T H E USE OF HEPARINIZED BLOOD I N STUDIES WITH CONGO RED
AND BROMSULPHALEIN
ISADORE E. GERBER AND MARYA FRYCZYNSKI
From the Laboratories of the Hudson County Tuberculosis Hospital, Jersey City, New Jerseyt
B. S. Pollak, M.D., Medical Director
The congo red dye test for amyloid disease introduced by Bennhold in 1923
consisted of a colorimetric comparison of the dye in the serum four minutes after
injection with that remaining at the end of 60 minutes. This served to determine the presence or absence of significant dye retention. Irregular results with
this technic led to modifications such as the precipitation of the serum proteins
with alcohol1 and subsequent comparison of the supernatant clear fluids. Taran 2
substituted acetone for alcohol with improved results and claimed that acetone
precipitated any hemoglobin released by hemolysis. This is only relatively true,
however, since many acetone-precipitated sera may often show a yellowish tint
if hemolysis is present.
The fact that the blood must be allowed to stand for some time to obtain
sufficient serum for colorimetric comparison led to the use of plasma. While
plasma was found to be adequate for congo red dye comparisons4 there were many
disadvantages, since the oxalates used as anticoagulants led to shrinking of the
red cells with resultant release of water and a tendency to hemolysis, particularly
if the specimen was allowed to stand for some time before centrifugalization.
In view of the difficulties presented by the use of serum or oxalated blood for
dye comparisons we felt the need for a more desirable method. Although the
acetone method represented a distinct improvement, we found that some of the
dye is precipitated with the protein leading to irregularities in the results of the
test. The use of acetone also introduces the error of dilution and adds an additional step to the procedure. Since similar deficiencies of the bromsulphalein
liver function test, using both serum and oxalated blood, were experienced we
were led to investigate the value of heparin as an anticoagulant.
The use of heparin has many advantages in that unlike the oxalates it has no
osmotic effect on the red cells preventing crenation or laking. It in no way
affects the character of the plasma or the dye content. Heparin is stabile and can
be evaporated to dryness in tubes at 110°C. or sterilized at 170°C. without destroying its anticoagulant activity. 3
MATERIAL AND METHODS
An isotonic solution of the sodium salt of heparin (Liquaemin*) was used. One-tenth
of a cubic centimeter suffices to prevent the coagulation of 10 to 12 cc. of blood. Smaller
quantities are unsatisfactory because of the tendency for the formation of a fibrin net in
the plasma. The blood, after being drawn, is rotated immediately in a clean bottle containing the Liquaemin. It can be allowed to stand without any fear of hemolysis, in contrast to the oxalates. The blood is then centrifuged for 10-15 minutes at approximately
* The Liquaemin was kindly furnished by Roche-Organon, Inc., Nutley, N. J.
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HEPARINIZED BLOOD FOR CONGO RED TEST
313
1500 revolutions per minute, following which the plasma is withdrawn into clean tubes and
then matched in the micro-colorimeter. If the plasma is cloudy further centrifugalization
will render it clear. Once the blood is centrifuged it is advisable to remove the plasma.
By this means the amount of plasma obtained is greater than the serum from a similar
quantity of whole blood. Hemolysis is readily detected since heparinized plasma has a
light orange color and is clear. Colorimetric comparison is thus facilitated.
CONGO RED TEST
In an extensive study on the value of the congo red tests for the demonstration
of the presence or absence of amyloid disease in tuberculous patients, we had the
opportunity of comparing the value of oxalates and heparin as anticoagulants.
Comparisons of the dye content of the respective 4 and 60 minute specimens were made
upon the plasma and frequently checked by the acetone method. With the use of oxalate
there were many discrepancies in our results, despite careful attention to the technique of
injecting the dye and withdrawal of the blood. It was then found that minimal degrees of
hemolysis could escape notice in the presence of a plasma stained red by the congo red dye.
Unfortunately, statistically we were not able to evaluate the incidence of hemolysis with
the oxalated blood. Subsequently, upon the introduction of heparin as an anticoagulant,
a more accurate check was kept. Four hundred and fifty-three heparinized specimens were
collected in over 200 tests. Of these, 43 were hemolyzed, varying from slight to marked.
At first glance, this may appear to be a high incidence; however, in a careful comparison
with serum or oxalated blood we feel that it is significantly lower. This was further confirmed in the bromsulphalein studies detailed below.
A comparison of results obtained in the use of the heparinized plasma for dye
estimation as against the acetone method on the same plasma indicates a great
discrepancy between the two. In 150 tests in which the direct colorimetric estimation of the plasma dye content was made as against the acetone method, 36
per cent gave lower readings with acetone, 40.6 per cent higher readings and 23.4
per cent identical readings. Of the latter, 26 of 35 specimens or 74 per cent
represented 100 per cent dye removal from the plasma. This can readily be
understood from the mere naked eye examination of the protein precipitated by
the acetone, in which there often can be clearly seen a marked variation in the
color of the proteins, evidencing a variable degree of removal of the dye by adsorption to the proteins. Furthermore, the dilution factor of the acetone must
also play a role in this variation.
BROMSULPHALEIN TESTS
Each patient was given 5 mgm. per kilogram body weight of dye and the specimens withdrawn 15 and 30 minutes following injection. Twenty cubic centimeters of blood were
removed each time and divided equally into a dry tube (for serum), into a tube containing
20 mgm. of dry potassium oxalate, and into a third tube containing 0.1 cc. of Liquaemin.
The serum was collected in the routine manner, and oxalated and heparinized specimens
were treated identically. Comparisons were made with bromsulphalein standards freshly
prepared. The color produced by the addition of sodium hydroxide in order to bring out
the bromsulphalein color was a clear purple shade with heparinized plasma, while that of
the serum and oxalated plasma was burgundy color. Comparison with the standards in a
Hynson, Westcott and Dunning comparator was easier with heparinized plasma. In the
presence of hemolysis the sodium hydroxide produced a brown color so that comparisons
were difficult or impossible.
314
ISADORE E. GERBER AND MARYA FRYCZYNSKI
In table 1 there is listed the incidence of hemolysis using serum, heparinized
plasma, and oxalated plasma. The superiority of the heparinized plasma is
readily evident.
TABLE 1
BROMSULPHALEIN
TESTS—COMPARISON OF SERUM,
PLASMA
OXALATED
AND
HEPARINIZED
NUMBER OF SPECIMENS HEMOLYZED
SPECIMEN
Number
Heparinized plasma
Serum
Oxalated plasma
3
33
41
Colorimetric
comparison
approximated
Colorimemetrie
comparison
unobtainable
1
27
2
6
4
37
TOTAL
NUMBER OT
SPECIMENS
119
123
118
Number of heparinized specimens hemolyzed when oxalated specimens and serum specimens not hemolyzed: 0.
Number of sera hemolyzed when oxalated specimens and heparinized specimens not
hemolyzed: 13.
Number of oxalated specimens hemolyzed when heparinized specimens and serum
specimens not hemolyzed: 21.
All three hemolyzed: 3.
COMMENTS
The value of a dye test is in proportion to the accuracy of its performance,
aside from the natural limitations of the test. In view of the fact that there are
certain evident limitations to both the congo red test and the bromsulphalein test
it is highly important that technical error be reduced to a minimum. By far the
greatest technical error introduced is that of hemolysis. The various causes of
hemolysis are the too rapid withdrawal of blood, the forcible ejection of the blood
from the syringe, the use of a wet syringe or needle, inadequate mixing of the
blood with the anticoagulant and the improper withdrawal leading to t h e formation of small clots in the syringe. All of these errors can be avoided by adequate
attention on the part of the individual performing the test and will, no doubt
contribute greatly to a more accurate determination. In addition, however,
hemolysis may result from the anticoagulant or from the handling of the blood in
the laboratory whether clotted or not. A superficial glance at a set of sera used
for the Wassermann test will suffice to show the casual observer the high incidence of hemolysis present in sera.
Although sera carefully prepared rarely show hemolysis, yet in the average
laboratory the need for speed often leads to too early removal of the clot, undue
shaking, etc. with resultant hemolysis. Potassium oxalate per se is hemolytic
and blood allowed to stand in the presence of oxalate will show gradual increasing
hemolysis, a fact which we have demonstrated to our satisfaction many times.
By contrast, however, heparinized blood may be allowed to stand for periods up
to 24 hours without any visible hemolysis. Unless unduly shaken or badly
handled heparinized blood, as is readily seen from the table, will show far less
hemolysis than either serum or oxalated blood.
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HEPARINIZED BLOOD FOE CONGO RED TEST
The apparent high incidence of hemolysis with the congo red tests may be due,
we believe, to the hemostyptic action of the congo red with tendency to clotting
in the syringe and the resultant need for forcible expression. We were particularly careful in estimating the presence of hemolysis and very liberal in its interpretation. In many instances slight degrees of hemolysis are overlooked in
the average laboratory since a slightly reddened blood may so merge with the red
color of the dye as to be imperceptible. This factor often leads to erroneous results in the evaluation of the amount of dye in the blood.
The objection in the past to the use of heparin because of its cost is no longer
valid today. A 10-cc. vial of Liquaemin is adequate for 95 to 100 specimens and
allowing 2 specimens per test, the cost would be approximately 6 cents per complete test. We believe that this is not high considering the loss of time and
inconvenience to patient, physician and laboratory through an inadequate test
resulting from hemolysis. We were not able to do studies upon other dye comparisons or other color compounds in the blood such as icterus index, etc. since
our institution is limited to patients with tuberculosis in whom jaundice is rarely
encountered. However, we believe that heparinized blood has a wide range of
usefulness in such fields, as for example, blood volume determinations, icterus
index, etc.
CONCLUSIONS
Heparinized blood used for the congo red and bromsulphalein tests requires less
time for the performance of these tests than the serum or acetone serum comparison methods. The incidence of hemolysis is markedly reduced with heparinized blood as against serum or oxalated blood. The advantages of heparin
over serum and oxalated blood for dye or color comparisons of the blood warrant
further extensive trials.
REFERENCES
(1) F R I E D M A N , M . M . , AND A U E R B A C H , O . :
An improved congo red test for a m y loidosis.
J . L a b . a n d Clin. M e d . ,
21: 93 (Oct.) 1935.
(2) TARAN, A . : F u r t h e r improved congo
red test for amyloidosis. J . L a b . a n d
Clin. Med., 22: 975 (June) 1937.
(3) J O R P E S ,
JERIK:
Heparin.
Oxford
Univ. Press, London: p . 54-64, 1939.
(4) DRAGSTEDT, C . A., AND M I L L S , A. M .
T h e employment of oxalated plasma
i n t h e bromsulphalein d y e retention
test. J . L a b . a n d Clin. M e d . , 2 1 :
1936.