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Diagnostic Testing Testing for Mononucleosis using Modied Latex Agglutination. Abstract The Epstein- Barr virus is an extremely common common human virus that is passed passed easily from person to person primarily primaril y through odily !uids. "t can cause seriou
Introduction The Epstein- Barr virus #EB$% #EB$% is one of of the most common human viruses in the &orld' and is most commonly spread through odily !uids such as saliva' lood and semen. "t is particularly particularly prevalent in teenagers and young adults' ut parents &ill often also pass on the virus to young children through the sharing of drin(s or through the use of dummies. dumm ies. The ma)ority of people infected &ith EB$ &ill only experience mild !u-li(e symptoms and recover in under * &ee(s' ho&ever it can also cause the proliferation of monocytes' resulting in infectious mononucleosis. +nce infected &ith the virus' a person can carry a latent form of it for the rest of their life' &ith symptoms only developing if the virus reactivates &hile the host is immunocompromised. "n this situation EB$ can cause much more serious illnesses such as hemiplegia' pneumonia and pancreatitis. These are life threatening and the early symptoms are very similar simi lar to many other viruses' so it is essential to rule out EB$ as a diagnosis early on. The presence of the EB$ stimulates the production of heterophile antiodies, so named ecause they are "gM antiodies that are reactive to proteins across species lines. "n the past' the aul Bunnel test &as used to diagnose acute EB$. EB$. "n this test' human serum is mixed &ith sheep erythrocytes' and a heterophile titre greater or eual to a */-fold dilution of the serum is counted as a diagnosis of acute EB$. EB$. 0ommercially availale test (its are availale &ith sensitivity of 1/-234 and specicity of 25-6//4' &ith a lo&er sensitivity in the rst t&o &ee(s after clinical symptoms egin. 7hile egin. 7hile the specicity of this test is high' a false positive result can e produced y other conditions such as ruella' ruell a' lymphoma and leu(aemia. The use of red lood cells in in the heterophile test may pose an issue as red red lood cells can lyse &ith age' &hich may e8ect the outcome of the test and thus the diagnosis of a patient. This can e overcome y instead using coated latex particles' as &as used in this study. study. The solution containing the latex particles has an even appearance that resemles mil(. 9o&ever' &hen they come into contact &ith antiodies such as in serum' the latex particles start to cross lin( and agglutinate' resulting in the liuid ecoming clearer &ith dispersed clumps of latex particles. This clumping should theoretically occur to some degree &hen the heterophile antiodies are present' no matter &hat the c oncentration. 9o&ever' this clumping may not e visile to the na(ed eye at lo&er serum antiody concentrations ut it should e visile under a light microscope. Thus' &hen examining the agglutination test under a light microscope' it should e possile to detect agglutination that is not visile to the na(ed eye' and thus oserve a positive result at a lo&er antiody serum concentration. Therefore' the hypothesis for the study is that &hen vie&ed under a light microscope' it should e possile to oserve a positive result from a heterophile agglutination test at a lo&er antiody serum concentration than &hen oserved &ith the na(ed eye.
Methods A serum sample &ith a (no&n antiody concentration of 6/// micrograms per millilitre #:g;ml% &as diluted 6 in < using saline solution. The sample &as then doule diluted &ith saline through four cycles' to give six di8erent antiody concentrations' as sho&n in tale 6. +riginal
6 in < dilution 63>
6 in 65 dilution 53.>
6 in =3 dilution =6.3>
6 in 5* dilution 6>.53>
6 in 63< dilution 1.<63>
0oncentrat 6/// ion of antiody serum sample #:g;ml% Tale 6 ? The concentration of antiody serum #:g;ml% compared to the dilution factor @a(ed eye latex agglutination tests &ere then carried out. 9E+C heterophile coated polystyrene particles of /.1 ? /.1 :m &ere used &ith this particular method. These latex particles &ere re-suspended y vigorously vortex mixing the mixture. 3/ :L of the latex suspension &as then pipetted onto microscope slides' along &ith > :L of &ater or serum' as sho&n in gure 6.
igure 6 ? ho&ing the latex suspension and serum drop orientation on the microscope slide efore mixing.
This &as performed on = slides' so that each dilution of the serum &as used' as &ell as &ater &hich acts as a negative control. The latex solution and serum' or &ater' &as then mixed to cover a circular area using a pipette tip' resulting in slides as sho&n in gure 3.
igure 3 ? ho&ing the latex suspension and serum drops after mixing.
The slides &ere then rotated in a circular fashion for three minutes so that the mixture in the area is (ept constantly in motion. This ensures that the latex remains in suspension' and therefore the mixture remains mil(y and evenly distriuted' unless agglutination occurs' in &hich case &hite clumps &ill start to form around the edge of the mixture or the mixture &ill ta(e on a granular form. igure = demonstrates the di8erence et&een a positive and a negative result. "f this agglutination occurred in under = minutes' a positive result &as recorded.
ositive
igure = ? sho&ing the typical agglutination test results.
The lo&est concentration of antiody at &hich a positive result could e oserved &as noted. "f all concentrations yielded a positive result then further antiody dilutions &ere performed until a negative result could e seen. The same latex suspension and antiody serum dilutions &ere then pipetted and mixed in the same fashion onto lysine coated 9endley-Essex =-+T multispot microscope slides. These &ere then oserved using a leit laorlux 66 microscope tted &ith a >63-556 specication microscope head. The mixtures &ere oserved at x*/ and x6// magnication so as to more accurately identify &hether there &as agglutination and thus a positive result. "f agglutination had occurred' clumping &ould e seen as in gure *' &hereas if there &as no agglutination and thus a negative result' no clumping &ould e oserved as in gure >.
igure * ? Expected oservation for a positive
igure > ? Expected oservation for a negative
Results The results for the original na(ed eye agglutination test' including the further dilutions' can e seen in gure 5. They sho& that all antiody serum dilutions except for 6 in 3>5 sho&ed positive results for the presence of the EB$ antiodies.
0oncentrati on of antiody serum sample #:g;ml% esult #F;-%
6 in < dilution
6 in 65 dilution
6 in =3 dilution
6 in 5* dilution
6 in 63< dilution
63>
53.>
=6.3>
6>.53>
1.<63>
6 in 3>5 dilution =.2/5=
F
F
F
F
F
-
igure 5 ? Tale sho&ing the results of the na(ed eye latex agglutination test The results from the altered method using lysine covered microscope slides and microscopy can e seen in gure 1. They sho& that the antiody serum dilutions of 6 in <' 6 in 65 and 6 in =3 sho& positive results for the presence of EB$ antiodies. The antiody serum solution dilutions of 6 in 5*' 6 in 63< and 6 in 3>5 sho&ed negative results for the presence of EB$ antiodies. 6 in < dilution 63>
6 in 65 dilution 53.>
6 in =3 dilution =6.3>
6 in 5* dilution 6>.53>
6 in 63< dilution 1.<63>
0oncentrat ion of antiody serum sample #:g;ml% esult #F;-% F F F igure 1 ? Tale sho&ing the results of the modied method
6 in 3>5 dilution =.2/5=
-
+verall' the original na(ed eye latex agglutination method sho&ed a positive result for the presence of EB$ antiodies at a lo&er titre
Discussion and Conclusion The hypothesis for the study &as that that &hen vie&ed under a light microscope' it should e possile to oserve a positive result from a heterophile agglutination test at a lo&er antiody serum concentration than &hen oserved &ith the na(ed eye. As sho&n from the results in gures 5 and 1' this &as disproved as a positive result &as oserved at an antiody serum concentration of 1.<63> µg;ml using the original agglutination test. 9o&ever' the lo&est concentration at &hich a positive result &as otained using the modied method &as at a serum antiody concentration of =6.3> µg;ml. 7hen these results &ere otained' the original slides of lo&er antiody serum concentration used in the unmodied method &ere also vie&ed using a light microscope. "t &as then discovered that &hat &as considered to e agglutination due to the presence of the antiody serum &as in fact residue due to the latex solution drying out. This conclusion &as reached as &hat &as oserved do&n the microscope did not resemle the clumping sho&n in gure *' ut instead sho&ed regular crystalline structure' &hich upon further research &ere agreed to e the dried latex solution. This may not have een seen in the modied method as lysine-coated microscope slides &ere used. These have een sho&n to improve the adhesion of tissues to the slides' &hich may have stopped the solution from spreading and thinning out and thus may have prevented the solution from drying out as uic(ly as in the original method. 7hen further analysing the results' the specicity and sensitivity of the modied method and the original method &ere calculated and then plotted on a eceiver +perating 0urve #+0 curve%. This can e seen in gure <.
igure < ? +0 curve comparing the original and modied method The graph sho&s that the original method is more e8ective to detect the EB$ than the modied method as sho&n y the higher curve on the +0 curve. This means that the original method reached a much higher sensitivity at a higher specicity than the modied method. This further that sho&s that the modied method did not improve on the original method as &ell as the fact that the study disproved the hypothesis. 7hile the modied method did not allo& a positive result to e oserved at a lo&er antiody serum concentration' it did demonstrate the enet of using lysine coated microscope slides. The use of these in the na(ed eye agglutination method may prevent the occurrence of false positive results eing recorded as happened in this study. This is due to their improved adhesion uality' &hich may prevent the solution from drying out as uic(ly &hich &ill allo& more time for it to e made certain that a result is due to the agglutination of the latex particles. The use of a light microscope may sho& to improve the original method once it has een made more clear &hat is a positive result in the na(ed eye test.
