Assignment

Course Subject Immunology

Course Code PPTC 6420302

Serial no. 56

Name Ong Jin Huey

Student ID P21100702

Year 2022

Faculty Faculty of Pharmacy

University AIMST University

Submission Date 9 June 2022

Course coordinator Dr. Geethaa Sahgal

1.0 Introduction

1.1 Dermatomyositis

Dermatomyositis is an infrequent inflammatory disorder that causes myasthenia and skin

rash. Both children and adolescent can be affected by this disease. In adults, dermatomyositis
typically arises between 40 and 60 years old, whilst in youngsters, it often appears around
aged 5 to 15. Besides, it affects women more frequently than men. Currently, there is no
treatment exists for dermatomyositis, yet the therapy can assist in eliminating the skin rash
and restoring muscular power and function.

The typical symptoms of dermatomyositis may develop abruptly or gradually over time,
including skin changes, violet-coloured or dusky red rash arise on the eyelids, face, knees and
elbows. Moreover, muscle weakness in which affects the muscles nearest to the trunk,
including those in the shoulders, hips, thighs, upper arms, and neck is one of the indications.
The origin of dermatomyositis is undetermined. However, the disease resembles autoimmune
disorders extensively in various ways, as the immune system targets body parts mistakenly.
Additionally, environmental and genetic factors such as viruses, UV exposure, certain drugs,
and smoking might cause dermatomyositis. (Mayo Clinic, 2020)

Diagram 1: Dermatomyositis.

1.2 Direct Immunofluorescence (DIF)

Primary immunofluorescence, commonly recognized as direct immunofluorescence,

encompasses the use of a singular antibody that has been attached directly to a fluorophore.
The antibody allows the attached fluorescent dye to be seen under a microscope when it binds
to the target molecule. This technique helps minimise concerns with antibody cross-reactivity
or non-specificity, resulting in increased background signal by decreasing the number of
stages in the staining process and making it faster. (Rockland, n.d.) It is an analytical method
for diagnosing skin, kidneys, and else organ system abnormalities by implying the application
of antibody–fluorophore conjugate molecules on biopsy tissue samples. These antibody–
fluorophore conjugates target the inappropriate protein depositions within that patient's tissue.
Afterwards, the fluorophore generates its wavelength of light, which can then be examined
under a microscope.

Direct immunofluorescence can rule out autoimmune illnesses, connective tissue diseases,
and vasculitis. The tissue staining patterns observed could be distinctive to a disease entity or
could ought to be evaluated concerning medical and histological data. Linear IgA bullous
illness and dermatitis herpetiformis are examples of autoimmune bullous disorders, while
lupus erythematosus including systemic, discoid, and subacute cutaneous variants, and
dermatomyositis are connective tissue diseases. Moreover, direct immunofluorescence can be
used to diagnose cutaneous vasculitis and a variety of other inflammatory skin diseases,
including photosensitivity rashes, small vessel vasculitis and lichen planus. (Zhang, 2017)

Diagram 2: Positive result of direct immunofluorescence.

2.0 Working Process of Direct Immunofluorescence

Firstly, 1 cm biopsy should be collected from a fresh blister or lesion, that is no more than
24–48 hours. The biopsy must be collected from an existing damaged tissue, typically in sun-
exposed locations.
 The first procedure in an immunofluorescence staining process is to fixate the specimen
by incubating it in a 4% paraformaldehyde at room temperature for 10 minutes. The cell must
then be permeabilized by incubating in detergent like Triton X-100 or Tween-20 with regard
to stain intracellular proteins. If permeabilization is neglected, the antibodies will be unable
to penetrate the cell’s lipid membrane. Moreover, blocking is accomplished by utilizing
serum from horse or lamb to limit the possibility of non-specific binding in which the
secondary antibody was produced, as well as bovine serum albumin or milk to diminish
intracellular or extracellular background signals.

Subsequently, primary antibody incubation is performed when doing multicolour staining

since distinct unique primary antibodies for the simultaneous discovery of multiple antigens
within the same sample should be developed in various hosts to minimise cross-reactivity. On
top of that, the final procedures before microscopy include counterstaining and mounting the
sample nuclei in a low-autofluorescence mounting medium. Ultimately, to acquire the
optimal outcomes, the microscopic investigation of the immunofluorescence staining should
be performed after the mounting. (ibidi, n.d.)

As a result, the staining patterns of specific skin disease, dermatomyositis, are shown in
granular IgM, IgG, C3 and cytoid bodies IgM and IgA. (Zhang, 2017)

Diagram 3: Fluorescence observed, where antigen is located.

3.0 Advantages and Disadvantages of Direct Immunofluorescence

3.1 Advantages

Since host species would not be a factor, direct immunofluorescence has the
advantage of being able to stain with several antibodies simultaneously. (Ken Lau, n.d.)
Direct labelling may be required when multiple antibodies produced in the identical species
are present, such as two mouse monoclonals. Direct approaches further reduce species cross-
reactivity if the fluorophore is attached to the primary antibody. (abcam, n.d.) Furthermore,
direct immunofluorescence processes are generally shorter since they only involve one
labelling step, owing to the lack of a secondary antibody incubation phase. Similarly, because
the absence of a secondary antibody results in minimizing background, secondary antibodies
can reveal significant non-specific binding. (Ken Lau, n.d.)

3.2 Disadvantages

Disadvantages of direct immunofluorescence include commercially accessible pre-

conjugated primary antibodies restrict the flexibility. Furthermore, the signal received in
direct approaches may appear faint compared to indirect immunofluorescence, as secondary
antibodies does not offer signal amplification. Additionally, primary conjugated antibodies
are typically more expensive than their non-conjugated equivalents. (abcam, n.d.)

4.0 Recommendation

A muscular biopsy is recommended to assist diagnose or detecting some

inflammatory muscle illnesses, including dermatomyositis. A needle biopsy involves
introducing a needle into the muscle and removing it, leaving a little tissue inside the needle;
an open biopsy entails cutting a small cut in the skin and the muscle. (UCSF Health, 2018)
An open biopsy is favoured over a closed-needle biopsy because it allows for larger
specimens and more remarkable preservation of muscle fibre orientation. Muscle biopsy
performed by a tiny incision with a local anaesthetic using a sharp-jawed medical device can
produce a diagnostically sufficient specimen with a low complication rate. Typically, the legs’
quadriceps, the deltoid or biceps in the arms are the muscle targets for a biopsy. (UpToDate,
2022) An abnormal result is obtained, indicating the existence of dermatomyositis, a muscle
illness characterised by inflammation and a skin rash. Muscle biopsy is preferred because it is
extremely precise (97.1%) and sensitive (86.5%) to identify myopathy. (National Library of
Medicine, 2018)
References
abcam. (n.d.). Retrieved from https://www.abcam.com/secondary-antibodies/direct-vs-
indirect-immunofluorescence

ibidi. (n.d.). Retrieved from https://ibidi.com/content/365-immunofluorescence-staining-a-

typical-workflow

Ken Lau, K. S. (n.d.). BioLegend. Retrieved from https://www.biolegend.com/en-

us/blog/immunofluorescence-vs-immunocytochemisty-vs-immunohistochemistry-
what-is-the-difference

Mayo Clinic. (2020, July 01). Retrieved from https://www.mayoclinic.org/diseases-

conditions/dermatomyositis/symptoms-causes/syc-
20353188#:~:text=Dermatomyositis%20(dur%2Dmuh%2Dtoe,late%2040s%20to%20
early%2060s.

National Library of Medicine. (2018, November 3). Retrieved from

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365764/

Rockland. (n.d.). Retrieved from https://www.rockland.com/resources/immunofluorescence-

technique/

UCSF Health. (2018, October 7). Retrieved from https://www.ucsfhealth.org/medical-

tests/muscle-biopsy

UpToDate. (2022, February 24). Retrieved from

https://www.uptodate.com/contents/diagnosis-and-differential-diagnosis-of-
dermatomyositis-and-polymyositis-in-adults#H5035136

Wix.com. (n.d.). Retrieved from https://zulkefflizan.wixsite.com/group13/form__map

Zhang, G. (2017, April). Dermnet NZ. Retrieved from https://dermnetnz.org/topics/direct-

immunofluorescence
 Plagiarism Checker X Originality Report
Similarity Found: 8%

Date: Wednesday, June 08, 2022

Statistics: 76 words Plagiarized / 992 Total words
Remarks: Low Plagiarism Detected - Your Document needs Optional Improvement.
-------------------------------------------------------------------------------------------

1.0 Introduction Dermatomyositis Dermatomyositis is an infrequent inflammatory

disorder that causes myasthenia and skin rash. Both children and adolescent can be
affected by this disease. In adults, dermatomyositis typically arises between 40 and 60
years old, whilst in youngsters, it often appears around aged 5 to 15.

Besides, it affects women more frequently than men. Currently, there is no treatment
exists for dermatomyositis, yet the therapy can assist in eliminating the skin rash and
restoring muscular power and function. The typical symptoms of dermatomyositis may
develop abruptly or gradually over time, including skin changes, violet-coloured or
dusky red rash arise on the eyelids, face, knees and elbows.

Moreover, muscle weakness in which affects the muscles nearest to the trunk, including
those in the shoulders, hips, thighs, upper arms, and neck is one of the indications. The
origin of dermatomyositis is undetermined. However, the disease resembles
autoimmune disorders extensively in various ways, as the immune system targets body
parts mistakenly.

Additionally, environmental and genetic factors such as viruses, UV exposure, certain

drugs, and smoking might cause dermatomyositis. (Mayo Clinic, 2020) / Diagram 1:
Dermatomyositis. Direct Immunofluorescence (DIF) Primary immunofluorescence,
commonly recognized as direct immunofluorescence, encompasses the use of a singular
antibody that has been attached directly to a fluorophore. The antibody allows the
attached fluorescent dye to be seen under a microscope when it binds to the target
molecule.

This technique helps minimise concerns with antibody cross-reactivity or non-specificity,