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Making a dilution and checking the reading

13 min

  • Calculate the specimen and diluent volumes for a chosen dilution
  • Decide whether a diluted reading falls inside the measuring interval before reporting

Read the full reference

Try first

Try first

You need 500 µL of a 1:10 dilution of serum. Which volumes make it?

The next section explains it.

Right. The next section explains why.

The next section explains it.

The next section explains it.

Get the idea

Plan the two volumes

A dilution starts from two numbers: the factor the procedure asks for and the final volume you want to make. The specimen is one part of that final volume, and the diluent is everything else:

specimen volume = final volume ÷ dilution factor

diluent volume = final volume − specimen volume

For 800 µL of a 1:4 dilution, the specimen is 800 ÷ 4 = 200 µL and the diluent is 800 − 200 = 600 µL.1

The common slip is to pour in the stated final volume as diluent. Putting 200 µL of specimen into 800 µL of diluent makes 1,000 µL at a factor of 5, and a result then multiplied by 4 reports 20% low.

Check the reading against the AMR

The diluted mixture then goes back on the analyzer, and its reading has to be one the method can measure.

  • Every quantitative method has an analytical measurement range (AMR), the span it measures directly, without dilution or other pretreatment. CLSI calls this span the analytical measuring interval.2,3
  • A reading outside it is an estimate. Multiplying an estimate by the factor produces a number the method never measured.
  • The laboratory's dilution scheme, verified against the manufacturer's claims, sets how large a dilution may go. That fixes the highest result the laboratory can report.3

Before multiplying, read the diluted result against the AMR:

Diluted readingWhat you do
Above the upper limitMake a larger dilution the scheme allows and rerun it
Inside the intervalMultiply by the factor and report the result with its dilution
Below the lower limitMake a smaller dilution the scheme allows and rerun it

A reading below the lower limit usually means the dilution was larger than the specimen needed. A larger dilution would push the reading further down, so the next attempt uses a smaller one.3

References
  1. Bishop ML, Fody EP, Van Siclen C, Mistler JM, Moy M. Clinical Chemistry: Principles, Techniques, and Correlations. 9th ed. Jones & Bartlett Learning; 2023.
  2. College of American Pathologists. Analytical Measurement Range. Accessed September 23, 2026. Applies to CAP-accredited laboratories.
  3. Clinical and Laboratory Standards Institute. Establishing and Verifying an Extended Measuring Interval Through Specimen Dilution and Spiking. 1st ed. CLSI guideline EP34. Clinical and Laboratory Standards Institute; 2018. Reaffirmed March 2023. Accessed September 23, 2026. https://clsi.org/media/sj3d0lue/ep34ed1e_reaffirmed_sample.pdf

Watch one

A creatine kinase (CK) reads above the analyzer's AMR of 50 to 1,000 U/L. You make 500 µL of a 1:5 dilution and the mixture reads 1,224 U/L. The laboratory's scheme allows dilutions up to 1:50. What do you report?

  1. Compare 1,224 U/L with the AMR: it is above the 1,000 U/L upper limit.

    The diluted reading decides whether the factor may be applied at all.

  2. Set the 1:5 result aside and plan a larger dilution the scheme allows: 1:10.

    Multiplying a reading outside the AMR would report 6,120 U/L from a number the method never measured.

  3. For 500 µL of a 1:10 dilution, pipette 500 ÷ 10 = 50 µL of serum and 500 − 50 = 450 µL of diluent.

    The specimen is one tenth of the final volume, and the diluent fills the rest.

  4. The 1:10 mixture reads 648 U/L, which is inside 50 to 1,000 U/L.

    Only a reading inside the AMR may be multiplied.

  5. Multiply by the factor of the mixture made: 648 U/L × 10 = 6,480 U/L. The out-of-range 1:5 reading would have reported the specimen too low.

    The analyzer measured a mixture ten times weaker than the specimen.

Report 6,480 U/L from the 1:10 dilution.

Your turn

Problem 1 of 3

You need 1,000 µL of a 1:5 dilution, defined as one specimen part in five total parts. Which volumes prepare it?

Correct. The specimen volume is 1,000 ÷ 5 = 200 µL. The remaining 800 µL is diluent.

Incorrect. These volumes total 1,000 µL, but 1,000 ÷ 250 gives factor 4.

Incorrect. These volumes total 1,200 µL. The factor is 1,200 ÷ 200 = 6.

Hint
  1. Start with the specimen: it is the final volume divided by the factor.
  2. The specimen is 1,000 ÷ 5 = 200 µL.
  3. The diluent is whatever the specimen leaves of the 1,000 µL.

Review Parts and dilution factor

Problem 2 of 3

The procedure asks for a 1:8 dilution, and you want 800 µL of it. How much diluent do you pipette?

Hint
  1. Find the specimen volume first: final volume ÷ factor.
  2. The specimen is 800 ÷ 8 = 100 µL.
  3. The diluent fills the part of the final volume the specimen does not.
Show the answer

700 µL

The specimen is 800 ÷ 8 = 100 µL, so the diluent is 800 − 100 = 700 µL. Adding 800 µL of diluent would make 900 µL at a factor of 9.

Review Parts and dilution factor

Problem 3 of 3

A creatine kinase (CK) reads above its AMR of 50 to 1,000 U/L. A 1:50 dilution is made, and the mixture reads 28 U/L. The scheme allows dilutions from 1:2 to 1:50. What is the next step?

That is 28 × 50, and 28 U/L is below the 50 U/L lower limit, so the method did not measure it reliably. The factor applies only to a reading inside the AMR.

Calculated an exact result from an out-of-range reading

The method measures accurately only inside its analytical measuring interval. A diluted reading above it needs a greater dilution, and one below it needs a smaller dilution or a report stated as below the limit. Multiplying an out-of-range reading by the factor produces a number the method never measured.

The scheme stops at 1:50, and a larger dilution would lower the reading further, to about 14 U/L, still below the lower limit.

The 1:50 mixture was too weak to measure. A 1:2 dilution of a specimen near 1,400 U/L would read about 700 U/L, inside 50 to 1,000 U/L, and that reading can be multiplied by 2.

The dilution scheme exists so that the laboratory can report a number. A smaller dilution inside the allowed range will give one.

Review Measuring intervals

Use it

Try this at the Dilution stationPendingCK · U/L>1000UNDILUTED · ABOVE AMR1:8 to 800 µLDilute specimen

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