Journal of Analytical Toxicology, Vol. 24, October 2000

Carbon Monoxide Stability in Stored Postmortem Blood Samples*

Gary W. Kunsman t, Carolyn t . Presses, and Paola Rodriguez Forensic Toxicology Laboratory, Bexar County Medical Examiner's Office, San Antonio, Texas 78229

Abstract

Carbon monoxide (CO) poisoning remains a common cause of both suicidal and accidental deaths in the United States. As a consequence, determination of the percent carboxyhemoglobin (%COHb) level in postmortem blood is a common analysis performed in toxicology laboratories. The blood specimens analyzed are generally preserved with either EDTA or sodium fluoride. Potentially problematic scenarios that may arise in conjunction with CO analysis are a first analysis or a reanalysis requested months or years after the initial toxicology testing is completed; both raise the issue of the stability of carboxyhemoglobin in stored postmortem blood specimens. A study was conducted at the Bexar County Medical Examiner's Office to evaluate the stability of CO in blood samples collected in red-, gray-, and purple-top tubes by comparing results obtained at the time of lhe autopsy and after two years of storage at 3~ using either an IL 282 or 682 CO-Oximeter. The results from this study suggest that carboxyhemoglobin is stable in blood specimens collected in vacutainer tubes, with or without preservative, and stored refrigerated for up to two years.

Introduction

Carbon monoxide (CO) is an odorless, colorless, and tasteless gas that readily mixes with air and is produced as a result of the incomplete combustion of organic compounds. Its toxic effects result from its combination with the hemoglobin (Hb) in red blood cells. The binding affinity of CO for Hb is approximately 200-300 times that of oxygen for Hb. Although the binding of CO to Hb is reversible, CO is not spontaneously displaced from Hb, but is ultimately displaced by the mass action of oxygen. The half- life of CO is 5-6 h at normal oxygen concentration, 30-90 rain at 100% oxygen, and 30 min under hyperbaric conditions (1,2).

CO poisoning is the leading cause of both accidental and sui- cidal deaths in the United States. Smoke inhalation is the most common source of CO exposure, and exposure to automobile exhaust is the most commonly used route of intentional CO poi- soning (3,4). For these reasons, the analysis of postmortem spec- imens to determine the percent carboxyhemoglobin (%COHb)

* Presented in part at the Society of Iorensi( Toxir 1999 Annual Meeting, San luan, Puerto Rico.

t Author to whonl ,c, rresponden(e should be addressed.

saturation is routinely performed in the forensic toxicology labo- ratory. These results are then used in the interpretation of the extent of carbon monoxide exposure and the degree of carbon monoxide toxicity in cases such as fire fatalities, suicides, and motor vehicle and industrial accidents. Although deaths related to CO exposure have been noted over a wide range of %COHb, most fatalities in which CO intoxication is determined to be the cause of death are associated with %COHb greater than 50%. CO exposure may be a contributing factor in deaths in which %COHb is 10-50% (2,5,6).

The analysis of CO in blood is most commonly performed using a spectrophotometric technique, using either a clinical analyzer such as the IL CO-Oximeter or an ultraviolet-visible (UV-vis) spec- trophotometer. Spectrophotometric methods measure CO as COHb and are, therefore, Hb dependent. A number of other tech- niques, such as a microdiffusion method and a gas chromatog- raphy (GC) method (7), are available for this analysis.

The ability to accurately measure CO content in the blood is a significant factor affecting the interpretation of carbon monoxide toxicity. Previous studies suggest that carbon monoxide stability is affected by the volume of headspace above the sample (the greater the surface area, the greater the loss of CO), storage tem- perature (increased temperatures result in greater CO loss), pres- ence of a preservative, and initial carboxyhemoglobin saturation (higher initial %COHb results in greater and more rapid loss of CO) (8-10).

The stability of carboxyhemoglobin was evaluated in post- mortem blood samples based on a comparison of %COHb results obtained as part of the initial toxicological analysis to %COHb results obtained upon a reanalysis of the specimen originally assayed, as well as all additional blood samples obtained at autopsy. All blood specimens were stored for two years at 3~ between the two analyses and all measurements were made using a spectrophotometric method.

Materials and Methods

Materials Sodium hydrosulfite was purchased from Sigma Chemical Co.

All other reagents used (CO-Oximeter/Oximeter Diluent, Variclean solution, Zeroing solution, Caldye reference standard,

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Journal of Analytical Toxicology, Vol. 24, October 2000

Multi-4 CO-Oximeter control--Level 1) were purchased from Instrumentation Laboratory Co. (IL). The Level 1 control, which has 17 g/dL total hemoglobin (THb) and 60%COHb, was used as a positive control All reagents listed were used with both the IL 282 and IL 682 CO-Oximeters. Bovine blood, with a %COHb less than 1% measured using the IL 682 CO-Oximeter, was used as a negative control with the second analysis assays.

Equipment This study was performed using the IL 282 CO-Oximeter for

the initial analysis of samples and the IL 682 CO-Oximeter for the second analysis of samples originally analyzed using the IL 282, as well as any additional blood specimens collected at autopsy. The CO-Oximeter is an automated visible spectrophoto- metric analyzer used for the identification and quantitation of various blood gases including %COHb and THb. Although designed as clinical analyzers for testing untreated whole blood samples, both instruments have been used extensively in the analysis of postmortem blood samples, and a series of compar- ison studies between the various models of IL CO-Oximeters has shown that the IL 682 and IL 282 provide quantitatively similar results (11,12). Both instruments were operated, calibrated, and maintained in accordance with the manufacturer's specifications (13).

Analysis Postmortem blood specimens from 64 Medical Examiner cases

submitted throughout 1996 were analyzed for carboxyhe- moglobin saturation as part of the initial toxicological evaluation. This initial assay was performed on a single specimen from each case using the IL 282 CO-Oximeter. These same 64 cases were then subjected to a second CO analysis after two years of refriger- ated storage at 3~ Blood specimens were collected at autopsy in red-top (no preservative), gray-top (NaF), and purple-top (EDTA) vacutainer tubes. All available blood specimens from each case (n = 189), both with and without preservative, were analyzed in this second analysis using the IL 682 CO-Oximeter.

Five-hundred microliters of sample was treated with 25 mg sodium hydrosulfite and an equal volume of CO-Oximeter diluent. Sodium hydrosulfite was added to reduce any oxyhe- moglobin and methemoglobin present in the sample to hemoglobin (COHb remains unaffected) and thereby prevent their interference with the absorbance of COHb and the subse- quent quantitation of the %COHb (14). The diluent solution was used to hemolyze red blood cells and facilitate the sampling of the blood specimens by the instrument. The sample was vortex mixed to ensure adequate mixing and then centrifuged for 5 min. The supematant was then subjected to CO-Oximeter analysis and sub- sequent THb and %COHb quantitation.

Results and Discussion

Blood specimens from 64 Medical Examiner cases, totaling 189 samples, were submitted to the second analysis following a two- year storage period (Table I). In 38 of those cases, the specimen used for the initial analysis was identified, allowing for a direct

comparison of first versus second analysis results for each of those specimens. In addition, all of the other blood specimens available were analyzed to provide a comparison of %COHb results for blood specimens stored with and without preservative: red top compared to gray top compared to purple top and all results compared to the initial %COHb determination. For 26 cases the initial blood specimen analyzed was not identified, so no direct comparison could be made between the first and second analyses of the same sample. For those cases, all blood specimens were analyzed and compared to the initial %COHb result so that even if the first analysis specimen tube was not identified, that sample would definitely be repeated in the second analysis and the reproducibility of the data be available for review. CO stability was defined in this study as a second analysis result that closely approximated the first analysis and did not result in a significantly different interpretation of the %COHb level.

The resulting data were divided into three groups based on the initial %COHb saturation for purposes of interpretation: (1) < 10%COHb, (2) 11-49%COHb, and (3) > 50%COHb. Blood spec- imens from 35 of the reviewed cases yielded %COHb results that were less than or equal to 10%. Of those 35 cases, the blood tube used for the initial CO analysis was identified for 22 cases; this allowed for the direct comparison of initial versus delayed analysis for 22 blood samples. All of the additional blood samples sub- mitted with those cases were analyzed as part of this study resulting in a total of 105 analyses performed for these 35 cases. Ninety-nine (94%) of the repeat analysis resulted in samples that, like the initial sample, yielded results less than or equal to 10%COHb. Six (6%) of those samples resulted in a second anal- ysis result greater than 10%COHb. In summary, for those sam- ples with an initial result less than or equal to 10%COHb, the second analysis results ranged from less than 1 to 12%COHb. There was also a large percent difference noted between many of the first and second analyses including repeat analysis of those cases for which the initial specimen was identified. Most impor- tantly, however, all of the second analysis results were either within range of the first analysis or fell within a range of %COHb results generally not considered to be of toxicological signifi- cance.

The second group of samples were those for which the initial %COHb result fell within the range between 11 to 49%COHb. For these cases, only 14 initial analyses were performed. The tube used for the initial analysis was identified for 9 of the 14 cases analyzed. All of the additional blood samples submitted with those cases were analyzed as part of this study, resulting in a total of 42 analyses performed for these 14 cases. Forty (95%} of the repeat analysis resulted in samples that, like the initial analysis, yielded results within the 11 to 49%COHb range. A single case resulted in a repeat analysis outside of the 11 to 49%COHb range; first analysis results were 2.2 g/dL THb and 27%COHb for that sample. The repeat analysis of the initial sample following refrigerated storage yielded a result of 3.5 g/dL THb and 16%COHb. The additional samples analyzed for these cases yielded the following results following the storage period: 10.1 g/dL THb and 7.9%COHb for an unpreserved blood sample and 12.2 g/dL THb and 7.1%COHb for an EDTA-preserved tube. The variation in results between the initial analysis and the anal- ysis of stored specimens is more consistent with the analysis of

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Journal of Analytical Toxicology, Vol. 24, October 2000

Table I. Carbon Monoxide Data--Initial versus Second Analysis Results

First Analysis Second Analysis Case # DOD* Date t Sample* THb s CO" Date* Sample * THbw CO"

96-0001 l-Jan-96 15-Apr-96 12.4 1.7% 9-Jul-98 RT 18.5 2.30% GT 15.2 < 1% PT 15.7 < 1%

960002 1-Jan-96 8-Jan-96 GT 17.9 39.4% 9-Jul-98 RT 23.5 41.0% GT 23 38.9% PT 23.7 38.4%

96-0003 1-Jan-96 8-Jan-96 GT 4.2 1.8% 9-Jul-98 RT 4.8 < 1% GT 6.4 < 1% PT 9.4 < 1%

96-0015 3-Jan-96 ~96 GT 22.8 5.2% 9-Jul-98 RT 19.1 4.9% GT 21.7 4.5% PT 19.3 4.2%

96-0022 5-Jan-96 8-Jan-96 GT 20.7 34.3% 9-Jul-98 RT 24.1 39.4% GT 22.8 31.7% PT 20.8 33.4%

9643043 6-Jan-96 8-Jan-96 GT 8.6 74.4% 9-Jul-98 RT 8.5 75.6% GT 13.1 73.3% PT 10 75.2%

96-0081 12-Jan-96 17-Jan-96 RT 22.6 0.4% 9-Jul-98 RT 14 < 1% GT 17.8 < 1% PT 15.6 < 1%

96-0096 16-Jan-96 17-Jan-96 12.6 80.6% 24-Jul-98 RT 13.6 81.0%

GT 16.4 80,6% PT 13 80.8%

96-0155 28-Jan-96 29-Jan-96 23.2 80.9% 24-Jul-98 RT 20.9 82.7% GT 19.4 81.3% PT 21.8 81.8%

96-0164 29-Jan-96 7-Feb-96 20.3 70.3% 24-Jul-98 RT 12.2 70.7% GT 13.9 72.7% PT 13.9 70.6%

96-0190 4-Feb-96 7-Feb-96 26.7 2.5% 24-Jui-98 RT 17.9 3.5% GT 30.9 < 1% PT 21.1 4.0%

96-0204 6-Feb-96 7-Feb-96 23.9 63.8% 24-Jul-98 RT 23.6 65,6% GT 24.4 64,1% PT 17.9 65.1%

96-0212 8-Feb-96 9-Feb-96 RT 13.2 35.1% 24-Jul-98 RT 12.2 36.8% GT 12.2 36.3% PT 12.6 35.5%

96-0252 15-Feb-96 6-May-96 21 1.3% 27-Jul-98 RT 23.5 2.6% GT 22.7 2.5% PT 33.5 3.7%

96-0267 17-Feb-96 20-Feb-96 17.2 68.0% 27-Jui-98 RT 23.7 71.1~ GT 23.7 67.6~ PT 22.7 66.9%

96..0356 5-Mar-96 7-Mar-96 15.7 65.0% 27-Jui-98 RT 14.7 66.4% GT 16,9 65.9% PT 15.6 65.3%

* DOD, dateofdeath. Date of analysis. Sample tube type: GT, gray top; PT, purple top; RT, red top. Total hemoglobin.

" Carboxyhemogiobin.

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Journal of Analytical Toxicology, Vol. 24, October 2000

Table I. (continued) Carbon Monoxide Data--Initial versus Second Analysis Results

First Analysis Second Analysis Case # DOD* Date* Sample t THbS CO" Date* Sample* THbS CO"

96-0402 14-Mar-96 18-Mar-96 RT 20.5 23.6% 27-Jul-98 RT 15.9 GT 13.1 PT 12.1

96-0410 15-Mar-96 18-Mar-96 RT 18,2 37.6% 27-Jul-98 RT 11.6 GT 13 PT 12.2

96-0415 16-Mar-96 18-Mar-96 15.4 21.8% 27-Jul-98 RT 11.3 GT 10.6 PT 11.1

96-0416 16-Mar-96 18-Mar-96 RT 18.9 39.9% 27-Jul-98 RT 16.3 GT 16.6 PT 15.2

96-0417 16-Mar-96 18-Mar-96 RT 18.6 72.9% 27-Jul-98 RT 14.8 GT 16.3 PT 14.4

96-0425 17-Mar-96 18-Mar-96 RT 24.4 82.6% 27-Jul-98 RT 23 GT 22.7 PT 23.3

96-0476 27-Mar-96 29-Mar-96 24.3 70.1% 27-]ui-98 RT 18.9 GT 18.6 PT 17,6

9643482 28-Mar-96 29-Mar-96 23.3 3.5% 27-Jui-98 RT 24.5 GT 10,9 PT 18.6

96-0526 4-Apr-96 21-May-96 RT 5.9 32.4% 27-Jul-98 RT 14.2 GT 13.9 PT 8

96-0574 11-Apt-96 15-Apr-96 1.5 1.2% 27-Jul-98 RT 6.8 GT 6.8 PT 7

96-0575 11-Apr-96 15-Apr-96 17.7 0.0% 27-Jul-98 RT 5.4 GT 11.5 PT 17.9

96-0609 16-Apr-96 6-May-96 RT 7,6% 27-Jui-98 RT 13.5 GT 10.5 PT 15.6

96-0613 17-Apr-96 6-May-96 RT 11.1 0.6% 6-Aug-98 RT 12 GT 9.7 PT 9.6

96-0772 18-May-96 21-May-96 RT 18.2 56.9% 6.-Aug-98 RT 16.4 GT 15.5 PT 15.5

96-0773 18-May-96 21-May-96 RT 22.7 74.0% 6-Aug-98 RT 19.4 GT 17.9 PT 18.6

96-0967 15-Jun-96 28-Jun-96 9.1 9.0% 6-Aug-98 RT I0 GT 9.5 PT 10,5

* DOD, date of death. t Date of analysis. * Sample tube type: GT, gray top; PT, purple top; RT, red top. w Total hemoglobin. " Carboxyhemoglobin.

26.1% 25.5% 25.4%

38.5% 39.0% 37.3%

24.3% 25.3% 24.6%

40.4% 41.3% 40.1%

74.6% 73.8% 73.3%

82.7% 82.1% 82.3%

70.6% 70.7% 80.2%

4.3% 5.0% 3.2%

25.4% 24.8% 25.7%

<1% 6.6% 1.5% <1% <1% 2.0%

9.5% 11.9% 9.7%

2,4% 3.3% 2.3%

58.1% 57.8% 55.3%

73.5% 73.2% 73.1%

11.3% 10.7% 11.7%

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Journal of Analytical Toxicology, Vol. 24, October 2000

Table I. (continued) Carbon Monoxide Data--Initial versus Second Analysis Results

First Analysis Second Analysis Case # DOD* Date t Sample ~ THbS CO" Date t Sample* THbw CO"

96-1259 3-Aug-96 6-Aug-96 24.9 0.0% 28-Aug-98 RT 10.5 < 1% GT 11.4 < 1% PT 11.3 <1%

96-1260 4-Aug-96 6-Aug-96 17.9 2 . 9 % 28-Aug-98 RT 21.2 3.1% GT 22.9 3.2% PT 12.4 3.0%

96-I 271 6-Aug-96 9-Aug-96 5.4 80 .8% 28-Aug-98 RT GT 9.8 76.8% PT 8.5 82.8%

96-1355 23-Aug-96 26-Aug-96 RT 14.4 0.0% 28-Aug-98 ~ 9.6 1.2% GT 9.3 1.5% PT 9.7 1.4%

96-1366 25-Aug-96 26-Aug-96 RT 11.4 1 . 0 % 28-Aug-98 RT 6.8 < 1% GT 24.2 < 1% PT 8.7 5.1%

96-I 455 12-Sep-96 12-Sep-96 9.6 19.0% I-0d-98 RT 9.3 20.8% GT 9.5 21.3% PT 9.2 21.4%

96-1458 12-Sep-96 30-Sep-96 2.2 27.2% 1 -Oct-98 RT 10.1 7.9% GT 3.5 16.0% PT 12.2 7.1%

96-1469 13-Sep-96 30-Sep-96 3.9 32 .7% 22-Oct-98 RT 6.4 37.2% GT 6.6 37.2% PT 6 32.6%

96-I 497 20-Sep-96 30-Sep-96 13.6 2.3% 2-0ct-98 RT 19.7 1.0% GT 15.7 < 1% PT 15.9 1.0%

96-I 524 23-5ep-96 30-Sep-96 I 1 .I 9.4% 2-0ct-98 RT 15.6 8.2% GT II.7 11.6% PT 13 8.4%

96-I 539 28-5ep-96 30-Sep-96 7.5 4.0% 2-0ct-98 RT 9.6 < 1% GT 8.3 1,2% PT 9.6 1.0%

96-I 546 29-Sep-96 30-Sep-96 16.3 27,3% 2-0ct-98 RT 15.8 29.0% GT 20.2 28.3% PT 16.4 28.1%

96-I 559 I-0ct-96 I-0ct-96 18 78.0% 2-Feb-99 RT 19,5 76.7~ GT 19.4 77.1% PT 18,4 76.9%

96-1580 5-Oct-96 10-Oct-96 GT 13.6 37 .1% 22-Oct-98 RT 11.8 38.6% GT 14.1 37.6% PT 14.2 37.5%

96-1615 11-Oct-96 14-Oct-96 GT 1.5 0.0% 22-Oct-98 RT 12.2 1.2% GT 12.5 2.4% PT 12.4 3.0%

96-1617 12-Oct-96 1443ct-96 GT 2.1 21 .6% 22-Oct-98 RT 13.1 21.7% GT 15.4 20.6% PT 14.9 19,7%

�9 DOD, date of death. ~" Date of analysis.

Sample tube type: GT, gray top; PT, purple top; RT, red top. Total hemoglobin,

" Carboxyhemoglobin.

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Journal of Analytical Toxicology, Vol. 24, October 2000

Table I. (continued) Carbon Monoxide Data--Init ial versus Second Analysis Results

First Analysis Second Analysis Case # DOD* Date t Sample* THD CO" Date t Sample* THbw CO"

96-I 681 24-Oct-96 29-Oct-96 GT 25.35 1.1% 8-Feb-99 RT 17.4 GT 18.9 PT 23.1

96-I 713 31-Oct-96 6-Nov-96 GT 17.8 7.6% 8-Feb-99 RT 13.6 GT 13.1 PT 13.7

96-1717 1-Nov-96 6-Nov-96 GT 18.8 1.1% 8-Feb-99 RT 10.7 GT 13.5 PT 13.6

96-1755 10-Nov-96 15-Nov-96 GT 8.1 7.1% 8-Feb99 RT 7.1 GT 5.6 PT 8

96-1768 14-Nov-96 15-Nov-96 GT 7 4.6% 8-Feb-99 RT 5.8 GT 8 PT 1 O.3

96-1799 20-Nov-96 11-Dec-96 GT 15 7.7% 8-Feb-99 RT 11.1 GT 14.8 PT 12.8

96-1836 26-Nov-96 11 -Dec-96 GT 16.8 0.1% 8-Feb-99 RT 9.9 GT 5.8 PT 7.6

96-1885 6-Dec-96 11-Dec-96 GT 17.7 0.2% 8-Feb-99 RT 10.4 GT 11 PT 9.8

96-1886 6-Dec-96 11-Dec-96 GT 21.6 0.1% 9-Feb-99 RT 10.1 GT 11.5 PT 7.8

96-1889 7-Dec-96 11-Dec-96 GT 16.8 1.3% 9-Feb-99 RT 10.1 GT 8 PT 15,4

96-1898 8-Dec-96 11-Dec-96 GT 15.4 0.0% 9-Feb-99 RT 14 GT 17.3 PT 17.2

96-1903 I0-Dec-96 11-Dec-96 GT 15.9 1.6% 9-Feb-99 RT 12 GT 6.6 Err 10

96-1906 I0-Dec-96 11-Dec-96 GT 16.2 0.1% 9-Feb-99 RT 9 GT 10.1 PT 10.2

96-1912 12-Dec-96 15-Jan-97 GT 8.5 1.6~ 9-Feb-99 RT 9.2 GT 13.2 PT 11.4

96-I 937 15-Dec-96 18-Dec-96 GT 19.5 4.9% 9-Feb-99 RT 9.7 GT 5.8 PT 7.8

96-2000 24-Dec-96 1S-Jan-97 GT 19.5 72.0% 9-Fei>99 krl " GT 9.8 PT

* DOD, date of death. t Date of analysis. * Sample tube type: GT, gray top; PT, purple top; RT, red top. w Total hemoglobin. " Carboxyhemoglobin.

1.2% 1.3% 1.2%

6.6% 6.9% 6.7%

<1% <1% <1%

<1% 2.5% 3.0%

5.2% 3.5% 2.4%

13.1% 9.4% 9.4%

<1% 2.8% <1%

<1% 1.1% 1.2%

<1% 3.6% <1%

1.0% 2.0% 1.6%

<1% <1% <1%

1.6% 3.9% 2.5%

<1% 2.2% <1%

2.0% 1.9% 1.6%

4.6% 4.5% 4.2%

70.4%

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Journal of Analytical Toxicology, Vol. 24, October 2000

low THb samples than with the loss of CO during storage (7). Summarizing the results of the analysis of the stored specimens, it was found that only 50% of second analysis results were within • 5% of initial result. Ninety-five (95%) of the second analysis results were within • 25% of initial result. More specifically, reviewing the repeat results for those cases for which the initial tube analyzed was identified, the second analysis results were within + 10% of the initial result for 8 (89%) of the samples and within • 25% of the initial result for one sample (11%). That case sample had an initial result of 32%COHb and a repeat result of 25%COHb. The range of %COHb values from 11 to 49% is generally considered to be toxic, but not lethal, except possibly in combination with other CNS depressants or in individuals with reduced pulmonary function. The results obtained for these samples exhibited a close correlation between the initial and second analyses. The interpretation drawn from either set of analyses concerning the significance of the measured %COHb level would be essentially the same.

The third group of samples were those for which the initial %COHb results were greater than or equal to 50%COHb. For these cases, 15 initial analyses were performed. The tube used for the initial analysis was identified for 5 of the 15 cases analyzed. All of the additional blood samples submitted with those cases were analyzed as part of this study resulting in a total of 42 analyses performed for these 15 cases. Forty-one (98%) of the samples tested had levels greater than or equal to 50%COHb and within • 5% of the initial result determined for each of those cases. The repeat analysis of the five identified initial samples were also within • 5% of the initial result. A single case had one specimen for which the result was 15% from the initial sample analyzed for that case, but the result was still greater than 50%COHb. There was very little variability noted between the initial and the second analysis results for these cases with elevated %COHb levels for which CO intoxication was determined to be the cause of death

Conclusions

The results of this study suggest that carboxyhemoglobin is stable in postmortem blood samples stored under the conditions described. Of those factors commonly thought to affect post- mortem COHb stability (storage temperature, presence of a preservative, volume of headspace above the sample, and initial %COHb), only the storage temperature was controlled in this study by storing specimens for two years at 3~ The initial %COHb values ranged from less than 1% to 82.6%, and both pre- served (NaF or EDTA) and unpreserved (red top) specimens were analyzed. The volume of headspace was essentially controlled for those specimens initially analyzed and then repeated because only 500 IJL of blood was removed in most cases, but was not con- trolled between blood samples from different cases or for the additional blood samples analyzed as part of the second analysis. In this study, preservative, volume of headspace, and initial

%COHb did not significantly affect the %COHb level in post- mortem blood specimens refrigerated for two years following specimen collection at autopsy. It is, therefore, acceptable to per- form a repeat analysis on a blood specimen, perform a second analysis on a case using a different blood specimen from that used in the initial analysis, or perform a first analysis on a stored post- mortem blood specimen up to two years after the specimens were collected if they were stored at 3~ over the entire storage period. It is recommended, however, that caution be used in the inter- pretation of such analyses with samples that have a THb less than 4 g/dL (7).

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Manuscript received March 31, 2000; revision received May 24, 2000.

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