Training Module for Vaginal Wet Preparation Provider Performed Microscopy (PPM)

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Training Module for Vaginal Wet Preparation
Provider Performed Microscopy (PPM)

• This training module must be completed by all
  individuals who will perform vaginal saline and
  KOH wet prep PPM testing at OSDH County Health
  Department sites.

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• In addition to completing this module, trainees
  must complete each of the following items before
  they can be deemed competent in saline and KOH
  wet prep procedures
• be appropriately trained in the collection,
  processing, and examination of vaginal samples
  and in the recording of results under the direct
  supervision of an individual who has been deemed
  competent to provide training
• read and understand the vaginal saline and KOH
  wet prep protocols
• provide a copy of each college transcript to be
  kept on-file at the County Health Department
• successfully complete a written test (80 or
  better is required to pass).

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• Once you are deemed independently capable of
  collecting vaginal samples and preparing and
  reading saline and KOH wet prep slides by your
  trainer, you will be required to participate in
  periodic reassessment of competency, including
• observation of you performing all steps of the
  wet prep procedure by a suitably trained
  individual, prior to 6-months from completion of
  initial training
• wet prep proficiency challenges periodically
  scheduled by the OSDH Public Health Laboratory.

• Your written test(s), 6-month evaluation,
  proficiency challenges, and copies of your
  transcripts must be kept on-site where testing is
  performed.

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The following slide presentation provides
examples of images commonly seen in saline and
KOH wet preps, including

• Normal vaginal epithelial cells
• Clue cells
• White and red blood cells
• Yeast cells
• Trichomonas
• Debris

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Epithelial Cells
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A majority of cells observed in a normal vaginal
smear are, not surprisingly, vaginal epithelial
cells. In addition, varying numbers of leukocytes
(white blood cells), erythrocytes (red blood
cells) and bacteria are usually evident. Small
numbers of other cells and microorganisms
sometimes can be observed.
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Squamous epithelial cells can be of varying
appearance depending on their stage of
development and position in the epithelium. When
immature they are smaller and more rounded. As
they mature in the epithelium they get larger,
flatter, and more irregular in shape.
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The most common epithelial cells are large, flat,
and polygonal (varied shape) - like the two big
cells in the center of the micrograph below. More
immature stages of epithelial cells are more
round in appearance (cell to the right green
arrow). Generally, a small, single nucleus is
present (red arrow). Notice how the nucleus can
be off-center in some cells. Cell borders of
normal epithelial cells are distinct and sharp.
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The micrograph below shows a typical mature
normal squamous epithelial cell irregular-shape
with sharp cell boundaries and a single nucleus
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Again 3 below is a normal squamous epithelial
cell with a typical large, flat appearance and
single, small nucleus. Note, at times, cells can
appear folded (red arrow)
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Not the best micrograph but.
these are examples of typical normal squamous
epithelial cells. The nucleus and the cell
boundaries are clearly observed. Again, one of
these cells looks as if it is folded (red arrow).

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Epithelial cells can appear as strings or sheets
of cells in preparations, as in the example below
(at least 4 normal squamous epithelial cells are
shown). Note the cell nucleus (red arrows) and
the distinct cell boundaries.
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The examples shown here are typical of normal
squamous epithelial cells. The cell nucleus and
the cell boundaries are distinct (some focusing
of this specimen would be required to view all
the cells clearly). Normal bacteria seen on some
cells.
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Clue Cells
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Clue cells are also epithelial cells of the
vagina but they appear fuzzy without distinctive
sharp borders. They get their stippled appearance
from being covered with bacteria. The nucleus in
this cell is still visible.
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To the left are examples of clue cells in a
saline prep. See how the cells are covered with
bacteria and have no distinctive edges to them?
By contrast ,the normal epithelial cells in the
micrograph below have distinctive borders.
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Clue cells are a sign of bacterial vaginosis
(BV), caused by various anaerobic bacteria.
Lactobacillus, present in normal vaginal fluids,
are replaced with high concentrations of
Gardnerella vaginalis, Mycoplasma hominis and
anaerobic bacteria, in particular Prevotella sp.
and Mobiluncus sp. Generally, when BV is present,
more than 20 of vaginal epithelial cells are
clue cells. BV is often associated with a
homogeneously thin, gray-white, or milky, vaginal
discharge that tends to adhere to the vaginal
wall and give a foul, fishy smell.
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Clue cells are squamous epithelial cells that are
covered with a thick matte of bacterial cells.
Often the bacterial overgrowth is so thick that
all cell details, including the cell nucleus, are
obscured (e.g., arrow 4 below).
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Clue cells have a heavy stippled or granular,
very refractile cytoplasm with shaggy or bearded
cell borders due to the heavy coating of
coccobacilli. Most of the cell surface should be
covered by bacteria for it to be identified as a
clue cell. The epithelial cells in this photo do
not exhibit this shaggy or bearded appearance,
and thus, should not be considered clue cells.
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This sample consists on two clue cells lying on
top of the other. The two cells can be
visualized by using the fine focus knob on the
microscope to focus up and down and adjust the
plane of view of the specimen. Notice the
stippled appearance of the cells and indistinct
borders.
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Cells labeled 3 4 in the two top micrographs
are clue cells
The cells in the bottom micrograph are not clue
cells. They show some stippling but the cell
borders are distinct. These are normal squamous
epithelial cells.
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White Blood Cells
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These micrographs have been stained with a
cytological dye and are not what would be seen in
a wet mount.
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In small quantities, white blood cells (WBCs) are
a normal component of the vaginal flora. A ratio
of one WBC for every epithelial cell is
considered within normal limits. WBCs are round,
equal in size to the nuclei of mature epithelial
cells, and appear dark and granular (1 arrow in
micrograph).
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An evaluation of the number of white blood cells
in vaginal discharge is essential to wet prep
interpretation. WBCs play a role in fighting
infections and can be elevated in numbers (WBCs
gt10/hpf) in some infections, especially in
Chlamydia, Trichomonas vaginitis, herpes, and GC.
Generally, a ratio of 5 WBCs to every 1
epithelial cell (51) indicates possible mild
inflammation. A ratio greater than 101 indicates
possible moderate to severe inflammation. The
micrographs below show WBCs and normal epithelial
cells.
Epithelial cell
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White blood cells and red blood cells can be
found in wet preps of vaginal discharge to
varying degrees. Note the slightly larger size
and granular appearance of the WBCs and the
highly refractile appearance of the RBCs.
RBC
WBC
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Yeast Cells
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Yeast are unicellular fungi that appear commonly
in vaginal discharge wet preps. Typically, they
appear as budding forms and pseudohyphae. Most
fungi are multicellular and grow by forming
tubular hyphae comprising one or more cells
surrounded by a cell wall collectively they form
a tangle of tubes called a mycelium. By contrast,
yeasts are unicellular. They divide by a process
called budding. They do not grow as hyphae.
Instead they form pseudohyphae - where a string
of cells remain incompletely attached to one
another after division.
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The KOH preparation helps in the identification
of yeast in wet preps 10 KOH destroys the
cellular material (WBCs, RBCs, trichomonads)
except for epithelial cells and yeast. The
epithelial cells appear enlarged and rounded. The
yeast becomes more prominent and easier to
identify. Yeast appears as thin, translucent
tubes that taper at various points and give a
pseudo segmented appearance - pseudohyphae.
Small, round yeast buds are often present in the
background.
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It is important to utilize high power to
differentiate yeast from various similarly shaped
forms, such as fibers, long lactobacilli, and
hair. For instance, it is difficult to identify
definitively the structures as yeast pseudohyphae
in the micrographs below, although there is some
apparent segmentation in some of the tubal
structures. Close examination is important.
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Candida and other yeasts are often associated
with a moderate, thick, white, cheesy and
adherent vaginal discharge. There is no amine
odor with KOH addition. The micrograph below is a
Gram-stained preparation. It shows segmented
pseudohyphae and yeast buds. Several epithelial
cells and numerous Gram-negative rods are in the
background.
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Trichomomas
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Trichomonas vaginalis, sometimes referred to as
trich, is a flagellated protozoan
(single-celled) parasite of the urogenital tract.
The cells are motile, have 4 flagella and a
single nucleus.
flagellae
nucleus
Trichomoniasis causes a vaginal discharge that is
frothy, yellow-green or brown, and bad-smelling.
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Saline preps need to be examined immediately
after preparation for optimum identification of
trichomonads, since cooling of the slide will
reduce the motility of the organism. Trichomonads
display directional, jerky motility and are
slightly larger than a WBC. Examine the entire
specimen for motile trichomonads. Suspicious
objects should be examined with the high power
(40x) objective.