IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. BME-32, NO.

5, MAY 1985 349

relates with brain size, standing waves, and they postulate a global is characterized by a button of agglutinated cells at the bottom cen-
wave theory for EEG [2]-all are supportive of a mechanical hy- ter of a well surrounded by a relatively clear supernatant, while a
pothesis. There is also a physiological reason: alpha has been known negative reaction is characterized by a uniform suspension of red
to return to normal after brain death with increased cardiac output. cells. A weakly positive reaction is seen as snmall clusters of cells
I have not squashed the cardiac electromechanical model but have spread across the bottom of a well. Examples of these reactions are
pursued patient data. In a recent study, cardiac periodicity in EEG shown in Fig. 1.
not related to ECG crosstalk has been observed. A paper presenting An automated microplate reader [4], which reads light absor-
this is now being prepared. bance vertically through each well at specific wavelengths, is used
Knowledge of the origin of EEG should improve the diagnostic in these systems to detect hemagglutination reactions. In some sys-
utility of this technique. As to the difference in concepts for the tems the average light absorbance is measured using a broad beam
origin, I paraphrase Jefferson and Freidman: ignorance is prefer- of light, while in other systems a narrow beam is used to measure
able to error and each should be free to choose his own model. light absorbance at an off-center position. In either case, low light
absorbance indicates a positive reaction while high light absorbance
REFERENCES indicates a negative reaction. If an intermediate absorbance is mea-
[1] R. Cohn, "Discovery of hidden data in EEG by power spectral density sured, the reaction is automatically classified as "questionable" and
and pattern recognition algorithm," Electroencephalogr. Clin. Neuro- an operator may be allowed to visually inspect the well to determine
physiol., vol. 58, p. 7, 1984. if the reaction is positive or not. After all of the reactions have been
[2] P. Nunez and R. Katznelson, Electric Fields of the Brain. Oxford, categorized as positive or negative, the pattern of reactions for a
England: Oxford University Press, 1981, pp. 249, 356, 385, 296. sample is compared to template patterns to determine the ABO/Rh
test results.
In a previous study [3], serological and microplate reader param-
eters were optimized for a system which measured absorbance at a
single location in each microplate well. In doing so, the noninter-
An Improved Method for Detection of pretation rate (i.e., the fraction of samples requiring either operator
input or retesting) was reduced from 30 percent to 7 percent. This
Hemagglutination Usintg an Automated rate was further decreased to 5.5 percent [5], a value which is com-
Microplate Reader parable to the noninterpretation rate of more expensive automated
systems. The majority of tests which were not interpreted auto-
MATTHEW L. SEVERNS AND LINDA M. KLINE matically contained weak reactions which were clearly distinguish-
able by the operator.
The goal of the present study was to attempt to more sensitively
Abstnwt--Several new automated microplate readers which can take detect weak reactions. This should decrease the number of samples
multiple readings in each well have, been commercially developed. Data which require operator intervention or retesting. In addition, im-
gathered from one of these readers have been analyzed to determine a proying the detection of weak hemagglutination reactions may al-
parsimonious algorithm for discriminating positive hemagglutination low the automation of other tests which routinely have weak positive
reactions from negative ones. The resulting classification algorithm results. Among these are tests for detection of irregular antibodies
provides more accurate classification of weak positive reactions than in sample plasma [6], [7], the crossmatching of donor and recipient
the one point measurements normally used with only a moderate in- blood [7], direct agglutination tests for rickettsiae, hemagglutina-
crease in computational complexity. tion-inhibition tests for a wide variety of viruses, and the TPHA
test for syphilis [8].
I. INTRODUCTION Since several commercially available automated microplate read-
ers can be easily modified to take readings at more than one position
Every year, over 12 million units of blood are collected in the in each well, we wanted to determine if additional information ex-
United States. For each donation, ABO/Rh test results must be de- ists in these measurements which would allow more sensitive de-
termined. Because of the serious complications which may arise if tection of weak positive reactions. One constraint which was placed
incompatible blood is transfused, it is important that each unit of upon this work was that the computational algorithm must be rela-
blood be tested correctly. Present methods of ABO/Rh testing in- tively simple. This is due to the fact that a microcomputer is used
volve mixing samples of donor red blood cells with several different to process the data, and it must be able to classify 80 reactions in
reagent antisera and observing each test to see if the red cells ag- less than 10 s in order to maintain adequate sample throughput.
glutinate, or clump together. For ABO determination, complemen- Therefore, only one-dimensional classification schemes were con-
tary tests are performed by mixing samples of donor plasma with sidered. In addition, the amount of redundant information which is
red blood cells of known antigenic specificity. The interpretation of measured should be minimized to further reduce computational
both sets of tests must agree before an ABO group can be assigned. complexity.
Until a few years ago, automated systems for ABO/Rh testing
were very expensive and could be cost justified only in major blood II. METHODS
collection facilities. Recently, inexpensive systems for automated All of the hemagglutination reactions used in this study were
ABO/Rh testing have been developed [1]-[3] which perform all of produced by ABO group antigens and antibodies. Microplates con-
the necessary tests in the 96 wells of a plastic disposable called a taining serologic reactions were prepared by first diluting A1, A2,
microplate. After a dilute suspension of donor red blood cells is and B cells to a 1.5 percent suspension in 0.9 percent saline solution
mixed with appropriate antisera and donor plasma is mixed with to which 0.02 percent Tween-20 (polyoxyethylenesorbitan laurate,
red cells of known antigenic specificity, the microplate is centri- Sigma Chemical Co., St. Louis, MO) was added. Weak positive,
fuged to pellet the red cells and then agitated to resuspend them. strong positive,and negative hemagglutination reactions were pro-
Cells which have agglutinated will settle to the bottom of a well duced in the wells of microplates by reacting appropriate plasma
within 2-5 min, while cells which have not agglutinated will remain samples (obtained by screening blood from random donors) with the
in suspension for 10 min or more. Thus, a strong positive reaction prepared red cells. Eight replicates of four weak positive reactions,
four strong positive reactions, and three negative reactions were
Manuscript received June 29, 1984. This work was supported in part by produced in each microplate. Plates were prepared by adding either
Dynatech Laboratories, Inc. 25 p.1 of plasma, 50 p.1 of plasma, or 25 IA1 of plasma plus 25 IA1 of
- The authors are with the Biomedical Engineering Laboratory, American saline to 25 p.1 of a 1.5 percent cell suspension. Alternatively, 35 1l
Red Cross Blood Services Laboratories, Bethesda, MD 20814. of plasma was added to 35 g1 of cell suspension. Preparing micro-

0018-9294/85/0500-0349$01.00 i 1985 IEEE

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350 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. BME-32, NO. 5, MAY 1985

TABLE I
CLASSIFICATION RATES FOR REACTIONS

Classifier: Optimum One Point

Percentage Classified as
Reaction Number in
Grade Test Set Positive Questionable Negative
Fig. 1. Drawings of a strong positive (grade = 4) hemagglutination reac-
tion, left; a weak positive (grade = 1) reaction, center; and a negative 0 1176 0.68 6.38 92.95
reaction, right. The line through the center of each drawing indicates the 0.25 288 10.76 42.71 46.53
approximate positions at which measurements were taken. 0.5 256 25.78 62.10 12.11
1 456 53.51 44.30 2.19
2 232 87.07 12.93 0.00
plates using different volumes of cells and plasma allowed us to 3 248 89.52 3.63 6.45
4 1208 94.95 4.80 0.00
evaluate the effects of operational variability. All of the reac-
tions were prepared in rigid styrene U-bottom microplates (Dyna- Classifier: Derived Canonical Variable
tech Laboratories, Inc., Alexandria, VA, or Flow Laboratories, Percentage Classified as
Inc., McLean, VA). Reaction Number in K
Microplates were centrifuged and then shaken to resuspend the Grade Test Set Positive Questionable Negative
cells using an established protocol [3]. After the resuspended cells
had settled for 4 min, the microplate was automatically read. In 0 1176 0.00 2.21 97.79
addition, a technologist graded each replicate of reactions using a 0.25 288 32.98 26.39 40.63
standard classification scheme which was expanded to include weak 0.5 256 81.64 13.67 4.69
and very weak categories. Reactions were graded as 4 (very strong), 1 456 98.90 0.88 0.22
3 (less strong), 2, 1, 0.5 (weak), 0.25 (very weak), or 0 (negative). 2 232 100.00 0.00 0.00
3 248 94.35 4.84 0.81
Wells which contained no cells were coded as -1 (empty). 4 1208 100.00 0.00 0.00
A prototype automated microplate reader (Dynatech Laborato-
ries, Inc., Alexandria, VA) was used to make 24 absorbance mea-
surements in a straight line through the center of each microplate
well. The data were transferred to a microcomputer (Applied Micro Each classification algorithm was evaluated by measuring its per-
Technology, Inc., Tuscon, AZ) and then to a VAX-11/780 (Digital formance on a set of 3864 reactions. The number of samples be-
Equipment Corp., Marlborough, MA) for subsequent analysis. longing to each reaction category is shown in Table I. The ability
It was necessary to characterize the microplate reader before of each classification variable to separate populations of reactions
analyzing the data from it. The principal variable of interest, the was measured using a normalized Mann-Whitney U score [12] of
ratio of light beam diameter to the distance between measurements, negative reactions versus positive reactions which were scored
was determined by measuring the absorbance profile which oc- manually as 2 or less. The normalized variable is given by
curred when a razor blade attached to a microplate was passed (2 U/np - 1) where U is the raw Mann-Whitney score, n is the
across the light beam. If the light beam is circular when the razor number of negative reactions, and p is the number of positive re-
blade has moved a distance x into a beam of radius r, the absor- actions. The normalized score has a value of 0.0 when the popu-
bance A is given by lations completely overlap and a value of 1.0 when the populations
A =0 x/r < 0
are completely separated. This measure of separability was chosen
over some of the more standard measures [13] because it does not
2- require the estimation of the density functions of the individual pop-
1
A'= -log -
7
sin-' xr +
x
r
0 < xlr < I ulations and it is not computationally intensive.
IV. RESULTS
A = do Fig. 2 shows the absorbance profiles of a strong positive (grade x/r > 1.
An interactive technique was used to estimate parameters from the 4), a weak positive (grade 0.5), and a negative reaction. The sudden
resulting absorbance data. The beam diameter was found to be steep rise in absorbance in the middle of the well for a strong pos-
2.4 times the distance between measurements (residual sum of itive reaction is caused by the dense central agglutinant button. The
squares = 0.00042; S.E.E. is 0.012). The beam diameter was also decrease in absorbance at the edges of the negative reaction is
measured using a micrometer and was found to be 0.76 mm. caused by a decreased light path due to the curvature of the well
and the meniscus of the liquid. The absorbance profile of the weak
III. DATA ANALYSIS positive reaction is caused by small agglutinants settling to the bot-
tom of the well and sliding toward the center. The absorbance mea-
A set of 600 reactions was selected for study. There were 336 surements at the far edges of the microplate well were not analyzed
negative, 40 grade 0.25, 48 grade 0.5, and 176 grade 1 reactions in because of large excursions which are apparently related to the in-
the data set. Data analysis was performed using the RS/1 (Bolt, teraction of the light beam with the verticaledge of the well. When
Beranek and Newman, Cambridge, MA) and BMDP (University of these points are discounted, a total of 19 data points remains.
California at Los Angeles) computer programs. Principal compo- If the absorbance of light within the microplate well were deter-
nent analysis [9] was conducted on the covariance matrix of the data mined in accordance with Beer's law, the mean of the absorbance
using BMDP4M to determine the underlying dimensionality of the measurements in a well should depend only upon differences in cell
measurements. The classification variables were derived using a suspension. In fact, positive reactiong have a lower average absor-
stepwise linear discriminant algorithm [10]. This algorithm first de- bance than negative reactions (p < 0.001, ANOVA with contrasts).
termines which of the available variables contribute significantly to This is apparently due to nonlinearities in the instrument above an
the differences between groups and then forms canonical variables absorbance of 2.0 and the relatively unpredictable effects of the light
from the selected variables. The canonical variables will optimally beam interacting with agglutinant particles whose sizes are of the
discriminate among groups provided that the assumption of nor- same order as the beam diameter. Linear regression of mean well
mality of distributions is not violated. A matrix of hypothesis con- absorbance by reaction grade showed that the mean absorbance de-
trasts [111 was specified to optimize separation of positive and neg- creased by 0.12 per reaction grade (p < 0.001, t test), but the cor-
ative reactions using a single variable without distinguishing relation coefficient of the regression is small (R2 = 0.368) due to
between grades of positive reactions. the large variability in absorbance for each reaction grade.

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IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. BME-32, NO. 5, MAY 1985 351

2.2 -

2.0

1.8-

18-

1.4-

83
c L.2- Negative
a0 -

.Positive
L
0 Weak Positive
U) j.o -- --

.0.

0.8

0.8

0.4

0.2 -

a lo
i
ii i2
-
i3
-
14
I
15
15 is18 1-
V7 is i9 20

Measurement Position
Fig. 2. Absorbance profiles of negative, strong positive (grade = 4) and weak positive (grade = 1) reactions measured using the reader.

Because of the beam overlap between measurements, it was ex- coefficients and measurement positions selected appear to be re-
pected that the underlying dimensionality of the data would be- less lated to the slope of the absorbance curve between measurement
than the number of measurements taken. The dimensionality of the positions 6 and 12.
data revealed by principal components analysis is substantially less The Mann-Whitney U test, when applied to each individual mea-
than can be explained by the measurement overlap alone. The first surement position, indicated that the best single measurement for
three principal components of the data accounted for 94.2 percent discriminating negative from positive reactions classed manually as
of the total variance; the first principal component accounts for 62.7 2 or less was A(6). The normalized Mann-Whitney score for S on
percent of the total. These components after "varimax" rotation the test set of 2408 weak positive and negative reactions was 0.975
correspond to measurements at the near edge of the well, measure- (raw Mann-Whitney U was 1430754 with 1176 negative and 1232
ments at the center of the well, and measurements at the far edge positive reactions). The corresponding score for the one point clas-
of the well. The inherently low dimensionality of the data indicates sifier on the same test data was 0.887 (raw Mann-Whitney U was
that a one-dimensional classifier should work reasonably well to 1366858). When the mean well absorbance was used to discrimi-
discriminate positive from negative reactions. nate reactions (roughly analogous to a wide beam of light illumi-
After initial data analysis, it became clear that attemptfs to clas- nating the entire well), the normalized Mann-Whitney score was
sify reactions as "positive," "negative," or "empty," using a sin- 0.650 on the test data (raw Mann-Whitney U was 1195461.5).
gle classification variable did not work well; derived variables which To further evaluate the performance of each classification algo-
performed well at separating weak positive and negative reactions rithm, an absorbance histogram such as the one shown in Fig. 3
poorly separated strong positive and empty wells. Therefore, a se- was generated. Upper and lower thresholds were estimated for each
quential classification scheme was employed. The data were first data set and every reaction was classified by comparing the derived
tested to see if the well was empty, then nonempty wells were tested measurement variable to these thresholds. Measurements which
for the presence of a positive or negative reaction. Because of the were less than the lower threshold were counted as positive reac-
variable size and location of the agglutinant buttons in strong pos- tions, measurements which were greater than the upper threshold
itive reactions, no single measurement position served as a reliable were counted as negative reactions, and measurements which fell
indicator of empty wells. Examination of the data led to a heuristic between the thresholds were classed as questionable. This proce-
algorithm: if the maximum absorbance in the well was less than dure mimics the current procedures for classification of hemagglu-
0.5, the well was classified as empty. tination reactions. The results of the classification.algorithms are
When the stepwise linear discriminant algorithm was applied to shown in Table I.
the selected data set, the following canonical variable was arrived
at: V. CONCLUSIONS
S =CIMR + C2A(6) + C3A(8) + C4A(9) + C5A(l1) It is possible to more sensitively detect weak positive reactions
where MR is the maximum absorbance in the well, A(n) is the ab- in microplate wells by measuring absorbance in multiple locations
sorbance measured at position n, and Ci are the coefficients of the in each well. The low dimensionality of the data revealed by prin-
equation. (For reference, position 12 is the center of the well.) The cipal components analysis indicates that only a few key parameters

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352 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. BME-32, NO. 5, MAY 1985

800 T

750 t

700 t

850 t
800 t
Liii] Grade = 4
550f
Grade = 3
500 t
U)
0) 450 Grade = 2
ao
c
02
L 400- Grade = I
L
a 350-1-
0 Grade = 0.5
300 ±
Grade = 0.25
250 ±
- Negative
200 -

150±
1oo t
50

0
.8 .8 1.0 1.2 1.4
.2 .4
Canonical Variable
Fig. 3. Histograms of the distribution of the derived canonical variable for each reaction class. Thresholds for separating positive
from negative reactions were set at -5.7 (upper positive limit) and -5.9 (lower negative limit). Reactions falling between these
thresholds were classed as questionable.

need to be established to describe a reaction. A one-dimensional sands of reactions necessary for this study, and L. I. Friedman who
sequential classification scheme was devised which first determines reviewed several' drafts of this paper.
whether a well contains a reaction and, if so, classifies it as positive REFERENCES
or negative. This method more sensitively detects reactions of grade
1 or less (p < 0.001, chi-square test) while misclassifying fewer
[1] A. Nieri and P. Innocenti, "Micromethods in immunohematology: The
automatic reading. Experience at the transfusional center of Treviso
negative reactions (p = 0.007, chi-square test) than do one point hospital," La Transfi del Sangue, vol. 27, pp. 65-69, 1982.
measurement classifiers. As illustrated in Table I, the new classi- [2] A. R. Bowley, I. Gordon, and D. W. Ross, "Computer controlled
fication algorithm properly detects 82 percent of all reactions automated reading of blood groups using microplates," Med. Lab.
classed as weak (grade = 0.5), while the optimum one point clas- Sci., vol. 41, pp. 19-28, 1984.
sifier detects only 26 percent. [3] M. L. Severns, S. L. Schoeppner, M. J. Cozart, L. I. Friedman, and
Although the distributions of the individual variables are not nor- M. S. Schanfield, "Automated determination of ABO/Rh in micro-
mal, and thus the derived canonical variable is not truly "optimal," plates," Vox Sang., vol. 47, pp. 293-303, 1984.
the procedures used are sufficiently robust that it is unlikely a more [4] V. M. Genta and J. H. Bowdre, "Evaluation of two photometers used
sensitive detection algorithm can be developed using the present in enzyme-linked immunosorbent assays," J. Clin. Microbiol., vol.
16, pp. 168-173, 1982.
instrument. Several attempts were made to include other derived [5] T. A. Kolb, D. Earp, and T. Wise, "ABO/Rh testing by automated
variables, such least-squares estimates of the slope of the absor- microplate using the Microbank system," in Proc. 18th Congr. Int.
bance curve at different points in the well. None of these variables Soc. Blood Transf., 1984.
by themselves provided better discrimination than a single off-cen- [6] Methods in Immunology and Immunochemistry, vol. IV, C. A. Wil-
ter measurement (as determined by the Mann-Whitney U test), and liams and M. W. Chase, Eds. New York: Academic, 1977, pp. 1-75.
several of them were so non-Gaussian (as determined by the Kol- [7] Technical Manual of the American Association of Blood Banks, F. K.
mogorov-Smirnov test) that they caused irregular behavior of the Widman, Ed. Washington, DC: American Association of Blood
statistical packages. Banks, 1981.
While detection sensitivity has been improved significantly, it [8] Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infec-
still falls short of the capabilities of a trained technologist. Thus, tions, E. H. Lennette and N. J. Schmidt, Eds. Washington, DC:
American Public Health Ass., 1979, pp. 25-26.
it is unlikely that critical hemagglutination tests which normally [9] C. Chatfield and A. J. Collins, Introduction to Multivariate Analy-
exhibit very weak reactions (such as the crossmatching of donor and sis. New York: Chapman & Hall, 1980, pp. 57-79.
recipient blood) can be automated using this technology. It is pos- [10] R. I. Jennrich, "Stepwise discriminant analysis," in Statistical Meth-
sible, however, that more sensitive serologic methods, for example, ods for Digital Computers, A. Ralston, Ed. New York: Wiley, 1977,
solid phase assays [14], could be utilized with this reader to perform pp. 76-95.
some of these tests. The modified reader and new algorithm should [11] BMDP Statistical Software, W. J. Dixon, Ed. Berkeley, CA: Uni-
be capable of automating-several other tests such as DU testing (a versity of California Press, 1983, pp. 679-681.
weak expression of the Rho antigen) and syphilis testing. In addi- [12] R. R. Sokal and F. J. Rohlf, Biometry. San Francisco: CA: Free-
man, 1981.
tion, the new classification algorithm should significantly reduce [13] W. Meisel, Computer-Oriented Approaches to Pattern Recogni-
the number of ABO/Rh tests which require operator intervention. tion. New York: Academic, 1972, p. 197.
ACKNOWLEDGMENT [14] R. E. Rosenfield, S. Kochwa, and Z. Kaczera, "Solid phase serology
for the study of human erythocytic antigen-antibody reactions," in
We would like to thank S. Schoeppner who prepared the thou- Proc. XII Congr. Int. Soc. Hematol., 1978, pp. 27-33.

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