+ All Categories
Home > Documents > Glucosa Antigua

Glucosa Antigua

Date post: 09-Dec-2015
Category:
Upload: felipe-munos
View: 246 times
Download: 1 times
Share this document with a friend
Description:
historia de determinacion de glucosa en sus inicios, incluyendo reactivos y procedimiento
15
Rapid System of Microchemical Analysis for the Clinical Laboratory Abraham Saifer, Shirley Gerstenfeld, and Michael C. Zymaris THE CLINICAL CHEMIST 111 charge of a routine biochemistry laboratory appears to be faced with an insoluble dilemma. At a time when there is an increasing demand from the medical profession for more bio- chemical tests per patient, which would seem to require the use of microprocedures, he finds it almost impossible to obtain personnel with the requisite technical skill. One way out of this dilemma would appear to be the use of completely automatic technics such as the autoanalyzer (1). The use of this kind of automation would solve the problem of performing the more frequent determinations, such as blood glucose, urea nitrogen, etc., in the larger laboratories. Because of their present high unit cost, their utilization in the smaller labora- tories is not economically feasible. In addition, automation does not solve the problem of increasing the efficiency of performance of the other less frequently requested biochemical tests. It is the purpose of this paper to present a system of analysis for the clinical laboratory which would permit the employment of microchemical procedures on a semiautomatic basis. This system is composed of five basic ele- ments: 1. The use of siliconated-heparinized plasma (2) in place of serum or whole blood, so that determinations may be started within a few minutes after the sample reaches the laboratory. 2. The use of the calibrated pipet tip-buret technic for measuring a constant volume of each plasma sample and of standards and blanks (3, 4). This enables measurements to be made on 0.20-nil. or less of fluid by personnel with little technical skill. From the Biochemistry Department, Isaac Albert Research Institute of the Jewish Chronic Disease Hospital, Brooklyn, N. Y. Received for publication Dec. 22, 1957. 127
Transcript
Page 1: Glucosa Antigua

Rapid System of Microchemical Analysis

for the Clinical Laboratory

Abraham Saifer, Shirley Gerstenfeld, and Michael C. Zymaris

THE CLINICAL CHEMIST 111 charge of a routine biochemistry laboratoryappears to be faced with an insoluble dilemma. At a time when thereis an increasing demand from the medical profession for more bio-chemical tests per patient, which would seem to require the use ofmicroprocedures, he finds it almost impossible to obtain personnelwith the requisite technical skill. One way out of this dilemma wouldappear to be the use of completely automatic technics such as theautoanalyzer (1). The use of this kind of automation would solve theproblem of performing the more frequent determinations, such asblood glucose, urea nitrogen, etc., in the larger laboratories. Becauseof their present high unit cost, their utilization in the smaller labora-tories is not economically feasible. In addition, automation does notsolve the problem of increasing the efficiency of performance of theother less frequently requested biochemical tests. It is the purpose ofthis paper to present a system of analysis for the clinical laboratorywhich would permit the employment of microchemical procedures ona semiautomatic basis. This system is composed of five basic ele-ments:

1. The use of siliconated-heparinized plasma (2) in place of serumor whole blood, so that determinations may be started within a fewminutes after the sample reaches the laboratory.

2. The use of the calibrated pipet tip-buret technic for measuringa constant volume of each plasma sample and of standards and blanks(3, 4). This enables measurements to be made on 0.20-nil. or less offluid by personnel with little technical skill.

From the Biochemistry Department, Isaac Albert Research Institute of the JewishChronic Disease Hospital, Brooklyn, N. Y.

Received for publication Dec. 22, 1957.

127

Page 2: Glucosa Antigua

128 SAIFER fT AL Clinical Chemistry

3. The use of the decantation principle (5) for making quantitativetransfers of the supernatant fluid from a precipitate.

4. The use of automatic syringe pipets (6) for making rapid, pre-cise volumetric measurements of solutions in place of ordinary volu-metric pipets.

5. The use of specific enzymatic procedures, wherever these arefeasible, for the quantitative determination of the desired constituentof the biologic fluids.

To carry out this system of analysis it is necessary to modify boththe composition of the reagents and the procedural operations pres-ently employed in most clinical chemistry laboratories. It is the pur-pose of this paper to describe the general way in which a method ismodified in order to conform to the basic tenets of the semiauto-matic system, and to illustrate its utility for the determination ofsome commonly analyzed biochemcial substances.

MATERIALS AND METHODS

GLUCOSE DETERMINATIONS (GLUCOSE OXIDASE METHOD)

The method to be described is taken from a recently developed pro-cedure by Saifer and Gerstenfeld (7). It is based on the abstracts byKeston (8) and Teller (9) describing the simultaneous use of twoenzymes in the following sequence of reactions:

glucoseGlucose + 02 > gluconic acid + 11202

oxidaseperoxidase

11202 + chromogenic oxygen acceptor ) chromogen(o-tolidine, o-dianisidine, etc.) (blue, orange, etc.)

This method presents an almost ideal example of the application ofthe five principles set forth in the introduction, that is, the utility ofthe proposed system for the routine analysis of an important bio-chemical constituent.

Reagents and Apparatus

1. Siliconated-Heparinized Plasma: Fill a series of test tubes tothe brim with a 1:10 dilution of a water-soluble sfficone1 in distifiedwater and let stand for about 3 minutes. Repour the sificone solutioninto its original container, rinse tubes in running tapwater, and dry

15j]j, product of Clay-Adams Co., New York, N. Y.

Page 3: Glucosa Antigua

Vol. 4. No. 2 RAPID SYSTEM OF MICROCHEMICAL ANALYSIS 129

tubes in an oven at 600. Add 0.10 ml. of a solution of sodium heparin(Merck) containing 1000 ,g./ml., to each tube and again dry in anoven at 60#{176}.5-10 nil, of freshly drawn venous blood when added to atube and mixed by gentle inversion will not clot for periods from 12-25 hours. Plasma obtained by centrifuging such tubes at 2000 rpmfor 5 minutes may be utilized for practically all biochemical determi-nations except for total protein and protein flocculation reactions.Oxalated plasma and heparinized whole blood may also be employedfor the glucose determinations.

2. Stock Glucose Standard: 1 ml. = 10 mg. of glucose. Weigh out10.000 Gm. glucose, reagent grade, and dilute to a liter with a 0.25%benzoic acid.

3. Dilute Glucose Standards: 100 and 200 mg. per 100 ml. Pipet10 and 20 ml. of stock standard into 100-mi. volumetric flasks anddilute each to mark with 0.2% benzoic acid.

4. Cadmium Sulfate: 0.25 Gm./100 ml. Dilute 100 ml. of 5%3CdSO4 .81120 to 2000 ml. with distilled water.

5. NaOH, 0.12N: This solution should be prepared by careful dilu-tion from a 1.OON NaOH solution which has been standardized againststandard acid using phenolphthalein as the indicator.

6. Glucose Oxidase Enzyme2 Reagent: Add 1 ml. methyl alcohol tothe o-dianisidine (chromogen) vial. To a 50-ml. graduated cylinderadd 2.5 ml. of phosphate buffer, pH 7.0, 0.4M, and approximately 35ml. of distilled water. Add, with shaking, the contents of the glucoseoxidase vial dissolved in small portions of distilled water. Then, addthe chromogen solution to the cylinder slowly and with constant shak-ing. Dilute with water to the 50-mi. mark and mix. Filter if not clear.Prepare enough solution as is needed for a day’s run.

7. H2S0, 0.5N: Dilute 13.5 ml. of concentrated acid to a liter.

8. Photoelectric Colorimeter: Klett-Sumnierson or equivalent in-strument.

9. Automatic syringe pipets8 for delivery of volumes up to 2.0 ml.10. Buret 50 ml., 0.10-mi. divisions with a 0.10-mi. calibrated tip.4

2Olueostat, product of the Worthington Biochemical Corporation, Freehold, N. J.8Product of the Beeton Dickinson Co., Rutherford, N. 3.4A complete self-contained unit for carrying out this system of analysis is obtainable

from Kopp Laboratory Supplies, 1680 Second Ave., New York 28, N. Y., as the ClinaiysizApparatus. This type buret is also obtainable from A. H. Thomas Co., Philadelphia, Pa., asthe Seligson Automatic Pipet.

Page 4: Glucosa Antigua

130 SAIFER fT AL. Clinical Chemistry

Glucose Oxidase M’ethod

1. Centrifuge blood to obtain plasma.2. Draw up into buret tip 0.10 ml. plasma.3. Rinse out tip with 7.0 ml. of CdSO4 from buret into a tube (or

preferably rinse out tip with 2.0 ml. CdSO4 solution into a tube con-taining 5.0 ml. of the same solution).

4. Add 1.0 mi.5 of NaOH and mix contents. Let stand 10 minutes.5. Centrifuge for 5 minutes at 3000 rpm.6. Carefully decant supernatant into another uncalibrated tube.7. Add 2 mi.5 of prepared glucose oxidase reagent, mix by inver-

sion.8. Place in 370 water bath for 30 minutes.9. Add 1 nil.5 of 0.5N H2S04, mix by inversion.

10. Let stand 5 minutes and read between 5 and 30 minutes in theKlett colorimeter with #42 ifiter after setting blank at the zero read-ing.

11. Blank: distilled 1120 treated exactly as plasma.12. Standards: 100 and 200 mg. per 100 ml.-each in duplicate,

treated exactly as plasma.

CalculationRd unk.

Rd st. X st. conc. = mg. per 100 ml. glucose

RATIONALE OF THE INCORPORATION OF A METHOD INTO THE SEMIAUTOMATIC SYSTEM

Each of the five basic tenets which have been incorporated into theabove-described glucose oxidase method will now be discussed inturn:

1. The utilization of plasma of any kind permits the requesteddeterminations to be started almost immediately after the blood sam-ple reaches the laboratory. However, the use of siliconated glasstubes, employing a water-soluble silicone, and which contain a mini-mal amount of heparin, a naturally occurring anticoagulant, has anumber of additional advantages over the use of oxalated or citratedplasma. It produces minimal changes in the electrolyte compositionof the sample and permits practically all other biochemical determi-nations, except total proteins and protein flocculation reactions, to beperformed on this plasma sample. The use of plasma is especiallyimportant for the determination of glucose, since it has been reported

5Syringe pipets were used for the addition of these solutions.

Page 5: Glucosa Antigua

Vol. 4, No. 2 RAPID SYSTEM OF MICROCHEMICAL ANALYSIS 131

that considerable glycolysis may occur during the time required forthe clotting process (10).

The rapid removal of plasma from the accompanying cells of wholeblood is not only important for glucose determinations but for manyother biochemical determinations as well, such as enzyme and electro-lyte determinations, and for C02, bilirubin, and cholesterol esteranalysis. The use of plasma not only permits much larger volumes offluid to be obtained for analytic purposes but also eliminates to alarge extent the possibility of hemolysis, which frequently occurs inattempting to obtain serum from a blood sample. This serves to re-duce the number of samples which have to be rejected for such deter-minations as potassium and certain enzymatic procedures becauseof the presence of hemolysis.

2. The measurement of the 0.10-mi. aliquot of the plasma is per-formed directly from the tube itself after centrifugation. Since theentire procedure consists of simply drawing up the aliquot of plasmaby means of gentle suction and closing off a stopcock, no special skillis required in this measurement step. The tip is then flushed out witha large volume of fluid, e.g., in the case of the glucose method with 2.0ml. (or 7.0 ml.) of the dilute cadmium sulfate solution. Because of thelarge volume employed in this step, there is little possibility of errordue to any plasma remaining in the buret tip itself, and this largevolume also permits a precise volumetric measurement by means ofthe buret. The remaining cadmium sulfate in the tip is then removedby suction and before the next sample is measured a minute amountof the specimen is drawn through the tip twice in order to measure thesample with the requisite precision. The measurements are then con-tinued until the entire run has been measured, both with respect to theplasma and the accompanying cadmium sulfate solution.

3. After the addition of the cadmium sulfate solution the necessaryamount of sodium hydroxide required to produce the pH necessaryfor protein removal, and for the enzymatic reaction, is added bymeans of a syringe pipet.

4. The contents of the tube are then mixed and centrifuged. Aftercentrifugation, the supernatant fluid is poured off into another un-calibrated tube. In this stage of the procedure care must be takento see that particles of the precipitate are not transferred into theother tube. The loss of small amounts of liquid in this stage is not ofgreat importance for the accuracy of the procedure. It is, however,

Page 6: Glucosa Antigua

-J©

0It

132 SAIFER fT AL. Clinical Chemistry

I

I-

Liz

r

Page 7: Glucosa Antigua

Vol. 4, No. 2 RAPID SYSTEM OF MICROCHEMICAL ANALYSIS 133

necessary that blanks and standards be run in a manner identical withthe unknown samples.

5. To the decantate is now added 2 ml. of the glucose oxidase re-agent, again by means of a syringe pipet, and the contents mixed andplaced in a water bath for 30 minutes at 37#{176}.After this period ofincubation, the 0.5N acid is added with a syringe pipet. This servesboth to stop the enzyme activity and to produce the yellow color whichis to be measured in the photoelectric colorimeter. After 5 minutesthe samples are read in the photoelectric colorimeter using a no. 42Klett filter or at a 390-mit wave length.

It should, perhaps, be emphasized at this point that the methodhas not employed a single calibrated volumetric pipet, neither havecalibrated tubes been used nor is it necessary to dilute the chromo-genic substance to a specific volume. The reasons such a proceduremay be used with little or no sacrifice in the analytic accuracy of themethod will be discussed later in this paper. The various steps in theglucose oxidase procedure which illustrate the five principles of thesemiautomatic system are shown graphically in Fig. 1.

APPLICATION OF THE ANALYTIC SYSTEM TO OTHER BIOCHEMICAL PROCEDURES

In addition to the method described above for glucose, the systemhas also been applied to the following biochemical determinations:(1) The determination of urea nitrogen with a photometric methodwhich employs urease as the specific enzyme (11’). (2) The determi-nation of sodium and potassium by means of flame photometry (12).(3) The determination of total protein in fluids using the biuret meth-od (13). (4) The photometric determination of phosphorus in plasma(5). (5) The determination of acid and alkaline phosphatase using abuffered phenyl phosphate as the substrate (14). (6) The determina-

FIg. 1. The five basic principles (I to V) of the semiautomatic system, all of which are

utilized in the glucose oxidase procedure: (I) The use of siliconated-heparinized plasma.

(II) The use of the calibrated-tip buret for the quantitative measurement and transfer ofthe plasma sample. (III) The use of the automatic syringe pipets for the precise measure-ment of volumes of solutions. (IV) The use of centrifugation and decantation for thequantitative transfer of aliquots of supernatant fluids from precipitates. (V) The use ofspeclife enzymatic methods for the analysis of the desired constituent of the plasma or otherbiologic fluid.

The system also requires that for precise results that no final dilution to a fixed volumebe made and that blanks and standards be run In an identical manner as are the unknowns.

The bureta employed In this system ail have an automatic zero leveling device with anoverflow to a waste bottle and a pressure bulb to fill the buret from the bottom opening.

Page 8: Glucosa Antigua

134 SAIFER ET AL. Clinical Chemistry

tion of calcium using a fluorescent indicator-EDTA titration proce-dure (15, 16).

The above methods are presently routinely employed in this labora-tory. However, a number of other procedures utilizing the semiauto-matic system are presently at various stages of development. Theseinclude, a photometric method for chloride analysis, a method for thecolorimetric determination of uric acid employing the specific eiizymeuricase, a method for the photometric determination of cholesteroland cholesterol esters, etc. Pertinent details for carrying out some ofthe procedures already in operation will be listed below.

Urea Nitrogen Determination

The method used in the semiautomatic system is essentially that ofHughes and Saifer (11) which has been modified in the followingmanner:

1. Centrifuge blood to obtain plasma.2. Draw off approximately 0.3 ml. plasma with a dropper into

small tubes (1/i” diameter).3. Add 1 small drop of urease solution. Mix and let stand 20

minutes at room temperature.4. Draw 0.10 ml. plasma into buret.5. Rinse with 10.0 ml. of 0.5% ZnSO4 . 71120 solution from buret

into a tube (or preferably rinse out tip with 2.0 ml. of the 0.5%ZnSO4. 71120 solution into a tube containing 8.0 ml. of the same solu-tion).

6. Add 2.0 ml. of 0.12N NaOH solution and mix. Let stand 10minutes.

7. Centrifuge for 5 minutes at 3000 rpm.8. Decant the supernatant into another uncalibrated tube.9. Add 1 drop of 2% sodium polyanethol sulfonate (Liquoid-

Roche).10. Add 0.5 ml. of Nessler ‘s solution and mix by inversion.11. Read after 10 minutes in the Klett colorimeter with #42 ifiter

(440 m/L) after first setting the blank at a zero reading.12. Blank: distilled 1120 treated exactly as the blood plasma.13. Standards: 15 to 30 mg. per 100 ml. of ammonium N, or prefer-

ably a standard serum6 of known urea N content, is run in duplicate,in exactly the same manner as the blood plasma.

0Versatol, warner-Chilcott Co., Morris Plains, N. 3., was used as the urea N standard inthis method.

Page 9: Glucosa Antigua

Vol. 4, No. 2 RAPID SYSTEM OF MICROCHEMICAL ANALYSIS 135

Calculation

Rd unk.Rd st X conc. st. = mg. per 100 ml. urea N

The original procedure should be consulted for the preparation ofthe reagents employed. The calibrated-tip buret used in this proce-dure is similar to that used for glucose determinations. The moreacid pH of this filtrate serves to eliminate any turbidity after Ness-lerization.

Sodium and Potassium Determinations (Flame Photometry)

The method for sodium and potassium using the flame photometerhas been modified from the original procedure described by Hald(12).

Reagents and Apparatus:

1. Lithium Sulfate Solution: 2.58 mEq./100 ml. Weigh 1.65 Gm. oflithium sulfate and dilute to a liter with distilled water.

2. Sodium Stock Standard: 100 mEq./I00 ml. Weigh 71.02 Gm. ofNa2SO4 and dilute to a liter with distilled water.

3. Sodium Working Standards: 137.5 mEq./l. Dilute 13.75 ml. ofthe stock standard to 100 ml. with distilled water. 150 mEq./l. Dilute15 ml. of the stock standard to 100 ml. with distilled water.

4. Buret: 0.05-mi. calibrated tip, 25-mi. capacity, graduated in0.10-mi. divisions.

5. Test Tubes: Acid wash and dry before use.

Procedure:

1. Centrifuge blood to obtain plasma or serum.2. Draw 0.05 ml. of plasma or serum into buret tip.3. Rinse tip with 2.5 ml. of lithium sulfate solution from the buret

into a tube.4. Add 10.0 ml. of lithium sulfate solution to the tube from the

buret used in the potassium procedure (see below).5. Mix tube contents after covering finger with a clean finger cot.6. Standards (137.5 and 150 mEq./l.) are treated in the same man-

ner as the serum.7. Blank: Water treated in the same manner as serum. The read-

ings of the standards, blanks, and unknowns are then made with theflame photometer in accordance with the manufacturer’s instructions.

Page 10: Glucosa Antigua

136 SAIFER fT AL. Clinical Chemistry

Potassium Determination

Reagents and Apparatus:1. Lithium Sulfate Solution: Same as for sodium determination.2. Potassium Stock Standard: 1 mEq./100 ml. Weigh 871.2 mg. of

K2S04 and dilute to a liter with distified water.3. Potassium Working Standards: 2.5 mEq./1. Dilute 25.0 ml. of

the potassium stock standard to 100.0 ml. with distified water. 5.0mEq./l. Dilute 50.0 ml. of the stock standard to 100 ml. with distifiedwater.

4. Buret: 0.20-mi. calibrated tip, 50 ml. capacity, graduated in 0.10-ml. divisions.

5. Test Tubes: Acid wash and dry before use.Procedure:1. Centrifuge blood to obtain plasma or serum.2. Draw 0.20 ml. of plasma or serum into buret tip.3. Rinse tip with 5.0 ml. of lithium sulfate solution from the buret

into a tube.4. Mix tube contents after covering finger with a clean finger cot.5. Standards (2.5 and 5.0 mEq./l.) are treated in the same manner

as the serum.6. Blank: Water treated in the same manner as serum. The read-

ings of standards, blanks and unknowns are then made with the flamephotometer in accordance with manufacturer’s instructions.7

Total Protein Determination

The method used is that by Reinhold (13) which has been modifiedfor the semiautomatic system in the following manner:

Reagents:1. Biuret Reagent (double strength): In a 1000-ml. volumetric

flask place 3 Gm. crystalline copper sulfate, 12 Gm. sodium potassiumtartrate (Rochefle salt), and approximately 500 ml. of distified waterand shake until dissolved; and with constant agitation of the flask add600 ml. of 2.5N NaOH and mix. Add 2 Gm. KI and shake until dis-solved; dilute to volume with distilled water.

2. Standard: Serum of known protein content8 as determined withthe micro-Kjeldahl method.

TThe flame photometer used in these studies was that manufactured by Process and In.struments Co., Brooklyn, N. Y.

8Versatol, warner-chileott Co., Morris Plains, N. 3., was used as the prepared proteinstandard In this procedure.

Page 11: Glucosa Antigua

Vol. 4, No. 2 RAPID SYSTEM OF MICROCHEMICAL ANALYSIS 137

3. Sodium Chloride: 0.85%. Dissolve 8.5 Gm. NaC1 in distilled wa-ter and dilute to 1000 ml.

Procedure:1. Centrifuge blood to obtain serum.2. Draw 0.10 ml. of serum into buret tip.3. Rinse tip with 3.0-mi. biuret reagent from the biuret into a

tube. This biuret is equipped with a Teflon stopcock.4. Add 2.0 ml. of distified water with an automatic syringe pipet

and mix contents.95. Blank: Saline treated in the same manner as serum.6. Standard: Known protein solution treated in same manner as

serum.7. Let tubes stand for 30 minutes and then read in a Klett col.

orimeter with #54 ifiter after setting blank at zero reading. Read ina flat-bottom Klett tube.

Serums with abnormal color (hemolysis, jaundiced, lipemic, etc.)must have a serum blank prepared to compensate for the additionalcolor. Prepare for each of these a separate tube containing serum,biuret blank reagent (without copper), and water. Run a blank andstandard in the same way. Subtract this reading from the biuretreading obtained above.

Calculation

RdunkRd st. X protem content standard solution (Gm./100 ml.) =

total protein (serum) in Gm./100 ml.

Phosphorus Determination

The method employed is essentially that described by Goldenberg(5) using a 25-mi. buret with a 0.2-ml. calibrated tip. The originalpaper should be consulted for the experimental details.

AcW and Alkaline Phosphatase Determination

The procedure used in the semiautomatic system has been modifiedfrom that described by King and Armstrong (14) using a 50-mi.buret with a 0.1-mi. calibrated tip. This method has been altered inaccordance with the five principles of the system so as to eliminate allvolumetric pipeting operations.

#{149}Forthe determination of cerebrospinal fluid protein the water is omitted and 2.0 ml. ofthe spinal fluid is substituted for the serum sample.

Page 12: Glucosa Antigua

138 SAIFER fT AL. Clinical Chemistry

Calcium Determination

The method used is that described by Ashby and Roberts (14)which uses an EDTA titration with a fluorescent indicator. For thismethod a 10-mi. graduated buret with a .25-mi. calibrated tip is used.The sample is measured with a calibrated tip and flushed out with theEDTA solution contained in the buret. The remainder of the proce-dure being carried out as discussed by the authors (15) except thatthe indicator employed is that proposed by Baron and Bell (16) whichgives sharp end points in diffuse daylight.

DISCUSSIONIt should be stressed at the beginning of this discussion that the

authors make no claim to originality with respect to any of the fiveprinciples employed in the “semiautomatic system” except for theirjuxtaposition into a practical working system for rapid microchemi-cal analysis. The use of siliconated-heparinized plasma samples asthe best fluid for routine biochemical analyses resulted from theextensive experimental work with anticoagulant agents of the lateDr. S. Losner of this laboratory (2). The use of the calibrated-pipet-tip buret technic was brought to our attention by Dr. David Seiigsonof the University of Pennsylvania Medical School (4) although thebasic principle had been previously described by Lowry (3) in 1916.The decantation principle as a precision step in colorimetric analysishas been adequately analyzed by Goldenberg (5) and for complete de-tails reference should be made to the original paper. Perhaps, how-ever, a brief discussion of this principle as applied to the proposedsystem is in order. The principle of decantation permits large lossesto be sustained in a transfer without appreciable effect on the finalphotometric readings. This is done by automatically correcting thedecantation error by reducing the total volume in proportion to thesample lost. Thus Goldenberg (5) has shown in his publication thatif for a supernatant volume of 10 ml. there was a loss of 0.50 ml. inthe decantation stop and 1.00 ml. of color-producing reagent werethen added, then the analytic error would be in the order of 0.5 percent. Since most colorimetric methods have an over-all error of 2 to5 per cent, the decantation process does not introduce an appreciableerror and is therefore a precision step. It has the further advantageof eliminating the volumetric measurements of aliquots and of not re-quiring dilutions to mark in calibrated tubes. In addition, decanta-tion makes use of the entire supernatant rather than a measured

Page 13: Glucosa Antigua

Vol. 4, No. 2 RAPID SYSTEM OF MICROCHEMICAL ANALYSIS 139

aliquot. It therefore serves either to increase the color yield ob-tained from a given amount of plasma or to reduce the amount ofplasma required for the same color yield. As stated by Golden-berg (5), “the decantation principle does not permit the chemist todispense with all precautions. Steps leading to the preparation of thesupernate must be carried out with analytic precision. It is only atthis stage, when the concentration of the test component and thesupernate has been fixed that rigor may be relaxed in transferring thesupernate for further treatment.”

Reasonable care must also be exercised in adding the reagents forcolor development when this is done with the syringe-pipet technic.The use of the Cornwall automatic pipeting units (syringe pipets), asrecommended in the paper by Dern and Pullman (6), has been shownto be rapid and accurate for the delivery of a constant volume of asolution. In colorimetric analysis it is usually not necessary for thedelivered volume to be an exact one, if blanks and standards are beingrun simultaneously with the unknowns. Therefore, once an approxi-mate volumetric setting is made for this pipet, it is to be kept con-stant throughout any particular run. We have found it most expedi-ent in this laboratory to use a different syringe pipet for each re-agent, keeping each one in a piece of plastic tubing alongside the re-agent bottle as is illustrated in Fig. 1.

The use of these automatic pipeting units in conjunction with thecalibrated-tip buret, while expensive at first, has reduced the volumeof pipets ordered in this laboratory to approximately 20 per cent orless of the previous yearly orders. In addition it has considerablyreduced the burden of washing and replacement of pipets on an al-ready overworked glassware-washing department. The savings onthe purchases of volumetric pipets, the reduction in the amount oftime spent in the cleaning and drying of these pipets as well as the useof uncalibrated test tubes in place of calibrated ones, has resulted inthe savings of many thousands of dollars annually in the laboratoryof this 800-bed hospital. The clinical chemist has been aware of theadvantage of specific enzymatic technics in biochemical analysis formany years, since the determination of urea nitrogen using ureasetechnics is a commonly employed procedure in many clinical labora-tories. However, recently the work of the enzyme chemist has madepossible the specific enzymatic determination of other important con-stituents of body fluids, e.g., the determination of glucose with glucoseoxidase (7, 8, 9), the determination of uric acid with uricase (17), etc.

Page 14: Glucosa Antigua

140 SAIFER fT AL Clinical Chemistry

The recent surge of enzymatic methods have led some investigatorsto suggest that specific enzymatic methods would eventually replacemost other procedures in the clinical laboratory (18).

In addition to the advantages of the semiautomatic system statedabove, it has a number of less-obvious advantages. For example, itpermits many more determinations to be performed from the samevolume of blood because of the larger volumes of plasma which can beobtained as compared to serum. It prevents the contamination ofstandard solutions since these are never pipeted directly from thebottle. Instead, a small volume of the standard solution is pouredinto a separate tube and measured with the calibrated-tip buret tech-nic in exactly the same manner as is the unknown. The excess ofstandard solution is then discarded. By the use of the semiautomatiosystem it has been found possible to perform as many as 50 glucoseand urea nitrogen determinations in a period of between 2 and 2#{189}hours. In practice this makes the time required per determinationalmost equivalent to that reported for the autoanalyzer. However,the proposed system has the further advantage that it may be em-ployed to increase the efficiency of a biochemical procedure whetherthe work load involved is a large or a small one.

The incorporation of a sixth step in semiautomatic system has beencontemplated. This is the addition of an automatic recording devicewhich can be attached to a photoelectric coiorimeter or a spectro-photometer. The purpose of such an innovation would be to eliminatethe laborious step of reading and recording the photometric results.However, such equipment would be relatively expensive comparedto the rest of the system and would presently not be practical for mostclinical laboratories.

SUMMARY

A rapid, semiautomatic system of microchemical analysis for theclinical chemistry laboratory has been proposed. Five basic elementsof this system are: (1) The use of siliconated-heparinized plasma.(2) The use of the calibrated-pipet-tip buret technic for measuringsmall (0.10-mi.) samples. (3) The use of the decantation principle asa precision step in making quantitative transfers. (4) The use ofautomatic syringe pipets for adding constant volumes of reagents,(5) The use of specific enzymatic methods, whenever these are appli-cable, for the determination of biologic constituents.

Page 15: Glucosa Antigua

Vol. 4, No. 2 RAPID SYSTEM OF MICROCHEMICAL ANALYSIS 141

The analytic system has already been applied to the determinationof such important biologic constituents as glucose, urea nitrogen,phosphorus, acid and alkaline phosphatases, sodium and potassium,calcium, and total protein.

The semiautomatic system permits the use of microprocedures in aclinical chemistry laboratory by persons of limited technical skill.

REFERENCES1. Skeggs, L. T. Jr., Am. 1. Chn. Path. 28, 311 (1957).

2. Losner, S., and Volk, B. W., Am. J. Med. Sci. 223, 75 (1952).3. Lowy, A., U.S. Patent No. 1,204,368, Nov. 7, 1916.

4. Seligson, D., Am. J. din. Path. 28, 200 (1957).5. Goldenberg, H., Anal. Chem. 28, 1003 (1956).6. Dern, R. 3., and Pullman, T. N., J. Lab. 4. GUn. Med. 36, 494 (1950).7. Saifer, A., and Gerstenfeld, S., J. Lab. 4 Clin. Med. 51, 448 (1958).8. Keston, A. S., Abstr. of Papers, 129th Meeting, ACS, Dallas, Texas, April 1956, p. 310.

9. Teller, 3. D., Abstr. of Papers, 130th Meeting, ACS, Atlantic City, N. 3., September1956, p. 69C.

10. Sunderrnan, F. w., MacFate, R. P., Evans, 0. T., and Fuller, J. B. Am. 3. dUn. Path.21, 901 (1951).

11. Hughes, J., and Saifer, A., .T. Lab. 4. Chn. Med. 27, 391 (1941).12. Hald, P. M., J. Biol. Chc’ir&.167, 499 (1947).13. Reinhold, 3. 0., Standard Methods of Clinical Chevnstrij. New York, Academic, 1953,

vol. 1, p. 88.14. King, E. J., and Armstrong, A. H., Gonad. Med. Assn. J. 31, 376 (1954).15. Ashby, H. 0., and Roberts, M., J. Lab. 4. Clin. Med. 49, 958 (1957).16. Baron, D. N., and Bell, 3. L., GUn. Chem. Acts 2, 327 (1957).17. Praetorius, B., and Poulsea, H., Scand. 3. Lab. 4. Clin. Invest. 5, 273 (1953).18. w6blki, F., Biochemical Biopsy via Body Fluids. New York, Sloan-Kettering In-

stitute for Cancer Research.


Recommended