1. Carboxyhemoglobin in the bodies of victims of the Tu-154M crash – preliminary analysis of results using the spectrophotometric method of Fretwurst-Meinecke
1.1 Limitations of the method
Examination of autopsy material, because of the nature of post-mortem changes, specifically rapid decomposition of blood has several limitations. Therefore, the main determinant of the reliability of analytic results when assessing carboxyhemoglobin (COHb) is the choice of analytic methodology. There are two main investigative methods to assess COHb used in contemporary forensic toxicology:
2) gas chromatography
From the descriptions contained in the Russian forensic-toxicology reports, in all cases examined for blood carboxyhemoglobin levels, except for rarely applied, imprecise tannin methods 3%, the second method used was the spectrophotometric analysis of Fretwurst-Meinecke.
This method uses the transformation of derivatives of hemoglobin, other than COHb in hemochromogen and measurement of absorbency of UV-light with a wavelength of about 576 nm through a blood sample before and after reduction with sodium dithionate in an alkaline environment. This method requires the addition to the blood of 30 ml of 0.1% solution of NH3 in a quantity sufficient to obtain a solution with UV-light absorbency in the range of 0.8-1.0. After 15 minutes, necessary for complete hemolysis to occur, the first measurement of absorbency is made (E1) . Next, 30 mg of sodium dithionate and 0.1 ml of 30% solution of sodium hydroxide (NaOH) are added and mixed. After 10 minutes the second measurement of absorbency is made (E2). The concentration of carboxyhemoglobin (CCOHb) is noted in reference to previously determined calibration using blood specimens with known COHb levels (0, 25, 50, 75 and 100%), according to the equation:
The main advantages of this method are:
-easy and quick preparation of specimens for analysis,
- short time of analysis,
- sensitivity (limit of detection 1%) is acceptable for forensic toxicology,
- low cost of analysis,
- possibility of automated assays.
Limitations of this method include:
- low accuracy for low concentrations (<5% COHb),
- risk of overestimation for concentrations >30% COHb,
- utility in the analysis of fresh, clean blood (without the effects of decomposition).
1.2 Russian results from toxicological tests for the presence of carboxyhemoglobin
Up to the time of examination and presentation of results in the Russian forensic medical reports on the 22nd of October 2014, made available by the families of four victims, full documentation describing examinations for the presence of COHb was found. Among those, the report on body No. 19 from sector 5 presents the only disclosed case in which Russian experts described in their concluding remarks, a positive result from the analysis of blood for the presence of COHb. Due to the amount of blood analyzed and the finding of 11% of COHb, as reported in the conclusion, this case became the reference for comparison of toxicological examinations on other Smolensk Victims.
An important aspect that warrants caution about the merits of final conclusions from toxicological tests arises from findings related to body No. 4 from sector No. 1. In the report from the survey of sector No.1 on the 10th of April, 2010, describing body No. 4 it was noted, “in the right pocket of a pair of trousers there was a Blackberry cell phone, 25 US dollars in denominations of 20 and 5 dollars, a package of SLIM cigarettes and a Bic lighter…” which indicates that the Victim may have been a cigarette smoker. In one of the investigative reports it is also noted, that the Victim smoked one cigarette half an hour before boarding the plane on the day of the crash. Also an interview with the family confirmed, that the Victim was a smoker. Except for this one case, the remaining Victims were non-smokers. Interviews with families excluded the possibility of having lived or spent time in a place, where contact with carbon monoxide was possible before departure for the ceremony of the 70th anniversary of the Katyn Massacre.
1.3 Analysis of quantitative data from the Russian toxicological reports.
After comparison of the quantitative results from the Russian toxicological reports, pertaining to body No. 19 from sector No.5 (see Fig.1) and body No.5 from sector No.3 (see Fig. 2), it was concluded, that the difference between values of E2/E1 in samples, and the value of E2/E1 pattern COHb 0% is exactly half of identical values found in the report on body No.19 from sector No.5, for which 11% COHb in examined blood was reported in the final conclusions.
Above: Fig.1. Excerpt from the translation of the toxicology report pertaining to carboxyhemoglobin (COHb). Body No. 19, sector No. 5
This indicates that in the concluding remarks on the toxicological examination of body No. 5 from sector No. 3 (see Fig.3), it should not have been stated that COHb was not found. Rather it should have been stated, that COHb was found at a level of 5.5%.
The quantitative results from all the available Russian toxicology reports and
Above: Fig.2. Excerpt from the translation of the toxicology report pertaining to carboxyhemoglobine (COHb). Body No. 5, sector No. 3
Fig.3 Excerpt from the translation of the toxicology report pertaining to carboxyhemoglobin (COHb). Body No. 2, sector No. 3
compared with the reference values indicate that the values E2/E1 from the examined blood samples are greater than the COHb 0% reference value, at the same time not exceeding the E2/E1 for the COHb 25% reference value. (see Fig.4). Thus, the values in relation to these reference points were used in the analysis.
where X represents the value from the blood sample.
1.1.4 Conclusions from the blood analyses
From the completed analyses using the reference points, it was established, that in the case of at least 4 Victims, the final conclusions in the report pertaining to COHb levels are “unreliable” and disagree with the results from the quantitative analyses found in those same reports.
Establishing the presence of carboxyhemoglobin in the Victims’ blood necessitates a determination of the source of carbon monoxide and whether it arose while the victim was still alive or whether it resulted from the effects of fire after death. Three of the five bodies were found at the crash site in an area remote from where there had been fire on the ground. Among them was body No. 2 from sector No. 3 in whom a level of 16.2% carboxyhemoglobin was found, even though the body lay a distance of about 16 meters from the edge of the area where there had been ground fire. However, body No. 5 from the same area lay half the distance away yet its COHb level was the lowest among all the analyzed bodies. Furthermore body No. 19 in sector No. 5, which was found directly in an area of ground fire (location of fire is shown in Fig.5), did not have a significant increase in its level of carboxyhemoglobin.
In this case, the measured concentration was lower than in body No. 2 from sector No. 3 by 4.1%. It can be clearly seen from these findings, that the carboxyhemoglobin levels in blood samples taken for forensic analyses on the 11th of April, 2010 do not correlate directly with the distance from any fire. Therefore, it can be assumed, that fire at the crash site was not the source of carbon monoxide found in the bodies. An additional reason to reject fires at the crash site as a cause of increased COHb levels in the Victims is provided by the results of the forensic examinations. Forensic specialists described burns affecting almost 20% of the body surface on a Victim having a COHb level of 16.2%. However, there were no such high thermal effects reported on the body of a Victim, who had a level of COHb of only 6% yet was located much closer to ground fire.
Likewise, forensic examinations of one of the Victims, in whom Russian toxicologists determined a COHb concentration of 11%, showed no correlation between direct contact with ground fire, and the COHb level. In this case the forensic report identified extensive internal damages such that there is no doubt that this person was lifeless at the crash site, with no signs of organ function, either of the heart or the lungs:
Heart- “in the area of the anterior surface of the pericardium there is a rupture with a fine-edged tear, 12 cm in length, facing diagonally upwards. In the pericardial space there is a large amount (5 ml) of dark blood. Moreover, on the boundary of the anterior wall of the right atrium and the interatrial septum there can be seen a diagonal-vertical complete rupture, 10 cm in length, which opens into the right atrial cavity”;
Parietal pleura - double-sided multiple trauma and in places fractured ribs and sternum. The damages are characterized by cracks from 0.5 to 4 cm in length;
Lungs - fill 1/3 of the lung cavity, pale-pink on the surface, with the consistency of cake.
From the above description, as air entered into the pleural space through the damaged parietal pleura, a pneumothorax developed preventing respiration. Therefore, contact with carbon monoxide arising from the fire zone would not result in inhalation so as to absorb CO by the Victim. Likewise, the circulation of CO throughout the body in the blood from which a sample of blood was taken for analysis would not have been possible given the extensive damage to the heart muscle wall.
Based on data from the literature, post-mortem absorption of carbon monoxide into the blood is possible only in very limited circumstances. The location of the bodies indicates that they were not exposed to such circumstances where they landed on the crash site. Therefore, it can be assumed, that contact of these Victims with carbon monoxide occurred earlier, while still alive, when there was momentary contact with air in which carbon monoxide was present. This means, it occurred while still on board the plane.
It is worth adding, that based on available information from the forensic examinations, of other Victims, in whom carboxyhemoglobin was found and similar extensive injuries were identified, the likelihood of absorption of carbon monoxide into the blood at the crash site is excluded.
Fig.4. Comparison of measured values using spectrophotometry and calculation of the true level of COHb in the blood of Victims.
Carbon Monoxide in the Blood of Victims
When carrying out forensic examination on the bodies of all victims of an air crash, an important aspect is securing materials for basic toxicological tests, related to changes in the human body caused by possible contact with products of incomplete oxidation (combustion) of organic substances (carbon monoxide).
Left: Fig.23. Copy of a translation of sections of cards 4 and 5 (199) of the Investigation Department of Smolensk Investigation Bureau at the Prosecutor’s Office of the Russian Federation. The document indicates partial or complete combustion of clothes and skin of 9 Victims from sector 5 [of the crash site].
A group of Polish specialists in forensic medicine, while in Moscow submitted to the Russians a request to conduct a wide range of tests to screen for the presence of carboxyhemoglobin (COHb) in the bodies of Victims. This was noted in the report (Fig.24 below) of the 10th of May, 2010 (signed doc. ZMS 205A/2010).
The Polish request was taken into account in part, which means, that in the documentation of five Victims (among ten available) no evidence was found to indicate that tests were conducted pertaining to COHb.
SUPPLEMENTAL DATA TABLES:
 Adamski Juliusz, Zuba Dariusz, „Oznaczenie karboksyhemoglobiny (COHb) w próbkach krwi sekcyjnej metodą chromatografii gazowej” Problems of Forensic Sciences 2012, vol. 89, 86–98
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