Note: Descriptions are shown in the official language in which they were submitted.
10~93Z
PROCESS FOR PREPARING BIOLOGICAL COMPOSITIONS FOR USE
AS A REFERENCE CONTROL IN DIAGNOSTIC ANALYSIS
The present invention is directed to a process for
preparing stable liquid human reference serums containing
biological materials, including such biologically active sub-
stances as found in blood serum or plasma e.g. enzymes;
metabolites; hormones; electrolytes; etc., in a manner which
will enable their storage in liquid form at temperatures ranging
from about 2 to 8C. for four to five weeks or -2QC. for 12
months and permit theîr direct use as reference standards in
instrumental analysis without the necessity for freezing and
thawing before use. This invention is also directed to the
improved analytical procedures using the compositions thus
prepared.
BACKGROUND, AND PREVIOUS ATTEMPT~ TO PROVIDE ~EFERENCE
STANDARDS FOR AUTOMATED ANALYSIS
Biologically active substances such as found in
serums: like enzymes; hormones, electrolytes and biologically
active metabolites, are used widely in the diagnosis of diseases.
They are used as reference standards for instrumental automated
colorimetric analysis since t~ey contain all or most of the
components of the unknown to be analyzed. Once the diagnosing
physician is aware of the patient' 5 concentration of components,
viz., differences versus normal mean ranges of concentration of
such components, the diagnosis can be made more ohjectively.
In their natural form, when separated from their normal biologi-
cal environment, such biologically active substances are unstable
and undergo undesirable changes under the influence of heat,
enzyme action, hydrolysis and other influences causing undesir-
able molecular transformations therein. In the past, severalmethods of preservation have been utilized for such labile
biological products.
One such procedure involves "freeze-drying" of the
:~k
-- 2 --
~09'~93Z
biological. The freeze-drying procedure essentially involves
rapidly reducing the temperature of the aqueous-containing
biological followed by dewatering it to a very substantial, if
not total, extent at reduced pressures. The freeze-dried
biologicals can be stored as a solid for varying lengths of
time depending on their compos;tion. Thus, for example such
dehydrated control material as blood serum or blood plasma can
be stored for one to two years when stored at 2 to 8C.
At present dehydrated freeze-dried ~iological
reference control compositions are reconstituted with distilled
water shortly before their intended use.
Once reconstituted their shelf life is usually from
three (32 to twenty-four ~241 hours when stored in liquid form
at 2 to 8C. Length of stability depends on the nature of
the biological material. Small ~iological molecules, e.g.,
electrolytes, will be among the most stable whereas large
molecules, such as enzymes, are among the least sta~le. Thus,
acid phosphatase can decompose within the first two hours after
reconstitution with water from the freeze-dried dehydrated state
whereas sodium and pota~sium can be stable for several days.
Customarily the unused portion of freeze-dried
reconstituted biological control compositions must be either
refrozen to solid form or di~carded at the end of each working
day. The first method of extending storage life is both
expensive (energy wise~ because refreezing requires temperatures
of -20 to -3QC. and time consuming ~ecause of the time
required for reth~wing. The second method is obviously wasteful
and hence expensive. The process of this invention offers an
inexpensive, convenient solution to the aforementioned problems.
The present invention extends the useful shelf
~storage) life of reconstïtuted freeze-dried biological reference
control compositions economically and permits them to be kept
at 2 to 8C. in the liquid state for periods of four to five
109 '193Z
weeks thus not only avoiding substantially the instability
problems encountered with refrigerator storage (at 2 to 8C.)
of compositions reconstituted in accordance with the prior art
but also avoiding the necessity of refreezing to solid form for
storage (at -20C) and rethawing for use. Significant improve-
ment in the shelf life at 2 to 8C. of freeze-dried reconstituted
biological reference control compositions have been attained
and instability arrested in such compositions containing uric
acid, glucose, ~ilirubin, a variety of enzymes, e.g., alkaline
phosphatase, etc. using the process improvement of this
invention.
PRIO~ ART
In general the preparation and use of biological
reference control compositions is well documented in the prior
art. For example, th.e following three pu~lications appearing
in professional analytical chemistry ]ournals indicate how such
: compositions are customarily prepared and/or evaluated: Teasdale,
P.R. Beamount, D & Pakee, J.; "~ialized pooled serum and control"
Clinica Chemica Acta 30, 535 (1970~; Hanok., A. Kuo, J. "The
Stability of a reconsti.tuted serum for the assay of 15 chemical
constituents", Clinical.Ch.emïstry, 14, 58 ~1968); and G.N. Bowers,
R.W. Burnett & R.B. McComb, "Preparation and use of Muman control
~: materials for monitoring precisi.on in Clinical Chemistry";
Clinical Chemistry, 21, 183~ C1975~.
In my prior U.S. Patent No. 3,876,375, a process is
disclosed for preparing refere~ce control compositions which are
storage sta~le by a sequence involving (1) partial dewatering
(.removing 20 to 40 wt. % of water~ and (2) adding C2 to C5
alkylene polyol to replace water removed.
3~ The present process dif~ers from that of Maurukas
U.S. Patent 3,87Ç,375 in two distinct respects (rendering the
two processes mutually exclusive one from the other);
(1) The present process requires the biological
,~
1094~32
reference composition to be substantially completely dewatered
(about 98 to about 99 wt. % of its original water is removed)
and
(2) The present process reconstitutes with a com-
position containing from about 20 to about 50 wt. % of a C2 to
C5 alkylene polyol with the remainder being water.
In accordance with the present invention the sub-
stantially completely dewatered reference biological can be
stored and shipped in its dewatered state thereby saving
shipping costs.
'COMP'OS'I':FI'ONS
From the compositional viewpoint, the freshly
prepared compositions resulting from the process of this
invention are not distinguishable ~y analysis from those
described and claimed in Maurukas U.S. Patent No. 3,876,375.
Thus said compositions are comprised in their non-biological
components of from about 5Q per cent to about 8Q weight per
cent water, from about 50 to about 2Q weight per cent of an
alkylene polyol having from 2 to 5 carbon atoms, the remainder
being chiefly at least one natural hiological material selected
from the such exemplary-classes as human or animal serum or
plasma, enzymes, proteins, hormones, metabolites, etc.
Suitable alkylene polyols which can be used are:
ethylene glycol, propylene glycol, butylene glycol, pentanediol
and glycerol. The alkylene polyol material preferably is
ethylene glycol, but other alkylene polyols can be utilized
individually or in admixture with ethylene glycol.
Usually these aqueous-containing biological composi-
tions contain in their non-biological components from about ~0
to about 80 weight per cent water, from a~out 20 to about 40
weight per cent of an alkylene polyol of the type indicated
hereinabove with the remainder ~eing comprised chiefly of the
aforementioned biological material.
109493Z
In some cases it is desirable to employ various
combinations of alkylene polyols. Thus it is desirable to
employ compatible mixtures of ethylene glycol and/or propylene
glycol and glycerol. While glycerol is often superior to
ethylene glycol in regard to keeping proteins in solution, it
does yield somewhat undesirable increases in viscosity of
biological fluids. Since viscous fluids are hard to pump and
introduce into certain equipment, e~g., pipettes, tu~es, etc.,
the utilization of ethylene glycol results in low viscosity and
at the same time the attainment of a good depression of the
freezing point to permit storage at low temperatures in the
liquid state. The use of ethylene glycol in concentrations
substantially in excess of 33 per cent by volume tends to
precipitate protein present in such labile biologicals however.
OVERALL PROCE5S CONSIDERATIONS
The process of this invention will be illustrated
by descri~ing how typical plasma and serum control material is
prepared for monitoring precision in clinical chemistry, viz.,
use as reference serums in analysis upon which diagnoses are
based. The procedure followed is that described in Clinical
Chemis*ry Volume 21, pp. 1830 et seq. (1975) ~y Bowers & Co.
Publishers.
Residual plasma or serum is collect~d into a frozen
pool (-5C to -20C~ of 25 to 35 liters. The term "residual
plasma" as used herein means several thousand hospital patients
serum sample; left-over, each from 2 to 5 ml, combined in "pool".
The pool is then defrosted Cat temperatures of 10C to 25C
over a period of 10 to 24 hours~ and the particulate matter is
filtered from the defrosted pool. Biological compounds, e.g.,
glucose, urea, enzymes, etc., are then added to the pool to
achieve the "target arithmetical values" along with stabilizers,
e.g., sodium azide, etc., and the thus prepared pool is
dispensed into lQ ml. vials Ctotal 2,500), subsequently freeze
.
~P~
1094932
dried at temperatures ranging from about -10C to -20C over
a two to ten hour time period at reduced pressures (for drying)
of from about 20 to about 1 millimeters o~ mercury and more
preferably from about 5 to lO millimeters of mercury. After
drying, the vials are stoppered and stored at temperatures
ranging from about 2 to 8C.
Commercial controls are prepared in similar fashion.
Dehydrated and st~pperedtubes can then be stored at temperatures
ranging from 2C to 8C for up to 2 years. In accordance with
conventional practice, the dried sample is redissolved in water,
alone, and consumed immedïately or discarded at the end of the
working day. In accordance wit~ acceptable practice only one
refreezing of reconstituted serum is allowed, repeated freezing
and thawing rapidly changes the quantitative composition of
serum controls. Keeping samples frozen is wasteful as is
discarding reconstituted ones which are unused the same day or
within a day or 50 of the day on which they are reconstituted.
These wastes are substantially ~liminated by the
process of the present invention since unused samples can be
stored for 4 to 5 weeks at 2C to 8C without having to refreeze
them. For example, reconstitution of freeze-dried serum with
water, alone, yields reference product with two (2~ days
refrigerator storage (:approximately 4C) life for glucose
determination compared with 4 to 5 weeks when serum from the
same freeze-dried pool is reconstituted with a mixture containing
33 wt. % ethylene glycol in water. Additionally the same
ethylene glycol-~ater reconstituted reference control composi-
tions can ~e stored at -2QC without becoming solid, thus
eliminating the necessity ~or time-consuming defrosting. This
; 30 is a clear advantage in today's ~usy laboratories.
AUTOMATED ANALYSIS AND USE OF THE STABLE REFERENCE
COMPOSITIONS AS ANALYTICAL STANDARDS IN COLORIMETRY
The present demands of clinical investigation on
- 7 -
10~ 32
analytical laboratory services reached the point requiring
automated clinical chemistry in place of prior manual methods,
with their inherent and cumulative analytic errors. Automated
procedures have been devised in which the sources of varia-
bility have been closely controlled by such means as the
substitution of dialysis for protein precipitation, the
combination of test fluid and reagents in flowing streams,
closely controlled heating and reaction times, and flow-through,
double-beam colorimeters coupled with recorders. Exemplary of
such automated analytical devices are the commercially available
TECHNICON AUTOANALYZERS available from the Technicon Instruments
Corporation, Chauncey, New York.
The sequence of operations for automated analysis is
similar to that in a manual method and involves measurement of
sample, removal of protein, addition of reagents, heating and
reaction timing, measurement of color, and calculation of results.
The automated system uses a pumping method in which plastic tubing
of various internal diameters is alternately compressed and
released by a set of rollers which imitate peristaltic action.
Since the rollers travel over the tubing at a constant rate, the
actual volume of fluid transported depends on the bore of the
tubing. Separation of one sample from the next is achieved ~y
insertion of air bubbles into the stream; this also gives some
"scrubbing" action which minimizes contamination of one sample
by the next. The segmentation of the fluid streams in this
manner also permits mixing by passing the stream through a rigid
glass helix, mounted horizontally, in which each small portion of
fluid is repeatedly tumbled as it passes along the coil.
Separation of protein from the samples is achieved by
3a dialysis. The dialyzer consists of two plates; in one surface
of each plate ;s cut a very accurately machined spiral groove,
forming a continuous channel with a semicircular cross-section.
When the two plates are secured with their grooves surfaces
-- 8 --
10949~32
facin~ each other and a very thin cellophane membrane separating
them, a continuous channel of circular cross-section, divided
along its whole length by the membrane, is formed. The total
length of the channel is about 87 inches. If serum is flowing
in one half of the channel and a reagent or other aqueous fluid
in the other half, dialyzable constituents of the serum will
pass across the membrane and enter the stream of reagent. Such
substances are urea, glucose, creatinine, uric acid, phosphate,
calcium, sodium, potassium, and chloride can by this means be
removed from serum and the large nondialyzable protein molecules
passed away to waste. It should be pointed out that only a
proportion of the small molecule~ and ions is transferred from
the sample stream to the reagent stream; but since, within limits,
the same proportion of the constituents of a standard solution
will also be transferred, the ratio of sample concentration to
standard concentration will be maintained. The automated
instrument operates on the accurate measurement of this ratio;
reactions do not have to be taken to completion, as in the manual
procedures, and thus reaction times can be shortened without
loss of accuracy.
If the procedure requires a heating or incubation
step, this is achieved by passing the mixture of reagent and
sample dialysate along a rigid glass helix immersed in a
suitable heating bath. The heating or incubation phase is
exactly determined by the time taken by the fluids to traverse
the coil when they are pumped at a constant rate. The baths
are completely enclosed and stirred continuously, permitting
accurate temperature control.
The colored solutIons resulting from the reaction
between the sample dialysate or standard dialysate and the
reagents are passed into the flow-type cell of a twin-beam
colorimeter which uses narrow band pass filters (about 17
millimicrons3. The air bubbles are removed by suitable venting,
_ g _
1094C~32
and the absorbance of the colored solution is converted to an
electrical signal by a photocell. A second photocell,
previously set to 100 per cent transmittance with a potentiometer,
serves as a reference. The difference in light absorbance between
the two beams is amplified and fed to the recorder, which shows
it as a peak on the tracing. Comparison of the height of the
peak produced by t~e sample with that produced by a standard
permits calculation of sample concentration.
The increase in demand for accurate chemical analysis
of many components of natural body biological fluids led to the
development of multichannel automatic analyzers. These machines
were also required to be capa~le of sequential operation in
order to handle the large volume of analytical samples to be
analyzed. Since each separate analysis (channel) had to be
performed colorimetrically in comparison with a standard of
known concentration ~by independent analysis); the desirability
of using a re~erence liquid containing each of the components
sought to ~e analyzed became apparent. For example, it is
present practice to analyze human blood for seventeen to eighteen
components, whose concentrations are reported in units as
follows: total proteîn "T. Protein" (gram per cent~; albumin
(gram per centl; calcium "Ca" Cmilligram per cent~; phosphorus
"P" (milligram per cent~; cholesterol Cm;lligram per cent);
uric acid Cmilligram per cent); creatinine (milligram per cent);
total Bilirubin "T. Bili." (milligram per centl; alkaline
phosphatase (International units per milliliter~; lactic
dehydrogenase "LDH" (International units per milliliter~;
Glutamic-oxaloacetic transaminase "GOT" (International units
per milliliter); creatinïne phbsphokinase "CPK" ~International
units per Milliliter~; chloride "Cl" (milliequivalents per
liter~; carbon dioxide "CO2" (milliequivalents per liter);
potassium "K" (milliequivalents per liter~; sodium "Na"
(milliequivalents per liter~; ~lood urea nitrogen "BUN"
-- 10 --
,~
109493Z
(milligram ~e.r cent);and glucose (milligram per cent).
The liquid reference biological standards (serums)
produced according to the process of this invention are used in
commercially available differential multichannel analyzers
based on colorimetry or spectrophotometry by placing said liquid
serum (of known concentration for each component by separate
independent analysis~ into one of the sample vials in the
machine permitting it to run through the machine and then hand
setting the printer or recorder to the known concentration of
each component as revealed by independent analysis. Hence, each
standardized component h.as its own întensity of color or optical
density and can serve as a reference standard for each analysis
(channel~. Once the opacity or shade or intensity of color has
keen equated with a mathematical unit' value of concentration
for each component, then analysis of the various unknowns can
proceed automatically to yield multichannel analytical print-out
: results on a large num~er of unknowns sequentially. The use of
the reference serum composition of this invention constitutes an
improvement because it permïts use of a stable reference standard
having a composition which i.s very close to that found in the
human body.
EXAMP:LE
Ten ~10~ liters of human serum obtained, frozen from
collecting stations was thoroughly mixed, strained through
several layers of cheese cloth and filtered at a pressure of
25 lb.s/inch2.
In order to ach;eve various target concentration
levels for the serum components, the following reagent grade
chemicals were added to the serum pool; glucose, lithium
car~onate, uric acid, bili.rubin, urea, di-sodium phosphate,
creatinine, and phosph.oric acid. The following enzyme prepara-
tions were also added: lactic dehydrogenase (from beef heart),
aspartate amino transferase (from beef heart), creatine
-- 11 --
10~4932
phosphokinase (from beef heart) and alkaline phosphatase (from
plant source).
The pool of 10 liters was dispensed into 10 ml vials
producing 1000 vials. It was then frozen to -20C to the solid
state. From 98 to 9~% by weight, of the water present in the
vials, was removed under vacuum from the frozen serum. The
vials were stoppered under vacuum and stored in the refrigerator
at 4C.
In order to compare the stability of the glycol-water
reconstituted serum with the one reconstituted with pure
distilled water, sufficient quantity of vials were reconstituted
with 33% ethylene glycol-water mixture (v/v) to its original
10 ml volume. The comparison controls were reconstituted with
distilled water only.
The tubes were capped with rubber stoppers and divided
into three equal quantities: (a) for storage at room temperature,
(b~ for storage in the refrigerator at 4C and (c~ for storage
in a freezer at -20C.
For analysis, one tube was removed from each storage
container. The samples were assayed for seventeen components:
total protein, albumin, calcium, inorganic phosphorus,
cholesterol, uric acid, creatinine, total bilirubin, alkaline
phosphatase, creatine phosphokinase, lactic dehydrogenase,
aspartate amino-transferase, chloride, potassium, sodium urea
nitrogen, and glucose. A commercially available "Hycel 17
Analyzer" ~manufactured and sold by "Hycel Inc." Houston, Texas)
was calibrated with "HyceI" reference serum and was used for
the abo~e mentioned assays. The samples ~ere assayed initially
at approximately daily and weekly intervals. As a result of
these tests, it was learned that a 33~ concentration of ethylene
glycol in water was a practical optimum probably due in some
part to the fact that the thus reconstituted product has a
viscosity similar to human serum.
- 12 -
1094~32
TABLE 1
STABILITY OF SERUM AT ROOM TEMPERATURE)
WEEK
ANALYTE INITIAL 1st 2nd 3rd 4th
Creatinine mg~ 2.9 3.5 2.5 3.1 3.0
Calcium mg% 10.2 9.8 9.7 8.5 8.0
Lactic Dehydrogenase
(I.U.) 175 198 190 187 ---
Phosphorus mg% 6.8 6.8 7.3 7.7 8.0
Creatine Phosphokinase
(I.U.) 91 70 60 75 50
Triglicerides mg%136 108 154 160 110
Transaminase ~I.U.~ 68 75 62 57 40
Alk. Phosphatase (I.U.).24 31 32 33 ---
Total Bilirubin mg% 3.6 3.4 2.9 2.4 ---
Sodium m Eq/L 148 151 154 152 152
Potassium m Eq/L5.6 5.2 5.5 5.5 5.6
Chloride m Eq/L 112 110 111 110 ---
Cholesterol mg% 200 185 202 193 200
Uric Acid mg% 8.0 7.7 7.7 7.7 8.0
Total Protein gm%6.9 6.6 7.1 7.0 6.8
Globulin gm% 2.7 2.7 2.8 2.6 2.7
Urea Nitrogen mg%28 27 27 27 26
Glucose mg% 186 179 181 187 185
- 13 -
,~," ,p.
~ .74 'i~
109493Z
TABLE 2
(STABILITY OF SERUM AT 2 to 8C)
WEEK
ANALYTE INITIAL 1st 2nd 3rd 4th
_ . . .
Creatinine mg% 4.0 3.2 2.6 4.2 3.5
Calcium mg% 9.9 10.0 10.2 10.0 9.9
Lactic dehydrogenase
(I.U.) 136 200 191 190 200
Phosphorus mg% 6.7 6.5 6.5 6.5 6.7
Creatine Phosphokinase
(I.U.~ 97 101 68 73 65
Triglyceride mg%154 98 138 138 140
Transaminase (I.U.~ 73 78 68 68 70
Alk. Phosphatase (I.U.) 25 26 27 27 27
Total Bi~irubin CI.U.~ 3.7 3.7 3.7 3.6 3.2
dium m Eq/L 153 149 154 156 154
Potassium m Eq/L5.4 5.1 5.4 5.7 5.4
Chloride m Eq/L lQ8 lQ7 110 107 110
Cholesterol mg% 188 187 199 190 188
Uric Acid mg% 8.1 7.5 7.6 7.7 7.5
Total Protein gm~6.7 6.8 6.Y 6.7 6.7
Globulin gm% 2.8 2.7 2.7 2.6 2.7
Urea Nitrogen mg%31 27 28 27 27
Glucose mg~ 185 173 178 187 177
1094932
TABLE 3
(STABILITY OF SERUM AT -13 to -20C)
WEEK
ANALYTE INITIAL 1st 2nd 3rd 4th
Creatinine mg% 3.0 3.2 2.6 3.4 3.0
Calcium mg% 10.3 10.010.2 10.910.2
Lactic Dehydrogenase
(I.U.) 180 188 180 184 180
Phosphorus mg% 6.9 6.8 6.5 6.8 6.5
Creatine Phosphokinase
(I.U.~ 89 70 68 84 70
Triglyceride mg% 111 100- 132 134 130
Transaminase ~I.U.l68 72 70 69 71
Alk. Phosphatase (:I.u.2 24 27 24 23 24
Total ~ilirubin (I.U.I 3.6 3.8 3.7 3.5 3.8
Sodium m Eq/L 148 148 153 151 150
Potassium m Eq/L 5.2 5.2 5.4 5.3 5.2
Chloride m Eq/L lQ7 110 111 -~
Cholesterol mg% 190 l9Q 193 179 180
Uric Acid mg% 8.0 7.5 7.5 7.3 ---
Total Protein gm%6.8 6.5 6.6 6.1 ---
Globulin gm% 2.8 2.8 2.7 2.4 ---
Urea Nitrogen mg% 27 29 28 27 ---
Glucose mg% 185 182 169 165 ---
- 15 -
4~32
Compositions prepared as described in this example
were tested for stability on refrigerator storage based on the
established permissible limits of deviation as shown below in
Table 4.
TABLE 4
PERMISSIBLE LIMITS OF DEVIATION
Component Normal Range 1 1 8%* .5SR**
T. Protein 6.0-8.0 g% .2 .3 .16 0.22
Albumin 3.5-5.0 g% .2 .2 .12 0.15
Ca 8.5-10.5 mg% .3 .4 .16 0.04
P 2.5-4.5 mg% .2 .3 .16 0.23
Cholesterol 150-300mg% lQ.018.0 12.00 17.00
Uric Acid 2.5-8.0 mg~ .3 .6 .44 Q.57
Creatinine 0-1.4 mg% .1 .2 .11
T. Bili. 0.2-1.0 mg% ----- .2 .06 -----
Alk. Phos. 30-85mU/ml 5.0 8.0 4.40 -----
CPK 25-145 mU/ml ~ 8.09.60 -~
LDH 100-225 mU/ml16.0 16.010.00 -----
GOT 7-40 mU/ml 2.0 2.02.60 -----
Cl 95-105 meq/l 3.0 4.0Q.80 0.9
C2 24-32 meg/l 2.0 2.0Q.64 0.8
K 3.5-5.0 meg~l .2 .20.12 0.14
Na 135-145 meg/l 3.0 4.00.8Q 0.5
BUN 10-20 mg% 1.0 1.00.80 1.5
Glucose 65-llQ mg% 6.0 ll.Q3.6Q 4.5
... '
*Ad Hoc Advisory Committee NIH (National Institute of Health~
Guidelines for Preparatïon of Control Materials: Class A
reference material guide line "95% confidence interval does not
exceed 8% of the 95% normal range"
** Cotlove Harris & Williams. Cl;n. Chem. 16 1028 (1970) ~5SR
"tolerable analytic variability" 1 acceptable deviations listed
for two commercial lyophilized products.
- 16 -
.~
10~ 93~2
If results obtained on analyses of the reconstituted
liquid reference serum are near or within these limits, it can
be concluded that the components were stable. Sample turbidity
(as examined periodically by visual inspection) presented no
analytical problems, viz., the samples remained clear to the
stability level indicated.
In addition to plasma and serum, the process of the
present invention is useful in extending the useful "shelf-life"
(viz., refrigerator shelf-life~ of hemoglobin standards; urine
controls and other biological reference controls or standards
for significant improvements in stability while eliminating the
need for refreezing to the solid state after reconstituting.
The stable, liquid, pooled reconstituted reference
serum described is suita~le for use as a calibration standard
or reference control for clinical chemical analyses by both
manual and automated analyzers capable of analyzing a plurality
of unknown blood or serum samples.
There has been described a process for preparing
stable reference serum from human serum and an improved procedure
for automated ch.emical analysi.s utilizing said serums.
While the invention has been described above chiefly
in relation to human blood serum, its applicability to other
human or animal biologically active substances, such as found
in serums: e.g. enzymes, hormones, electrolytes, and biologically
acti.ve metabolites used widely in the diagnosis of diseases, both
h.uman and non-human, and other naturally occurring biologically
liquids for use as analytical reference controls or standards,
will be appreciated by those skilled in the art.
~'
