Understanding blood analysis in dui and traffic homicide investigations

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0.18-0.30 Confusion

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Exaggerated emotional states (fear, rage, grief, etc)

Disturbances of vision (diplopia, etc.) and perception of color, form, motion, dimensions

Increased pain threshold

Increased muscular incoordination; staggering gait; ataxia

Apathy, lethargy
0.25-0.40 Stupor

General inertia; approaching loss of motor functions

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Marked muscular incoordination; inability to stand or walk

Vomiting; incontinence of urine and feces

Impaired consciousness; sleep or stupor

0.35-0.50 Coma

Complete unconsciousness; coma; anesthesia

Depressed or abolished reflexes

Subnormal temperature

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Possible death

0.45+ Death

Probable death from respiratory arrest


Gas Chromatography

The oldest and most fundamental chemical test for intoxication is a test for ethanol in blood. Blood-alcohol analysis is commonly performed in driving under the influence (DUI) arrests and investigations of serious injury or fatal traffic accidents. This analysis is undertaken with whole blood samples collected from the suspect, as well as from any deceased driver or passenger. There are a variety of laboratory methods to determine the alcohol concentration in a biological specimen. However, the criminal justice practitioner should be familiar with the two most common methods: gas chromatography and enzymatic assay.

In General: Forensic ethanol analyses typically employ a scientific process known as gas chromatography, which is a widely-used technique in modern analytical chemistry. Known as a separation science, gas chromatography is an instrument-based technology that separates mixtures of molecules based upon their chemical and/or physical properties. The instrument is called a gas chromatograph (commonly abbreviated as GC). Components of all GCs include a stationary medium where the actual separation takes place, an injection device for introducing the sample mixture to the stationary medium, a carrier gas to move molecules trough the stationary medium and a device to detect the separated molecules, all enclosed in a temperature-controlled oven. These components are connected in series to create a closed, tubular pathway for the molecules and gas to travel through the system.

GC Operation: Separations occur with molecules in the gas state, which requires that most substances be vaporized during the analysis. This is accomplished by “injecting” via syringe a liquid solution of the molecules into a very hot (200°C to 250°C), glass-lined chamber (10 cm long x 4 mm diameter) where the molecules are vaporized into the necessary gas state. Pressurized carrier gas (typically helium) flows through this chamber and carries the vaporized molecules to a stationary, porous, inert powder such as silica packed into a long narrow stainless steel tube or column (6 feet long x 1/8 inch diameter). This packed material is called the stationary phase because it remains stationary within the column as the molecules are carried through by the pressurized gas stream. The vaporized molecules “stick” to the stationary phase based upon their chemical or physical attraction in a process called adsorption. The column is then heated (hence the oven) to a temperature where molecules begin to “boil away” or dissociate from the stationary phase to be carried downstream by the gas (called the moving phase) in a process called elution.

As these molecules flow downstream, they actually undergo repeated cycles of re-adsorption and dissociation with the stationary phase in a process called partitioning. The stronger the attraction is between molecules and the stationary phase, the more frequently they re-adsorb and remain “stuck” and the slower they will elute from the column. This is not unlike people stepping on and off a moving sidewalk, where the speed of travel depends upon the time spent on the moving versus stationary platforms. A current alternative stationary phase to the solid porous powder packing is a waxy or resinous coating applied to the inner surface of a long coil of flexible glass capillary tubing (30 meters long x 100 µ to 530 µ inside diameter). This coating serves the same purpose as the solid porous powder packing by offering a surface to which molecules may repeatedly adsorb and dissociate as they flow through the column. In a properly designed system, the end result of partitioning is the elution of a succession of molecules separated into groups with similar or identical chemical and/or physical properties and, hence, structure.

Eluted molecules leave the column and flow into a detection device. The device typically used for ethanol analyses is the flame-ionization detector (FID), which generates an electrical signal in proportion to the mass of molecules passing through. The successive waves of separated molecules eluting from the column and passing through the detector provide a time chart (called a chromatogram) which appears as a series of Gaussian (bell-shaped) peaks, each representing a group of eluted molecules. The time taken by each group of molecules to elute is called the retention time and is an identifiable characteristic of the molecules.

Individual substances may be quantified by measuring the size of the peaks eluting with the retention time characteristic for the substance. The GC is calibrated with a series of samples or calibrators containing known amounts of substances and establishing the detector responses for each substance separated in the mixture. These responses are then used with linear regression mathematics to calculate the mass of each eluted substance.

Headspace Gas Chromatography

Ethanol is a small molecule that readily evaporates into a gas state at ambient temperature, even from solution in water. This volatility lends ethanol to a special type of analysis called headspace GC, which is a process well suited for the analysis of gases. Headspace analysis refers to the analysis of the air (head) space above a liquid or solid in a container. This is an indirect analysis because the vapors emitted from the sample are tested rather than the sample itself. This is not unlike identifying wine from its aroma. Headspace GC differs from conventional GC in that a vapor rather than a liquid sample is introduced into the GC. Similarly-volatile substances such as methanol, acetone and 2-propanol (isopropanol) may also be separated and measured with headspace GC.

The contents of a liquid’s headspace reflects the contents of the liquid itself because Henry’s Law (1803) states “At a constant temperature, the amount of a given gas dissolved in a given type and volume of liquid is directly proportional to the partial pressure of that gas in equilibrium with that liquid.” In other words, in a sealed vessel and at equilibrium, volatile substances will be present in the vapor state above a liquid at concentrations in proportion to their respective concentrations in the liquid. Therefore, if a specimen is placed in a sealed vial, then one may determine the concentration of a volatile substance in the liquid by analyzing the equilibrated headspace above the liquid. With headspace GC, only volatile substances are analyzed so the potential universe of interferences is drastically limited. Furthermore, because the non-volatile substances in the specimen are not injected into the GC (like dirt in a modern automobile fuel system), the longevity of the GC is extended and necessary maintenance reduced. It is not uncommon for a headspace GC to operate properly for years without requiring maintenance or repair.

Specimens are prepared for headspace GC analysis by dispensing a small volume (typically 100 µL) into a glass vial (typically 20 mL) and adding a diluent (typically 1.0 mL of a saturated sodium fluoride solution containing 1-propanol or tert-butanol as internal standard). Vials are sealed by crimp-cap and placed into the headspace injector where they are gently heated with gentle mixing at 40°C to 70°C for 5 min to 15 min. During this period, volatile substances in the liquid equilibrate with the headspace in the vial above the liquid. The vial is then pressurized with carrier gas, after which the gas flow is reversed so that the pressurized vapor in the vial may flow to the column through a transfer line. This is not unlike blowing into a balloon then having it blow back. The process of sample equilibration, mixing and transfer and GC analysis is automated so the analysis may proceed unattended. Specimens are typically analyzed in batches along with quality control samples to allow monitoring of the accuracy and precision of the process.

The diluent used in sample preparation is vital to the accuracy and reliability of headspace GC. First, the internal standard, a substance of similar chemical and physical properties as the target analyte, provides a retention time marker and a scale against which the quantity of the substance is normalized. This is not unlike a ruler in a photograph providing a reference for object size.

Second, the saturated salt, typically sodium fluoride, promotes volatilization of substances from the liquid specimen, thereby increasing the sensitivity of the analysis. By diluting the specimen with such an overwhelming mass of salt, any variability in specimen composition or consistency is equalized, thereby improving the precision of the analysis.

A recent innovation in blood alcohol analysis involves splitting the injected specimen vapor into two parallel capillary columns with somewhat different stationary phases and in which target substances are expected to elute with somewhat different retention times. Assurance that ethanol is correctly identified and quantified is improved because the eluted peak identified as ethanol must meet the characteristic retention times for both columns in the same analysis. Whereas use of dual-capillary column headspace GC improves confidence in results, it does not render obsolete single-column analyses7.

The accuracy of any quantitative analysis should be monitored with the use of control materials. Control materials, like calibrators are samples which contain known amounts of substance and which are analyzed concurrent to specimens. If the control materials yield the expected results (each analysis should have acceptance standards for this), then the analysis is deemed to be “in control”. Otherwise, the analysis is “out of control” and specimen results should be rejected. Out-of-control analyses should be inspected to determine the problem then re-calibrated. In reality, analyses do not require periodic re-calibration as long as they are properly controlled and demonstrate that the system has experienced no fundamental change. Therefore, whereas it is customary to periodically re-calibrate an analysis, this is not scientifically necessary as long as concurrent control materials meet acceptance standards. Performance of control materials is the most reliable indicator of the accuracy of the analysis.

Hospital Analysis

Clinical and hospital laboratories also conduct ethanol determinations but typically do so with serum rather than whole blood. This is because clinical laboratories are engaged in diagnostic testing, which is focused primarily on a vast universe of substances in serum of which ethanol is only one. Furthermore, rather than using the headspace GC, hospital and clinical laboratories use an enzymatic method to distinguish and quantify ethanol in serum. A typical enzymatic method relies upon the enzyme, alcohol dehyrogenase (ADH) to distinguish ethanol in a biological specimen. ADH oxidizes alcohol to acetaldehyde in the presence of a coenzyme, nicotinamide adenine dinucleotide (NAD+), which is reduced to NADH. The NADH is then measured as it is produced in proportion to the concentration of ethanol in the specimen.

[Ethanol + NAD+ → Acetaldehyde + NADH + H+]8

The primary reason hospital laboratories use an enzymatic rather than a GC method is because an enzymatic method is easily adapted to existing clinical laboratory instrumentation. Furthermore, enzymatic methods may be used for individual specimens and can be accomplished in as short as 20 minutes whereas GC analyses are typically conducted with batches of specimens and may require many hours. However, enzymatic methods lack the precision of GC methods (2.5% deviation common in analysis) and may not be entirely specific for ethanol. Such non-specificity may allow other substrates (e.g. isopropyl and butyl alcohols) to contribute to the end result.

Another difference is that clinical laboratories typically express ethanol results as milligrams of ethanol per deciliter of specimen (mg/dL). This difference, however, reflects only a difference in the units of expression and not the actual content of the specimen. If a hospital or clinical result is to be used for evidentiary purposes, the impact of the different methodologies and specimen on the interpretation of the result should be examined.

Despite any technological differences between GC and enzymatic methods for ethanol analysis, the requirements for proper calibration and verification through controls remain to insure the delivery of accurate results.


Who Can Draw Blood?

Under the Code of Alabama, 1975, section 32-5A-194 (a)(2), “only a physician or a registered nurse (or other qualified person)” is authorized to take a blood sample for use as evidence in civil and criminal cases. See, McGough v. Slaughter, 395 So. 2d 972 (Ala. 1981). See, also, Lankford v. Redwing Carriers, Inc., 344 So. 2d 515 (Ala. Civ. App. 1977): The purpose of allowing only physicians, registered nurses, or duly licensed clinical laboratory technicians to withdraw blood samples is to ensure that standardized procedures and equipment is used, thereby preserving the validity of the test. “Strict compliance with the Chemical Test for Intoxication Act is required.” Lankford, supra.

Alabama Code section 32-5A-194 (a)(2) mandates that only certain licensed persons may draw blood samples. By statute, all licensed physicians and registered nurses are presumed competent to draw evidentiary blood samples. The term “other qualified person” is not further defined within the Code, but several prior court decisions held that a licensed tab technologist is qualified to draw blood. See, McGough v. Slaughter, 395 So. 2d 972, 975 and Rehling v. Carr, 330 So. 2d 423 (Ala. 1976).

In Powell v. State, 515 So. 2d 140 (Ala. Cr. App. 1986), the defendant submitted to a blood sample drawn by a licensed medical laboratory technician. Defense counsel later objected to the blood draw, but the Court specifically held the lab technician “was therefore qualified to draw blood samples” in accordance with the statute. Powell, 515 So. 2d at 1446. In the later case of Ingram v. State, 720 So. 2d 1036, 1041 (Ala. Cr. App. 1998), where the blood sample was drawn by a licensed medical technologist working as a medical laboratory technician, no objection was made to the technician’s credentials or qualifications9.

However, it is instructive to note that all of the above cited cases, except Ingram, were decided prior to the comprehensive revision of the pre-existing statute to the current 32- 5A-194, commonly known as the “Chemical Test for Intoxication Act.” The original statute was enacted in 1969 and was codified at Title 36, section 155. The original statute was worded more exactly than the current statute. In the prior Title 36, section 155, in paragraph (C), the statute stated the following:

Only a physician, registered nurse, or duly licensed clinical laboratory technologist or clinical laboratory technician acting at the request of a law enforcement officer may withdraw blood for the purpose of determining the alcoholic content therein.”

The current Code section was enacted in 1988, and upon revision, concerning the appropriate persons authorized to draw blood samples, retained the terms “physician” and “registered nurse” but replaced “licensed clinical laboratory technologist” and “clinical laboratory technician” with the words “other qualified person.” The term “other qualified person” is not further statutorily defined10. Presumably, the Alabama Department of Forensic Sciences has the authority under the Alabama Administrative Code to determine appropriate qualifications or set standards for credentialing for persons to meet the term “other qualified person,” but as of this writing, DFS has not done so. Therefore, the term “other qualified person” is left open to the sound discretion of the trial court to determine the proper training, certification, and credentials of the individual that drew the blood sample.

Custody of the Sample:

By statute and decisional law, the state must identify the person and offer into evidence the credentials of the duly authorized person who drew the blood sample from the defendant. The blood sample cannot be presumed to have been taken in the correct manner unless the blood draw is established by the person who took the sample. The law of blood test admissibility in Alabama courts is extensive and clear: blood test evidence must be established by both predicate and chain of custody. These two requirements are properly subject to thorough cross-examination by defense counsel.

The leading Alabama case in this area regarding admissibility of the results of laboratory samples is Ex parte Holton, 590 So.2d 918 (Ala. 1991) which examined in detail the theory of chain of custody11. In order to establish a proper chain, the State must show to a reasonable probability that the object is in the same conditions, and not substantially different from, its condition at the commencement of the chain. The court requires that proof be shown on the record with regard to exact chain of custody of the sample.

The chain of custody is composed of “links.” A link is anyone who handled the item. The State must identify each link from the time the item was seized. In order to show a proper chain of custody, the record must show each link and also the following with regard to each link’s possession of the item: 1) the receipt of the item; 2) the ultimate disposition of the item, i.e., transfer, destruction, or retention; and 3) the safeguarding and handling of the item between receipt and disposition. If the State, or any other proponent of demonstrative evidence, fails to identify a link or fails to show for the record any one of the three criteria as to each link, the result is a “missing” link, and the item is inadmissible. If, however, the State has shown each link and has shown all three criteria as to each link, but has done so with circumstantial evidence, as opposed to the direct testimony of the “link,” as to one or more criteria or as to one or more links, the result is a “weak” link. When the link is “weak,” a question of credibility and weight is presented, not one of admissibility. See, also, Lee v. State, 748 So. 2d 904 (Ala. Cr. App. 1999)12.

In regards to blood samples, all three Alabama appellate courts have adhered to the ‘link’ analysis for establishing the chain of custody. In Creel v. State, 618 So.2d 132 (Ala. Cr. App. 1992), a vehicular homicide case where chain of custody of the blood sample was questioned, the Court found the state did not establish a chain of custody with respect to vials of blood drawn from the defendant following an automobile accident. The transmittal forms accompanying vials upon their arrival at Department Forensic Sciences in Auburn were not signed or initialed by person who shipped blood from Dothan, and forensic sciences investigator in Dothan who collected blood from investigating officers and placed it in a refrigerator with the transmittal forms could not unequivocally testify that he was person who shipped blood.

The Courts generally apply a “reasonableness” test in regards to maintaining security over the blood samples. The case of Wallace v. State, 574 So.2d 968 (Ala. Cr. App. 1990) is instructive. In that case, the nurse on duty drew two blood samples at the hospital and handed two sealed samples to the investigating police officer. The officer then placed the vials inside a sealed Styrofoam box (referred to in the Court’s opinion as ‘a DUI evidence kit’) in a refrigerator at City Hall where the kit remained over the weekend. The refrigerator was not locked or secured and was accessible to any number of city employees. The following Monday morning, the officer retrieved the still- sealed kit and delivered it to the forensics lab for analysis. The forensic analysis testified that there was nothing to indicate the kit had been tampered with. The Court found the chain of custody of blood samples was sufficient despite evidence indicating some carelessness in storage of the samples13.

The Court noted: “Although the evidence indicates some carelessness in the storage of the blood samples, we find that the evidence of the test results was properly admitted. ‘[I]t is presumed that the integrity of evidence routinely handled by governmental officials was suitably preserved “[unless the accused makes] a minimal showing of ill will, bad faith, evil motivation, or some evidence tampering.” United States v. Roberts, 844 F. 2d 537, 549-50 (8th Cir.). Applying those principles to the facts of this case, we find that the State proved to a reasonable probability that the blood samples were the same as, and not substantially different from, the samples as they existed at the beginning of the chain. Ex parte Williams, 548 So. 2d 518, 520 (Ala. 1989); Suttle v. State, 565 So. 2d 1197 (Ala. Crim. App. 1990).”

Another example of circumstantial evidence to support the chain of custody requirement was found in Bartlett v. State, 600 So. 2d 336 (Ala Cr. App. 1991), the appellant’s blood was drawn by a hospital nurse and the blood sample vial shortly thereafter transported to the hospital laboratory for analysis. The nurse drawing the blood labeled the vial with the appellant’s name and placed the sample in a pre-vacuum sealed vial. The lab technician responsible for the analysis testified that he would not have accepted the sample for analysis had it not been in a sealed condition upon arrival at the hospital lab. The fact that a ward clerk transported the sample to the laboratory for analysis did not defeat the chain of custody. In Bartlett, the Court stated:

To establish a sufficient predicate for admission into evidence it must be shown that there was no break in the chain of custody. Identification and continuity of possession must be sufficiently established to afford ample assurance of the authenticity of the item. Ex parte Yarber, 375 So. 2d 1231, 1234 (Ala. 1979). ‘A party need not negative the remotest possibility of substitution, alteration or tampering with the evidence.” Whetstone v. State, 407 So.2d 854, 859 (Ala. Cr. App. 1981) (emphasis in original).

Likewise in Moorman v. State, 574 So.2d 953 (Ala.Cr.App. 1990), the Court found the chain of custody sufficient where, in prosecution for criminally negligent homicide following fatal automobile collision, the chain of custody for a blood sample taken from the defendant was sufficiently established even though to “links” in the chain (the unit secretary at the hospital who sent the sample to the laboratory and the person from the laboratory who picked up the sample) did not testify14. The evidence was sufficient to establish chain of custody for victim’s body, even though person who transported body to morgue and county coroner who received body did not testify.

However, in Suttle v. State, 565 So.2d 1197 (Ala. Cr. App. 1990), the chain of custody not established, and therefore the blood sample was deemed inadmissible. The appellant’s conviction for vehicular homicide was reversed because the state failed to account for the whereabouts of the blood samples drawn from the defendant during the four days between the time the samples were taken by the nurse and the time they were received by the state’s forensic expert. The nurse who gave the blood samples to the trooper who did not testify. The forensic expert received the blood through the U.S. mail. The toxicologist who received the samples could not testify where the samples had been located during the previous four days. The court held it was reversible error to allow test results conducted on a blood sample when there was not a sufficient chain of custody for the sample.

The importance of proving the chain of custody of a blood sample was demonstrated in Miller v. State, 484 So. 2d 1203 (Ala. Cr. App. 1986) where the investigating state trooper in a traffic fatality case secured blood samples from the defendant at the local hospital, then took the blood sample vials to the Jacksonville state trooper office, “put it in the envelope, sealed it and initialed it” then placed the sample in the department’s outgoing mail, not the U.S. mail. Three days later, the sample was delivered to the Department of Forensic Science lab in Birmingham for analysis. There was no accounting for the location or security of the blood samples for the three days prior to delivery at the DFS lab.

Although the use of the U.S. Mail attaches a legal presumption that materials are delivered in substantially the same condition as when placed in the mailbox or post office, no such presumption is attached to “regular outgoing mail” delivery service used by a state agency. “To establish a sufficient predicate for admission into evidence it must be shown that there was no break in the chain of custody. ... Where ‘missing links’ are involved in the chain of custody the question presented is one of admissibility rather than credibility.” (emphasis in original), citing Whetstone v. State, 407 So. 2d 854, 859- 60 (Ala. Cr. App. 1981).

In the case of Green v. Alabama Power Company, 597 So. 2d 1325 (Ala. 1992), a wrongful death case where the defense was contributory negligence on part of the decedent, fluid samples were taken during the autopsy which, after analysis, allegedly showed the presence of a controlled substance. The plaintiff objected to admissibility of the sample where the analysis of blood and other body fluid samples were shipped by U.P.S. delivery service and subsequently analyzed at the DFS laboratory.

In Green, the Alabama Supreme Court held:

In chain-of-custody cases involving “specimens taken from the human body,” the proponent of the evidence must demonstrate “where and by whom the specimen was kept and through whose hands it passed.” J. Richardson, Modern Scientific Evidence, 13.14a ( Ed. 1974). Gothard v. State, 452 So. 2d 889, 890 (Ala. Cr. App.), cert. striken, 450 So. 2d 479 (Ala. 1984).” Suttle v. State, 565 So. 2d 1197, 1199 (Ala. Cr. App. 1990) (reversing vehicular homicide conviction for failure of prosecution to account for blood sample during four day interval between delivery of unsealed sample to police officer and reception at laboratory.)”

The Supreme Court held in Green that a similar four day gap between the date of the blood draw and the subsequent delivery to the forensic laboratory, without explanation as to the sample’s location or control, would render the sample inadmissible into evidence.

The case of Jones v. City of Summerdale, 677 So. 2d 1289 (Ala.Cr.App. 1996) is illustrative of the requirement for live witness testimony to establish both the manner of the blood draw and establishment of the chain of custody. In Jones, the Court of Criminal Appeals held conformity with evidentiary predicate was required for the admission of blood tests as well as compliance with chain of custody requirements.

The Jones case holds that results of a blood test administered to determine blood alcohol content may be received into evidence, provided a proper predicate is laid. The state must first lay a sufficient predicate in support of such evidence to indicate its reliability. A lab report indicating the results of a blood alcohol test, without any supporting testimony, invites reversible error. In Jones, the state did not present y testimony regarding the blood test performed on the appellant. The Court of Criminal Appeals held if the State elects to offer the results of blood alcohol test into evidence, the State must comply with the rules of evidence.

The Court’s opinion stated:

In this case the state offered the blood test into evidence without any testimony indicating the reliability of the test, who performed the test, or the circumstances under which the test was performed. The trial court received the test without any foundation whatsoever having been established. The trial court erred to reversal when it incorrectly received the blood evidence into evidence. Jones v. City of Summerdale, 677 So. 2d at 1291.

In the case of Nelson v. State, 551 So. 2d 1152 (Ala. Cr. App. 1989), citing the prior case of Kent v. Singleton, 457 So. 2d 356 (Ala. 1984), the Court of Criminal Appeals held it fundamental to establishing admissibility that blood evidence must demonstrate the chain of custody requirement. Without establishing a strict chain of custody, the sample results are inadmissible into evidence. The evidence in the Nelson case did not disclose the identity of the person who withdrew the blood sample at the hospital. The trial court properly refused to admit the test results under § 32-5A-194. The results were not admissible under general evidence principles as there was no proof that the test performed on the defendant was conducted according to accepted scientific methods and there was no proof of the qualifications of the person who withdrew the blood sample. The Court further held the mere fact that the blood sample was taken at a hospital does not insure its reliability.15

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