Acta path. microbiol. scand. Sect. C. 86: 227-236. 1978.

THE REACTIVITY OF HUMAN PLATELETS IN THE 251-LABELLEDSTAPHYLOCOCCAL PROTEIN A TEST RII'M'A KEKOMAKI Finnish Red Cross Blood Transfusion Service, Helsinki, Finland

Kekomaki, R. The reactivity of human platelets in the I2sI-labelled staphylococcal protein A test. Acta path. rnicrobiol. scand. Sect. C, 86: 227-236. 1978. Stored platelets have been employed in the testing of various antiplatelet antibodies and platelet unrelated immune complexes. The antisera included sera containing ABO-, HLA- or platelet specific (Zwa) antibodies and serum from an adult patient with idiopathic thrombocytopenic purpura (ITP). Use was made of I2X-labelled staphylococcal protein A (IPA) for the detection of platelet bound IgG. The reactivity of cells with the ITP serum and sera from healthy blood donors increased after storage for a few days, and declined after a highly reactive period extending over some weeks. In some cases predilution of the serum increased the probability of discovering weak antibodies. The normal components of the serum modified the platelet-antibody reaction, but all the various kinds of platelet reactive activities could be detected by this single test. Key words: Platelets; immune complexes; staphylococcal protein A; radioimmunoassay Riitta Kekomaki, Finnish Red Cross Blood Transfusion Service, Kivihaantie 7, SF-003 10 Helsinki 3 I , Finland.

Received 14.ii.78

Accepted 8.v.78

Stored platelets are used in some of the methods employed for the detection of specific antibodies reactive with platelets. Complement-fixing HLAantibodies have been tested by means of the platelet complement furation test (3, 23). In the platelet indirect radioactive Coombs (PIRC) test (22). platelets stored in azide solution for one year could be used, but that test is applicable for special purposes only. Metabolically-inactive platelets have been employed by Dixon et a!. (5) for the measurement of platelet-bound IgG in a quantitative complement lysis-inhibition assay. However, this test was described as being too complex for routine testing, mainly because of the high demands imposed on immunological reactants (5). Recently, a radioimmune antiglobulin test with platelets has been introduced (PRAT; 17). This has been used for the detection of both drug-associated and HLAantibodies (1 8). For reasons not clearly understood, different tests adapt well for the detection of limited specificities of antiplatelet antibodies only.

The aim of this study is to characterize the interaction of stored human platelets and various antibodies by a single test: the platelet-~2SI-labelled protein A (PIPA) test (12). A rabbit immune complex serum, characterized previously in the platelet aggregation sedimentation pattern test ( 1 3). and human gammaglobulin polymers were used as additional references. MATERIALS AND METHODS Separarion and Preservation of Plarelets Human venous blood from healthy volunteers of blood group 0 (or A, if indicated) was collected in blood bags (Code No. FPR 05 12, Travenol Laboratories S.A., Castlebar, Country Mayo, Ireland; citrate-phosphatedextrose (CPD) anticoagulant). From 40 to 60 buffy coats were harvested in an equal volume of acid citrate-dextrose-NaCI (ACD-NaCI, I : 5 , v/v) anticoagulant. The platelets were then separated by differential centrifugation. The cells were washed four times in EDTA-Tris-saline (0.009 M ethylene-diaminetetra-acetate (EDTA). 0.067 M Tris-hydroxymethylamino-

221

methane (Tris, Sigma Chemical Corp., St. Louis, MO., USA), 0.1 M saline, pH 7.0, and once with citrate buffered saline (CBS, 0.006 M citrate, 0.15 M saline, pH 7.2) before final suspension in the same buffer. Microscopic examination was made to ascertain the purity of platelet preparations.NaN3 (Merck AG, Darmstadt, W. Germany) was added to a final concentration of 0.01 per cent. A proportion of each fresh platelet preparation was fixed with 4 per cent formaldehyde, as described by Allain et al. ( I ) . After three washings with physiological phosphate buffered saline (PBS; 0.093 M phosphate 0.15 M saline, pH 7.4). the preparation was treated with an equal volume of I M aquous ethanolamine (Fluka AG, Buchs SG, Switzerland) for one hour in order to bind the remaining aldehyde groups (24). Finally, NaN3 was added as above. The platelets were preserved in small capped aliquots at + 4OC. These were used at a mean age of approx. four weeks, if not otherwise stated. Small samples of some batches were frozen rapidly in a cold bath at -2OOC without cryopreservative and used immediately after thawing at 37OC.

Radio-iodination of Protein A Protein A (PA) from Staphylococcus a u r a s was purchased from Pharmacia Fine Chemicals, Uppsala, Sweden and carrier-free Nal25I from the Radiochemical Centre Ltd.. Amersham, England. The iodination of PA was effected by the chloramine-T method (91, as modified by Dorval et a/ . (6). An amount of 20 p1 of the preparation contained approximately 4 x 10s cpm. and the specific activity of the iodinated PA (IPA) was approximately 10 pCi/pg. IPA was stored at -2OOC and used within six weeks.

Test System The platelets were suspended in a solution of low ionic strength (LIS), (15); with a conductance of 3.7 mmho at 37OC, and an osmolality of approx. 305 mosm/kg). The suspension contained I x 106 platelets/pI, as calculated in a Coulter Counter model Fn. The aperture diameter was 70 pm. Varying threshold values were also employed for estimation of the relative number of platelet aggregates in test suspension. Various dilutions of the sera were tested, the most commonly used being 1:8. To 0.1 ml of serum dilution was added an equal volume of platelets, and incubation was carried out for one hour at an ambient temperature. After two washings with one ml of CBS, 20 pl of IPA was added. The incubation time was half-an-hour at ambient temperature. The platelets were then washed twice with CBS and resuspended in one ml of the same buffer. A sample of 0.9 ml was transferred to a counting vial and counted for 30 s. The test could also be made on microtitre plates with 96 U-bottom wells of 250 pl (Meda, Helsinki). A quantity of 15 pl of cell suspension was added to 20 p1 of serial serum dilutions. The mixtures were incubated for 60 min at an ambient temperature. Two washings were made, with aliquots of I50 pl CBS per microwell, and 20 pl of IPA diluted I :4 was added to each well. Incubation

228

for 30 min at ambient temperature was followed by two washings and resuspension in 150 pl CBS. Finally, samples of I00 pl were transferred to counting vials. The results are expressed as the percentage of the total l2Sl added which was bound by the platelets (bound/total). All assays were carried out in duplicate.

Sera and Reagents Used to Characterize the PIPA test In each test setting, the following three controls were included: (1) platelets incubated with CBS instead of serum (platelet control); (2) a normal serum, selected for representation of the mean value of 14 sera obtained from healthy blood donors (normal serum control); (3) one control serum obtained from an adult patient with a clinical diagnosis of idiopathic thrombocytopenic purpura (ITP). This serum had been tested for the presence of immune complexes by the platelet aggregation sedimentation pattern test ( 19) with negative result. The undiluted serum or serum diluted I :2 reacted weakly in a microcytotoxicity test (2) with anti-HLA-B 15 related specificity. The same serum displayed a weak but specific binding of IPA in dilution 1:8 which exceeded the random variance of the binding induced by normal sera (Fig. 1 ). The ability of the test to distinguish between this positive serum and the normal serum was taken as the descriminative ability of the test. The titre of the serum is expressed as the highest dilution of the serum which exdeeds the IPA-binding induced by normal serum at the particular dilution in which its maximal binding is displayed. The following other specific reagents were included as indicated: (1) One normal rabbit serum; (2) Rabbit serum, known to contain in vivo induced platelet-unrelated soluble immune complexes (rabbit-IC), was used for identification of the IC-binding sites (Fcreceptors) on platelets ( 1 3); (3) For the same purpose, purified human gammaglobulin ( 1 6 % ; Finnish Red Cross Blood Transfusion Service, Helsinki, Finland) was aggregated with heat (63OC for 10 min). Immediately after centrifugation (3 000 g/30 m i d , the soluble polymers (heat-aggregated IgG, HA-IgG) were separated with acrylamide agarose gel-filtration (Ultrogel AcA 22, LKB Ltd., Industrie Biologique Francaise) in Tris-HCI buffer (0.I M TrisHCI, 0.2 M NaC1, 0.05% NaN3), pH 7.6. It was found that the isolated polymers reacted with platelets in the platelet aggregation sedimentation pattern test ( 19). even at a concentration of 1 pg/pI (unpublished data); (4) Human immune anti-A serum obtained from the Finnish Red Cross Blood Transfusion Service. This had a specific antibody titre in excess of 2500, as determined by the indirect Coombs agglutination technique employing red blood cells; (5) Platelet-specific antiserum (containing C-fixing anti-Zwa antibodies) generously supplied by Professor A . Svejgaard, Tissue Typing Laboratory, Rigshospitalet, Copenhagen, Denmark;

( 6 ) Hypogammaglobulinaemic serum: IgG concentration: < I g/l. (7) Anti-HLA-A2 serum provided by Dr. Anja Tiiiikainen. FRC Blood Transfusion Service. This had an antibody titre of 20 as determined by the microcytotoxicity test (2). All the sera and HA-IgG were stored in small aliquots at -2OOC. Measurement of Serum IgC Mancini's immunodiffusion technique ( 14) was employed. The standard human serum was purchased from Behringwerke, Marburg a/L, W. Germany. The rabbit anti-human IgG serum from FRC Blood Transfusion Service was diluted for the measurement of IgG with a minimum detection limit of 1 g/l. Fractionution of the Seru (1 ) Ion-exchange chromatography (QAE-Sephadex, Pharmacia Fine Chemicals, Uppsala, Sweden) was used for isolation of the main immunoglobulin G (IgG) fraction from the serum. The remaining fraction, immunoglobulin G depleted serum (IgG-dS) ( I O), contained no more than traces of IgG. (2) Alternatively, precipitation with saturated ammonium sulphate at + 4°C was employed. Fractionation of the serum was monitored by means of electrophoresis on cellulose acetate. Folin-Ciocalteau's technique was used for measurement of the protein concentrations. (3) Quantities of 0.2 ml of specific control sera, diluted to one half, were run as indicated in density gradient of 5 ml volumes. A linear sucrose gradient from 12.5% to 37% w/v was applied. Separation was effected in a Spinco SW 50.1 rotor for I8 hours, 30 000 g/min at + 4OC. Paul-Bunnel positive serum was the I9 S reference marker and rubella positive serum the 7 S marker. These prepatations were made at the Department of Virology, University of Helsinki. Treatment of PIuteiets with Enzymes Three ml of platelet suspension was treated with neuraminidase (from Ciostridium perfringens; N 9 I 30, 30-50 U/mg, Sigma Chemical Corp., St. Louis, MO.. USA) for 60 min at 37%, pH 7.0. The minimum final activity of the enzyme was adjusted to 0. I or 0.0 I U/I x 109 platelets. The treatment with trypsin (82 13, I30 000 U/g; Merck AG, Darmstadt, W . Germany) was carried out at 37OC for 30 min. The enzyme activity was adjusted to 200 or 40 U/I x l o 9 platelets. For retardation of the reactions and the removal of traces of enzymes, the platelets were washed twice with ten volumes of cold ( + 4OC) CBS.

RESULTS

A. Normal and Pathologic Sera, Specific Reagents and Isolated Serum Fractions in the PlPA Test A . l . Eflect of normal human sera. The amount of direct binding of iodinated protein A on to washed platelets varied only slightly, even when different batches of platelets were utilized.

Platelets treated with sera from healthy donors bound more IPA than platelets treated with dilution buffer alone. The normal variation of this binding was small, the SD being approximately 20 per cent of the mean (Fig. 1.). In each test setting, one of these sera was used as negative control. The binding of IPA was augmented by the dilution of normal serum from 1 : 2 to 1 :8. There was correlation between the results obtained with these two serum dilutions (r = 0.65, n = 11). The amount of IPA bound by the platelets after the pretreatment of platelets with hypogammaglobulinaemic serum was within the normal range. As a rule, duplicates gave results which fell within a narrow range. The test was repeated if the results showed a difference of more than 20 per cent. The reproducibility of the results obtained in respect of normal sera was high (r = 0.64, n = 12). In addition, repeated measurements with different platelet preparations and different lots of 8/T, % 9

8

7

6

5

4

T

3 2

1

0

I

I

7

14

I

I

28 t , days Fig. 1 . Variation of the IPA-binding related to the age of one platelet preparation. The mean and 1 S.D. (n = 14) are indicated for the two dilutions of normal sera. Symbols: open circle = normal serum, solid circle = ITP serum and triangle = platelet control. 0

21

229

platelet-associated activity was seen to move with the 7 S marker in density-gradient centrifugation. Minimal antiplatelet activity was located in the 19 S region (Fig. 41, as measured by the PIPA test. Different behaviour was observed with rabbit serum containing in vivo induced platelet unrelated immune complexes. As regards the fractions of ionexchange chromatography, the main activity of unfractionated serum was in IgG-dS (Table I). In density gradient centrifugation, it sedimented as fast as or faster than the I9 S marker, as reported earlier (12). To minimize the bias resulting from the 0 1.0 1.5 2.0 2.5 possible formation of aggregated gammaglobulin B I T , % during the chromatography, normal human or rabbit sera were carried as simultaneous controls Fig. 2. Positive correlation between the 1PA-binding infor the ITP and rabbit IC-sera, respectively. No duced by normal serum diluted I :8 and the concentration more than traces of platelet reactive activity were of IgG in the undiluted serum (n = 13; r = 0.59). observable in the IgG fractions of normal human or rabbit sera (Table I). IPA showed that none of the normal sera augmented IPA binding to the specific binding level evaluated in every test setting by the weakly positive serum (ITP). The amount of IgG attached to the platelets and the potent of fixing IPA might be a function of the total amount of serum IgG. When tests were made with undiluted serum or even serum diluted to one half, no correlation was detectable between the IgG concentration of serum and the binding of IPA. When serum diluted 1:s was examined, there was correlation (r = 0.59. n = 13) between IPA binding and IgG (Fig. 2). A.2. EJfect of pathologic sera and specific reagents. In some instances, the discriminative ability of the test was found to be enhanced when serial dilutions of serum were tested. In particular, when ITPserum or anti-HLA serum was diluted 1:4 to 1:s. the binding of IPA by the platelets was promoted. At higher dilutions, the binding diminished. More avid or concentrated antibodies, anti-Zwa, and antiA or HA-IgC, gave the most marked reaction when used undiluted (Fig. 3). The rabbit serum, rich in IC, reacted in a similar manner. The IPA binding was seen to decline with increasing serum dilutions. When dilution was plotted against binding, the slope varied individually for different sera (Fig. 3). A.3. Effrct of isolated serum fractions. Anionexchange chromatography (QAE-Sephadex) was performed for concentration of the IgG fraction from the serum. Both IgG and IgG-dS were tested for reactivity. In ITP serum, the presumed antiplatelet antibody was generally found in the IgG fraction (Table I). In accordance with this, the 230

B. Role of the Platelets in PIPA Test B . l . Age of the platelet preparation. When the platelets were tested six hours after blood donation, they were unable to discriminate between the weak specific and non-specific reactions (Table 2). It was found that even one day’s storage augmented the binding, and storage of from three to four days resulted in further enhancement of the binding of IPA by the platelets (Fig. 1). During a period of one to three subsequent weeks (Fig. I), the sensitivity of the PIPA test remained essentially unchanged. The increment in the binding of IPA coincided with a spontaneous formation of platelet aggregates. Their number increased during the first 3-4 days of preservation. Subsequently, the ratio of single cells to aggregates remained almost stable, the aggregates representing approximately 3 0 per cent of the total number of particles. A small decrement in the number of aggregates was observable after a storage period of several weeks. An increase was discernible in the non-specific binding from normal serum during succeeding months. Mainly for this reason, the discriminative ability of the test weakened (Fig. 5). The specific binding ability of cells was not affected by further storage. The platelets were found to be reactive even after storage for ten months. 9.2. ABO-group of pluteiets. The amount of plateletbound IgG from ITP-serum was independent of the blood group of the platelets (Table 3). B.3. Reactivity of platelets fixed with formaldehyde. Platelets treated with formaldehyde displayed properties essentially similar to those of platelets stored in sodium azide alone. However, the direct IPA-

80

ITP

7 -

8-

6 -

7 -

V ant i - H LA - A 2

6 -

5 -

5 4 4 -

3 3 -

2 -

2 -

A

1 -

1 -

0 -

n

0 111

1/2

1/4

118

1/16

1/32

1/64

I

I

I

I

112

118

1/32

11256

dilution of the serum

dilution of the serum 01'

%

Q anti-Zwa 40

30

15

-

20

10

-

\

A

10

5 -

A

a 0

In0

I

I

l/2 0

1/40

I

I

I

I

1/80 1/160 1/320 V 6 4 0

dilution of the serum

o l

1 1 1 1 1 112

114

118

1/16

1/32

dilution of the serum

Fig. 3a. Effect of dilution of the serum on IPA-binding induced by various antisera. Symbols as in Fig. 1 and as indicated in separate figures.

23 1

WT, %

V B/

96

HA-1gG

20 0 rabbit-IC

20

15 15

10

5

5

A 0

1

1

1

'

1/1

1/10

1/20

1/40

1

1

1

1

I

0

1/80 1/160 11320 1/640 V1280

dilution of t h e solution

dilution 01 the serum

Fig. 3b. Effect of dilution of the serum on IPA-binding induced by rabbit immune complex serum (rabbit-IC) or heataggregated gammaglobulin (HA-IgC). Protein concentration of the undiluted HA-lgC = 3.84 g/l. Symbols: triangle = platelet control. Other symbols as indicated in separate figures.

5 r

3

li

2 1 -

@;

A 0-UJ

19s

7s

A

A

a

Fig. 4 . Fractionation of the ITP serum by density gradient centrifugation. The inset (solid circle) gives the IPA-binding

of the serum dilutions indicated. The fractions (open circle) were tested as I :2 dilutions.

232

TABLE 1. IPA-binding Activity of the IgC and IgC-depleted Fractions of Human and Rabbit Sera Fractionated by lonExchange Chromatography

Type of serum

Protein concentration gll

CPS diluted

Normal human

diluted

ITP patient

Normal rabbit

diluted

Rabbit-IC

diluted

24 I 296

I :8 IgG IgG-dS 1:8 IgG IgG-dS 1:8 IgG IgG-dS

2.1 6.5

118

440 814 496 205 I96 87 I170 I86 1272 96

1:8

IgG-dS Platelets

2.6 15.8 1 .o 5. I

1 .o 6.5

Total counts added per test tube approx. 8300 cps. ITP, idiopathic thrombocytopenic purpura. Rabbit-IC, rabbit serum containing immune complexes formed in vivo

binding of the platelets was increased, with a diminished discriminative ability as the net result (Table 3).

BIT, %

"r 7 -

B.4. Reactivity of platelets stored in frozen state. The discriminative ability of the test for ITP serum remained almost unaltered if the platelets were stored either in sodium azide or in frozen state. The Fc-receptors were preserved in both forms of storage. The reactivity of the platelets was weakened if the cells were frozen and thawed three times.

B.5. Reactivity ofplatelets treated with enzymes. The number of single cells in test suspension was diminished after treatment with any of the enzymes.

TABLE 2. Non-reactivity of a Fresh Platelet Preparaiion

6 -

5 4 -

3 2 -

0

LI 0

Type of serum

Dilution of serum

Age of platelet preparation < I day 16 days ~

~~~~

CPS Normal human ITP patient Platelets

undiluted diluted 1:8 undiluted diluted I :8

51 73 63 70 80

CPS 48 176 48 I 84

Total counts added per test tube approx. 7900 cps. ITP, idiopathic thrombocytopenic purpura.

I

I

J

50 100 150 age of platelet preparation, days

Fig. 5 . Variation of the IPA-binding related to the age of six different platelet preparations in one simultaneous testing. Symbols as in Fig. I .

In experiments with neuraminidase, the binding from normal or ITP serum, both undiluted and diluted, was enhanced (Table 4a). Trypsin treatment of the platelets diminished the dilution-induced enhancement of IPA-binding by normal and ITP-

233

sera (Table 4b). None of the treatments exerted more than a minimal effect on the binding of IPA to platelets induced by rabbit IC-serum.

DISCUSSION

ITP, idiopathic thrombocytopenic purpura. 9 cps Total counts added per test tube approx. 7500 cps.

Stored washed platelets are adaptable (3, 5,12, 17, 2 2 ) to the detection of platelet-associated IgG with a view to avoiding the monitoring of the metabolic changes in fresh cells (8, I I , 20). The use of stored platelets has circumvented some additional problems encountered with fresh preparations. The daily separation of platelets has been precluded, and as pooling has been made feasible, the variation induced by interindividual differences in cells could be minimized. Although the possible bias caused by HLA allotypia cannot be eliminated, the very rare antigens are presumably diluted by pooling. As specific marker of platelet-associated IgG, I *SI-labelled staphylococcal protein A was taken. This is known to react with Fc-parts of all human IgG subgroups except IgG3, and to react with rabbit IgG (6). In this study, the non-reactivity of IPA with IgG3 is of no more than marginal significance, as the results obtained with a few known sera were compared throughout the study. As it is known that human platelets carry several types of membrane antigens and immunological receptors (i.e., ABO-, HLA- and platelet specific antigens and Fc-receptors), both specific antibody reactions with platelets and what are termed Fc-

TABLE 4a. Effeci nf Treatrncnt of Plateleis ( I X 109 Cells) M,itli Ncuraminidase (0.1 U or 0.01 U .for 60 Minuic.s ai 37’C, p H 7.0)on IPA-binding and on Nitnihrr n f Platelets in Test Suspension

TABLE 4b. Effeci qf Treatment qf Platelets (1 x 109 Cells) w5iih Trypsin (200 U or 40 U .for 30 Minuies ai 37’C, p H 7.0) on IPA-binding and on Number nf Platelets in Test Suspension

TABLE 3. IPA-binding qf Various Control Sera in Dilution 1:8 as Studied with Platelets Preserved in Two Dqferent Ways and qf Two Diffirent Blood Groups Method of preservation Formaldehyde treatment and azide

Azide

Blood group

Blood group

Type of serum A

Normal human anti-A ITP patient Platelets

0

198a) 190 1102

407 76

-

553 69

A

0

274 1085 404 254

234 380 238

Neuraminidase Type of serum

Dilution of the serum 0.1 U

Try psi n

0.01 U control

Type of serum

Dilution of the serum 200 U

I18 211

188

736 910

725 1016

294 509

ITPpatient

1077

1373

1004

Rabbit-IC

Patelets

I65

136

130

Patelets

Total number of platelets: Number of aggregates:

5 I4 246

603 295

905 409

Total number of platelets: Number of aggregates:

Normal human

I:2 1.8

ITP patient

I:2 1:8

Rabbit-IC

1:20

179a) 274

96

a) cps Total counts added per test tube approx. 7700 cps. ITP, idiopathic thrombocytopenic purpura. Rabbit-IC. rabbit serum containing immune complexes formed in vivo.

234

Normal human

40 U

control

1.2 1:8

270a) 286

268

124

270

161

1:2 1:8

603 507

518 517

310 490

1:20

828

967

752

78

74

52

414 112

309 152

830 228

a) cps Total counts added per test tube approx. 7900 cps. ITP, idiopathic thrombocytopenic purpura. Rabbit-IC, rabbit serum containing immune complexes formed in vivo.

mediated reactions between unrelated immune complexes and platelets have been used to characterize the measuring properties of platelets in this test system. The reactivity of the platelets in the test, as characterized by the reaction of the weakly positive and normal sera, showed a constant pattern which was dependent on the age of cells. The initial hyporeactivity of the first few days, exploited by Svejgaard (231, was followed by a more reactive period. It is known that platelets display irreversible shape changes even after 24 hours at + 4OC (4). It is possible that swelling and partial lysis make accessible antigenic sites and receptors that were previously unexposed. In 1954, Moreau & Andre suggested that only a minor part of the A- or Bantigen is present on the surface of the platelet, and that the major part is more deeply located (16). In agreement with this concept, in the present series good binding of anti-A was demonstrable in platelets even after months of storage. The period of high reactivity lasted for several weeks. Finally, although the change to increased reactivity coincided with cessation of the spontaneous formation of platelet aggregates, the aggregation per se could not explain the alteration in reactivity without simultaneous membrane changes in single cells. Attempts were made to induce changes in the reactivity of platelets by the treatment of fresh cells with enzymes. Treatment with neuraminidase, with only one hundredth of the enzyme activity reported by Glynn & Phil (7), led to enhancement in the IPA binding ability of the test cells. It is known that treatment with neuraminidase diminishes the serotonin binding capacity (7). It could accordingly uncover receptors which were previously masked. To preclude false positive reactions, treatment with neuraminidase has not been used in routine testing to date. The enhancement in binding attained with trypsin-treated platelets is in agreement with the results obtained by Pfueller & Liischer (21). They suggested that the proteolytic digestion of platelets unmasked Fc receptor sites on them. In addition, the enzymatic digestion of complement receptors could eliminate the possibility of these binding sites for preference being occupied by normal serum components. The binding from undiluted normal serum, or serum diluted to one-half, was not found to correiate with the total amount of IgG in the serum. When the normal control serum was diluted to 1 :8, the binding of IPA was enhanced. A definite correlation now existed between the IPA-binding and the concentration of IgG in serum. A similar enhancement of IPA-binding by serum dilution was

discernible for weak specific reactions ( I 2). This corroborates the assumption that, in addition to IgG, some other factods) control the binding of antibody on to platelets in undiluted serum. All the specific control sera could be titrated in the PIPA test, as suggested previously by us ( 1 2), and the results compared quantitatively. To date, this has been impracticable when using a single technique. The search for optimal binding conditions of weak antiplatelet antibodies necessitates extended investigations.

REFERENCES I . Allain, J. P.. Cooper, H.A.. Wager, R . H. & Brinkhous, K . M . : Platelet fixed with paraformaldehyde: a new reagent for assay of von Willebrand factor and platelet aggregating factor. J . Lab. Clin. Med. 85: 318-328. 1975. 2. Amos, D. B., Bashir, H . , Bode, W.. M U C Q U ~MI. ,& Tiilikainen, A , : A simple microcytotoxicity test. Transplantation 7: 220-222. 1969. 3. Aster, R . H.. Cooper, H . E. & Singiv, D. L.: Simplified complement fixation test for the detection of platelet antibodies in human serum. J . Lab. Clin. Med. 6 3 : 161-172, 1964. 4. Behnke, 0.:Some possible practical implications of the lability of blood platelet microtubules. Vox Sang. 13: 502-507, 1967. 5 . Dixon, R . , Rosse, W . & Ebbert, L.: Quantitative determination of antibody in idiopathic thrombocytopenic purpura. N. Engl. J . Med. 2Y2: 230-236. 1975. 6 . Dorval, G., Welsh. K. 1. & Wigzrll, H , : A radioimmunoassay of cellular surface antigens on living cells using iodinated soluble protein A from Staphylococcus aurrus. J. Immunol. Meth. 7: 237250, 1975. 7. Clynn, M . F. X . & Phil, D.:The role of the platelet membrane in platelet function. 11. 5-hydroxytryptamine (serotonin) uptake. A.J.C.P. 60: 636-643. 1973. 8 . Hirschman, R . J. & Shulmun. N . R . : The use of platelet serotonin release as a sensitive method for detecting antiplatelet factor in patients with idiopathic thrombocytopenic pupura. Br. J. Haemat. 24: 793-802, 1973. 9. Hunter. W . M . & Greennvod, F. C.: Preparation of Iodine- I 3 1 labelled human growth hormone of high specific activity. Nature 194: 495-496, 1962. 10. Joustra, M . & Lundgren, H.: XVIIth Annual Colloquium >)Protides of the biological fluidscc. Brugge, 17: 5 1 1-515. 1969. I I . Karpafkin, S . & Siskind, G. W . : In vitro detection of platelet antibody in patients with idiopathic thrombocytopenic purpura and systemic lupus erythematosus. Blood 33: 795-8 12, 1969. 12. Kekomaki, R . : Detection of platelet-bound IgC with I25I-labelled staphylococcal protein A . Med. Biol. 54: 112-1 14, 1977.

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