Download Agglutination of an EDTA Blood Sample Caused by an EDTA

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TAK.EMORI ET AL.
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Agglutination of an EDTA Blood Sample Caused by an
EDTA-Dependent Panagglutinin
MARION E. REID, M.S., FIMLS, LINDA K. BOTTENFIELD, MT(ASCP), PEARL TCY TOY, M.D.,
SANDRA S. ELLISOR, M.S., MT(ASCP), AND CAROL A. HART, MT(ASCP)
A novel example of an EDTA-dependent panagglutinin is
described. A blood sample drawn into EDTA for a routine
hematologic workup demonstrated strong agglutination due to
EDTA-dependent panagglutinins. Previous examples have been
detected because of discrepant ABO results. This is the first
report of an EDTA-dependent panagglutinin that caused agglutination in the EDTA sample collection tube and a false
positive direct antiglobulin test. (Key words: Antibody EDTAdependent; Agglutination; EDTA-dependent panagglutinin) Am
J Clin Pathol 1985; 83: 534-535
ANTIBODIES to additives in commercially prepared
reagents have been implicated in anomalous blood
typing results,5 and several examples of antibodies that
react in the presence of ethylenediaminetetraacetic acid
(EDTA) have been reported.1"4,6 These EDTA-dependent
panagglutinins were detected by discrepant ABO results.
This is the first report of a patient whose serum had an
EDTA-dependent antibody that was detectd by a high
mean cell volume (MCV) and subsequent visual agglutination in the EDTA Vacutainer® tube and not by an
ABO discrepancy.
Report of a Case
A 58-year-old white woman with no history of transfusion or
pregnancy was seen by her physician for an annual physical examination.
She gave a history of anxiety, depression, and insomnia. Physical
Received June 26, 1984; received revised manuscript and accepted
for publication September 26, 1984.
Address reprint requests to Ms. Reid: American Red Cross Blood
Services, Central California Region, 333 McKendrie Street, San Jose,
California 95110.
American Red Cross Blood Services, Central California
Region, San Jose and Drs. Simard and Hoops Medical
Laboratories, Salinas, California
examination was unremarkable. Medications were Synthroid®, Sinequan®, Hygroton®, and monthly estrogen injections. The patient was
referred to a laboratory for routine tests. The blood sample for a
complete blood count (CBC) was collected in K3EDTA and cycled
through a Coulter S-Plus®. This instrument indicated a mean corpuscular
volume (MCV) of 115 mm3 (normal range 81-99). Repeat analysis of
the same sample, within a few minutes, showed an MCV of 136 mm3.
The blood collection tube was examined visually, and the sample
appeared agglutinated. The patient was called back to the laboratory
for repeat analysis of blood samples collected into heparin and EDTA.
The same phenomenon was observed using blood from the EDTA
tube, however, reliable results were obtained with blood from the
heparinized sample for the MCV, red blood cell count, and indices.
Materials and Methods
ABO and Rh typings and a DAT were performed on
both EDTA and clotted specimens according to standard
methods found in the AABB Technical Manual.7
The paient's serum was tested against group A, B,
and O red blood cells, to which 0.1 M K3 EDTA had
been added. Antibody detection tests were carried out
at room temperature, 4 °C, albumin 37 °C, and antiglobulin (AHG). Serial twofold dilutions of serum were
prepared and studied in the presence of 0.1 M EDTA
(Na2) at room temperature, 4 °C, and 37 °C test phases
in parallel with two other sera in which EDTA-dependent
panagglutinins had been identified previously.
535
CASE REPORTS
Vol. 83 • No. 4
Results
ABO, Rh, and DAT results on this patient's red blood
cells from the EDTA specimen could not be interpreted
because of spontaneous agglutination. In fact, the EDTA
tube appeared like a clotted specimen. This finding was
reproducible both on repeat testing and on a repeat
sample. The spontaneous agglutination was not dispersed
by either cooling the tube to 4 °C or warming it to
37 °C. Red blood cells from the clotted sample caused
no problems and typed as AB, Rh positive. The DAT
on red blood cells from the clot tube was negative.
No serum alloantibodies were detected in any test
phase. The patient's serum only agglutinated red blood
cell samples if EDTA was present. The hemagglutination
titers and scores (7) of EDTA-dependent panagglutinins
in sera from this and two other patients are shown in
Table 1. Parallel tests without the addition of EDTA
were nonreactive.
Discussion
Previous examples of EDTA-dependent panagglutinins
have caused ABO discrepancies because of the presence
of EDTA in some commercial reverse grouping cells.'"6
The present example illustrates that such agglutinins
also can be a pitfall to the Hematology laboratory by
causing difficulty in cell-counting procedures.
The two examples of EDTA-dependent panagglutinins
previously tested in our reference laboratory were most
reactive at low temperatures and were detected because
of discrepant ABO results. Other reported examples
have had similar serologic characteristics.2,X6 The example described in this article had higher titer and score
at 37 °C, which would explain why warming did not
disperse the agglutination. We speculate that this particular EDTA-dependent panagglutinin had a high affinity,
since washing the agglutinated red blood cells with saline
also failed to disperse the agglutination. EDTA-dependent
panagglutination has been shown to depend on free
polycarboxyl groups for the agglutination activity.1,6
Although this case was not deiected by an ABO
discrepancy, it would have caused one if ABO reverse
typing cells had contained EDTA. This case is reported
to warn hematology laboratory staff of another cause of
Table 1. Hemagglutination Titers and Scores of the
Three Anti-EDTA Antibodies
Test Phase
Patient
'» Anti-EDTA #1
Anti-EDTA #2
37 °C
RT
4°C
8/27*
2/11
2/9
4/19
1/3
0
4/19
4/12
4/14
* Titer/score.
in vitro agglutination. This in vitro phenomenon can be
bypassed by collecting the blood sample into an anticoagulant other than EDTA.
Acknowledgments. The authors thank Margie Enger, Terry O'Day,
and Lauren O'Brien for comments during preparation of this article.
They also acknowledge the technical assistance of Margie Enger and
Terry O'Day and the secretarial skills of Margaret Rees-White.
Addendum. A second example of the phenomenon described in the
above article has been examined in our reference laboratory. A 51year-old man was admitted to hospital for a colectomy. A blood
sample, drawn into EDTA, was submitted to the laboratory for routine
hematology evaluation. On automated testing, the MCV was 102. The
hematology technologist then noted that the red blood cells were
agglutinated and appeared as a blood sample from a patient with cold
agglutinin disesae. However, tests in the transfusion service revealed a
negative direct antiglobulin test and no cold agglutinin activity. Blood
collected into heparin did not agglutinate. The patient's serum was
shown to have a panagglutinin active in the presence of EDTA. The
titer and score of this agglutinin were as follows: RT 2/15, 4C 8/31,
37C2/13.
References
1. Beck ML, Freihaut B, Henry R, Pierce S, Bayer WL: A serum
haemagglutinating property dependent upon polycarboxyl
groups. Br J Haematol 1975; 29:149-156
2. Gillund TD, Howard PL, Isham B: A serum agglutinating human
red cells exposed to EDTA. Vox Sang 1972; 23:369-370
3. Gunson HH: A serum agglutinin inhibited by ionized calcium.
Vox Sang 1969; 17:514-524
4. Henry R, Freihaut BH, Pierce SR, Bayer WL, Beck ML: A second
example of an agglutinin for red cells exposed to EDTA
(Abstract). Transfusion 1973; 13:345
5. Howard PL: Blood Bank reagents: Some problems related to
preservatives and dyes. Transfusion 1976; 16:166-169
6. Howe SE, Sciotto CG, Berker D: The role of carboxylic acids in
EDTA-dependent panagglutination. Transfusion 1982; 22:111114
7. Technical Manual. Eighth edition. Washington, D.C., American
Association of Blood Banks, 1981