Download ELECTRONIC DEMONSTRATION OF BLOOD CELL AGGLUTININS

T H E AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Vol. 35, No. 2, pp. 105-108
February, 1961
Copyright © 1961 by The Williams & Wilkins Co.
Printed in U.S.A.
ELECTRONIC DEMONSTRATION OF BLOOD CELL AGGLUTININS
MARK J. HALLORAN, M.D., WILLIAM J. HARRINGTON, M.D., VIRGINIA MINNICH, M.S.
AND GRACE K. ARIMURA, B.S.
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri
Visual reading of serologic agglutination
reactions is subject to the bias of the observer. The availability of electronic instruments capable of distinguishing between
particles according to size provided an
opportunity for eliminating this subjective
factor.4 Antibodies for each of the 3 formed
elements of the blood were tested; readily
reproducible results which correlated well
with predicted values were obtained. The
study was designed chiefly as a means of
studying platelet agglutinins, however,
and this paper includes detailed data only
on these.
with the following modifications: (1) the
silicone used for coating glassware was diluted 1 to 20 with petroleum ether to minimize contamination with droplets of this
agent; (2) all distilled water was passed
through an ion exchange column primarily
to remove any particulate matter, and for
the same reason the ethylene diamine tetraacetic acid (disodium) was filtered through
Celite Analytical Filter Aidf and Whatman
No. 50 paper; (3) 30 rather than 20 min.
at 150 g were given to centrifugation of the
blood for preparation of the platelet-rich
plasma in order to assure complete separation of leukocytes and erythrocytes; (4) All
test serums were centifuged at 2000 X g
for 20 min., and if lipemic, their clear central portions were carefully pipetted into a
clean test tube (a precaution necessary only
on samples that had been stored in the
cold or frozen, inasmuch as thereafter any
neutral fat present tended to separate by
flotation). Three-tenths milliliter of a 1:1
dilution of test serum in isotonic saline
solution was added to 0.1 ml. of the platelet suspension which contained 300,000 to
400,000 platelets per cu. mm. The mixture
was stored for 18 hr. at 5 C , then agitated
at 80 r.p.m. on a rotary platform for 5 min.
One-tenth milliliter was diluted 1:100 in 10
ml. of isotonic saline solution.
METHODS
The apparatus used was the Coulter
Counter* outfitted with standard equipment for the enumeration of blood cells.1
This instrument records the number and
size of particles suspended in an electrolyte
solution. As 0.5 ml. of the suspension flows
through an aperture, 100 fi in diameter, a
change in the resistance between an electrode
on each side produces a voltage pulse of a
magnitude proportional to the particle
size. With appropriate scaler settings all
particles admitted, the larger or only the
largest may be recognized.
Platelet agglutination reactions were
performed as previously described,6 but
Received, May 16, 1960; revision received,
July 5; accepted for publication October 6.
Dr. Halloran is Assistant in Medicine, Dr. Harrington is Associate Professor of Medicine, Virginia Minnich is Research Assistant Professor,
and Grace Arimura is Research Assistant.
Presented at the annual meeting of the Southern Section, American Federation for Clinical Research, New Orleans, La., January 21, 1960.
Supported by Grant No. H22 (C14), United
States Public Health Service, and by gifts given
in memory of Mark Edison, Phillip Hunkel, Bill
Burns, and Frederick Jostes.
* Model A, Coulter Electronics Company,
Chicago, Illinois.
Studies on erythrocyte antibodies were
performed on a 2 per cent cell suspension.
Two-tenths milliliter of appropriate antiserum was added to 0.1 ml. of the suspension prepared either for Rh testing or for
the antiglobulin reaction. Standard technics
were used. Dilution for reading on the instrument was performed as described for
the platelets.
Tests for leukocyte agglutinins were
conducted according to the method of
Brittingham. 2 Leukemic leukocytes were
t Johns-Manville Products, New York City,
New York.
105
106
H A L L O R A N ET
Vol. 85
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TABLE 1
ELECTBONIC D E T E C T I O N OF BLOOD C E L L
AGGLUTININS*
I n s t r u m e n t Settings and Readings
Visual Readings
15/1
A.
B.
C.
D.
E.
29 control patients
11 patients with I T P
3 patients with I T P
3 control patients
22 patients with I T P
1 patient with drug-induced T P
(quinine)
Without quinine
With quinine
+
+
+
36
66
395
182
650
±
±
±
±
±
15/2
6
18
151
69
195
95
298
101
195
1122
788
1663
±
±
±
±
±
15/3
19
66
360
307
403
765
988
3971
2422
5823
±
±
±
±
±
170
500
130
412f
422J
1105§
750
911
210H
I n s t r u m e n t Settings and Readings
Erythrocytes
Anti-Rho serum
Rh-negative cells
Rh-positive cells
Antiglobulin serum
Autoimmune hemolytic
erythrocytes
Albumin control
Antiglobulin serum
Visual Readings
30/1
40/1
50/1
+
109
3362
73
1928
40
1107
+
342
1322
124
708
96
410
disease
I n s t r u m e n t Settings a n d Readings
Leukocytes
Normal serum
Isoimmune serum
Visual R e a d i n g s
+
40/1
50/1
60/1
90
1241
67
415
40
163
* The d a t a are a basis for comparison of visual and electronic readings of blood cell immunologic
reactions. With appropriate scaler settings, aggregates of blood cells diluted in t h e suspending medium
may be recognized, whereas individual cells of the particular line under study are not counted. The
figures given indicate the number of aggregates, in 0.5 ml., with a size greater t h a n t h e minimum predetermined by each combination of settings of threshold and aperture current.
In the instrument values on platelets 3 points are notable: (1) the lack of significant difference
between results with serums of control patients and most patients with I T P whose serums do not contain visually demonstrable platelet agglutinins; (2) the highly significant difference between either
control serums or I T P serums which are negative on visual reading, when compared to the I T P serums
graded as positive by the visual method; and (3) the finding of 3 positive I T P serums on instrument
readings which were negative by t h e standard method.
As noted in the text, t h e significance of the platelet aggregation is not established either by the
present method or any other technic currently used, but presumably in I T P most instances reflect
antibody-mediated agglutination of platelets.
t d for average A & B , p > 0.1.
t d for average A & E, p < 0.01.
§ d for average B & E, p < 0.01.
H The instrument setting for these two determinations was 70/1.
Feb.
1961
ELECTRONIC DETECTION OF ANTIBODIES
used in order to minimize erythrocyte contamination; the final concentration of cells
was 20,000 per cu. mm. Here also the final
reaction mixture was diluted 1:100.
Various scaler settings for threshold and
aperture current were observed to be necessary in order to make allowance for a range
in size of agglutinates in a given sample,
and to exclude recognition of unagglutinated
cells. For platelets, 3 aperture currents, 1, 2,
and 3, were used with a single threshold
setting, 15.J Additional threshold settings
of 20 and 25 were generally not of value although an exception to the lack of usefulness
of higher thresholds in platelet studies will
be noted later. For erythrocytes and leukocytes a single setting for aperture current, 1,
was used with threshold settings of 30, 40,
and 50 for the former, and 40, 50, and 60
for the latter cells.
In all instances conventional microscopic
evaluations were performed independently
on the undiluted samples and later compared with the results obtained on the instrument.
RESULTS
Platelet agglutinins. The results on serums
from 32 randomly selected medical admissions are compared in Table 1 to those obtained on 36 patients with idiopathic
thrombocytopenia (ITP) and 1 patient
with thrombocytopenia caused by quinine.
Two points are notable: (1) the great
spread among the values on ITP serums
between the 14 samples graded as negative
on visual reading and the 22 samples with
positive reactions; (2) the little difference
between the negative control group and the
negative I T P serums. There were three
t Empiric settings. Not necessarily interchangeable with other instruments of the same
model. Principles discussed in reference 6; with a
population of particles of uniform size a decrease
in threshold can be precisely off-set by a parallel
increase in aperture current. With the systems
used herein this reciprocal relation should also
obtain; however, when only one setting at a time
is varied a predictable change in count does not
take place, although it would be possible to determine with a high degree of precision the distribution in size of agglutinates if systematic variation
in a setting is made.
107
"false positives" 3 by both methods in the
control samples (1 sample from a woman
with systemic lupus erythematosus and 2
serums from patients with advanced
hepatic cirrhosis). Of possible significance was
the finding that 3 out of 14 ITP serums
graded negative by visual readings yielded
instrument values within the positive range
(15/2 readings of 853, 1007, and 1525),
whereas there was no increase in the number of positive reactions among control
serums when instrument values were compared to visual gradings.
Two trials were performed on serum from
a patient with quinine-induced thrombocytopenia. As is characteristic, the agglutination when the drug was present was
gross and composed of few but very large
clumps. Inasmuch as there were so few
agglutinates, the instrument count was
low, although the amplitude of each pulse
was high; however, when the threshold
was increased from 15 to 70 the background
values were minimized, thereby accomplishing a spread that was not present at the
lower threshold between the samples without quinine and those to which it had been
added.
Red cell antibodies. Listed also in Table 1
are the values obtained on erythrocytes.
Although only 2 trials were made, it is
evident that agglutination reactions involving these antibodies may also be easily
detected.
Leukocyte antibodies. Agglutination of
white blood cells from a patient with chronic
myelocytic leukemia was demonstrated
when serum from a patient who had received over 100 transfusions was tested
(Table 1). Here also the trial was performed
only to test a premise.
DISCUSSION
The use of instruments to replace visual
readings in laboratory procedures is desirable if certain criteria are fulfilled. Accuracy
and ease of operation are among the major
considerations. Both are achieved by the
method described herein. The study was
designed, however, not with the objective
of replacing present methods, but rather
with the intent of adding one more precau-
108
H A L L O R A N ET
tion against any pitfalls inherent in technics
involving subjective factors. The method
should not supplant microscopic readings,
inasmuch as extraneous particles or extraordinary size of aggregates which can be
simply evaluated microscopically are sources
of error in instrument readings. Nor does
the method distinguish between nonimmunologic aggregation of cells and agglutination owing to antibodies; in this respect a
major shortcoming of agglutination reactions in general remains unresolved.
A possible advantage of the technic is
suggested by the finding of an increased
incidence of positive reactions among I T P
serums when tests with the instrument are
compared to visual readings. Many more
serums must be examined, however, before
the platelet agglutination as recognized
only by the instrument can be related to
clinical data and interpreted.
Finally, as a corollary to some of the
observations described herein it should be
noted that aggregation of platelets in blood
samples from patients with thrombocytosis
introduces a significant error in the electronic enumeration of erythrocytes. Although platelet clumping is common in most
blood samples prepared for counting of
erythrocytes, the number of aggregates
which approximate or exceed the size of red
blood cells is generally insignificant unless
the platelet count is extremely high. When
the latter obtains "red blood cell counts,"
several hundred thousand per cubic millimeter above the true value may result.
Vol. 85
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SUMMARY
Agglutinins for blood cells can be readily
and reproducibly demonstrated on an
electronic instrument now available in many
hematology laboratories. Advantages and
limitations of its adaptation to serology are
discussed.
SUMMARIO I N
INTERLINGUA
Agglutininas pro cellulas de sanguine pote
esser demonstrate prestemente e reproducibilemente per medio de un instrumento
electronic que es currentemente disponibile
in multe laboratorios de hematologia. Es
discutite le avantages e le limitationes de su
adaptation a objectivos de serologia.
REFERENCES
1. B B B C H E B ,
G.,
SCHNEIDEEMAN,
M.,
AND
WILLIAMS, G. Z.: Evaluation of an electronic
blood cell counter. Am. J. Clin. Path., 26:
1439-1449, 1956.
2. B R I T T I N G H A M , T .
E.
AND C H A P L I N , H.,
JR.:
Febrile transfusion reactions caused by sensit i v i t y to donor leucocytes and platelets.
J . A. M . A., 165: 819-825, 1957.
3. DAUSSET, J . : Les thrombo-anticorps. Acta
Haemat., 20: 185-194, 1958.
4. HALLOBAN, M .
J.,
H A B E I N G T O N , W.
J.,
M I N N I C H , V., AND ARIMIIBA, G. K . : Elec-
tronic demonstration of platelet agglutinins.
Clin. Res., 8: 53, 1960. (Abstract.)
5. H A B B I N G T O N ,
W.
J.,
MINNICH,
V.,
AND
ARIMURA, G. K . : The autoimmune thrombocytopenias. In TOCANTINS, L. M . : Progress
in Hematology, Vol. 1. New York: Grune
and Stratton, 1956, p p . 166-192.
6. M A T T E R N ,
C.
F.
T.,
BRACKETT, F .
S., AND
OLSON, B . J . : Determination of number and
size of particles by electrical gating: blood
cells. J. Appl. Physiol., 10: 56-70, 1957.