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1. Many
serological tests are qualitative
a.
The test primarily indicates the
presence or absence of antibody in patient's serum and thus identifies the
disease process.
2. The
majority of qualitative tests require that, at a given temperature, specified quantities
of a known antigen are added to a patient's serum. The two are incubated for a
period of time to permit a reaction (if any) to take place.
3. Resultant visible reactions (agglutination or precipitation)
indicate that specific antibody is present.
4. Although
this qualitative test for a specific antibody is positive, the physician needs
to know more, i.e., how much antibody is present.
5. The
amount of antibody is often associated with the severity of infection; e.g., a
large amount sometimes indicates a severe infection, whereas a small amount
means less severity.
6. The
serologist must therefore determine the quantity of antibody present.
1. To
determine the amount of antibody, the serologist must perform a Quantitative Test in which the patient's serum is diluted by a
standardized method in 5 to 10 test tubes, using a pipet.
2. The
10 test tubes are prepared so that each contains half the quantity of antibody
(serum) that is found in the previous tube.
3. Identical
quantities of antigen are added to each tube.
4. The
tubes are incubated for a specified period of time, after which the pattern of
agglutination or precipitation is observed and recorded.
5. The
Quantitative dilution procedure is similar to that for the Qualitative Test
except for two primary differences:
a. In
the qualitative procedure only the undiluted serum is tested, while in the
Quantitative Test, 10 test tubes containing different quantities of antibody
(serum) are tested. These tubes are
subjected to the same mixing, the same quantity of antigen, and the same
incubation period.
b. Ten
tubes containing different quantities of antibody will reveal different
agglutination patterns. A few of the
first tubes may be strongly positive, but weaker reactions follow. The Qualitative Test, however, is strongly
positive. This is how the strength of
the antibody is determined. The
reciprocal of the dilution of the last tube in which agglutination is seen is
referred to as the "titer" or strength of the antibody.
6. The
"titer" of an antibody indicates the quantity of that antibody
present in a serum. The only way to determine its quantity is to dilute out the
antibody until no more agglutination is visible.
The standard dilution
technique most frequently used in the serology laboratory is the twofold
dilution. The guiding principle for this
dilution type is the fact that each tube contains half serum and half diluent (usually saline).
The amount of antibody is directly proportional to the quantity of
serum. The first tube of the twofold
dilution series usually contains specified quantities of undiluted patient's
serum - the same undiluted serum originally found to cause agglutination in the
Qualitative Test. The second tube
contains half the amount of serum and therefore half the amount of antibody;
the third tube contains a quarter of the amount of antibody, the fourth tube
contains an eighth, the fifth contains a sixteenth, the sixth a thirty-second,
the seventh tube a sixty-fourth, etc.
The twofold dilution procedure should be performed in the following
manner:
1. Place
10 clean test tubes in a test-tube rack.
2. Add
1.0 ml of 0.9% saline to tubes 2 through 10, using a 5.0 ml pipet.
3. Add
1 ml of patient's serum to tubes 1 and 2.
4. With
the same pipet, mix the contents of tube 2 by drawing
the contents up into the pipet. The process of drawing up and blowing out is
considered one mixing. To be thorough,
mix four times. Note that tube 1
contains only undiluted serum and tube 2 contains half the amount of the serum
because 1.0 ml of undiluted serum was diluted with 1.0 ml saline. When serum is diluted to half the original
quantity, it is said to be diluted 1:2.
This means that there is one part of undiluted serum and one part of diluent, which equals two parts or two volumes.
5. With
the same pipet that was used in step 4, transfer 1.0
ml of serum from tube 2 to tube 3 which also contains 1.0 ml of saline. Mix four times as described above. The half quantity of serum in tube 2 has
been diluted to one-quarter in tube 3.
When serum is diluted to one-quarter the original quantity, it is said to
be diluted 1:4. This means that 1 volume
of a 1:2 serum diluted with 1 volume of saline is equal to 2 volumes. Examine these dilutions carefully. If a serum is diluted 1:4 directly from
undiluted serum, this may be accomplished by adding 1 ml of serum to 3 ml of
saline for a total of 4 volumes.
1 + 3 = 4
or 1 volume of
serum diluted to 4 volumes
or 1:4 dilution
But a 1:4 dilution has been
prepared from a 1:2 dilution, not from undiluted serum. This is done by diluting 1 volume of a 1:2
dilution with 1 volume of saline to make a 1:4 dilution.
6. With the same pipet
used in step 5, transfer 1.0 ml of serum from tube 3 (1:4) to tube 4 to prepare
a 1:8 dilution. Mix four times as
described above.
If one were to prepare a 1:8 dilution from undiluted serum, one could do so by adding 1 volume of serum to 7 volumes of saline to get 8 volumes.
1 + 7 = 8
or 1 volume of serum diluted to
8 volumes or 1:8
But because a twofold dilution
is being prepared, one merely dilutes 1 ml of a 1:4 with 1 volume of saline to
get a 1:8 dilution, or
1:4 diluted 1:2 = 1:8
7. Continue to transfer 1 ml of each serum dilution from tube 4 to tube 5, mix well; take 1 ml from tube 5 and place it in tube 6, mix well; take 1 ml from tube 6 and place it in tube 7, etc., to tube 10.
8. Mix tube 10 and discard 1 ml from that
tube. Identical volumes in all tubes
have been maintained. Each contains
exactly 1 ml total volume.
EXAMPLE PROBLEM: Twofold
Dilution
Given 0.6 ml of serum,
diagrammatically prepare a twofold dilution, using 0.3 ml of serum in tubes 1
and 2.
Answers:
1 + 1 = twofold dilution
X 0.3 ml (multiply both sides by
0.3 ml)
0.3 ml serum + 0.3 ml saline = 0.6 ml
of a 1:2 dilution
It should be pointed out at
this time that if a twofold dilution series were being performed to quantitate antibody, one would be required to complete the
test dilutions out through each of ten tubes.
Specified quantities of specific antigen would be added to each tube,
the 10 tubes would be shaken to mix the contents, then
incubated, and finally each tube would be examined for visible
agglutination. An antibody titer would
then result.
Rarely is a five or tenfold
dilution required, except perhaps for problem-solving or research
purposes. The standard method previously
described is for the preparation of a twofold dilution. Note the following facts about these ranges:
* The second test tube in the five and tenfold series
contains less serum than that in the twofold series.
* The dilution in the fourth tube of the twofold dilution
(1:8) is between the first and second tubes of the ten fold (1:10) and between
the second and third tubes of the fivefold (1:5).
The significant point is
that the dilution increments in the twofold series are less than that in the
five or tenfold dilution series. In
other words, the serum undergoes less dilution from one tube to the next in the
twofold than in the five or tenfold series. Because of the facts presented in
the preceding paragraph, if one wanted to use a dilution technique that offers
a more accurate endpoint, one would choose the twofold dilution series (the dilution
increments are smaller). This is why the
twofold dilution series is the method of choice for serological purposes.
However, there are occasions when a greater dilution range is needed. Imagine a patient who is known to have a
particular disease. You know that his
last titer was high and the physician wants the patient's antibody levels quantitated. You
will have to set up a broad enough dilution series to detect an endpoint as
quickly as possible. The twofold dilution span may not be broad enough because
the patient may have an antibody titer beyond 1:512. Thus, a five or tenfold dilution series would
be preferred. Using the fivefold format,
the dilution increment is greater than that of the twofold. Therefore, the antibody endpoint may be approximated
using fewer test tubes.
1. How would you set up a 1:1000
dilution from undiluted serum?
Answer:
Format: 1 volume undiluted serum + 999 volumes of
diluent = 1000 volumes or 1:1000 dilution
Obviously, if one actually
had to prepare this dilution, 1000 ml of a 1:1000 dilution would be too great a
volume to be practical. It can be
decreased while maintaining the 1:1000 dilution as
follows:
1 + 999 = 1000 or 1:1000
X 0.1 ml
0.1 ml + 99.9 = ml of 1:1000 (100 ml is still too large a quantity for
practicality)
or
1 + 999
X 0.01 ml
0.01 ml serum + 9.99 ml saline = 10
ml of 1:1000
This is a practical volume of
serum with which to work. Plan to measure small quantities such as 0.01 ml of undiluted serum
with a 0.01 ml serologic pipet to insure accuracy. Note:
When the decimal point is moved to the left on the left side of the
formula (0.1 to 0.01), it must also be moved to the left on the right side
(99.0 to 9.99) in order to maintain the 1:1000 dilution. The decimal must be moved the same distance
on both sides.
2. Prepare a 1:80 dilution from
an undiluted serum.
Answer:
Format 1 + 79 = 1:80
1 volume of undiluted serum + 79
volumes of saline = 80 volumes of a 1:80 dilution. Since 80 ml is too great a volume with which
to work, use 0.1 ml undiluted serum + 7.9 ml
saline = 8.0 ml of a 1:80
dilution.
3. Prepare a 1:8 dilution directly
from 1:1 or undiluted serum.
Answer:
1 volume of serum + 7 volumes of diluent = 8
volumes
or
1 volume of serum is
diluted to 8 volumes
or the serum is diluted 1:8
The above example indicates how
a 1:8 dilution is prepared. Now, how can
a 1:8 dilution be made if there is only 0.1 ml of undiluted serum with which to
make that dilution?
First:
multiply each side of the basic formula (1 and 7) by 0.1 ml serum.
1+ 7 = 8
X 0.1 ml
0.1 ml serum + 0.7 ml diluent = 0.8 ml total volume
Thus, by adding 0.1 ml of undiluted serum
to 0.7 ml saline, 0.8
ml of a 1:8 dilution has been prepared.
4. Prepare a dilution of 1:64
using 0.5 ml of serum.
Answer:
1 volume of serum + 63 volumes of diluent = 64
volumes or 1:64
Multiply each side of the formula (1 and
63) by 0.5 ml serum
1 + 63 = 64 or 1:64
X 0.5 ml
0.5 ml serum + 31.5 ml diluent = 32 ml of a 1:64 dilution
Note that one can also prepare a dilution
of 1:512 on paper similarly:
1 volume of serum + 511 volumes of saline
= 512 volumes of 1:512 dilution
Or if 0.1 ml of serum is
used,
0.1 ml serum + 51.1 ml saline = 51.2 ml of
a 1:512 dilution.
5. Prepare a twofold dilution - any volume
When a twofold dilution was
described above in STEP 7, the final volume in each tube was 1.0 ml; however,
any final volume can be prepared. If one
used 0.2 ml of each serum dilution instead of 0.1 ml, the final volume in each
tube would be 0.2 ml. This is how a
twofold dilution with a final volume of 0.2 ml would be prepared:
1 + 1 = 2 (1:2) twofold
X 0.2 ml
0.2 ml serum + 0.2 ml saline = 0.4 ml
If each side is multiplied
(1 and 1) by 0.2 ml, a result of 0.2 ml serum + 0.2 ml of diluent
is obtained, which equals 0.4 ml. the result is 0.4 ml of a twofold
dilution. Now if one wishes to set up a
twofold dilution using 0.2 ml of each serum dilution, after mixing, 0.2 ml must
be removed, leaving 0.2 ml of the dilution in the tube. In this way, all test tubes have a final
volume of 0.2 ml. Note that to set up a
twofold dilution using 0.2 ml of serum in tube 2, a total volume of 0.4 ml
undiluted serum is needed (0.2 ml for tube 1 and 0.2 ml for tube 2). The less serum required, the better, since
remaining serum may be used for other tests, if requested.
6. Prepare a twofold
dilution-minimum serum volume.
To economize without adversely
affecting accuracy, perform steps 1-7 by using 0.1 ml of serum in tubes 1 and
2, stipulating the format on paper, and diagrammatically prepare the twofold
dilution to determine how one would actually perform this procedure using this
quantity of serum.
1 + 1 = 2 (1:2)
X 0.1 ml
0.1 ml of serum + 0.1 ml saline = 0.2
ml of a 1:2 dilution
7. You have a frozen 1:10 dilution of serum and
you need to prepare from this a 1:35 dilution for an antibody test. Show how you would do this.
Answer: Step 1.
Find the working dilution
to be prepared:
1:35 = 1:3.5
1:10
A dilution of 1:35 is 3.5
times more dilute than the 1:10. Note
that a 1:3.5 of a 1:10
gives
a final dilution of a 1:35.
Step 2. Write
the format: 1:3.5 = 1 + 2.5 = 3.5 ml of
a 1:3.5 dilution
Step 3. Write
the directions: Add 1 ml of a 1:10
dilution to 2.5 ml saline, to prepare a 1:3.5 dilution.
8. Given a 1:3 dilution, prepare a 1:60
dilution.
Step 1. Working dilution:
1:60 = 1:20
1:3
A dilution of 1:60 is 20 times more dilute than the 1:3. Note that a 1:20 of a 1:3 gives a final
dilution of 1:60.
Step 2. Format:
1:20 = 1 + 19.0 = 20 ml of a 1:20
Step 3. Directions
Add 1 ml of a 1:3 to 19 ml of saline, to prepare a 1:60
dilution or 0.1 ml of a 1:3 to 1:9 ml
of saline = 2.0 ml final volume.
9. Given
a 1:60 dilution, prepare a 1:100 dilution.
Step 1. 1:100
= 1:1.66
1:60
Step 2. 1:1.66
= 1 ml + 0.66 ml of diluent = 1.66 ml
Step 3. Add 1
ml of the 1:60 dilution to 0.66 ml of saline to
prepare a 1:100 dilution.
Answer the following, using
the approach demonstrated previously:
10. Given a 1:5 dilution, prepare a 1:50 on paper and show your
mathematical calculations:
Step 1. Working
dilution:
Step 2. Format:
Step 3. Directions:
11. Given a 1:100 dilution, prepare a 1:10,000 dilution on paper and
show your mathematical calculations:
Step 1. Working
dilution:
Step 2. Format:
Step 3. Directions
12. Given a 1:20 dilution,
prepare a 1:65 dilution on paper and show your mathematical calculations:
Step 1. Working
dilution:
Step 2. Format:
Step 3. Directions:
ANSWERS TO PROBLEMS 10-12
10. Step 1. Working dilution: 1:50 = 1:10
1.5
Step 2. Format: 1:10 = 1 + 9 ml = 10 ml of a 1:10 dilution
Step 3. Directions: Add 1 ml of the 1:5 dilution
to 9 ml saline to prepare a 1:50 dilution.
11. Step 1. Working dilution: 1:10,000
= 1:100
1:100
Step 2. Format: 1:100 = 1 ml + 99 diluent
= 100 mil of 1:100 dilution
Step 3. Directions: Add 1 ml of the 1:100 to 99 ml of diluent to prepare a 1:10,000 dilution.
12. Step 1. Working dilution: 1:65
= 1:3.25
1:20
Step 2. Format: 1:3.25 = 1 ml + 2.25 ml of diluent = 1:3.25
Step 3. Directions: Add 1 ml of the 1:20 dilution to 2.25 ml of
saline to prepare a 1:65 dilution.
In order to demonstrate your
problem-solving expertise, go back and see how you solve problem 10 if you had
only 0.2 ml serum. Show your
mathematical calculations.
Format:
1:10 dilution (1:50 is a 1:10 dilution of a 1:5)
1 + 9 = 10 ml of a 1:10
X 0.2 ml
0.2 ml + 1.8 ml = 2.0 ml of a 1:10
Directions: Add 0.2 ml of the 1:5 to 1:8 saline to prepare 2.0 ml of a 1:50.
1. Given
0.5 ml of serum, diagrammatically prepare a twofold dilution format using 0.25
ml of serum in test tubes 1 and 2.
2. What
are the only differences between the above format and the previous ones?
3. Has
the twofold dilution format been maintained?
Explain.
4. What
is the final volume in each test tube?
5. How
much serum did you need for this format?
6. Of
the previous examples, which format is ideal from the standpoint of the volume
of serum required to perform the twofold dilution?
7. Has
the twofold dilution format been maintained in each tube?
8. How
much saline has been added to each of the nine tubes using this format?
ANSWERS TO ADDITIONAL PROBLEMS
1. 1 + 1 = 2 volumes of a 1:2
X 0.25 (multiply each side by 0.25 ml)
0.25 + 0.25 = 0.5 ml of a 1:2
Tube No. 1 2 3 4 5 6 7 8 9 10
Serum 0.25 0.25
0 0 0 0 0 0 0 0
(ml)
Saline 0 0.25 0.25
0.25 0.25 0.25 0.25 0.25
0.25 0.25
(ml)
Transfer 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25
Fin. Vol. 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25
Dilution 1:1 1:2
1:4 1:8 1:16 1:32 1:64 1:128
1:256 1:512
2. The quantities of serum and saline
3. Yes
4. 0.25 ml
5. 0.5 ml
6. The serum volume of 0.1 ml in tubes 1 and 2