Diagnostic Testing for Mononucleosis using Modified Latex

Agglutination.
Abstract
The Epstein- Barr virus is an extremely common human virus that is passed
easily from person to person primarily through bodily fluids. It can cause seriou

Introduction
The Epstein- Barr virus (EBV) is one of the most common human viruses in the
world, and is most commonly spread through bodily fluids such as saliva, blood
and semen. It is particularly prevalent in teenagers and young adults, but
parents will often also pass on the virus to young children through the sharing of
drinks or through the use of dummies. The majority of people infected with EBV
will only experience mild flu-like symptoms and recover in under 4 weeks,
however it can also cause the proliferation of monocytes, resulting in infectious
mononucleosis. Once infected with the virus, a person can carry a latent form of
it for the rest of their life, with symptoms only developing if the virus reactivates
while the host is immunocompromised. In this situation EBV can cause much
more serious illnesses such as hemiplegia, pneumonia and pancreatitis. These
are life threatening and the early symptoms are very similar to many other
viruses, so it is essential to rule out EBV as a diagnosis early on. The presence of
the EBV stimulates the production of heterophile antibodies; so named because
they are IgM antibodies that are reactive to proteins across species lines. In the
past, the Paul Bunnel test was used to diagnose acute EBV. In this test, human
serum is mixed with sheep erythrocytes, and a heterophile titre greater or equal
to a 40-fold dilution of the serum is counted as a diagnosis of acute EBV.
Commercially available test kits are available with sensitivity of 70-92% and
specificity of 96-100%, with a lower sensitivity in the first two weeks after clinical
symptoms begin. While the specificity of this test is high, a false positive result
can be produced by other conditions such as rubella, lymphoma and leukaemia.
The use of red blood cells in the heterophile test may pose an issue as red blood
cells can lyse with age, which may effect the outcome of the test and thus the
diagnosis of a patient. This can be overcome by instead using coated latex
particles, as was used in this study. The solution containing the latex particles
has an even appearance that resembles milk. However, when they come into
contact with antibodies such as in serum, the latex particles start to cross link
and agglutinate, resulting in the liquid becoming clearer with dispersed clumps
of latex particles. This clumping should theoretically occur to some degree when
the heterophile antibodies are present, no matter what the concentration.
However, this clumping may not be visible to the naked eye at lower serum
antibody concentrations but it should be visible under a light microscope. Thus,
when examining the agglutination test under a light microscope, it should be
possible to detect agglutination that is not visible to the naked eye, and thus
observe a positive result at a lower antibody serum concentration. Therefore, the
hypothesis for the study is that when viewed under a light microscope, it should
be possible to observe a positive result from a heterophile agglutination test at a
lower antibody serum concentration than when observed with the naked eye.

Methods
A serum sample with a known antibody concentration of 1000 micrograms per
millilitre (g/ml) was diluted 1 in 8 using saline solution. The sample was then
double diluted with saline through four cycles, to give six different antibody
concentrations, as shown in table 1.
Original

1 in 8
dilution
125

1 in 16
dilution
62.5

1 in 32
dilution
31.25

1 in 64
dilution
15.625

1 in 128
dilution
7.8125

Concentrat 1000
ion of
antibody
serum
sample
(g/ml)
Table 1 The concentration of antibody serum (g/ml) compared to the dilution
factor
Naked eye latex agglutination tests were then carried out. SPHERO heterophile
coated polystyrene particles of 0.7 0.7 m were used with this particular
method. These latex particles were re-suspended by vigorously vortex mixing the
mixture. 20 L of the latex suspension was then pipetted onto microscope slides,
along with 5 L of water or serum, as shown in figure 1.

Figure 1 Showing the latex suspension

and serum drop orientation on the
microscope slide before mixing.

This was performed on 3 slides, so that each dilution of the serum was used, as
well as water which acts as a negative control. The latex solution and serum, or
water, was then mixed to cover a circular area using a pipette tip, resulting in
slides as shown in figure 2.

Figure 2 Showing the latex suspension

and serum drops after mixing.

The slides were then rotated in a circular fashion for three minutes so that the
mixture in the area is kept constantly in motion. This ensures that the latex
remains in suspension, and therefore the mixture remains milky and evenly
distributed, unless agglutination occurs, in which case white clumps will start to
form around the edge of the mixture or the mixture will take on a granular form.
Figure 3 demonstrates the difference between a positive and a negative result. If
this agglutination occurred in under 3 minutes, a positive result was recorded.

Positive
Negative
Figure 3 showing the typical agglutination test
results.

The lowest concentration of antibody at which a positive result could be

observed was noted. If all concentrations yielded a positive result then further
antibody dilutions were performed until a negative result could be seen.
The same latex suspension and antibody serum dilutions were then pipetted and
mixed in the same fashion onto lysine coated Hendley-Essex 3-SPOT multispot
microscope slides. These were then observed using a leitz laborlux 11
microscope fitted with a 512-661 specification microscope head. The mixtures
were observed at x40 and x100 magnification so as to more accurately identify
whether there was agglutination and thus a positive result. If agglutination had
occurred, clumping would be seen as in figure 4, whereas if there was no
agglutination and thus a negative result, no clumping would be observed as in
figure 5.

Figure 4 Expected
observation for a positive
result

Figure 5 Expected
observation for a negative
result

Results

The results for the original naked eye agglutination test, including the further
dilutions, can be seen in figure 6. They show that all antibody serum dilutions
except for 1 in 256 showed positive results for the presence of the EBV
antibodies.

Concentrati
on of
antibody
serum
sample
(g/ml)
Result (+/-)

1 in 8
dilution

1 in 16
dilution

1 in 32
dilution

1 in 64
dilution

1 in 128
dilution

125

62.5

31.25

15.625

7.8125

1 in
256
dilution
3.9063

Figure 6 Table showing the results of the naked eye latex agglutination test
The results from the altered method using lysine covered microscope slides and
microscopy can be seen in figure 7. They show that the antibody serum dilutions
of 1 in 8, 1 in 16 and 1 in 32 show positive results for the presence of EBV
antibodies. The antibody serum solution dilutions of 1 in 64, 1 in 128 and 1 in
256 showed negative results for the presence of EBV antibodies.
1 in 8
dilution
125

1 in 16
dilution
62.5

1 in 32
dilution
31.25

1 in 64
dilution
15.625

1 in 128
dilution
7.8125

Concentrat
ion of
antibody
serum
sample
(g/ml)
Result (+/-) +
+
+
Figure 7 Table showing the results of the modified method

1 in 256
dilution
3.9063

Overall, the original naked eye latex agglutination method showed a positive
result for the presence of EBV antibodies at a lower titre

Discussion and Conclusion

The hypothesis for the study was that that when viewed under a light
microscope, it should be possible to observe a positive result from a heterophile
agglutination test at a lower antibody serum concentration than when observed
with the naked eye. As shown from the results in figures 6 and 7, this was
disproved as a positive result was observed at an antibody serum concentration
of 7.8125 g/ml using the original agglutination test. However, the lowest
concentration at which a positive result was obtained using the modified method
was at a serum antibody concentration of 31.25 g/ml. When these results were
obtained, the original slides of lower antibody serum concentration used in the
unmodified method were also viewed using a light microscope. It was then
discovered that what was considered to be agglutination due to the presence of
the antibody serum was in fact residue due to the latex solution drying out. This
conclusion was reached as what was observed down the microscope did not
resemble the clumping shown in figure 4, but instead showed regular crystalline
structure, which upon further research were agreed to be the dried latex
solution.
This may not have been seen in the modified method as lysine-coated
microscope slides were used. These have been shown to improve the adhesion of
tissues to the slides, which may have stopped the solution from spreading and
thinning out and thus may have prevented the solution from drying out as
quickly as in the original method.
When further analysing the results, the specificity and sensitivity of the modified
method and the original method were calculated and then plotted on a Receiver
Operating Curve (ROC curve). This can be seen in figure 8.

Figure 8 ROC
curve comparing the original and modified method
The graph shows that the original method is more effective to detect the EBV
than the modified method as shown by the higher curve on the ROC curve. This
means that the original method reached a much higher sensitivity at a higher
specificity than the modified method. This further that shows that the modified
method did not improve on the original method as well as the fact that the study
disproved the hypothesis.
While the modified method did not allow a positive result to be observed at a
lower antibody serum concentration, it did demonstrate the benefit of using
lysine coated microscope slides. The use of these in the naked eye agglutination
method may prevent the occurrence of false positive results being recorded as
happened in this study. This is due to their improved adhesion quality, which may
prevent the solution from drying out as quickly which will allow more time for it
to be made certain that a result is due to the agglutination of the latex particles.
The use of a light microscope may show to improve the original method once it
has been made more clear what is a positive result in the naked eye test.

how would we change method

whats the next step

References