Blood Discovery Cheat Sheet by Justin McShane
The Following Items Concern General Matters:

1.	A copy of any accreditation certificates for the laboratory that were in effect at the time of the analysis.

Comment as to item 1:

What is it? There are many different accrediting bodies that exist that cover various aspects of a testing and calibration laboratory. Some of the common ones include ASLCD/LAB, FQS-I, A2LA, ABFT, NAME, COLA, CLIA and CAP. Also, there may be state specific accrediting bodies such as the various Departments of Health. Each certificate of accreditation should be dated noting when the accreditation was achieved and the length of the accreditation. Most importantly, most of the certificates will outline what the scope of the accreditation covers in terms of what specific forensic science discipline or form of testing.  

Why does it matter? While accreditation does not equate to automatic quality in results, it means that a laboratory adheres to some sort of external Quality Management System (QMS). How worthwhile that QMS is versus what is necessary in the general scientific community to insure true results is debatable. Just determining whether or not a laboratory is accredited and by whom is the very beginning of assessing the quality of the reported and alleged test result. It should be remembered that these standards are minimum expressions of acceptable practices. A laboratory could and should decide to employ greater standards than the minimum to insure quality of the results issued.

2.	The laboratory’s overall policies as to testing and calibration.
3.	The laboratory’s overall protocols as to testing and calibration.
4.	The policies that applies to the section of the laboratory where this particular testing or calibration event occurred.
5.	The procedures that applies to the section of the laboratory where this particular testing or calibration event occurred.

Comment as to items 2, 3, 4, and 5:

What is it? Each laboratory should be divided into separate sections. A laboratory should have a written set of documents that outline the overall testing and calibration philosophy of the laboratory and/or the section.

Why is it important? These documents contain information such as the laboratory’s and the section’s statements as to the need for its personnel to be well-trained, well-educated, and well-monitored. In addition, within these documents there should be language as to the need for every person in the laboratory to follow the laboratory’s and the section’s policies, procedures and instructions in order to achieve a valid and true (meaning actual) result. In addition, these documents should reveal the consequences for failure to follow the directions of the laboratory such as warning, suspension, retraining and firing.

The Following Items Concern Pre-analytical Matters:

6.	Validation studies (both internal and external) that prove the validation of the method and instructions used.
7.	The policy that applies to the assay performed in this particular test or calibration event that covers the calibration or the achieving of a calibration curve.
8.	The procedure that applies to the assay performed in this particular test or calibration event that covers the calibration or the achieving of a calibration curve.
9.	The instructions that apply to the assay performed in this particular test or calibration event that covers the calibration or the achieving of a calibration curve.

Comment as to items 7, 8, 9:

What is it? The act of calibration (and bias) is a procedure that imperfectly transforms the testing (the response from the analytical instrument) into a useful measure. Calibration and bias have to do with precision and accuracy respectively. The true issue surrounds analytical drift and other basics of metrology. Over time, regardless of use (however, heavy throughput does exacerbate the problem and so too does also lack of use) all analytical devices lose their sensitivity, their precision and their accuracy. A calibration curve is not a curve, despite its name. Rather, if well preformed, it is a straight line as determined by various measures of linearity. The calibration curve is the analytical method that results in a plot of calibration data. It is a general method for determining the concentration of a substance in an unknown sample by comparing the unknown to a set of standard samples of known concentration.

Why is it is important? The consequence of failing to achieve acceptable calibration is that the quantitation (how much do we have of what we measured) will always be wrong.

10.	The calibration curves and all chromatograms generated on the batch on the machine on which the sample in this case was tested.

Comment as to item 10:

What is it? For a discussion as to what a calibration curve is, please see the ‘‘Comment as to item 7, 8, 9:’’ above. A chromatogram is the written record of the testing event. Think of it as the receipt of the analysis. It can be likened to the breath test ticket that prints out at the end of an evidentiary breath test. This request requests the laboratory to produce proof that the policy, procedure and instructions outlined in requested item number 9 was actually conducted. This request includes all logs, reports, spreadsheets, control charts, and other documents, in whatever form, reflecting the calibration of all equipment used in the testing and the preparation of the sample at issue in this case as well as the preparation of all samples within the batch.

Why is it important? The consequence of failing to achieve acceptable calibration is that the quantitation (how much do we have of what we measured) will always be wrong.

11.	The identification and source of all internal standards, standards, standard mixtures (separation matrix), verifiers, blanks, and controls that were run within the batch in which the sample in this case was run.

Comment as to item 11:

What is it? An internal standard is a compound purposefully added to a sample in known concentration to facilitate the qualitative identification and/or quantitative determination of the unknown sample tested. ‘‘Controls’’ refers to the series of materials that are used for the assessment of the performance of an analytical procedure. They are standards. Controls that appear before the unknowns are called calibrators. Controls
that appear after the unknowns are known as verifiers. Certified Reference Materials are a type of extremely high quality controls used to check the quality and traceability of products. This request includes all certificates of traceability for the standards, and controls used to analyze this sample. This request should include any quality control certificate provided by the supplier or manufacturer with, or applicable to, such solutions
including reporting of uncertainty in them, control charts during their use and the raw data giving evidence of this. In addition, there should be documents reflecting the expiration date of all externally purchased solutions or reagents used in the batch in which the sample in this case was tested.

Why is it important? Part of the integrity of any testing of unknowns has to do with the veracity of the sources of knowns against which the unknowns are compared. For if we do not have certainty in the knowns and
the references used, then there cannot be any confidence in our conclusions of the analysis of unknowns.

12.	All records reflecting internal testing or quality control testing of all solutions, reagents, or standard mixtures used as, as part of, or in relation to internal standards, controls, standard mixtures, or standards in the batch in which the sample in this case was run.

Comment as to item 12:

What is it? An important feature of achieving a true test result and one that is valid is not taking another’s word for it. Simply relying upon a ‘‘trusted’’ manufacturer’s or vendor’s certification is insufficient.

Why is it important? Even the most ‘‘trusted’’ manufacturer or vendor can and do make mistakes in the manufacturing or packaging of its products that it allows into commerce. This is why it is essential for the receiving laboratory to verify independently that these items are what they are purported to be. Without performing this essential step, then the validity only is presumed and cannot be said to be proven even if all other aspects are correct.

13.	All refrigeration logs, reports, or other documents in whatever form, for all refrigerated compartments in which this sample, other unknowns within the run, internal standards, controls, standard mixtures, standards, and reagents used in or in relation to the analysis in this case were stored or kept at any time.

Comment as to item 13:

What is it? All of the testing materials including the unknowns must be stored in the correct environment.

Why is it important? The failure to maintain the samples or the controls and other materials used in the testing within a narrow temperature range can lead to abhorrent results in the quantitative reporting. For example,
in the case of testing those who are accused of a DUI where the accused’s blood is collected, if the blood exceeds the normative temperature as prescribed by the manufacturer, and if Candida Albicans is present in the sample or the environment, then the neo-formation of EtOH can occur. In simpler words, if the blood sample is not refrigerated, then the blood can breakdown resulting in a higher reported BAC that is not correct.

14.	All proficiency testing results for the section of the laboratory testing the sample in this case as well as for the person who conducted the testing in this case --- since the last date of accreditation inspection preceding the test, and for any such testing since the testing in this case. This specifically includes the summary report of expected results for the proficiency testing (and the manufacturer’s information sheet) against which the proficiency test results are judged.

Comment as to item 14:

What is it? Proficiency Testing (PT) is defined as a means of evaluating a laboratory's performance under controlled conditions relative to a given set of criteria through analysis of unknown samples provided. The best
practice is for the PT testing to be blind where the analyst and the laboratory do not know they are being tested.

Why is it important? If the laboratory has no method to prove that its laboratory or its analysts can actually test the samples correctly, then the testing involving the accused is suspect. The records may show that the analyst is not testing perfectly.

15.	Quarterly balance quality control records on any balance instrument related to the calibration of the EtOH standard solution or the preparation of knowns or unknowns used in the blood alcohol testing of the samples in this case. The records reflecting the calibration of weights on any balance or instrument related to this case as well as the control charts kept.

Comment as to item 15:

What is it? If the laboratory performs any sort of independent verification of vendor provided material or creates its own internal solutions from stock materials or performs its own pippetting calibration, an analytical
balance (otherwise known as a scale) is needed to do all of this. Control charting is a graphical and empirical statistical tool used to detect excessive process variability to try to identify specific assignable causes that
can be corrected.

Why is it important? The calibration of any analytical balance is only as tight as the items that are used to check and correct it. Control charting serves to determine whether a process is in a state of statistical control; that is, the extent of variation of the output of the process does not exceed that which is expected based on the natural statistical variability of the process. Control charting is a great way to identify the source of statistical outliers where a machine can be pulled, an environment checked or an inappropriate operator stopped or re-trained.

The Following Items Concern Analytical Matters:

16.	The instructions that apply to the assay that was used in this particular testing or calibration event occurred.

Comment as to item 16:

What is it? This request covers that which was perhaps referred to as ‘‘Standard Operating Procedures.’’

Why is it important? Any deviation from a valid method, even a seemingly small one, from the instructions leads to a non-validated result. See the ‘‘Comment as to item 6’’ above.

17.	The employee training record, curriculum vitae, and resume for any person listed on chain of custody documents in this case or who performed the analysis.

Comment as to item 17:

What is it? This request is self-explanatory.

Why is it important? An analyst is only as good as his or her training. A personnel file can outline work related deficiencies and lack of proficiencies.

18.	Indentify the make, model, and brand/manufacturer of the instruments and other supporting instruments (i.e. balance, pipette, etc.) used during the analysis and/or preparation of the samples in this case and the variables used in its installation and operation.

Comment as to item 18:

What is it? This should identify the method of detection for the chromatographic method used in this particular testing (e.g., headspace injection versus direct injection, manual injection versus automated injection, split versus splitless injection port (in the case of Gas Chromatography), Wall Coated Open Tubular columns (capillary columns) versus packed columns, Electronic Pressure Control versus manual flow control, installation of a Mass Spectrometer versus a Flame Ionization Detector versus UV/Vis DAD system) as well as the conditions used (e.g. type of column and dimensions of the column; the length of the time of the run; the integration parameters, and the carrier and makeup gas and its flow; inlet flow/pressure rate (split ratio), oven temperature, incubation time/injector time, injector temperature, detector temperatures, equilibrium times).

Why is it important? All of this information is necessary to arrive at the quantitative result and the qualitative result. An analytical device can be likened to an automobile. Every automobile comes with various options that can either increase the performance of the automobile or make it not as good as the top of the line model. The same is true with analytical devices. Some are better than others. Some have been upgraded to make them top of the line in terms of performance, while others are subpar. In addition, just as every automobile may be driven differently, each analytical instrument has variables as to its operation. Just as in the case of the automobile where one can control its use through its variables (for example, fuel efficiency by how one steps on the gas pedal), so can one change the fidelity of the result by changing the variables as to the analytical device used. This request focuses on how ‘‘good’’ the device itself is as well as how well it is being used according to its published method.

19.	The policy concerning the sample selection criteria used in this particular case.
20.	The procedure concerning the sample selection criteria used in this particular case.
21.	The instructions concerning the sample selection criteria used in this particular case.

Comment as to items 19, 20, and 21:

What is it? Generally across all of forensic science, only a remarkably small part of the whole, called an aliquot, is actually tested by any analytical device. This choice is referred to as ‘‘sample selection.’’ A policy, procedure, and instructions should be in place to outline the sample selection process.

Why is it important? In essence, what happens in the laboratory when an aliquot is prepared is an exercise a massive amount of truly subjective discretion by selecting a ‘‘pinch of this’’ or a ‘‘section of that’’ from the whole unknown sample submitted for examination. Sample selection is perhaps the single biggest area of potential uncertainty and error in obtaining true results. It remains one of the areas that goes largely unexposed in the courtroom. As a result of this non-totally consuming testing method where only a part of the whole is examined, it becomes crucial and vitally necessary for the laboratory to ensure homogeneity in the aliquot tested. This is a supreme challenge. Even with a policy, procedure, and instruction in place, it is clear that by performing sample selection massive representation errors with respect to non-colloidal  (nonhomogeneous) mixtures can occur. This is sometimes referred to as ‘‘random sampling error.’’ It is a very large potential source of error and at worst results in erroneous results. If the laboratory received more than one vial or container of blood or other substance, records reflecting which vial was selected for testing in this case should exist based upon these policies, procedures and instructions. Further, how the selection and preparation of the particular aliquot from the whole tube was arrived at should likewise be justified.

22.	The source and type of all consumables used in collection, preparation, and analysis of the samples run in the batch.

Comment as to item 22:

What is it? Just like an automobile, each analytical instrument has consumables.  Instead, of air filters, lights, sparkplugs, windshield whipers, there are items such as inlet liners, septa, solvents, golden seals, tubing, vials, o-rings, ferrules and other items.

Why is it important? The failure to replace consumables can affect performance, just as the case with failing to replace worn out sparkplugs.

23.	If a Gas or Liquid Chromatograph is used, the reporting of t0 time according to the method.

Comment as to item 23:

What is it? t0 represents the time that it takes for the carrier gas to elute through the column.

Why is this important? t0 is the essential measure that enables an external auditor of the testing event to determine whether or not each test in a batch was performed properly and uniformly throughout the run. t0 is a means to verify retention time and relative retention time. Retention time is used to examine the validity of the qualitative measurement.

The Following Items Concern Reporting Matters:

24.	The particular records for this testing or calibration event.

Comment as to item 24:

What is it? This request calls for the production of all test specific information.  It includes all laboratory notes, case files, case reports, or bench notes, by whatever name they are known, and in whatever form. It also includes all records contained in the testing folder specific to the test in this case. This includes a copy of the case or testing folder itself if it contains any notations or entries. All data and notes including the chromatograms produced from all of the samples in this batch and run should be produced. This calls not simply for this one unknown sampled and tested that is attributed to the accused, but also for the records, such as the chromatograms, for all of the controls (calibrators, verifers), standard mix (separation matrix), blanks, and other unknowns in the batch in which this sample in this case was run. It should include all chain of custody records specific to the specimen(s) in this case.

Why is it important? The need for these records should be self-evident.
25.	The quality control policy and protocol for the laboratory, the section, and the assay performed.
26.	The quality assurance policy and protocol for the laboratory, the section, and the assay performed.

Comment as to item 25 and 26:

What is it? This refers to the written policy, procedure and instructions for the reporting of unexpected results as applies to this laboratory, this section and for this assay. This should include the information for the discarding or non-reporting of data or results (regardless of whether the data or results were or are made during the analysis or before or after the analysis).

Why is it important? Mistakes will happen in the laboratory. The question is what does the laboratory do when mistakes are caught before they are reported outside of the laboratory and what does the laboratory do when mistakes are caught after they are reported outside of the laboratory is relevant.

27.	The full reporting and the underlying validation of the valuation of the uncertainty measurement (UM) in the ultimate reported result.

Comment as to item 27:

What is it? A whole body of science is devoted to measurement. It is called metrology. A portion of metrology is uncertainty measurement (UM). UM answers the question of ‘‘how off is the test.’’ Every testing event and every testing process has some inherent variability due to systemic and random factors over which there is no control and which cannot be eliminated economically. 

Why is this important? As can be the case, a laboratory’s stated but unproven error (often expressed as +/- x% or some fixed number) grossly understates reality. Without knowing the uncertainty of the measure, then the measure is not relevant.

28.	If a Mass Spectrometer is used, then the following additional materials should be provided:
28.1	If a spectral library is used to examine spectra and elucidate spectra, the source of the library spectra.
28.2	The hit list, and the hit histogram for the testing.
28.3	All ‘‘tune’’ reports ran within one year if a MS detector was used.

Comment as to items contained in 28:

What is it? Mass spectrometry (MS) is a specific type of analysis.  Mass Spectrometry is the branch of science dealing with all aspects of mass spectroscopes and the results obtained with these instruments. It measures the mass-to-charge ratio of charged particles. It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules. The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measurement of their mass-to-charge ratios. The result from the analysis of the unknown is compared against the known. In MS analysis, there is a library of knowns.

Why is it important? In mass spectrometry, the spectral library is crucial. It enables the analyst to compare the unknown to a known to arrive at an opinion that is often expressed as a conclusion. There are many different commercial sources for these libraries. Some are better than others are. In fact, some systems allow the analyst to exercise his or her discretion in employing his or her own standards as opposed to the commercially available ones that are derived from CRMs. Electron Ionization (EI) based Mass Spectrometry has been so fantastically removed from its base in science, that it has been simplified to accommodate the masses. In the modern crime laboratory, EI-based MS has been reduced to simple computer-assisted pattern recognition. Sadly gone are the days of acid-based chemistry to elucidate mass spectral
patterns. Instead, most analysts just let the computer do it. What the computer produces is a list of likely matches as compared against the library of reference spectra. This computer generated matching and evaluation produces match scores, probability scoring, and other grading methods. The analyst with this data, then uses his or her own discretion based upon these lists to these lists, grading systems and scores to opine what the substance is.