Rose Bengal is a laboratory test based on an antigen-antibody reaction for the diagnosis of brucellosis. The technique allows the detection of specific antibodies against the Brucella abortus bacterium in human serum samples. The result can be reported qualitatively or semi-quantitatively.
The qualitative form expresses whether the patient is positive or negative for the test, that is, whether or not there are antibodies. Meanwhile, the semi-quantitative report is reported in IU / ml and indicates the approximate amount of antibodies present. It should be noted that antibodies will only be produced if the patient has been in contact with the microorganism.
Rose Bengal reaction in an agglutination plate. Source: taken from the video
Due to its great simplicity, high sensitivity and specificity, it is one of the seroagglutination techniques most used in medicine as an initial test for the diagnosis of this disease.
Some researchers have compared the efficacy of the Rose Bengal test with other techniques, such as seroagglutination (febrile antigens) and have observed that although there is good correlation, they saw isolated cases in which the febrile antigen test was negative and Rose de Positive flare.
The difference obtained was due to the fact that these patients had a subclass of IgG antibodies against Brucella abortus with better binding capacity at acidic pH, therefore they could react with the Rose Bengal reagent but not with that of febrile antigens.
In this sense, they have suggested that the reagents of the febrile antigen technique be modified to an acidic pH so that they can detect this type of case.
Basis
The Rose Bengal Reagent consists of an antigenic suspension. It consists of the S99 strain of Brucella abortus, diluted in an acidic lactate buffer (pH 3.6), plus phenol and the Rose Bengal dye.
Therefore, in the sample what is sought are anti-Brucella antibodies, if these are present they will react with the reagent antigen and a macroscopically visible agglutination reaction will be observed. The test detects either IgM antibodies or IgG antibodies.
This means that it can detect the disease both in its acute stage where IgM antibodies prevail or in its chronic stage where IgG antibodies predominate.
This represents an advantage since it increases the sensitivity of the test, but at the same time it is a disadvantage since it does not discriminate between one stage and another, since the reaction with IgM and IgG antibodies are identical.
The test must always be performed together with a negative control and a positive control. The negative control contains animal serum without antibodies and the positive control contains serum of animal origin with 50 IU / ml of anti-Brucella antibodies.
Use
Brucellosis is a serious disease that tends to be chronic and dangerous, so it is very important that it be diagnosed early. It is a zoonosis and humans can become infected by direct contact with contaminated material, the most vulnerable people being veterinarians and animal caretakers.
Infection can also occur by eating infected raw meats, among other forms of infection.
This disease attacks locally or systemically. The systemic form is the most serious, since various organs can be affected, including the reticulum endothelial system (liver, spleen, bone marrow), skin (cellulitis and lymphadenopathy), respiratory system (pneumonia), musculoskeletal system (arthritis, sacroiliitis and spondylitis), among others.
The Rose Bengal test is a very useful technique to carry out an initial screening, because it is very inexpensive, easy to use, and has great specificity and sensitivity.
Cases of false negatives and false positives are very rare, and can occur in people with very low antibody titers (<25 IU / ml) or extremely high (> 1000 IU / ml) respectively.
Process
materials
-Rose Bengal Kit
-Agglutination plate white background
-50 µl pipette
-Rotator (optional)
-Vortex
Technique (qualitative method)
Commercial Rose Bengal kits come with ready-to-use reagents.
-Temperate the reagents before starting to work.
-The agglutination plates have circles drawn, each one for a different sample. Use 3 circles, the first for the negative control, the second for the sample, and the third for the positive control.
-Place a drop or 50 µl of the controls and the sample in their corresponding circle.
-Mix the Rose Bengal reagent using a vortex. Place a drop next to the previously placed ones.
-Mix with wooden toothpicks (use one for each specimen or control). Make circular motions and spread so that you cover the entire circle.
Source: images taken from Video: https://youtu.be/igAVQ-GiyGY. Edited image layout.
-Place the plate on an automatic rotator at 80 to 100 RPM or rotate manually for 4 minutes. Read the proof at the end of this time.
-Verify that the controls gave as expected. Compare the reaction of the test sample with the controls. Report as positive if agglutination is observed and as negative if there is no agglutination.
A positive test indicates that the patient has an amount equal to or greater than 25 IU / ml of anti-Brucella antibodies.
Interpretation of a positive (with agglutination) and negative (without agglutination) reaction. Source: Citrulline. Edited image.
Technique (semi-quantitative)
If in the qualitative reaction the sample is strongly positive, it can be semi-quantified. For this, serial double dilutions of the sample are made with physiological saline solution. The procedure previously described is carried out with each dilution.
It is also interpreted by observing the agglutination macroscopically. The result will be the titer of the highest dilution in which a positive result was observed.
To calculate the approximate value of anti-Brucella antibodies, the following formula is used:
25 IU / ml x reaction titer = IU / ml
Example, if a patient in the semi-quantitative test tests positive at the ½, ¼ and 1/8 dilution and begins to be negative from the 1/16 dilution onwards, this means that the patient has a titer of 8.
Applying the formula:
25 IU / ml x 8 = 200 IU / ml
QA
-The kits have an expiration date and it must be respected. Should not be used if the reagent has expired.
-During use, make sure that the reagent does not contain solid particles, as this is a sign of deterioration.
-Keep between 2 and 8 ° C.
-Do not freeze, this act damages the reagent irremediably.
-Always carry out the test together with the negative and positive controls.
-The technique tolerates serum samples with a certain degree of lipemia and hemolysis, however it is not recommended to use excessively lipemic and hemolyzed sera, both conditions alter the results of the test.
-Always bring reagents to room temperature before starting the analysis.
-Do not interpret reactions that take longer than the recommended time, as this generates the report of false positives, since after a certain time the reagent precipitates, simulating a positive reaction.
-The technique is 100% sensitive and has a specificity of 98%.
-After a semi-quantification of 1000 IU / ml there is the possibility of observing a prozone effect (false negative due to excess antibody compared to the amount of antigens).
References
- Rubio M, Barrio B and Díaz R. Value of Rosa de Bengal, Coombs and counter-immunoelectrophoresis tests to diagnose cases of human brucellosis in which serum agglutination is negative. Department of Microbiology. Clinical Microbiology Service. University Clinic. University of Navarra. 406-407. Available at: elsevier.es/es-revista-enfermedades-infecciosas
- "Brucellosis." Wikipedia, The Free Encyclopedia. 6 Dec 2019, 14:37 UTC. 18 Dec 2019, 18:09 en.wikipedia.org.
- Monlab Laboratories. Monlab Bengal Rose - test. 2016.Available at: monlab.es/
- Carrillo C, Gotuzzo E. Brucellosis. Rev. Peru. Med. Exp. Public Health 1997; 14 (1): 63-66. Available at: scielo.org
- Morales-García R, García-Méndez N, Regalado-Jacobo D, López-Merino A, Contreras-Rodríguez A. Clinical, serological and polymerase chain reaction follow-up of a family with brucellosis. Rev. chil. Infectol. 2014; 31 (4): 425-433. Available at: scielo.conicyt.