Path 415 – Agglutination is right, but how do you know that your determination is right?

Hi everyone! Shout outs to Curtis, Muskaan and Caitlyn who reached out, and all on the right track!

Agglutination, yup, that’s it. Why does it work?

What’s in antisera? Antiserum is blood serum that contains specific antibodies against an infective organism or poisonous substance. Antiserum (plural: antisera) typically contains polyclonal antibodies and is used to pass on passive immunity to many diseases.

How will they work? Think of what we know about antibodies that bind to antigens, and how they cause the formation of clumps. Be aware that there are also OTHER factors in antisera! Serum includes all proteins not used in blood clotting (coagulation) and all the electrolytesantibodiesantigenshormones, and any exogenous substances including complement proteins – the ones that bind to antibodies

Slide agglutination serogrouping (SASG) test for serogrouping N. meningitidis isolates

  • Reference laboratories must isolate, identify, and characterize the serogroup of isolates of N. meningitidis.
  • Gives public health authorities means to identify outbreaks controllable by vaccination campaigns, recognize serogroups causing sporadic disease, and detect emergence of new outbreak strains.

The CDC Website http://www.cdc.gov/meningitis/lab-manual/chpt07-id-characterization-nm.html provides the outline of how the assay can be performed, and what to look for.

**Most importantly, you will need isolates from each of the patients (grown up – think back to microbiology) that are relatively pure to be tested. The CDC gives important tips on using formalin-fixed isolates in the assay, can you think of why?

Please remember you will need positive and negative controls (from your own historical samples, where you have isolates from Group B seropositive and seronegative isolates/people). You will also want to include saline as a negative control to detect nonspecific autoagglutination. Looking below, you can see the different degrees of responses.

Reading the SASG results

  1. Rating the intensity of the agglutination reaction

    Agglutination occurs when the antisera bind to the bacterial cells causing the cells to agglutinate or clump together, thus making the cell suspension appear clearer. The intensity of the agglutination reaction may vary according to the density of the cell suspension or the antisera used. A description on the intensity ratings shown in Figure 6 are listed below.

    4+ All of the cells agglutinate and the cell suspension appears clear
    3+ 75% of the cells agglutinate and the cell suspension remains slightly cloudy
    2+ 50% of the cells agglutinate and the cell suspension remains slightly cloudy
    1+ 25% of the cells agglutinate and the cell suspension remains slightly cloudy
    +/- Less than 25% of the cells agglutinate and a fine granular matter occurs
    0 No visible agglutination; the suspension remains cloudy and smooth

    Figure 6 is a picture showing the rating the intensity of the agglutination reaction.

    Figure 6. Rating the intensity of the agglutination reaction

  2. Determining the serogroup
    • A positive result is designated by a 3+ or 4+ (strong agglutination) within 1-2 minutes, except for serogroup B, which is considered positive with a rating of 2+ or greater.
    • A negative result is designated by a 0 (saline), +/-, 1+ or 2+ (weak agglutination).
    • The serogroup is determined when a positive result occurs with only one of the antisera and not with the saline.
    • If a serogroup is not determined, the isolate is considered NG. The following result combinations are all reported as NG:
      • Agglutination in the saline, regardless of strong reactions with other antisera, characterizes the culture as autoagglutinating.
      • Agglutination with more than one serogroup-specific antisera in the absence of agglutination in saline characterizes the culture as polyagglutinating or cross-reactive.
      • No agglutination with any of the antisera or the saline characterizes the strain as non-reactive.