☘️Antinuclear Antibodies (ANA)☘️
Here's a 🧵 on another #rheumatology test which can confuse>help if ordered without a 💭 (part 1)
Let's peek into the #immunology lab to learn more about the lab aspect of ANA 🥼🧪👀
(1/12)
#MedTwitter #LupusAwarenessMonth #MedEd
Here's a 🧵 on another #rheumatology test which can confuse>help if ordered without a 💭 (part 1)
Let's peek into the #immunology lab to learn more about the lab aspect of ANA 🥼🧪👀
(1/12)
#MedTwitter #LupusAwarenessMonth #MedEd
🤔 What do we mean by ANA??
🔹As the name suggests, they are antibodies to various nuclear components
🔹These Antigens(Ag) can be related to
1. DNA🧬: DNA, histones, nucleosomes
2. RNA: Smith, RNP, Ro, La
3. Others: ribosomal P, Ku etc
(2/12)
🔹As the name suggests, they are antibodies to various nuclear components
🔹These Antigens(Ag) can be related to
1. DNA🧬: DNA, histones, nucleosomes
2. RNA: Smith, RNP, Ro, La
3. Others: ribosomal P, Ku etc
(2/12)
Which one of the following is the oldest test to detect ANA? 📜
(3/12)
(3/12)
1️⃣LE cells indeed!
Discovered in 1948,
LE cells are nothing but phagocytes that have eaten up the antibody bound nuclear material of another cell
(4/12)
pubmed.ncbi.nlm.nih.gov
Discovered in 1948,
LE cells are nothing but phagocytes that have eaten up the antibody bound nuclear material of another cell
(4/12)
pubmed.ncbi.nlm.nih.gov
🤷How do we test for LE cells?
💉🩸➡️ Shake 🪇 vigorously for leukocytes to rupture & release nuclear material ➡️incubate for ANAs to bind to this material & phagocytes to eat🍴 these up➡️centrifuge➡️buffy coat ➡️stain and observe🔬
(5/12)
💉🩸➡️ Shake 🪇 vigorously for leukocytes to rupture & release nuclear material ➡️incubate for ANAs to bind to this material & phagocytes to eat🍴 these up➡️centrifuge➡️buffy coat ➡️stain and observe🔬
(5/12)
LE cell assay has now given way to more sophisticated tests⏬
2️⃣Indirect immunofluorescence assay(IFA) is an old test but 🧓is🪙!
📜Initially, rodent liver cells were used but now Hep2 cells are used for the assay 🐀
surl.li
(6/12)
2️⃣Indirect immunofluorescence assay(IFA) is an old test but 🧓is🪙!
📜Initially, rodent liver cells were used but now Hep2 cells are used for the assay 🐀
surl.li
(6/12)
🤷What do we mean by IFA??
Let's break it down
🔹Hep2 cells are fixed on a slide
🧐Why hep2 cells: large nuclei + presence of dividing cells✅ for ANA🔬
🔹+ serum in appropriate dilution (remember pro/post zone phenomena!)
ANA if present will bind to the Hep2 cells
(7/12)
Let's break it down
🔹Hep2 cells are fixed on a slide
🧐Why hep2 cells: large nuclei + presence of dividing cells✅ for ANA🔬
🔹+ serum in appropriate dilution (remember pro/post zone phenomena!)
ANA if present will bind to the Hep2 cells
(7/12)
🔹+ fluorescent tagged anti-human or 'secondary' antibodies (YES, that's why it's called indirect immunofluorescence!!)
4. Observe under a special fluorescent 🔬
(8/12)
4. Observe under a special fluorescent 🔬
(8/12)
It tells you 3️⃣things
1. Titre- dilution at which ANA is positive, usually done in 1:80 or 1:100
2. Intensity of staining
3. Pattern- a specific staining pattern helps identify the type of antigen
ps: it's like fireflies on a slide 😍
(9/12)
1. Titre- dilution at which ANA is positive, usually done in 1:80 or 1:100
2. Intensity of staining
3. Pattern- a specific staining pattern helps identify the type of antigen
ps: it's like fireflies on a slide 😍
(9/12)
IFA can also detect Ab to cytoplasmic Ag hence ANA is better called 'Anti Cell antibodies'
IFA is the 🪙 standard but results depend on
🔹Quality of cell fixation
🔹Reagents
🔹Observer dependent
Hence we have newer tests (ELISA, CIA, FEIA, ALBIA etc)
(10/12)
IFA is the 🪙 standard but results depend on
🔹Quality of cell fixation
🔹Reagents
🔹Observer dependent
Hence we have newer tests (ELISA, CIA, FEIA, ALBIA etc)
(10/12)
3️⃣ ELISA
A mix of nuclear Ag coated on a plate
Hep2 extract: 👍 sensitivity, 👎 specificity
Mix of separate Ag: 👎 sensitivity, 👍 specificity
Why?
🔹Different Ag may compete for binding🍽️
🔹3D structure of Ag may change
Also, specific Ag can't be determined
(11/12)
A mix of nuclear Ag coated on a plate
Hep2 extract: 👍 sensitivity, 👎 specificity
Mix of separate Ag: 👎 sensitivity, 👍 specificity
Why?
🔹Different Ag may compete for binding🍽️
🔹3D structure of Ag may change
Also, specific Ag can't be determined
(11/12)
In summary, IFA helps detect ANA positivity, the titre, intensity and the ANA pattern! Hence it's the method of choice for ANA testing.
We'll be dealing with patterns and diseases in part 2 of ANA!!
📖nature.com
📖nature.com
(12/12)
We'll be dealing with patterns and diseases in part 2 of ANA!!
📖nature.com
📖nature.com
(12/12)
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