· A serial dilution is any dilution where the concentration decreases by the same quantity in each successive step.
· In the clinical lab, doubling dilutions are common. This is a series of 1:2 dilutions. Each successive tube will have the total volume of the previous tubes but will be half as concentrated.
· When performing serial dilutions in the clinical lab, one must know the dilution of each tube. If you know the dilution factor, you multiply each successive tube by it and can easily determine the concentration of each tube.
· Titers are reported out as the reciprocal of the dilution in the last tube giving a positive reaction. The titer is expressing the degree to which the solution can be diluted and still produce an observable reaction.
1. In a series of 1:2 dilutions, what is the dilution of tube 6? (Assume you begin with a 1:2 dilution in tube 1)
2. Assume the series in question one began with a 1:10 dilution in tube 1. After tube 1,a series of 1:2 dilutions were made. What would the dilution be in tube 6?
3. What is the dilution factor when 0.2 mL of patient specimen is added to 3.8 mL of diluent?
4. Start with the tube described in question 3 and carry out the same dilutions for 4 tubes. What is the dilution in tube 4?
5. Using the same dilution series, what is the titer if tube 3 demonstrates a positive reaction and tube 4 is negative?
6. The first tube of a 3-fold serial dilution is 1:3. What is the dilution in the 4th tube?
7. Add 5 mL of diluent to 5 mL of a 1:10 dilution. What is your new dilution?
· Sometimes, paired serial dilutions are assayed to help diagnose current and previous infections.
· Typically, the physician orders an Acute Specimen – collected during the acute phase of the illness. The Convalescent Specimen may be ordered for days to weeks after recovery.
· The Acute Specimen is always assayed AT THE SAME TIME, by the same technologist as the Convalescent Specimen. That is, the acute specimen is drawn and then held for the arrival of the second specimen for testing together.
· When comparing the two titers, there must be at least a 2-tube (4-fold) change to be considered clinically relevant. Less than a 2-tube (4-fold) difference is considered within the realm of acceptable error in the assay. In other words, if the titer in one series is 16 and the titer in the other is 32, THEY ARE CONSIDERED TO BE THE SAME from a clinical standpoint.
· Paired titers are also useful in determining a patient’s immune status, especially when a specific class (IgG or IgM) is assayed. Knowing the difference between the immune reaction in the primary immune response versus the secondary immune response is required to determine if a patient has a current infection, or merely demonstrating evidence of previous exposure. Useful video: https://youtu.be/u7w7eqziAZU
Evaluate the following test results using acute and convalescent titers for antibodies to Dengue virus:
8. Mary B: Acute IgM = 2; Convalescent IgM = 256
Does Mary have a current Dengue infection? Why or why not?
9. Tom A: Acute IgG = 64; Convalescent IgG = 256
Does Tom have a current Dengue infection? Why or why not?
10. Sylvia P: Acute IgM = 4; Convalescent IgM = 8
Does Sylvia have a current Dengue infection? Why or why not?
The following graph should help you interpret numbers 8-10