Brain death is clinically defined by the irreversible cessation of all cortical functions, including the brainstem reflexes, motor responses and respiratory drive in a normothermic, unsedated comatose patient with an irreversible major brain injury and no contributing metabolic disorder . When brain death is clinically suspected, an important component of the clinical diagnosis is the apnoea test, although not always required by guidelines and/or law throughout the world .
When the apnoea test is performed, an increase in arterial carbon dioxide partial pressure (PaCO2) above 60 mmHg is the widely accepted threshold in order to check for the absence of spontaneous respiratory movements . The diagnosis of respiration is usually based on the clinical observation of spontaneous chest or abdominal excursions that produce adequate tidal volumes . During apnoea testing, the mechanical ventilator must be completely disconnected from the patient to obtain an appropriate assessment of breathing, because the ventilator's sensors may give false readings . Nevertheless, when the result is in doubt, a spirometer can be connected to the patient to confirm that tidal volumes are effectively absent . Elsewhere, besides these variables, the continuous recording of the capnography could be used during the apnoea test in order to detect CO2 washout. Indeed, CO2 washout is an indirect sign of respiratory drive and, to the best of our knowledge, capnography monitoring for assessment of spontaneous respiratory movements during the apnoea test in brain-dead patients has not yet been investigated.
During the apnoea test in brain-dead patients, the rate of PaCO2 increase is slow and biphasic with a decline in the increase rate throughout the duration of the apnoea test [5-10]. Nevertheless, the rate of PaCO2 increase has been reported as very erratic, and moreover unpredictable, because of CO2 washout, atelectasis, cardiac-induced ventilations and other potentially unknown factors . Elsewhere, to the best of our knowledge, the mean value of the gradient between arterial and end-tidal CO2 (etCO2) before and at the end of an apnoea test performed through a complete disconnection from the ventilator has not been previously reported.
Therefore, the aim of this study was first to investigate the relevance of capnography monitoring, and secondly to evaluate the PaCO2-etCO2 gradient, during the apnoea test in brain-dead patients.
This prospective study was conducted in accordance with the Helsinki declaration, and the protocol was approved by our local Ethics Committee (Comité de Protection des Personnes se Prêtant à la Recherche Biomédicale Pitié-Salpêtrière, Paris). Since only routine care was performed, informed consent of patient's families was waived by our Ethic Committee. Sixty patients clinically suspected of brain death were investigated prospectively over a 2-yr period. All of them had been admitted to the ICU for severe coma resulting mainly from spontaneous intracranial haemorrhage, head injury or cerebral anoxia. The cause of coma was established for every patient. Reversible abnormalities (drug and metabolic intoxications, hypothermia <35°C and shock) were excluded as the causes of coma. Because of the severity of their cerebral lesions at the time of admission into the ICU, all these patients were potentially expected to develop brain death. Care of the patients confirmed to standard procedures in our ICU for severely comatose patients. The patients were monitored with an arterial pressure catheter, enabling samples for arterial blood gas measurements. etCO2 was continuously measured with an infrared mainstream capnograph, which was daily calibrated (CO2 monitor M1016A; Hewlett Packard, Boeblingen, Germany).
At the time of investigation, all the patients were in a deep, unresponsive coma. They lacked all bulbar reflexes (pupillary (light), corneal, oculo-cardiac and oro-pharyngeal (gag and cough) reflexes), had no spontaneous breathing movements and usually showed vasoplegia and diabetes insipidus. All these findings strongly indicated brain death .
The apnoea test
Since the apnoea test has been shown to be deleterious in some patients and may therefore limit organ procurement for transplantation [10-13], we decided in our ICU to perform the apnoea test only after brain death had been confirmed by electrocortical silence on one electroencephalogram (EEG) with maximal amplification. On the other hand, when EEG could not be contributive for the confirmation of brain death, mainly because of residual relevant blood concentration of sedative drugs (barbiturates, benzodiazepines and/or opioids), the diagnosis requires the absence of intracerebral blood flow on a four-vessel cerebral angiography. However, angiography is not only potentially deleterious, but also risky because of transportation of the patient to the radiology department, especially when there is major haemodynamic instability [14,15]. Therefore, when four-vessel cerebral angiography is mandatory, we usually perform the apnoea test before angiography. In such cases, before the apnoea test, we always verify the absence of intracerebral blood flow by transcranial Doppler ultrasonography . Indeed, in contrary to other countries such as Spain, transcranial Doppler ultrasonography is not legally accepted in France for brain-death diagnosis .
The apnoea test was performed after a 20-min preoxygenation period with an inspired oxygen concentration of 100%. After the ventilator was disconnected, 8 L min−1 oxygen flow was delivered via a tracheal cannula attached to the endotracheal tube. The capnograph was attached to the end of the endotracheal tube, allowing oxygen to flow freely. The patient was then closely observed for 20 min for respiratory efforts, while the capnogram was continuously recorded and printed and the CO2 concentration analysed. If spontaneous respiratory efforts or complications (major haemodynamic instability despite catecholamine administration and/or severe hypoxaemia) occurred, then the patient was immediately reconnected to the ventilator. Otherwise apnoea was observed for 20 min and afterwards the patient was reconnected to the ventilator at the end of the 20-min period. The apnoea test was considered positive if there were no respiratory efforts and if PaCO2 was 60 mmHg or higher at the end of the test [1,4].
Clinical characteristics, aetiology of brain death, confirmatory test performed (EEG or four-vessel angiography) and haemodynamic variables (heart rate (HR), systolic arterial pressure (SAP) and oxygen peripheral saturation (SPO2)) were recorded. Complications during the apnoea test were recorded as hypotension or hypertension (respectively defined as a decrease or increase in arterial pressure of more than 20% of baseline value) and severe hypoxaemia (defined as a decrease of SPO2 < 90%). Arterial blood gases were sampled immediately before the apnoea test for comparison of the PaCO2 (PaCO2 Baseline) to the contemporaneous etCO2 (etCO2 Baseline). Similarly, arterial blood gases were sampled at the end of the apnoea test for comparison of the PaCO2 (PaCO2 Apnoea End) to the contemporaneous etCO2 (etCO2 Apnoea End) immediately after reconnection of the patient to the ventilator.
Data are expressed as mean ± SD. Comparison of two means was performed using the t-test. All P-values were two-tailed and a P-value of less than 0.05 was considered significant. The NCSS 2001 statistical program (Statistical Solutions Ltd, Cork, Ireland) was used for all statistical analyses.
The apnoea test was performed 60 times in 60 patients, 39 males and 21 females, with mean age 47 ± 16 yr. Causes of brain death were cerebral haemorrhage (n = 35), blunt head trauma (n = 14), cerebral anoxia (n = 7) and cerebral gunshot injury (n = 4). The confirmatory test of brain death was EEG in 43 patients, whereas the 17 other patients required cerebral angiography because of significant residual relevant blood concentration of sedative drugs. The apnoea test was completely performed in 57 patients and none of them showed any spontaneous respiratory movements, whereas the capnograph displayed the absence of CO2 release during the 20-min apnoea test (Fig. 1). In one patient, the apnoea test was discontinued after 10 min because of occurrence of both hypotension and hypoxia, requiring major increase in epinephrine administration and reconnection to the ventilator. Nevertheless, since this patient did not show any spontaneous respiratory movements during the 10-min apnoea period and since PaCO2 before being reconnected to the ventilator was above the targeted threshold of 60 mmHg, this apnoea test was considered as valid and the result as positive. In two patients, for whom EEG was inappropriate because of significant residual relevant blood concentration of benzodiazepines following previous sedation (respectively, 3 and 6 days sedation after spontaneous massive cerebral haemorrhage and traumatic subdural haematoma), the apnoea test was negative: spontaneous respiratory movements occurred within a few minutes after disconnection of the patient from the ventilator. Interestingly, the continuous recording of the capnograph showed small but significant CO2 releases (Fig. 2), a short time before the spontaneous respiratory movements became obvious to the bedside clinician. The apnoea test was therefore immediately stopped and those patients were reconnected to the ventilator. Nevertheless, those two patients evolved later to brain death but were not investigated again in the present study.
Figure 3 shows the evolution of PaCO2, etCO2 and PaCO2-etCO2 gradient during the apnoea test in the 57 completed patients. The mean PaCO2 Baseline and etCO2 Baseline before the apnoea test were, respectively, 40 ± 7 and 31 ± 6 mmHg. The mean PaCO2 Apnoea End and etCO2 Apnoea End at the end of the apnoea test were, respectively, 97 ± 19 mmHg (P < 0.001 vs. baseline) and 68 ± 17 mmHg (P < 0.001 vs. baseline). None of the 57 completed patients showed a PaCO2 Apnoea End below 60 mmHg, which means that the apnoea test was positive in all of them. The mean PaCO2-etCO2 gradient significantly increased from 9 ± 4 mmHg before the apnoea test to 29 ± 10 mmHg at the end of the apnoea test (P < 0.001 vs. baseline). Considering the PaCO2-etCO2 gradient as a percentage of the PaCO2 value, it significantly increased from 22 ± 8% to 30 ± 9% (P < 0.001). According to the Bohr dead-space equation, this means an increase in the ratio of dead space to tidal volume (VD/VT) from 0.22 to 0.29 . The rate of increase of PaCO2, etCO2 and PaCO2-etCO2 during the apnoea test were, respectively, 2.8 ± 0.9, 1.8 ± 0.7 and 1.0 ± 0.4 mmHg min−1.
Clinical complications due to the apnoea test were observed in 19 (32%) patients: 16 (27%) patients showed significant hypotension and three (5%) patients showed significant hypertension. One patient, previously described, showed significant hypoxaemia accompanied with hypotension, requiring premature reconnection to the ventilator after 10 min of apnoea.
In this study, we have shown that: (1) capnography could be useful during the apnoea test in clinically suspected brain-dead patients by detecting significant CO2 releases contemporaneously with the occurrence of spontaneous respiratory movements; (2) printing of the capnography recordings may be easily performed, which could be eventually useful for medico-legal purposes; (3) there is a major increase in the PaCO2-etCO2 gradient, with a rate of approximately 1 mmHg min−1 during the apnoea test.
When an apnoea test is performed to confirm brain death, the absence of spontaneous respiratory movements is usually diagnosed only on clinical observation for chest or abdominal excursion and when the result is in doubt, on the absence of tidal volume on spirometry. However, the patient should be completely disconnected from the ventilator during the apnoea test, since cardiac contractions may sufficiently decrease the airway pressure to trigger the ventilator and therefore lead to false negative apnoea test . In the present study, we have shown that capnography, besides clinical observation, could be useful for detection of spontaneous respiratory movements. Indeed, among the 60 patients investigated, the apnoea test was positive for 58 patients and none of them showed either spontaneous respiratory movement or release of CO2 during apnoea (Fig. 1). Conversely, in two patients, the apnoea test was negative, as assessed by simultaneous spontaneous respiratory movements and releases of CO2 during the apnoea (Fig. 2). Nevertheless, one major advantage of capnography during the apnoea test is the continuous recording and printing of etCO2, which may be eventually useful for medico-legal considerations in some countries (Figs 1 and 2).
Among the 60 investigated patients, the two negative apnoea tests occurred in two apparently clinically brain-dead patients requiring cerebral angiography because EEG was inappropriate since significant residual relevant blood concentrations of benzodiazepines were still present following previous sedation. According to our guidelines, we had decided for those two patients to perform first the apnoea test. But since this test was negative, this result allowed us to cancel the transportation of those patients to the radiology department. This was especially helpful in one of those two patients who was haemodynamically unstable since transportation to the radiology department would have been dangerous. Moreover, our practice has avoided one useless contrast injection for each patient, which increases the risk of nephrotoxicity. Nevertheless, one should keep in mind that our guidelines are relevant only when a confirmatory test of brain death is mandatory or required by law, as for example in France. Indeed, such confirmatory tests are not required in many countries, sometimes because these devices are not available on a timely basis . At last, the relevancy of performing an apnoea test when significant residual relevant blood concentration of sedative drugs are present should be questioned: indeed, according to French law, the apnoea test is legally mandatory to clinically confirm brain death whatever the sedative drugs that are present in the blood. Conversely, only the choice of which confirmatory test to be used (EEG or 4-vessels cerebral angiography) depends on those blood samples .
The mean rate of increase of PaCO2 during the 20-min apnoea test in our study was 2.8 ± 0.9 mmHg min−1, higher than that previously reported by Bruce and colleagues  (1.7 mmHg min−1), close to that reported by Ropper and colleagues  (2.6 ± 0.8 mmHg min−1) and Orliaguet and colleagues  (2.75 mmHg min−1), but lower than that reported by Goudreau and colleagues  (3.7 ± 1.8 mmHg min−1) and Benzel and colleagues  (3.7 ± 2.3 mmHg min−1). However, as previously reported, individual rates of increase of PaCO2 were highly variable (1.6-4.6 mmHg min−1) from one patient to another and unpredictable because of CO2 washout, atelectasis, cardiac-induced ventilations and other potentially unknown factors . This variability, associated with the 20-min duration of the apnoea test we have chosen, fully explains why PaCO2 Apnoea End was very high in some of our patients (PaCO2 Apnoea End > 120 mmHg in nine patients). On the other hand, a shorter apnoea time such as 10 min has been reported as insufficient to reach the threshold of 60 mmHg in some patients [9,18,19]. Similarly, estimation of the required apnoea test duration to reach the threshold of 60 mmHg was inefficient because of the unpredictable increment of PaCO2 during the apnoea test [5,20]. Indeed, although some factors such as temperature, basal PaCO2 and duration of the apnoea test have been weakly correlated with the rate of increase of PaCO2, they were clearly insufficient to allow a good prediction of the required apnoea test duration to reach the threshold of 60 mmHg . On the other hand, several authors have reported that brain-dead paediatric patients may take spontaneous breaths during the apnoea test at PaCO2 levels of >60 mmHg [21-23]. Therefore, we consider for our clinical practice that 20 min for the apnoea test in adult patients is a more efficient duration to be certain to substantially overcome the threshold of 60 mmHg.
The mean PaCO2-etCO2 gradient before the apnoea test was 9 ± 4 mmHg in our study. This is similar to the 7 ± 4 mmHg gradient reported by Sharpe and colleagues  in brain-dead patients. Nevertheless, the mean PaCO2-etCO2 gradient in ICU-ventilated patients is highly variable from one patient to another and is weakly predictable [24-26]. Indeed, many factors including duration of ventilation and presence of respiratory diseases, which were present in some of our 60 patients, may noticeably increase this gradient . At the end of the apnoea test, the mean PaCO2-etCO2 gradient had increased to 29 ± 10 mmHg. This is dramatically higher than the 9 ± 6 mmHg gradient reported by Sharpe and colleagues . However, since these authors induced an increase in PaCO2 by exogenous CO2 administration, their result could not be relevant for the standard apnoea test commonly recommended throughout the world, which usually requires an increase in PaCO2 through a complete disconnection from the ventilator [1,4]. To the best of our knowledge, our study is the first one that investigated the variation of the PaCO2-etCO2 gradient during a standard apnoea test in brain-dead patients. Elsewhere, we observed a high variability in the rate of increase in the PaCO2-etCO2 gradient at the end of the apnoea test. This variability precludes any extrapolation of the PaCO2Apnoea End from the etCO2Apnoea End following reconnection of the patient to the ventilator (Fig. 3c). When expressed as a percentage of the PaCO2 value, the PaCO2-etCO2 gradient was significantly more important at the end of the apnoea test than that before the apnoea test. According to the Bohr equation, this increase in dead space is perfectly explained in lungs not being ventilated for 20 min by the lower increase in etCO2 than in PaCO2 during apnoea and may probably be due to the occurrence of atelectasis and ventilation/perfusion mismatches related to the apnoea . Moreover, the tracheal insufflation of oxygen during the apnoea test could have noticeably participated in carbon dioxide elimination from the tracheal tube and anatomical dead space through a passive carbon dioxide-washout phenomenon and therefore must have contributed to limiting the rise of the alveolar CO2 tension . On the other hand, we cannot rule out the hypothesis that cardiac-induced ventilations allowed some alveolar gaseous exchange and therefore also partly limited the rise of PaCO2 during the apnoea [29,30]. Indeed, such cardiogenic oscillations per se have been reported to generate significant flow or pressure, sufficient, for example, to trigger the ventilator during an apnoea period .
Significant complications were observed during the apnoea test among the 60 investigated patients. We reported 16 patients (27%) showing hypotension, which is similar to the 24% and 23% hypotension, respectively, reported by Goudreau and colleagues  and Saposnik and colleagues , but lower than the 39% hypotension reported by Jeret and colleagues . Hypotension during the apnoea test in brain-dead patients results mainly from a decrease in peripheral vascular resistance and possibly from myocardial depression, both linked to the severity of hypercapnia and acidosis . Furthermore, the apnoea test may exceptionally induce a sudden and irreversible cardiac arrest which may prevent any organ donation [10,32,33] and could have legal implications since the diagnosis of brain death had not been previously established . However, no significant myocardial complication usually occurs during the apnoea test providing that oxygenation is maintained (SPO2 > 95%) . Indeed, physicians should keep in mind that complications may be noticeably reduced by correcting, before the apnoea test, the predisposing factors such as hypoxaemia, acidosis and arterial hypotension [10,13]. On the other hand, we observed only one severe hypoxic episode associated with hypotension, which required premature reconnection of the patient to the ventilator. Indeed, the 20-min preoxygenation period with FiO2 100% and the 8 L min−1 oxygen insufflation inside the endotracheal tube during the apnoea test, both performed in our study, may dramatically limit the occurrence of significant hypoxaemia during the apnoea test [4,10,31]. On the other hand, higher oxygen flow had been previously recommended to prevent hypoxaemia during the apnoea test but may be responsible for barotrauma complications [5,12,34].
Finally, we have to point out some limitations of our study. First, the apnoea test is not required by law throughout the world . Secondly, if the apnoea test is mandatory, the end-point may be different from one country to another: clinical observation of only apnoea, with target PCO2 defined, which could be either a 50 or 60mmHg threshold, or a 20 mmHg increase from the normal baseline PCO2 . Thirdly, one could argue that the 20-min apnoea test period that we have chosen may be rather long, especially according to the rate of complications we observed. Indeed, either early repeated blood samples for PaCO2 measurement or new monitoring such as transcutaneous PCO2 measurement may enable us to reduce the apnoea time and consequently the rate of complication . Finally, new methods such as continuous positive airway pressure may also reduce desaturation during the apnoea test .
In conclusion, capnography may be systematically monitored and recorded during the apnoea test in brain-dead patients, which could be useful for medico-legal considerations. The absence of ventilation is responsible for a major increase in the PaCO2-etCO2 gradient at the end of the apnoea test through a major increase in the physiological dead space. Nevertheless, the high variability in the rate in increase in the PaCO2-etCO2 gradient precludes any extrapolation of the PaCO2 from the etCO2 at the end of the apnoea test.
The authors are indebted to Dr David Baker DM, FRCA (Department of Anesthesiology and Critical Care, Hôpital Necker-Enfants Malades, Paris) for reviewing the paper. This study was supported solely by departmental sources.
Conflict of interest.
The authors declare that they have no conflict of interest.
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