The piston, usually in the form of a thumb plunger, is depressed to a stop position on the pipetting device. The tip is placed in the liquid to be measured, and then the plunger is slowly allowed to rise back to the original position (Fig. 8-4). This will fill the tip with the desired volume of liquid. The tips are usually drawn along the inside wall of the vessel from which the measured volume is drawn, so that any adhering liquid is removed from the end of the tip. These pipette tips are not usually wiped, as is done with the manual pipettes, because the plastic surface is considered nonwettable. The tip of the pipette device is then placed against the inside wall of the receiving vessel and the plunger is depressed. When the manufacturer’s directions for the device being used are followed, sample delivery volume is judged to be extremely accurate.

Figure 8-4 Steps in using piston-type automatic micropipette.
A, Attaching proper tip size for range of pipette volume, and twisting tip as it is pushed onto pipette to give an airtight, continuous seal. B, Holding pipette before use. C, Follow instructions for filling and emptying pipette tip. (From Kaplan LA, Pesce A: Clinical chemistry: theory, analysis, correlation, ed 5, St Louis, 2010, Mosby.)

The pipette tips are usually made of disposable plastic, so no cleaning is necessary. Various types of tips are available. Some pipetting devices automatically eject the tip after use. These will also allow the user to insert a new tip and remove the used tip without touching it, minimizing infectious biohazard exposures.

The problems encountered with automatic pipetting depend largely on the nature of the solution to be pipetted. Some reagents cause more bubbles than others and some are more viscous. Bubbles and viscous solutions can cause problems with the measurement and delivery of samples and solutions.

Micropipettors contain or deliver 1 to 500 µL of solution. It is important to follow the individual manufacturer’s instructions for the device being used; each may be slightly different. In general, the following steps apply for use of a micropipettor:

1. Attach the proper tip to the pipettor and set the delivery volume.

2. Depress the piston to a stop position on the pipettor.

3. Place the tip into the solution and allow the piston to rise back slowly to its original position (this fills the pipettor tip with the desired volume of solution).

4. Some tips are wiped with a dry gauze at this step and some are not. Follow the manufacturer’s directions.

5. Place the tip on the wall of the receiving vessel and depress the piston, first to a stop position where the liquid is allowed to drain and then to a second stop position where the full dispensing of the liquid takes place.

6. Dispose of the tip in the waste disposal receptacle. Some pipettors automatically eject the used tips, minimizing biohazard exposure.

Automatic Dispensers or Syringes

Many types of automatic dispensers or syringes are used in the laboratory for repetitively adding multiple doses of the same reagent or diluent. These devices are used for measuring serial amounts of relatively small volumes of the same liquid. The volume to be dispensed is determined by the pipettor setting. Dispensers are available with a variety of volume settings. Some are available as syringes and others as bottle top devices. Most of these dispensers can be cleaned by autoclaving.

Diluter-Dispensers

In automated instruments, diluter-dispensers are used to prepare a number of different samples for analysis. These devices pipette a selected aliquot of sample and diluent into the instrument or receiving vessel. They are primarily of the dual-piston type, with one used for the sample and the other for the diluent or reagent.

Dilutions

It is often necessary to make dilutions of specimens being analyzed or to make weaker solutions from stronger solutions in various laboratory procedures. Clinicians must be able to work with various dilution problems and dilution factors. They often need to determine the concentration of antibody in each solution, the actual amount of material in each solution, and the total volume of each solution. All dilutions are a form of ratio. Dilution is an indication of relative concentration.

Diluting Specimens

In most laboratory determinations, a small sample is taken for analysis and the final result is expressed as concentration per some convenient standard volume. In a certain procedure, 0.5 mL of blood is diluted to a total of 10 mL with various reagents, and 1 mL of this dilution is then analyzed for a particular chemical constituent. The final result is to be expressed in terms of the concentration of that substance per 100 mL of blood.

Dilution Factor

A dilution factor is used to correct for having used a diluted sample in a determination rather than the undiluted sample. The result (answer) using the dilution must be multiplied by the reciprocal of the dilution made. For example, a dilution factor by which all determination answers are multiplied to give the concentration per 100 mL of sample (blood) may be calculated as follows.

First, determine the volume of blood that is actually analyzed in the procedure. Using a simple proportion, it is evident that 0.5 mL of blood diluted to 10 mL is equivalent to 1 mL of blood diluted to 20 mL:

0.5mL blood10mL solution=1mL bloodxmL solution

x=1mL blood×10mL0.5mL=20mL

The concentration of specimen (blood) in each milliliter of solution may be determined by the use of another simple proportion to be 0.05 mL of blood per milliliter of solution:

1mL blood20mL solution=xmL blood1mL solution

x=1mL×1mL20mL=0.05mL

Because 1 mL of the 1:20 dilution of blood is analyzed in the remaining steps of the procedure, 0.05 mL of blood is actually analyzed (1 mL of the dilution used × 0.05 mL/mL = 0.05 mL of blood analyzed).

To relate the concentration of the substance measured in the procedure to the concentration in 100 mL of blood (the units in which the result is to be expressed), another proportion may be used:

100mL(volume of blood desired)0.05mL(volume of blood used)=concentration desiredconcentration usedor determined

Concentration desired=100mL×concentration determined0.05mL

Concentration desired=2000×value determined

The concentration of the substance being measured in the volume of blood actually tested (0.05 mL) must be multiplied by 2000 to report the concentration per 100 mL of blood.

The preceding material may be summarized by the following statement and equations. In reporting results obtained from laboratory determinations, one must first determine the amount of specimen actually analyzed in the procedure and then calculate the factor that will express the concentration in the desired terms of measurement. Thus, in the previous example, the following equations may be used:

0.5mL(volumeofbloodused)10mL(volumeoftotaldilution)=xmL(volumeofbloodanalyzed)1mL(volumeofdilutionused)

x=0.05mL(volumeofbloodactuallyanalyzed)

100mL(volumeofbloodrequiredforexpressionofresult)0.05mL(volumeofbloodactuallyanalyzed)=2000(dilutionfactor)

Single Dilutions

When the concentration of a particular substance in a specimen is too great to be determined accurately, or when there is less specimen available for analysis than the procedure requires, it may be necessary to dilute the original specimen or further dilute the initial dilution (or filtrate). These single dilutions are usually expressed as a ratio, such as 1:2, 1:5, or 1:10, or as a fraction, ½, ⅕, or 110. These ratios or fractions refer to 1 unit of the original specimen diluted to a final volume of 2, 5, or 10 units, respectively. A dilution, therefore, refers to the volume or number of parts of the substance to be diluted in the total volume, or parts, of the final solution. A dilution is an expression of concentration, not volume; it indicates the relative amount of substance in solution. Dilutions can be made singly or in series.

To calculate the concentration of a single dilution, multiply the original concentration by the dilution expressed as a fraction.

Example of Calculation of Concentration of a Single Dilution

A specimen contains 500 mg of substance per deciliter of blood. A 1:5 dilution of this specimen is prepared by volumetrically measuring 1 mL of the specimen and adding 4 mL of diluent. The concentration (C) of substance in the dilution is calculated as follows:

C=500mg/dL×15=100mg/dL

Note that the concentration of the final solution (or dilution) is expressed in the same units as that of the original solution.

To obtain a dilution factor that can be applied to the determination answer and express it as a concentration per standard volume, proceed as follows. Rather than multiply by the dilution expressed as a fraction, multiply the determination value by the reciprocal of the dilution fraction. In the case of a 1:5 dilution, the dilution factor applied to values obtained in the procedure would be 5, because the original specimen was five times more concentrated than the diluted specimen tested in the procedure.

Use of Dilution Factors

A 1:5 dilution of a specimen is prepared and an aliquot (one of a number of equal parts) of the dilution is analyzed for a particular substance. The concentration of the substance (C) in the aliquot is multiplied by 5 to determine its concentration in the original specimen. If the concentration of the dilution is 100 mg/dL, the concentration of the original specimen is:

C=100mg/dL×5(dilutionfactor)=500mg/dLinblood

Serial Dilutions

Dilutions can also be made in series, in which the original solution is further diluted. A general rule for calculating the concentrations of solutions obtained by dilution in series is to multiply the original concentration by the first dilution (expressed as a fraction), this by the second dilution, and so on, until the desired concentration is known.

Several laboratory procedures, especially serologic methods, make use of a dilution series in which all dilutions, including or following the first one, are the same. Such dilutions are referred to as serial dilutions (Table 8-1). A complete dilution series usually contains 5 or 10 tubes, although any single dilution may be made directly from an undiluted specimen or substance. In calculating the dilution or concentration of a substance or serum in each tube of the dilution series, the rules previously discussed apply.

Table 8-1

Example of Preparation of a Serial Dilution

	Tube
	1	2	3	4	5	6	7	8	9	10
Saline (mL)	1	1	1	1	1	1	1	1	1	1
Patient serum or preceding dilution (mL)	1	1 of 1:2	1 of 1:4	1 of 1:8	1 of 1:16	1 of 1:32	1 of 1:64	1 of 1:128	1 of 1:256	1 of 1:512
Final dilution	1:2	1:4	1:8	1:16	1:32	1:64	1:128	1:256	1:512	1:1024

A twofold dilution may be prepared as follows (Fig. 8-5). A serum specimen is diluted 1:2 with buffer. A series of five tubes are prepared, in which each succeeding tube is rediluted 1:2. This is accomplished by placing 1 mL of diluent into each of four tubes (tubes 2 to 5). Tube 1 contains 1 mL of undiluted serum. Tube 2 contains 1 mL of undiluted serum plus 1 mL of diluent, resulting in a 1:2 dilution of serum. A 1-mL portion of the 1:2 dilution of serum is placed in tube 3, resulting in a 1:4 dilution of serum (½ × ½ × ¼). A 1-mL portion of the 1:4 dilution from tube 3 is placed in tube 4, resulting in a 1:8 dilution (¼ × ½ × ). Finally, 1 mL of the 1:8 dilution from tube 4 is added to tube 5, resulting in a 1:16 dilution ( × ½ × 116). One milliliter of the final dilution is discarded so that the volumes in all the tubes are equal.

Figure 8-5 Schematic of a twofold serial dilution. (From Turgeon ML: Linné & Ringsrud’s clinical laboratory science: the basics and routine techniques, ed 6, St Louis, 2012, Mosby, p. 166.)

Note that each tube is diluted twice as much as the previous tube, and that the final volume in each tube is the same. The undiluted serum may also be given a dilution value, 1:1.

The concentration of serum in terms of milliliters in each tube is calculated by multiplying the previous concentration (mL) by the succeeding dilution. In this example, tube 1 contains 1 mL of serum, tube 2 contains 1 mL × ½ × 0.5 mL of serum, and tubes 3 to 5 contain 0.25, 0.125, and 0.06 mL of serum, respectively.

Other serial dilutions might be fivefold or tenfold; that is, each succeeding tube is diluted 5 or 10 times. A fivefold series would begin with 1 mL of serum in 4 mL of diluent and a total volume of 5 mL in each tube. A tenfold series would begin with 1 mL of serum in 9 mL of diluent and a total volume of 10 mL in each tube. Other systems might begin with a 1:2 dilution and then dilute five succeeding tubes 1:10. The dilutions in such a series would be 1:2, 1:20 (½ × 110 × 120), 1:200 (120 × 110 × 1200), 1:2000, 1:20,000, and 1:200,000.

Antibody Testing

In obtaining specimens for serologic testing, it is important to consider the phase of the disease and the condition of the patient at the time of specimen collection. This is especially important in assays for diagnosis of infectious diseases. If serum is being tested for antibody levels with a specific infectious organism, generally the blood should be drawn during the acute phase of the illness—when the disease is first discovered or suspected—and another sample drawn during the convalescent phase, usually about 2 weeks later. Accordingly, these samples are called acute and convalescent serum. A difference in the amount of antibody present, or the antibody titer, may be noted when the two different samples are tested concurrently. Some infections, such as Legionnaires’ disease or hepatitis, may not manifest a rise in titer until months after the acute infection.

Antibody Titer

A central concept of serologic testing is the manifestation of a rise in titer, or concentration, of an antibody. The antibody titer is defined as the reciprocal of the highest dilution of the patient’s serum in which the antibody is still detectable. That is, the titer is read at the highest dilution of serum that gives a positive reaction with the antigen. If a serum sample has been diluted 1:64 and reacts positively with the antigen suspension used in the testing process, and if the next highest dilution of 1:128 does not give a positive reaction, the titer is read as 64. A high titer indicates that there is a relatively high concentration of the antibody present in the serum.

Determination of the concentration of antibody (titer) for a specific antigen involves the following two steps:

1. Preparing a serial dilution of the antibody-containing solution (e.g., serum)

2. Adding an equal volume of antigen suspension to each dilution

A high titer indicates that a considerable amount of antibody is present in the serum. For most pathogenic infections, an increase in the patient’s titer of two doubling dilutions, or from a positive result of 1:8 to a positive result of 1:32 over several weeks, is an indication of a current infection. This is known as a fourfold rise in the antibody titer.

CASE STUDY

JJ, a 9-year-old boy, was taken to the emergency department with a sore throat. On examination, he had redness of the throat and slightly swollen glands. The physician assistant ordered a throat culture and blood drawn for an antistreptolysin-O antibody (ASO). An antibiotic was prescribed for a 10-day period. His mother was told to make an appointment with his pediatrician for a follow-up.

At the follow-up visit 2 weeks later, the results of the laboratory test revealed a throat culture with a few colonies of β-streptococci. The qualitative ASO test result was reported as positive. The acute serum was frozen at the time of testing. The pediatrician ordered a convalescent specimen to be tested semiquantitatively in parallel with the acute specimen for an ASO titer.

The acute and convalescent specimens were prepared as twofold serial dilutions of each specimen (see table).

	Tube
	1	2	3	4	5	6
Saline (µL)	—	50	50	50	50	50
Serum (µL)	50	50	50 (1:2)	50 (1:4)	50	50
Mix and transfer to next tube		50	50	50	50	50
Dilution/titer	1:1	1:2	1:4	1:8	1:16	1:32
IU/mL	200	4008	800	1600	3200	6400