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Anesth Analg 1999;88:258
© 1999 International Anesthesia Research Society

PEDIATRIC ANESTHESIA

Platelet Hyporeactivity in Young Infants During Cardiopulmonary Bypass

Fumito Ichinose, MD*, Shoichi Uezono, MD*, Rika Muto, MD{dagger}, Haruo Uchida, MD{dagger}, Fumimaro Hatori, MD{dagger}, Katsuo Terui, MD*, Yoshinari Niimi, MD, PhD*, Takahisa Goto, MD*, Yoshinori Nakata, MD, MBA*, and Shigeho Morita, MD*

Departments of Anesthesia, *Teikyo University Ichihara Hospital and {dagger}Chiba Children's Hospital, Ichihara, Chiba, Japan

Address correspondence and reprint requests to Fumito Ichinose, MD, Department of Anesthesia, Massachusetts General Hospital, Fruit St., Boston, MA 02114.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Platelet dysfunction is one of the most important factors contributing to a postoperative hemorrhagic diathesis in children with congenital heart disease undergoing operations requiring cardiopulmonary bypass (CPB). However, very little is known about the influence of CPB on platelets in neonates and young infants. We studied 16 patients—8 young infants (<2 mo old) and 8 children (>12 mo old)—with congenital heart disease undergoing CPB. Surface density of an important platelet adhesive receptor, glycoprotein Ib, and degree of platelet activation, indicated by p-selectin positivity, were measured by whole blood flow cytometry in samples obtained at seven time points during the operations. We found that the percentage of p-selectin–positive platelets increased significantly in children, but not in young infants, during CPB. The young infant group exhibited a significantly smaller reduction of glycoprotein Ib than the child group during CPB (21.0% ± 12.0% vs 32.7% ± 18.1%; P < 0.05). Lack of CPB-induced increase of p-selectin and a smaller decrease of glycoprotein Ib in young infants in the current study suggest reduced platelet reactivity in young infants and neonates during CPB. The clinical significance of the reduced platelet reactivity in young infants and neonates remains to be determined.

Implications: Platelets of young infants are less reactive than those of children during cardiopulmonary bypass, as determined by the cardiopulmonary bypass-induced alterations in platelet membrane adhesive receptors.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Postoperative hemorrhagic diathesis is a well known problem associated with neonatal or pediatric cardiac surgery involving cardiopulmonary bypass (CPB) (1–3). Multiple factors, such as platelet dysfunction, thrombocytopenia, and decreases in coagulation factors, are believed to contribute to the hemostatic defect (3,4). However, one of the most important factors contributing to the hemostatic defect associated with CPB is platelet dysfunction (5–7).

It has been reported that there are structural and functional changes in circulating cells in adult and pediatric patients during CPB (8–11). Platelets are activated, release granule contents, and express neoan-tigens such as CD62 and CD63 on the cell membrane. In addition, surface expression of the major platelet von Willebrand factor receptor, glycoprotein Ib, decreases during CPB. Postoperative coagulopathy associated with adult cardiac surgery is at least partly due to the alteration of the platelet membrane receptors, and the bleeding tendency can be corrected by aprotinin or tranexamic acid, presumably by decreasing the platelet damage (2,12–14). Because of the relatively large volume of CPB circuit compared with their small blood volume, it has been assumed that the degree of cellular activation and defects of membrane receptors would be greater in neonates and young infants than in adults (1,15). However, few studies have examined the influence of CPB on platelets of neonates and young infants.

Whole blood flow cytometric technique allows the examination of large numbers of individual cells from small amounts of whole blood, circumventing the methodological problems of other techniques (16). The purpose of the current study was to examine the influence of CPB on platelet membrane receptors in young infants and neonates undergoing open heart surgery. Platelet surface expressions of p-selectin and glycoprotein Ib of young infants and neonates were measured by whole blood flow cytometry and compared with those of children older than 12 mo.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
After institutional approval by the Human Investigation Committee of the Teikyo University and the Chiba Children's Hospital and informed parental consent, we studied 16 consecutive patients with congenital heart disease undergoing open heart surgery requiring CPB at the Chiba Children's Hospital. They were divided into a young infant group (age <2 mo, n = 8) and a children group (age >12 mo, n = 8). CPB was conducted with a membrane oxygenator (Masterflo Module 1500; Dideco, Rome, Italy) using comparable flow rates at temperatures determined by the complexity of the planned surgical procedure (Table 1). Deep hypothermic cardiac arrest was not used in any patients in the current study. No antifibrinolytic drugs were administered. One milliliter of whole blood samples was taken from the radial artery catheter, and 100 µL of each sample was immediately fixed in 1 mL of 1% paraformaldehyde. Sampling points were as follows: after induction of general anesthesia before the start of the operation, after systemic heparinization before the start of CPB, 15 min after the start of CPB, 60 min after the start of CPB, 90 min after the start of CPB (when applicable), 10 min after protamine administration, and 2–4 h after termination of CPB. Blood samples were studied for platelet surface density of glycoprotein Ib, and the degree of platelet activation (p-selectin expression).


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  		Table 1. Indices of Cardiopulmonary Bypass (CPB)
 
Three fluorescein-labeled monoclonal antibodies were used in this investigation. Phycoerythrin (PE)-conjugated monoclonal antibody CD62 (PE-anti-CD62, clone AC 1.2; Becton Dickinson Immunocytometry Systems, San Jose, CA) is specific for P-selectin. P-selectin, also referred to as CD62 or GMP-140, is a component of the {alpha}-granule membrane of resting platelets that is only expressed on the platelet surface membrane after platelet degranulation and secretion. Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody CD42b (FITC-anti-CD42b, clone SZ2; Coulter/Immunotech, Westbrook, ME) is directed against von Willebrand factor binding site on the amino terminal domain of platelet membrane glycoprotein Ib {alpha}. Peridnin-chlorophyll protein (PerCP)-conjugated monoclonal antibody CD61 (PerCP-anti-CD61, clone RUU-PL 7F12; Becton Dickinson) recognizes glycoprotein IIIa, the common ß-subunit of the glycoprotein IIbIIIa complex that is expressed on all normal resting and activated platelets.

A whole blood flow cytometric method was used. All whole blood samples were fixed in 1% paraformaldehyde for 2 h at 4°C, then washed three times in phosphate-buffered saline containing 0.1% sodium azide (CellWash; Becton Dickinson). Subsequently, 50 µL of each sample was incubated with saturating concentrations of FITC-anti-CD42b (final concentration 9.1 µg/mL), PE-anti-CD62 (0.27 µg/mL), and PerCP-anti-CD61 (0.55 µg/mL) for 20 min, washed, and resuspended in 1 mL of CellWash for three-color flow cytometric analysis.

Samples were analyzed on a FACScan flow cytometer (Becton Dickinson). The instrument was equipped with a 15 mW air-cooled argon ion laser operated at 488 nm, and fluorescein fluorescence data were collected at 530 nm for FITC, 585 nm for PE, and 680 nm for PerCP. Data were stored in list mode files on a computer system. The surface density of glycoprotein Ib and the percentage of platelets expressing p-selectin were determined as previously described (9,17). Briefly, platelets were identified in the whole blood samples by PerCP-positivity and characteristic forward and side light scatter. PerCP-conjugated CD61 was used as the platelet-specific monoclonal antibody. After identification of platelets by PerCP-positivity and light scatter gates, binding of PE-anti-CD62 and FITC-anti-CD42b was determined by analyzing 10,000 individual platelets, using CELLQuest software (Beckton Dickinson). Antibody binding was expressed as the mean fluorescence intensity for FITC-anti-CD42b and as the percentage of platelets positive for PE-anti-CD62. Isotype- and fluorochrome-matched control monoclonal antibodies were used in each experiment to determine nonspecific background antibody binding.

Data are presented as mean ± SD unless otherwise specified. Systat software (version 5.2.1; SPSS, Chicago, IL) was used for statistical analyses. Repeated-measures analysis of variance (ANOVA) followed by Dunnett or Bonferroni multiple comparison post hoc tests or unpaired Student's t-tests were used where appropriate.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The demographics of young infants and children are shown in Table 2. Except for age and weight, no variables differed significantly. Five young infants and four children had cyanotic heart diseases. One patient in the children group and two patients in the young infant group had a minimal temperature below 25°C, although the average lowest temperature during CPB did not differ between groups (Table 1). The platelet count decreased from 28.9 ± 4.0 to 10.0 ± 5.0 (x104/µL) in the child group (65%) and from 32.1 ± 11.1 to 9.3 ± 3.4 (x104/µL) in the young infant group (71%) between the baseline and 2–4 h after CPB, but the magnitude of reduction in the two groups was not significantly different.


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  		Table 2. Patient Demographics
 
In the child group, the percentage of p-selectin–positive platelets increased significantly (P < 0.001, ANOVA), peaking at 90 min of CPB (21.1% ± 6.1%), then decreased 2–4 h after CPB (Figure 1). The percentage of activated platelets did not increase significantly in the young infant group at any time (P = 0.257, ANOVA), showing a marked difference from the child group (P < 0.001, repeated-measures ANOVA).



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  		Figure 1. Percentage of circulating activated platelets in the child (•) and the young infant ({circ}) groups as determined by surface expression of p-selectin. CPB = cardiopulmonary bypass. Data are presented as mean ± SE. # Values significantly differ from baseline and from values of infants at the same time point (P < 0.05).

 
The baseline surface expression of the major platelet von Willebrand factor receptor glycoprotein Ib did not differ between the young infant group and the child group. The glycoprotein Ib expression significantly decreased after 60 min of CPB in children and after 90 min of CPB in young infants and reached the lowest values 2–4 h after CPB in both groups (P < 0.001 for each group, ANOVA) (Figure 2). Although absolute values of platelet surface density of glycoprotein Ib did not differ significantly between children and young infants, the percent reduction in the platelet surface expression of glycoprotein Ib from baseline values in each group was greater in the child group than in the young infant group (32.7% ± 18.1% vs 21.0% ± 12.0%; P = 0.025, repeated-measures ANOVA). A post hoc analysis by Bonferroni multiple comparisons test revealed that the magnitude of the percent reduction of glycoprotein Ib expression in children was significantly greater than that in young infants at 60 and 90 min of CPB.



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  		Figure 2. Changes in surface expression of platelet glycoprotein Ib in the child (•) and the young infant ({circ}) groups. Mean fluorescence intensity of FITC-anti-CD42b binding to platelets was measured in whole blood samples. CPB = cardiopulmonary bypass. The fluorescence intensity of platelets of children at baseline was assigned a value of 100 units. # Values significantly differ from baseline of young infants (P < 0.05). * Values significantly differ from baseline of children (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The major findings of the current study are that children demonstrated a significant increase of p-selectin–positive platelets during CPB; that p-selectin–positive platelets did not increase in young infants during CPB; and that platelets of young infants showed a smaller percent reduction of glycoprotein Ib expression during CPB than platelets of children. These results seem to suggest that platelets of neonates and young infants with congenital heart disease were less affected by CPB than those of children.

The nature of the putative platelet function defect associated with CPB is unclear. The contact of the platelet with synthetic material, shearing stress, suction of the surgical field, and activation of the complement system and fibrinolysis during CPB all may injure the platelet to some extent. Several types of intrinsic platelet defects, including expression of p-selectin associated with degranulation and defects of platelet surface glycoproteins Ib and IIbIIIa, have been reported to occur in adults and children during CPB (11,18). Because of the extremely small blood volume and a proportionally large synthetic surface area of CPB circuit in neonatal cardiac surgery, it has been assumed that the magnitude of such CPB-induced platelet defects would be greater in neonates, possibly contributing to the severe hemostatic defects often seen in these patients after cardiac surgery (15). Therefore, our findings that there were fewer such CPB-induced platelet defects in neonates and young infants than in children are rather surprising. An explanation for this apparent platelet hyporeactivity of neonates and young infants may be age-related intrinsic differences of platelets.

Platelets of newborn infants have specific morphological features that may affect cellular function (19,20). Platelets of preterm and term infants have less pseudopods, smaller glycogen deposits, less visible microtubular structures, and markedly less {alpha}-granules, which indicate that platelets undergo an age-dependent maturation process (21,22). The response of neonatal platelets to agonists leading to platelet activation is different than that of adult platelets. A study using whole blood flow cytometry showed that neonatal platelets are clearly hyporeactive to several platelet agonists, probably because of an intrinsic defect (23). Because CPB-induced increase of p-selectin and decrease of glycoprotein Ib are considered to be a form of platelet activation, our finding that platelets of young infants showed significantly less platelet activation than those of children is consistent with the hypothesis that neonatal platelets are intrinsically hyporeactive.

Children with congenital heart disease and, in particular, cyanotic congenital heart disease, are likely to have preexisting platelet defects that result in a prolonged bleeding time (24,25). A study by Rinder et al. (9) showed that the platelet surface expression of glycoprotein Ib was less at baseline and subsequent time points during CPB in cyanotic pediatric patients than in noncyanotic children, which may explain the relatively high prevalence of a prolonged bleeding time in patients with cyanotic heart disease. Because both groups in the current study had a similar number of cyanotic patients (five young infants and four children), it is unlikely that the differences between groups were due to the influence of cyanotic heart disease on platelets. Interestingly, the reduction of glycoprotein Ib in cyanotic and noncyanotic patients was almost parallel in the study by Rinder et al. (9), whereas we observed a significant difference in the magnitude of glycoprotein Ib reduction in young infants and children. Therefore, we speculate that an age-dependent maturation process and the presence of cyanotic heart disease have different effects on the expression and regulation of glycoprotein Ib.

There are considerable differences among studies regarding the effects of CPB on platelet activation. In fact, some studies report no change in the expression of p-selectin and glycoprotein Ib (8), whereas others, including the current study, report significant degranulation and up-regulation of p-selectin and reduction of glycoprotein Ib during CPB (11,18). Furthermore, different results have been reported regarding the duration of platelet p-selectin positivity after CPB (11,26). Although these differences are inherently difficult to reconcile because of considerable differences in techniques of surgery, perfusion, and blood sampling among institutions, an explanation for these discrepancies may be the fate of platelet p-selectin. Although it has been postulated that surface p-selectin–positive (degranulated) platelets are rapidly cleared from the circulation by leukocytes, Michelson et al. (27) reported that circulating degranulated platelets rapidly lose surface p-selectin to the plasma pool but continue to circulate and function. Therefore, depending on the timing of platelet activation and blood sampling, various amounts of p-selectin–positive platelets could theoretically be detected, making comparisons among studies more difficult. Although neonatal platelets in the current study may also have had an increase of p-selectin during CPB and may have lost p-selectin rapidly, it is unlikely because the sampling method and frequency were identical between young infants and children (27).

In summary, compared with platelets of children, those of young infants and neonates demonstrated a marked hyporeactivity during CPB, showing an absence of CPB-induced increase of p-selectin and a smaller decrease of glycoprotein Ib. These findings are consistent with the hypothesis that neonatal platelets are intrinsically hyporeactive to various stimuli. Further studies with a larger sample size are required to clarify the clinical significance of this platelet hyporeactivity in neonates and young infants undergoing CPB.


   Footnotes
 
This study was partly supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan, and by a grant from Daido-Hoxan Co., Tokyo, Japan.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication November 5, 1998.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press