The skin smear
is a valuable, cost-effective tool
in the routine management of the
Hansen's disease patient.
The smear is a means of estimating
the number of acid-fast bacteria
present, reported as the Bacterial
Index (BI), and is important in
determining the type and severity
of disease as well as assessing
the response to treatment. |
GENERAL:
- Initial skin smears are usually taken
from 6 “routine sites” (both
earlobes, elbows, and knees) as well
as several typical lesions from the
patient. Repeat smears are obtained
from 3 to 4 of the most active sites
previously tested to evaluate progress.
- The time interval between repeat
smears is determined by the physician,
but in general, annual smears are
adequate for monitoring response to
treatment and during the following-up
period to detect any evidence of relapse.
- All microscopic slides on which skin
smears are made should be precleaned
in 70% alcohol, acetone, or alcohol-acetone
to remove amorphous debris. The
slides are wiped dry with a clean hand
towel. Blades that are used in
smear taking are likewise cleaned.
- Slides should be air-dried and NEVER
heat fixed.
- They may be sent in protective mailers
to:
National Hansen's Disease Programs
Attention: Clinical Laboratory – Skin
Smears
1770 Physicians Park Drive
Baton Rouge, La. 70816
Phone: (225) 756-3735
PROCEDURE FOR OBTAINING
SMEARS : |
1.
|
Universal
precautions should be observed
in obtaining skin smears.
|
2.
|
The
skin is cleansed with 70%
alcohol and air-dried or wiped
dry with cotton. (Zepharin
tends to make the skin too
slippery and is not recommended.)
|
3.
|
A
fold of skin is made relatively
avascular by pinching or mild
clamping. If the skin
cannot be grasped by pinching,
it can be compressed.
A surgeon's glove may aid
in grasping. |
4.
|
Local
anesthesia is generally unnecessary.
(If there is not adequate
decrease in sensation, obtain
local anesthesia with 1% Xylocaine
or Ethyl Chloride spray can
be carefully applied.)
The compression of the skin
by pinching aids in the anesthesia.
|
5.
|
An
incision 3-5 mm long and 2-3
mm deep is made with a alcohol
cleansed, single-edged razor
blade. A scalpel with
a #15 Bard-Parker blade may
also be used. Mild pressure
to maintain relative avascularity
is continuously applied to
the area until an adequate
smear has been obtained. |
6.
|
A
small amount of blood does
not interfere with the reading,
but large amounts should be
avoided and can usually be
controlled by the amount of
pressure of the pinch.
If excessive bleeding occurs,
it can be wiped away with
a cotton swab. |
7.
|
After
the incision is made, and
before the blade is withdrawn,
the inner surface of the wound
is scraped with the blade
held at a right angle to the
incision. Upon scraping,
tissue fluid and dermal tissue
are obtained. |
8.
|
The
material is transferred to
the cleaned microscope slide.
A moderately thick smear,
with a visible uniform opacity
is made. The smear is
made in a circular manner
on the slide, no larger
than a pencil eraser (5-7
mm) , beginning peripherally
and ending in the center,
leaving a central “button”
(2-4 mm) which can be easily
focused upon with the microscope.
Slides should be properly
labeled as shown below in
the sample diagram for 3 routine
sites. |
|
|
9.
|
A
Band-Aid is generally sufficient
to protect the smear site.
|
10.
|
A
single technician takes all
smears to insure more uniform
and consistent results. |
11.
|
The
smears are then sent to the
National Hansen's Disease
Programs for reading. |
12.
|
A
chart to diagram sites of
the skin smears is linked
from the top of this page.
|
|
STAINING OF SKIN SMEARS
: |
1.
|
Dry
the slide with smear at room
temperature. DO
NOT HEAT FIX . |
2.
|
Place
slides on a staining rack
and flood with 10% formalin
for 15 minutes for fixation.
|
3.
|
Gently
rinse well with tap water. All
formalin must be removed to
prevent the formation of precipitates.
|
4.
|
Flood
slides with Ziehl-Neelsen
carbol-fuchsin for twenty
minutes. The carbol-fuchsin
must be filtered before each
use. Filtering can be accomplished
by placing pre-cut filter
paper strips on the slide
prior to the addition of stain
and left in place for the
full twenty minutes. |
5.
|
After
removing and discarding filter
paper strips, gently rinse
slides well with tap water
to remove excess stain. |
6.
|
Decolorize
with 2% acid
alcohol for 1 minute. This
is best accomplished by placing
slide into a two-slide plastic
slide mailer filled with acid
alcohol. Occasional up and
down movement of the slide
in the acid alcohol should
remove all excess carbol fuchsin. |
7.
|
Gently
rinse slides thoroughly
with tap water. |
8.
|
Counterstain
with alkaline methylene blue
for 30 seconds to 1 minute.
|
9.
|
Gently
rinse well with tap water
and air dry. |
NOTE: Positive and negative
control slides must be used each day
for quality control purposes.
|
Z-N Carbol Fuchsin Stain:
Basic fuchsin ---------------------------
1.0 gm.
Phenol crystals (melted)----------------5.0
mls.
95% ethanol ---------------------------
10.0 mls.
Water, to make ----------------------
100.0 mls.
Dissolve stain in alcohol, and then add
phenol/water mixture. Let stand
overnight before use. Store in dark
brown bottle. Stable for 1 year.
Acid alcohol:
Conc. HCl ------------------------------
2.0 mls.
95% ethanol --------------------------
98.0 mls.
Alkaline Methylene Blue:
KOH (10%) --------------------------
0.10 mls.
Methylene blue ----------------------
0.35 gms.
95% ethanol ---------------------------30.0
mls.
Water to make -----------------------100.0
mls.
Dissolve the stain in the alcohol, then
add the KOH and water mixture and allow
to sit overnight. Filter before
use.
MICROSCOPIC EXAMINATION
OF SKIN SMEARS : |
The stained smears
are examined with a quality microscope
using the oil immersion objective
(x100) to determine the total number
of bacilli. The same individual
should read all smears for the purpose
of consistency. The smear
will have similar numbers of bacilli
throughout. However, four
separate quadrants of the smear
are examined and averaged to establish
the Bacterial Index. |
REPORTING THE BACTERIAL
INDEX : |
The
results are reported on a 0 to 6+
semi-logarithmic scale using a descriptive
phrase or numerical code.
This is an indicator of the total
bacillary load of the patient.
It falls about 1 point per year
during effective treatment as dead
bacilli undergo lysis and are absorbed.
|
Very Numerous
|
( +6 ) |
over 1000 bacilli
per oil immersion field. |
Numerous
|
( +5 ) |
100 to 1000 bacilli
per oil immersion field. |
Moderate
|
( +4 ) |
10 to 100 bacilli
per oil immersion field. |
Few
|
( +3 ) |
1 to 10 bacilli
per oil immersion field. |
Very
few |
( +2 ) |
1 to 10 bacilli
per 10 fields. |
Rare
|
( +1 ) |
1 to 10 bacilli
per 100 fields. |
None
found |
( NF ) |
No AFB seen on
entire site. |
|
Revised by Clinical
Lab 11/06/2008 |
|