Factors Affecting the Loss of Carbon Monoxide from Stored Blood Samples*

Transcription

1 Factors Affecting the Loss of Carbon Monoxide from Stored Blood Samples* Donald H. Chace**, Leo R. Goldbaum, and Nicholas T. Lappas*** The Department of Forensic Sciences, The George Washington University, Washington, D.C Abstract The loss of carbon monoxide (CO) from whole blood or hemolysates has been investigated. Blood samples were exposed to the atmosphere or to a limlted volume of air for various storage periods at three temperatures. The nitlal hemoglobin (Hb) concentration and the percent carboxyhemoglobln (%COHb) saturation were varied in separate experiments. In addition, the effect of repeated exposure of blood to air was evaluated. The %COHb saturation decreased from 80 to 50% following storage of a 1-mL blood sample with 49 ml of air In a sealed container at room temperature for 45 hr. Greater decreases in the %COHb saturation were observed In samples which were exposed to the atmosphere. Lesser, but significant, losses occurred when samples were stored In a refrigerator or freezer. The concentration of Hb In the samples as well as the Initial %COHb saturation were found to influence the decrease In the %COHb saturation. Introductio..n Accurate detection and quantitation of drugs in postmortem samples may be influenced by the postmortem storage of these samples during either the phase I or phase il storage periods. Phase I storage period refers to the storage of cadavers between death and autopsy. Phase I1 storage period refers to the storage of body fluids and tissues between the time they are obtained at autopsy and the time they are analyzed. Although little can be done by the toxicologist to alter potential problems of storage during the phase I period, various precautions may be taken during the phase II period to minimize or completely eliminate alterations in drug concentrations. Previous studies have demonstrated that the percent carboxyhemoglobin (%COHb) saturation of postmortem blood samples may be decreased during phase I1 storage when those samples are "A partial report of this research was presented at the mooting of the Mid-Atlantic Association of Forensic Science, Baltimore, MD, April 1983 and at the S=xth Internat=onal Biodetedoration Symposium, Washington, D.C., August **In partial fulfillment of the degree requirements for the Master of Sc=ence in Forensic Science. "'*Author to whom requests for reprints should be addressed. exposed to air (1-5). However, these studies generally were not conducted systematically, but rather in a haphazard manner in which important parameters such as storage temperature, hemoglobin (Hb) concentration, initial %COHb saturation, volume of air, and surface area either were not carefully controlled or were not reported. Because the determination of the cause of death in a person exposed to carbon monoxide (CO) is based largely on the analytical result obtained by the forensic toxicologist, it was felt that the effect of several parameters on the stability of the COHb during phase II storage should be evaluated in a systematic manner. This paper describes the results of several COHb phase II storage studies in an attempt to expand and clarify previous work as well as to quantitate the decrease in the %COHb saturation. Experimental Preparation of Storage Samples A working standard of hemolysate containing 100% COHb was prepared by a modification of a procedure reported by Collison et al. (6). Approximately 10 ml of freshly drawn blood were collected in tubes containing EDTA (Becton-Dickinson #6537). The blood was transferred into another tube and was diluted with 50 ml of saline (0.85% NaCI w/v). The diluted blood was mixed by inverson and centrifuged at 2000 rpm for I0 rain. The diluted serum was discarded and the red blood cells (RBC) were resuspended in 25 ml of normal saline and recentrifuged. The supernatant was discarded. To each volume of resultant-packed RBC, three volumes of a ph 8.4 borate buffer (6.2 g sodium tetraborate dissolved in 55 ml of 0.2N NaOH and diluted to a final volume of 500 ml with water; ph adjusted to 8.4 with 0.1N NaOH or 0. IN HCI, if necessary) and 0.1 volumes of octylphenoxydecaethanol (Diluent 182, Instrumentation Lab., 126 g in 1 L water) were added. The solution was mixed for 5 rain to ensure complete hemolysis. The hemolysate was centrifuged at 3000 rpm for 5 min to remove cellular debris. Approximately 20 ml of the supernatant hemolysate was transferred to a 50-mL plastic syringe (B-D) and all the air expelled. A three-way Teflon stopcock fitted on the tip of the syringe was closed to the environment. Approximately 30 ml of high purity CO (Matheson) were added to the 50-mL syringe containing approximately 20 ml of hemolysate. The syringe was rotated for 15 min in order to saturate the Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission. 181

2 Journal of Analytical Toxicology, VoL 10, September/October 1986 hemolysate with CO. The excess CO gas was expelled following the mixing period and a fresh 30-mL sample of CO added and mixed for 10 min to ensure complete saturation. After the excess CO gas was expelled, approximately 40 ml of nitrogen was added to the syringe. The hemolysate was mixed with nitrogen for I min to remove excess dissolved CO. The gases were expelled and the hemolysate was stored in the syringe in the absence of any gas at 5~ The CO content of the presumably 100% COHb-saturated hemolysate was determined by spectrophotometry according to the method by Collison et al. (6). Hemolysates used for preparation of a CO standard curve were prepared from packed RBC obtained from a local hospital in a manner similar to the procedure described above. The hemolysate was divided into two portions, one of which was saturated with CO by means of the method described above. Known volumes of the CO-saturated hemolysate were diluted with the CO free hemolysate to produce putative COHb saturations of 100, 50, 25, 10, and 5%. The hemolysates were stored in air-tight syringes without air at 5~ Triplicate aliquots of each of these CO-saturated aliquots were analyzed by the method described below. The hemolysates used in the storage studies were prepared from fresh packed RBC which were diluted with saline and hemolysing agent to produce a hemoglobin concentration of 21, 13, 12, and 6 g/100 ml of hemolysate. The hemolysates were separated into groups based upon hemoglobin concentration. The hemolysates at each concentration of Hb were divided into two portions, one of which was saturated with CO by means of the method described previously. CO-saturated hemolysates at each hemoglobin concentration were diluted with CO-free hemolysate with the same Hb concentration to produce a final CO saturation of 80%. Within the 13 g/100 ml hemolysate group, additional COHb saturations of 50 and 25% were also produced. All samples were stored in air-free syringes without an observable air space at 5~ Whole blood used in the storage studies was prepared by diluting packed RBC with saline to the desired hemoglobin concentration of 13 g/100 ml of blood. One portion of the blood was saturated with CO as described previously and diluted with non-co-saturated blood to a desired COHb saturation of 80%. The blood was stored without air in a syringe at 5~ Postmortem blood samples used in the storage studies were prepared by adding 2 to 3 drops of hemolysing agent for every 1 ml of blood. The hemolysates were stored in the absence of air at 5~ Determination of the %COHb Saturation of Samples The gas chromatographic (GC) method of CO analysis as described by Goldbaum et al. (7) was used throughout this study. The CO binding capacity was determined using a cyanmethemoglobin procedure (8) with modifications. The %COHb saturation was determined by calculating the ratio of the CO content and the CO-binding capacity of a sample expressed as a percentage (Equation i): CO content (vol. %) CO binding capacity (vol. %) x loo = %COHb saturation Eq.l One milliliter of an acid liberating agent (2:2:1 lactic acid, HCI, and water) was injected into a reaction chamber (Figure 1) with the stopcock positioned so that dissolved gases were purged from the mixture. After the liberating agent was stirred for 1 min, the stopcock was closed. A sample of blood, either 50, 100, 150, or 4 -Way Stopcock Liberating Solution -- Hetium Carrier Gas Inlet t ion Port Figure 1, Diagram of reaction chamber, To G.C 250 p.l, was injected into the chamber and mixed with acid liberating agent for 5 min. Following this mixing period, the stopcock was positioned so that helium was allowed to purge the vessel and sweep liberated gases onto the GC column. The response of the detector was recorded by an integrator capable of determining the area of peaks produced on the chart recorder. The CO content (I.,,L/100 ml) of a sample of blood was determined by comparison with a standard CO-saturated hemolysate with a known CO content. The total hemoglobin concentration of the blood samples was determined by a modified cyanmethemoglobin method. A 20-p,L aliquot of hemolysate or blood sample was diluted with 5 ml of Drabkin's reagent and incubated at room temperature for at least 90 min to allow for complete conversion of COHb and other hemoglobin derivatives to cyanmethemoglobin. The absorbance of the unknown was compared to a standard curve of cyanmethemoglobin concentrations and comparative absorbances. The resultant hemoglobin concentrations were expressed as g/100 ml blood. The CO binding capacity of a 100% saturated blood sample was assumed to be 1.39 (ml CO/g Hb) at standard temperature and pressure (9). Following the determination of hemoglobin concentration and CO content of a sample, the %COHb saturation was calculated using Equation 1. Storage Studies All hemolysates or whole blood samples used in these studies, unless otherwise noted, had an 80% COHb saturation and a hemoglobin concentration of 13 g/100 ml. At the end of each storage period, the CO content, hemoglobin concentration, and the %COHb saturation were determined in each of two aliquots taken from each stored sample. Duplicate samples were stored under each storage condition. All control samples were stored at 5~ in sealed syringes in the absence of air. Storage of Hemolysatea Exposed to the Atmosphere Effect of temperature. One-milliliter aliquots of the hemolysate 182

3 were delivered into the barrels of 50-mL plastic syringes from which the plungers had been removed. Three-way valves fitted to the tip of the syringe were closed. The samples stored in the syringe barrels had a surface area of 4.5 cm 2. All samples were stored in a vertical position at either 23 ~ 5 ~ or -20~ Samples were stored at 5 ~ and 23~ for periods of l, 3, 6, 10, 20, or 45 hr or at -20~ for l, 2, 5, or l0 days. Effect of surface area and volume. One-milliliter aliquots of the hemolysate were stored either in a syringe barrel or test tube resulting in sample surface areas of 4.5 and 1.3 cm 2, respectively. Five-milliliter aliquots of hemolysate were also stored in a syringe barrel resulting in sample surface areas of 4.5 cm 2. A surface area to blood volume ratio (SA/V) of 0.9 resulted when 5 ml of hemolysate were stored in the 50-mL syringe barrel. An SA/V value of 1.3 corresponds to 5 ml of hemolysate stored in a test tube; the SA/V value of 3.7 corresponds to 5 ml of hemolysate stored in a sample cup; and the SA/V value of 4.5 corresponds to 1 ml of hemolysate stored in a 50-mL syringe. All samples were stored in a vertical position for periods of 1, 3, 6, 9, 24, and 48 hr at 23~ The samples stored in the cups were placed in a humid chamber, when the storage period exceeded 10 hr, to prevent drying of the hemolysate. Free air exchange was allowed to occur during storage. Effect of the initial %COHb saturation. Hemolysates with COHb saturations of 80, 50, and 25% were used in this study. Onemilliliter aliquots of the hemolysates were delivered into the barrels of 50-mL syringes from which the plungers had been removed. Three-way valves fitted to the tip of the syringes were closed. The samples stored in the syringe barrel had a surface area of exposure of 4.5 cm 2. All hemolysate samples were stored in a vertical position for 3, 6, 24, or 48 hr at 23~ Storage of Hemolyeates Exposed to a Limited Volume of Air Effect of storage temperature and volume of air. One- or fivemilliliter aliquots of hemolysate were delivered into 50-mL sytinges fitted with three-way valves. The syringe plungers were adjusted so that 1 ml of hemolysate was exposed to either 0, 4, 9, 24, or 49 ml of headspace air in the syringe. The 5-mL hemolysate was exposed to 45 ml of air. The syringes were stored in a vertical position for either 1, 3, 7, 10, 20, or 45 hr at 23 `= or 5~ One-milliliter samples stored at -20~ were exposed to 49 ml of air and stored for as long as 10 days. In all samples the three-way stopcock was closed to the environment. Effect of the initial %COHb saturation. Hemolysates with carboxyhemoglobin saturations of 80, 50, and 25% were delivered into 50-mL syringes fitted with three-way valves. The syringe plunger was adjusted so that 1 ml of the hemolysate was exposed to 49 ml of air. The three-way valves were closed upon addition of the hemolysates. The syringes were stored in a vertical position at 23~ for 4, 8, 24, and 48 hr. Effect of the initial hemoglobin concentration. Hemolysates with a COHb saturation of 80% and hemoglobin concentrations of either 6, 12, 13, or 21 g/100 ml of hemolysate were used in this study. One-milliliter aliquots of these hemolysates were delivered into 50-mL syringes fitted with three-way valves. The syringe plunger was adjusted so that 1 ml of hemolysate was exposed to 49 ml of air. The three-way valves were closed upon addition of the hemolysate. The syringes were stored in a vertical position for 24 hr at 23~ Storage of Whole Blood Samples Non-physiological samples. Whole blood with a COHb saturation of 80% and a hemoglobin concentration of 13 g/100 ml of blood, prepared as described above was used in this study. One- milliliter aliquots of this blood were delivered to 50-mL syringes fitted with three-way valves. The syringe plunger was adjusted so that 1 ml of blood was exposed to 49 ml of air. Upon addition of the blood samples, the three-way valves were closed. The sytinges were stored in a vertical position for 5, 24, and 48 hr at 23~ Postmortem samples. Postmortem blood samples with COHb saturations of 86, 77, and 87%, and hemoglobin concentrations of 18, 23, and 22 g/100 ml, respectively, were used in this study. One-milliliter aliquots of the postmortem samples were delivered into 50-mL syringes fitted with three-way valves. The syringe plunger was adjusted so that 1 ml of the postmortem sample was exposed to 49 ml of air. Upon addition of blood samples, the three-way valves were closed. The samples were stored in a vertical position at 23~ for 24 hr. Miscellaneous Storage Studies (Storage of Hemolyaatea Exposed to Nitrogen, Oxygen or Air) Hemolysates with a COHb saturation of 80% and a hemoglobin concentration of 13 g/100 ml of hemolysate were used in this study. One-milliliter aliquots of the hemolysate were delivered to 50-mL syringes fitted with three-way valves. Nitrogen, air, or a mixture of 95% oxygen and 5% carbon dioxide, were delivered to the syringes containing l ml of hemolysate so that the hemolysates were exposed to 49 ml of one of these gases. The threeway valves were closed immediately following addition of the hemolysate and gases. The samples were stored in the vertical position for 24 to 48 hr at 23~ Results and Discussion Carboxyhemoglobln Analysis The determination of the CO content of blood samples was simple, accurate, and precise as reported elsewhere (7). A chromatogram of blood gases and methane is presented in Figure 2. A CO content of 10.1 ml/100 ml blood for the 100% COHb saturated hemolysate was determined using a spectrophotometric B!!!!!! ; ; retention time in minutes Figure 2. Typical chromatogram. The gases represented are (A) oxygen; (B) nitrogen; (C) methane; and (D) carbon monoxide. D 183

4 method described by Collison et al. (6). Calculation of the CO content of this same standard by determining the theoretical CO binding capacity based on Hb concentration as described previously yielded a CO content of 10.4 ml/100 ml blood. No detectable change of the CO content in the fully saturated hemolysate was observed during the entire course of this research, approximately eight months, when the sample was stored in a syringe without air at 5~ Collison et al. (6) reported similar stability of CO-saturated hemolysates stored under similar conditions. The estimation of hemoglobin concentration was accurate and precise for blood containing CO, provided sufficient time, i.e., at least 90 min, was allowed for the complete conversion of COHb and other hemoglobin derivatives to methemoglobin. Reaction times of less than 90 min using a standard cyanmethemoglobin procedure for estimation of highly CO-saturated hemolysates may result in significant analytical error (10). The analytically determined %COHb saturations of all blood or IIO , "c 9 -zo hemolysates prepared for experimental use were within 1 to 3% of the assumed saturations in all cases. The COHb saturation of these "stock" hemolysates or blood samples remained constant throughout the experiments when stored in an air-free syringe at 5~ The CO content of postmortem blood samples also remained constant under proper storage conditions. Results of Storage of Hemolysates to the Atmosphere Effect of temperature. The results obtained from the atmospheric exposure of l-ml aliquots of a hemolysate with a COHb saturation of 80% are presented in Figures 3 and 4. The %COHb saturation decreased significantly in samples stored for a short period of time at either 23 ~ or 5~ i.e., p<0.05 for storage periods equal to or greater than 1 hr at 23~ and storage periods greater than or equal to 10 hr at 5~ when compared to control samples. The %COHb saturation in samples stored at - 20~ was decreased significantly after a storage period of 45 hr or more; i.e., p<0.05 for storage periods equal to or greater than 45 hr at 23~ when compared to control samples. It was observed that the hemoglobin concentration increased during the atmospheric exposure of hemolysates. The hemoglobin concentrations increased as much as 42, 45, and 10% following 20 hr of storage at 23 ~ 5 ~ and -20~ respectively. Based on the authors' findings that the rate of CO loss was inversely proportional to hemoglobin concentration (see below), it appears that the decreases in COHb saturation observed in this study may have been less than would have occurred had the hemoglobin concentration remained constant. The large standard deviations observed in the data obtained from the samples stored at 5~ were presumably a result of the inability to maintain the constant temperature of 5~ in the refrigerator due to the fact that the refrigerator was multi-purpose and was opened frequently during the day. Therefore, it may be suspected that the losses were somewhat greater than would have been the case at a constant 5~ temperature. However, in practical situations most blood samples are stored in a refrigerator which is heavily used. Effect of surface area and volume. The results obtained from the atmospheric exposure of l- and 5-mL hemolysate aliquots with,~) ;o ]'o,'o s'o Figure 3, The decrease of %COHb saturation in hemolysates stored at 23 ~ 5 ~ and - 20~ and exposed to the atmosphere. The means - 1 standard deviation are plotted. 60- iq cm ~l o IO-- I/. 7 o - ~ j ~ to. ~ ~ ~ 4 ~ ;,~",'0 2'0 3'0,~ s~ Figure 4. The decrease of %COHb saturation in hemolysates stored for several days at -20~ and exposed to the atmosphere. The means 1 standard deviation are plotted. o Figure 5. The decrease of '/ocohb saturation resulting from the atmospheric exposure of hemolysates with different surface area to blood volume ratios. The means _+ 1 standard deviation are plotted. 184

5 .o-1 9 o- \" 20-!!o Sur ace orea/volqme Figure 6. Decreases in %COHb saturation in hemolysates exposed to the atmosphere at 23~ as a function of the surface area to b~ood volume ratio. The means _ 1 standard deviation are plotted. 180" t I,n,hell ~COHb 9 80'4 6O 4o i 20 0 ~lwrbl/ofl 9 21S% $~oea~! lime (k~,,) Fioure 7. The decrease of %CDHb saturation resultino from the atmospheric exposure of hemolysates with different initial %COHb saturation. The means _ 1 standard deviation are plotted. surface area to volume ratios of 0.9, 1.3, 3.7, and 4.5 are presented in Figure 5. A significant (i.e., p<0.05) decrease in the %COHb saturation occurred at all SA/V ratios at storage periods of 3 hr or more. Figure 6 presents the decrease in %COHb as a function of SA/V for three different storage periods. This graph demonstrates that the rate of decrease of the %COHb saturation of the samples is a linear function of the SA/V ratio of a sample stored at 5~ and exposed to the atmosphere. It was observed that the hemoglobin concentration increased in all the samples; e.g., a 41% increase resulted when the 5-mL hemolysate was stored in the sample cup, a 42% increase when 1 ml was stored in a syringe, a 20% increase when 5 ml were stored in a syringe, and 12% when 1 ml was stored in a test tube. As discussed previously, an increase of the hemoglobin concentration may cause a slower rate of decrease in the %COHb saturation. In samples with a large SA/V ratio, the increase of the Hb concentration was greater than in samples with small SA/V ratios. Therefore, the actual rate of decrease of the %COHb saturation of samples with large SA/V ratios may be underestimated. Effect of the initial %COHb saturation. The results obtained from the exposure of 1 ml of 80, 50, and 25% COHb saturated hemolysates to the atmosphere are presented in Figure 7. It was determined that the %COHb saturation decreased significantly in all samples stored for short periods at 23~ i.e., p<0.01 for storage periods equal to or greater than 3 hr when compared to control samples. Therefore, in this study a significant loss of CO occurred when blood samples were exposed to the atmosphere for a period of at least 3 hr, when the initial percent saturation of COHb was between 25 to 80%. Storage of Samples Exposed to a Llmlted Volume of Alr Effect of storage temperature and volume of air. The results obtained from the exposure of 1-mL aliquots of a hemolysate with a COHb saturation of 80% to several different volumes of air at 23 ~ 5 ~ and - 20~ are presented in Figures 8, 9, and 1O. These data demonstrate that the loss of CO from stored samples is dependent upon the headspace volume in the storage container as well as the temperature during storage. Generally the rate of CO loss from these samples increased as either or both of these variables were increased. Significant CO losses from a 1-mL sample occurred in as little as 3 hr when a l-ml sample was stored with 9 ml of air at 23~ Decreasing the headspace volume to 4 ml or decreasing the temperature to - 20~ resulted in no significant CO loss over a storage period of approximately two days. With all variations of headspace volume and storage temperature studies, a constant %COHb saturation was attained in the stored samples. Presumably this occurred when equilibrium was established between the CO in the headspace and the CO in the hemolysates. The time required to establish equilibrium was inversely proportional to both temperature and headspace volume. In addition, the %COHb saturation of the hemolysates at equilibrium was dependent upon the headspace volume. This relationship is demonstrated in Figure 11 in which the %COHb saturation after 20 hr of storage (after equilibrium had been established in all cases) is plotted as a function of the headspace volume of air. The data obtained from exposure of 5 ml of blood to 45 ml of air at 230C are compared to the data obtained from the exposure of 1 ml of blood to 5 ml of air at the same temperature in Figure 12. These data demonstrate that it is the ratio of blood volume to air volume rather than the absolute value of either which can be related to the loss of CO from stored hemolysates. Effect of the initial %COHb saturation. The results obtained from exposure of l-ml aliquots of hemolysates in which the %COHb saturation was either 25, 50, or 80% to 49 ml of air at 23~ is presented in Figure 13. The %COHb saturation decreased significantly in all samples stored for a minimum of either 3 or 5 hr at 23~ (p<0.05). 185

6 The relative decrease in the %COHb saturation appeared to be related to the initial %COHb. For example, at equilibrium, samples with initial COHb saturations of 80, 50, and 25% demonstrated relative decreases of approximately 40, 26, and 22%, respectively. These differences in the relative % decrease of %COHb saturations may be due to the differences of CO binding at the three saturation levels studied. It is known that the dissociation of CO from COHb occurs more readily when the degree of saturation increases. For example, the loss of one CO molecule from Hb which is 100% saturated is more likely than when the Hb is 25% saturated due to the cooperativity effect. Effect of the initial hemoglobin concentration. The results obtained from the exposure of l-ml hemolysates with a COHh saturation of 80% and a hemoglobin concentration of either 6, 12, 13, or 21 g/100 ml to 49 ml of air at 23~ for 24 hr, is presented in Figure ML OJ Ale g 70- ],2 60" 70" IR t J , ol!! I f S STORAGE ( dov s ) PERIOD Figure 10, The decrease of %COHb saturation in 1-mL hemolysates exposed to 49 ml of air at - 20~ The means - 1 standard deviation are plotted. ]- o i~ 2'o ~'o,'o 5'o Figure 8. The decrease of %COHb saturation in 1-mL hemolysates exposed to varying volumes of air and stored at 230C, The means -1 standard deviation are plotted, 9 4g.! ~J J,l 60- Slorog* T~perat ~f9 ('c) t,~, Ikeurd Figure 9. The decrease of %COHb saturation in 1-mL hemolysates exposed to varying volumes of air and stored at 5~ The means -*-1 standard deviation are plotted. 2~ t I i I I I V,~ume ~ A*r l ml l Figure 11. Decreases in the %COHb saturation at equilibrium in hemolysates stored at three temperatures as a function of the headspace volume of air. The means +_. 1 standard deviation are plotted. 186

7 The decrease in the %COHb saturation was significant in all samples regardless of the hemoglobin concentration, i.e., p<0.01 when compared to controls. The results indicate that the decrease in the %COHb saturation of a carboxylated hemolysate exposed to a limited volume of air following 24-hr storage is dependent upon the hemoglobin concentration. It is apparent that the relationship between the final %COHb saturation and Hb concentration is linear over the range of Hb concentrations examined, i.e., r = (Figure 15). However, the linearity of this relationship apparently does not hold at low Hb concentrations, e.g., less than 6 g/100 ml, since the extrapolated y-intercept of this plot is approximately 22% COHb saturation rather than the theoretical y-intercept of 0% COHb. It is important to note that as the loss of CO from the hemolysate causes the %COHb to fall below 50%, the binding characteristics of the carboxyhemoglobin change as the %COHb saturation falls to even lower values as would be predicted in the case of a dilute carboxylated hemolysate exposed to large volumes of air. The ability for remaining CO to dissociate becomes more difficult, and therefore more air is needed to dissociate the complex. Such a mechanism may account for the apparent lack of linearity at low Hb concentrations. 70~ [r,' 60- H MOLYIAI (~) All (.i) ~lt I $ Storage of Whole Blood Samples Non-physiological samples. The results obtained from the exposure of 1 ml of whole blood samples, with COHb saturations of 80%, to 49 ml of air at 23~ for periods of either 5, 24, or 48 hr is presented in Table I. These data demonstrate that the %COHb saturation decreased significantly in samples stored for a short period of time at 23~ i.e., p < 0.05 for storage periods equal to or greater than 5 hr when compared to controls. However, there was no difference between the data obtained at 24 and 48 hr of storage. These results indicate that the loss of CO from the blood sample was complete after 24 hr. Apparently, an equilibrium was established between the CO in the air and in the blood. A comparison of the decrease in %COHb saturation obtained when 1-mL samples of either whole blood samples or hemolysates with initial %COHb saturations of 80% were exposed to 49 ml of air for 24 hr is presented in Figure 15. This comparison demonstrates that under these conditions of storage, the loss of CO is not influenced by the type of sample used. ~ ~ 2b 3'o,~ s~, hm9 Ikovfd 80- Figure 12. The decrease of %COHb in hemolysates with the same air volume to hemolysate volume ratio. The means -+ 1 standard deviation are plotted., ZCOHb In,u,al I % 9 50%.g 411! YxI.gX ", 23 t=096s N 'o 2b 3'0 4'0 sb ~l~,lue 1,~ thou,~j Figure 13. The decrease of %COHb saturation in 1-mL hemolysates with different initial saturation exposed to 49 ml of air at 23~ The means _+ 1 standard deviation are plotted. o,~ ~ h 1~, 2b Hemaglobm (gin / looml) Figure 14. Final %COHb saturation of t-ml hemolysates with different hemoglobin concentrations exposed to 4g ml of air at 23~ for 24 hr. The means _ 1 standard deviation are plotted. 187

8 80' 70" 60~..o,,,,+oo IqlMOtViAII Postmortem samples. The results obtained from the exposure of 1-mL aliquots of CO containing postmortem blood samples to 49 ml of air at 23~ for 24 hr are presented in Table II. These data demonstrate that the percent saturation of all of these samples decreased significantly, i.e., p < 0.0l when compared to controls. The rate of decrease in the %COHb saturation in these samples appears to be slower than that observed in whole blood samples to which CO was added (Table I), This apparently slower rate of CO loss in these postmortem samples may be due to the greater concentration of Hb in these samples than in the whole blood samples to which CO was added. SO. 40- n /= o 1'o ='0 3'0 4'0 5'0 $TOIAOI llmi (kouf*) Figure 15. The decrease of %COHb saturation in 1-mL whole blood samples exposed to 49 ml of air and stored at 23~ The means --1 standard deviation are plotted. Table I. The Decrease of %COHb Saturation in 1-mL Whole Blood Samples Exposed to 49 ml of Air and Stored at 23oc Storage Relative Period %COHb Standard Decrease (hr) (mean) Deviation p* (%) < < < "Compared to zero time storage; p determined by Student's t-test. Table II. %COHb Saturation of 1-mL Postmortem Blood Samples Exposed to 49 ml of Air and Stored for 24 Hr at 230C Sample Condition Hb concentration (9/100 ml) %COHb saturation (initial) %COHb saturation (final) Standard deviation p* <0.01 <0.01 <0.0t Relative % decrease ~ Final %COHb saturation compared to initial %COHD saturation', p determined by Student's t-test, Table III. %COHb Saturation of Hemolysates Exposed to Nitrogen, Oxygen, or Air end Stored for 24 Hr at 230C Gas of Exposure Condition Nitrogen Air 95% Oxygen %COHb saturation (initial) %COHb saturation (final) Standard deviation p 9 >0.99 <0.01 <0.01 Relative % decrease Final %COHb compared to Initml %COHb; p determined by Student's t-test. Effect of Nitrogen, Oxygen, and Air on CO Loss The results obtained from exposure of l-ml aliquots of a hemolysate with a COHb saturation of 80% to 49 ml of either nitrogen, a mixture of 95% oxygen and 5% carbon dioxide, or air at 23~ as described above is presented in Table III. These data demonstrate that the %COHb saturation decreased significantly when samples were exposed to air or oxygen, e.g., p < 0.01 when compared to control hemolysates following a 24-hr storage period. These data also demonstrate that exposure of a hemolysate to nitrogen results in no significant decrease in the %COHb saturation. A greater decrease in the %COHb saturation was found in the samples exposed to oxygen than in those exposed to air. These findings demonstrate that the loss of CO from stored blood samples is dependent upon the total amount of oxygen available. Therefore, when the amount of oxygen is increased (e.g., exposure to high concentrations of oxygen) the loss of CO will be increased. Conclusions Exposure of CO-containing blood to the atmosphere will result in a decrease in the %COHb saturation. The rate of decrease is related to the ratio of the surface area to the volume of blood exposed'to the atmosphere, temperature, and initial %COHb saturation. Carboxylated blood may loose nearly all of its bound CO if the exposure period is sufficiently long. Storage of carboxylated blood in a sealed container with a limited volume of air may result in a significant decrease of %COHb saturation. The decrease is related to the ratio of volume of air to volume of blood ratio, temperature, initial %COHb saturation, and hemoglobin concentration. The loss of CO will continue until an equilibrium has been established between the CO in the air and the CO in the blood. It has been determined that the stability of COHb is decreased by the presence of oxygen in the air, and therefore every effort must be made to prevent exposure of carboxylated blood to air. The authors have found that a syringe fitted with a three-way valve is suitable for storage of CO-containing blood samples. All air may be removed once a sample is drawn into a syringe and the syringe may be sealed to prevent contamination with air. When a sample is needed, it may be withdrawn from the syringe without exposing the entire volume of blood to air. The syringe then may be stored in a refrigerator until further analysis is required. If longterm storage is required, CO-containing blood should be stored without air in a freezer, preferably in a syringe. References 1. A.C. Maehly. Quantitative determination of carbon monoxide. In Methods of Forensic Science, Vol. 1. F. Lundquist, ed. Interstate Publishers, New York,

9 2. H. Hartridge. The action of various conditions on carbon monoxide and hemoglobin. J. Physiol. 44:22-33 (1912). 3. E.A, Gaensler, J.B, Cadigan, M.F. Ellicott, R,H. Jones, and A. Marks, A new method for rapid precise determination of carbon monoxide. J. Clin. Lab. Med. 49: (1957). 4. L.R. Goldbaum and F.L. Rodkey. Studies on the Evaluation of Problems Associated with the Measurement of Low Concentrations of Carboxyhemoglobin. National Technical Information Service, Document #PB (1979). 5. A. Ocak, J.C, Valentour, and R.V. Blanke. The effects of storage conditions on the stability of carbon monoxide in postmortem blood. J. Anal Toxicol. 9:202-6 (1985). 6. H.A. Collison, F.L. Rodkey, and J.D. O'Neil. Determination of carbon monoxide in blood by gas chromatography. Clin. Chem. 14: (1968). 7. L.R. Goldbaum, D.H. Chace, and N.T, Lappas. Determination of carbon monoxide in blood by gas chromatography using a thermal conductivity detector. J. Forensic Sci. 31: (1986), 8. The Quantitative Colorimetric Determination of Total Hemoglobin in Whole Blood at nm. Sigma Technical Bulletin No. 525, May K.M. Dubowski and J.L. Luke. Measurement of carboxyhemoglobin and carbon monoxide in blood. Ann. Clin. Lab. ScL 3: (1973). 10. J.D. Taylor and J.D. Miller. A source of error in the cyanmethomoglobin method of determination of hemoglobin concentration in blood containing carbon monoxide, Am. J. Clin. Pathol. 43: (1965). Manuscript received April 25, 1986; revision received June 17,