Rapid System of Microchemical Analysis

for the Clinical Laboratory

Abraham Saifer, Shirley Gerstenfeld, and Michael C. Zymaris

THE CLINICAL

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 biochemical 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 laboratories 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 elements:
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.
CHEMIST

111 charge

From the Biochemistry

Department,
Isaac
Chronic Disease Hospital, Brooklyn, N. Y.
Received for publication
Dec. 22, 1957.
127

Albert

Research

Institute

of

the

Jewish

128

SAIFER fT AL

Clinical Chemistry

3. The use of the decantation principle (5) for making quantitative

transfers of the supernatant
fluid from a precipitate.
4. The use of automatic syringe pipets (6) for making rapid, precise volumetric measurements
of solutions in place of ordinary volumetric pipets.
5. The use of specific enzymatic procedures,
wherever these are
feasible, for the quantitative
determination
of the desired constituent
of the biologic fluids.
To carry out this system of analysis it is necessary to modify both
the composition of the reagents and the procedural
operations
presently employed in most clinical chemistry laboratories.
It is the purpose of this paper to describe the general way in which a method is
modified in order to conform to the basic tenets of the semiautomatic system, and to illustrate
its utility for the determination
of
some commonly analyzed biochemcial substances.
MATERIALS AND METHODS
GLUCOSE DETERMINATIONS (GLUCOSE OXIDASE METHOD)

The method to be described is taken from a recently developed procedure by Saifer and Gerstenfeld
(7). It is based on the abstracts by
Keston (8) and Teller (9) describing
the simultaneous
use of two
enzymes in the following sequence of reactions:
glucose
Glucose +

02

>

gluconic acid +

11202

oxidase
peroxidase
11202

+ chromogenic oxygen acceptor

(o-tolidine, o-dianisidine,
etc.)

chromogen
(blue, orange,

etc.)

This method presents an almost ideal example of the application

of
the five principles set forth in the introduction,
that is, the utility of
the proposed system for the routine analysis of an important
biochemical constituent.
Reagents and Apparatus

1. Siliconated-Heparinized
Plasma: Fill a series of test
the brim with a 1:10 dilution of a water-soluble
sfficone1 in
water and let stand for about 3 minutes. Repour the sificone
into its original container, rinse tubes in running tapwater,
15j]j,

product of Clay-Adams

Co., New York, N. Y.

tubes to
distified
solution
and dry

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 an
oven at 60#{176}.
5-10 nil, of freshly drawn venous blood when added to a
tube and mixed by gentle inversion will not clot for periods from 1225 hours. Plasma obtained by centrifuging
such tubes at 2000 rpm
for 5 minutes may be utilized for practically
all biochemical determinations except for total protein and protein flocculation
reactions.
Oxalated plasma and heparinized
whole blood may also be employed
for the glucose determinations.
2. Stock Glucose Standard:
1 ml. = 10 mg. of glucose. Weigh out
10.000 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. Pipet
10 and 20 ml. of stock standard
into 100-mi. volumetric
flasks and
dilute 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 dilution from a 1.OON NaOH solution which has been standardized
against
standard acid using phenolphthalein
as the indicator.
6. Glucose Oxidase Enzyme2 Reagent: Add 1 ml. methyl alcohol to
the o-dianisidine
(chromogen)
vial. To a 50-ml. graduated
cylinder
add 2.5 ml. of phosphate buffer, pH 7.0, 0.4M, and approximately
35
ml. of distilled water. Add, with shaking, the contents of the glucose
oxidase vial dissolved in small portions of distilled water. Then, add
the chromogen solution to the cylinder slowly and with constant shaking. Dilute with water to the 50-mi. mark and mix. Filter if not clear.
Prepare enough solution as is needed for a days run.
7. H2S0, 0.5N: Dilute 13.5 ml. of concentrated
acid to a liter.
8. Photoelectric
Colorimeter:
Klett-Sumnierson
or equivalent instrument.
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 Clinaiysiz
Apparatus.
This type buret is also obtainable from A. H. Thomas Co., Philadelphia, Pa., as
the Seligson

Automatic

Pipet.

130

SAIFER fT AL.

Clinical Chemistry

Glucose Oxidase Method

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 containing 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 inversion.
8. Place in
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 the
Klett colorimeter with #42 ifiter after setting blank at the zero reading.
11. Blank: distilled 1120 treated exactly as plasma.
12. Standards:
100 and 200 mg. per 100 ml.-each
in duplicate,
treated exactly as plasma.
370

Calculation
Rd 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 the
above-described
glucose oxidase method will now be discussed
in
turn:
1. The utilization
of plasma of any kind permits the requested
determinations
to be started almost immediately
after the blood sample reaches the laboratory.
However, the use of siliconated
glass
tubes, employing a water-soluble
silicone, and which contain a minimal amount of heparin, a naturally
occurring anticoagulant,
has a
number of additional advantages
over the use of oxalated or citrated
plasma. It produces minimal changes in the electrolyte
composition
of the sample and permits practically
all other biochemical determinations, except total proteins and protein flocculation reactions, to be
performed
on this plasma sample. The use of plasma is especially
important for the determination
of glucose, since it has been reported
5Syringe

pipets

were

used

for the addition

of these

solutions.

Vol. 4, No. 2

RAPID SYSTEM OF MICROCHEMICAL

ANALYSIS

131

that considerable
glycolysis may occur during the time required for
the clotting process (10).
The rapid removal of plasma from the accompanying
cells of whole
blood is not only important for glucose determinations
but for many
other biochemical determinations
as well, such as enzyme and electrolyte determinations,
and for C02, bilirubin,
and cholesterol
ester
analysis. The use of plasma not only permits much larger volumes of
fluid to be obtained for analytic purposes but also eliminates
to a
large extent the possibility of hemolysis, which frequently
occurs in
attempting
to obtain serum from a blood sample. This serves to reduce the number of samples which have to be rejected for such determinations
as potassium
and certain enzymatic procedures
because
of the presence of hemolysis.
2. The measurement
of the 0.10-mi. aliquot of the plasma is performed directly from the tube itself after centrifugation.
Since the
entire procedure consists of simply drawing up the aliquot of plasma
by means of gentle suction and closing off a stopcock, no special skill
is required in this measurement
step. The tip is then flushed out with
a large volume of fluid, e.g., in the case of the glucose method with 2.0
ml. (or 7.0 ml.) of the dilute cadmium sulfate solution. Because of the
large volume employed in this step, there is little possibility of error
due to any plasma remaining in the buret tip itself, and this large
volume also permits a precise volumetric measurement
by means of
the buret. The remaining cadmium sulfate in the tip is then removed
by suction and before the next sample is measured a minute amount
of the specimen is drawn through the tip twice in order to measure the
sample with the requisite precision.
The measurements
are then continued until the entire run has been measured, both with respect to the
plasma and the accompanying
cadmium sulfate solution.
3. After the addition of the cadmium sulfate solution the necessary
amount of sodium hydroxide required to produce the pH necessary
for protein removal, and for the enzymatic reaction, is added by
means of a syringe pipet.
4. The contents of the tube are then mixed and centrifuged.
After
centrifugation,
the supernatant
fluid is poured off into another uncalibrated
tube. In this stage of the procedure
care must be taken
to see that particles of the precipitate
are not transferred
into the
other tube. The loss of small amounts of liquid in this stage is not of
great importance
for the accuracy of the procedure.
It is, however,

SAIFER fT AL.

132

Clinical Chemistry

-J

I-

Liz

0
It

Vol. 4, No. 2

RAPID SYSTEM OF MICROCHEMICAL

ANALYSIS

133

necessary that blanks and standards be run in a manner identical with

the unknown samples.
5. To the decantate is now added 2 ml. of the glucose oxidase reagent, again by means of a syringe pipet, and the contents mixed and
placed in a water bath for 30 minutes at 37#{176}.
After this period of
incubation, the 0.5N acid is added with a syringe pipet. This serves
both to stop the enzyme activity and to produce the yellow color which
is to be measured in the photoelectric
colorimeter.
After 5 minutes
the samples are read in the photoelectric
colorimeter
using a no. 42
Klett filter or at a 390-mit wave length.
It should, perhaps, be emphasized
at this point that the method
has not employed a single calibrated
volumetric
pipet, neither have
calibrated tubes been used nor is it necessary to dilute the chromogenic substance to a specific volume. The reasons such a procedure
may be used with little or no sacrifice in the analytic accuracy of the
method will be discussed later in this paper. The various steps in the
glucose oxidase procedure which illustrate
the five principles of the
semiautomatic
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 system

has also been applied to the following biochemical determinations:
(1) The determination
of urea nitrogen with a photometric
method
which employs urease as the specific enzyme (11). (2) The determination of sodium and potassium by means of flame photometry
(12).
(3) The determination
of total protein in fluids using the biuret method (13). (4) The photometric
determination
of phosphorus
in plasma
(5). (5) The determination
of acid and alkaline phosphatase
using a
buffered phenyl phosphate as the substrate
(14). (6) The determinaFIg.
utilized

1. The five basic principles

in the

glucose

oxidase

(I to V) of the semiautomatic
system, all of which are
procedure:
(I) The use of siliconated-heparinized
plasma.

(II) The use of the calibrated-tip

buret for the quantitative
measurement
and transfer
of
the plasma
sample. (III)
The use of the automatic
syringe
pipets for the precise measurement of volumes of solutions.
(IV)
The use of centrifugation
and decantation
for the

quantitative transfer of aliquots of supernatant

fluids from precipitates.
(V) The use of
speclife enzymatic methods for the analysis of the desired constituent
of the plasma or other
biologic fluid.
The system also requires that for precise results that no final dilution
to a fixed volume
be 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 an
overflow to a waste bottle and a pressure bulb to fill the buret from the bottom opening.

134

SAIFER ET AL.

Clinical Chemistry

tion of calcium using a fluorescent indicator-EDTA

titration
procedure (15, 16).
The above methods are presently routinely employed in this laboratory. However, a number of other procedures utilizing the semiautomatic system are presently at various stages of development.
These
include, a photometric method for chloride analysis, a method for the
colorimetric
determination
of uric acid employing the specific eiizyme
uricase, a method for the photometric
determination
of cholesterol
and cholesterol esters, etc. Pertinent details for carrying out some of
the procedures already in operation will be listed below.
Urea Nitrogen

Determination

The method used in the semiautomatic

system is essentially that of
Hughes and Saifer (11) which has been modified in the following
manner:
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 solution).
6. Add 2.0 ml. of 0.12N NaOH solution and mix. Let stand 10
minutes.
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
(LiquoidRoche).
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 preferably a standard serum6 of known urea N content, is run in duplicate,
in exactly the same manner as the blood plasma.
.

0Versatol,

this method.

warner-Chilcott

Co., Morris

Plains,

N. 3., was used as the urea N standard

in

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

of
the reagents employed.
The calibrated-tip
buret used in this procedure is similar to that used for glucose determinations.
The more
acid pH of this filtrate serves to eliminate any turbidity after Nesslerization.
Sodium and Potassium Determinations

(Flame Photometry)

The method for sodium and potassium using the flame photometer
has 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. of
lithium sulfate and dilute to a liter with distilled water.
2. Sodium Stock Standard:
100 mEq./I00 ml. Weigh 71.02 Gm. of
Na2SO4 and dilute to a liter with distilled water.
3. Sodium Working Standards:
137.5 mEq./l. Dilute 13.75 ml. of
the stock standard to 100 ml. with distilled water. 150 mEq./l. Dilute
15 ml. of the stock standard to 100 ml. with distilled water.
4. Buret: 0.05-mi. calibrated
tip, 25-mi. capacity,
graduated
in
0.10-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 manner as the serum.
7. Blank: Water treated in the same manner as serum. The readings of the standards,
blanks, and unknowns are then made with the
flame photometer in accordance with the manufacturers
instructions.

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. 5.0
mEq./l. Dilute 50.0 ml. of the stock standard to 100 ml. with distified
water.
4. Buret: 0.20-mi. calibrated tip, 50 ml. capacity, graduated in 0.10ml. 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 readings of standards, blanks and unknowns are then made with the flame
photometer
in accordance with manufacturers
instructions.7
Total Protein Determination

The method used is that by Reinhold (13) which has been modified
for 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 potassium
tartrate
(Rochefle salt), and approximately
500 ml. of distified water
and shake until dissolved; and with constant agitation of the flask add
600 ml. of 2.5N NaOH and mix. Add 2 Gm. KI and shake until dissolved; dilute to volume with distilled water.
2. Standard:
Serum of known protein content8 as determined
with
the micro-Kjeldahl
method.
TThe flame photometer used in these studies was that manufactured
by Process
struments Co., Brooklyn, N. Y.
8Versatol, warner-chileott
Co., Morris Plains, N. 3., was used as the prepared
standard In this procedure.

and In.
protein

Vol. 4, No. 2

RAPID SYSTEM OF MICROCHEMICAL

137

ANALYSIS

3. Sodium Chloride: 0.85%. Dissolve 8.5 Gm. NaC1 in distilled water 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.9
5. 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 in
a flat-bottom Klett tube.
Serums with abnormal color (hemolysis, jaundiced,
lipemic, etc.)
must have a serum blank prepared to compensate
for the additional
color. Prepare for each of these a separate tube containing
serum,
biuret blank reagent (without copper), and water. Run a blank and
standard
in the same way. Subtract
this reading from the biuret
reading obtained above.
Calculation
Rdunk
Rd st.

X protem content standard solution (Gm./100

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

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 original
paper should be consulted for the experimental
details.
AcW and Alkaline Phosphatase Determination

The procedure used in the semiautomatic

system has been modified
from that described by King and Armstrong
(14) using a 50-mi.
buret with a 0.1-mi. calibrated tip. This method has been altered in
accordance with the five principles of the system so as to eliminate all
volumetric pipeting operations.
#{149}For
the determination
of cerebrospinal
fluid protein
the spinal fluid is substituted
for the serum sample.

the water is omitted

and 2.0 ml. of

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 this
method 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 the
EDTA solution contained in the buret. The remainder
of the procedure being carried out as discussed by the authors (15) except that
the indicator employed is that proposed by Baron and Bell (16) which
gives sharp end points in diffuse daylight.
DISCUSSION
It should be stressed at the beginning of this discussion that the
authors make no claim to originality
with respect to any of the five
principles employed in the semiautomatic
system except for their
juxtaposition
into a practical working system for rapid microchemical analysis.
The use of siliconated-heparinized
plasma samples as
the best fluid for routine biochemical
analyses resulted
from the
extensive experimental
work with anticoagulant
agents of the late
Dr. S. Losner of this laboratory
(2). The use of the calibrated-pipettip buret technic was brought to our attention by Dr. David Seiigson
of the University
of Pennsylvania
Medical School (4) although the
basic principle had been previously described by Lowry (3) in 1916.
The decantation
principle as a precision step in colorimetric
analysis
has been adequately analyzed by Goldenberg (5) and for complete details reference should be made to the original paper. Perhaps, however, a brief discussion of this principle as applied to the proposed
system is in order. The principle of decantation
permits large losses
to be sustained in a transfer without appreciable
effect on the final
photometric
readings.
This is done by automatically
correcting
the
decantation
error by reducing the total volume in proportion
to the
sample lost. Thus Goldenberg (5) has shown in his publication
that
if for a supernatant
volume of 10 ml. there was a loss of 0.50 ml. in
the decantation
stop and 1.00 ml. of color-producing
reagent were
then added, then the analytic error would be in the order of 0.5 per
cent. Since most colorimetric
methods have an over-all error of 2 to
5 per cent, the decantation
process does not introduce an appreciable
error and is therefore a precision step. It has the further advantage
of eliminating the volumetric measurements
of aliquots and of not requiring dilutions to mark in calibrated
tubes. In addition, decantation makes use of the entire supernatant
rather than a measured

Vol. 4, No. 2

RAPID SYSTEM OF MICROCHEMICAL

ANALYSIS

139

aliquot.
It therefore
serves either to increase the color yield obtained from a given amount of plasma or to reduce the amount of
plasma required
for the same color yield.
As stated by Goldenberg (5), the decantation
principle does not permit the chemist to
dispense with all precautions.
Steps leading to the preparation
of the
supernate must be carried out with analytic precision.
It is only at
this stage, when the concentration
of the test component
and the
supernate has been fixed that rigor may be relaxed in transferring
the
supernate for further treatment.
Reasonable care must also be exercised in adding the reagents for
color development
when this is done with the syringe-pipet
technic.
The use of the Cornwall automatic pipeting units (syringe pipets), as
recommended
in the paper by Dern and Pullman (6), has been shown
to be rapid and accurate for the delivery of a constant volume of a
solution. In colorimetric
analysis it is usually not necessary for the
delivered volume to be an exact one, if blanks and standards are being
run simultaneously
with the unknowns.
Therefore,
once an approximate volumetric
setting is made for this pipet, it is to be kept constant throughout
any particular
run. We have found it most expedient in this laboratory
to use a different syringe pipet for each reagent, keeping each one in a piece of plastic tubing alongside the reagent bottle as is illustrated
in Fig. 1.
The use of these automatic pipeting units in conjunction
with the
calibrated-tip
buret, while expensive at first, has reduced the volume
of pipets ordered in this laboratory
to approximately
20 per cent or
less of the previous yearly orders.
In addition it has considerably
reduced the burden of washing and replacement
of pipets on an already overworked
glassware-washing
department.
The savings on
the purchases
of volumetric
pipets, the reduction in the amount of
time spent in the cleaning and drying of these pipets as well as the use
of uncalibrated
test tubes in place of calibrated ones, has resulted in
the savings of many thousands of dollars annually in the laboratory
of this 800-bed hospital.
The clinical chemist has been aware of the
advantage
of specific enzymatic technics in biochemical analysis for
many years, since the determination
of urea nitrogen using urease
technics is a commonly employed procedure in many clinical laboratories. However, recently the work of the enzyme chemist has made
possible the specific enzymatic determination
of other important
constituents of body fluids, e.g., the determination
of glucose with glucose
oxidase (7, 8, 9), the determination
of uric acid with uricase (17), etc.

140

SAIFER fT AL

Clinical Chemistry

The recent surge of enzymatic methods have led some investigators

to suggest that specific enzymatic methods would eventually replace
most other procedures in the clinical laboratory
(18).
In addition to the advantages
of the semiautomatic
system stated
above, it has a number of less-obvious advantages.
For example, it
permits many more determinations
to be performed
from the same
volume of blood because of the larger volumes of plasma which can be
obtained as compared to serum. It prevents the contamination
of
standard
solutions since these are never pipeted directly from the
bottle. Instead, a small volume of the standard
solution is poured
into a separate tube and measured with the calibrated-tip
buret technic in exactly the same manner as is the unknown.
The excess of
standard solution is then discarded.
By the use of the semiautomatio
system it has been found possible to perform as many as 50 glucose
and urea nitrogen determinations
in a period of between 2 and 2#{189}
hours. In practice this makes the time required per determination
almost equivalent to that reported for the autoanalyzer.
However,
the proposed system has the further advantage
that it may be employed to increase the efficiency of a biochemical procedure
whether
the work load involved is a large or a small one.
The incorporation
of a sixth step in semiautomatic
system has been
contemplated.
This is the addition of an automatic recording device
which can be attached to a photoelectric
coiorimeter
or a spectrophotometer.
The purpose of such an innovation would be to eliminate
the laborious step of reading and recording the photometric
results.
However, such equipment would be relatively
expensive compared
to the rest of the system and would presently not be practical for most
clinical laboratories.
SUMMARY

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

Vol. 4, No. 2

RAPID SYSTEM OF MICROCHEMICAL

ANALYSIS

141

The analytic system has already been applied to the determination

of 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 a
clinical chemistry laboratory
by persons of limited technical skill.
REFERENCES
1. 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., September
1956,
10.
11.
12.

13.
14.

15.
16.
17.
18.

p. 69C.

Sunderrnan,
F. w., MacFate, R. P., Evans, 0. T., and Fuller, J. B. Am. 3. dUn. Path.
21, 901 (1951).
Hughes,
J., and Saifer, A., .T. Lab. 4. Chn. Med. 27, 391 (1941).
Hald, P. M., J. Biol. Chcir&.167, 499 (1947).
Reinhold, 3. 0., Standard Methods of Clinical Chevnstrij.
New York, Academic, 1953,
vol. 1, p. 88.
King, E. J., and Armstrong,
A. H., Gonad. Med. Assn. J. 31, 376 (1954).
Ashby, H. 0., and Roberts, M., J. Lab. 4. Clin. Med. 49, 958 (1957).
Baron, D. N., and Bell, 3. L., GUn. Chem. Acts 2, 327 (1957).
Praetorius, B., and Poulsea, H., Scand. 3. Lab. 4. Clin. Invest. 5, 273 (1953).
w6blki,
F., Biochemical
Biopsy via Body Fluids.
New York, Sloan-Kettering
Institute for Cancer Research.