Forensic Laboratories

The proper operation of a forensic laboratory is a complex process that integrates management functions and scientific activities.

The forensic process begins with the proper collection of the specimen, and continues through to the reporting of results. Thus, the proper management of a laboratory is important. However, the performance of the forensic process can have an even more significant impact on the reliability and quality of the analysis of evidence.

Private organizations, the federal agencies (e.g., the Department of Transportation, the Substance Abuse and Mental Health Services Administration, and the Department of Health and Human Services), and State and County crime labs all have procedures in place to cover all aspects of the process.

All laboratories, whether clinical or forensic, develop procedures for the smooth and efficient operation of their laboratory. These guidelines are generally documented in the laboratory’s Standard Operating Procedure (“SOP”).

At minimum, a forensic laboratory must have guidelines for the following:

• Preparation of the evidence collection kits.

• Collection of the evidence.

• Chain of custody.

• Receipt of the evidence at the laboratory.

• Storage and security of the evidence.

• Analysis of the evidence.

• Reporting of the analytical results.

• Any related activity.

These requirements are often spelled out in State regulations. In fact, the laboratory may be required to receive a license from the State to perform evidential testing. For example, California requires a laboratory not only to follow the State guidelines, but to submit the SOP for approval prior to issuance of a license.

In addition, the results of the analysis must follow general data acceptance criteria. The laboratory must establish guidelines that clearly outline what constitutes an acceptable analysis, and how the result is to be reported.

The final step is the expression of the result in court. The laboratory analyst must be adequately trained to effectively and fairly express an opinion based on the analytical results and state of knowledge in the industry.

Clinical Laboratories

A clinical laboratory operates in a slightly different manner than a forensic laboratory.

The end result of a clinical lab analysis is not litigation, but rather the quick evaluation of a patient who may need urgent care. As such, the methods employed in a clinical laboratory generally entail time-saving analytical procedures that may not have the same quality control and quality assurance procedures employed in a forensic laboratory.

However, since clinical test results occasionally find their way into the forensic arena, the scientist and legal professional must be aware of the limitations and differences in order to perform a reliable interpretation.

Good Laboratory Practice (GLP) Guidelines

The umbrella under which the laboratory operation is conducted is a set of guidelines published by the procedures of Good Laboratory Practice (GLP). The GLP sets out the following:

• A director who oversees the project or testing procedure.

• Quality Assurance (QA).

• Qualified personnel.

• Maintenance of raw data.

• A Standard Operating Procedure (SOP).

• Regulation of reagents and controls.

• Maintenance and calibration of the instruments.

The GLP guidelines help ensure that the laboratory’s results are accurate and reliable.

The Forensic Laboratory

A.     Collection of the Specimen

Evidence Collection Kits

In the forensic laboratory, alcohol is predominantly tested in the blood, urine or breath. The basic foundation of the reliability of the test result lies in the collection of the specimen. If the specimen is not collected properly, the entire result may be compromised.

The laboratory often prepares an official biological specimen collection kit. Once the specimen is collected, the specimen is sent to the laboratory for analysis, usually on a gas chromatograph. After analysis, the analytical data is reviewed and the result reported to the prosecutor’s office for adjudication of the case.

If breath is being measured, the breath sample is collected directly from the subject into the breath machine, which prints out a breath card with the results of the test. In breath testing, there is generally no official collection kit, unless a portion of the breath is captured and saved by a special device.

Collection kits for blood or urine testing are either purchased from commercial sources, created in the laboratory, or assembled as a kit from purchased materials. Most kits contain alcohol-free cleansing swabs, such as povidone iodine, a urine collection container with preservative, and/or a blood collection tube with preservative and anticoagulant. An evidence submission envelope with an accompanying chain of custody form, and a sample vial tamper-evidence seal should also be included in the kit. These kits are distributed to the police or collection agencies, which then collect the biological specimen.

Importance and Regulation of the Collection Process

The following factors all impact the identity and integrity of the specimen:

• The manner in which the specimen is collected.

• The safeguards against specimen tampering during the collection.

• The correct identification of the specimen.

• The accompanying chain of custody.

Therefore, the collection is the foundation of the process, and can affect the reliability and effectiveness of the entire analysis.

When evaluating a blood alcohol case, scientists should consider the methods of blood (or urine) collection, the amount of preservative in the sample, storage conditions, and evidence of alcohol-producing organisms. The scientific community, as well as some State regulations requires that the integrity of the sample must be maintained through collection to analysis and reporting. Thus, the collection process is the beginning of the chain of integrity of the specimen

For example, the California Code of Regulations provides the following requirements:

• Blood samples must be collected by venipuncture from living individuals as soon as feasible after an alleged offense and only by persons authorized by the Vehicle Code. [17 Cal. Code Reg. §1219.1(a).]

• Sufficient blood must be collected to permit duplicate determinations. [17 Cal. Code Reg. §1219.1(b).]

• Alcohol or other volatile disinfectants may not be used to clean the skin. Aqueous benzalkonium chloride (zephiran), aqueous merthiolate or other suitable aqueous disinfectanct shall be used. [17 Cal. Code Reg. §1219.1(c).]

• Blood samples must be collected using sterile dry hypodermic needles and syringes, or using clean, dry vacuum type containers with sterile needles. Reusable equipment, if used, shall not be cleaned or kept in alcohol or other volatile organic solvent. [17 Cal. Code Reg. §1219.1(d).]

• The blood sample must be deposited into a clean container which is closed with an inert stopper. [17 Cal. Code Reg. §1219.1(e).]

• Alcohol or other volatile solvents must not be used to clean the container. [17 Cal. Code Reg. §1219.1(e)(1).]

• The blood must be mixed with an anticoagulant and a preservative. [17 Cal. Code Reg. §1219.1(e)(2).]

• In order to allow for analysis by the defendant, the remaining portion of the sample shall be retained for one year after the date of collection. [17 Cal. Code Reg. 1219.1(g).]

The Collection Procedure

Collection of the urine or blood specimen generally starts with the collector/phlebotomist opening up a pre-packaged laboratory collection kit. For blood specimens, the blood kit generally contains one or two “grey-top” blood collection tubes, a non-alcoholic swab for cleaning the injection site prior to blood withdrawal, a checklist delineating the steps the collector must follow, labels to put on the blood vials, and an evidence collection envelope, upon which the chain of custody and donor information are placed. For urine specimens, the kit generally contains a commercial urine collection jar, or one made up by the laboratory, along with a checklist, labels to put on the container, and an evidence collection envelope.

All biological specimens must be preserved properly. The blood vials must contain a preservative and an also an anticoagulant. The preservative helps maintain the integrity of the specimen by hindering the growth of microorganisms, and the anticoagulant keeps the blood from clotting. Both of these chemicals are white powders or granules which can be easily seen by the phlebotomist prior to the blood draw. The urine containers do not need an anticoagulant, since urine is all liquid. However, preservative is still necessary to keep microorganisms, which can easily enter the collection container, from producing ethanol. (See Preservative and Anticoagulant, below)

Several types of commercial tubes can be employed. However, more often than not, the commercial 10 mL “grey-top” tubes are employed for blood collection. These tubes generally contain 100mg sodium fluoride and 250 mg potassium oxalate. Thus, when 10 mL of blood are collected, the resulting concentration in the blood of preservative is 1%. Occasionally, yellow, green, red or purple top tubes are collected. However, these tubes are generally employed in other areas of the forensic lab other than toxicology (e.g., typing or DNA). The following chart shows the use of different tubes:

Blood Collection Tube Guideline

Color Top	Additive	Required Mixing (180o Inversion)	Uses
Grey	Potassium oxalate and sodium fluoride	8	Blood alcohol
Yellow	SPS or SPD	8	Blood culture (SPS) or DNA (ACD)
Lavender	Liquid K3EDTA	8	Hematology
Red	None	None	Serum testing

[http://www.bd.com/vacutainer/pdfs/plus_plastic_tubes_wallchart_tubeguide_VS5229.pdf.]

Practice Tip

Failure to invert (shake or mix) the collecting tube after collecting the sample is not uncommon. Even more common is the failure to invert the tube the recommended number of times. Point out to the jury that it does not do much good to have the proper amount of preservative and anticoagulant if they are not mixed with the biological sample.

Phlebotomists are trained to draw different tubes in a particular order when multiple tubes of different types are to be drawn. Tubes with anticoagulants, such as those for blood alcohol “should be drawn last so that they can be properly mixed as quickly as possible.” Any tube with anticoagulants must be inverted 8 times to mix the additives with the blood. [http://www.upstate.edu/phlebotomy/pages/venipunc/venitec4.htm.]

This mixing is also required by the manufacturer of the Vacutainer™ grey-top tubes. The company requires that the tubes be inverted (180° inversions) 8 times for the proper mixing of the additives to the blood. [http://www.bd.com/vacutainer/pdfs/plus_plastic_tubes_wallchart tubeguide_ VS5229.pdf]

Contamination During Collection

If the phlebotomist does not adequately clean the injection site, bacteria and other microorganisms can contaminate the specimen. Since the integrity of the specimen is vital to the forensic analysis, the collection must be performed in a particular manner.

The first step in the blood collection process is to decontaminate the area of the intended puncture. A prepackaged 70% isopropyl alcohol pad is the preferred antiseptic for clinical use. However, povidone-iodine (Betadine) is used as an alternative to the isopropyl alcohol for forensic blood alcohol draws. Regardless of which antiseptic is used, the phlebotomist must carefully rub the puncture site, working in concentric circles from the inside out. This procedure helps ensure that the site will be clean. The procedure must sometimes be repeated in some subjects. [http://www.upstate.edu/phlebotomy/pages/venipunc /venitec4.htm]

Since iodine solutions can deteriorate some rubber materials, excess iodine must be wiped off stoppers of tubes. [http://www.upstate.edu/phlebotomy/pages/ venipunc/venitec4.htm] This is especially important in blood alcohol testing, since the stopper must be properly sealed to inhibit loss of alcohol, or increase in alcohol due to contamination. [For more on contamination, see §15:20, Contamination by Microorganisms.]

§15:14  Checklist of Collection Issues

Scientists must be certain that the proper protocols were followed in the collection and transportation of the specimen to the laboratory. The scientist should evaluate the evidence, or ask the blood draw technician:

• Was the blood specimen collected using sterile needles?

• Was the blood specimen deposited in a clean, dry container?

• Was the blood draw from the vein, artery, or capillary?

• Was the blood adequately mixed with the anticoagulant and preservative?

• Did the collector note if the blood tube contained the proper amount of preservative?

• Was the vacuum seal on the tube intact (if a vacuum tube was used)?

• Was the skin cleansed properly prior to collection?

• Was the disinfectant used to clean the skin full strength or diluted?

• Did the disinfectant contain alcohol?

• Was any of the equipment used reusable, or disposable only?

• Was the identification label placed on the collection tube prior to the draw?

• Was the sample placed immediately into a sample envelope and sealed?

• When was the chain of custody signed?

B.     Contamination

Contamination by Microorganisms

One of the greatest risks to the integrity of a forensic sample is the growth of microorganisms in a sample that has not been properly collected or preserved. Proper blood draw techniques are crucial to the integrity of the sample.

The human body naturally contains a variety of microbes on the skin and in the body. The amount and variety of microbes on the skin varies from individual to individual. For example, an average person has between 100 to 100,000 microbes per square centimeter, depending on the time of day and whether the person has been exercising. Thus, the prospective puncture site must be properly cleaned with a non-alcoholic swab and with proper mechanical movement to minimize the drawing of microbes into the syringe.

Despite cleaning the arm before puncture, micro-organisms living in skin pores or the skin layers may still survive. A needle puncture to draw the blood may pick up a plug of skin that will introduce microbes into the blood sample. Clinical inspection of the blood for these microbes may reveal survival even after cleansing.

Contamination Produces Alcohol

Candida albicans uses the glucose in a biological sample to produce ethyl alcohol. The amount that is produced depends on the amount of glucose available. The Sacramento County Crime lab produced a report regarding a urine specimen (which normally should not contain glucose) that had increasing levels of alcohol. The conclusion was that urine alcohol levels of 0.20% could be realized from specimens that were negative when first collected. [Ronald Briglia et al. 31st Semiannual Seminar, California Association of Criminalists, Long Beach, CA, May 16-18, 1968.]

Another study concluded that within 12 hours yeasts such as Candida albicans will produce significant alcohol in the presence of glucose. [Saady et al., Production of Urinary Ethanol after Sample Collection, Journal of Forensic Sciences, Vol. 38, No. 6, November 1993, pp. 1467-1471.]

Since blood naturally contains glucose, a specimen contaminated with Candida albicans will produce alcohol. There are scores of scientific articles on the changes in alcohol level on contaminated specimens. Whether the studies are ante or post mortem, the condition for ethanol production is clear: the combination of an organism (such as Candida albicans) and glucose can produce alcohol in the sample. Microorganisms that affect the true BAC include:

• Candida albicans.

• Streptococcus fecalis.

• Klebsiella oxytoca.

• Pseudomonas maltophilia.

• Klebsiella pneumoniae.

• Enterobacter agglomerans.

• Enterobacter cloacae.

• Enterobacter aerogenes.

• Serratia liquifaciens.

• Serratia vulgaris.

• Proteus vulgaris.

• Morganella morganii.

• Morganella mirabilis.

• Citrobactobacter fruendii.

• E. coli.

Mixing the Preservative

Proper mixing of the preservative into the blood upon collection is vital. Without proper mixing, the granular/powdered additives may remain at the bottom of the tube, and not adequately mix into the entire blood sample. As a result, small clots can form in the tube and the preservative concentration is too low in a large portion of the blood.

The simple example of making lemonade is a good analogy to improper mixing. If sugar is placed in the bottom of the lemonade pitcher, and then water and lemons are added, without mixing, the top portion of the lemonade is inordinately sour. The mixture needs to be stirred for the sugar granules to dissolve and for each glass from the pitcher of lemonade to taste the same. Similarly, without proper mixing in the blood tube the preservative and anticoagulant will sit like sludge in the bottom of the tube, inadequately performing their intended function on the blood contained in the vial.

Refrigeration and Sodium Fluoride Levels

A study has been made of the efficiency of several different concentrations of sodium fluoride in the prevention of the formation of ethanol by Candida albicans. Aliquots of urine specimens containing glucose were inoculated with Candida albicans, and each aliquot given a different concentration of sodium fluoride. Urine aliquots containing sodium fluoride levels of between .1% to 2% were allowed to stand either at room temperature (22 degrees C) or in a refrigerator (4 degrees C) for up to 34 days. The researchers found that 1% to 2% sodium fluoride was necessary to prevent the production of ethanol by the microorganism. In addition, they found that storing the specimens at 4 degrees C, even without sodium fluoride also hindered the production of ethanol. Without the proper amount of preservative or refrigeration, levels of up to 0.025% (2 days) to .450% (8 days, with 1% w/v glucose). [Jones, A.W., Hylen, L., Svensson, E., Helander, A., Storage of Specimens at 4 Degrees C or Addition of Sodium Fluoride (1%) Prevents Formation of Ethanol in Urine Inoculated with Candida Albicans, J Anal Toxicol, 1999, Sep;23(5):333-336.]

Certain organisms survive even in the presence of a preservative such as sodium fluoride. Even in a blood tube containing sodium fluoride, Candida albicans, the most common microbial culprit of ethanol production in blood samples, can produce ethanol. [Blume P., Laktua D.J., Bacterial Contamination on BAC Stability, Am. J. Clin. Path., 1973, 60:700-702.]

One researcher has stated that he has “sometimes isolated large numbers of yeasts from preserved samples taken either from live subjects or post-mortem.” In addition, when the yeasts were able to multiply, they affected the alcohol levels. [Corry, J., Methods of Assessing the Effects of Microbes in Blood and Urine on Ethanol Levels, Proceedings, 8th International Conference on Alcohol, Drugs and Traffic Safety, Sweden, June 15-19, 1980.]

However, not all microorganisms increase the alcohol concentration. The bacteria Serratia marcescens and Pseudomonas sp. are capable of growing at ambient temperature in blood containing sodium fluoride at 1% w/v concentration. They degrade alcohol in specimens in preserved blood, and lower the alcohol concentration. [Dick, G.L., Stone, H.M., Alcohol Loss Arising From Microbial Contamination of Drivers’ Blood Specimens, Forensic Sci Int., 1987, May-Jun; 34(1-2): 17-27.]

Determination of Contamination

Not all microorganisms are created equal. Some thrive in warm conditions, in the presence of air, away from the presence of air, and with different ideal nutrients for survival. Often, one microorganism will thrive, only to die out and be overgrown by another microorganism. Sometimes one or several of these microorganisms can be identified via a blood culture.

The typical clinical culture (one growth medium, 5-day incubation) is not the best method to use to help revive these microorganisms. In order to have the best chance to revive the microorganism, and allow it to grow so that it can be identified, several different growth media and growth conditions need to be explored. Once even one microorganism is identified, the scientist should consider the possibility of other, previously thriving microorganisms.

The presence of microbes in the blood indicates that the blood draw procedure was not adequate to prevent contamination of the specimen. Contaminated specimens are inherently unreliable, and should not be used forensically.

If the blood cannot be cultured, the presence of sodium fluoride at a concentration less than 1% (10 mg/mL) may give evidence of one or more of the following:

• The laboratory’s procedures are not being followed.

• The blood draw technician did not mix the vial properly.

• The tube, as received from the manufacturer, did not contain the proper amount of preservative.

• The blood was filled past 10mL in a 100 mg sodium fluoride tube.

In any event, the concentration of the sodium fluoride in this situation is less than is adequate to help prevent growth of microorganisms. (Keep in mind the sodium fluoride does not kill the microorganism, it just inhibits growth.)

C.   Chain of Custody

Documentation

Chain of custody documentation must be maintained at all times, and the chain of custody must remain intact.

In forensic science, the specimen label, specimen envelope, and the laboratory’s internal chain of custody all help establish the integrity of the specimen.

The Federal Government requires that their standardized Custody Control Form be used, and the control and accountability of specimens be maintained at all times.

Regardless of the agency, the requirements are universal: every individual in the chain must be identified, and the date and purpose for access to the specimen must be documented by the individual handling the specimen. Since the chain of custody begins from the start of the blood draw, procedures must be established to safeguard the identity of the specimen vial and to prevent any possible mix-up of specimens in multiple-draw situations.

Proper chain of custody documentation shows who handled the specimen and where the specimen was located at all times, using the “Z formation.” The Z formation is named because of the zigzag path the chain follows. That is, the format states that custody goes from A to B, from B to C, from C to D, etc. Thus, the Z formation always has the same name or location in the “to” entry as in the immediately following “from” entry.