Serology

ASO TEST
INTRODUCTION:
Most of the strains of Streptococcus which are pathogenic to human
beings belong to group A. All such organisms produce an exotoxin known
as streptolysin O. It is a good antigen and induces the formation of antistreptolysin O (ASO) in the person suffering from streptococcal injections,
which is helpful in the diagnosis of streptococcal infections. It provides a
simple screening method of detecting an elevated ASO titre.
AIM:
To perform the anti-streptolysin O test in the given patient serum both
by qualititative and quantitative methods.
PRINCIPLE:
This
is
slide
agglutination
test.
The
reagent
contains
an
aqueous
suspension of polystyrene latex particles which are sensitized with
streptolysin O. these particles agglutinate in the presence of ASO present
in patient serum.
Polystyrene latex particles are coated with purified and stabilized
streptolysin O (antigen) which react with its corresponding antistreptolysin O (antibody) in the test sample resulting in the agglutination
of latex particles.
INTERPRETATION:
•
Agglutination visible within two minutes is to be interpreted as
positive reaction.
•
No agglutination is to be considered as a negative reaction.
RESULT:
The ASO titre of the given test serum is 200 IU/ml, indicating past or
present acute streptococcal infection.
ASO (ANTI STREPTOLYSIN O)
1.What is ASO?
Anti Streptolysin O
2. What is Streptolysin O?
It
is a
hemolysin O,
heat
labile protein produces complete
hemolysis, strongly antigenic and induces brisk antibody response,
produced by group A, C, & G.
3.Name the conditions in which ASO titre is elevated?
1.Rheumatic fever
2.Glomerulonephritis
3.Scarlet fever
4.Why this hemolysin is known as Streptolysin O?
Because it is Oxygen – labile
5.What is the significant ASO titre?
More than 200 Todd units /ml (or) 200 IU/ml
6.What is the significant titre indicates?
It indicates recent /recurrent infection with streptococci
7.Why plasma should not be used for ASO test?
Fibrinogen in the plasma can lead to non specific agglutination of the
latex particles
8.What are the supporting evidences for acute Rheumatic fever?
•
Positive throat culture for group A streptococci
•
Recent scarlet fever
•
Increased titre of ASO, CRP and ESR
9.What are the disadvantages of ASO?
ASO titre also rises in C and G infections
10.Why ASO titre is not raised in skin infections?
ASO response following skin infection is poor because inactivation of
antigen by the cholesterol present in the skin.
11.WhyASO increased in Rheumatic fever?
Antigenic cross reactions between Streptococci and heart tissues.
12.What are the infections caused by Streptococcus pyogenes?
a) Suppurative:
•
Respiratory infections – scarlet fever, pharyngitis,
tonsillitis, otitis media and Streptococcal pneumonia
•
Skin and soft tissue infections – Erysipelas, impetigo,
cellulitis, Necrotizing fasciitis
b) Non Suppurative:
•
Acute glomerulo nephritis.
•
Acute rheumatic fever.
13.What are the sequalae in post Streptococcal infections?
•
Acute rheumatic carditis
•
Nephrotic syndrome
14.What is agglutination?
When a particulate antigen reacts with its antibody in the presence
of electrolytes at an optimum temperature and pH, forms an
insoluble clumps.
15.What is precipitation?
When a soluble antigen reacts with its antibody in the presence of
electrolytes at an optimum temperature and pH, forms an
insoluble precipitate.
16.Which factors inhibit the activity of Streptolysin O?
•
Cholesterol in the serum
•
Bacterial contamination of the serum
•
Chemical treatment of the serum
1. Anti-deoxyriboniclease B increased titre is significant in which
condition?
Skin infection in retrospective diagnosis
2. What is the significant titre of anti-deoxyribonuclease B?
Titres higher than 300 or 350 are significant
C REACTIVE PROTEIN – LATEX TEST
AIM:
Toperform C-reactive protein latex test for the detection of C-reactive
protein in human serum and to determine the titre of C-reactive protein
in serum.
PRINCIPLE:
C-reactive protein in patient’s sera has been found in association with
acute infections and a variety of inflammatory disorders. There is a
strong correlation between serum levels of CRP and onset of the
inflammatory process. Monitoring the levels of CRP in patient’s sera
indicates the effectiveness of treatment and the assessment of patient
recovery. CRP latex particles are coated with antibodies to human CRP.
When the latex suspension is mixed with serum containing elevated CRP
levels on a slide, clear agglutination is seen within two minutes if CRP
concentrations are greater than 6mg/L. If it is less than 6 mg/L then no
agglutination is observed.
INTERPRETATION:
•
Agglutination visible within two minutes is to be interpreted as
positive reaction.
•
No agglutination is to be considered as a negative reaction.
•
Sensitivity of the test is 6 mg/L.
RESULT:
The given test serum may show agglutination if the CRP levels are more
than 6mg/L.
If agglutination observed, it is further proceeded for a quantitative test.
RESULT:
The approximate serum CRP level is 6mg/L
C – REACTIVE PROTEIN (CRP
1. What is CRP?
CRP is an abnormal protein (β-globulin)
2. What is the nature of this protein?
β-globulin
3. Where CRP is synthesised?
Hepatocytes in the liver
4. What is the principle of CRP test?
Passive agglutination test
5. Why it is known as C-reactive protein?
Because it precipitates with the C antigen of Pneumococci
6. Which are the carrier particles used other than latex?
Bentonite, gelatine and RBC
7.What is the significant titre of CRP?
6mg/dl
8.What is the diagnostic importance of CRP?
Detects the activity of inflammation and also prognostic indicator
9.Name the test based on the principle of latex agglutination?
ASO, CRP, RF and HCG detection
10. What are the conditions in which CRP level is elevated?
Bacterial
infection,
inflammation,
malignancy
and
tissue
destruction
RHEUMATOID (RA) FACTOR TEST
INTRODUCTION:
Rheumatoid factors are anti-globulins of the IgG, IgA or IgM which forms
immune complexes in serum or joint fluids. This result in complex
deposition in tissues where blood flow is restricted resulting in dermatitis
(skin), nephritis (kidney), synovitis and arthritis (joints), vasculitis (vessels
in many tissues).So testing for RF has a high diagnostic value.
AIM:
To detect the rheumatoid factor (auto-antibody) in the given patient’s
serumand to quantitate them if they are present.
PRINCIPLE:
Latex particles (polystyrene particles) coated with human gamma
globulin (IgG) are agglutinated when mixed with samples containing
rheumatoid factors. Distinct agglutination indicates the presence of
rheumatic factors (RF).Patients specimens that do not react with the
latex particles contain either no rheumatoid factors or concentration
below 20 IU/ml. Elevated RF titres often indicate a severe course and a
generalized pathological process.
.
INTERPRETATION:
•
Agglutination visible within two minutes is to be interpreted as
positive reaction.
•
No agglutination is to be considered as a negative reaction.
•
Sensitivity of the test is 20 IU/ ml.
•
The following conclusion may be drawn depending upon the
observation as follows:
RESULT:
The given test serum is positive/negative for RF depending on the
observation.
Marked agglutination indicates the presence of rheumatoid factor (≥
20IU/ml)
If the given serum is positive for RF, it is further proceeded for a
quantitative test.
CLINICAL SIGNIFICANCE:
The rheumatoid factor test is done in order to detect the presence of
rheumatoid factors in the serum of patients with rheumatoid arthritis.
Higher titre of RF is usually related to the severity of rheumatoid
arthritis. Rheumatoid factor may be present in low concentrations in 3.5
percent of the normal population.This percentage increases with the age
of population. This factor is non specific. Positive results may occur
occasionally in various pathological disease states including systemic lupus
erythematosus,
hepatitis,
cirrhosis,
lymphomas,
hyper-
gammaglobulinaemia, scleroderma and sarcoidosis. Quantitative analysis
helps to monitor the therapy of rheumatoid arthritis.
RHEUMATOID (RA) FACTOR
1. What is Rheumatoid factor?
It is an anti gamma globulin autoantibody acts as an antibody to
human IgG
2. Rheumatoid factor belongs to which class of immunoglobulin?
Mostly IgM, may also be IgG or IgA.
3. What are auto antibodies?
These are antibodies produced against self antigens.
4. What is the principle of this test?
Passive agglutination
5. Name the disease in which RF is used as the diagnostic test?
Rheumatoid arthritis
6. Name the other conditions where RF test is positive?
SLE, hepatitis, dermatomyositis, cirrhosis, syphilis
7. What is nature of the latex Rheumatoid factor antigen?
It is an aqueous suspension of polystyrene particles coated with
human IgG
8. Which test was used earlier for detection of RF?
Rose waaler test
9. What is amboceptor?
Sub agglutinating dose of rabbit anti sheep erythrocyte antibody
10.
What is Rheumatoid arthritis?
Symmetrical polyarthritis with muscle wasting with subcutaneous
nodules with involvement of small joints with residual deformity
and disseminated lesions like serositis, myocarditis
11.
What is the significant titre for RF test?
≥20 IU/ml
12.
Classify Autoimmune diseases?
a) Localised (organ specific) – Hashimoto’s disease, Graves disease,
Addison’s disease, pernicious anaemia , Myasthenia gravis
b) Systemic (non-organ specific) – SLE, Rheumatoid arthritis,
polyarthritis nodosa, Sjogren’s syndrome.
c) Hemocytolytic – Autoimmune haemolytic anaemia.
13.
What are the differences between Rheumatic and Rheumatoid
arthritis?
•
Rheumatic arthritis – migrating polyarthritis involving major
joints, without deformity
•
Rheumatoid arthritis – symmetric polyarthritis involving
minor joints with muscle wasting and deformity
RPR TEST
AIM:
To screen primary syphilis by detecting the reagin antibodies in the
given patient serum.
PRINCIPLE:
It is a non-treponemal test used to detect early stages of primary
syphilis. This test involves an RPR antigen suspension which is a carbon
containing cardiolipin-lecithin antigen. It detects the regain antibody
present in the serum of individuals with syphilis. When a specimen
contains antibody, flocculation occurs due to co-agglutination of carbon
particles of the RPR antigen which appears as black clumps against the
white
background
macroscopically.
of
the
card.
This
co
agglutination
is
read
INTERPRETATION:
After 8 minutes rotation, inspect the card in good light. Turn or
tilt the card to see whether there is clumping (reactive result).Test
cards include negative and positive control circles for comparison.
Reaction type
Appearance
Interpretation
Non-reactive
No clumping
Non-reactive for
syphilis.
Reactive
Highly visible
Reactive for syphilis
clumping
Weakly
Minimal clumping
Reactive for syphilis.
reactive
INTERPRETING TEST RESULTS:
RPR
RPR TITRE
Active infection
+
>1:8
Latent syphilis
+
Often