/ - - . . I SCI. MED. J. CAI. MED. SYND. VOL. 8, NO. I, Jan., 1996.

PLATELET AGGREGATION IN BETA (p) THALASSEMIA

Alua Al-Zayat, Omar Khashab, Mervat Mattar, Elham Saleh and Nadia Hassaballuh*

Departments of Internal Medicine anti Clinical Pathology*, Cairo Ur~iversity

ABSTRACT Both throt~~OoenzOolic and Izenlorrhagic tendencies have been reported in P thalasse-

ruin. Platelet Junction tests tnight help elucidate its role in these events. In vitro platelet aggregation to ADP, collagen and ristocetin togetlter with platelet count rasios ( F / E) were measured in 30 /3 thalassetnia adults. They were cornpared to 20 matched controls. Results showed defective platelet aggregation in all patients.

INTRODUCTION & AIM OF THE WORK

'I'brombo - embolic ~vents, which iue associated with significant morobidity and mortality occur in beta thalassemic pa- tients (Micheali et al., 1992). These in- clude recurrent and transient ischemic ce- rebral manifestations and strokes (Paolini, et al., 1983). Also a high frequency of thrombotic lesions in thc pulmorlary artcr- ies has been observed at autopsy (Sonakul et al., 1980).

On the other hand, ElQor et. a'., (1989) have noticed a significant decrease in plate- let aggregation responses to various ago- nists. This may explain the rare hemor- rhagic tendency in this disease but, as noted by them, cannot acount for the in- creased risk of thrombo - embolism.

The airn of his work is to study and compare in vivo and in vitro platelet func- tions in beta thalassemia major patients.

SUBJECTS AND METHODS 'Thirty adult beta thalasse~nia major Consistillg of 16 non splenectomized

patients were included. They comprised cases. 17 males (and 13 females wilt] age rallgc All patients wcre chosen at least 6 " l2 - 25* were sub-divided lllollths after their last hemolytic crisis and group A : 2 weeks after their last blood transfusion.

(lonsisting of 14 splcncctomizecl cases They were all undcr regular dcsferroxa- :u~d group B : mine therapy for Inore than 6 months.

38 - - - - - - . ,,,,,,,.Alas Al-Zayat, O~nur Khashab et id., ................................................

Twenty healthy age matched controls were chosen for comparison. None of the included subjccts recieved aspirin or any other platelet function inhibitor at least 7 days before testing.

Full history and clinical examination were undertaken in every subject with stress on tiirombo - embolic or bleeding manifestations.

Investigations : -

Urine and stool analysis, liver and re- nal functions, complete blood count with direct platelet count, serum iron and total iron binding capacity (TIBC) together with hemoglobin electrophoresis were performed lo all cases.

An ECG and chest x-rays were also routinely performed. Clotting time, pro- thrombin time (PT), Partial ffirombo plas- tin time (PTT) and fibrinogen were done. In vitro platelet aggregation studies were performed in all groups in response to : ADP (N : 65 - loo), collagen (N : 75 - loo), Ristocetin (N : 85 - 100) (Bauer, 1982).

In vivo platelet aggregation studies

were also performed in all groups by cal- culation of platelet count ratios according to Wu and Hoak (1974).

This method compares platelet count fourid in a blood sample added to buf- fered E.D.TA. / formalin mixture, to an- other samplc added to E.D.T.A. solution alone after centrifugation.

Platelet count in the sample added to E.D.T.A. 1 formalin ~nixture would be lower than that in Ule samplc added to E.D.T.A. alone if platelet aggregates were present. This is because platelet aggre- gates, when found, are fixed and centri- fuged down in the E.D.T.A. / formalin mixture but are broken down in E.D.T.A. alone. Platelet count ratio (FE) are cal- culated as follows :

l'latelet aggregate ratio = (F 1 E).

(F) Platelet count in E.D.T.A. / formalin platelet rich plasma

(E) Platelet count in E.D.T.A.

Normally, the ratio would be close to one. If platelet aggregates are present, the ratio would be much less Uian one (Wu and I loak, 1974).

RESULTS

Clinically, all patients showed the was elevated in all paticnts with a mean of characteristic facial changes of thalassem- 3.6 mg/dl f 1.4. ic patients with growth retradation and Serum iron was significantly higher in diffMnt degYees he~flfos~lenomegal~. patients ( 1 93.7 "g / dl 5 1.7) ban in con-

One patieril showed bilateral loss of trols (1 14.7 ug / dl f 15.6) and Hb level terminal phalanges of both ring and little was signil'icantly lower (7.5 gm 94~ * 1.3) fingers. Another complained of mild re- with platelet count within the normal range current epistaxis. in all groups i~lcluding the spleneclornized

seen in table ( I ) , serum bilirubill group A. HbF was 45.5% + 17.7 among pa- tients.

,;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;; Platelet Aggregation . . . . <;<<;;;;i /

Groups A + B showed a mean P.T. of 77.4 %. Bleeding time (BT) was pro- longed in 3 patients, only one of whom suffered of mild recurrent epistaxis. Clot- ting time, PIT and fibrinogen were within normal.

In vitro platelet aggregation studies showed significant reduction in platelet aggregation to ADP in groups A (29%) and B (21.6%) versus controls (91.4%) (see figure 1).

In all in vitro aggregation studies there was no significant difference between splenectomized and non - splenectomized patients.

In vivo platelet function studies showed a significant reduction in platelet count ratio (FIE) in both groups A (0.5) and B (0.6) versus control group (0.9).

There was no statistical significance betwecn splenectolnizd and non splenec- Lomized patients (see figure 2).

A similar significant reduction in ag- A positivc correlation was found in all gregation to collagen in groups A (18-6%) patients between iron level and platelet an? B (15.6%) versus contfols (88%) was aggregatio~l with ADP, collagen and ris- ali6 noted (se figure 1). Platelet aggrega- tocetin and iron level. However no such tion to ristocetin was dso significantly re- was found with platelet count duced in group A (26%) and B (22.5%) ratio. versus co~ltrols (89%) (See figure 1).

DISCUSSION

Platelets play a prunoridial role in he- mostasis. Variation in platelet function is . hcld to be an important factor in arterial thrombosis (O'Brien et al., 1974).

Platelet functions in beta thalassemia was a point of controversy as both throm- botic and hemorrhagic events have been reported (Eldor ct al., 1989).

Platelet aggregation using an aggre- gometer was reported by Dacie and Lewis (1985) to be the best single in vitro test of platelet function. Eldor et al., (1989) no- ticed a significant d e ~ ~ e b c in platelet ag- gregation responses to various agonists. Platelet aggregation defccts had also been previously noted by Hussierl et al., (1979).

In this work, collagen and rcstocetin were used a exanples of strong agonists for in vitro platelet aggregation, while

ADP was used as an examplc of weak ago- nists.

In vivo platelet activation was mcas ured by the platelet count ratio technique which detects circulating platelet aggre- gates.

In all our 30 thalassemic patients clini- cal findings were identical to lhose report- ed in Egyptians (Sabry, 1973, and Mokhtar et al., 1983).

Blood picture among patients showed marked anisocytosis and poikilocytosis with target cells and normoblasts. Averagc hemoglobin was 7.5 gm + 1.3 similar to other reports (Ozsolu et al., 1973 and Mokhtar et al.. 1985). Morphological red cell changes was more evident in splencc- tomized patients (group A). This was also observed by Polliack et al., 1974 and Rach- milewitz et al., (1985).

Table (1) : mean value (* SD) of all patients group

I1 I I I I I1

P < 0.05 significant

P = highly significant c 0.001

Mean

SD

Mean (SD) all patients.

Mean (SD) plenect. (gp.A).

Mean (SD) Nan - plenect. (gp.B).

P value a l l patients versus control

P value Splencet versus Non - splenet

Age

Y

18

43

ug I dl

193.7 (5 1.8)

194.8 (+55.5)

192.8 (+50.1)

< 0.00 1

NS

Fe

T. BBI

mg

3.6

1.4

ugldl

315 (54.5)

357 (62.4)

302 (56.5)

> 0.05

NS

I TIBC

Ind. HII

mg

2 4

1.2

gm % 7.5 (+1.3)

6.1 (+2)

7.6 (+1.3)

< 0.01

NS

HB

I cm 265000 (+127000)

261800 (+108.300)

269400 (+145200)

< 0.001

NS

SCOT

iul

622

30.2

Plat

SGPT

i u l % % L L L % %

61.7

313

AIL. phm.

u l

279.1

63.6

urea

nlg

247

5.6

f-t

mg

0.7

0.3

RBC

/an

2800000

1300000

TLC

Ion

9879

487

HI

%

25

d.2

Rotle

%

6.3

U

HbA

%

50.6

1 7

HbAl

%

3.9

3.1

HbF

%

45.5

17.7

- BT

min

25

0.8

CT

min

4.9

1.1

PT

%

77.4

166

PTT

%

35.9

7.7

FIb

mg %

250

41

41 .----------.----.-----------------.-------------------------- . Platelet Aggregation . . . . ;<ii<G

Table (2) : Results of platelet aggregation in response to ADP, collagen & ristocetin in both groups of patients.

Group (A) Splenectomized

Group (I%) Non - Splenectomized

Table (3) : Results of ciculating platelet aggregates (platelet count ratio FIE) in both groups of patients.

Group (A) Splenectomized

Group (S) Non - Splenectomized

F I E

0.6

0.4

0.4

0.6

0.4

0.2

0.8

0.7

0.9

0.8

0.8

0.3

0.7

0.5

0.6

0.8

no

2

3

5

8

9

10

12

13

14

16

22

23

24

25

27

30

no

I

4

6

7

11

15

17

18

14

20

21

26

28

29

E x lO3/cm

56

229

285

242

658

260

174

160

134

90

123

190

25 1

261

F 103/cm

14

98

166

105

28 1

192

79

108

90

37

58

54

121

157

E x 103/cm

304

49

293

1 26

113

222

27

25

20

141

207

116

298

427

347

200

F I E

0.3

0.4

0.6

0.4

0.4

0.7

0.5

0.7

0.7

0.4

0.5

0.3

0.5

0.6

F x l03/cm

188

20

108

79

42

34

22

27

18

107

165

3 1

205

227

222

168

43

________________*__*----.-----------------------------*-----. ------ .,.,,,,..,,...,..,,. .. . , ..,.,. ..,, ........ ... Platelet Abgregnti'"' a ... x-....

Fig. t2:)

Platelet Count Ratio (FIE) In Controt Group A & Group 3.

tB ~ r o u p A ~ r o u p B control Group (A) # Group (B) P = NS.

Control Group B Group A

0,9 0 4

,-- 0,5

45 ........................................................... Platdet Aggregation . . . . ;;;,

Fig. (3) : Correlations between Platelet aggregation with ADP and Iron level.

All Patients

0 Significant ( r =03)

Fig. (4) : Correlations between Platelet aggregation with Collagen and Iron level.

9

t

* C 0 All Patients _--

Significant ( r = ~ a

46 .------. ,,,,,,.ALaa AL-Zayat, Omar Khashab et al., ................................................

Fig. (5) : Correlations between Platelet aggregation with Ristocetin and Iron level.

ALL Pat ients

Significant ( r =a)

Fig. (6) : Correlations between Platelet count ratio and Iron level.

All Patients

Non Significant

Reticulocytic count among patients showcd a mean of 6.2% f 2.3. Thc lower than expected count was noted by Pearson and Benz, (1984) and attributed to inef- fective erythropoiesis. HB12 mean value was 45.5% * 17.7 in patients.

While serum iron was significantly higher among patients tharl~~ contisls (P < 0.01), TIBC was withn normal in the ma- jority oS cases. These findings agree with Arthur ct al., (1984) and confirm the role of fenitill as a more accurate marker of iron over load.

I'rothroinbin concentration was below normal arnong the patients group (77.4% + 16.6). This can be attributed liver affec- tion either due to transfusion related infec- tions or to hepatic iron deposition.

Bleeding time was within normal 2.5 + 0.8 minutes except in 3 patients, o~ic of whom complained of epislaxis. M"1', clotting time and fibrinogen were within normal levcls in all patients. These re- sults disagree with those reported by I-Iussicn ct al., (1979) who observed mild prolongation of IYI' and PTT atnorig their cases.

A normal platelct count among groups A and B in comparison to controls contradicts France et al., (1981) who found increased platelet counts among their splenectomizcd group.

In vitro platelet function tests in our study showed defective in vitro plarelet aggregation to ADP, collagen wd risloce- tin. These defects wcre noted among both the splerlectornized and non splenecto- mized patients and were signiticantly low- er than controls. No signillcant dill'crcnce

47 ---------- ,,,,,,,,,, Platelet Aggregation . .. . ;;;;G

was noted between the splenectomized and the non splenectomized group A.

Our findings confinn previous studies documenting significant defect in vitro platelet aggregation in beta thalassemic patients (Eldor, 1978). However, these studies related platelet function defects to the sevetity of he~norrhagic symptoms in thcir patients (Qdor et al., 1989). This could riot be applied in our work.

In vivo, increased circulating platelet aggregates as evidenced by a low platelet count ratio was found in all our patients (group A and B). Platelet count ratio was significantly lower Lhan controls with no difference between splencto~nized and non - splenectomized groups.

Tissue hypoxia due to chronic anemia may damage the endothelium causing the interaction of platelets with thc vessel wall (Morrison et al, 1977). This would result in the formation of circulating ag- gregates by the more active platelets with the less active ones detected in vitro as poorly aggregable. An in vitro refractory state induced by preexposed platelets to agoriists has been suggested (Pareti et al., 1980).

Our Sindings suggest increased in vivo platelet aggregation among thalassemic patienls which may subject them to an in- creased risk of thrombosis.

Wc recommend a wider scale study of both in vivo and in vitro of platelet func- tions in beta thalassernic patients with the conlctnplation of the possible role of platclct inhibitor drugs in these patients.

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.--------------*-------------------------.------------------- - - - - - - c~~~~~~~~~IIIxIxIIIII,, ,~KKkkk~~~~~kkkk~kkkk~k~kkkkk~~k~kk~,+ Platelet Aggrggntion . . . . ...=

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