Ethylenediaminetetraacetic acid-dependent ...

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JCP Online First, published on May 30, 2013 as 10.1136/jclinpath-2013-201545 Short report

Ethylenediaminetetraacetic acid-dependent pseudomacrocytosis Jose Manuel Vagace,1 Miguel Ángel Rodriguez,2 María Dolores de la Maya,1 Guillermo Gervasini3 ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jclinpath-2013-201545). 1

Department of Pediatric Hematology, Materno Infantil Hospital, Badajoz, Spain 2 Service of Immunohematology, Transfusion Center, Madrid, Spain 3 Department of Surgical & Medical Therapeutics, Division of Pharmacology, University of Extremadura, Badajoz, Spain Correspondence to Dr Jose Manuel Vagace, Department of Pediatric Hematology, Materno Infantil Hospital, C/ La Violeta 4, Badajoz 06010, Spain; [email protected] Received 15 February 2013 Revised 2 April 2013 Accepted 2 May 2013

To cite: Vagace JM, Rodriguez MiguelÁ, de la Maya MD, et al. J Clin Pathol Published Online First: [ please include Day Month Year] doi:10.1136/ jclinpath-2013-201545

ABSTRACT We investigated the case of a 14-year-old girl with an ethylenediaminetetraacetic acid (EDTA)-dependent haemagglutination detected by macrocytosis, which was only evident by an abnormal red blood cell (RBC) population in the histogram. Investigations included haemograms with different anticoagulants and experimental conditions. Immunohaematological studies were performed using a gel-based technology. At admission, the patient had a low RBC count and an increased mean corpuscular volume with normal haemoglobin. A double population appeared in the RBC histogram. However, the peripheral blood smear was normal and macrocytosis was absent when heparin or citrate was used instead of EDTA. Later studies revealed that the patient’s serum was able to induce macrocytosis of control RBC only in the presence of EDTA. An EDTAdependent panagglutinin was then indentified that produced mixed field agglutination. These findings provide evidence of a haemagglutination induced by EDTA as a source of pseudomacrocytosis. Red blood cell (RBC) agglutination induced by ethylenediaminetetraacetic acid (EDTA) is a rare phenomenon usually attributed to IgM autoantibodies, although the exact mechanism remains uncertain. More than 20 years ago, when it was common practice to add EDTA to commercial reagent cell A and B to prevent haemolysis during testing, these haemagglutinins were described as a cause of errors in the ABO typing.1 These errors are rarer now but the agglutinins can still originate pseudomacrocytosis. The case we present here is one such example. A 14-year-old Spanish Caucasian girl was referred to our haematology service for macrocytosis detected in a routine analysis (mean corpusculr volume (MCV) 115.5 fl, table 1). The girl was asymptomatic. Platelets and leucocyte levels were within the normal range. No abnormalities were observed in the peripheral blood smear and the levels of folic acid, vitamin B12 and homocysteine were all normal. Cryoglobulins and cold agglutinins were negative; reticulocytes were normal (0.9% of RBC) and biochemical parameters were all within the normal range. In order to elucidate whether the observed elevated MCV was an artefact, a new analysis was performed using a different anticoagulant. Table 1 shows that the sample collected into citrate tubes had a normal MCV (85 fl). In addition, the sample collected with EDTA produced a double population in the RBC histogram that was absent with citrate (table 1). An artefact related to EDTA-dependent agglutination was then suspected

Vagace JM, et al. J Article Clin Pathol 2013;0:1–4. doi:10.1136/jclinpath-2013-201545 Copyright author (or their employer) 2013.

and further studies were conducted to characterise this phenomenon.

METHODS Blood samples were collected in tubes containing K3-EDTA, 0.129 M sodium citrate or heparin (Venosafe, Leuven, Belgium). Cold agglutinins titre was conducted using 3% commercial RBC (Surgiscreen, Raritan, New Jersey, USA) as described elsewhere.2 Immunohaematological studies were performed using a gel-based technology (DiaMed, Cressier, Switzerland). Reticulocytes were isolated by density gradient centrifugation. IgM inactivation and flow cytometry were conducted as described elsewhere.3 4 Briefly, after incubation with the patient’s serum in the presence of EDTA, control RBC were washed three times in phosphate-buffered saline (PBS) and resuspended at 1% haematocrit in PBSA. One hundred microliters of RBC suspension were incubated with 10 ml of fluorescein isothiocyanate (FITC)-labelled anti IgG or IgM. Cells were washed once with 1 ml of PBSA and once with 1 ml of PBS, and resuspended in 200 ml of PBS. Cell suspensions were analysed immediately on a flow cytometer (Cytomics FC 500, Beckman Coulter).

RESULTS Subsequent studies showed that haemoglobin values were again within normal ranges and the differences in haemogram results according to the anticoagulant used were still present (table 1). This table also shows that samples with EDTA had a lower RBC count and increased MCV, mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC). In addition, a double population was observed in the RBC histogram. These abnormalities were magnified after incubation of the samples at 4°C for 1 h and disappeared after washing the RBC with saline (data not shown). Refrigeration did not modify the haemogram resulting from the patient’s sample collected into citrate tubes (table 1). The use of heparin as an anticoagulant showed similar results to those of citrate (data not shown). Next, we incubated the patient’s serum in the presence of EDTA with two samples of salinewashed, O negative control erythrocytes at 4°C for 1 h. Table 2 shows that the MCV of both control samples was substantially increased compared with their basal values. Furthermore, a double population could be observed in both histograms after incubation. The immunohematological study did not yield ABO typing discrepancies as the patient’s blood

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Histogram

138 3.2 115.5 43.5 37.6 12.5 Haemoglobin, g/l RBC count×1012/l MCV, fl MCH, pg MCHC, g/dl RDW, %

Arrows mark the double population. EDTA, ethylenediaminetetraacetic acid; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; MCV, mean corpuscular volume; RBC, red blood cell; RDW, red cell distribution width.

122 4.2 85.8 29.1 34 13.6 135 3.1 116.6 43.9 37.6 13.2

EDTA

129 4.6 80.4 28.0 34.8 13.0

134 3.7 96.6 36.1 37.3 13.1

122 4.1 84.5 29.5 34.9 12.7

Citrate EDTA Citrate EDTA

Room temperature Room temperature

Citrate

Second study (26 months later) First study (at admission)

Anticoagulant

Table 1

Changes in the patient’s haemogram and histogram according to the anticoagulant utilised and temperature in two different studies

4°C

Short report type was O negative. A direct antiglobulin test and antibody screening performed on a gel card were also negative. However, a mixed agglutination was observed by adding EDTA (figure 1). Under these conditions, the antibody panel showed no specificities, as all the cells were equally agglutinated by the patient’s serum when EDTA was added (see supplementary figure S1, available online only). The agglutination disappeared when serum was diluted 1:2 with saline. Flow cytometry using anti IgG and IgM antibodies was negative. The low agglutinin titre precluded the use of DTT as IgM inactivating agent and therefore the nature of the agglutinin involved in the process could not be determined.

DISCUSSION The case described here was diagnosed as an artefact able to modify corpuscular parameters in the haemogram. All the analytical parameters suggested the implication of a cold agglutinin, even more so when changes were magnified after incubation at 4°C.5 However, the cold agglutinin titre was negative and analytical parameters returned to normal when heparin or citrate were used. Furthermore, no significant RBC aggregates were observed in the peripheral blood smear. Finally, there was no anisocytosis (the red cell distribution width (RDW) was normal) and only the double RBC population in the histogram was indicative of RBC agglutination. The haematological analysers utilised in the present work quantify RBC distribution using impedance technology. Validated pulses between 36 and 360 fl are classified as RBC, including small RBC aggregates that are considered as a sole pulse, thus increasing the calculated MCV.5 Therefore, it is tempting to speculate that the small size of these aggregates could produce a double population in the RBC histogram, while going unnoticed in the peripheral blood smear. In the same manner, the analyser would not detect anisocytosis, because the extra peak appearing in the histogram is out of the area used in the calculations for RDW (for a technical explanation see http:// www.beckmancoulter.com/wsrportal/bibliography? docname=9617_Red_Cell_Parameters.pdf). In the only available case report of a patient presenting with an EDTA-dependent antibody that was detected by macrocytosis,6 the sample appeared like a clotted specimen; in contrast, no macroscopic agglutination was observed in our sample. The haematological analyser used in the cited study reported different MCV values after repeating the analysis within minutes, which promptly suggested the idea of an artefact. Yasuda et al7 later reported another EDTA-dependent spurious macrocytosis detected by discrepant typing results. In that case, visible RBC agglutinates were also present. The results of the RBC histograms were not described in any of these two studies. The patient’s serum also induced pseudomacrocytosis in normal RBC controls when EDTA was present, producing a double population in the histogram. The immunohaematological study demonstrated that the pseudomacrocytosis was produced by a panreactive, EDTA-dependent cold agglutinin. It should be mentioned that, although most of EDTA-dependent agglutinins are panreactive, other specificities such as anti-H or anti-B have also been described.7 Different types of EDTA-dependent agglutinins have been reported depending on the role of calcium.1 7–10 In the present case, collection of blood into citrate did not induce Vagace JM, et al. J Clin Pathol 2013;0:1–4. doi:10.1136/jclinpath-2013-201545


Short report Table 2 Normal RBC parameters of two O negative controls (basal haemogram) Control 1

Haemoglobin g/l RBC count×1012/l MCV, fl MCH, pg MCHC, g/dl RDW, %

Control 2

Basal

After incubation

Basal

After incubation

141 4.5 88.8 30.9 34.8 13.7

121 3.0 109.4 40.9 37.3 13.4

143 4.8 85.9 29.6 34.5 13.3

109 2.5 112.3 42.8 38.1 13.2

Histogram

The same parameters are shown after washing the RBC with saline and subsequent incubation with the patient’s serum at 4°C in the presence of EDTA (haemogram after incubation). EDTA, ethylenediaminetetraacetic acid; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; MCV, mean corpuscular volume; RBC, red blood cell; RDW, red cell distribution width.

haemagglutination, suggesting that this phenomenon was probably more linked to the presence of EDTA than to the absence of calcium. Alterations on the surface of erythrocyte membranes may expose cryptic antigenic sites that react with the EDTA agglutinin.11 It seems plausible that these cryptoantigens could be heterogeneously distributed in the RBC population, which would explain a double pattern of agglutination. To test whether the RBC age could play a role in this heterogeneous distribution, we incubated control reticulocytes with the patient’s serum and EDTA, but the double pattern did not significantly differ from that of the older RBC (see supplementary figure S2, available online only). In summary, this is the first reported case of pseudomacrocytosis in a child caused by an EDTA-dependent agglutinin. In our case, only the observation of the RBC histogram allowed for the identification of this artefact. We propose that a change in the anticoagulant should be considered in the study of patients with isolated macrocytosis without anaemia in order to identify this phenomenon and prevent unnecessary studies.

Key messages ▸ We present the first case of EDTA-induced pseudomacrocytosis in a child. ▸ Our study demonstrates that this was produced by an EDTAdependent cold agglutinin. ▸ Only the observation of the RBC histogram made us suspect of this artifact, which disappeared when we used a different anticoagulant.

Acknowledgements The authors would like to thank Drs Ahmed Bentahar, Eduardo Muñiz and Nieves Alonso for their valuable critical input, and Francisco Moreno for his excellent technical assistance. Contributors JMV and GG drafted the manuscript, MAR performed cytometry analyses, MDM and JMV carried out the immunohaematological studies. JMV and GG are responsible for the overall content of this work.

Figure 1 The patient’s serum only agglutinated cross-matched isogroup adult control erythrocytes when EDTA was added (well 1, control 1 with EDTA; well 2, control 1 without EDTA; well 3, control 2 with EDTA; well 4, control 2 without EDTA). EDTA, ethylenediaminetetraacetic acid.

Vagace JM, et al. J Clin Pathol 2013;0:1–4. doi:10.1136/jclinpath-2013-201545

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Short report Funding This work has been supported in part by grants GR10022 from Junta de Extremadura, Mérida, Spain and grant PRIS11003 from FUNDESALUD, Mérida, Spain. Competing interests None.

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Patient consent Obtained. Provenance and peer review Not commissioned; externally peer reviewed.

REFERENCES 1 2 3 4

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Howe SE, Sciotto CG, Berkner D. The role of carboxylic acids in EDTA-dependent panagglutination. Transfusion 1982;22:111–14. Mollison PL, Engelfield CP, Contreras M. Blood transfusion in clinical medicine, 10th edn. Oxford: Blackwell Scientific Publications, 1997. Pirofsky B, Rosner ER. DTT test: a new method to differentiate IgM and IgG erythrocyte antibodies. Vox Sang 1974;27:480–8. Schaefer DM, Priest H, Stokol T, et al. Anticoagulant-dependent in vitro hemagglutination in a cat. Vet Clin Pathol 2009;38:194–200.

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Zandecki M, Genevieve F, Gerard J, et al. Spurious counts and spurious results on haematology analysers: a review. Part II: white blood cells, red blood cells, haemoglobin, red cell indices and reticulocytes. Int J Lab Hematol 2007; 29:21–41. Reid ME, Bottenfield LK, Toy PT, et al. Agglutination of an EDTA blood sample caused by an EDTA-dependent panagglutinin. Am J Clin Pathol 1985;83:534–5. Yasuda H, Ohto H, Motoki R, et al. An EDTA-associated anti-B agglutinin: the role of ionized calcium. Transfusion 1997;37:1131–6. Beck ML, Freihaut B, Henry R, et al. A serum haemagglutinating property dependent upon polycarboxyl groups. Br J Haematol 1975;29:149–56. Gillund TD, Howard PL, Isham B. A serum agglutinating human red cells exposed to EDTA. Vox Sang 1972;23:369–70. Gunson HH. A serum agglutinin inhibited by ionized calcium. Vox Sang 1969;17:514–24. Forstner J, Manery JF. Calcium binding by human erythrocyte membranes. Biochem J 1971;124:563–71.

Vagace JM, et al. J Clin Pathol 2013;0:1–4. doi:10.1136/jclinpath-2013-201545


Ethylenediaminetetraacetic acid-dependent pseudomacrocytosis Jose Manuel Vagace, Miguel Ángel Rodriguez, María Dolores de la Maya, et al. J Clin Pathol published online May 30, 2013

doi: 10.1136/jclinpath-2013-201545

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