Immunodiagnostics changing the practice of Rheumatology:
Past and a Peep into future
Owing to advances in immunology, molecular biology, instrumentation and demand for high throughput results , there has been a revolution in the field of immunodiagnostics. Managing patients with rheumatic diseases without immunology laboratory results is unthinkable, be it acute phase reactants such as CRP, C3 or CRP or autoantibodies like RF, ANA, ANCA or both. Results interpretation when the clinical picture matches is satisfying however the interpretation is extremely challenging or even confusing when there is a discordant clinical picture. The purpose of this piece is to highlight some immune biomarker has changed the practice of rheumatology and to give a view of the cutting edge techniques as we are heading to a personalised medicines
Autoantibodies detection and quantifications have significantly changed modern day practice of rheumatology.
One of the major translational values of immunology are detection of autoantibodies in both diagnosis and prognosis of rheumatic diseases.
Today the major workload of laboratory tests are based on antibodies due to their high specificity. Be it an ELISA for Beta-HCG (Human Chorionic Gonadotropin), to an (IHC) Immunohistochemistry for colon cancer, the antibody is the basic element in the test that detects the antigen or target of interest. Nowadays there are so many diverse tests in Immunology and in Rheumatology, whose uniqueness is matched only by the ingenuity in designing such a test. This short write-up cannot do justice to all the tests, but is aimed at providing the reader a taste of the variety of tests available. First let us see how antibody detection helped in the diagnosis of RA and Lupus:
Auto-antibodies in RA:
Named as the Waaler-Rose test after the discoverers (1), the Rheumatoid factor is often mislabelled in our country as “RA factor”. The original test was a haemagglutination slide test using sheep RBCs. This was complemented by the antibody against cyclic citrullinated peptides (Anti-CCP). These are basically ELISA based tests with the actual antigen not revealed by the patent holders. However, later generations of this test targets even non-cyclic citrullinated peptides, and the better name for the test is anti-citrullinated peptide antibodies (ACPA). There are now many autoantibodies described to various modified peptides that are found in the RA synovium including anti-perinuclear factor (APF), anti-keratin antibodies (AKA) and anti-filaggrin antibodies (AFA). These have not been able to surpass or even compete with RF and ACPA. The anti-Carbamylated peptide (CarP) antibodies are available commercially and might be positive in around 10% of rheumatoid arthritis patients negative for RF and ACPA.
Autoantibodies define SLE and related disease:
The LE cells were first discovered in the bone marrow of a patient with systemic lupus erythematosus. These were actually mature polymorphonuclear leukocytes which had phagocytosed the liberated nuclear material of another leukocyte (with the help of some “anti-nuclear” antibodies. Less than a decade later, George Friou in 1957, described the immunofluorescence(IF) technique of ANA detection(3). It is now the gold standard since it is comparatively cheap, highly sensitive and specific, and also the different patterns provide additional information about the type of ANA. The diversity of ANA pattern, homogenous, coarse and fine speckled, centromere is due to a host of cytoplasmic and nuclear antigens, known by names more literally than scientific such as Sm, U1RNP, Ro (SSA) and SSB.
The diversity of ANAs initially based on labour intensive techniques like immunodiffusion, counter immunoelectrophoresis and western blot is credited to Eng Tan (4) for their work in the 1970s. This led to discovery of antigenic targets like Sm, U1RNP, Ro (SSA), La (SSB), Jo-1, Scl -70, RNA polymerase III (collectively called Extractable nuclear antigens or ENA) among others provided confidence to clinical diagnosis of connective tissue diseases. Almost parallel to this development, detection of antibodies to double strand DNA moved from the liquid phase radioimmunoassay (Farr) to solid phase ELISA which became widely available and was as sensitive but less specific. The overall impact of both these antibody systems unravelled a subset of clinical features associated with specific autoantibodies which have been tapped and made a serological confirmation to the diagnosis where the clinical situation was confusing.
Table I Autoantibodies used as diagnostic or prognostic marker
Autoantibodies to |
Disease /clinical features Association |
Techniques |
Ds DNA |
SLE , Nephritis |
ELISA, Radioimmunoassay |
Sm |
SLE
|
Line blot |
U1-RNP |
SLE, MCTD, IIM |
Line blot |
Ro and La |
SLE, Sjogren's Syndrome, Myositis, Neonatal lupus, Congenital heart block. |
Line blot, ELISA |
Jo-1, PL-7, PL-12 |
Anti Synthetase syndrome |
Line blot, ELISA |
Antiphospholipid antibodies define “Hughes Syndrome
Graham Hughes Laboratory at Hammersmith Institute, London described for the first time the presence of anticardiolipin antibodies in SLE that is one of the phospholipid and its association with vascular thrombosis, recurrent abortions and thrombocytopenia (4) Subsequently, Lupus anticoagulant another antibodies in this category based on in vitro prolongation of prothrombin dependant clotting pathways was found to be strongly associated with the triad mentioned. Both aCL and LA are pathogenic when these target Beta 2 GP1, antibodies to β2 GP1 can be measured by ELISA and the presence of all three have a strong pathogenic effect.
Presence of Autoantibodies (ANCA) replaced Biopsy for diagnosis of Small vessel Vasculitis
GPA (Granulomatous polyangiitis), MPA (Microscopic polyangiitis) and EPA (Eosinophilic polyangiitis) are rare but potentially fatal diseases, required histological confirmation from not so easily available tissues (lung /kidneys) for diagnosis. The discovery of ANCA in the 1980s (5,6)and its antigenic specificity c-ANCA and p-ANCA to be of high sensitivity and specificity (90-100%) has replaced biopsy in most cases.
Immunoprecipitation led to discovery of Myositis specific autoantibodies
Detecting antibodies to small RNA molecules and RNA proteins complex by precipitating antibodies with radiolabelled cell extracts revealed a myriad of a myriad of antigenic targets that were associated with Inflammatory Myositis and /or interstitial lung disease. Subsequently, detection of these myositis specific autoantibodies are available, using commercial kits, some of these have characteristic association antibodies which have clinical relevance. Association of antibodies to Antisynthetase associated with ILD, antibodies to MDA5 associated with a rapidly progressive ILD, anti TIF1γ associated with malignancy related myositis and antibodies to Mi2 is associated with characteristic DM rash are examples .
Cutting edge
Large scale genetics, linkage analysis and Next generation sequencing:
Classically, the gold standard for gene sequencing is Sanger sequencing or DNA sequencing with chain-terminating inhibitors. Next generation sequencing, also known as massively parallel or deep sequencing, can be carried out on different platforms based on a principle of “sequencing by synthesis”. Now, instead of sequencing thousands of overlapping genes one by one, all can be sequenced “parallelly” in “massive” numbers. The interpretation requires bioinformatics analysis and today’s computers can analyse these Gigabytes of data (per whole exome or genome) within hours.
Linkage analysis is the study of linkage disequilibrium that is when two genes may be associated across individuals or families more than it can be accepted by chance (Mendel’s law of independent assortment). Usually, genes that are physically near on the same chromosome will exhibit linkage disequilibrium. This principle has been used to find candidate genes for congenital disorders, for example, the discovery of the MEFV gene as the causative agent in Familial Mediterranean Fever took years to be located on the short arm of Chromosome 16 (3). It took another 5 years to know that MEFV encodes the protein pyrin. Today, NGS can map the whole exome of a family within a day and in silico protein structure prediction tools can be used to find the effect of various mutations.
FACS
Fluorescence-activated cell sorting (FACS) or flow cytometer is predominantly a research tool but can also help in diagnostics such as CD4-positive cell counts or B cell depletion after use of drugs like Rituximab. It is based on fluidics. The basic principle is to maintain such a flow such that only one cell flows through the counter at one time. The size and granularity of the cell is responsible for “forward scatter” and “ side scatter” respectively. Also, various chromatophores tagged antibodies can be used to identify different cell surface markers, or even intra-cellular markers.
Multi-omics and hypothesis free approach
Taking a leaf out of the book of genomics, various other -omics have sprung up: transcriptomics, methylomics, metagenomics, proteomics, metabolomics, and so forth. While transcriptomics deals with RNA sequencing, methylomics is about detecting methylated Cytosine residues (that imply the gene activation state). Metagenomics is the detection of various microbiota associated with skin, oral mucosa, nasal mucosa, lung, gut or even vagina. Like genomics, transcriptomics, methylomics and metagenomics use NGS platforms with some modifications.
Proteomics and metabolomics use different technologies. But common ones are use of time of flight (TOF) along with mass-spectrometry detection (e.g. MALDI TOF with MS-MS), or gas or liquid chromatography. Also, nuclear magnetic resonance (NMR) using radioisotopes can be used for both metabolite detection of different biological fluids or 3-dimensional structural study.
The use of these technologies allow for a “hypothesis free approach”. In classical science we need to have a hypothesis that molecules A, B and C are associated with this disease condition. Three different experiments need to be set up for each A, B and C. If the three hypotheses are proven untrue, the researcher is almost back to zero. However, using hypothesis free approaches, hundreds of thousands of molecules (proteins, metabolites or gene transcripts or genes themselves) can be tested in 1 single experiment. This reduced the number of experiments and is more likely to find positive results. Though there are various statistical errors the researchers need to be aware of, currently the cost of these investigations is within the reach of the average researchers.
Single cell omics
An approach like transcriptomics provides gigabytes of information that is a summary of the gene expression patterns of various different cells. In peripheral blood, there may be theoretically hundreds of such cells. Similarly, for metabolomics, serum provides the summation of the metabolic processes of thousands of cells. This may lead to predominant cell types to foreshadow the effects of smaller populations of cells that may have a more important role. Technology advancement in the last decade has led to formulation of techniques such as single cell transcriptomics or single cell metabolomics.
An example is the elucidation of the PRIME (pre-inflammatory mesenchymal cells) cells that may potentially precipitate flares in rheumatoid arthritis(4). These were detected via the application of single cell transcriptomics.
Microfluidics: the future of medicine
Microfluidic platforms can be the future of diagnostic medicine. These combine the properties of fluidics and convert antibody antigen reactions to electric signals. Thus, the specificity of antibodies can be applied for point of care diagnosis. A single chip or even paper based microfluidic device can take a drop of blood and pipe it into different microchannels, each with detection of a specific antigen or marker. Since these can be “printed” using cheap material using 3-D printers, they are expected to provide cheap and point-of-care diagnostic devices.
Thus, the world of immunodiagnostics has been growing exponentially with major breakthroughs in the last couple of decades. This short write-up is only aimed at providing a glimpse of this vast world to the reader. The authors are interested in these and would love to have any questions or elaborations. Each of the techniques mentioned here deserve a complete write-up each! Also, feedback from the reader would enable us to focus on these or more basic research methodologies like confocal microscopy or CRISPR based diagnostics.
References:
Ramnath Misra, Sakir Ahmed
Professor & Associate Professor
Clinical Immunology and Rheumatology,
Kalinga Institute of Medical Sciences,
Bhubaneswar.