Thread by @PanktiMehta24

☘️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 🥼🧪👀

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#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

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Which one of the following is the oldest test to detect ANA? 📜

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1️⃣LE cells indeed!
Discovered in 1948,
LE cells are nothing but phagocytes that have eaten up the antibody bound nuclear material of another cell

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pubmed.ncbi.nlm.nih.gov/18921142/

🤷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🔬

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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/gubjw

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🤷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

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🔹+ fluorescent tagged anti-human or 'secondary' antibodies (YES, that's why it's called indirect immunofluorescence!!)

4. Observe under a special fluorescent 🔬

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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 😍
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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)

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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

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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/articles/nrrhe…

📖nature.com/articles/s4158…

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