Pseudothrombocytopenia Due to Platelet Aggregation and Degranulation in Blood Collected in EDTA

Scand J Haematol(l975) 15, 161-170

Pseudothrombocytopenia Due to Platelet Aggregation

and Degranulation

in Blood Collected in EDTA

M. J. Mmr, M.B., J. C. G. DQERY, M.D.,M.sc., J. GAULDIE, PH.D.

& H. SWrS, M.D.

Divisions of Haematology, Departments of Laboratory Medicine, St. Joseph’s Hospital (Chief, J . Hirsh) and Hamilton General Hospital (Chief, M . A. M . Ali),

Hamilton, Ontario, Canada, Departments o f Pathology (Chief, J . F. Mustard) and Medicine (Chief , E . J . M . Campbell),

McMaster University, Hamilton, Ontario, Canada and

Division o f Haematology (Chief, J . R. Hill), Department o f Laboratory Medicine, University of Alberta Hospital, Edmonton, Alberta, Canada

Three patients are described in whom platelet aggregation andor degranulation occurred in blood collected into EDTA. These changes resulted in spurious thrombocytopenia and morphological changes similar to those observed in some thrombocytopathies. The abnormalities were dependent on the presence of EDTA and did not occur in citrate, oxalate or heparin anticoagulants. In two patients the abnormality was shown to be due to a plasma factor which was not IgG, IgM, fibrinogen or albumin. The most likely explanation is that these patients have an unidentified abnormal plasma component which, on exposure to EDTA, develops ‘anti-platelet activity’. Although relatively uncommon a prospective study of the incidence of these phenomena indicates that they are probably more common than either platelet cold agglutinins or platelet satellitism. They have practical significance with respect to the diagnosis of thrombocytopenia and also to the interpretation of abnormal platelet morphology.

Key words: pseudothrombocytopenia - EDTA

Accepted for publication June 18, 1975

Correspondence to: Dr. M. J. Mant, Division of Clinical Haematology, Department of Medicine, Clinical Science Building, Room 7-108, University of Alberta,

Edmonton, Alberta, Canada T6G 2G3

A preliminary reposrt of part of this study was published in Abstr Clin Res (1972) 20, 934.

Scand J Haematol(l975) 15 11

162 M. J. MANT, J. C. G. DOERY, J. GAULDIE & H. SIMS

Ethylene diamine tetraacetic acid (EDTA), a heavy metal chelator, inhibits platelet aggregation and causes platelet sphering. These properties, together with its anticoagulant effects, have led to its adoption as the anticoagulant of choice for routine blood counts and film preparation. Spurious thrombocytopenia due to blood being taken into EDTA (Gowland et al 1969, Shreiner & Bell 1973, Kjeldsberg & Hershgold 1974) has been reported and has both theoretical and practical implications. We have recently investigated 3 such patients who form the subject of this report.

PATIENTS AND METHODS

Index patients

Patient 1 , a 42-year-old male, presented with tran- sient rectal bleeding. There was no personal or family history to suggest a bleeding diathesis. Physical examination was normal and no cause for the bleeding ws established. A routine blood film using EDTA as the anticoagulant showed apparent severe thrombocytopenia yet a platelet count using EDTA blood was normal (200,000/~~1). A subsequent blood film in EDTA blood showed gray agranular platelets with mild thrombocytopenia. The patient had received no blood transfusions.

Patient 2, a 16-year-old female, presented with a lifelong history of mild easy bruising and recent epistaxis. Slight bleeding had followed prior ton- sillectomy and tooth extraction. The history was of doubtful significance and there was no family history to suggest a bleeding diathesis. Blood collected into EDTA gave a platelet count of 27,000/ p l and the blood film showed moderate thrombocytopenia with apparently giant agranular platelets. The patient had received no transfusions.

Patient 3, a 68-year-old male, presented with al- coholic cirrhosis of the liver with jaundice, edema and ascites but without a palpable spleen. 24 years previously a partial gastrectomy had been performed for a bleeding peptic ulcer. There was no personal or family history to suggest a bleeding

diathesis. Physical examination showed no evidence of abnormal bleeding. Blood collected into EDTA gave a platelet count of 13,00O/,ul and discrete identifiable platelet aggregates were seen on the blood film. Several blood transfusions had been administered previously.

Platelet and coagulation studies

Blood was collected by clean venipuncture and immediately anticoagulated. Blood films were stained with Wright’s and May-Griinwald-Giemsa stains. Abnormalities of platelet morphology were confirmed on at least four separate occasions. Platelets were counted by Coulter Counter (Bull et a1 1965) and by a manual method (Brecher & Cronkite 1950) (normal: 150,00-400,000/pl). Methods for the bleeding time, platelet adhesiveness to glass beads, platelet aggregation studies, prothrombin consumption index, fibrinogen, partial thromboplastin, prothrombin and thrombin times have been previously described (Mant et a1 1973).

Anticoagulants and incubation studies

The routine anticoagulant for preliminary investi- gations was 1/100 vol 0.35 M tripotassium EDTA (Vacutainer 3204 QS, Becton Dickinson & Co., Ontario, Canada). Other anticoagulants used were: 1/10 vol 0.05 M disodium EDTA with 0.1 M NaCI, 1/10 vol 0.05 M ethylene glycoltetra- acetic acid (EGTA) with 0.1 M NaCl, 1/10 vol 0.15 M sodium citrate, 1/10 vol 0.1 M sodium oxalate and heparin 50 U/ml (final concentration). Whole blood was incubated with each anticoagulant for 4 h at room temperature and also at 37O C with EDTA and at 4O C with sodium citrate. Aliquots were removed at intervals for blood films and for platelet counts. Simultaneous control studies with normal blood were performed. In addition noa-anticoagulated capillary blood films were made from each patient.

Electron microscopy

Fresh unanticoagulated whole blood or blood incubated with anticoagulants was fixed and pro- cessed essentially as described by Grottum et a1 (1969). Thin sections were examined with a Phil- lips 300 electron microscope.

PSEUDOTHROMBOCYTOPENIA 163

Mixing studies

Citrated whole blood from 2 compatible normal donors was incubated at room temperature for 4 h with an equal volume of the following plasma fractions obtained from each of the first two patients: citrated platelet poor plasma (PPP), serum, a gamma globulin fraction, a purified IgG fraction, serum from which albumin had been removed and serum from which IgM had been removed. Control incubations contained citrate only and test incubations also contained 1/10 vol 0.05 M EDTA in 0.1 M NaC1. Aliquots were removed at intervals for preparation of blood films.

Gamma globulin fractions were prepared by precipitation in 50 % saturated ammonium sul- phate followed by dialysis against 0.15 M NaCl. Purified IgG fractions were prepared as previously described (Mant et a1 1973). IgM and albumin were removed from serum by immunoprecipita- tion using rabbit antihuman globulin and antihuman albumin respectively with confirmation of removal by double radial immunodiffusion against specific anti-IgM and anti-albumin.

Additional experiments were performed in which CaClz or MgClz were added to 0.05 M EDTA in 0.1 M NaCl prior to its addition to normal blood/patient plasma mixtures. Heparin (50 U/ml - final concentrations) was also added to prevent coagulation. Positive and negative controls for these studies were identical samples without CaClz and MgClz and samples in which citrate replaced EDTA.

Nucleotide and LDH assays

Specific enzymatic assays for adenosine triphosphate (ATP) (Greengard 1965) and adenosine diphosphate (ADP) (Adam 1965) were carried out on a Turner 430 spectrofluorometer; excitation wavelength 340 mp, emission wavelength 460 m,u. Samples for nucleotide assay were deproteinized by the addition of an equal volume of 7 % per- chloric acid and neutralized just prior to assay by the addition of solid potassium bicarbonate. Platelet rich plasma (PRP) was used for total nucleotide determination while nucleotide release was assessed using samples of PPP prepared by centrifugation of PRP for 1.5 min in an Eppen- dorf centrifuge after exposure to EDTA or citrate.

Lactic dehydrogenase (LDH) assays were performed in a total volume of 2.5 ml at 25O C as previously described (Doery et a1 1970). Total LDH was determined using a sonicated sample of PRP and release of platelet LDH was determined from the increase in LDH activity in PPP after exposure of PRP to citrate or EDTA. Simultaneous studies on normal blood served as controls.

Prospective incidence studies

200 patients on whom platelet counts had been requested had blood collected into both EDTA and citrate anticoagulants. Blood films and platelet counts were performed immediately after col- lection and after standing for 4 h at room temperature. In addition, 11W blood films from blood collected in EDTA, referred for a haematological opinion over a 16 month period, were examined specifically for evidence of platelet aggregation and/or degranulation. When either phenomenon was observed blood was recollected in both citrate and EDTA anticoagulants and studied as above.

RESULTS Index patients

The effect of various anticoagulants on the appearance of platelets in peripheral blood films from patients 1, 2 and 3 are shown in Table I. Each of the phenomena observed occurred only in the presence of EDTA or EGTA, platelets appearing normal in other anticoagulants. Platelets from patient 1 did not degranulate immediately on exposure to EDTA or EGTA but by 15 min most platelets were partially or completely degranulated (Figure 1 a) and by 75 min degranulation was virtually complete. The normal blue/gray cytoplasmic staining was lost more slowly and most platelets gradually became undetectable by light microscopy although occasional ‘ghosts’ remained visible. Elec- tronic and manual platelet counts in EDTA and EGTA remained normal (156,000-

11.


TABLE I Influence of anticoagulants on platelets of index patients*

Anticoagulant Patient 1 I Patient 2 I Patient 3

Aggrega- Degranu- Aggrega- Degranu- Aggrega- D ; : ~ ~ - I tion I lation I tion I lation I tion

None EDTA 2 2 O C and 3 7 O C

and EGTA 22O C

oxalate 2 2 O C and heparin 2 2 O C Citrate 2 2 O C and 4 O C -

* In citrate, oxalate and heparin anticoagulants some platelet aggregation occurred in all experiments including the normal controls with which results were compared.

a b C Figure 1. Blood films from patients 1 (Figure a), 2 (Figure b) and 3 (Figure c) obtained from

blood anticoagulated with 0.005 M EDTA and incubated at 2 2 O C for 15 min (patient l), 30 min (patient 2) and 1 h (patient 3) (x 250). Figure a shows degranulated platelets (arrows) resembling those described in the ‘gray platelet syndrome’. Figure b shows large platelet aggregates in which individual platelets cannot be identified and in which degradat ion has occurred (arrows). The appearances are similar to the giant agranular platelets seen in some thrombocytopathies. Figure c shows platelet aggregates in which individual platelets are readily identifiable and have not lost their granules (arrow).


165,OOO/pl) for up to 24 h. The EDTA induced changes persisted over 8 months of observation.

On exposure to EDTA or EGTA platelets from patient 2 immediately formed small aggregates in which individual platelets were readily identifiable. These aggregates rapidly enlarged, individual platelet outlines were lost and platelet degranulation occurred (Figure 1 b). Simultaneous electronic platelet counts in EDTA and citrated blood were 22,OOO/pl and 306,00O/pl respectively. Manual platelet counts were similar, the EDTA induced platelet aggregates not being readily identifiable as such in the counting chambers. The EDTA in-

duced changes persisted over 2 months of observation and were unaltered during 2 weeks of prednisone treatment.

In patient 3 EDTA and EGTA caused immediate platelet aggregation. Individual platelets remained readily identifiable in these aggregates which slowly enlarged (Fig- ure 1 c). Platelet degranulation did not occur over a 4 h period. The degree of platelet aggregation and electronic platelet counts in EDTA varied over 2 weeks of observation. Platelet counts in EDTA remained consistently lower than those in citrate, the most marked difference being 38,OOO/pl (EDTA) and 180,00O/,~l (citrate). Most platelet aggregates were readily identifiable

Figure 2. Platelet ultrastructure in patient 1 after incubation of whole blood anticoagulated with 0.005 M EDTA at room temperature for 4 h. This electron micrograph shows extensive platelet degranulation. (The empty spaces are artefact.) ( x 10,200)


TABLE I1 Total platelet nucleotides and nucleotide release on exposure to E D TA in

patients I and 2"

I I I I Patient # 2 Patient # 2 Control

Patient # 1 Control Patient # 1

Total ATP and ADP 14.26 15.36 7.2 8.9 Release at 10 min 0.21 (2.4 %) Release at 15 min 3.41 (23.9 %) 0.08 (0.5 %) Release at 30 min 0.80 (11.1 %) 0.23 (2.6 %) Release at 4 h 4.51 (31.6 %) 0.16 (1.0 %)

0.14 ( 1.9 %)

* These experiments were performed simultaneously on each patient and their respective control on PRP (patient 1) and whole blood (patient 2) anticoagulated with 0.005 M EDTA in 0.01 M NaCI, and incubated at 22O C. ATP and ADP were assayed individually although only total results in ,uM/ 1011 platelets are given. Timing is from venepuncture. Nucleotide release in citrate anticoagulant was much less than in EDTA.

in manual counting chambers with counts of discrete platelets being similar to those obtained electronically.

Platelets from patients 1 and 2 showed normal ultrastructure after immediate fixa- tion of whole blood at 3 7 O C and also after 4 h at 22O C in citrate anticoagulant. After 4 h in EDTA platelets from patient 1 showed marked degranulation (Figure 2). Satisfactory specimens in EDTA could not be obtained from patient 2. In both patients 1 and 2 total platelet nucleotides were normal but on exposure to EDTA considerable nucleotide release occurred (Table 11). Pla- telet LDH release was studied in patient 2 only and was negligible after 3 h in both EDTA and citrate, being similar to the normal control. Addition of 1/10 vol 0.05 M EDTA to this patient's citrated PRP in an aggregometer induced a small aggregation wave over 30 min not observed with saline, nor on addition of EDTA to normal citrated PRP.

The effect of various plasma fractions from patients 1 and 2 on normal platelets in whole blood was as follows: Platelet degranulation could be induced by plasma only in the presence of EDTA and not in citrate alone and was completely inhibited

by CaClz in equivalent concentration, but only partially inhibited by MgClz in twice equivalent concentration. Serum, a crude gamma globulin fraction and serum from which either IgM or albumin had been removed induced platelet degranulation whereas purified IgG fractions did not. In these studies platelet aggregation with plasma fractions from patient 2 was inconstant although platelet degranulation occurred consistently. Plasma from patient 3 had no effect on normal platelets.

Two of our first patient's 3 children and the mother and only sibling of patient 2 were available for study. Platelet changes in EDTA could not be demonstrated in any of these close relatives, each of whom had normal platelet counts.

Additional studies in patients 1 and 2

The following tests of haemostasis were normal in both patients 1 and 2: bleeding time, platelet adhesiveness, prothrombin consumption index, prothrombi time, partial thromboplastin time, thrombin time and quantitative fibrinogen. Platelet aggregation on exposure to ADP, adrenaline and collagen was normal in patient 2 while pa-

PSEUDOTHROMBOCYTOPENI A 167

tient 1 had normal aggregation to ADP, but an absent secondary wave to adrenaline and subnormal aggregation to collagen on 1 of 4 occasions.

Additional tests giving normal results in both patients were: serum calcium, protein electrophoresis, immunoelectrophoresis, LE- cell preparations and immunofluorescent studies for anti-nuclear, anti-parietal cell, anti-thyroid, anti-mitochondrial and anti- smooth muscle antibodies. Direct and in- direct Coombs tests in the presence and absence of EDTA were negative.

Additional studies were performed in an attempt to identify either an antibody against EDTA or a plasma component whose properties were altered in the presence of EDTA or which could be shown to bind EDTA in abnormal amounts. Double immunodiffusion on Ouchterlony plates was performed using patient’s plasma and the crude gamma globulin fractions against EDTA. No precipitin lines were identifiable. Serum protein electrophoresis in the presence and absence of 0.005 M EDTA showed no change in the mobility of any plasma proteins. Serum protein electrophoresis on cellulose acetate was also performed in the presence of 0.005 M EDTA containing tracer amounts of 14C- EDTA, the individual protein bands identified, excised, eluted and counted in a liquid scintillation counter. No consistent peak of radioactivity could be identified in either patient as compared to normal controls. 1/10. vol of 0.05 M EDTA containing a tracer amount of I4C-EDTA was added to serum from each patient and a crude gamma globulin fraction prepared. No significant radioactivity could be identified in the re- dissolved precipitate in either patients or normal controls.

Prospective incidence studies

4 patients with EDTA induced platelet changes were identified in a prospective study of 1300 hospitalized patients. Details are given in Table 111. 3 patients showed aggregation only and one showed degranulation only. Several EDTA blood films obtained from patient 4 on an admission 2 months previously showed no gregation. The EDTA induced gregation observed in patient 5 after 2 weeks treatment with cyclophosphamide and cytosine At this time platelet counts

platelet ag- platelet ag- disappeared prednisone, arabinoside.

in EDTA (52,OOO/pl) and citrate (46,OOO/,d) were similar. EDTA induced platelet changes were not present in either parent or 2 sib- lings of patient 7.

DISCUSSION

3 patients are described in whom platelet aggregation andlor degranulation occurred in blood anticoagulated with EDTA. Plate- let degranulation alone occurred in the first patient, aggregation and degranulation in the second and aggregation alone in the third. These initial impressions obtained from examination of peripheral blood films and from platelet counts were confirmed in the first two patients by combinations of electron microscopy, platelet nucleotide and LDH release studies and platelet aggregation studies. In each case platelets from unanticoagulated blood and from blood anticoagulated with citrate, oxalate or heparin appeared normal although in EGTA platelet changes were identical to those in EDTA.

Similar in vitro phenomena have been in- frequently reported and include 9 patients

168

Platelet changes

in EDTA

M. J. MANT, J. C. G. DOERY, J . GAULDIE & H. SIMS

Platelet counts (electronic)

Pat. no.

‘ggre- granu- lation I De-

TABLE 111 Clinical details o f 4 patients found to have E D TA induced platelet changes during

prospective study of I300 hospitalized patients

EDTA Citrate

Sex

F

M

M

M

Diagnosis

Cirrhosis Hypersplenism

Acute myeloid leukaemia Adenocarcinoma prostate Iron deficiency anaemia

Bleeding

Yes (oesophageal

varices) No

N o

No

Pre- vious

transfusions

bloomd

Yes

Yes

Yes

No

+ - 17,000 51,000

+ - 119,000 246,000

+ - 74,000 143,000

- + 175,000

(Gowland et a1 1969, Shreiner & Bell 1973, Rhee 1974) and a miniature pig (Ragan 1972). In all except one instance platelet aggregation alone was observed, these cases thus resembling our third patient. In one patient platelet lysis, in addition to aggregation was recorded (Gowland et a1 1969). However, the electron micrographs in this latter publication indicate that degranulation rather than lysis probably occurred, this patient’s findings thus resembling those in our second patient. The platelet degranulation, without aggregation, observed in our first patient may be unique. However, one patient has been described with similar light microscopic findings which were attributed to a serum platelet stain preventing factor (Stavem & Berg 1973). Platelet degranulation as a cause of this phenomenon does not appear to have been excluded in this report.

The finding that a plasma factor was responsible for the EDTA induced platelet changes in 2 of our 3 patients is consistent with similar findings in most previously reported patients with EDTA induced platelet changes (Gowland et a1 1969, Shreiner &

Bell 1973, Stavem & Berg 1973). It appears probable that a plasma factor is responsible for all three types of EDTA induced platelet alteration, with failure of its detection in rare instances. The mechanism by which such a plasma factor might operate remains uncertain. EDTA dependence could be ex- plained by alteration of the plasma factor or by structural alteration of platelets ren- dering them susceptible to it. Gowland et al (1969) speculated that a complement fixing antiplatelet auto-antibody found in their patient caused platelet lysis, dependence on EDTA being attributed to unmasking of platelet antigens by its effect on platelet structure. Studies in two of our patients provided strong evidence against this hypo- thesis and it seems probable that the antiplatelet antibody identified by Gowland et a1 was an isoantibody which was not responsible for the platelet changes observed. Another possible explanation for these phenomena is the presence of an anti- EDTA antibody acting on platelets via im- mune complex formation. Our experiments provided no support for this possibility


which would be an inhererdiy unlikely occurrence.

The third possibility, which we consider most likely, but which must remain specula- tive, is that EDTA acts physicochemically on some abnormal plasma component to alter its structure or cause complexing, this change resulting in acquisition of ‘anti-platelet activity’. Experiments with calcium and magnesium additicn indicated that chelation was involved, as did observations that similar changes occurred in EGTA. Thus, the plasma component probably contains a heavy metal which is most likely to be calcium. Although attempts to identify the plasma factor responsible for the EDTA induced platelet changes were unsuccessful our studies indicated that it was not IgG, IgM, fibrinogen or albumin in two cases. Shreiner & Bell (1973) identified the plasma factor in one of their reported cases as an IgG immunoglobulin whereas we were unable to implicate this protein. Thus, although similarities between the plasma fac- tors responsible fcr EDTA induced platelet changes can be demonstrated it is unlikely that they are identical in all patients.

The three patients initially studied presented over a six month period. We therefore speculated that these phenomena might be more common than the rarity of previous reports indicated. In a prospective study of 1300 hospitalized patients 4 examples of EDTA dependent platelet changes were identified. The abnormalities are therefore not extremely rare and our data, together with previous reports, suggests that platelet aggregation without degranulation is the most frequent of the changes described. It is of interest that no patients with platelet cold agglutinins (Watkins & Shulman 1970) or platelet satellitism (Kjeldsberg & Hersh- gold 1974) were identified during this pro-

spective study suggesting that they are probably less common occurrences. Although several patients had abnormal bleeding it is unlikely that the in vitro phenomena described have any in vivo clinical sign& cance.

The occurrence of EDTA induced platelet aggregation in a patient in whom this phenomena was previously absent and its disappearance in another patient suggests an acquired abnormality. However, an in- herited abnormality in some instances cannot be excluded. The Occurrence of these in vitro phenomena in otherwise healthy individuals indicates that they need not be secondary to another disease process.

The changes described are of theoretical interest because EDTA normally prevents platelet aggregation to most aggregants and at least partially inhibits platelet degranulation. In addition, our observations have two practical implications. Firstly, particularly in view of the apparent moderate frequency of these phenomena, they reinforce the desir- ability of performing both a peripheral blood film examination and a platelet count when thrombocytopenia is suspected with either procedure. Secondly, the morphologic changes observed in our first two patients were strikingly similar to those seen in the grey platelet syndrome (Figure 1 a) (Rac- cuglia 1971) and in some familial thrombo- cytopenic thrombocytopathies with giant agranular platelets (Figure 1 b) (Hirsh et a1 1950). Although other findings indicate that these two platelet abnormalities are real en- tities many reports of abnormal platelet morphology do not include details of anticoagulants used. Our findings indicate that apparent morphological abnormalities observed by light microscopy, particularly in blood collected in EDTA, should not be considered to represent in vivo changes

170 M. J . MANT, J. C. G. DOERY, J. GAULDIE #& H. SIMS

without further investigation. The abnormalities described may be simply identified and may also be disinguished from platelet cold agglutinins by the use of unanticoagulated blood films and anticoagulants other than EDTA. These procedures also permit reasonably accurate platelet counts when platelet aggregation occurs in EDTA.

ACKNOWLEDGEMENTS

We wish to thank Dr. D. Hughes, Dr. W. E. C. Wilson and Dr. S. Miller for permission to study three of these patients and their families, Dr. J. Hirsh and Dr. J . Bienenstock for their interest and advice and Dr. M. A. Blajchman for performing the immunofluorescent studies. J. C. G. Doary is supported by a Canadian Heart Foundation Med- ical Scientist Fellowship.

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Pseudothrombocytopenia Due to Platelet Aggregation and Degranulation in Blood Collected in EDTA

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