Serial DilutionsSerial Dilutions

MLAB 1335 MLAB 1335 Immunology/SerologyImmunology/Serology

Terry KotrlaTerry Kotrla

IntroductionIntroduction

Many of the laboratory procedures involve the use of dilutions.

It is important to understand the concept of dilutions, since they are a handy tool used throughout all areas of the clinical laboratory.

These dilutions have to be considered as they make a quantitative difference in what is going on.

Serial DilutionsSerial Dilutions

A serial dilution is any dilution where the concentration decreases by the same quantity in each successive step.

Serial dilutions are mutiplicative.

What Does This What Does This Mean??Mean?? If a solution has a 1/10 dilution

the number represents 1 part of the patient sample added to 9 parts of diluent.

So the volumes used would be 10-1= 9.

This represents 1 part patient sample added to 9 parts of diluent.

DilutionsDilutions

If a 1/8 dilution of the stock solution is made followed by a 1/6 dilution what is the final dilution.

The final dilution is: 1/8 x 1/6 = 1/48

These type of dilutions are trickier These type of dilutions are trickier and not used very frequently in and not used very frequently in the clinical lab.the clinical lab.

Doubling DilutionsDoubling Dilutions

“Doubling dilutions” are very popular.

This is a series of ½ dilutions. Each successive tube will ½ the amount of the original concentrated solution.

If this is done 6 times this is what you would end up with:

Doubling Dilution 6 Doubling Dilution 6 TimesTimes 1st dilution = 1 /2 2nd dilution = 1 /2 x 1 /2 = 1/4 3rd dilution = 1/4 x 1 /2 = 1/8 4th dilution = 1/8 x 1 /2 = 1/16 5th dilution = 1/16 x 1 /2 - 1/32 6th dilution = 1/32 x 1 /2 = 1/64 This results in a series of dilutions, each

a doubling dilution of the previous one

Dilution FactorDilution Factor

The dilution factor is the final uses the formula volume/aliquot volume.

EXAMPLE: What is the dilution factor if you add 0.1 mL aliquot of a specimen to 9.9 mL of diluent?– The final volume is equal to the aliquot

volume PLUS the diluent volume: 0.1 mL + 9.9 mL = 10 mL

– The dilution factor is equal to the final volume divided by the aliquot volume: 10 mL/0.1 mL = 1:100 dilution

PracticePractice

Problem: What is the dilution factor when 0.2 mL is added to 3.8 mL diluent?

Set Up The ProblemSet Up The Problem

dilution factor = final volume/aliquot volume

0.2 +3.8 = 4.0 total volume 4.0/0.2 = 1:20 dilution

Problem ContinuedProblem Continued

Remember that serial dilutions are always made by taking a set quantity of the initial dilution and adding it successively to tubes with the same volume.

So each successive dilution would be multiplied by the dilution factor.

Problem ContinuedProblem Continued

So in the above problem all successive tubes would have 3.8 mLs of diluent.

You would then transfer 0.2 of the initial diluted sample into the next tube, mix transfer 0.2, mix and so on.

If you had 4 tubes what would be the final dilution of tube 4?

Solving the Problem - Solving the Problem - *Calculate DF of tube 1*Calculate DF of tube 1

TubeTube 11 22 33 44

AliquotAliquot 0.20.2 0.20.2 0.20.2 0.20.2

DiluenDiluentt

3.8 3.8 3.8 3.8

MathMath *4/0.*4/0.22

1/20x1/201/20x1/20 1/400x1/21/400x1/200

1/8000x1/201/8000x1/20

DilutioDilutionn

1:201:20 1:4001:400 1:80001:8000 1:160,0001:160,000

Solving the ProblemSolving the Problem

Or if you simply wanted to know Or if you simply wanted to know the dilution of the final tube you the dilution of the final tube you could just multiply them together:could just multiply them together:

1/20 x 1/20 x 1/20 x 1/20 = 1:160,000

NextNext

What if we added the indicator system What if we added the indicator system to the test system?to the test system?

You must create additional rows, if you You must create additional rows, if you are showing your work, to take into are showing your work, to take into consideration the dilutional effect that consideration the dilutional effect that will have on the dilution.will have on the dilution.

When an indicator is added it is When an indicator is added it is CRITICAL to remove an aliquot from CRITICAL to remove an aliquot from the last tube.the last tube.

Adding Indicator CellsAdding Indicator Cells

TubeTube 11 22 33 44

DilutioDilutionn

1:201:20 1:4001:400 1:80001:8000 1:160,0001:160,000

RBCsRBCs 0.20.2 0.20.2 0.20.2 0.20.2

VolumVolumee

3.8 3.8 3.8 3.8

DFDF 4/14/1

MathMath 4x204x20 4x4004x400 4x80004x8000 4x160,0004x160,000

DilutioDilutionn

1:801:80 1:8001:800 1:32,0001:32,000 1:640,0001:640,000

Serial DilutionsSerial Dilutions

When performing serial dilutions in the clinical laboratory one must know the dilution of each tube so a chart is always created to indicate 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 to create your chart.

TitersTiters

TITERS are reported out as the reciprocal of the last tube giving a positive reaction.

So if tube 2 was the endpoint, the dilution is 1:800 the titer is reported out as 800/1= 800.

Other ApplicationsOther Applications

Sometimes when analyzing a Sometimes when analyzing a blood sample the results are blood sample the results are above the linearity (or accuracy) above the linearity (or accuracy) of the machine.of the machine.

When this happens the blood When this happens the blood must be diluted, retested, then must be diluted, retested, then the result multiplied by the the result multiplied by the dilution factor.dilution factor.

ExampleExample

A blood glucose of 800 mg/dL was A blood glucose of 800 mg/dL was obtained. obtained.

According to the manufacturer According to the manufacturer the highest glucose result which the highest glucose result which can be obtained on this particular can be obtained on this particular instrument is 500 mg/dL.instrument is 500 mg/dL.

The sample must be diluted.The sample must be diluted.

ExampleExample

The serum was diluted 1:10 and The serum was diluted 1:10 and retested.retested.

The result is 80 mg/dL.The result is 80 mg/dL. THIS IS NOT THE REPORTALBE THIS IS NOT THE REPORTALBE

RESULT!RESULT! You must multiply by the dilution You must multiply by the dilution

factor of 10.factor of 10. 10 x 80 = 800 mg/dL10 x 80 = 800 mg/dL