Elijah W. Muriithi

Heparin, Platelet Aggregation, Neutrophils, and Cardiopulmonary Bypass

UNLABELLED Cardiopulmonary bypass (CPB) is associated with both neutrophil activation and failure of platelets to form large stable aggregates. We aimed to determine the effects of heparin and of neutrophil activation on platelet aggregation in whole blood. Fourteen patients undergoing routine aortocoronary bypass grafting and NSAID-free for over 7 days were studied before and after heparinisation, and at end-CPB. Whole blood, anticoagulated with rHirudin, was stirred for 3 minutes, and macroaggregation in response to collagen (0.6 microg. mL(-1)) or the neutrophil stimulant fMLP (10(-7)M) was determined by whole blood impedance aggregometry. Microaggregation was measured by counting unaggregated single platelets (corrected for haemodilution). The blood of volunteers was studied in vitro. PATIENTS Before CPB, heparin effectively abolished platelet macroaggregation induced by collagen (20.5 to 1.4 Ohms) or fMLP (3.9 to 0 Ohms (p<0.0001). CPB had no additional effect. Heparinisation also reduced the platelet count from 127 (110-170) to 95 (64-117). The inhibition of macroaggregation could not be reversed by ex vivo heparinase. VOLUNTEERS In vitro, the same heparin concentration, as measured in vivo (4 micromL(-1)), inhibited collagen-induced macroaggregation (20.3 to 14.7 Omega), but this effect was less than that observed ex vivo and was reversed by heparinase. In vitro heparin promoted fMLP macroaggregation (2.9 to 8.6 Omega). The inhibition of macroaggregation resulted from heparinisation, per se, rather than CPB and was insensitive to heparinase. There was less inhibition by in vitro heparin, which was reversible by heparinase, indicating a direct effect of heparin in vitro. The disparate findings are suggestive of an indirect action by heparin in vivo on macroaggregation, although heparin had a small direct stimulatory action on microaggregation.

Platelet aggregatory responses to low-dose collagen are maintained in hirudin-anticoagulated whole blood for 24 h when stored at room temperature

Whole blood from 15 volunteers was anticoagulated with hirudin (200 U/l) and the response to a known submaximal concentration of collagen (0.6 w g/ml) was tested by impedance aggregometry. In 8 volunteers platelet counts were also taken before and after the maximum aggregatory response. These tests were repeated when the samples had rested for 24 h at room temperature. The median [interquartile range] aggregatory response immediately after sampling was 17.3 [16.7-18.4] ohms. At 24 h it was 17.7 [15.8-19.3] ohms ( p = 0.88) although variance was increased ( p = 0.006). The immediate platelet count before collagen exposure was 438 [381-510] 2 10 9 /l and 258 [227-297] 2 10 9 /l post-collagen. At 24 h the platelet count was 448 [443-473] 2 10 9 /l ( p = 0.224 versus immediate count) but variance was not increased ( p = 0.215). After full aggregation the count fell to 284 [234-304] 2 10 9 /l ( p = 0.592 versus early post-collagen). Variances were similar ( p = 0.558). Aggregate response ratios increased non-significantly after 24 h from 0.59 [0.53-0.62] to 0.64 [0.51-0.68] although variance was increased ( p = 0.021). Full macroaggregatory responses by impedance aggregometry were seen after 24 h storage of whole blood with hirudin at room temperature. This suggests both that distant assessment of platelet function using a standardized method is possible and a potential role of thrombin inhibition for platelet storage.

Quantitative detection of platelet aggregates in whole blood without fixation

Platelet counting detects lesser degrees of platelet aggregation than conventional aggregometry. In order to prevent progressive platelet aggregation or disaggregation after sampling it is customary to fix blood samples. However fixation may introduce other artefacts. We first compared stability of platelet counts in EDTA-, citrate- and r-hirudin-anticoagulated blood from healthy volunteers. Second, the stability of platelet counts in unfixed EDTA- and hirudin-anticoagulated blood was compared with glutaraldehyde-fixed blood in the same anticoagulants. Third, the effect of in vivo heparin administration on platelet counts in EDTA- and hirudin-anticoagulated blood was studied. Platelet counts within 2 h of collection were significantly higher in EDTA than in hirudin- or citrate-anticoagulated blood ( P =0.002 vs. hirudin and P =0.001 vs. citrate). Twenty-four hour counts in hirudin and EDTA were unchanged ( P =0.3 and P =0.2, respectively, vs. earlier counts). Counts in citrate increased significantly ( P =0.007; n =10). Platelet counts in fixed blood did not differ significantly from those in unfixed blood. Heparin administered for cardiopulmonary bypass reduced platelet counts in hirudin-anticoagulated blood from (mean +/-1 standard deviation) 180+/-45 to 162+/-30 x 10 9 l -1 ( P =0.01; n =14), without significantly lowering counts with EDTA-anticoagulation, consistent with increased platelet aggregation. Hirudin and EDTA provided stable platelet counts, suggesting that fixation is unecessary.Platelet counting detects lesser degrees of platelet aggregation than conventional aggregometry. In order to prevent progressive platelet aggregation or disaggregation after sampling it is customary to fix blood samples. However fixation may introduce other artefacts. We first compared stability of platelet counts in EDTA-, citrate- and r-hirudin-anticoagulated blood from healthy volunteers. Second, the stability of platelet counts in unfixed EDTA- and hirudin-anticoagulated blood was compared with glutaraldehyde-fixed blood in the same anticoagulants. Third, the effect of in vivo heparin administration on platelet counts in EDTA- and hirudin-anticoagulated blood was studied. Platelet counts within 2 h of collection were significantly higher in EDTA- than in hirudin- or citrate-anticoagulated blood (P = 0.002 vs. hirudin and P = 0.001 vs. citrate). Twenty-four hour counts in hirudin and EDTA were unchanged (P = 0.3 and P = 0.2, respectively, vs. earlier counts). Counts in citrate increased significantly (P = 0.007; n = 10). Platelet counts in fixed blood did not differ significantly from those in unfixed blood. Heparin administered for cardiopulmonary bypass reduced platelet counts in hirudin-anticoagulated blood from (mean +/- 1 standard deviation) 180 +/- 45 to 162 +/- 30 x 10(9) l-1 (P = 0.01; n = 14), without significantly lowering counts with EDTA-anticoagulation, consistent with increased platelet aggregation. Hirudin and EDTA provided stable platelet counts, suggesting that fixation is unnecessary.

Erythrocyte-containing versus crystalloid cardioplegia in the rat: effects on myocardial capillaries

BACKGROUND The purpose of this study was to investigate the effects of crystalloid and erythrocyte-containing cardioplegia on capillary morphology of the isolated erythrocyte-perfused rat heart. METHODS Hearts from adult Sprague-Dawley rats were perfused throughout with resuspended sheep erythrocytes and subjected to the following protocols (n = 6, all groups): (1) 15 minutes nonworking and 30 minutes working heart mode (control; group 1); (2) as for group 1, with 30 minutes erythrocyte-containing (BL) or crystalloid (CR) cardioplegic arrest without reperfusion (groups 2BL and 2CR); (3) as for group 2, with 30 minutes nonworking reperfusion (groups 3BL and 3CR); and (4) as for group 3, with 30 minutes working heart mode (groups 4BL and 4CR). After each protocol troponin I from coronary effluent was measured. Corrosion casts were then made of the coronary microvasculature. Cast density was calculated as cast volume per left ventricular dry weight. Casts also underwent scanning electron microscopy. Analysis was by analysis of variance. Values are mean +/- standard deviation. RESULTS Prearrest working heart coronary flow averaged 15.1 +/- 4.7 mL/min without any differences among groups. Coronary flow in group 4 working hearts was the same before and after either cardioplegia. Cardiac outputs were similarly consistent in all groups. Cast density in group 1 (control) was 9.60 +/- 1.17 x 10(-2) mm3/mg. It was unaltered by erythrocyte-containing cardioplegia, but after crystalloid cardioplegia (group 2CR), it was 6.52 +/- 0.93 x 10(-2) mm3/mg (p = 0.0001 versus group 1 and p = 0.0007 versus group 2BL). With 30 minutes of nonworking reperfusion (group 3CR, there was slight improvement in cast density at 7.60 +/- 0.90 x 10(-2) mm3/mg (p = 0.0072 versus group 1; p = 0.0242 versus group 3BL). No further improvement was seen in group 4CR. Electron micrographs showed circumferential angularities or narrowings in crystalloid-perfused, arrested hearts, consistent with ischemic damage. Troponin I rose significantly after reperfusion in all groups, but it was higher in crystalloid-perfused, arrested hearts: 0.054 +/- 0.013 microg/L versus 0.024 +/- 0.017 microg/L (p = 0.0273). CONCLUSIONS Erythrocyte-containing cardioplegia maintained capillary density and morphology. Crystalloid cardioplegia produced capillary loss, visible abnormalities, and higher troponin I release. These hearts may be more vulnerable to myocardial damage during reperfusion than hearts perfused with erythrocyte-containing cardioplegic solution.

Postoperative bleeding after coronary artery bypass surgery with cardiopulmonary bypass.

We thank Drs. Seubert and Gravenstein for their appreciation of our findings and their comments. We can only agree with their comment that movement of previously entrained air entering the circulation during other surgery at risk for air embolism (such as hip arthroplasty), at the end of surgery, or after surgery due to changing ventilation patterns (such as positive end-expiratory pressure release), or change of patient positions can severely impair the patient’s hemodynamic stability. Intraoperative transesophageal echocardiography (TEE) is not only the most sensitive monitor of venous air embolism, but also the most useful monitor to visualize directly the cardiac performance. It is this double function of TEE, the recognition of venous air embolism in the heart and the possibility to observe to what degree the cardiac function is affected, which makes TEE so useful in our daily practice. We can only hope that the increasing perception of how frequent venous air embolism really is and that it can occur with so many different types of surgery will lead us to further advance the distribution of TEE devices and to train not only cardiac anesthesiologists in this immensely important technique. The essential message of our investigation is to use TEE in all surgery at risk for venous air embolism and to continue TEE monitoring after surgery until the patient is in the supine position and positive end-expiratory pressure has been released to ensure that sudden hemodynamic problems due to reoccurrence of air in the circulation are not missed.