Semiautomatic Pipetting of Ultramicro Volumes of ...

Vol. 4S, No. 5 Printed in U.S.A.

THE AMERICAN JOURNAL OF CLINICAL PATHOLOGY

Copyright © 1967 by The Williams & Wilkins Co.

SEMIAUTOMATIC PIPETTING OF ULTRAMICRO VOLUMES OF SAMPLE AND REAGENT

OLE SIGGAARD-ANDERSEN, M.D., AND BRIAN S. BULL, M.D. Division of Clinical Pathology and Laboratory Medicine, and the Research and Development Laboratory University of California, San Francisco Medical Center, San Francisco, California; and Clinical Pathology Department, Clinical Center, National Institutes of Health, Bethesda, Maryland

DESIGN OF THE ULTRAMICRO PIPETTE AND DILUTOR The ultramicro dilutor consists of a gastight Hamilton* syringe provided with a Received December 12, 1966. Dr. Bull's present address is Department of Hematology, Postgraduate Medical School of London, Ducane Road, London, W.12, England. * Available from Hamilton Co., Inc., Whittier, California.

special syringe attachment which allows accurate and reproducible positioning of the plunger of the syringe. Gas-tight Hamilton syringes with Teflontipped plungers are available in volumes of 50 nl through 100, 250, and 500 nl, all with the same outer diameter of the barrel (0.300 to 0.310 in.). All parts, including the Teflon tips, are exchangeable. After prolonged use the syringe may begin to leak, but this is usually easily remedied by spinning out the edges of the Teflon tip with a nail. The barrel of the syringe is fixed in an aluminum holder (Fig. 1). The steel plunger of the syringe, which is quite fragile in the small syringes, is elongated by a sturdy stainless steel rod. The thumb end of the plunger is cut off and the end of the plunger is turned down to 0.05S in. o.d. for a length of Yi in. (the plunger of the 50-LII. and 100-/ul. syringes does not need to be turned down). A polyethylene tube of 0.023 in. i.d. and 0.038 in. o.d. (Clay Adams, New York City, P E 50) usually makes an airtight fit when it is pushed into the tip of the syringe. The polyethylene tube is heated gently and pulled to a fine tip suitable for the volumes in question. The finer the tip, the more reproducible the surface at the end of the tip, and thus less fluid adheres to the outer surface. The tube should be long enough to contain three times the volume of sample to be drawn up into the tip. This prevents any of the sample from entering the barrel of the syringe. The tube should be long enough (10 to 15 in.) to reach into all of the test tubes in the test tube rack. I t is easier to bring the tip into the tubes than to bring the tubes to the tip. No change in volume is observed when the polyethylene tip is moved from one position to the other. A small piece of adhesive tape is conveniently attached to the polyethylene tube at a given distance from

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Several authors have described ultramicro methods for the clinical chemistry laboratory and some ingenious technics have been developed.12 Conversely, other workers have discredited ultramicro procedures for routine laboratory use because of the deficiencies of presently available ultramicro pipettes. 6 An improved ultramicro pipette should not only be less susceptible to operator error but also perform adequately with ultramicro methods that require only 5 to 10 pi. of sample. The claim has been made that methods using 50 to 100 ii\. per test are adequate for ordinary clinical uses,10 but every drop of cutaneous blood is difficult to obtain, particularly if the peripheral circulation is poor. Frequently, a battery of tests is wanted and if the amount of blood needed exceeds that which is readily obtained from a single puncture wound there is the temptation to squeeze out more blood, contaminating it with tissue juice and invalidating the entire set of test results. In a previous communication, an ultramicro dilutor for pipetting sample volumes of 5 to 10 /*l.,s was described. This pipette has now been in use for almost 1 year, during which time it has undergone several modifications. I t is the purpose of this paper to describe the design of this pipette and its applications in micro chemistry in greater detail.

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PIPETTING ULTRAMICRO SAMPLES AND REAGENTS

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C D

D

o£o t r

fFiG. 2. Details of the ultramicro dilutor. For explanation of symbols, see Figure 1.

FIG. 1. Ultramicro pipette and dilutor. An airtight Hamilton syringe is fixed in an aluminum holder (C) by means of a nylon setscrew. The plunger of the syringe (B) is elongated by a sturdy stainless steel rod (E), sliding in a Teflon bushing (0) inside an aluminum holder (£>). The plunger may slide through the Teflon bushing, as well as rotating between two stainless steel side arms (F and G). By means of set collars on the plunger (L) and the side arms (M and N), several well defined positions of the plunger may be obtained. The ultramicro dilutor is fixed horizontally to a laboratory stand by means of a holder (H). A polyethylene tube (A) tapered to a fine tip is attached to the tip of the syringe. The syringe and the polyethylene tube are filled completely with diluent and the set collar

the tip to serve as a holder and to indicate how far the tip should be inserted into the test tubes. The syringe attachment* is easily machined in a laboratory workshop with a reasonably good lathe and drill press (Figs. 1 to 3). The front piece and end piece (C and D) are machined from a 1-in. aluminum rod, the pieces K, L, M, andiV from a H-in. aluminum rod. The side bars, F and G (and T, L is set against the upper edge of the set collar N. A given volume of sample is sucked into the polyethylene tip by pulling the plunger (L) from N to D. A given total volume of sample plus diluent is expelled when the plunger is rotated to the other side arm and pushed to the stop at M.


Q:

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reagent -•-sample

I}

reagent _f sample

• diluent

K>-> FIG. 3. Sample plus reagent dilutor. By means of two set collars on the same side arm, fluid can be sucked up in two steps. The sample is sucked up when the plunger is pulled from N to X. Reagent is sucked up when the plunger is rotated and pulled from X to D. Sample plus reagent plus diluent are delivered when the plunger is pushed from D to M. The dilutor is refilled with diluent, and the plunger (L) is reset to the starting position.

aluminum plate (R) and a 3^6"U1- aluminum sheet (S). The Teflon bushing is made from a 0.75-in. Teflon rod. The springs are 0.04in. piano wire. The Tygon tubing (AutoAnalyzer tubing) has approximately 0.1-in. o.d. and 0.020-in. i.d. for reagent volumes of 30 to 60 n\. For smaller volumes tubing with smaller inner diameters should be used. The reproducibility can be checked by pulling air into the completely water-filled pipette. When the air is expelled, the meniscus should return exactly to the tip of the pipette. The slightest deviation from this is easily observed when a very fine threadlike polyethylene tip is used. OPERATING PROCEDURE The syringe is filled completely with the diluent. This is accomplished by means of a fine polyethylene tube reaching all the way into the lumen of the syringe (Clay Adams, PE 10). The air in the syringe may also be expelled by slowly filling the syringe with diluent, emptying it rapidly with the tip upward, and repeating this procedure several times. The completely filled polyethylene tip is attached inside of the tip of the full syringe. No air should appear inside the syringe when it is correctly used, i.e., when it is filled slowly to avoid degassing the fluids by vacuum. When it is used as an ultramicro dilutor the set collar (L) on the plunger is set against the upper edge of the set collar (N) on the side arm (Fig. 1). The polyethylene tip is dipped into the sample and a given volume of sample is sucked into the tip by pulling the plunger from set collar, N, to the end piece, D. The polyethylene tip is then placed in the test tube and the sample plus diluent is expelled by rotating the plunger and pushing it all the way to the set collar, M. The polyethylene tip is then dipped in the diluent and the plunger is reset to the starting position. The syringe is refilled with diluent in the process. If, by mistake, the syringe is refilled with anything except the diluent, the syringe and polyethylene tip must be carefully rinsed and refilled with pure diluent. When the tip of the polyethylene tube is fine and it is not dipped too far into the sample or diluent, adherence of fluid to the outer surface is minimal and no


Fig. 4), are 3ir m - stainless steel rod, 5 in. in length. The Teflon bushing is machined from a %-h\. Teflon rod. The bushing must not project outside the aluminum holder (D). It is essential that the three holes in the front piece and end piece (C and D) for side bars, syringe, and plunger be exactly parallel. The plunger (2?) must be exactly in line with the syringe. All screws should be interchangeable, for example, size 3-56. The reagent pipette consists of the diluting syringe plus a valve attachment that requires no operation other than moving the plunger of the syringe. This could have been accomplished by two unidirectional flow valves, but as the available flow valves seemed to present leakage problems, it was decided to utilize the simple and reliable principle of clamping tubes. The valve attachment (Fig. 4) is machined from a ]/i-m..

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to delivery tip

bottle

FIG. 4. Valve attachment for reagent pipette. A polyethylene tubing inserted into the tip of the syringe connects to a T tube. Lengths of polyethylene tubing lead from the two arms of the T tube to the reagent bottle and a delivery tip, respectively. Each polyethylene tubing is divided and the cut ends are inserted into a small piece of pliable Tygon tubing (P), which may be closed by an aluminum wedge fastened to a metal spring (Q). The metal spring is not shown in side projection. The tubes and springs are attached to an aluminum plate (R), which is fixed to the end piece (D) of the ultraniicro pipette by means of a set screw; D is shown clotted in the side projection but not in the end projection. The tubes (7-'), normally closed, are opened when the lever (S), revolving, on a Teflon bushing on the end piece (D), pushes the spring back. The side arm (2') slides within a slit cut into the lever (S) so that rotation of the plunger rotates the lever to open either Tygon tubing (P). This can be accomplished in any position of the plunger, so that the Tygon tubing can be opened at either end of the stroke. No position allows both tubes to be open simultaneously. Filling and emptying of the syringe and opening and closing of the valves are all achieved by operating the plunger of the syringe. wiping of the tip is necessary. The tip may be easily washed on the outside alter the sample is drawn up by being dipped into a vial containing diluent. The absolute volume of sample aspirated and of sample plus diluent delivered is determined by the position of the set collars, M and N, in relation to the end piece (D). The volumes may be approximately adjusted by using the divisions on the Hamilton syringe. For a more accurate adjustment a micro balance is preferable. The absolute volumes are seldom important because standards most frequently are used for calculation of the unknowns. For an accurate adjustment of the dilution factor a caliper may be used to adjust the distance traveled by the plunger on the long side arm in order to make it exactly as long as the distance traveled on the short side arm multiplied by the dilution factor. Once the volumes and dilution factor are fixed, the front piece (C) with the syringe may be detached and put back into position without affecting the adjustment of the micro dilutor.

To ensure complete washout of the sample from the tip, the dilution factor should not be less than five. To ensure a reasonable distance on the short side arm (between N and D) a dilution factor of more than 100 should not be attempted. If large dilutions are necessary, two syringes should be used. In our laboratory the ultraniicro dilutor is used as described for the following tests. Sodium and potassium (Instrumentation Laboratories flame photometer). The diluent is a lithium solution, the sample volume is approximately 7.5 M'-, and the dilution factor is 10. An additional 1.5 ml. of lithium diluent is delivered from another syringe, giving a final dilution factor of 200. The dilution is made in a disposable AutoAnalyzer cup. The coefficient of variation on 30 consecutive determinations of a serum pool (Na 139 mEq./liter, K 4.9 mEq./liter) was 0.16 % for sodium, 1.0% for potassium. Each dilution was measured twice on the flame photometer and the average reading was used for the calculation.


reagent

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Chloride (coulometric titration with the kom10). The diluent is water. Sample volCotlove chloride titrator). The diluent is ume and reagent volume (concentrated subwater, the sample volume is approximately strate-buffer mixture) are each 10 /ul., and a 10 ttl., and the dilution factor is five. Up to total volume of 100 til. is delivered into a 20 samples may be successively titrated in micro polyethylene tube for incubation at the same vial. The coefficient of variation 37 C. The coefficient of variation on 30 when a serum sample was titrated (102.5 determinations on a serum sample (1.4 U / mM/liter) 20 times was 0.15 %.13 liter) was 2.4%. Glucose (hexokinase method, Calbiochem When used as an ultramicro pipette withreagents). The diluent is water, the sample out dilution of the sample, the syringe is volume is approximately 2 ttl., and the dilu- filled with water and the plunger (set collar tion factor is 10. The sample is delivered L) is set a little beyond the set collar M on into a micro polyethylene test tube contain- the side arm. Air is then sucked into the ing a predispensed (frozen) 130-td. volume polyethylene tip and the plunger is set to the of adenosinetriphosphatase, hexokinase, glu- upper edge of set collar M. The sample is cose-6P-dehydrogenase, reduced adenosine- sucked up by pulling the plunger from M to diphosphatase reagent. The optical density D, and delivered into the test tube by pushat 340 run. is read on the Gilford 300 spectro- ing the plunger back to M. The air interphotometer provided with a 5-mm. Beck- phase prevents mixture of the sample with man/Spinco micro cuvette and a special the water. The volume of air should be as cuvette-filling device.14 The coefficient of small as possible, (i.e., 10 to 20% of the variation calculated from 30 determinations" sample volume) so that small changes in on a serum pool (10.6 miVI/liter) was 0.S %. the volume of the air due to varying surface Alkaline -phosphatase (method of Babson1 tensions or temperature changes represent using phenolphthalein monophosphate sub- a small fraction of the sample volume. The strate). The diluent is water, the sample syringe should be filled and emptied slowly volume is approximately 4 id., and the dilu- to allow the fluid to drain off the sides of the tion factor is 10. The coefficient of variation polyethylene tube because there is no washon 30 determinations on a serum pool (133 ing with this method of pipetting. Between samples the air may be expelled and the U/liter) was 1.4%. Spinal fluid protein (turbidometry accord- pipette may be washed by sucking water ing to Henry). 9 The diluent is 31 Gm./ back and forth several times. The following liter of trichloracetic acid, the sample vol- are examples of this application of the piume is approximately 30 til., and the dilution pette. factor is five. The coefficient of variation on Serum bilirubin (method of Michaelsson 30 measurements on a serum dilution (2.1 and associates11)- The pipette is set for 30 Gm./liter) was 1.5%. id. on one side arm (F) and 6 jul. on the other In some instances it is an advantage to (Fig. 1). For slightly icteric sera, 30 tn\ are suck up sample plus reagent and deliver pipetted without dilution. For more icteric both into the test tube. This ensures good sera (bilirubin over 170 tiM/liter) 6 jul. of mixing of sample and reagent. In this case serum are used and diluted 5 times. The two set collars are used on one side arm to coefficient of variation from 30 determinaallow fluid to be drawn up in two steps tions on a sample with 77 tiM/liter was (Fig. 3). Examples of this application follow. 1.8%. Serum phosphorus (method of Dryer and Serum urea (Berthelot reaction, Chaney and Marbach 6 ). The diluent is water. Sample colleagues7). The pipette is set for 50 ix\. on volume and reagent volume (concentrated one side arm, 10 til. on the other. The serum urease solution) are each 5 til. A total volume is pipetted, with 10 id- and a 5-fold diluof 50 til. is delivered. The coefficient of tion used. The supernatant from the trivariation on 30 determinations on a serum chloracetic acid precipitation is pipetted with 50 /xl. used without dilution. The copool (S.5 mM/liter) was 1.5%. Acid phosphatase (method of Van Gor- efficient of variation calculated from 30 de-

Nov. 1967


DISCUSSION Several micro pipettes of different design are available; they include a tube to be filled to a mark, a tube to be filled to a constriction (Linderstrom-Lang-Levy type), and a tube to be filled completely (Sanz type). The tube may be made from glass or plastics, and may be filled by mouth suction, artificial suction, or capillary action. In all cases the sample is taken into a tube which originally contains air, and visual control of the filling operation is necessary because of the risk that air bubbles will remain in the tube. In these instances the volume of sample is defined by the volume of the tube containing the sample. The volume sample may also be defined by a syringe, i.e., not the tube in which the sample is contained. The syringe may be filled with air or with a fluid. An air-filled

syringe equipped with a plastic tip may be used to pipette and deliver the sample.2 The Eppendorf ultra-micro pipette is based on this principle and uses disposable plastic tips to prevent carryover. This principle is simple and rapid for pipetting samples when no dilution can be allowed (or in cases where the dilution factor must be less than five). Unfortunately, the reproducibility does not match the reproducibility of devices utilizing the dilution principle, as a result of variations in the volume of the air interphase. In the design described here, where water is used in the syringe and the air interphase is small, this source of variation is minimized. Bull3 has described a special microliter syringe with a side inlet that functions as a one-way valve when the Teflon tip of the plunger slides by the inlet. In procedures where a high dilution factor is desired, the diluent can be admitted through this side inlet, thus eliminating the air space and providing a rinse at the same time. If the diluent is metered by a motor-driven syringe, a semiautomatic dilutor results. This device is useful when a specific dilution with the same diluent must be performed many times, but the additional convenience is obtained at added expense. If no dilution is required, the sample may be sucked into the syringe by vacuum; a highly accurate and efficient way of pipetting samples results. This method, however, requires an excess of sample to fill the syringe and to rinse, and is thus not usually applicable to sample pipetting in microchemistry. although it is ideally suited to the handling of venous blood samples in hematology.4 Feichtmeir and co-workers, in 1961, described the use of the dilution principle for pipetting of samples.8 Since the appearance of their Autodilutor, many variations of this principle have been utilized commercially. The commercial dilutors are generally designed for sample volumes of 20 to 500 /A. and dilutions of up to 500 times. The present dilutor is primarily designed for sample volumes of less than 10 fii. and dilution factors of 5 to 20, although larger volumes may also be used, depending on the size of the syringe. The dilution principle is considered to be the


terminations on a serum pool (2.4 mM/liter) was 2.0%. For the dispensing of reagents directly from the reagent bottle, the end piece (D) is provided with a valve attachment (Fig. 4). The syringe is filled with reagent, with the plunger rotated to the side which opens the tube leading to the reagent bottle. When the plunger is rotated to the other side, first the tube leading to the reagent bottle closes, and by further rotation the tube leading to the delivery tip opens. The decompression of the Tygon tube causes a small but reproducible amount of air to be sucked into the polyethylene tip. The reagent is delivered by pushing the plunger all the way down. The last portion of the reagent is delivered when the plunger is rotated and the Tygon tube is again compressed, i.e., the delivery operation must include the rotation step and closing of the tubing. The amount of fluid displaced by compression of the Tygon tubing should be kept as small as possible by using small-bore Tygon tubing and sharp occlusion. The volume delivered is determined by the position of the holder for the side arm (T) on the plunger (E). The coefficient of variation during the pipetting of 10.2 p\. of mineral oil 30 times onto the pan of the Cahn Gram Electrobalance was 0.18%.

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and easily attached to the sample dilutor and pipette. SUMMARY An airtight syringe is provided with an attachment which allows an accurate and highly reproducible setting of the plunger of the syringe in several positions. The syringe is filled with water or diluent. A given volume of sample or reagent or both is sucked into a polyethylene tip. The most accurate pipetting is achieved when the syringe is used as an ultramicro dilutor and the aspirated sample or reagent is washed out with at least four times its volume of diluent. Sample plus several reagents may be pipetted and diluted in one step. The coefficient of variation during pipetting of 7.5 /*1. of serum is 0.16% or less. When no dilution is wanted, mixing between the water in the syringe and the sample is prevented by a small air interphase. A valve attachment allowing the pipetting of reagent directly from the reagent bottle is described. REFERENCES 1. Babson, A. L.: Phenolphthalein monophosphate, a new substrate for alkaline phosphatase. Clin. Chem., 11: 789, 1965. 2. Brun, G. C : Personal communication, 1960. 3. Bull, B. S.: A semiautomatic micro sample dilutor. Am. J. Clin. Path. (Tech. Sect.), 47: 545-548, 1967. 4. Bull, B. S., and Siggaard-Andersen, O.: The hem-aliquanter: a dispenser dilutor for hematologic tests. Am. J. Clin. Path., to be published. 5. Chaney, A. L., and Marbach, E. P.: Modified reagents for determination of urea and ammonia. Clin. Chem., 8: 130-132, 1962. 6. Clayton, B. E., and Jenkins, P.: Micro methods and micro apparatus for chemical pathology with special reference to paediatrics. J. Clin. Path., 19: 293-297, 1966. 7. Dryer, R. L., Tammes, A. R., and Routh, J. I.: The determination of phosphorus and phosphatase with Ar-phenyl-p-phenylenediamine. J. Biol. Chem., 825: 177-183, 1957. 8. Foichtmeir, T. V., Jenkins, K. D., and Baer, D. M.: A device to pipet and dilute fluid semi-automatically. Am. J. Clin. Path., 35: 378-382, 1961. 9. Henry, R. J.: Clinical Chemistry. Principles and Technics. New York: Harper and Row, 1964, pp. 186-189. 10. Mabry, C. C, Gevedon, R. E., Roekel, I. E., and Gochman, N.: Automated submicro chemistries. A system of rapid submicro chemical analysis for the measurement, of sodium, potassium, chloride, carbon dioxide, sugar, urea nitrogen, total and direct-reacting bilirubin, and total protein. Am. J. Clin. Path., 46: 265-282, 1966. 11. Michaelsson, M., Nosslin, B., and Sjolin, S.:


most reproducible and efficient way of pipetting ultramicro samples. No visual control of the filling of the pipette is necessary, and carryover is eliminated by the complete washout of the sample from the pipette. The reproducibility for the ultramicro tests mentioned here (Na, K, CI, CSFprotein, glucose, basic and acid phosphatase, urea, phosphorous, and bilirubin) corresponds closely to the reproducibility of the same macro procedures. For determination of the reproducibility of each test, samples giving optical densities between 0.3 and 0.6 were selected. Chloride and sodium were determined with the smallest coefficient of variation (0.15 and 0.16%, respectively). The greater coefficient of variation found with the other tests indicates that the major contribution to the variability does not lie in the pipetting step but in the sensitivity of the measuring device (potassium) or the chemical reaction (glucose, cerebrospinal fluid protein). The serial pipetting of a reagent directly from the reagent bottle is desirable for several reasons. Under these circumstances there is no reagent adherent to the outside of the pipette, no waste of reagent, no need for reagent beakers, less possibility of using the wrong reagent, and, most important, there is increased speed of operation. A number of commercial reagent dispensers utilizing syringes and unidirectional flow valves are available. When gravity is used as the force to close the valve, the efficiency of the valve becomes dependent on the specific gravity of the reagent, and for very heavy reagents the valve may float instead of sink. Furthermore, the pipette cannot be provided with a long tip to reach into the test tubes without a risk of leakage due to hydrostatic pressure. A reagent dispenser for ultramicro quantities with a spring-loaded unidirectional flow valve has recently been announced (Microchemical Specialties Inc., Berkeley, California). It may well prove to be the ideal reagent dispenser for ultramicro volumes, but it has not yet become commercially available. The advantage of the present design is that leaks in the valves can be efficiently prevented, and that it can be manufactured in the laboratory workshop

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Plasma bilirubin determination in the newborn infant. A methodological study with special reference to the influence of hemolysis. Pediatrics, 85: 925-931, 1965. 12. Sanz,M. C: Ultramicro methods and standardization of equipment. Clin. Chem., 8: 406419, 1957. 13. Siggaard-Andersen, 0.: A simple semiautomatic ultramicro pipet. Scandinav. J. Clin. Lab. Invest., 18: 666-667, 1966. 14. Siggaard-Andersen, 0.: Laboratory sugges-

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tion: Alternative way of filling the ultramicro cuvet of the Beckman/Spinco spectrocolorimeter. Am. J. Clin. Path., 47: 243-245, 1967. 15. Siggaard-Andersen, 0.: Ultramicro determination of chloride in biologic fluids with the Cotlove chloridetitrator. Am. J. Clin. Path., 45-444-446, 1967. 16. Van Gorkom, 0. W. H.: De bepaling van prostaat zure fosfatase in serum. Ned. Tdschr. Geneesk., 106: 297-301, 1962.
