Immunol Res (2014) 58:7074

DOI 10.1007/s12026-013-8480-1

Autoimmune hemolytic anemia with gel-based

immunohematology tests: neural network analysis
Marco Lai Valerio De Stefano Raffaele Landolfi

Published online: 27 December 2013

European Union 2013

Abstract In a previous report, we investigated the capability of commercially available immunohematology tests
based on gel technology to add useful information for the
diagnosis of autoimmune hemolytic anemia (AIHA). In
this report, we analyzed the same casuistic to find useful
information on the importance of different immunohematology tests for the AIHA diagnosis, but using the artificial
neural network (ANN) analysis. We studied 588 samples
with a positive direct antiglobulin test (DAT), of which 52
samples came from patients with AIHA. The samples were
analyzed with the ANN using the multilayer perceptron
with the backpropagation algorithm. Using the ANN in the
observed data set, the predictive value for the presence of
AIHAs was 94.7 %. The rate of DAT-positive cases that
were not AIHA and that were correctly classified was
99.4 %. The receiver operating curve area for the model
was 0.99. The independent variable importance analysis
found that the gel centrifugation test anti-IgG titer was an
important contributor to the network performance, but
other variables such as the IgG subclasses can also be
considered important. The use of the ANN permitted us to
identify immunohematology tests that were hidden with
the common statistical models used previously. This was

M. Lai (&)
Internal Medicine Department, Transfusion Centre, Catholic
University, Largo A. Gemelli 8, 00168 Rome, Italy
e-mail: marco_lai@fastwebnet.it
V. De Stefano
Clinical Hematology Institute of Hematology, Catholic
University, Rome, Italy
R. Landolfi
Internal Medicine Department, Catholic University of Sacred
Heart, Rome, Italy

123

the case for the IgG subclasses. However, it is very likely

that the information given to the network from those tests is
quantitative rather than qualitative.
Keywords Immunohematology tests in auto immune
hemolytic anemia
Artificial neural network analysis

AIHA
Direct antiglobulin test

Introduction
The immunohematology tests for warm autoimmune
hemolytic anemias (AIHAs) were previously investigated
with the aim of improving their diagnostic ability. In these
reports, the authors studied the quality of the proteins on
red blood cells (RBC) in AIHA cases. These investigations
arose because the direct antiglobulin test (DAT), which is
the laboratory gold standard for AIHA diagnosis, is often
positive in the absence of autoimmune hemolysis. The
authors argued that qualitative or quantitative factors are
present on red blood cells, which could suggest the presence of autoimmune hemolysis.
The IgG subclasses were extensively investigated, but
with discordant results. In previous studies using tube
methods [1] and gel technology, [2, 3] the quantitative factors seemed to prevail over the qualitative ones. However,
even though some tests do not show a direct relationship with
the diagnosis, the same tests can increase or enhance the
resolution of those most strongly correlated, such as the antiIgG titer or the DAT strength. Analysis systems like the
artificial neural network (ANN) allow us to make these
assessments. Therefore, we reanalyzed a data set published
previously in which we used logistic regression and chisquared automatic interaction detector (CHAID) [3]. In the
results obtained with the ANN analysis, we observed not

Immunol Res (2014) 58:7074

only a higher predictive rate for an AIHA diagnosis, but also

a set of variables that contribute to this result. The utility of
those variables was completely missed in previous studies
using traditional methods for statistical analysis.

71
Table 1 Results of the classification with the ANN. The AIHA cases
and not AIHA cases with positive DAT
Classification
Sample

Observed

Predicted
AIHA

NO
AIHA

Percent
correct (%)

Materials and methods

Training

Patients
The study included 588 blood samples from 588 patients.
All of these samples yielded a positive-DAT result. Blood
specimens and analyses were as previously described [3].
All the immunohematology tests were performed with
commercially available gel centrifugation test (GCT) cards
as previously described. A diagnosis of AIHA was made
following the rules previously detailed. Samples from
AIHA patients were taken at diagnosis.

Statistical analysis
We used the multilayer perceptron network architecture
with the backpropagation algorithm and a second phase of
training with the conjugate gradient descendent algorithm.
The 588 samples were randomly assigned by the program
to the training sample (67.5 %, 397 cases) and to the test
sample (32.5 %, 191 cases). The selected activation function for hidden layers was hyperbolic tangent. The selected
activation function for output layer was sigmoid, which
introduces nonlinearity in the network. Otherwise, the
network could only learn functions that are linear combinations of the inputs. The error function was sum of
squares.

Testing

AIHA

27

81.8

NO AIHA

357

98.1

Overall percent (%)

8.6

91.4

96.7

AIHA

18

94.7

NO AIHA

171

99.4

Overall percent (%)

9.9

90.1

99.0

The results are detailed for the training and the testing phase
Dependent variable: AIHA

classified as not AIHA by ANN analysis. Of the 19 patients

in the test sample diagnosed with AIHA, 18 (94.7 %) were
correctly diagnosed with AIHA by the ANN analysis.
Table 1 shows the complete data for the classification
obtained by the ANN analysis in the training and testing
phases. Figure 1 shows the MLP network graph in which
the input variables are indicated: the two hidden layers and
the classifier dichotomous variable AIHA (AIHA, NO
AIHA). Figure 2 shows the ROC curve. The area for the
model is 0.99.
The independent variable importance analysis (Table 2;
Fig. 3) gave the following results: GCT anti-IgG titer
100 %; IgG1 strength at dilution 100, 79.6 %; IgG3
strength at dilution 100, 62.8 %; IgG3 strength, 50.2 %;
IgG strength, 15.7 %; C3d strength, 10.1 %; IgG1 dilution,
7.1 %; IgG1 strength, 5.9 %; IgG3 dilution, 4.3 %; DAT
strength, 1.9 %; C3d, 1.6 %; IgG3, 0.9 %, and Mult_Ig,
0.6 %.

Results

Discussion

We analyzed 588 samples [3] of which 52 were from

patients diagnosed with warm AIHAs. ANN analysis was
performed by entering the following input predictors: DAT
strength, GCT anti-IgG titer, IgG1 (positive or negative).
IgG3 (positive or negative), IgG strength, C3d strength,
IgG1 dilution (1, 100), IgG3 dilution (1, 100), IgG1
strength, IgG3 strength, IgG 1 strength at dilution 100
(IgG1 strength at 100), IgG3 strength at dilution 100 (IgG3
strength at 100), multiple immunoglobulins (positive or
negative; Mult_Ig), and C3d (positive or negative).
The statistical software randomly assigned 397 cases to
the training sample and 191 cases to the test sample.
Between the 172 patients (test sample) who were DATpositive without AIHA, 171 (99.4 %) were correctly

Investigations of RBC surface proteins in patients with

warm AIHAs have been inconclusive. The same IgG class,
IgG subclass, and complement composition were detected
in the DAT-positive cases in the absence of AIHA. It seems
that the quantity (quantitative factor) of immunoglobulins
(Ig) is more correlated with the occurrence and severity [4,
5] of warm AIHAs. In our previous report [3] using gel
technology, we found that the probability of having AIHA
using the GCT anti-IgG titer increased for a titer [100.
Other authors [611] suggested that differences in the Ig
class and IgG subclass (the qualitative factor) could play a
role in the autoimmune RBC hemolysis.
Most likely, the correlation of some immunohematology
tests with AIHA diagnosis can only be seen when two or

123

Immunol Res (2014) 58:7074

only a higher predictive rate for an AIHA diagnosis, but also

a set of variables that contribute to this result. The utility of
those variables was completely missed in previous studies
using traditional methods for statistical analysis.

71
Table 1 Results of the classification with the ANN. The AIHA cases
and not AIHA cases with positive DAT
Classification
Sample

Observed

Predicted
AIHA

NO
AIHA

Percent
correct (%)

Materials and methods

Training

Patients
The study included 588 blood samples from 588 patients.
All of these samples yielded a positive-DAT result. Blood
specimens and analyses were as previously described [3].
All the immunohematology tests were performed with
commercially available gel centrifugation test (GCT) cards
as previously described. A diagnosis of AIHA was made
following the rules previously detailed. Samples from
AIHA patients were taken at diagnosis.

Statistical analysis
We used the multilayer perceptron network architecture
with the backpropagation algorithm and a second phase of
training with the conjugate gradient descendent algorithm.
The 588 samples were randomly assigned by the program
to the training sample (67.5 %, 397 cases) and to the test
sample (32.5 %, 191 cases). The selected activation function for hidden layers was hyperbolic tangent. The selected
activation function for output layer was sigmoid, which
introduces nonlinearity in the network. Otherwise, the
network could only learn functions that are linear combinations of the inputs. The error function was sum of
squares.

Testing

AIHA

27

81.8

NO AIHA

357

98.1

Overall percent (%)

8.6

91.4

96.7

AIHA

18

94.7

NO AIHA

171

99.4

Overall percent (%)

9.9

90.1

99.0

The results are detailed for the training and the testing phase
Dependent variable: AIHA

classified as not AIHA by ANN analysis. Of the 19 patients

in the test sample diagnosed with AIHA, 18 (94.7 %) were
correctly diagnosed with AIHA by the ANN analysis.
Table 1 shows the complete data for the classification
obtained by the ANN analysis in the training and testing
phases. Figure 1 shows the MLP network graph in which
the input variables are indicated: the two hidden layers and
the classifier dichotomous variable AIHA (AIHA, NO
AIHA). Figure 2 shows the ROC curve. The area for the
model is 0.99.
The independent variable importance analysis (Table 2;
Fig. 3) gave the following results: GCT anti-IgG titer
100 %; IgG1 strength at dilution 100, 79.6 %; IgG3
strength at dilution 100, 62.8 %; IgG3 strength, 50.2 %;
IgG strength, 15.7 %; C3d strength, 10.1 %; IgG1 dilution,
7.1 %; IgG1 strength, 5.9 %; IgG3 dilution, 4.3 %; DAT
strength, 1.9 %; C3d, 1.6 %; IgG3, 0.9 %, and Mult_Ig,
0.6 %.

Results

Discussion

We analyzed 588 samples [3] of which 52 were from

patients diagnosed with warm AIHAs. ANN analysis was
performed by entering the following input predictors: DAT
strength, GCT anti-IgG titer, IgG1 (positive or negative).
IgG3 (positive or negative), IgG strength, C3d strength,
IgG1 dilution (1, 100), IgG3 dilution (1, 100), IgG1
strength, IgG3 strength, IgG 1 strength at dilution 100
(IgG1 strength at 100), IgG3 strength at dilution 100 (IgG3
strength at 100), multiple immunoglobulins (positive or
negative; Mult_Ig), and C3d (positive or negative).
The statistical software randomly assigned 397 cases to
the training sample and 191 cases to the test sample.
Between the 172 patients (test sample) who were DATpositive without AIHA, 171 (99.4 %) were correctly

Investigations of RBC surface proteins in patients with

warm AIHAs have been inconclusive. The same IgG class,
IgG subclass, and complement composition were detected
in the DAT-positive cases in the absence of AIHA. It seems
that the quantity (quantitative factor) of immunoglobulins
(Ig) is more correlated with the occurrence and severity [4,
5] of warm AIHAs. In our previous report [3] using gel
technology, we found that the probability of having AIHA
using the GCT anti-IgG titer increased for a titer [100.
Other authors [611] suggested that differences in the Ig
class and IgG subclass (the qualitative factor) could play a
role in the autoimmune RBC hemolysis.
Most likely, the correlation of some immunohematology
tests with AIHA diagnosis can only be seen when two or

123

72

Fig. 1 Multilayer perceptron network graph

123

Immunol Res (2014) 58:7074

Immunol Res (2014) 58:7074

73

Fig. 2 ROC curve for the AIHA model obtained with the ANN
analysis

Table 2 Importance analysis and the normalized importance analysis

given by the ANN for the input variables
Independent variable importance
Importance

Normalized importance (%)

IgG3

.003

0.9

C3d

.005

1.6

Mult_Ig
DAT_strength

.002
.005

0.6
1.9

GCT_anti_IgG_titer

.293

100.0

IgG_strength

.046

15.7

C3d_strength

.030

10.1

IgG1_dil

.021

7.1

IgG3_dil

.013

4.3

IgG1_strength

.017

5.9

IgG1at100

.234

79.6

IgG3_strength

.147

50.2

IgG3at100

.184

62.8

more tests are taken into account simultaneously. To find

hidden and multifaceted relationships between an AIHA
diagnosis and the immunohematology test, we used the
ANN analysis. The importance variable tool indicated that
as in our previous report [3], the GCT anti-IgG is one of the
tests most related to the AIHA diagnosis. However, the
ANN found that other variables are also important for the
network to provide high efficiency results. These are (in

decreasing importance) the IgG1 strength at dilution 100,

IgG3 strength at dilution 100, IgG3 strength, IgG strength,
C3d strength, IgG1 dilution, IgG1 strength, IgG3 dilution,
DAT strength, C3d, IgG3, and Mult_Ig.
The importance of the new relationships found with the
ANN comes from the higher efficiency in indicating AIHA
when compared with our previous report [3]. With the
ANN, the efficiency was 94.7 % (18 of the 19 AIHA cases
were detected in the test sample). Between the 19 cases
predicted as AIHAs, 18 were AIHAs (94.7 %). For the
DAT-positive cases without AIHA, the efficiency of the
ANN in predicting that the case was not AIHA was 99.4 %
(171 of the 172 cases in the test sample).
With the CHAID analysis performed in the same casuistic [3], DAT strength [4? predicted 50 of the 52 AIHAs
(96.1 % compared with 94.7 % with the ANN), but the
node was highly contaminated by the NON-AIHAs; i.e.,
25 of the 75 cases (AIHAs were 66.7 %). The CHAID,
adding the GCT anti-IgG titer to the DAT strength (4 ?),
selected 47 cases of which 38 were AIHA cases (80.9 %);
these correspond to 73.07 % of the 52 AIHAs compared
with 94.7 % of the ANN in this study (test sample).
A remaining question concerns the importance of the
qualitative factor in the autoimmune hemolysis [1]. The
adjunctive important variables detected with the ANN
belonged to the IgG subclasses. Some previous reports
underlined the effectiveness of the different IgG subclasses
in generating RBC destruction [7, 9]. Other authors, however, found that none of the IgG subclasses could be
associated, in absolute terms, to the destruction of RBC [1,
12]. We agree with this thinking. The new relationships
found with the ANN for IgG1 and IgG3 may be rolled back
to a quantitative factor. In a previous report, the authors
found that there was a significant relationship between the
amount of IgG on RBC and the presence of IgG3 and C3d
[13] The new relationships found with the ANN may add
quantitative information capable of increasing the efficiency of the network to indicate which ones are AIHAs
cases. The importance of the DAT strength was resized
when compared with our previous report [3] because this
new model gives a set of nonlinear relationships instead
of single linear correlation. Of note, the variables with low
significance in the analysis cannot be eliminated from the
analysis. This is because their elimination reduces the
efficiency in indicating the AIHAs cases (data not shown)
and the importance of the observed correlations is then
reduced.
This study indicates a new approach for evaluating the
relationship between immunohematology tests and the
AIHA diagnosis. Compared with linear models, [3] the
ANN model showed higher efficiency in correctly classifying cases of AIHA. The study indicated hidden variables that when combined in a nonlinear model show a

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Immunol Res (2014) 58:7074

Fig. 3 Importance variables

analysis. IgG1at100 is for IgG1
strength at dilution 100.
IgG3at100 is for IgG3 strength
at dilution 100. IgG1_dil is for
IgG1 dilution. IgG3_dil is for
IgG1 dilution. Mult_Ig is for
multiple immunoglobulins

strong relationship with the diagnosis of AIHA. This is

because those hidden variables can be observed only when
they can be driven by other variables, such as the GCT
anti-IgG titer or the DAT strength. Of course, the diagnosis
of AIHAs remains a clinical one because it requires the
integration of clinical and laboratory data. However, the
relationship between AIHA and the immunohematology
tests is a multifaceted and complex condition, but further
information can be obtained with new analysis approaches.

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