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Many practitioners, when receiving a new DUI client, can easily conceive of the challenges they could mount if the client took a breath test: Mouth Alcohol, Radio Frequency Interference, and Interfering Substances, to name a few. But if the client took a blood test, these same prac¬ titioners throw up their hands and con¬ cede the chemical test result. It does not have to be this way. There are a number of weaknesses in the collection and analysis of blood samples that can be exploited to create doubt as to the accuracy of the blood test result. This article will provide the practitioner with a variety of tools to challenge blood test results. One systematic way to present these strategies is to track the blood sample from collection to analysis to reporting. Collection Materials Start by looking at the materials that were used to collect the blood specimen. It makes a difference if inappropriate materials were used. Most agencies that draw blood for forensic analysis use some type of partially evacuated blood collection tube, such as the Vacutainer from Becton-Dickinson. The tubes are sold with a variety of additives inside depend¬ ing on the ype of laboratory test for which the sample is being collected.’ The appropriate tube for blood ethanol analy¬ sis is a tube containing a mixture of sodi¬ um fluoride and potassium oxalate. These tubes are intended for blood glu¬ cose determinations, and typically have a gray stopper. The stoppers for other types of tubes are different colors. The sodium fluoride in the tube with the gray stopper is present as an anti-gly- colic; it inhibits the formation of glucose by microbes that could be in the blood or contaminating the blood draw. The potas¬ sium oxalate is present to prevent clotting. If the blood alcohol result is derived from one of the tubes designed for a different type of analysis, the result maybe inaccu¬ rate due to interference with the different chemicals inside these tubes, or from the separation of the blood into plasma or serum. Plasma is blood minus the blood cells. Additionally, remove the clotting factors and serum is the result. Always check what type of tube was used for the collection. Plasma and serum have a high¬ er percentage of water than whole blood, and since alcohol dissolves in water, the alcohol results in serum or plasma will be higher than with the whole blood sample from which they came.2 The plasma to whole blood ratio ranges from 1.09:1 to 1.17:1, and serum to whole blood ranges from 1.09:1 to 1.18:1.3 These tubes have a shelf life and are stamped with an expiration date. After the date has expired, the vacuum may have depleted, which may result in con¬ tamination with microbes containing room air. This will result in less than the full amount of blood being drawn into the tube, a so-called “short draw.”4 A short draw may also result from the technician pulling the tube off the needle before it has fully illed, which again could result in the introduction of microbe-contaminat¬ ed room air being introduced into the tube. In a blood case, always check the expiration date and the volume of blood collected to see if it is the appropriate amount for the size of the tube. If it is less, could lead to problems with the accu¬ racy of the blood ethanol result. A short draw may cause hemolysis,5 which is a rupturing of the blood cells that can affect the water content of the blood and change the blood test results. Skin Prep and Alcohol Swabs Prior to drawing blood for alcohol determinations, the skin should be cleansed with a swab that does not con¬ tain alcohol. In this case, the term “alco¬ hol” means not only ethanol, but other alcohols such as isopropanol or rubbing alcohol. The swab is used to kill or remove any microbes that could contam¬ inate the sample. The swabbing should be done in an outward spiral to move the microbes away rom the puncture site. Ask the person who drew the blood to demonstrate his or her technique; many technicians just rub the swab back and forth over the draw site. Using a swab that contains alcohol can cause contamination of the sample in a variey of ways. Peek et al. found that swabbing the arm with ethanol caused signiicant contamination, even when the arm was allowed to dry before collecting the sample.6 Isopropanol had no effect when similarly applied. Dubowski found that using alcohol swabs and evacuated specimen tubes caused substantial con¬ tamination when the needle was with¬ drawn while the swab was held over the injection site and there was still vacuum in the tube.7 One method of uncovering the type of swab used is to subpoena the pur¬ chase records for the skin prep swabs from the faciliy or agency that drew the blood. Then consult with the manufacturer to see if there is any alcohol or other volatile compound in the swab. The recommen¬ dation is against having any volatile com¬ pound in the swabs used prior to blood alcohol sample collection to guard against any potential contamination.8 Location of the Blood Draw The blood sample should not be taken from the same arm in which an IV is being given. This may cause dilution of the alcobol result.’ Intravenous luids contain¬ ing Ringers Lactate may also interfere with accurate blood alcohol analysis if the analysis is done by enzymatic analysis.10 Similarly, the blood draw should not be taken from an injured limb.” Sample Collection Another concern is the size of the needle used to collect the blood. A needle that is too small may cause the blood cells to rupture, called hemolysis, which will raise the fluid percentage in the blood and alter the apparent blood alcohol con¬ centration. This is more common when a butterfly needle or other small-bore nee¬ dle is used to take blood from places such as the back of the hand when the techni¬ cian has difficuly drawing from one of the veins in the pit of the elbow.L2 Hemolysis may also be caused by the angle that the needle enters the vein. Blood collection needles are beveled to make insertion easier. If the beveled edge sits against the wall of the vein, this may disrupt the flow of blood into the needle and cause rupture of the blood cells.13 Immediately after the blood is drawn into the tube, the tube should be repeat¬ edly inverted to mix the chemicals with the blood. If this is not done, the chemi¬ cals may not be effective in performing their function. The blood may partially clot and microbes may produce addition¬ al alcohol in the sample. One manufac¬ turer’s mixing guideline speciies eight inversions to properly mix the chemicals with the blood in the tube with the gray top.14 Inversions are done slowly. Vigorous shaking of the tube can also cause hemolysis.15 The blood sample, now collected, must be properly preserved prior to analy¬ sis. One of the critical factors is tempera¬ ture. Microbes such as Candida albicans, a common yeast capable of producing alco¬ hol by fermentation in blood samples, grow better in warmer temperatures. Chang and Kollman studied the effect of temperature on ethanol production in blood samples inoculated with Candida* They found that there was significant pro¬ duction of ethanol in tubes kept at room temperature, even in the presence of sodi¬ um fluoride. Refrigeration was very effec¬ tive in preventing the fermentation rom taking place, both in tubes with and with¬ out the preservative. It may be helpful to have a portion of the blood sample released to a private laboratory for independent testing. Testing can be done for the ethanol result, the preservative amount, and the pres¬ ence of microbial contamination. The blood can also be tested for DNA if the identiy of the subject is an issue. The subject of DNA testing brings up another factor to be considered: the track¬ ing of the blood sample from collecion to analysis. The agency that draws the blood should have procedures in place to identi¬ fy the sample and the person rom whom it was drawn. Some jurisdictions go to the trouble of taking a thumb print from the subject and placing it on the vial label or the container the vial is shipped in. In busy jails or hospitals, it is quite possible for a sample to be mislabeled as the tech¬ nician draws blood rom several people in succession before the identifying informa¬ tion has been completely writen on the labels. This can result in the wrong label being applied to the vial. Independent DNA testing can reveal if the wrong blood was attributed to the subject. Analysis Issues Now that the sample is at the labora¬ tory where it will be analyzed, there are more places where mistakes and other factors can compromise the accuracy of the results. It is important to remember, however, that even the most accurate analysis may be completely unreliable because of problems in the collection and preservation of the sample. To begin with, the laboratory should have a procedure for recording the arrival of the sample and its condition. Any sign of leakage, damage, clotting, or other problem should be documented. The vol¬ ume of blood in the tube should be meas¬ ured and recorded. Counsel should rou¬ tinely request copies of these records to see what problems the laboratory noted on arrival. If your inspection of the tube later reveals evidence of problems, either they occurred while the sample was in the lab’s custody or the lab failed to note them on arrival. Either way, the lab looks bad. Laboratory analysis typically is con¬ ducted by gas chromatography (direct injection or headspace methods) or by enzymatic analysis. Each is subject to problems speciic to the method, and each is also subject to common problems of procedure. One of the problems common to all methods is qualiy assurance. Some labo¬ ratories will report a result based on a sin¬ gle analysis. There is no way to detect a problem occurring with that sample, such as accidentally swapping it with another sample somewhere during the analytical process, or a problem in the sampling and preparation for analysis. A slight improvement is to conduct repli¬ cate analysis, where the same sample is run twice in the same set of samples. This helps eliminate random errors in sampling that would manifest as differences between the replicate results, but it would not detect systematic errors with the per¬ son or equipment doing the sampling and analysis. A better procedure is for the laboratory to perform duplicate analysis. In duplicate analysis, the blood is: (1) sampled by two different analysts using different sampling devices, and (2) ana¬ lyzed on different instruments, preferably using different methods of analysis or dif¬ ferent gas chromatograph columns. Some laboratories will run a single sampling through two gas chromato¬ graph columns rom a single injection. While this is a form of replicate analysis, it will not detect problems with the sam¬ pling and preparation for analysis. Sampling problems can occur prior to either type of analysis. Most modern analytical methods use a very small amount of blood. A small variance in the amount of blood used for analysis can make a signiicant difference in the result. Sampling variances can result rom bub¬ bles in the blood caused by too vigorous mixing prior to sampling, changes in vis¬ cosiy that happen as blood begins to clot, sampling device inaccuracies, and techni¬ cian technique. These ypes of problems can be detected by independent analysis of the blood sample. Be sure to obtain a copy of the labo¬ ratory methods for analysis to see where problems could go undetected. Have these reviewed by an independent expert famil- ■< iar with proper laboratory protocols. Standards and Calibration Any method of analysis for alcohol needs to be calibrated to insure accuracy. Calibration involves submitting a known quantity of alcohol to the analy¬ sis and measuring the response. Assuming that no alcohol gives no response, a calibration curve can be gen¬ erated with only one alcohol standard. The value of an unknown sample is pre¬ sumed to be proportional to the response for the one known sample. This can be an unwarranted assumption if the response of the method is not lin¬ ear. A better procedure is to use multiple r _ standards spread out across the range of expected unknown values. The calibra¬ tion curve is then mathematically fitted to the points, either point to point, with another formula such as linear regres¬ sion. With any calibration, the accuracy of the calibration is only as good as the accuracy of the standards themselves. If the lab’s standards are off, the calibration may look acceptable, but still produce inaccurate results. Calibration should be checked against another standard trace¬ able to a national standard, such as those Standards and Technology. Even a properly calibrated system is subject to random variation in the results. Part of this variation comes from each point in the sampling or analysis where error or variability can be introduced, including the collection, storage, sampling, and analysis. Laboratories should have conducted an analysis of these sources of variabiliy and know the magnitude of these potential variations. These various sources of variability combine to orm an uncertainy in the result, sometimes referred to as a margin of error. The margin of error should be equally dis¬ tributed, such that the true value may be either higher or lower than the reported value. If the true value is consistently on one side or the other of the reported value, a bias has developed in the system and needs to be corrected. When dealing with margin of error or uncertainty, some experts have recommended sub¬ tracting the measure of uncertainty from the reported value so that the inal value is beyond a reasonable doubt the correct value or higher for that subject, but that the true value would not be expected to be any lower.17 issues With Enzymatic Analysis Both gas chromatography and enzymatic analysis are subject to inaccu¬ racies inherent in the method. With enzymatic analysis, one of the most sig¬ niicant problems is with interfering substances. As already discussed above, some types of enzymatic analyses are subject to interference from Lactate and I.actate Dehydrogenace, which can be present in the body from certain intra¬ venous fluids, and also become elevated after death as well as in severely ill patients.1″ Some enzymatic methods are also subject to interference from iso¬ propanol.1” Isopropanol can be in the blood sample from an improper skin prep swab,” from ingestion of iso¬ propanol,21 and also from bioconver- sion” in diabetic patients or dieters pro¬ ducing ketones.23 Issues With Gas Chromatography While microclotting and salting out are discussed frequently as sources of error in gas chromatography, espe¬ cially in the headspace variety, problems in the chromatography itself are a source of error worthy of examination. There are a number of different chro¬ matography problems that may lead to inaccuracies in the blood alcohol result. One of these is carryover. Carryover is caused when a previous, typically high concentration, sample is not completely cleared from the system, often from somewhere in the autosampler.24 The remaining sample can then be picked up with the subsequent sample and add to the result. Carryover may also occur when a peak with an unusually long retention time comes off the column during the time for the analysis of the subsequent sample and may interfere with the quantitation of the alcohol or internal standard peaks. To check for carryover of the irst variety, the lab should be running a blank sample after the highest standard. Peak tailing is another problem that should be examined by the defense. Peak tailing is an elongation of the backside of the peak such that it takes longer for the response line to return to the baseline value. This can cause differ¬ ences in quantitation of the peaks depending on the peak measurement system. Chromatography peaks can be measured by height or area. Peak tailing has more effect on peak area calculations. Similar to peak tailing is baseline elevation, where the baseline value after the peak is higher than it was before the peak. This can cause inaccuracies depending on where and how the peak is measured. The defense should also check for the possibility of co-elution. Co-elution occurs when two or more compounds come off the column at the same time. In this case, they cannot be distin¬ guished, and their peak value will be ele¬ vated by the addition of the two com¬ pounds. If the retention time of two compounds is nearly the same, there might be split peaks, which could also compromise accurate measurements. When reviewing a blood case with gas chromatography, be sure to request printouts of the chromatograms for the subject sample, as well the calibration standards, quality control standards, lin¬ earity checks, and any sample composed of a mixture of compounds designed to show separation. Have these reviewed by an independent expert familiar with chromatography. Also determine what type of column is being used, and check with manufacturer’s data to see if there are any known issues with co-elution, which can be spotted as compounds with similar retention times. Determine if the client has been exposed to any of these compounds. Salting Out Blood is largely water. Ethanol, when it dissolves in water, its in between the spaces between the water molecules. When a salt, such as sodium fluoride, is subsequently dissolved in the blood, it also takes up some of the space between the water molecules. This pushes some of the ethanol molecules out of the blood and into the air above the blood. If this occurs in a headspace vial during gas chromatography, the result of the head- space analysis will be too high. Jones reported observing this phenomenon when comparing headspace analysis blood preserved with sodium fluoride to blood preserved with Heparin.15 The samples preserved with sodium fluoride had higher values. Other reports have failed to ind the same results, and it appears to depend on the ype of internal standard employed.26 Post-Analytical Issues Even after the analysis, there are places where problems can occur and places where the defense needs to look. After a group of samples is run, the lab analyst should have a list of acceptability criteria to check before accepting the run as valid. It is better if a second analyst or supervisor also checks the criteria. The defense needs to have the same list of cri¬ teria and make sure that all of the criteria were met. The defense should also have an independent expert check the criteria to make sure that they are the appropriate criteria to prove the validiy of the run. When the run criteria are not met, the defense should know what the lab’s response should be. Make sure to request the data for any unacceptable run of samples that also contained the client’s sample. Ultimately, the lab will issue a report with the inal result. Labs typically have an administrative review process to catch clerical or other errors in the reporting before the report leaves the lab. The, defense should be familiar with this process and make sure it was followed. Discovery Plus There is a lot of documentation that a defense attorney should have in order to properly evaluate the lab’s result and the sample collection process. If an attor¬ ney is only reviewing the lab’s report, it is like seeing only the outside of the onion. Underlying the report are the results of the run, including the calibration ajid the quality control checks. Furthermore, underlying these results is the chro¬ matography that led to the results. The next items to review are the preparation and determination of the values of the standards and quality controls upon which the accuracy of the calibration depends. And, underlying those are the accuracy of the equipment used to deter- – mine those values and the maintenance records of the instruments involved, including gas chromatographs, gas gen¬ erators, dilutors, and pipettes. Each step has a method or procedure to follow, and probably a log to record the results. In addition, there will be records of the training the analyst underwent in order to qualify to perform that work. Only by peeling the onion, delving layer by layer into the lab’s documenta¬ tion, can an attorney ind some of these process errors. Be aware that if an attor¬ ney requests and receives the laboratory “method,” there may be a host of other documents that the attorney was not given that are referred to by other names, such as standard operating pro¬ cedures, standards, guidelines, proce¬ dures, and manuals. A defense attorney should look for references to other doc¬ uments in the documents obtained and request these documents as well. In addition, a typical forensic laboratory will keep a host of records and logs. Most forensic laboratories operate on the maxim of “if it isn’t written down, it didn’t happen.” Therefore, when cross- examining a laboratory scientist about procedures, be sure to ask where both the procedure and the result are written down, and get a copy. Be prepared for a ight, and use every discovery tool available, including state and federal Freedom of Information or Open Records Act requests, third party subpoenas, and Brady v. Maryland (it is exculpatory if the lab made a mistake that could affect the accuracy of their reported results) to augment standard discovery. With these documents in hand, the defense will be able to show, rather than just speculate, that sloppy technique and laboratory errors have caused inaccuracies in the blood test result. If the blood test is inaccurate, then there is a reasonable doubt as to whether the client is guilty Happy hunting.

Article by: BY   BARRY   T.   SIMONS   AND   RON   MOORE