An immunohistochemical method differentiates cryptococcus neoformans varieties and serotypes in formalin fixed and paraffin-embedded tissues.

1. NAME : PRISCILLA ANAK NARI
MATRIC NO : 131642
IDENTITY CARD : 930115135900
TITLE:
AN IMMUNOHISTOCHEMICAL METHOD THAT DIFFERENTIATES CRYPTOCOCCUS NEOFORMANS
VARIETIES AND SEROTYPES IN FORMALIN-FIXED PARAFFIN-EMBEDDED TISSUES
Kingdom : Fungi
Phylum : Basidimycota
subphylum : Basidimycotina
Order : Sporidiales
Family : Sporidiobolaceae
Genus : Filobasidiella (Cryptococcus)
Species : Filobasidiella neoformas (Cryptococcus neoformans)
Keywords : Cryptococcus Neoformans, Cryptocococis, Cryptococcus Neoformans var gatti, Immunohistochemical

2. INTRODUCTION
Immunohistochemistry (IHC) analysis is a method for demonstrating the presence and location of
proteins in tissue sections
Immunohistochemical detection of molecules of interest such as receptors, neurotransmitters, or
intracellular signaling molecules is an important techniques to determine the distribution of those
molecules in tissues
Obviously, single staining protocols and multiple staining protocol show similarities, but multiples staining
protocols are more complex. To avoid target cross reactivity, complex protocol may be necessary.
Differentiation of stain colors may be difficult, especially if the targets are co-localized.
Then, in many cases, the best antibody concentration and the most appropriate visualization systems to
distinguish the target and to obtain the topographic information desired are necessary.
Cryptococcosis is an important disease throughout the world and the most common systemic mycosis
affecting animals and humans in Australia
This paper describes a method that detects C. neoformans and differentiates C. n. var. neoformans and C.
n. var. gattii in formalin-fixed tissues of cryptococcosis cases by the use of immunohistochemical labelling.
The method will permit retrospective analysis of archival material with particular emphasis on comparing
and contrasting host–parasite interaction of the two varieties in disease.

3. EXPERIMENT PROCEDURE (AFTER TREATMENT)
a) Sample preparation b) Immunostainning protocol
Blocking
Incubation with the
primary antibody
Incubation with the secondary
antibody
Adding enzyme substrate (for
colometric detection)
Coverslip and
observation
Fixation
Embedding
Sectioning
Antigen retrieval
Publication Analysis
Immunostaining
Protocol
Sample
Preparation
Sample
Treatment

4. MATERIALS AND METHODS
a) Case Selection b) Animal Tissues c) Primary Antibodies
To be included in this study, a positive
histological diagnosis of cryptococcosis based
on characteristic organism morphology and a
positive microbiological diagnosis to the
varietal level were both required.
Round to oval yeast cells (2-15 µm diameter)
with variably size capsules and narrow necked
budding.
Isolated initially cultured on ‘Saboraud’s
dextrose agar’. C neoformans identified
basedon brown colour effect at 28°C and
37°C. then, unrease production and presence
of a capsule.
Isolated send to the Australia National
References Laboratory in Medical Mycology
where species identify was confirmed using
the API ATB 32C identification system
(Biomerieux, Mercy L’Etoile, Frence)
Variety determined using L-Canavanine glycine
bromothymol blue (CGB) agar.
Tissue were obtained from animals with
naturally occurring 30/31or experimentally
produced (1/31) cryptococcosis over a B-year
period (1988-2000).
The specimen included a range of tissues
(including upper and lower respiratory tract,
skin, subcutis and brain from various animal
species (20 cats, four dogs, five koalas, one
ferret, one rat)
Tissues were process with histology routine
fixation in 10% neutral buffered formatim and
embedded in paraffin. Paraffin blocks were
stored at room temperature.
Section for histology cut at 5 µm, mounted on
glass slide and staining using haematoxylin and
eosin (H&E)
Commercially available polyclonal serotyping
antibodies
(Crypto-Check, Iatron Laboratories, Tokyo,
Japan) were utilized at varying dilutions from
1:50 through to 1:4000 (especially factors 1, 5
and 7 from the Crypto-Check system)

5. MATERIALS AND METHODS
d) Monoclonal antibodies
e) Immunohistochemical Staining Method
f) Controls
Monoclonal antibodies raised against cryptococcal capsular
antigens in previous investigations were screened for their
usefulness in immunohistochemistry. Table 2 provides details of the
monoclonal antibodies used.
Sections for
immunohistoche
mical staining
were cut at 4 µm,
mounted onto 3-
aminopropyltriet
hoxysilane
coated glass
slides, dried at
37°C and stored
at room
temperature
Before staining, sections
were deparaffinized in
xylol and rehydrated
through successive
ethanol dilutions to
water. All incubations
were carried out at
room temperature
(21°C). Thorough
washing in phosphate-
buffered saline (PBS)
was performed between
each step.
Endogenous
peroxidase activity
was quenched by
incubating
sections for 30
min in a 2%
peroxide solution
in 50:50 mix of
methanol and
PBS.
Antigen retrieval was
enhanced by boiling in
0.01 M tri-sodium
citrate for 6 min in a
microwave oven. Non-
immunological binding
of antibody was
minimized by pre-
incubating the sections
in 5% normal goat
serum in PBS in a
humified chamber for
30 min.
Secondary antibody
was applied to the
sections and allowed to
incubate for 60 min.
The secondary antibody
used was 1:100 dilution
of biotinylated goat
anti-rabbit/mouse
immunoglobulin.
Sections were counterstained
with haematoxylin, dehydrated
through graded alcohols to
100% ethanol and xylol and
then cover-slipped (Depex
Mounting Medium Gurr, BDH
Chemicals, Poole, UK). Sections
were examined for evidence of
specific antibody binding.
Duplicates
for each
antibody
tested and
two negative
controls
The first negative control had
the primary antibody
substituted by normal mouse
immunoglobulin matched for
isotype and subclass to test for
untargeted species-specific
binding.
Second negative control
had the primary antibody
omitted to test for non-
immunological binding.
Sections acted as their own
positive controls.
The whole process was
repeated at different
times for many of the
specimens, to ensure
consistency of results over
the time course of this
investigation.
Normal tissues from animals
with no histopathological
evidence of cryptococcosis from
with other fungal infections
were also run as negative
controls against the test
antibodies.

6. RESULTS
Polyclonal antibodies used were neither consistent nor accurate in determining serotype and it was
concluded that these antibodies were unsuitable for immunohistochemical labelling.
Five mAbs evaluated, 471 and 302 were found to be the most useful, both providing consistent and
specific staining of a specific range of cryptococcal capsular antigen types.
MAb 471 labelled C. neoformans capsular antigen of all serotypes and mAb 302 labelled C. n. var.
neoformans (serotypes A, D and AD) capsules.
These antibodies permitted inferential identifications with a high degree of congruence to the
correct varietal status of C. neoformans isolates.
MAb 12A1 labelled only cryptococcal capsular antigens, but the antigens labelled were not variety-
specific (data not shown). Therefore, this antibody was not useful for our purposes.
MAb 2D10 proved to be a general marker of C. neoformans capsule; however, mAb 471 was superior,
giving stronger capsular staining.
Positive capsular staining with the mAb 471 was considered definitive in identifying organisms in
tissue sections as C. neoformans. All cases with a positive tissue diagnosis of cryptococcosis had
demonstrable organisms whose capsules were positively labelled by mAb471.
The type of tissue in which yeast cells were located had no effect on the ability to label
cryptococcal capsular antigen.
Tissues infected with other species of fungi tested (Sporothrix schenckii, Aspergillus sp., Candida
albicans, Histoplasma capsulatum, Coccidioides immitis) did not show positive capsular labelling
with mAb 471 although a light, non-immunogenic staining of yeast and hyphal cell walls was
sometimes apparent.

7. RESULTS
Fig. 1 a, C. neoformans var.
gattii infected lung labelled
by mAb 471
Fig. 1 b, C. neoformans
var. neoformans serotype
A infected meninges
labelled by mAb 471
Fig. 1 c, C. neoformans var.
gattii infected brain
labelledby mAb 302
Fig. 1 d, C. neoformans
var.neoformans serotype
A infected meninges
labelled by mAb 302
Fig. 1 e, C. neoformans var.
neoformans infected
subcutis labelled by mAb
CRND8
Fig. 1 f, C. neoformans var.
neoformans serotype A
infected meninges labelled
by a negative antibody
control (primary antibody
omitted)
a) There is a slight degree of labelling of dispersed soluble components of cryptococcal
capsule and some uptake by host cells. Immunoperoxidase x300.
b) There is a slight degree of labelling of dispersed soluble components of cryptococcal
capsule and some uptake by host cells. Immunoperoxidase x300.
c) Absence of tissue labelling. There is a slight non-specific staining of the yeastcell wall.
Immunoperoxidase x300.
d) The strong capsular staining (arrow). There is a slight degree of labelling of dispersed
soluble components of cryptococcal capsule and some uptake by host cells.
Immunoperoxidase x300.
e) The positive labelling of cryptococcal capsule (arrow). Immunoperoxidase x300.
f) The absence of capsular staining (arrow). Immunoperoxidase x300.
mAb 471 labelled both varieties (Fig. 1a, b), labelling was more
intense in cases that were subsequently classified as C. n. var. gattii.
Positive capsular staining with mab 302 (fig. 1d) was considered
indicative of a C. N. Var. Neoformans infection whereas a negative
result indicated a C. N. Var. Gattii infection (see table 3)
A further breakdown of cases positively labelled as caused by C. n. var.
neoformans (serotypes A, D, AD) was made using the mAb CRND8 (Fig.
1e), which positively labelled C. neoformans serotype D, with negatively
staining organisms considered to be serotype A.
It was not thought possible, however, to distinguish serotype D
organisms from yeast cells of serotype AD, as the latter serotype
displays capsular antigens of both serotypes A and D.

8. RESULTS
From Table 4 it can be seen that of 31 cases presented
here with a tissue and mycological diagnosis of C.
neoformans, 14 were categorized
immunohistochemically as C. n. var. neoformans and 17
as C. n. var. gattii.
Results of biotyping of cultured isolates, from the same
cases, using CGB agar yielded 15 as C. n. var.
neoformans and 16 as C. n. var. gattii.
In all cases bar one, the variety determined by the
immunohistochemical method matched the variety
determined by culture on CGB agar.
The sensitivity of the immunohistochemical method to
determine cryptococcal variety in formalin fixed
tissues was initially determined to be 97% (1/31 false
negative) and the specificity was rated as 100% (0/31
false positive), using the CGB agar method as the gold
standard.
Two C. n. var. gattii isolates were not available for
serotyping. All C. n. var. gattii isolates serotyped were
serotype B. A further isolate, determined to be C. n.
var. neoformans on CGB agar, was also found to be
serotype B.
It also had microscopic and colony morphology
consistent with C. n. var. gattii. As this isolate would
actually appear to be C. n. var. gattii, the sensitivity of
immunohistochemistry to determine cryptococcal
variety was 100% (no false negatives). Apart from this
isolate, all C. n. var. neoformans isolates were found to
be serotype A.

9. DISCUSSION
• The aim of this study was to devise a method of reliably identifying yeast cells as C. neoformans in formalin-fixed tissues and
determining their variety. An immunohistochemical approach was devised to provide additional information on the distribution of
capsular material in tissues and on its interaction with the host.
• The immunohistochemical method described in this paper proved to be consistent and accurate in our hands. The important
factors underlying the accuracy of this method include the speciŽcity of the primary antibodies, the success of antigen retrieval and
the use of appropriate dilutions and staining conditions.
• The present investigation has shown mAbs 471 and 302 to be most useful in this immunohistochemical context to biotype C.
neoformans in tissue sections. The relevant antigenic sites seem to be abundant and easily retrieved from formalin-fixed paraffin-
embedded tissues using standard immunohistochemical techniques.
• The technique currently appears capable of distinguishing between the varieties of C. neoformans and possibly also to
differentiate between serotypes A and D, although it is inherently unable to distinguish between serotype D isolates and serotype
AD isolates. There was some disparity between the results of the Crypto-Check kit and the immunohistochemical method with
regard to isolates immunoreactive for serotype D epitopes. At one laboratory, five isolates displayed reactivity to Factor 7
(serotype A specific) and weak reactivity to Factor 8 (serotype D specific) using the Crypto-Check kit, whereas at another
laboratory the same isolates displayed serotype A reactivity only.
• Consequently, these isolates were designated as serotype A in the present study. However, of those five cases, three were
positively labelled by mAb CRND8 using immunohistochemistry, indicating the presence of serotype D capsular antigens.
Although Factor 8 (Crypto-Check kit) and mAb CRND8 have been shown to have equivalent specificity of antigenic site [47], it is
possible that the immunohistochemical method has greater sensitivity than a slide agglutination method, because individual yeast
cells can be identified as displaying the appropriate antigenic site. This could explain the discrepancy between
immunohistochemistry and the Crypto-Check kit in identifying serotype D isolates.
• Additionally, we are currently attempting to develop molecular techniques to be used as an adjunct to immunohistochemistry to
further characterize C. neoformans isolates in formalin-fixed tissues.
• In conclusion, this study has validated the use of immunohistochemistry to identify and differentiate varieties of C. neoformans in
formalin-fixed paraffin embedded tissues. This provides a number of avenues in which to investigate host–parasite interactions
using archival material, especially where culture was not performed. This will allow an accumulation of data on the prevalence of
infection by the different C. neoformans varieties amongst animal species.