RAPID TESTS: AGGLUTINATION AND IMMUNOLOGY TESTING

RAPID AGGLUTINATION LATEX TESTS - from blood:

- ASLO;

- CRP;

- RF.

HEPATITIS: A, B, C, D and E:

- HEPATITIS A VIRUS - from blood;

- HEPATITIS B VIRUS - from blood ;       

- HEPATITIS C VIRUS - from blood;

- HEPATITIS D VIRUS - from blood;

- HEPATITIS E VIRUS - from blood, stool. 

SYPHILIS - treponema pallidum; from blood and other fluids:

- VDRL;

- RPR;

- TPHA.

 

HIV TESTING - from blood, oral fluid, urine.

TROPONIN LEVEL TESTING - from blood. 

HELICOBACTER PYLORI DETECTION: 

- ANTIBODY DETECTION - from blood;

- ANTIGEN TEST - from stool.

 

FAECAL OCCULT BLOOD (FOB) TEST - from stool.

GIARDIA LAMBLIA - ANTIGEN DETECTION - from stool.

IMMUNOGLOBULIN E - from blood.

CORONAVIRUS - from blood, nose/throat swab. 

ASLO, CRP, RF: rapid agglutination tests

Serology - Immunology

Agglutination Kits & Serology Reagents: there are many options of latex serology kits on the market. Common serological tests to detect in serum:

- ASLO/ASO: Anti-streptolysin-O;

- CRP: C-Reactive Protein;

- RF: Rheumatoid Factor.

The latex assays are available in different kit sizes, with positive and negative controls provided. The full kits include all the required consumables. They use the common agglutination method for efficient, reliable testing, to ascertain the causes of inflammation, rheumatoid disorders and streptococcal infections. Latex agglutination testing is sometimes referred to as latex serology. 

Agglutination occurs when an antigen comes into contact with its corresponding antibody. The rapid test kits are used to identify streptococcal infections, levels of C-reactive protein that would indicate high levels of inflammation in the body and rheumatoid factors that would indicate a diagnosis of rheumatoid arthritis.

Latex serology tests are used to ascertain whether specific antigens or antibodies are present in a sample. This is done by applying the samples to the latex beads. If the suspected substance is present in the sample, the latex beads clump together (agglutinate). The results of latex serology tests can be obtained within 15 minutes to an hour, allowing a rapid diagnosis to be made, which is obviously a significant advantage.

 

These slide agglutination assay tests are used for the qualitative and semi-quantitative determination of ASLO, CRP and RF in human serum. The simple, five-step procedure provides easy-to-read results in 2 minutes. No initial dilution of patient samples is required. Kits usually include ASLO/CRP/RF latex reagents, reactive control, nonreactive control, 100 disposable stirrer pipettes and disposable cards. The latex reagent does not require additional preparation. MATERIAL REQUIRED: automatic pipettes, mechanical rotator, adjustable at 100 r.p.m, laboratory alarm clock.

QUALITY CONTROL: Positive and negative controls should be run daily following the steps outlined in the Qualitative Test, in order to check the optimal reactivity of the reagent. The positive control should produce clear agglutination. If the expected result is not obtained, you must not use it. 

ASO/ASLO: slide agglutination test for the qualitative and semi-quantitative detection of streptolysin antibodies in human serum. Analytical sensitivity: 200 (+/-50) IU/mL. 

CRP: slide agglutination test for the qualitative and semi-quantitative detection of C-reactive protein in human serum. Analytical sensitivity: 6 mg/L. 

RF: slide agglutination test for the qualitative and semi-quantitative detection of rheumatoid factors in human serum. Analytical sensitivity: 8 IU/mL. 

SOURCES OF ERROR: Bacterial contamination of controls and specimens as well as freezing and thawing of the ASLO/CRP/RF-latex reagents may lead to false positive results. Traces of detergent in the test cards may give false positive results. Preferably the used cards would not be reused. If cards are reused, wash them first under tap water until all reactants are removed and then with distilled water. Allow to air dry, avoiding the use of organic solvents as they may impair the special finish on the slide. 

The ASLO/CRP/RF-latex antigen must not be used beyond its expiry date because a prolonged storage can affect the sensitivity of the suspension. Do not allow reagents in the kit to get in contact with skin or mucous membranes.

Reagents are stable if stored at 2-8ºC up to expiration date. Allow the suspension to reach to room temperature and mix gently prior to use. Do not freeze. Frozen reagents could change the functionality of the test. Reagent and Controls are ready for use and stable until the expiry date stated on the label. Do not use haemolysed, lipaemic or contaminated serum for testing. Hemoglobin (<10 g/L), bilirubin (<20 mg/dL) and lipemia (<10 g/L) do not interfere. Other substances (rare) may interfere. SAMPLES: must be fresh, clear serum. After the clear serum has been separated it may be stored at 2-8ºC for up to one week or longer periods at -20ºC, before testing. Undiluted samples should be used. Do not use plasma since fibrinogen can form non-specific agglutination. The sensitivity of the test may be reduced at low temperatures. The best results are achieved at 15-25ºC. 

PROCEDURAL STEPS for the QUALITATIVE TEST: 

1. Bring the test reagents and samples to room temperature. 

2. Transfer 50µl/1 drop of the patient's serum into one of the circles of the test card. Dispense 1 drop of positive control and 1 drop of negative control into two additional circles. 

3. Gently shake the suspension of the latex reagent (ASLO/CRP/RF), then using the pipette, aspirate dropper several times to obtain a thorough mixing. Add 1 drop/50µl reagent of the suspension to the same test circle with the patient's serum and to those with the controls. 

4. Using the stirrers, mix the serum and the latex reagent and spread them. Mix the contents of each circle while spreading over the entire area enclosed by the ring. Use separate stirrers for each mixture. 

5. Rotate the test card/slide for 2 minutes by means of a mechanical rotator (100r.p.m.) and observe immediately under a suitable light source for any degree of agglutination. 

READING THE LATEX SLIDES:

- Nonreactive: smooth suspension with no visible agglutination, as shown by the negative control. 

- Reactive: any degree of agglutination visible macroscopically (best to check under a suitable light). 

The delays in reading the results may generate in overestimation of the antibody present.

For the SEMI-QUANTITATIVE TEST:

- Dilute the sample to be tested following the 2-fold dilutions, as follows: 1/2, 1/4, 1/8, 1/16, 1/32. Follow the other steps as in the quantitative test.

How to make the dilutions: it is up to the biologist, for as long as it is correctly diluted mathematically. 

Example of dilution 1/2: 50µL non-diluted sample + 50µL dilution liquid (physiological serum/distilled water), then transfer 1 drop/50µL from the ASLO/CRP/RF latex reagent over the mixture. The other steps are as in the quantitative test.   

Reading is the same for both the Qualitative and for the Semi-Quantitative Test:

- Negative/Non-reactive: no agglutination; 

- Positive/Reactive: visible agglutination.  

The titer of the specimen for the Semi-Quantitative test is reported as the highest dilution that shows reactivity. The next higher dilution should be negative. If the highest dilution tested is reactive, repeat the test using the next dilution. 

The results obtained for the Semi-Quantitative Test do not replace the fully-automated quantitative tests available on the market. The levels obtained following the Semi-Quantitative Test procedure are considered only "approximate", not exact values, hence the naming of "Semi-Quantitative Test". 

The approximate ASLO/CRP/RF levels (IU/mL) present in the sample may be obtained by multiplying the titer of the last positive dilution by the minimum detectable unit (analytical sensitivity) corresponding to each reagent type. 

Reading the Semi-Quantitative tests:

ASLO: 

- 1/2 dilution shows reactivity => ASLO level: 2 x 200 = 400 IU/mL

- 1/4 dilution shows reactivity => ASLO level: 4 x 200 = 800 IU/mL

- 1/8 dilution shows reactivity => ASLO level: 8 x 200 = 1600 IU/mL

- 1/16 dilution shows reactivity => ASLO level: 16 x 200 = 3200 IU/mL

1/4 reactive and 1/8 non-reactive => ASLO level: 800-1600; ASLO>800 and <1600.

CRP: 

- 1/2 dilution shows reactivity => CRP level: 2 x 6 = 12 mg/L

- 1/4 dilution shows reactivity => CRP level: 4 x 6 = 24 mg/L

- 1/8 dilution shows reactivity => CRP level: 8 x 6 = 48 mg/L

- 1/16 dilution shows reactivity => CRP level: 16 x 6 = 96 mg/L

1/2 reactive and 1/4 non-reactive => CRP level: 12-24; CRP>12 and <24.

RF: 

- 1/2 dilution shows reactivity => RF level: 2 x 8 = 16 IU/mL

- 1/4 dilution shows reactivity => RF level: 4 x 8 = 32 IU/mL

- 1/8 dilution shows reactivity => RF level: 8 x 8 = 64 IU/mL

- 1/16 dilution shows reactivity => RF level: 16 x 8 = 128 IU/mL.

1/8 reactive and 1/16 non-reactive => RF level: 64-128; RF>64 and <128.

ASLO

Antistreptolysin O (ASO/ASLO)

 

- ASLO/ASO latex kit test: an indirect agglutination kit for the detection of anti-streptolysin-O in human serum using latex particles coated with streptolysing-O. 

ASO titer is adviced for the diagnosis of post infectious glomerulonephritis, scarlet fever, streptococcus infections and others. The Streptolysin O is antigenic and cause hemolysis of RBCs. So there is antibody formation (Antistreptolysin-Ab) in the blood. 

Anti-Streptolysin O (ASO or ASLO) is the antibody made against streptolysin O, an immunogenic, oxygen-labile hemolytic toxin produced by most strains of group A and many strains of groups C and G streptococci. 

In the course of streptococcal infections, the extracellular products of the bacteria act as antigens to which the body responds by producing specific antibodies. Streptolysin O (ASLO) is one of two hemolysins (the other being Streptolysin S) produced by virtually all strains of Streptococcus pyogenes.  

Principle:

Patient serum with dilution is mixed with a fixed amount of streptolysin O- Ag and a reaction takes place. Then add Ab-coated RBC. There will be hemolysis in the tube, where streptolysin O is free.

Antistreptolysin (ASO) leading to hemolysis

The result is reciprocal of the highest dilution where hemolysis starts. Units are international units (IU).

Antistreptolysin (ASO) titer procedure

Normal value: below 200.

Definitive value: 400 or more.

Rising titer from 200-250 is significant.

ASLO-Latex Test: The assay is performed by testing a suspension of latex particles coated with streptolysin O antigen against unknown serum. The presence or absence of visible agglutination indicates the presence or absence of ASLO in the samples tested. 

REAGENT COMPOSITION ASLO-Latex Antigen. Suspension of polystyrene latex particles coated with stabilized streptolysin O in a buffered saline solution.

CONTROL + Human serum with an ASLO activity > 200 IU/mL.

CONTROL - Animal serum with an ASLO activity < 100 IU/mL. 

Precautions: Components of different human origin have been tested and found to be negative for the presence of antibodies anti-HIV 1+2 and anti-HCV, as well as for HBsAg.  Do not allow to contact with skin or mucous membranes.  

EXPECTED VALUES: 95% of healthy adults have ASLO titers of 200 IU/mL or less, the highest titers been found in school children with titers up to 250 IU/mL. Since a single ASLO determination does not provide much information unless it is high, titrations at bi-weekly intervals for 4 to 6 weeks of the doubtful cases are advisable to follow the evolution of the disease.

CLINICAL SIGNIFICANCE: Elevated ASLO serum titers occur in response to infection with hemolytic streptococci of Group A, C and G, producers of streptolysin O, an extracellular protein of enzymatic character with strong antigenic properties. Immunochemical assay of these specific antibodies to streptococcal metabolites provide valuable information to establish a diagnosis of streptococcal infections (acute rheumatic fever, glomerulonephritis). ASLO testing has a high diagnostic value on a tentative diagnosis made on the basis of case history and clinical findings. 

ANALYTICAL PERFORMANCE: The minimum detectable unit (analytical sensitivity) is of approximately 200 IU/mL (± 50 IU/mL). 

LIMITATIONS OF PROCEDURE: Positive reactions do occur in conditions other than glomerulonephritis, in which the production of ASLO is especially high. In scarlet fever, early and acute periods of rheumatoid arthritis, healthy carriers, complicated and no complicated tonsillitis and various streptococcal infections increased ASLO levels have been found. Biologically false negative reactions can occur in early infections and during the early years of life (from six months to 2 years). The sensitivity of the test may be reduced at low temperatures. The best results are achieved at 15-25ºC. Delays in reading the results may result in over-estimation of the antibody present. 

CRP

C-reactive protein (CRP)  

Definition

    1. CRP is γ-globulin (in the γ-region); is found in various inflammatory diseases. This is also called acute-phase protein. 

    2. CRP is produced in the liver.

    3. CRP name is derived from the reaction with streptococcal capsular (C) polysaccharide. 

                                                                C-Reactive Protein (CRP)

CLINICAL SIGNIFICANCE: C-reactive protein is an acute phase protein present in normal serum, which increases significantly after most forms of tissue injuries, bacterial and virus infections, inflammation, and malignant neoplasia. CRP contributes to non-specific defense by complement activation and accelerating phagocytosis. CRP testing has a high diagnostic value on a tentative diagnosis made on the basis of case history and clinical findings. 

Principle:

1. CRP is opsonin and it activates the complement system and ultimately leads to lysis.  

                         C-reactive protein (CRP) leads to lysis by the activation of complement 

                                      C-reactive protein (CRP) role in Complement activation

2. Serum of the patient (CRP) + Somatic C polysaccharide of pneumococci mixed and gives rise to a precipitate.

3. Practically CRP is injected into the rabbit when the anti-CRP antibody is produced. Now take serum of the patient (CRP) + Mix anti-CRP. This will give precipitation. 

                                                                              

CRP + Anti CRP = Precipitation

C-Reactive Protein (CRP) formation 

Causes of CRP

    1. Produced in various bacterial diseases.

    2. Produced by injured myocardial muscle in myocardial infarction.

    3. Positive in acute and chronic rheumatic fever.

CRP is also positive in:

    1. Sydenham's Chorea.

    2. Myocardial infarction and negative in angina.

    3. Many malignancies.

    4. Rheumatoid arthritis.

    5. Gout.

    6. Viral infection like Viral Hepatitis.

    7. Bacterial Pneumonia.

    8. Active Tuberculosis.

    9. Lepromatous Leprosy.

    10. Acute Tonsillitis, Scarlet fever, and Mumps.

PRINCIPLE: CRP-Latex Test is a rapid slide agglutination procedure, developed for the direct detection and semi-quantitation of C-reactive protein (CRP) in serum. The assay is performed by testing a suspension of latex particles coated with anti-human CRP antibodies against unknown serum. The presence of a visible agglutination indicates an increase of the CRP level above the upper limit of the reference interval in the samples tested. 

REAGENT COMPOSITION - CRP-Latex Reagent: Suspension of polystyrene latex particles coated with specific anti-human C-reactive protein antibodies in a buffered saline solution. CONTROL + Human serum with a CRP concentration > 15 mg/L. CONTROL - Animal serum with a maximum concentration of human CRP of 1 mg/L. Precautions: Components of different human origin have been tested and found to be negative for the presence of antibodies anti-HIV 1+2 and anti-HCV, as well as for HBsAg. However, the controls should be handled cautiously as potentially infectious. 

EXPECTED VALUES: While the C-reactive protein concentration is generally below 5 mg/L in the sera of healthy adults, in a number of disease states these values often exceeded within 4 to 8 hours after an acute event and reach levels up to 500 mg/L. Since an elevated CRP level is always associated with pathological changes, determination of CRP is of great value in diagnosis, treatment and monitoring of inflammatory conditions. 

ANALYTICAL PERFORMANCE The minimum detectable unit (analytical sensitivity) is of approximately 6 mg/L (5-10 mg/L). Hemoglobin (<10 g/L), bilirubin (<20 mg/dL) and lipemia (<10 g/L) do not interfere. Rheumatoid factors (>100 IU/mL) interfere. Other substances may interfere. 

LIMITATIONS OF PROCEDURE  The presence of rheumatoid factors (RF) in a serum sample may cause false positive reactions. The sensitivity of the test may be reduced at low temperatures. The best results are achieved at 15-25ºC. Delays in reading the results may result in over-estimation of the CRP concentration. In case of positivity, use the titration/dilution procedure, as presented above, at the "ASLO/CRP/RF" section.   

RF

Rheumatoid Factor (RF)

The diagnosis of Rheumatoid arthritis is based on clinical assay, but the laboratory and radiological tests are useful to confirm clinical diagnosis and to evaluate the seriousness and the phase of the disease. Rheumatoid Factor (RF) in serum is one of the main clinical markers for rheumatoid arthritis. RF is a term used to describe a group of antibodies (IgM, IgG and IgE) directed towards the antigenic site on the Fc portion fragment of the human and animal body's own IgG antibodies. 

Rheumatoid Factor Testing: clinical testing supplies designed to identify the presence of Rheumatoid Factor (RF) in patient blood samples and aid in diagnosing rheumatoid arthritis. RF-latex serology kit test is an indirect agglutination kit for the detection of Rheumatoid Factor (RF) in human serum using latex particles coated with human IgG. 

PRINCIPLE: RF-Latex Test is a rapid agglutination procedure, developed for the direct detection and the semi-quantitation on a slide of rheumatoid factors (RF) in serum. Imuno Latex RF is a very sensitive test and is performed by testing a suspension of polystyrene latex particles coated with purified and stabilized human gamma globulin IgG and suspended in glycine buffer pH 8.2, against unknown serums. The reagent is based on an immunological reaction between human IgG bound to biologically inert latex particles and rheumatoid factors in the test specimen. The presence or absence of a visible agglutination indicates the presence or absence of RF in the samples tested. 

When serum containing rheumatoid factors is mixed with the latex reagent, visible agglutination occurs. The suspension of latex is mixed on a test card with serum containing elevated rheumatoid factor levels; a clear agglutination is seen in 2 minutes. 

The RF latex reagent sensitivity has been adjusted to detect a minimum of 8 IU/mL of rheumatoid factors according with the WHO International Standard without previous sample dilution. The regular kit contains: suspension of latex, positive control serum, negative control serum, stirrers, test card, instructions for use.

REAGENT COMPOSITION - RF-Latex Reagent: Suspension of polystyrene latex particles coated with human gamma globulin in a buffered saline solution. CONTROL + Human serum with an activity equivalent to appr. 25 IU/mL. CONTROL - Animal serum with an activity < 5 IU/mL. Precautions: Components of different human origin have been tested and found to be negative for the presence of antibodies anti-HIV 1+2 and anti-HCV, as well as for HBsAg.

EXPECTED VALUES: Of those patients with a clinical diagnosis of rheumatoid arthritis approximately 70-80% are seropositive for rheumatoid factor. Positive results were shown for nearly all patients with variants of rheumatoid arthritis. A positive result can be expected in less than 5% of healthy individuals, while in the population aged 60 years and older as many as 30% may be seropositive using latex tests for the detection of rheumatoid factor. 

CLINICAL SIGNIFICANCE: Rheumatoid factors are found in the sera of most patients with rheumatoid arthritis as well as in a variety of other diseases. Rheumatoid factors testing has a high diagnostic value on a tentative diagnosis made on the basis of case history and clinical findings. 

ANALYTICAL PERFORMANCE: The minimum detectable unit (analytical sensitivity) is of approximately 8 IU/mL (6-16 IU/mL).  

LIMITATIONS OF THE PROCEDURE: Positive reactions do occur in conditions other than rheumatoid arthritis such as mononucleosis, hepatitis C, syphilis, various other infections and in elderly patients. When tested by the quantitative test, however, most of these specimens give very low results.