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= Agglutination test = An agglutination test is a procedure requested by a doctor or other medical professional to detect the presence of a specific antibody or antigen in a range bodily fluids (saliva, blood, urine and cerebrospinal fluid etc.). This test is performed by a scientist or lab technician in the laboratory who will then provide the agglutination test results to the doctor or medical professional. There are different forms of agglutination tests available, and each test is used according to the sensitivity, effort, timing, and whether it is qualitative or quantitative.

History
Typhoid fever was a major public health problem during the 1800s whereby, the diagnosis of this disease remained unclear due to its similar fever-like symptoms as observed in other conditions such as malaria, hepatitis and dengue

Georges Fernand Isidore Widal (1862–1929) developed the first agglutination test in 1896 (Widal test) to detect the presence of the Salmonella typhi (S. typhi) bacteria in a patients blood serum. This diagnostic test involved the extraction of a bodily fluid such as blood or urine in a suspected S. typhi-infected patient with fever-like symptoms. These samples were definitively tested for the presence of the agglutin (antibody) in regards to the sample type.

The presence of S typhi antibodies in the patient’s sample detects the presence of Salmonella typhi flagella (H) and somatic (O) antigens, which results in agglutination.

This method provided a quick and straightforward analysis of the disease, thus, helping to alleviate the public health concern at the time, due to the importance a fast and accurate clinical diagnosis of this disease plays in its effective management and treatment.

The discovery of the Widal test further aided in the development of successive qualitative and quantitative agglutination tests used in current medical diagnosis.

Background
The process of agglutination develops due to the formation of a stable lattice network which involves a two-phase reaction known as the sensitisation (primary phase) and the lattice formation (secondary phase).

The primary phase involves antibody (Ab) and antigen (Ag) binding, which involves antibodies reacting with a single antigenic determinant site (epitope) on the surface of an antigen such bacterial cells.

The secondary phase occurs after the primary phase and involves the rapid formation of cross-linkages between antibodies and antigens. During cross-linkages, the gaps between particles are bridged as the Fab portion of the antibody attach onto the antigenic determinants of two adjacent antigen particles. These multiple cross-linkage formations result in the structure of a lattice, which can be visually observed as clumping. This secondary phase is dependent on environmental conditions such as ionic strength of milieu, pH and temperature.



Extraction
There are multiple methods of bodily fluid extractions used during agglutination tests, and each is used regarding the antigen or antibody of interest.

Saliva
The extraction of saliva is a method used to detect and test the presence of specific bacteria in a patient’s serum.

This procedure involves gathering the patient’s saliva (enough to fill a saliva collection tube) for laboratory analysis.

Examples of agglutination tests involving saliva extraction include the rapid and invasive-free detection of Streptococcus gordonii.

Blood
The extraction of blood is a standard method used for agglutination tests.

This method commonly involves the use of a venipuncture to extract a patient’s blood sample. This extraction method is used in many agglutination tests, which detects the presence of bacterial antigens in a patient’s blood.

The use of blood testing via agglutination tests provides a fast and easy method to detect the presence of antigens, thus, serving an important role in clinical diagnosis.

The agglutination test also provides information regarding the blood typing of individuals required during donor and recipient blood transfusions.

Blood extraction is also used in agglutination tests to detect the presence of white blood cells (leukoagglutination) as seen in the detection of Pneumonia (Mycoplasma pneumoniae).

Urine
The extraction of urine is a method used to detect and test the presence of specific bacteria in a patient’s serum.

This procedure involves the collection of the patient’s urine (enough to fill a urine collection vial) for laboratory analysis.

Examples of agglutination tests involving urine extraction include the invasive-free rapid detection of visceral leishmaniasis and the presence of invasive bacterial infection in children.

Cerebrospinal fluid
The extraction of cerebrospinal fluid (CSF) through a lumbar puncture is a method used to gather a patient’s sample for agglutination test analysis for the detection of specific bacteria in a patient’s serum.

This test involves the insertion of a spinal needle between the lumbar vertebrae to extract cerebrospinal fluid from the subarachnoid space of a lying or seated patient.

Although this procedure is invasive, the spinal level is highly selective, which prevents the occurrence of spinal injuries.

Another method used for the extraction of CSF is spinal anaesthesia which involves the removal of a patients CSF in a seated position, the use of this procedure is implemented in special clinical circumstances to prevent the formation of scoliosis or unreliable anatomical landmarks in obese patients.

In children, both a lying flexed position and seated position can be used when obtaining CSF, however, due to the anatomical differences of children's spines to adults, the lumbar punctures are inserted at different spinal locations while using different methods such as the Cincinnati method.

The CSF collection of lumbar punctures are sent to laboratories for testing and diagnosis via agglutination tests.

Examples of antibody-mediated tests for CSF include the detection of antigens in common bacterial pathogens, the diagnosis of neurosyphilis, Lyme disease, and the detection of specific antibodies in patient samples.

Slide agglutination test
Slide agglutination tests are used in many laboratory settings to detect the presence of an antigen by using a known antibody which binds to the specific antigen of interest.

This test involves placing a specified concentration and volume of the killed antigen of interest with the patient’s serum sample on a slide, plate, or card. This mixture is then carefully rocked side to side which allows both the primary and secondary phases of agglutination to occur over a specified period (seconds to minutes)

The diagnosis of the results is made though naked eye observations of clump formation in the mixed samples.

Slide agglutination tests are also used for the serotyping of bacterial strains, such as Salmonella for the diagnosis of a disease or illness. Additionally, this test is also commonly used to identify the ABO blood type group of a patient due to its rapid qualitative, inexpensive, and straightforward methodological approach.

The slide agglutination test does not provide a quantitative analysis.

Tube agglutination test
The tube agglutination test (also known as the serum agglutination test (SAT)) is a laboratory test which involves the serial dilution and testing of a patient’s serum. The test is performed by the preparation of different dilutions of a patient’s serum (doubling dilutions) at increasing concentrations. Specific defined amounts of antigen are then added to each of these tubes and incubated at 37ºC for approximately 20 hours. After incubation the tubes are then qualitatively observed to detect the presence of clumps at the bottom of each test tube. This test is analysed using the 50% agglutination method to obtain the titer in relation to its relative agglutination amount. This test only provides a semi-quantitative analysis, is less time consuming and requires small volumes of antigen and serum. This test is laborious, time consuming, requires a number of reagents and additional confirmatory ELISA tests. Examples of the use of this method can be seen when detecting the presence of Brucellosis.

Passive agglutination test
The passive agglutination test is used in laboratory settings to detect the presence of a specific antibody through the use of particles or cells (without antigenic markers) coated with a known antigen (insoluble). These coated particles (soluble) such as red blood cells, latex, bentonite or charcoal are mixed with the patient’s serum (or bodily fluid sample). This mixture is then left to allow primary and secondary phases of agglutination to occur over a specified period (seconds to minutes). The clumping of the mixture indicates the presence of the antibodies which are specific to the antigen of interest. This procedure provides a qualitative analysis which is rapid and simple, but does require laborious preparation and is expensive. Examples of the use of this method involve the detection of rheumatoid factor, antinuclear antibodies in LE cell test and group A streptococcus.

Microscopic agglutination test
The microscopic agglutination test (MAT) is common laboratory test used for the serodiagnosis of leptospirosis in a patient’s serum sample.

This procedure involves the preparation of serial dilutions of a patient serum. These dilutions are then mixed with a specified amount of the live strain of the antigen of interest. The presence of leptospires in a patient’s sample are observed by the presence of agglutinated clumps under dark-field microscopy. The titer is determined by qualitative comparison to the control after 2 weeks to determine the point of 50% agglutination.

This qualitative method requires the maintenance of live cultures, paired sera for optimal results, is time consuming and is less sensitive than ELISA.

Examples of its use include diagnosis and epidemiological serological characterisation of leptospiral isolates.

Haemagglutination test (HAT)
The haemagglutination test is used in the laboratory setting to detect the presence of a specific viral or microbial antigens or for the ABO grouping of a patient’s blood.

Viral and microbial antigens contain proteins which bind to the surface of red blood cells resulting in agglutination. The detection of viral antigens in a patient’s blood are tested though the process of serial dilutions into an assay tray (microtiter plate). Standard defined amounts of the antigen of interest are then added to each of these wells and left for approximately 30 minutes to allow primary and secondary phases of agglutination to occur.

This semi-quantitative method provides a quick and inexpensive method for the estimation of the number of viral particles. Examples of the use of this method involve the detection of Newcastle disease virus (NDV), adenovirus type 3, Avian influenza virus (AIV), Avian infectious bronchitis virus (IBV) and mycoplasma synoviae.

This test is also used for blood typing of a patient’s blood by using antibodies which bind to the A or B blood group antigens in a patient’s blood with the addition of a direct Coombs test in the presence of confounding antibodies.