1. AUTOIMMUNITY AND AUTOANTIBODY TEST
INTRODUCTION TO
AUTOIMMUNITY AND AUTOANTIBODY TEST
By Madiha Hamid
Horror autotoxicus: Literally, the horror of self-toxicity.
Tolerance to self Ags is maintained by central and peripheral mechanisms.
Dysregulation in these mechanisms will trigger autoimmune disease.
Autoimmunity is the failure of an organism to recognize its own constituent parts as ''self'', which allows an immune response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease. 
Prominent examples include Coeliac disease, diabetes mellitus type 1 (IDDM), systemic lupus erythematosus (SLE), Sjögren's syndrome, Churg-Strauss Syndrome, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, andrheumatoid arthritis (RA).
Horror autotoxicus: Literally, the horror of self-toxicity.
A term coined by the German immunologist Paul Ehrlich ( ) to describe the body's innate aversion to immunological self-destruction.
A family of 80 chronic and disabling diseases
Affects about million people in the USA.

2. CAUSES Autoimmunity Genetic Factors Symptoms
Three main sets of genes are suspected in many autoimmune diseases. These genes are related to:
Immunoglobulins
T-cell receptors
The major histocompatibility complexes (MHC).
Autoimmunity Environmental Factors
Following factors can trigger autoimmunity:
Pathogens,
drugs,
hormones,
toxins
Symptoms
Common symptoms may be Fever, muscle ache, fatigue, joint pain.
Each disease has specific symptom

3. Autoimmunity Can be classified into clusters that are either organ-specific or systemic
Organ-specific diseases
Damage is confined to the organ against which the immune response is mounted
Non-organ-specific diseases
Immune response against antigens which are not associated with the organ involved
Lungs of a patient with Goodpasture’s

4. Autoantibody test Mainly done for the diagnosis of autoimmune disease.
The Antinuclear antibody (ANA) test is often ordered first. ANA is a marker of the autoimmune process –
it is positive with a variety of different autoimmune diseases but not specific.
If an ANA test is positive, a panel of 4 or 6 autoantibody tests called extractable nuclear antigens (ENA) is typically ordered. , it is often followed up with other tests associated with arthritis and inflammation, such as a rheumatoid factor (RF), an erythrocyte sedimentation rate (ESR), a C-Reactive Protein (CRP), and/or complement protein complement levels.
The AMA test is ordered to help diagnose primary biliary cirrhosis (PBC).These abs are detedtced by indirect immunoflourescence in > 90% of patients.Reference range is < units.
Autoantibody tests may be ordered as part of an investigation of chronic progressive arthritis type symptoms and/or unexplained fevers, fatigue, muscle weakness and rashes.
Specific autoantibodies are usually present in a percentage of people with a particular autoimmune disorder. For instance, up to 80% of those with SLE will have a positive anti-double stranded DNA (anti-dsDNA) test, but only about 25-30% will have a positive anti-ribonucleoprotein (anti-RNP). Some individuals with an autoimmune disorder will have negative autoantibody test results but, at a later date as the disorder progresses, autoantibodies may develop
AMA TEST: antimitochondrial antibody test,
APA TEST: antiphospholipid antibody test,
ANTI CCP : anti-cyclic citrullinated peptide
APA TESTING: Depending on a person's signs and symptoms and medical history, a doctor may order one or more types and classes of these tests to help detect the presence of antiphospholipid antibodies and to help diagnose antiphospholipid syndrome (APS):
Cardiolipin antibodies (IgG, IgM, and sometimes IgA) are frequently ordered as they are the most common antiphospholipids. 
Lupus anticoagulant assay (e.g., RVVT) if a person has a prolonged PTT test
Anti-beta2 glycoprotein I and anti-phosphatidylserine testing may be ordered along with the other antiphospholipid antibodies to detect their presence and to provide the doctor with additional information.
APA testing is used to help determine the cause of :
Inappropriate blood clot formation (unexplained thrombotic episode, excessive clotting)
Recurrent miscarriage
Low platelet count (thrombocytopenia)
Prolonged PTT test
A cyclic citrullinated peptide (CCP) antibody test may be ordered along with or following a rheumatoid factor (RF) test to help diagnose rheumatoid arthritis (RA) and to assess the severity and probable course of the disease (prognosis).

5. C-Reactive Protein Complement Levels
C-reactive protein (CRP) test: The C-reactive protein (CRP) test can be used to monitor inflammation, which is associated with autoimmune disorders. A high or increasing amount of CRP in the blood suggests that the patient has an acute infection or inflammation. In a healthy person, CRP is usually less than 10mg/L. Most infections and inflammations result in CRP levels above 100mg/L.
While the test is not specific enough to diagnose a particular disease, it can be used to help physicians monitor inflammation and determine if current treatments are effective
Gul Sanober

6. C-reactive protein (CRP) test
a non-specific marker produced by the liver
increases during episodes of acute systemic inflammation
measured by blood tests.
Some forms of arthritis
Autoimmune diseases, such as lupus or vasculitis
a CRP level of 10 mg/L or lower is considered "normal."
However, a low CRP level does not always mean that there is no inflammation present. Levels of CRP may not be increased in people with rheumatoid arthritis and lupus. The reason for this is unknown.

7. Complement Levels most commonly C3 and C4 Synthesized in the liver
Measured through blood tests
Normal levels C3
Males: mg/dL
Females: mg/Dl C4
Males: mg/dL
Females: mg/dL
Levels decrease in autoimmune disorders(Hypocomplementemia)
The complement system is a complex series of some 30 proteins, two-thirds of which circulate in the plasma while the remainder are present on cell and tissue surfaces. Complement components are rapidly synthesised and released into the circulation following trauma or inflammation and elevated concentrations are common in many acute illnesses. Complement consumption can also take place in certain disease states, and complement concentration can be useful in diagnosis and monitoring progress. When present, low concentrations indicate increased consumption or decreased synthesis.
C3 and C4 concentrations should be requested in patients with renal disease, joint disease, and multi-system disorders with evidence of vasculitis. A low complement concentration would suggest an immunological basis for the symptoms. C3 and C4 concentrations are often low in patients with SLE.

8. Diagnose and monitor:
 acute or chronic autoimmune diseases such assystemic lupus erythematosus (SLE)
immune complex-related diseases and conditions such as glomerulonephritis, serum sickness, rheumatoid arthritis, and vasculitis
Individual complement components ordered when the total complement activity (CH50 or CH100) is abnormal to help determine which of the component(C1 to C9)s are deficient or abnormal

9. Considerations Serum is preferred
 All samples must show no signs of deterioration and lipaemic samples should be avoided.
Samples may be stored at 4°C prior to analysis
Acute malarial infection can cause false-positive results
Complement component 3 has been shown to interact with Factor H.

10. Rheumatoid factor - RF
By: Namra Haq

11. Rheumatoid factor explained
Rheumatoid factor is an immunoglobulin (antibody) which can bind to other antibodies. Antibodies are normal proteins found in the blood which function within the immune system. Rheumatoid factor though is not normally found in the general population (only found in about 1-2% of healthy people). The incidence of rheumatoid factor increases with age and about 20% of people over 65 years old have an elevated rheumatoid factor.
A blood test is used to detect the presence of rheumatoid factor. The blood test is commonly ordered to diagnose rheumatoid arthritis. Rheumatoid factor is present in 80% of adults who have rheumatoid arthritis but there is a much lower prevalence in juvenile rheumatoid arthritis. The incidence of rheumatoid factor increases with duration of disease in rheumatoid arthritis: at 3 months the incidence is 33%, while at one year it is 75%. Up to 20% of rheumatoid arthritis patients remain negative for rheumatoid factor (also known as "seronegative rheumatoid arthritis") throughout the course of their disease.

12. Conditions not associated with RF
Other conditions in which RH is positive apart from rheumatoid arthritis
Other autoimmune diseases can also be positive rheumatoid factor including:
Sjogren’s Syndrome, Systemic Lupus Erythematosus, Scleroderma, Polymyositis, Dermatomyositis, Mixed Connective Tissue Disease
Other infections or conditions which can be associated with positive rheumatoid factor include:
Bacterial Endocarditis, Osteomyelitis, Tuberculosis, Syphilis, Hepatitis, Mononucleosis, Liver Cirrhosis, etc.
Conditions not associated with RF
Rheumatic conditions NOT associated with elevated rheumatoid factor include:
Osteoarthritis, Gout, Reiter's syndrome / Reactive arthritis, etc.

13. How is RF factor measured?
The amount of rheumatoid factor in blood can be measured by:
Agglutination tests
One method mixes the patient's blood with tiny latex beads covered with human antibodies (IgG). The latex beads clump or agglutinate if rheumatoid factor (IgM RF) is present. Another method mixes the patient's blood with sheep red blood cells that have been covered with rabbit antibodies. The red blood cells clump if rheumatoid factor is present.
A titer is an indicator of how much the agglutination test blood sample can be diluted before rheumatoid factor is undetectable. A titer of 1:20 indicates that rheumatoid factor can be detected when 1 part of blood is diluted by up to 20 parts saline. The lab value for rheumatoid factor of 1:20 or less is considered normal.

14. Nephlometry test
This method mixes the patient's blood with antibodies that cause the blood to clump if rheumatoid factor is present. A light is passed through the tube containing the mixture and an instrument measures how much light is blocked by the mixture. Higher levels of rheumatoid factor create a more cloudy sample and allow less light to pass through, measured in units. The lab value for rheumatoid factor of 23 or less units is considered normal.
When analyzing lab results the following should be considered:
A rheumatoid factor more than 23 units and a titer more than 1:80 is indicative of rheumatoid arthritis but may also occur in other conditions.
False positive results can occur when the blood is high in fats.
Inaccurate results can be caused by improper handling of the blood specimen.
A negative test result for rheumatoid factor does not exclude the diagnosis of rheumatoid arthritis.

15. Erythrocyte Sedimentation Rate Test (ESR / Sed Rate)
Marker of Inflammation-autoimmune diseases like lupus or rheumatoid arthritis cause inflammation.
Nonspecific Test
Not used for screening purpose (low senstivity and specificity)
Inexpensive
Uses:
a. Diagnosis(auto-immune diseases ,rheumatoid arthritis, chronic kidney diseases)
b. Disease severity
c. Monitoring response to therapy ( inflammatory diseases such as temporal arteritis, polymyalgia rheumatica and rheumatoid arthritis)
Significnace: Although ESR is neither specific nor absolutely accurate test, it is very useful in inflammatory process monitoring.  The cause of ESR increase remains unexplained in 5% of cases. 
Reasons
ESR blood testing is called for when there is a suspicion of an inflammatory process underway in the body. The problem with the ESR test with blood is that it doesn't really state what exactly the cause of the inflammation is.
(Pediatric Crohn's Disease Activity Index),
The clinical usefulness of ESR is limited to monitoring the response to therapy in certain inflammatory diseases such as temporal arteritis, polymyalgia rheumatica and rheumatoid arthritis

16. Erythrocyte Sedimentation Rate Test (ESR / Sed Rate)
Measure of the settling of red blood cells in a tube of blood during one hour.
When abnormal proteins are present in the blood, typically due to inflammation or infection, they cause red blood cells to clump together and sink more quickly.
ESR is said to be nonspecific because increased results do not tell the doctor exactly where the inflammation is in the body or what is causing it, and also because it can be affected by other conditions besides inflammation. For this reason, the ESR is typically used in conjunction with other tests.
The erythrocyte sedimentation rate essentially measures the rate at which erythrocytes sediment to the bottom of a tube. Normally, erythrocytes sediment slowly because red cells have a net negative charge and repel each other. On the other hand, acute-phase reactants such as fibrinogen and other positively charged proteins can neutralize the surface charge of erythrocytes and promote aggregation. As a result, red cell aggregation increases the rate of sedimentation.

17. Erythrocyte Sedimentation Rate Test (ESR / Sed Rate)
Autoimmune Disorders
Higher than Normal
Factors That May Influence ESR
Increase ESR
Old Age
Pregnancy
Anemia
Macrocytosis
Decreased ESR
Extreme leukocytosis
Polycythemia
Red blood cell abnormalities
Very high ESR levels
Allergic vasculitis
Giant cell arteritis
Polymyalgia rheumatica
Reference Age
Adults Upper limit of reference range (mm/hr)
Age < 50 years
Men to 15
Women 0 to 20
Age > 50 years
Men to 20
Women 0 to 30
The immune system helps protect the body against harmful substances. In autoimmune disorder is a condition that occurs when the immune system mistakenly attacks and destroys healthy body tissue. ESR is often higher than normal in people with an autoimmune disorder.
Any condition that elevates fibrinogen (e.g., pregnancy, diabetes mellitus, end-stage renal failure, heart disease, collagen vascular diseases, malignancy) may also elevate the ESR. Anemia and macrocytosis increase the ESR. In anemia, with the hematocrit reduced, the velocity of the upward flow of plasma is altered so that red blood cell aggregates fall faster. Macrocytic red cells with a smaller surface-to-volume ratio also settle more rapidly.
A decreased ESR is associated with a number of blood diseases in which red blood cells have an irregular or smaller shape that causes slower settling.

18. Secret Blood Test of Autoimmune Disease
TH1 / TH2 Cytokine Test 
The immune system works like a seesaw. On one side you have Th1 cytokines that initiate the first line of defense. On the other side you have Th2 cytokines which help product antibodies to protect you from future invasions. However, when one side goes up, the other side goes down. This can contribute to a weak immune system and potentially promote autoimmune conditions. 

19. Complete blood count in primary care

20. PRINCIPLE
This test measures red blood cells and white cells. The test includes: red blood cell count, hemoglobin, hematocrit, MCV, MCHC, RDW, platelet count, white blood cell count, neutrophils, lymphocytes, monocytes, eosinophils and basophils.

21. Phisiology:
Whole blood collected in EDTA (purple top tube) is the only acceptable specimen. The
specimen must be kept and transported at room temperature. For best results, the
collection tube should be full and no less than half full. Mix the specimen slowly for 2
minutes.
Unacceptable specimens include:
Any sample not collected in an EDTA tube.
Any tube less than half full
Any tube that is not labeled properly
Hemolyzed specimens
Specimens older than 24 hours
Specimens that are clotted
METHOD: VCS Technology, Hemoglobinometry

22. Significance: Screen for a wide range of conditions and diseases
Help diagnose various conditions, such as anemia, infection, inflammation, bleeding disorder or leukemia, to name just a few
Monitor the condition and/or effectiveness of treatment after a diagnosis is established
Monitor treatment that is known to affect blood cells, such as chemotherapy or radiation therapy
A CBC is a panel of tests that evaluates the three types of cells that circulate in the blood and includes the following:
Evaluation of white blood cells, the cells that are part of the body's defense system against infections and cancer and also play a role in allergies and inflammation
Evaluation of red blood cells, the cells that transport oxygen throughout the body
Evaluation of platelets, cell fragments that are vital for normal blood clotting

23. ULTRASOUND* >20,000 Hertz OR more
Diagnostic Medical Ultrasound - use of high frequency sound waves to aid in the diagnosis and treatment
Frequency ranges -> MHz
Piezoelectric Effect : The principle of converting energy by applying pressure to a crystal
Pulse-echo principle, ultrasound transducers; [convert 1 type of energy to another]
– Electricity into sound = pulse {machine to tissue}
– Sound into electricity = echo {tissue to machine}
Echoes are interpreted and processed by the ultrasound machine

24. X-RAYS*
Digital Radiography, High Frequency Radiography Unit help in diagnosis
Electro magnetic radiations {ELECTRONS}
High-speed flow of electrons bombarding the anode target surface, 99% converted to heat, to produce X-ray - 1% ; {(50Hz or 60Hz)}
Useful in detection of pathology of the skeletal system
Bones contain much calcium hence absorb x-rays efficiently ; reduces amount of X-rays reaching detector in the shadow of the bones {making them clearly visible on the radiograph}
In medical diagnostic applications, low energy (soft) X-rays are unwanted {since they are totally absorbed by the body, increasing the radiation dose without contributing to the image}
A thin metal sheet, often of “aluminium”, [an X-ray filter], usually placed over the window of the X-ray tube, absorbing the low energy rays. Center of the spectrum shifts toward higher energy {hardening the beam ; Hard X-rays}

25. BIOPSY*
Medical test performed by surgeon involving sampling of cells or tissues for examination. [TISSUE SAMPLING]
Medical removal of tissue from a living subject to determine the presence or extent of a disease.
Frequent biopsies are taken to assess activity of disease and to assess changes that precede malignancy.
Examined under a microscope or analyzed chemically by a pathologist.
Entire lump or suspicious area is removed {Excisional biopsy}.
Sample of tissue is removed with preservation of the histological architecture of the tissue’s cells {Incisional biopsy or core biopsy}.
Scanner's images help doctors determine exact position of the needle in the targeted tissue {CT-guided biopsy}.
An ultrasound scanner helps a doctor direct the needle into the lesion. {Ultrasound-guided biopsy}.
A needle withdraws material out of a mass. {Aspiration biopsy}.

27. THANK YOU