ENA + specific exam questions

Question 1

Antigens detected in ENA

Answer 1

U1-nRNP, Sm, SSA (Ro), SSB-La, Scl70, PM-ScI, Jo-1, Centromeres, PCNA, dsDNA, Nucelosomes, histones, ribsomal p-proteins, AMA-M2

Question 2

associated diseases to antigens

U1-nRNP, Sm, SSA (Ro), SSB-La, Scl70, PM-ScI, Jo-1, Centromeres, PCNA, dsDNA, Nucelosomes, histones, ribsomal p-proteins, AMA-M2,

Answer 2

U1-nRNP: Sharp syndrome, SLE, systemic sclerosis, poly/dermamyositis

Sm: SLE

SSA (Ro): Sjogrens, SLE, neonatal lupus erythmatosus

SSB-La: Sjogrens, SLE, neonatal lupus erythmatosus

Scl70: systemic scleorosis

PM-ScI: Systemic scleorosis
polymoyostis/Systemic scleorosis overlap

Jo-1: poly/dermamyositis

Centromeres: Systemic sclerosis, PBC

PCNA: SLE

dsDNA: SLE

Nucelosomes: SLE

histones: SLE, RA,Drug induced lupus erythmatosus

ribsomal p-proteins: SLE

AMA-M2: PBC

Question 3

What do the antibodies target

Answer 3

Ab target nuclear antigens are directed against various cell components (nucleic acids, cell nucleus proteins, ribonucleoproteins)

Question 4

Purpose of ENA

Answer 4

An extractable nuclear antigen (ENA) panel detects the presence of autoantibodies in the blood that react with proteins in the cell nucleus

Question 5

ENA vs ANA

Answer 5

The ANA tests for the presence or absence of autoantibodies, while the ENA panel evaluates which proteins in the cell nucleus the autoantibodies recognize. The ENA panel helps diagnosis, distinguish between, and monitor the progression of autoimmune diseases

Question 6

ANA Test principle, purpose, results

Answer 6

Purpose:
Diagnosis of rheumatic diseases
Detecting Autoimmune and Connective Tissue Diseases: The primary purpose of the ANA test is to screen for the presence of antinuclear antibodies (ANA) in the blood. These antibodies target various components within the cell nucleus, such as DNA, RNA, and nuclear proteins. The presence of ANAs can be indicative of autoimmune and connective tissue diseases, including systemic lupus erythematosus (SLE), Sjögren’s syndrome, rheumatoid arthritis, and others.

Test Principle: INDIRECT IIF
The ANA test is typically performed using an immunofluorescence assay. In this method, a patient’s blood sample is exposed to a set of different cells or tissues (Hep-2000) on a microscope slide. Hep 2000 cells are genetically engineereed ‘typical’ Hep2 cells (mitotic human epitheloid cells) which have been transfected with human cDNA for the SSA/Ro antigen. This transfecrion allow the Hep 2000 cell to produce native SSA-Ro antigen. Only 10-15% of the hep2000 cells will hyperexpress the SSA antigen, allowing other ANA patterns to be observed on the remaining cells.

Diluted patient serum is incubated with the Hep2000 cell substrate, this allow the specific binding of ANA’s to the nuclear material. This binding causes the formation of Ag-Ab complexes and during the washing process, unbound and non-specific autoantibodies will be removed. If these bound antibodies are present in incubation, then detected using fluorescently labeled secondary antibodies (fluroescein labelled anti-human antibody conjugate) as this conjugate will bind to the ag-ab complex during incubation. After washing off excess conjugate, the complexs can be seen under a UV micorscope. If the ag-ab complex is present, the fluroscence will bright apple green and have a specific pattern. If the sample is negative, there will be no clearly detectable fluroscent pattern in the cell nucleus and the non-chromosome region of the mitotic cells will show brigther staining.

Results:
ANA test results are reported as titers and patterns. Titers indicate the concentration of ANAs in the blood, while patterns describe how the ANAs bind to specific nuclear antigens. The pattern can be homogeneous, speckled, nucleolar, or centromere, among others. The pattern can sometimes help in diagnosing specific autoimmune diseases or guiding further testing.

Question 7

ENA Test principle, purpose, results

Answer 7

Purpose:
Identifying Specific Autoimmune Diseases: The ENA test is used to follow up on a positive ANA test by identifying and quantifying specific antibodies that target extractable nuclear antigens. These antigens are proteins found within the cell nucleus. The ENA test helps in diagnosing and differentiating between specific autoimmune diseases, such as Sjögren’s syndrome, systemic sclerosis (scleroderma), polymyositis, dermatomyositis, and mixed connective tissue disease.

Test Principle: Line immunoassay
The test uses test strips that are coated with parrallel lines of antigens which have been purified by affinity chromatography. The strip contains 18 different antigens (eg RNP, Sm, Ro, SSB, Scl-70, Jo-1, dsDNA, AMA-M2 etc). In the first reaction step, diluted patient sample are incubated with the immunoblot strips. In pos samples, the specific IgG antibodies and IgA or IgM will bind to the corresponding antigen site. To detect them, a second incubation occurs with an enzyme labelled anti-human IgG conjugate which is capable of inducing a colour change.
Thus, detect and quantify antibodies against specific extractable nuclear antigens. Each antibody target (e.g., SS-A, SS-B, Sm, RNP, Jo-1) is tested separately to determine its presence and concentration in the patient’s blood.

Results:
ENA test results specify which extractable nuclear antigens’ antibodies are present and at what concentrations. The presence of particular antibodies can help in diagnosing specific autoimmune diseases. Interpretation of ENA test results is crucial for identifying the underlying autoimmune condition accurately.

In summary, the ANA test screens for the presence of antinuclear antibodies, which are indicative of autoimmune and connective tissue diseases. The ENA test follows up on a positive ANA test and identifies specific antibodies targeting extractable nuclear antigens, aiding in the diagnosis and differentiation of specific autoimmune diseases. Both tests are essential tools in diagnosing and managing autoimmune and connective tissue disorders.

Question 8

Challenges in diagnosis of AID

Answer 8

1.Diverse Symptoms:
Autoimmune diseases can affect virtually any part of the body, leading to a wide range of symptoms. These symptoms can be vague and overlap with those of other conditions, making it challenging to pinpoint the underlying cause. For example:
Rheumatoid arthritis can cause joint pain, stiffness, and swelling, which are also symptoms of other musculoskeletal disorders.
- Gastrointestinal symptoms like diarrhea and abdominal pain can occur in conditions such as Crohn’s disease and celiac disease
- Fatigue and joint pain can be indicative of various autoimmune diseases, including rheumatoid arthritis and lupus

2.Variability in symptoms:
Autoimmune diseases can manifest differently in different individuals due to factors of environmental triggers, genetics, age, gender, lifestyle, co-existing diseases. This variability can make it difficult to identify a common set of symptoms or markers for diagnosis. For instance:
- Genetic Factors: Genetic predisposition plays a significant role in the development of autoimmune diseases. Different genetic variations can lead to variations in how the immune system responds to self-antigens. Individuals with different genetic backgrounds may have different patterns of symptom expression. Example: In the case of type 1 diabetes, genetic variations play a significant role. Type 1 diabetes is strongly influenced by variations in the HLA-DQ genes. Different combinations of HLA-DQ alleles can affect the age of onset and the severity of diabetes symptoms in affected individuals.
- Environmental Triggers: Autoimmune diseases often have environmental triggers, such as infections, exposure to toxins, or hormonal changes. These triggers can vary from person to person, leading to differences in symptom onset and severity. For example, in multiple sclerosis (MS), viral infections have been linked to disease exacerbations in some individuals. Example: Systemic lupus erythematosus (SLE) can be triggered or exacerbated by exposure to sunlight. However, not all individuals with SLE will have the same sensitivity to sunlight, leading to differences in skin rashes and other sun-induced symptoms.
- Comorbid Conditions: Many individuals with autoimmune diseases have comorbid (coexisting) conditions that can influence symptomatology. These comorbidities may contribute to the complexity of symptoms. For example, a person with lupus may also have fibromyalgia, which can cause additional pain and fatigue.
- Lifestyle and Diet: Lifestyle factors, such as diet, stress levels, and physical activity, can impact the severity and expression of autoimmune disease symptoms. Some individuals with RA report changes in symptom severity related to fasting or specific diets. Intermittent fasting or certain anti-inflammatory diets (e.g., Mediterranean diet) may have a positive impact on joint pain and inflammation for some RA patients, but not all.
- Age and Gender: Some autoimmune diseases have a predilection for certain age groups or genders. For instance, rheumatoid arthritis is more common in women, and the age of onset can vary widely. These demographic factors can influence the presentation of symptoms.Example: Systemic scleroderma is more common in women, but the age of onset can vary widely. Symptoms and disease progression may differ between a young adult and an older individual with the same condition.

3. Fluctuating Symptoms:
   Autoimmune diseases often have symptoms that come and go or vary in intensity over time. This variability can make it difficult to diagnose, as patients may not exhibit symptoms during medical appointments. For instance:
   Multiple sclerosis (MS) can have periods of remission and relapse, making it challenging to identify during a single doctor’s visit.
   Coeliac - only with gluten (hard to isloate that cause)

4.Overlapping Diseases:
Many autoimmune diseases share common features and can co-occur in the same individual. This complicates the diagnostic process, as one autoimmune disease may mask or be mistaken for another. For example:
Celiac disease and type 1 diabetes often occur together and can have similar gastrointestinal symptoms. For instance, Hashimoto’s thyroiditis can mimic the symptoms of depression and chronic fatigue syndrome, leading to incorrect initial diagnoses.

5.Lack of Specific Biomarkers:
Autoimmune diseases typically lack specific laboratory tests or biomarkers that definitively diagnose the condition. Diagnosis often relies on a combination of clinical signs, symptoms, and lab results, which can be inconclusive. While there are diagnostic tests available for many autoimmune diseases, no single test can definitively confirm all autoimmune conditions. Some tests, like the antinuclear antibody (ANA) test, may be positive in multiple autoimmune diseases, but further evaluation is required to pinpoint the specific condition. A positive ANA test alone does not suffice for diagnosis. In fact, low levels of ANAs can be found in healthy patients. Given that ANAs are present in up to 30% of the average healthy population, there are inherent challenges against using them to diagnose autoimmune connective tissue disorders. Positive results must be interpreted with the existing clinical manifestations to establish a diagnosis.
For instance:
In SLE, diagnosis is based on a combination of clinical criteria, including skin rashes, joint pain, and positive autoantibody tests, but no single test can confirm the disease.
Eg: Sjogrens - need 2/3 of the criteria

6.Delayed Diagnosis:
Many autoimmune diseases have a gradual onset and may take years to develop fully. This delayed progression can result in a delayed diagnosis, as symptoms may be subtle or dismissed initially.
Patients may delay seeking medical care until their symptoms become more pronounced or debilitating. The symptoms of Hashimoto’s thyroiditis can be subtle and nonspecific, including fatigue, weight gain, hair loss, and cold intolerance. These symptoms can easily be attributed to other factors, such as stress or aging)

Normal Thyroid Function: In the early stages of Hashimoto’s thyroiditis, thyroid function tests may show normal or borderline thyroid hormone levels. It may take time for these levels to change enough to clearly indicate hypothyroidism, leading to delays in diagnosis.

7.Rare Autoimmune Diseases:
Some autoimmune diseases are exceptionally rare, making them even more challenging to diagnose due to limited awareness and expertise. For example, diseases like Stiff Person Syndrome are extremely rare and often misdiagnosed as other neurological conditions.

8.Diagnostic criteria: Autoimmune diseases often have diagnostic criteria that require meeting a certain number of criteria or exclusion of other conditions. This can make the diagnostic process complex and time-consuming

Question 9

ANA patterns and antigen and disease

Answer 9

1. Homogenous
   dsDNA (SLE)
   Histones (DLE, RA)
2. Rim
   Centromere (CREST)

3.Speckled
RO-SSA (Sjogrens syndrome)
SSB La (Sjogrens syndrome)
RNP Miced connective tissue disease)
Sm (SLE)
Scl70 (scleroderma)

4.Nucelolar
Scl-70 ( Scleroderma)

5.Cytoplasmic
Jo-1 (Poly/dermamyositis)
Mitochondria (PBC)
Smooth muscle (autoimmune hepatitis)

Question 10

ENA autoantibody and associated CTD

Answer 10

Sm - SLE
RNP - SLE, MCTD
Scl-70 - systemic screlrosis
SSA - SS, SLE
SSB - SS, SLE
Centromere - CREST
Jo-1 = poly/derma

Question 11

ENA detects what 6 proteins in the cell nucleus

Answer 11

These proteins are known as “extractable” because they can be removed from cell nuclei using saline and represent six main proteins (Ro, La, Sm, RNP, Scl-70 and Jo1).

Question 12

ANA patterns and diseases

Answer 12

Homogenous: SLE and drug induced lupus
Nucleolar: scleroderma and polymusositis.
Speckled: SLE, Sjogrens, scleroderma,
Centromere: CREST

Question 13

three major outcomes when the complement system is activated

Answer 13

1.cell lysis upon assembly and insertion of the terminal membrane attack complex (MAC)
2. complement mediated opsonization
3.Release of anaphylatoxins that enhance local inflammation

Question 14

ENA method type

Answer 14

Line Immunoassay

Question 15

define titre and serial dilution

Answer 15

A serial dilution is any dilution where the concentration decreases by the same quantity in each successive step.

The titer is expressing the degree to which the solution can be diluted and still produce an observable reaction. Titers are reported out as the reciprocal of the dilution in the last tube giving a positive reaction. Eg 1:20 diltuion - titre of 20.

the highest dilution of a viral suspension that still causes agglutination of red blood cells

Question 16

MHC
Roles
Why called MHC?

Answer 16

role in immune response
role in organ transplantation
role in predisposition to disease

The term “Major Histocompatibility Complex” actually refers to a region of the genome that encodes a number of genes (hence Complex) that play an important (hence Major) role in tissue transplantation and antigen presentation (hence Histocompatibility).
Essentially, they are called MHC because this the main protein that expresses antigens to specific cells, both self and non-self antigens to T cells (hence the major part as all APC cells use MHC for antigen presentation to cause immune response). The MHC ensures that everything existing in the body is able to occur together without conflict (nothing foreign present to disrupt the harmony in the body), as if a foreign antigen is presented this indicates a possible break in this compatible state of the body and therefore needs to be eradicated. A key example used is in organ transplants (hence the histo-compatibility part). This MHC protein is the major protein used in the body for antigen presentation and therefore must vary in characteristics to support the presentation of all antigen types and presentation to specific cells (hence the complex part). This forms the complexity observed by their being 3 classes of MHC formed.
Class I = A, B and C (also called HLA-A, HLA-B and HLA-C)
- Ag (peptide) presentation to CD8+ cells

Class II = DP, DQ and DR (also called HLA-DP, HLA-DQ and HLA-DR)
- Ag (peptide) presentation to CD4+ cells

Class III = Complement proteins, Tumor necrosis factor

They are also complex in terms of their structure and the differences in strucutre between classes
MHC 1:
Open peptide binding cleft
3 Alpha chain domains
1 β2-microglobulin chain domain

MHC: 2 alpha, 2 beta, closed peptide binding cleft

The term “major” in Major Histocompatibility Complex refers to the fact that these molecules are a significant part of the immune system’s ability to recognize foreign substances, such as pathogens or transplanted tissues.

“Histocompatibility” implies compatibility between tissues from different individuals, which is particularly relevant in the context of organ transplantation.

“Complex” signifies that this group of genes encodes a variety of proteins with diverse functions, including the MHC class I and MHC class II molecules.

Question 17

MHC restricted T cells meaning, examples and IMPORTANCE

Answer 17

“restricted” = “recognizes antigen on…”)

MHC restriction, refers to the fact that T cells are restricted to only interacting to self MHC, specific MHC and only responding to ag on MHC. For example, Tc are restricted to recongising MHC class 1 and TH are resticted to recognising MHC class 2.

CD4+ T cells are class II MHC-restricted i.e. they must recognize antigen presented on class II MHC.
CD8+ T cells are class I MHC-restricted i.e. they must recognize antigen presented on class I MHC

MHC restriction also implies that T cells are restricted to only responding to the antigen when it is bound to a particular MHC molecule.

Generally, T cells must recognize antigen on a self MHC allele and so are said to be self-MHC restricted. This is because T cells are “tuned” to recognize antigen complexed with self-MHC during T cell maturation in the thymus (central tolerance). Those which do NOT recognise the self MHC or those with TOO much affinity will not undergo positive selection and will undergo apoptosis.

This restriction results from a process of positive selection during T cell development in the thymus. In this process, those immature T cells that will be capable of recognizing foreign peptides presented by self MHC proteins are selected to survive, while the remainder undergo apoptosis. Thus, MHC restriction is an acquired property of the immune system that emerges as T cells develop in the thymus

IMPORTANCE:
MHC present antigens (foriegn and self) and this allows for the T cells to surviellance the host for any potential threats (pathogens, viral infections etc) and to distinguish ‘self’ and ‘foreign’ molecules (preventing autoimmunity when T cells leave for the periphery). Therefore, this restriction is important because it allows this surviellance and the recognition of self and non-self cells which prevents T cells reacting against self antigens and also allows for an immune response to occur if a foreign antigen is presented.

Question 18

Defination of neutralisation, opsonisation and complement activation

Answer 18

Neutralisation - Antibodies can bind and neutralise the effects of toxins produced by pathogenic microbes or the ability of antibodies to block the site(s) on bacteria or viruses that they use to enter their target cell.

Opsonisation - antigens with antibodies bound to their surface are easily recognised by macrophages for phagocytosis. Opsonization is an immune process which uses opsonins to tag foreign pathogens for elimination by phagocytes.

Complement activation – antibodies activate the complement system by coating a bacterial cell. Cascade of plasma proteins that can be activated directly by pathogens or indirectly by pathogen-bound antibody, leading to a cascade of reactions that occurs on the surface of pathogens and generates active components with various effector functions

Question 19

What is the role of IANZ in terms of medical lab

Answer 19

To provide technical input into government standard policies to help with export trade

To assess a laboratories competence against relevant standards (ISO 15189:2012 - Medical laboratories – Requirements for quality and competence) and provide endorsement of accreditation alongside audit outcomes of Recommendations
Corrective action request.

They assess five key areas:
Competence and experience of staff
Integrity and traceability of equipment and materials
Technical validity of methods
Validity and suitability of results
Compliance with discipline specific management systems standards

IANZ requires the establishment of quality indicators to monitor and evaluate laboratory performance throughout critical aspects of pre-examination, examination, and post-examination processes

Question 20

How does MHC occur from central tolernce (T cells)

Answer 20

During T cell development, T cells go through a selection process in the thymus to ensure that the T cell receptor (TCR) will not recognize MHC molecule presenting self-antigens, i.e that its affinity is not too high. High affinity means it will be autoreactive, but no affinity means it will not bind strongly enough to the MHC. The selection process results in developed T cells with specific TCRs that might only respond to certain MHC molecules but not others. The fact that the TCR will recognize only some MHC molecules but not others contributes to “MHC restriction”

Enforcing the restriction that T cells are activated by peptide antigens only when the antigens are bound to self-MHC molecules, MHC restriction adds another dimension to the specificity of T cell receptors so that an antigen is recognized only as peptide-MHC complexes

Question 21

main cause of transplant rejection

Answer 21

Rejection is mediated mainly by T cells, which react against genetically “foreign” versions of cell-surface proteins (MHC)

T cells respond to foreign MHC proteins in the same way they respond to self MHC proteins that have foreign antigen bound to them.

Question 22

define QA and QC

Answer 22

QA:
all the planned and systematic activities implemented within the quality system that can be demonstrated to provide confidence that a product or service WILL FULFILL requirements for quality

QC:
the operational techniques and activities USED TO FULFILL requirements for quality

Question 23

Why are T cells MHC restricted
- central tolerance criteria
- what happens once it reaches periphery (what is it able to do due to central tolerance)
-Biological reason
-Generally why MHC restriction is significant for T cell function

Answer 23

MHC restriction is present in T cells due to the selection methods for T cells in the thymus during central tolerance. Central tolerance involves the positive selection of T cells which are selected for that meet a criteria of:
-demostrate specific levels of affinty to self MHC (not too much where it causes an immune response or too little that it doesnt recognise the protein)
-and the appropiate affinity level to the self antigen (has to have some interaction but not strong interaction).

Therefore, once the T cell reaches the periphery it is able to differeniate between self and non-self antigens (which are presented on MHC) due to being able to recognise the protein presenting it. This is why MHC restriction exists, to let T cells perform their function of recognising self vs non-self T cells. Another key purpose of MHC restriction is to prevent The biological reason of MHC restriction is to prevent supernumerary wandering lymphocytes generation (T cells which are non-fucntional (do not have any affinity to MHC or self antigens, thus no ability to mount immune response as can not recognise MHC to bind to it) do not become in such excess volumes to the point where there are so many T-cells which roam aimlessly throughout the body and prevent activity of functional t cells)

MHC restriction is significant for T cells to function properly when it leaves the thymus because it allows T cell receptors to bind to MHC (which presents antigens) and detect cells that are infected by intracellular pathogens, viral proteins and bearing genetic defects.

Question 24

Explain this statment:
The biological reason of MHC restriction is to prevent supernumerary wandering lymphocytes generation

Answer 24

This means that the strict selection process in central and peripheral tolerance using MHC restrcition means that there is prevention of excessive circulating T cell population. This is important because if non-functional T cells (no affinity to self antigens or MHC) were allowed into circulation, they would crowd out functional T cells and slow down the rate at which adaptive immune responses are formed.

This strict selection means that 98% of immature T cells are killed, with 2% progressing to peripheral tolerance. This selection process not only limits the T cell population to prevent over crowding (supernumerary wandering lymphocytes) but also ensures that the T cells in the periphery are functional MHC restricted T-cells (able to recognise MHC and the subsequent antigen presented as self or foreign).

Therefore, MHC restrcition plays a pivotal role in this vigorous central tolerance by presenting the antigens to T cells and thus demostrating what T cells are self-reactive T cells and therefore preventing them from being released into the bloodstream and creating large T cell populatios wandering in the periphery that are able to cause an autoimmune reaction.