semen analysis for the evaluation of the infertile
couple and adequacy for artificial insemination procedures requires additional procedures including assessment of physical semen characteristics, sperm concentration and motility grading, sperm morphology,
sperm viability, and bacteriologic studies.23,65,67,69
Although the percent of sperm moving forward is
used to assess sperm mobility and quality, it is not
highly correlated with the fertilizing capacity of semen samples.70,71 To successfully fertilize an egg,
sperm must pass through a substantial cumulus cell
layer, then the thick extracellular matrix, the zona
pellucida, and finally the plasma membrane.72 To
complete this journey, sperm are hyperactivated, resulting in an increase in flagellar bend amplitude and
beat asymmetry.73 At a biochemical level, hyperactivation requires the opening of a voltage-dependent
Ca2+-channel (CatSper).74 CatSper mRNA and protein are only found in testis and the Ca2+-channel is
located on the plasma membrane of the principal
piece of sperm. It is required for normal sperm motility and for sperm penetration of the zona pellucida.74 The evaluation of the presence of sperm and
sperm motility represents the first screen for genetic
mutations that may result in male sterility (mutations
in calmodulin-dependent protein kinase, Camk4,
which phosphorylates protamine-2) or complete arrest of late spermiogenesis and increased germ cell
apoptosis (mutations in TATA-binding proteinrelated protein, TLF, which regulates transcription
during mammalian spermatogenesis).75
Eosinophils in nasal smears
Fig 12
Viable sperm in semen (bright field, original magnification x 10).
28
F. L. Kiechle et al
Increased eosinophils in nasal smears are associated
with seasonal or airborne latex allergic rhinitis76–79
Point of Care, vol. 2, no. 1
and nonallergic chronic nasal disorders, like nasal polyposis.80 In the allergic rhinitis group, the nasal eosinophil scores significantly correlated with the clinical signs score.77 Over a 3-year study period, school
children with early onset nasal hypereosinophilia
were more likely to develop nasal symptoms later.78
In the chronic nonallergic nasal disorder group, the
average nasal eosinophil count was 1.5 ± 2.3% compared with controls, 5 ± 2.6%.80 The degree of nasal
eosinophilia correlated with the number of eosinophils detected in surgically removed nasal polyps.80
Eosinophils release granules, which contain cytotoxic
proteins by two morphologic degranulation patterns,
piecemeal degranulation, or eosinophil cytolysis.81
These patterns can be distinguished in tissue biopsies
using transmission electron microscopy. Nasal biopsies from patients with allergic rhinitis show the greatest degree of eosinophil degranulation and nasal polyps revealed the second greatest degree compared
with allergic asthma.81 These findings support the concept that the inflammatory response in the airway is unified and that allergic rhinitis of the upper airway is associated with eosinophilia of the lower airway in associated allergic bronchitis or asthma.82 Allergic rhinitis is
also associated with increased transcription and translation of the inducible isoform of nitric oxide synthase
and nitric oxide production79,83,84; increased expression of GATA-3 which is a transcription factor which
regulates interleukin-5 gene expression 85; and increased interleukin-5 in nasal lavage fluid.79
Nasal smears are prepared by transferring a thin
smear of the nasal mucus collected on a cotton swab
onto a glass slide3 or by impression cytology.86 Air
dry the smear and stain with either Hansel or WrightGiemsa stains. The smear should be examined under
low and high power using a bright field microscope.
Polymorphonuclear leukocytes will have a segmented
nucleus with 2 to 5 lobes which will stain blue and the
cytoplasm will be clear and homogeneous (Hansel) or
neutral pink staining secondary granules (WrightGiemsa). Eosinophils will have bilobed blue nuclei
and deep red granules in the cytoplasm or outside the
cell if released from degranulated eosinophils (Fig
13). To estimate the number of eosinophils, locate an
area that is well-stained and representative of the slide
that contains approximately 50 to 100 cells. If three
eosinophils are observed in a field of 50 polymorphoPoint of Care, vol. 2, no. 1
Fig 13
Bilobed eosinophil (arrow) and three polymorphonuclear leukocytes in nasal smear (Wright-Giemsa stain, oil immersion, original magnification x 50).
nuclear leukocytes plus eosinophils, 6% eosinophils
would be reported. A grading system may be used as
illustrated below.
Eosinophils/% Eosinophils
None seen/0%
Rare/1% to 9%
Few/10% to 40%
Many/more than 40%
No quality control is available; however, a peripheral blood smear with increased eosinophils stained at
the same time will monitor the staining quality.
Conclusion
Provider-performed microscopy procedures provide
a method for diversification of personnel performing
laboratory tests.87 In this case, the tester may be a
physician, nurse practitioner, nurse midwife, physician assistant, or dentist. In a hospital setting, the
CMS license to perform PPM is best obtained by a
physician responsible for the clinic or subspecialty
performing the PPM procedures.6 This policy removes this activity from the central laboratory’s CMS
license, but allows the point-of-care coordinators to
provide assistance in program design and monitoring.6 The second advantage of this organizational
structure is that because both the central laboratory’s
primary inspection organization, CAP, and the hospital’s primary inspection organization, JCAHO, differences in PPM regulations are minimized. 88
Because the CAP does not distinguish between the
different types of test complexity described by
Provider-Performed Microscopy
29
invasive techniques for measuring cellular elements
or chemical constituents.100,101
References
Fig 14
Artifacts introduced by scratches on the glass coverslip during urine microscopy (bright field, original magnification
x 10).
CLIA’88, the checklist questions for competency and
training for any point-of-care test would apply for
personnel performing PPM procedures. Examples of
competency indicators for PPM have been described3,9 and Web-based competency testing has
been reported.8,89
Efforts to reduce fatal or nonfatal medical errors in
diagnoses, surgery, and prescriptions are under investigation, especially the more numerous prescription
errors.90 A comparison of the emergency physicians
and laboratory “technicians” to perform urine microscopy reported good agreement for red cell detection
but poor agreement on detection of white cells and
bacteria.91 These findings reinforce the need for adequate training and retraining to achieve optimal
skills. There are a variety of printed, intranet, or internet sources available for reviewing images of
formed elements in urine.8,89,92–94 Artifacts may be
present and should be identified. Fig 14 illustrates the
artifact introduced by scratches on the coverslip used
for urine microscopy, which may be mistaken for fungal hyphae. Two or more procedures may be superior
to a single procedure in detecting disease. For example, the microscopic examination of both spun
urine and vaginal fluid specimen yields an improved
detection rate of Trichomonas vaginalis.95 Algorithms
may also be used that will recommend the next step
following the completion of a screening PPM procedure,96 which should include the evidence-based
logic for the algorithm’s selection.97–99 The future of
point-of-care test procedures, in general, rests on the
successful development of noninvasive or minimally
30
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