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DUI LIBRARY | Blood Alcohol Concentration Testing

BLOOD ALCOHOL TESTING | DUI

The Definitive Guide

Blood Alcohol Testing DUI

If you are arrested for DUI, Extreme DUISuper-Extreme DUI, or Aggravated DUI and seeking to challenge the results of a blood alcohol test then here are some things you need to know:

 LET’S GO.

Introduction

FUNDAMENTALS

Measurements

The Measurand

Measurement Criteria

The Scientific Method

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Quality Assurance

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Fit for Purpose

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The Equation

PRE-ANALYTICAL

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Garbage In & Out

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Representativeness

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Neogenesis Alcohol

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Collection & Storage

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Impound

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Equilibrium

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Pipetting

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Loading

ANALYTICAL

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Gas Chromatgraphy

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Reading Chromatograms

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Traceability

POST-ANALYTICAL

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Technical Review

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Uncertainty

Big Take Aways

INTRODUCTION

The Most Important Thing...

There is an essential truth about blood alcohol testing. 

It’s found in this picture. The sentence below the image, translated from French to English, says “this is not a pipe.”  The artist, René Magritte, was making a point.  Images can fool us.

If someone asks “what do you see?”, and you answer it’s “a pipe“, then you are mistaken.  Of course, when you think about it, the correct answer is obvious: it’s merely a picture of a pipe.

Depictions can make us believe, they are the same thing, as the object they depict. When in reality, they are merely a one sided copy, that our brain has transformed into a three dimensional subjective replica.

By juxtaposing a warning label with an image Magritte reminds about this self-deception. That we have a tendency to substitute perception for truth…even when it’s staring us right in the face.

Simply put, I believe the big lesson here is: an image is not the same – as the things – in it. If you want me to to believe you, show me the real thing. Not merely what you have chosen to depict.

And yes, this is the most important thing you need to know about blood alcohol testing. It’s the controlling idea that you must keep in mind when seeking truth.

That is because:

"This is not a true value"

Blood Alcohol Test Results

Let’s spend some time discussing why.

FUNDAMENTALS

If we had two stone tablets and carved a list scientific commandments on them, then this would have to make the list:

Science is more about questions than answers. At the heart of scientific inquiry is discovering what are the meaningful questions? Without knowing the right questions, you can’t know if you can trust the answers. Answers that someone is asking you to rely on.

Understanding a question is half an answer.

Socrates

What You Don't Know

Science tries to determine the breadth and scope of what you don’t know.  It only after those boundaries are revealed can you know how confident (or not) you can be about the answer you have found.

As Socrates also points out, the more we learn about a subject the more we realize what we don’t know. He is attributed with explaining “the only true wisdom is in knowing you know nothing.” Ironically, the more you know, makes you realize the more you don’t know.

Uncommon Knowledge

When people in a courtroom (like jurors) are asked to rely on science to make a decision they do not possess common knowledge.  Their level of comprehension at the beginning of an inquiry, determines how much you have to teach them, before they are even able to solve the problem.

You must take into account that each of them arrives at the courthouse with a different level of scientific literacy.

At The Start

They don’t know the same things, about the same things. Nor do they know all know what they need to know. They must be taught. 

This knowledge gap is compounded by the fact everyone also arrives to the problem with their own misconceptions that must be dispelled.

The Right Questions?

To help them reveal the meaningful questions, that lead to solving the inquiry, calls for teaching them the fundamentals. 

They must learn the scientific nuts and bolts of the concepts that underpin the problem.

A Method

The nuts and bolts of scientific inquiry is more than acquiring data.  It requires understanding a process of how to give meaning to data. The scientific method attempts to distill data into scientific knowledge. The essential feature of scientific literacy is learning how to analyze the information you obtain.

Scientific Method

Without understanding how to properly evaluate data it has no value.  Even more concerning, it can be misleading.

The Precision Example

Take DUI case where a blood sample was analyzed. The government attempts to measure ethanol concentration in the blood. Discovering the relevant questions for this inquiry requires understanding specific fundamental concepts, processes, and principles of more than one area of science.

A primary area of science involved is metrology. The government commonly focuses only on the technique gas chromatography used to analyze blood. However, when the required knowledge and methods of metrology are applied to the inquiry, the government’s results look much he different than presented. Their results will appear more uncertain.

How To Measure

Metrology originates from the Greek words “metron” and “logos.” These translate into the study of measurement. Today metrology is still considered the the science of measurement.  At it’s  core, metrology attempts to identify and quantify the amount of uncertainty in a measurement by comparing an unknown quantity of thing with a known quantity.

Metrology requires that measurements must be analyzed using multiple criteria. A measurement can meet specification in metric but be unfit in others.  

Precision is Not Accuracy

Within metrology are the concepts of accuracy and precision. Accuracy is asks whether you hit the target? Precision asks, how close were your repeated attempts were – to each other? Not the target. Precision can be mutually exclusive from accuracy.

It Looks Accurate

A blood sample in a DUI case is often tested in duplicate. Two portions of the same sample are tested at the time. That is, the same person, using the same equipment, in the same testing process uses a machine to analyze the two portions of the same sample. Under such conditions you would expect the both measurements to be close.

Blood alcohol test results of .1108 and .1108 are precise, but that does not mean they are accurate.

Test Results Close Together

Under such conditions you would expect the both measurements to be close.

However, getting blood alcohol measurements close together is not accuracy. Blood alcohol measurements can be close together but miss the target.  They can be precise but not accurate.

But Really...

In DUI cases, crime labs often taut the precision of their test results. Prosecutors stand before juries and tell them, that the test blood results are so close together, the results must be correct. Without knowing that numerical closeness, does not prove accuracy, a person (such as a juror) would likely conclude the results are accurate.

To properly evaluate these measurements a juror must possess the knowledge that precision is not accuracy. They must also know how to apply this information.

How To Process

Often overlooked in courtrooms is the fact the science is a method of inquiry. The scientific method provides for how information should be obtained, from whom and when to give it weight.

For example, the proponent of an opinion, conclusion or measurement has the burden prove his/her claim. 

Burden Shifting

The proponent can never shift this burden.  If they do that voids any scientific validity to their claim. Learning this required methodology, is just as important to scientific inquiry, as the is difference between the concepts of precision and accuracy.

Measurements

A blood alcohol test result is a measurement.  So, what are the fundamental principles of measurements, we need to understand, in order to rely on any measurement? 

What is a Measurement?

Measurements are not true values. The true value of anything you attempt to measure is unknowable.

Measurements are estimations. Some are better than others. This is true whenever a lab measures the amount of alcohol in a blood or breath sample in a DUI case.

Blood Alcohol Test

Constrain Uncertainties

If a true value is unknowable, then what is knowable?  You can measure with the uncertainties of your measurement system. The only thing that can be done in a lab to constrain how uncertain that is, but at some level it will always be uncertain.

AGAIN, this is true whenever a lab measures the amount of alcohol or drugs in a blood (or breath) sample in a DUI case.

Metrology

As stated above in the precision example, metrology is the science of measurement. Metrology defines measurement as the process of comparing an unknown quantity of thing with a known quantity.

Metrology has several primary  activities. These include (1) defining units of a measurement; and (2) establishing traceability which links measurements to reference standards.

The Measurand

A measurand are the unknown properties of the thing you intend to measure. It is only after this determination is made can a measurement assign some kind of value(s) to what you are seeking to measure (i.e. the measurand).

To illustrate

So, the government claims to have a blood test result above the legal limit.  That is, they took a blood sample.  

The government claims they measured the amount of alcohol in the blood sample

Blood Alcohol Test Results

…And, the result shows an alcohol concentration above .08.

The First Step

But…did they measure, what they were required, to measure? Or, did they measure something that just looks like it?

The first step in making a measurement is to identify the measurand.

Measurand DUI

Defining The Goal

A measurement must define its goal. Defining what is to be measured, and its intended use, helps ensure the measurement will fit for its purpose. This also helps determine how much rigor is needed in the measurement process to achieve the measurement’s goals.

Measurand?

A measurand is formally defined as ‘‘quantity intended to be measured.” See The International Vocabulary of Metrology, (VIM), 3rd ed. A measurand is specific to the properties of a target. 

In most DUI cases alcohol is the chemical target. However, that is not the measurand. The measurand is more narrowly defined.  

The Analyte

The term analyte is often erroneously used to interchangeably with the term measurand. An analyte is the substance (or constituent) of interest that is the subject of measurement. Alcohol is merely the chemical analyte in a DUI case.

However, the measurand are the properties (i.e. mass and volume) that have been chosen. These properties are both qualitative (specific to an attribute, quality, or characteristic of something) and quantitative (numerical). 

Qualitative

A measurand has different kinds of qualities. It is qualitative (specific to an attribute, quality, or characteristic of something). Qualitative analysis attempts to identify an unknown. For example, a specified kind of molecule, gender, or nationality.

Quantitative

A measurand is also quantitative. Quantitative analysis provides numerical data. Examples quantitative data are weight, length, mass, volume or speed.

Before you can quantitate – you must qualitate.  Put simply,  before you determine how much there is of “something,” you must identify that “something.” 

Why Do You Care?

DUI and impaired driving allegations often rest on the premise that the government has a measurement that proves guilt. The is usually an ethanol concentration above a prohibited limit. In such cases, you must determine if the government actually measured the thing violates the law – often they do not.

How Do You Know?

How do you know if the required measureand was measured or not? If the measureand defined by law, then you start by looking at the statute, that is alleged to be violated. Arizona has such a statute.

"Alcohol Concentration"

Arizona law states an “alcohol concentration” in blood is defined: “The number of grams of alcohol per one hundred milliliters of blood.”

See A.R.S. 28-101.

The Defined Measurand

The statutorily defined measurand is grams of alcohol per 100 milliliters of blood.The measurand is a combination of properties of that target intended to be measured: (1) mass of alcohol, per (2) volume of the blood.

Narrowing The Measurand

Arizona’s DUI statute further specifies the measurand. It requires that an alcohol concentration “results from alcohol consumed.” See A.R.S. 28-1381(A)(2). This further specification of the measurand is commonly overlooked.

Therefore, the measurand is not:

  • Alcohol by itself – alcohol is the analyte.
  • Alcohol in water – the matrix defined by the statute is blood.
  • Less and a gram per 100 milliliters – the required properties of the target chemical are mass and volume in specific amounts. 
  • Alcohol that has formed in the vial after the blood sample was drawn – an alcohol concentration must result from alcohol consumed. 

A measurand must be more than a value.

Measurement Criteria

The government typically frames the relevant question in DUI cases as whether the test results are accurate. This is a misleading frame.

Properly evaluating a measurement require looking at all of its essential dimensions: (1) Accuracy, (2) Reliability, and (3) Validity.

Accuracy

The accuracy of a measurement is the “closeness of agreement between a measured quantity value and a true quantity value of a measurand.” See Section 2.13, International vocabulary of metrology, 3rd edition.

Reliability

Reliability is about trustworthiness.  As the court in Daubert noted: “[O]ur reference here is to evidentiary reliability— that is, trustworthiness…” See FOOTNOTE 9, Daubert v. Merrell Dow Pharmaceuticals, 509 U.S. 579 (1993).

Validity

The concept of validity refers to whether a test or a measurement, actually measures what it claims to measure. See Kelly TL, Interpretation of Educational Measurements. New York, NY: MacMillan 1927;14 (“…a test is valid if it measures what it purports to measure.”).

Scientific Method

How Science Works

Science is more than knowledge. It is how you obtain knowledge. The method used to obtain what you have learned is just as, if not more, important than answers found. Science uses a process to determine what we know and what we don’t.  The scientific method is a fundamental process using logical steps to answer questions.

But, in order to qualify as “scientific knowledge,” an inference or assertion must be derived by the scientific method.

Daubert v. Merrell Dow Pharm., Inc.,
509 U.S. 579, 590, 113 S. Ct. 2786, 2795 (1993)
The Underlying Principals

The scientific method is often expressed a list of general steps in order to get get an answer to a question.

However, in the case of forensic blood alcohol testing, the principals that underpin the scientific method are the tools that are most effective in revealing if a result is trustworthy.

Proving Yourself Wrong

Science spends time trying to prove yourself wrong. That’s one of the primary premises of the scientific method. The proponent of an opinion, conclusion or measurement has the burden of proof in science.

Testable

A hypothesis must be testable. This means that it’s possible to make observations that agree or disagree with it. If a hypothesis cannot be tested by making observations, it is not scientific. For example, declaring a red fish is better than a blue fish is not a testable hypothesis. 

“Better” is subjective and lacks a specific meaning.  Therefore, it can’t be tested.

Falsifiable

Is there a possible negative answer to the hypothesis? Science is about things that can be shown to be false. If you are not able to show something is false it can’t  to be false then it is not science. Using this type of thinking you approach finding true things – without being able to show that anything actually is true.

For example, take the hypothesis “all fish are blue.” This statement is falsifiable, because if some observes a fish that is not blue (e.g. a red fish), that shows it to be false.

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The Scientific Method
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Quality Assurance

Quality is the degree to which an item satisfies the given need it was designed to fulfill. Is it fitness for purpose? Demonstrating that the essential steps in a measurement system followed fall under the categories of quality assurance and quality control

Quality Assurance

Quality assurance is utilizing a methodology to prove accuracy, reliability, and validity to the people inside the lab.  This includes things such as establishing an acceptable limits of uncertainty; procedures in place that will lead to results within the specified uncertainty limits…

…and demonstrating the ability to perform measurements within the specified uncertainty.

Quality Control

Quality control is to prove it to people outside the lab. Quality control is used to check that specified requirements (such as protocols and consensus standards like ISO 17025) have been fulfilled. Quality control is an integral part of Quality Assurance.

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Fit for Purpose

The Wrong Question

When presented with the results of a forensic test we ask: is it accurate?  However, that’s often an unknowable thing.   That’s the wrong question. Instead, we must determine if it’s good enough…for what what we are trying to determine.

If You Can't Know

When you measure an unknown amount of something the best science can do is provide us with an estimate. Thus, the end result is an estimate.  It is not intended to represent a true value. 

The true value is unknowable.

Then...What Can You Know?

Therefore, asking if the result accurate, isn’t the right question.  We need to know if we can trust the measurement system (to work as well as when we are testing a known value).  We must determine if the measurement system is good enough to rely on.

With all the above in mind, we see why asking is it accurate? is not the right question.  When presented with a forensic measurement we have to focus on the measurement system, not the result.  We have to ask – is it fit for its purpose? 

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Fit For Purpose
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The Equation

The blood alcohol testing process in DUI cases can be thought of as a two part an equation.

The first part of the equation is the human part.  This variable is includes all the variables that a human being that must do correctly to get the intended measurement.  

H = Human

The measurement process starts long before the gas chromatograph is actually turned on.  The blood must be collected, identified, stored and transferred properly before the sample is put into the gas chromatograph.

Even the best machines cannot account for, or identify, that a sample has been corrupted. 

M = Machine

The measurement process completed by the machines and their software is largely automated. Some malfunctions and errors will be visible.  Many others are not.

Every step in the equation (i.e. the measurement process) must have been done correctly. 

DUI Blood Alcohol Concentration Testing

The government must prove each and every step was done correctly.

PRE-ANALYTICAL

With the above concepts in mind, let’s look at our list and some of the associated issues.  Our list is divided into three categories: Pre-AnalyticalAnalytical and Post-Analytical

Pre-analytical refers to steps in the measurement process before the sample is put into a machine called a gas chromatograph. Errors in this phase are just as damaging, if not more, than errors in the analytical phase.

Forensic Science DUI

For example, if the person drawing the blood mislabels a blood tube, or the sample is contaminated, the gas chromatograph will not be able to identify the error.

The pre-analytical steps commonly performed prior to using a gas chromatograph in Arizona crime labs and their underlying principals are discussed below.

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Garbage In & Out

Software does what a human programs it to do. The results of the software’s function rely on a human.  Unreliable human input, results in an untrustworthy result (output) of the gas chromatograph’s software’s, even if the software performed perfectly.

The term garbage in / garbage out is sometimes expressed as garbage in, gospel out. It reflects our propensity unjustifiably trust in whatever computers tell us are so.

Blood seized without a warrant DUI

Contamination

Contamination is accidental introduction of foreign substance.  The introduction can materially distort the results of experiments or a measurement. This is particularly true where small samples are used.

Identification Limitations

The machine’s software will only identify what you teach it identify.  Thus, some measurement (shown as a peak on a graph) will not be labeled by the machine.  It will simply remain an unknown.

Known Contaminates

Some of the more commonly encountered contaminants found in the results of blood alcohol analysis by crime labs are: methanol and acetone.

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Representativeness

When measuring an alcohol concentration, representativeness refers to how well a portion of a sample (a “subsample”) reflects the entire sample. If evidence for representativeness is not presented, the data produced by a measurement process cannot be characterized as effective for decision-making. See Crumbling, 2001.

Representative of What?

Evaluating representativeness can only be done in the context of the question the measurement intended answer. Here, this brings back to the concept of a measurand.  The thing intended to be measured.

The Thing To Be Measured

Arizona defines what must be measured in a blood alcohol test. It is grams of alcohol per 100 milliliters. However, police do not test that large of a sample of blood.  The take a sample smaller than that from a person and then test a smaller subsample.

A Subsample

The subsample of blood is a fraction of even one gram.  Its usually either 100 or 250 micro-litters taken from a grey top vial used to store the blood. It is removed from the blood tube through a process called pipetting.

Is That Enough?

On it’s face, 100 (or 250) micro-litters is an insufficient amount. However, the idea extrapolate the measurement from a representative sample.  That is, a subsample which represents the entire sample.

If everything is the measurements process is not correctly, then yes its enough.  If not shown to be done correctly, then no it’s not.

Does It Tell The Whole Story

Think about representativeness like reading a book.  How many pages do you have to read in order to get the full story?

The Story

J.K. Rowling’s book, Harry Potter and the Sorcerer’s Stone, has 17 chapters that divide approximately 76,944 words.  Each chapter moves the plot forward.  Paragraphs include dialogue that build on each subsequent paragraph to develop the characters. Thus, if you want to understand the story you need to read the whole book.

A Few Sentences

Let’s compare the grams / milliliter ratio to words / whole book in Harry Potter.  How many words would be equivalent sampling 250 micro-liters?  The math works out just 192 words.  Does reading about a one and half paragraphs tell you Harry Potter’s whole story (which is 76,944 words)?

Reading only part of of the first page Harry Potter and The Sorcerer’s Stone doesn’t even tell you what a muggle is…for the record it’s you.

Small Problems Big Consequences

Extrapolation

An extrapolation is the process extending something in to the future.  It’s an estimation based on what you know.  You examine the data you have attempting to find a trend.  Then you estimate, if this trend continues into the future, what will you expect to occur?  Essentially you are making a prediction outside the what you know.

Deviations From True Values

When something varies from what is expected it is called a deviation. Extrapolations are based on known data.  When the perceived value of data deviates from its true value, predictions from that data will be erroneous.

Only One Degree

What happens when you are off course by 1 (one) degree when flying? For every sin­gle degree you fly off course, you will miss your tar­get land­ing spot by 92 feet for every mile you fly. That amounts to about one mile off tar­get for every sixty miles flown.

After 1 Mile

If you were only flying a few miles you would not end up that far off course.  You may not even notice being 92 feet off course. 

However, the initial error (one degree from the intended direction) scales. The further you travel the greater from your true destination you end up.

JFK to LAX

On a flight from JFK to LAX, that one degree error might have you landing in the pacific ocean rather than LAX.  Thus, being off by one degree may make you test whether your seat cushion will really work as a personal flotation device or not.

The same concept is applies to small errors in the measurement of an alcohol concentration.  When labs test a fraction of the required amount of sample, then extrapolate the results, small errors scale.

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Alcohol Formation Post-Draw

The seizure of a blood sample from a driver and the subsequent testing of that sample by a crime laboratory is a common practice in DUI cases. For this process to provide an accurate, reliable and valid measurement requires a rigorous testing methodology. One well established danger that must be prevented is an artificially higher result created by the neo formation of ethanol. That is, alcohol produced after the blood sample has been drawn from the driver.

Neogenesis

The amount of ethanol created after the sample is removed from the human body is influenced by many factors. these include:

  • The species of microorganisms present
  • The available substrates
  • Temperature 
  • Time of storage
  • The presence of preservatives added to the samples

A large number of common microorganisms are capable of generating alcohol. Neogenesis of ethanol is byproduct of bacterial decomposition processes.

The Blume Study

On page 701, the first paragraph after “Results” shows the problem. In a contaminated blood sample without NaF, and refrigerated, significant ethanol production over night.

Clotting

In order to understand neo formation of ethanol in blood you first need to know a little about the clotting process that occurs in blood.

Coagulation

Coagulation is the process of making blood clot. During this process the components of blood work together slow bleeding.

Platelets

Blood contains cells called platelets. Blood platelets are smaller cell fragments. They are essential for clotting to occur. Platelets will locate and fill holes in blood vessels.

Fibrons

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What is a Clot?

Platelets form a plug. When a blood vessel is damaged platelets are activated by triggers released when a blood vessel is damaged. They stick to the walls in the area and each other, changing shape to form a plug that fills in the broken part to stop blood from leaking out. When activated, platelets also release chemicals to attract more platelets and other cells, and to set off the next step.

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Collection & Storage

The grey top tubes containing the blood samples are supposed to be stored in a manner that slows sample degradation. The sample is dynamic, and chemical processes will continue after collection. Proper storage and collection will help slow the process, but will not prevent it.

DUI Blood Draw by Police

The Blood Draw

The blood draw begins the process. Some of the issues commonly encountered that may affect the number the machine prints out at the end of the process are:

Volume

Is there an adequate volume in the blood tube used to collect the blood sample? The vacutainer tubes are designed to collect just above 9 mls of blood. If there is less than that amount then why that occurred must be accounted for by the person who drew the blood.

DUI BAC Testing

Tube Integrity

Did the “vacutainer” have the proper amount of anticoagulant and preservative?

Inversion

Was the correct amount of inversions done?  At the correct number of degrees?

Labeling

The blood sample must be correctly labeled to ensure the rights test result is matched with the correct person.

The goal of sample collection is to ensure that a representative blood sample is loaded into a gas chromatograph for analysis. Put another way, the sample should be in substantially the same condition when analyzed, as it was when drawn from the person’s arm.

Temperature

Temperature is an essential factor in the proper storage of a blood alcohol sample.

Hemolysis in DUI cases

Hemolysis

Hemolysis is when red blood cell membranes break.  It results in causing the release of internal components into the surrounding fluid. 

Causes of Hemolysis

  • A short draw (i.e. the tube not filling with the minimum amount of sample) can cause hemolysis.
  • Prolonged tourniquet application or fist clenching.
  • Drawing from a venipuncture site which is not dry.

  • Improper mixing of the sample with anticoagulant and preservatives.

  • Drawing the plunger back too forcefully.

Effects Of Hemolysis

Hemolysis can result in an artificially inflated alcohol concentration.

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Impound & Inspection

After blood is drawn an officer is required to impound the sample(s) with property and evidence section of the police department. The officer’s request for a blood alcohol measurement is entered into law enforcement’s information system. Eventually, a computer processes the data and generates a type of pick-up list and work-list.

Impound

After an officer collects a blood sample it must be transported and impounded into evidence.  This is typically done at a police station’s property and evidence section.

The Crime Lab

When it is ready to be tested, the sample is taken from the refrigerator (in the property room) and brought to the lab. Because testing is done in a batch multiple samples must be transported together.

A batch means that multiple samples will be tested in the same run. Accordingly, about 30 to 40 blood samples will be taken to the lab. The concern in transportation to making sure the sample maintains its integrity.

Visual Inspection

Once the blood samples are transported to the lab, the lab tech will do a visual inspection. These observations of the lab tech are documented and result in paperwork which should be discoverable. For example, during this inspection, the volume of the blood sample in the tube is measured and documented.

Sample Preparation

Prior to being analyzed by the instrument, the blood sample must be brought to room temperature and mixed to make the homogeneous. Many crime labs such a the City of Phoenix will place the blood tube on a rocker for approximately an hour.

Failure to prepare the sample in this manner may result in an artificially high blood alcohol measurement.

Some of the issues to be examined here are:

• How long was the sample without refrigeration?
• Is the chain of custody accounted for?
• How was the sample assigned and identified for analysis by the lab?

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Equilibrium

Homeostasis refers to a relatively stable equilibrium between interdependent elements within something.

Distributed Components​

The goal of the blood test is to determine an alcohol concentration, in a person, at the time the sample is drawn.  To accomplish this the sample must be tested in a condition similar to when it was drawn.

Effects of Storage​

Blood samples are stored at low temperatures in a refrigerator.  This causes the sample to be denser than at room temperature. 

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Pipetting

In its simplest form, a pipette is a slender graduated tube. It is by chemists for measuring and transferring quantities of a liquid substance from one type of container to another kind of container.

Vacutainers

A blood sample is collected in a “grey top” blood tube. In order for it to be analyzed in a gas chromatograph, a small portion of the sample must be transported into a headspace vial. The headspace vial, containing the blood sample, is what is loaded into the gas chromatograph. 

Pipetting is the process used to transfer the blood sample into the headspace vial.

Headspace Vials

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Human are also responsible for teaching the machine what an alcohol concentration.  The machine does not come out of the box knowing any specific alcohol concentration.  Typically a lab will purchase approximately four (4) different alcohol concentrations from a vendor.  For example, .01, .10, .20, .40 are often used to calibrate the machine. 

These samples are put into the machine and the analyst programs the machine’s software to using these values.  If the analyst tells the machine a sample if a .40 but it is really a .30, the machine cannot tell the difference.  Ensuring a calibrator is what it purports to be is known as traceability.

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Before the gas chromatograph can extrapolate a measurement from a blood sample, a portion of the sample must be removed from the grey top tubes. Pipetting is a process where a small amount of the sample is removed from the grey top tube and placed in a smaller headspace vial.

A pipettor works in a manner similar to an eyedropper or a syringe. The pipettor is an instrument with a narrow tube that is inserted into the grey top blood collection tube and draws up a specific, and small, sample amount. Subsequently, the pipettor is put into the top of the headspace vial to deposit the sample.

Grey Top to Head-Space

Some lab techs pipette directly from the blood tube into a headspace vial. However, many techs use a cuvette to aid them in this transfer.

A cuvette is a small container sealed at one end designed to hold a portion of the blood sample. If a cuvette is used it sanitation must be insured. Reusing a cuvette without properly sanitizing it could lead to cross-contamination.

The device used to transfer the blood sample is called a pipettor dilutor. After the lab tech uses the pipettor dilutor to draw a small amount of blood (approximately 100-250 ml) from the grey top tube used for collection, it is then used to deposit the sample into a headspace vial. While depositing the blood sample, it also deposits an internal standard into the headspace vial.

An internal standard, if properly used, attempts to minimize error. An internal standard (sometimes called a surrogate) is another chemical substance whose analytical behavior is well known and similar to the substance you are quantifying. A specific and consistent concentration of it is added to the subject samples, blank, calibrators and controls. The response from the analyte peak is compared to the internal standard. In blood alcohol testing, N-propanol is commonly used by Arizona crime labs as an internal standard. This because N-propanol has a similar structure and reacts similarly to ethanol in the testing process, at the same time it is not expected to be found in a blood ethanol sample.

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Loading

As previously stated, a full batch of samples will typically mean the gas chromatograph will be processing over one hundred samples. Because each sample is stored in a headspace vial that also means that the analyst will have to manually load over one hundred headspace vials into a companion instrument called an autosampler.

The Autosampler

The autosampler is connected to the gas chromatograph. As the name implies, the autosampler is a device that helps automate the testing process. The device is controlled through software. The software commands the autosampler to inject (and then remove) a specific volume from a headspace vial, in a specific position on the autosampler’s tray of vials. All of the autosampler’s components (including the syringe and injection needle) are automated and programmable through the software.

The gas chromatograph is not able to detect human error when loading the samples. If subject samples are accidentally loaded into the incorrect slot, the machine has no flashing light or siren that will go off. Depending on the circumstances, a review of the data produced may indicate incorrect vial placement.

However, if duplicate subject samples are loaded into the incorrect slots, then there will be no indication in the printed data. That mistake cannot be detected after the fact.

Vials Into The Machine

Arizona crime labs manually load the headspace vials into the gas chromatograph’s autosampler. Loading the wrong vials into the wring slot can cause the machine to print out a measurement belong to a different subject. Accordingly, great care is required in the loading process. Some labs, such as the City of Phoenix’s Crime Lab has a second person double if the correct vials were loaded into the correct slot. However, others like the Scottsdale Crime Lab have no such safeguard.

A good practice is to keep the headspace vials in a tray and only handle one subject at a time. As you can see from the illustration below, Arizona crime lab label the headspace vials – not with a bar code label – but with a sharpie marker. While this reduces costs, this practice is not recommended to prevent misidentification of a sample.

Eventually, the headspace vials are placed into the autosampler for processing. As you see below the autosampler can hold many vials. A full run will hold approximately 110 for most of the units in Arizona crime labs. The slots are not labeled. The identification process is controlled by software and the data that has been manually entered lab personnel.

ANALYTICAL

The primary instrument used by crime labs in the United States to measure an alcohol concentration within a blood sample is headspace gas chromatography. The process is designed to be largely automated.

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Gas Chromatography

Definitions

Gas ChromatoGRAPHY

The technique performed by a gas chromatograph machine to separate and quantify substances.

Gas ChromatoGRAPH

A machine that separates molecules, and then measures, the amount of the various components in a sample.

Gas ChromatoGRAM

The graphical representation of the data produced by the gas chromatograph.

Calibrator

Water solutions having a known ethanol concentrations used to calibrate the gas chromatograph.

Control

A mixture of blood and​ ethanol.

Blank

A sample without the analyte of interest used to determine contamination or sample degradation.

Batch Data

Group of tests executing sequentially one by one is known as batch testing.

Headspace Gas Chromatography

A process that uses a machine that separates molecules, and then measures, the amount of the various components in a sample.

General Principles

Some big pictures things about gas chromatography:

  • If done properly, gas chromatography is a reliable way to measure the amount of blood in an alcohol sample.  However, automobiles are also reliable, but there are still thousands of car wrecks every day.  There is no presumption of reliability simply because a gas chromatograph was used.
  • The measurement process has both human components and machine components.  All steps in the process must be done correctly for the measurement to be trusted.

Indirect Measurement

Gas chromatography is an indirect measurement. The machine does not test liquid portion of a blood sample.  In headspace gas chromatography, the machine converts substances to a gas, and then it must separate the different types of molecules in the sample.  After separation, a microscopic amount of the gas is measured by software.

BAC Testing

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Separating Before Measuring

The machine must demonstrate it is able to separate different types of molecules before it can measure If it cannot properly separate different categories of molecules, then its measurements may appear artificially higher.

Testing In Batches

Samples are tested in batches. This is similar to production-line chemistry. A full batch of 110 vials can take over 10 hours to process. An analyst will commonly observe only about 10 to 20 minutes of the process. Thus, it is critical to ensure that the instrument and its software are working reliably if the test results are to be trusted.

Result = Estimation

Like most State’s, Arizona has a legal definition of an alcohol concentration. A.R.S. 28-101(2)(a), provides an alcohol concentration is defined as “[t]he number of grams of alcohol per one hundred milliliters of blood.” However, the State does not test 100 millimeters of blood. They merely use a blood sample between 100-250 micro-liters. A sample of this size will appear to be just smaller than a piece of M&M candy.

A Gas Chromatograph Does Not Have a Brain

We sometimes forget that even the most sophisticated and expensive gas chromatographs require human beings to produce a measurement.

The machine has no brain.   Therefore, human beings must use their own when operating the instrument.

Production Line Chemistry

Gas chromatography is done in manner like a production line. Multiple samples (usually over 100 vials) are being processed in a “batch.”  It is essential to safeguard against the wrong information being assigned to the wrong sample.

Anatomy of the Machine

The Machine's Essential Functions

During the Analytical phase, the instrument must perform two essential functions: (1) separating different substances, and then (2) quantifying them. Ultimately, it is during the analytical phase that these things occur. However, the analytical phase begins well before the machine is turned on. It starts with teaching the machine to know specific alcohol concentrations.

Calibration

A gas chromatograph does not come out of the box inherently knowing any alcohol concentration. While the device can separate different substances, it does not have the ability to identify them. It must be taught. This process is known as calibration.

For blood alcohol measurements, gas chromatography is calibrated by using known alcohol concentration. That is, they purchase commercially made ethanol concentrations (i.e.40, .20., .10 and .02) build a calibration curve. Think of it like building a ruler. The calibrator is like specific points of measurement on the ruler and the instrument’s software connects them. Subject samples are compared to the virtual ruler. A measurement is then derived from the comparison.

Two Machines

There are actually two devices. The gas chromatograph and autosampler. These devices and their corresponding software are commonly used to measure blood alcohol concentrations. The process is automated and the lab tech is not present for the majority of the machine’s work.

It Samples Air Not Blood

The machine does not analyze your blood. Recall that the sample is contained in a headspace vial which has been loaded into the machine. On top of that headspace vial is a rubber septa.

After it heats the vial, the gas chromatograph injects a syringe through the rubber septa. It then obtains a small sample of the air above the liquid in the vial. Thus, no blood is directly sampled, only the air above the blood sample.

A Small Portion

The machine starts its analysis after a small portion (less than the size of the a single M&M) of each blood sample is put into a headspace vial. The headspace vials (usually over 100) containing the sample are loaded into part of the machine called the autosampler.  

Heating the Sample

The samples are then heated (in a headspace tube) forcing molecules in the liquid portion of the sample to rise.  After the molecules are vaporized, a needle punctures the top of the headspace vial and extracts a microscopic portion of the gas above the liquid.

Molecules Into Two Columns

Humans have intestines to help process what it put in their bodies.  Gas Chromatographs have columns.  The machine contains long thin tubes for substances to pass through. The mixture of the blood sample and the internal stand vapor in the headspace vial is transported by a carrier gas (usually hydrogen) through a column.

The capillary column is contained within an oven.

Duel Columns

These vaporized molecules are pushed through long thin columns by a carrier gas (hydrogen or helium).

Separation In Each Column

Thin “capillary” columns have a coating in their internal wall. Based upon a variety of factors (temperature, flow rate, the affinity of each substance for parts of the column’s internal coating), different types of substances travel through the column at a different rate. Accordingly, each substance will start to separate from each other, and then come out the other end of the column at a different time.

Carrier Gas

These thin columns have a chemical coating inside them designed to interact with the molecules passing through them.  The carrier gas moves at a constant pressure.  This results in different molecules in the gas to group together (e.g. ethanol with ethanol, methanol with methanol).  Each molecule group, such as ethanol, has a unique rate of speed.  This accounts for the separation of the each substance in the columns.

Elution

The unique time each substance exists the column is called the time when a substance “elutes.” To illustrate, if ethanol elutes at 1.5 minutes (the time it takes to travel through the column) anything that comes out the column at that time will be identified as ethanol by the machine – even if it not ethanol.

After each molecule group is pushed out of the column, they will be pushed to a detector.  The time when is substance exits the column is called the time it elutes

The Flame After The Columns

The flame ionization detector (FID) is connected to the gas chromatograph by the columns discussed above. After a sample’s trip through the column, where it separates into its different substances, it exits the column and goes into the FID. This is where the substance is quantified.

The Detector

The detector’s software has been programmed to identify different substances by the time they elute from the column.  The Flame Ionization Detector, as the name implies, then burns each molecule group and then measures how much is burned.

Burning Creates Ions

The sample stream (the gas) from the column passes through a flame in the FID. The compound is burned by the flame. Burning a compound containing carbon creates ions by removing electrons. If you recall science class, an ion is a molecule with a net electric charge. As ethanol contains carbon it is capable of being ionized.

Electrons Measured

The newly freed electrons are then measured. The device works on the premise that the electrical current will be in direct proportion to the concentration of the hydrocarbons burned. Current will pass between electrodes (an electrometer) located in the FID close to the flame. The ions are attracted to the detector and eventually become a recordable digital signal. In the FID, the signal is amplified and converts to parts-per-million.

Raw Data

The software gathers the “raw data” and then processes it. 

The Data Is Graphically Represented

After detection, the signal is processed by software. The end result of this process is called a chromatogram. A chromatogram is a visible representation of the data in a graph. It illustrates the results of separating the substances and their quantification.

Comparison Made

The software does its analysis by comparison. It compares the known concentrations (calibrators and internal standard) to the unknowns (subject samples). A subject sample chromatogram will always have at least two peaks. Recall the headspace vial, shown above, contains both a blood sample and an internal standard. If ethanol is detected that will be represented by a peak, as will the internal standard.

Chromatograms Produced

The end result of the measurement process is a graphical representation of the analysis in the form of a chromatogram. As the name suggests a chromatogram puts the information in a graph with vertical and horizontal lines. A chromatogram from an Arizona crime lab typically has two graphs on one page. This is because there are two columns in the gas chromatograph’s oven each performing an analysis at the same time. Thus, the processed data illustrates the results of both the “A” and “B” columns.

Results Only From The A Column

The “A” at the top of the chromatogram is the reported measurement. Crime Lab commonly program the software not to report the results of the B column.  They refer to the B column as merely a confirmation.

Moreover, the lab does not actually test the 100-250 micro-liters liquid. The sample is heated and a portion of the gas above it is captured to be analyzed by the machine. As such, the process estimates what the result would be if 100 millimeters were analyzed. It is an indirect measurement relying on presumptions and extrapolations.

These samples are put into the machine and the analyst programs the machine’s software to use these values.  If the analyst tells the machine a sample is a .40 but it is really a .30, the machine cannot tell the difference.  Ensuring a calibrator is what it purports to be is known as traceability.

Like most State’s, Arizona has a legal definition of an alcohol concentration. A.R.S. 28-101(2)(a), provides an alcohol concentration is defined as “[t]he number of grams of alcohol per one hundred milliliters of blood.” However, the State does not test 100 millimeters of blood. They merely use a blood sample between 100-250 microliters. A sample of this size will appear to be just smaller than a piece of M&M candy.

Moreover, the lab does not actually test the 100-250 micro-liters liquid. The sample is heated and a portion of the gas above it is captured to be analyzed by the machine. As such, the process estimates what the result would be if 100 millimeters were analyzed. It is an indirect measurement relying on presumptions and extrapolations.

How a GC works

Henry’s Law

Microscopic amount – extrapolated

Small deviation = big consequence

Data Flow

Graphical representation v. Data

Formula computing variables

Data Flow & JusticeTrax & S. Key

Rule – Section 5.5.7

Lab’s quality manual

Rule – Section 4.11.2

“Unexpected” and “Unspecified” Results

When errors discovered?

Self-Repairing?

Can’t merely rely on the printouts

Raw Data

The software gathers the “raw data” and then processes it. 

Processed Data

The “process data” is graphically represented in something called a chromatogram.    The measurement is found here

How a GC works

 

Indirect measurement

Microscopic amount – extrapolated

Small deviation = big consequence

Data Flow

Graphical representation v. Data

Formula computing variables

Data Flow & JusticeTrax & S. Key

Rule – Section 5.5.7

Lab’s quality manual

Rule – Section 4.11.2

“Unexpected” and “Unspecified” Results

When errors discovered?

Self-Repairing?

Can’t merely rely on the printouts

A Gas Chromatograph Does Not Have a Brain

Even the most sophisticated and expensive gas chromatographs require human beings to produce a measurement. The machine has no brain; therefore, human beings must use their own brain when operating the instrument. Simply put, the machine does not work without human beings. Similarly, the machine also does not work reliably without reliable human beings.

The human part of the process starts with the collection of the blood sample. There are numerous requirements for the proper drawing of blood that must be followed. An evacuated tube system is used where a needle is inserted into the subject, and a tube containing a vacuum is attached causing the blood to be drawn into the tube. In many cases, blood samples used by law enforcement are stored in grey top glucose tubes. There are specific amounts of preservative and anti-coagulant in these storage tubes to prevent the loss or gain of ethanol. The manufacturer’s corresponding documentation can often provide valuable information regarding the requirements for using these glucose tubes.

The grey top tubes containing the blood samples are supposed to be stored in a manner that slows sample degradation. The sample is dynamic, and chemical processes will continue after collection. Proper storage and collection will help slow the process, but will not prevent it.

Loading the Headspace Vials Into the Autosampler

Creating a methodology that complies with minimum requirements of the scientific community can accomplish this task.  These minimum requirements are found in what are known as consensus standards.  The seminal consensus standard for creating a trustworthy measurement is ISO 17025.  Accordingly, all the steps and procedures on our list must substantially comply with this consensus standard.

Teaching the Machine Regarding a Blood Alcohol Concentration

It's Not as Simple as Pushing a Button

Cross-examining a crime lab analyst about a blood test should include dispelling the notion that just because a gas chromatograph was used, that automatically equates to an accurate and reliable measurement.

Does using a gas chromatograph, to measure an alcohol concentration in blood, create a presumption of reliability?  NO.

Who says so? The United States Supreme Court has found this to be the case.

DUI Blood Alcohol Concentration Testing

Footnote one in the case of Bullcoming v. New Mexico states:

Although the State presented testimony that obtaining an accurate BAC measurement merely entails “look[ing] at the [gas chromatograph] machine and record[ing] the results,” App. 54,
authoritative sources reveal that the matter is not so simple or certain. “In order to perform quantitative analyses satisfactorily and . . . support the results under rigorous examination in court, the analyst must be aware of, and adhere to, good analytical practices and understand what is being done and why.” Stafford, Chromatography, in Principles of Forensic Toxicology 92, 114 (B. Levine 2d ed. 2006)...
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Bullcoming v. New Mexico,

Nor is the risk of human error so remote as to be negligible. Amici inform us, for example, that in neighboring Colorado, a single forensic laboratory produced at least 206 flawed blood-alcohol readings over a three-year span, prompting the dismissal of several criminal prosecutions. See Brief for National Association of Criminal Defense Lawyers et al. as Amici Curiae 32–33. An analyst had used improper amounts of the internal standard, causing the chromatograph machine systematically to inflate BAC measurements…

Bullcoming v. New Mexico,

It is difficult to debate the risk of error in the blood alcohol measurement process based upon this authority from the United States Supreme Court.