J. clin. Path. (1969), 22, 460-464

Agglutination of platelets by a serum factor in thepresence of EDTA

E. GOWLAND, H. E. M. KAY, JUDY C. SPILLMAN, AND J. R. WILLIAMSON

From the Department of Clinical Pathology, Royal Marsden Hospital, and Chester Beatty Research Institute,Institute of Cancer Research, London

SYNOPSIS The platelets of a patient with reticulum-cell sarcoma were found to exhibit agglutinationand cytopathic changes when her blood was treated with EDTA but not with other anticoagulants.Investigation showed that the phenomenon was caused by a serum factor that was probably an

antibody.

The occasional development of autoantibodies inpatients with neoplastic disease of the reticulo-endothelial system is well documented (Dacie, 1962).In this case report we describe the discovery of anantiplatelet antibody in a patient with reticulum-cellsarcoma. The case is remarkable in that a reactionbetween platelets and antibody was observed onlywhen the anticoagulant employed was EDTA(ethylenediamine tetra-acetate).

CASE REPORT

A 70-year-old woman was admitted with a history oflumps in the right groin for 18 months. Biopsy of anenlarged inguinal lymph node revealed a reticulum-cellsarcoma of pleomorphic-cell type. She had once receiveda blood transfusion 15 years previously. In a routinecount from a venous sample with EDTA the plateletswere found to number 45,000/cmm. These were estimatedusing the Coulter model A counter by the method des-cribed by Williamson (1967). Examination of the bloodfilm, however, showed many clumps of pale-stainingplatelets, mostly towards the tail of the film, and arepeated blood count using a capillary sample withoutEDTA gave a platelet concentration of 249,000/cmm,which seemed to be in accordance with the numbers inthe blood film.

INVESTIGATIONS AND RESULTS

The phenomenon was further investigated usinga variety of anticoagulants: heparin (Pularin)l 12-5units per ml of blood; 3-8% wv sodium citrate; di-sodium and dipotassium EDTA, 2% wv, I volumeto 9 volumes of blood. Commercially prepared tubes

'Evans Medical Ltd.

Received for publication 27 November 1968.

460

containing EDTA2 were also used. Polystyrene con-tainers were employed throughout.The effect of EDTA on the patient's platelets is

illustrated in Figure 1. In this experiment sequentialplatelet counts were performed on each of threespecimens of blood treated with EDTA, citrate, orheparin and also on a specimen to which no anti-coagulant had been added. Specimens from threenormal individuals, of which one is illustrated,served as controls. In specimens without an anti-coagulant the counts fell gradually for six to eightminutes and then decreased rapidly as clottingoccurred. In the citrate and heparin samples therewas a progressive fall in the apparent platelet con-centration probably due to clumping, but thisdecrease was not of a significantly greater magnitudethan that of normal controls. In normal circum-stances only EDTA effectively prevents this pro-gressive clumping as is illustrated by the results inthe control blood sample. However, when thepatient's blood was treated with EDTA, in the formof either the sodium or the potassium salt, the plate-let count was severely and rapidly reduced.

In slides of the patient's blood with EDTAstained by May-Grunwald-Giemsa the clumps ofplatelets were seen to be palely basophilic, andazurophilic granules were very scarce, whereas inthe absence of EDTA the platelets were morpho-logically normal.

These changes were observed in greater detail byelectron microscopy. Specimens of the patient'sblood were treated with EDTA and citrate andallowed to stand at room temperature for about 90minutes. The platelet-rich plasma was then harvestedfrom each, and, after centrifugation, the platelet2Stayne Laboratories Ltd.

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Agglutination ofplatelets by a serum factor in the presenice of EDTA

250

200

EE

° 150

X

0Q 100a-0

0

50

0 4 8 12 16 0

MinutesFIG. 1. Effect on apparent platelet count of different anticoagulants.

pellets were fixed in glutaraldehyde. The electronmicrographs prepared from these specimens, togetherwith those made from similar preparations ofplatelets from a normal subject, are shown inFigures 2 to 5. These show no abnormality of thepatient's platelets when incubated in citrated plasma(Fig. 2) but there are fairly profound differencesfrom the normal in EDTA preparations. The controlplatelets remain discrete, irregularly rounded, andshow little loss of the dense a-granules which char-acterize platelets (David-Ferreira, 1964). A few plate-lets contain small or medium-sized vacuoles (Fig. 3).The patient's platelets, by contrast (Figs. 4 and 5),

are swollen, with consequent disorganization ofstructure; some platelets contain many smallvacuoles and in almost all there is a loss of densegranules that is particularly conspicuous whencompared with the platelets in citrate. Platelet aggre-

gation in EDTA has taken the form of focalclustering as distinct from the fairly uniform'crazy paving' effect in citrate.

Clearly the abnormal reactivity of the patient'splatelets could be either intrinsic to the plateletsthemselves or result from the action of a factor inthe plasma. The absence of bleeding manifestationsand the ability of the platelets to cause normal clotretraction could be interpreted either way, and theanswer was provided very simply by testing thepatient's serum with normal platelets.

4 8 12 -16

Serum from the patient was added to an equalvolume of blood from each of five individuals of thesame group (group 0). Both blood and serum weretreated with EDTA and then placed on a rollermixer for 30 minutes, after which a platelet countwas determined. The results, which are presented inTable I, show platelet counts that were much lowerthan would have been expected had no reactionoccurred, whereas in control tests with serum formnormal subjects the counts obtained were close toexpected values. Tests performed with the patient'sserum diluted with saline to 1/8 showed a reductionin platelet count of more than 50%. From theseresults it appeared that the patient's serum con-tained a factor that caused clumping of normalplatelets in the presence of EDTA. This conclusionwas confirmed by the results of an experiment inwhich the test outlined above was repeated usingplatelet-rich plasma from normal subjects instead ofwhole blood. Microscopic examination of thespecimens after 30 minutes on the roller mixerrevealed marked clumping of the platelets that hadbeen treated with the patient's serum, but not ofthose treated with control serum.On the premise that the 'antiplatelet' factor present

in the patient's serum might be an antibody, wetested protein fractions of her serum for activity.Two fractions were tested: the first comprised theglobulins precipitated at 33% saturation with

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462 E. Gowland, H. E. M. Kay, Judy C. Spillman, and J. R. Williamson

Cv , a

FIG. 2.

FIG. 2. Patient's platelets in citrated plasma x 5,000.

FIG. 3. Normal platelets in EDTA plasma x 5,000

FIG. 4. Patient's platelets in EDTA plasma x 5,000.

FIG. 3.

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FIG. 5. Patient's platelets in EDTA plasma x 25,000.

ammonium sulphate ('euglobulin' fraction) and thesecond those globulins precipitated when the con-centration of ammonium sulphate was increasedfrom 33% to 50% saturation ('pseudoglobulin'fraction). The 'euglobulin' fraction showed markedactivity when treated for its capacity to lower the

platelet count of a specimen of normal blood,whereas the 'pseudoglobulin' fraction was inactive(Table II).These findings suggest that the factor involved

was an antibody. Supportive evidence was providedby Dr K. L. Goldsmith at the MRC Blood Group

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E. Gowland, H. E. M. Kay, Judy C. Spillman, and J. R. Williamson

TABLE IEFFECT OF NORMAL AND PATIENT S SERUM ON PLATELETS FROM FIVE NORMAL SUBJECTS

Initial Platelet Serum Added Platelet Count after Percentage ChangeCount Addition of Equal Volume

of Serum

1 160,000 Normal 82,000 t 31 160,000 Patient's 19,000 762 132,000 Normal 69,000 m 42 132,000 Patient's 14,000 -793 134,000 Normal 72,000 + 73 134,000 Patient's 13,000 -814 149,000 Normal 74,000 - I4 149,000 Patient's 23,000 -695 185,000 Normal 77,000 - 175 185,000 Patient's 17,000 - 82

TABLE ILEFFECTS OF PATIENT'S DILUTED SERUM OR SERUM FRACTIONS ON NORMAL PLATELETS

Initial Platelet Added Serum Platelet Count afterCount of Blood or Fraction Adding Equal Volume Percentage Change

of Serum or Fraction

Normal serumPatient's serumPatient's serum diluted 1 2Patient's serum diluted I 4Patient's serum diluted 1 8Patient's serum diluted 1 16Normal 'euglobulin'Patient's euglobulinPatient's euglobulinPatient's 'pseudoglobulin'

77,00017,00013,00022,00038,00066,00094,00016,00018,00082,000

- 17-82-86-76-59-29+ 2-84-78+ 5

Reference Laboratory where an antiplatelet antibodyin the patient's serum was demonstrated by a com-

plement-fixation technique. It does not, of course,follow from this observation that complementfixation is involved in the phenomenon describedabove; although the morphological changes suggesta complement-mediated lysis it is, in fact, improbablethat complement is involved in view of the anti-complementary activity of EDTA.

Three hypotheses may be forwarded to explainthe fact that a reaction between antibody andplatelets was observed only in the presence ofEDTA.The first is that EDTA acts directly on the antibodyto bring about its activation. The second proposesthat EDTA destroys an inhibitor of the antibody.Neither of these seems likely in the light of presentknowledge. The third hypothesis holds that thesurface structure, and hence the antigenic deter-minants of the platelets, might be modified by theanticoagulant. Ethylenediamine tetra-acetate is wellknown to affect the structure of platelets, causingthem to become more spherical and to remaindiscrete (Zucker and Borrelli, 1954). Furthermorethe survival of EDTA-treated platelets after trans-fusion is poor compared with citrate-treatedplatelets (Aster, 1966) and some direct or indirecttoxic effect upon normal platelets can be presumed.In this instance they were apparently rendered

susceptible to the action of an autoantibody of widebut undetermined specificity.The phenomenon is of interest as a manifestation

of autoimmunity in neoplasia of the reticulo-endothelial system but is not otherwise of patho-logical importance. In practice EDTA remains themost satisfactory anticoagulant for platelet estima-tions, but the chance of anomalous agglutination,as in this case, makes examination of the blood filmsa wise precaution.

ADDENDUM

More recently a second example of this phenomenonwas identified in an 82-year-old man with a pepticulcer previously transfused once five years before.As before, the agglutination of the platelets, thoughless pronounced, occurred only in the presence ofEDTA. The phenomenon could not in this instancebe shown with plasma fractions.

REFERENCES

Aster, R. H. (1966). Transfusion (Philad.), 6, 32.Dacie, J. V. (1962). The Haemolytic Anaemias, 2nd ed., pt. 2. p. 343.

Churchill, London.David-Ferreira, J. F. (1964). Int. Rev. Cytol., 17, 99.Williamson, J. R. (1967). Progr. med. Technique, 4, 118.Zucker, M. B., and Borrelli, J. (1954). Blood, 9, 602.

BloodDonor

185,000185,000185,000185,000185,000185,000185,000185,000156,000156,000

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