As stated in the Quality Management Program Quality Manual v2.3/4 (QMPQM v2.3/4), an employee has the following responsibilities...

5.1.1. To actively participate in the quality management program and follow policies and procedures outlined in this Manual, applicable procedure manuals, and other policy and procedure documents;

and...
5.1.3. To immediately bring to the attention of their Supervisor, Section Chief, or Director any situation which potentially compromises the integrity of work performed or reported by Institute personnel;

As such, the following document is being presented in support of the belief that the Serology Procedures Manual v1.0 (SPM v1.0), as they are currently written, do not allow an employee to fulfill the requirements of the QMPQM v2.4:
5.1.2. To diligently and accurately perform the duties of their position;

Like any other lab, the SWIFS Forensic Biology Unit (FBU) is a highly adaptive, dynamic, and constantly changing entity. Older lab equipment is replaced with new lab equipment. Different lab reagents and consumables are introduced into the lab to replace more costly or lower quality reagents and consumables. Personnel leave SWIFS to pursue other opportunities and new personnel are employed. Since March 2008, 7 new Forensic Biology analysts have been hired giving a total of 18 members of the FBU. However, the Forensic Biology Unit's SPM v1.0 is significantly and critically seven (7) years out-of-date. Since its implementation in November 2001, the SPM v1.0 has had 4 content-changing memos (and 1 non-content changing memo) and a few content-changing emails. Unfortunately, a large number of procedures stated in the SPM v1.0 are obsolete, erroneous, and in some instances, contradictory to the procedures expected to be performed by the serologists. To compound the problem, there are also a number of policies and changes in protocol procedures which, over the years, have been verbally communicated to the analysts. These additions and changes have not been documented via Memo, email or any other official SWIFS document as is required by the QMPQM 2.3/4:
11.4.3. Revisions and corrections to procedures are summarized in the front of each manual including effective date of revision, description of the change, and initials of individual authorizing the change. 11.4.4. Revisions may be made by memo or by updating procedures; the effective date of implementation must be noted.

This means of communicating "protocol" to the analysts has lead to dissemination of incorrect and incomplete information, and misinterpretation of correct lab procedures

by members of the lab. Consistency and reproducibility in lab procedures vanishes as one analyst performs their tasks differently than another analyst. The lax in enforcement of the QMPQM has also given the analysts the perception that introduction of procedural steps or use of unsecured documents/templates (for their own convenience, for example) is an acceptable practice. What may be perceived as an inconsequential alteration in a procedure (a change made for convenience to the analyst, for example) may actually have a substantial and detrimental impact on SWIFS's greater responsibilities such as maintaining ASCLD accreditation, providing protection and security of submitted evidence, and upholding fiscal accountability to Dallas County. As the serological protocol "drifts" from analyst to analyst (and generation to generation), logical explanation and rational reasoning behind the steps and procedures of the original 2001 protocol become lost, misunderstood, or convoluted by the analysts. And it goes without saying that verbally communicated procedures and protocols are impossible to archive once a new protocol is issued, as required by the QMPQM v2.3/4....
11.4.6. Archived procedures are maintained by the Quality Manager. 11.4.7. Out-dated procedures are immediately removed from general accessibility.

These problems with SPM v1.0 have been directly related to the frustration of the new forensic biologists undergoing Serology Training. The trainees are tested on those written procedures (in a written exam), yet are expected to perform those procedures that are verbally communicated to them, which at times, is contradictory to those procedures in print. Thus, at any given moment, a trainee may be breeching protocol by ignoring what is in print (in order to do what they are told), or performing the protocol as dictated by the SPM v1.0 (and disregarding the trainer's instructions.) The Serology Training Guide v1.1 (STG v1.1), also, has not been updated sine 11-27-01. Today's new trainees follow the same the training guidelines presented within. However, it too needs to be updated to match present-day SWIFS operations and the current serology laboratory activities.

Original date 11/09/01 Revisions and Corrections: 08/16/02 Memo-Deletion of Acid Phosphatase Quantitation Assay from Manual 12/11/02 Replace P30 Cross-over test with P30 ABACard test 1/16/03 Addition - Post-Conviction Case Research Guidelines 4 pages 09/03/03 Add One-Step ABA HemaTrace for the Forensic ID of Human Blood 01/24/08 Memo - Individual pdf procedure files were consolidated into a single pdf collection document. No content change to the individual procedures

Technical Oversights (due to incorporation of the SPM v1.0 into the Web Portal) Pages not numbered...Table of contents not correct. Post-conviction Case Research Chapter not listed in Table of Contents. Memos not listed in Table of Contents. Security watermark "Not a Controlled Document" printed in center of page obliterates text in some of the procedures. Security watermark ("Not a Controlled Document") not printed on every page. Security watermark concerning "controlled document" printed twice on some pages (2 different locations).

(Pages 4-5)
"Brentamine Fast Blue B Test for Acid Phosphatase"

The protocol mentions nothing of the procedure for QCing the Brentamine Reagent prior to use, and there is no reference to Appendix I for the recipe or the correct QC procedure, or recording QC results, dates, etc. on paperwork. As stated in the SPM v1.0 (Appendix 1, page 40)...
"...Each preparation of the Brentamine Reagent must undergo quality control (QC) before initial use. This is performed by adding a drop of the reagent to a control swab containing semen at a 1:1000 dilution and observing a color change within 15 seconds..."

Nowhere in the SPM v1.0 is there a description of how the stock semen is qualified to be considered a standard or positive control for QC of the Brentamine Reagent. The stock semen used does not fit the definition of a standard as set forth in the QMPQM 2.3/4:...
10. Quality Assurance of Standards, Controls, and Reagents

10.1.2. Standard or Reference Standard A preparation which has known concentration and/or physical or chemical composition used for the purpose of calibrating equipment and/or as an experimental control. 10.1.3. Control A standard of comparison for verifying or checking the finding of an experiment. Positive controls verify the presence of analytes or conditions; negative controls verify the absence of analytes or conditions. 10.5. Standards 10.5.1. Standards should be obtained from reputable suppliers and be of known purity and/or chemical or physical composition. 10.5.2. A certificate of analysis will serve to establish the quality of a chemical standard and should be obtained from the chemical supplier where possible. When a certificate of analysis is not available from the manufacturer, standard composition should be verified internally by an applicable analytical technique. 10.5.2.1. A log must be kept of all standard solutions prepared including the name of the standard, the initials of preparing analyst, date of preparation, method of preparation, and lot number.

Typically, the stock semen used as a standard in the lab is donated by an unknown individual, deposited into a 50mL conical, and stored in the lab -20C freezer. There is no historical log or record keeping of the stock semen (who, when, or performance of QC). Since there is no identifiable individual associated with the stock semen, there is no accountability for the contents of the 50mL conical. Therefore, it can not be confirmed that the 50mL conical actually contains semen (i.e., other biological reagents in the lab can also give an identical color change with the Brentamine Reagent), that it does not contain other compromising components inadvertently introduced during the collection of semen (e.g. lubricants, saliva), or that it has a known amount of seminal acid phosphatase enzymatic activity (the cause of the color change in the Brentamine Reagent). For the QC of new stock semen (the only test performed), previously QC'd Brentamine Reagent (against the OLD stock semen) is used against a 1:1000 dilution of the new stock semen. A color change in the swab within 15 seconds means that the new stock semen is acceptable to use as a standard. However, because the enzymatic activity of the tested protein (seminal acid phosphatase) is potentially susceptible to freeze-thaw cycles, vortexing, room temperature dilutions, and otherwise "normal" day-to-day (mis)handling of the semen, at any given moment the QCing of the Brentamine Reagent may fail. According to the protocol, this failure can only be attributed to an incorrect preparation of Brentamine Reagent, and not the semen standard. As such, a failure in QC of the Brentamine Reagent requires a second preparation of Brentamine Reagent. Failure in QC of a second preparation of Brentamine Reagent leads to preparation of new Brentamine Buffer, followed by the purchasing of new stock chemical reagents for the Brentamine Reagent, etc. The only means

of successfully QCing the Brentamine Reagent (pass, color change within 15 seconds of a 1:1000 semen dilution on a swab) is to replace the "bad" stock semen with ANOTHER stock semen. (Then arises the question, how does one QC freshly made Brentamine Reagent with an non-QC'd stock semen?) It is also not widely known among the serology analysts that, because of the long-standing use of the Brentamine Reagent protocol in SWIFS's lab, the protocol for the Brentamine Reagent has been "grandfathered" (per ASCLD) as an acceptable protocol and is exempt from normal protocol validation studies (as stated in the QMPQM). This type information should be stated in the SPM v1.0 to alert serologists to the historical context of the protocols used in the lab.
2. Newly prepared standard solutions must meet quality control criteria established by each laboratory to ensure proper response.

Although the SPM v1.0 states that a positive control 1:1000 dilution of semen on a swab is used to QC freshly prepared Brentamine Reagent, it does not state the procedure for making the 1:1000 positive control swab. This omission had lead to an "unauthorized" protocol that the serologists have created for the batch preparation of 1:1000 positive control swabs (a task which is rotated among serologists periodically.) Briefly, this protocol suggests making 40mL of a 1:1000 solution from stock semen, moistening a batch of sterile swabs (500+) with this dilution, drying the swabs in the hood overnight, then storing them in the freezer at -20C until use (one at a time). An alternative to this protocol (verbally communicated) is to store 40uL aliquots of the 40mL 1:1000 dilution in microfuge tubes at -20C, each of which would be removed from the freezer to prepare a single positive control swab the day of use. While the endpoints to each of these protocols are the same, the means by which the endpoint is obtained are different. Freezer storage of pre-made swabs is different from storage of aliquots. There are even debates among lab members as to which protocol yields greater swab-to-swab consistency and affords longer storage-life. This same "protocol" also describes the batch preparation of the positive control swabs (1:10,000 blood) for the QC of the LMG Reagent (see below).

(Page 7)
"Microscopic examination of smears for spermatozoa." II. Interpretation of Results A. Results recorded...

The name of the microscope used for the sperm search needs to be recorded. The coordinates of the sperm on a slide are specific to the microscope.
B. If spermatozoa are seen on the smear...

There is no definition/term for a smear that has 8-14 sperm. This range has been erroneously omitted from the list.

There is also no explanation as to the reasoning for quantifying the description of the sperm (why is it important for knowing the number of heads versus intact spermatozoa) There is also the question as to why ALL slides don't require a second analyst (experimentally blinded from the results of the first analyst). Unfortunately, in practice, second reads (pertaining to negative slides) tend to be biased in that the second reader may proceed at a quicker rate (less thorough than a first read) knowing that the first reader already scrutinized the smear. The requirement of a second reader to read a blind slide (unknown results from the first reader) would eliminate the preconceived assumption by the second reader that the slide is negative, thereby requiring an equally thorough second read of the smear. The requirement of a second analysts's read/confirmation would also be analogous to the other confirmatory tests which require second readers (Ouchterlony, HemaTrace and p30 cards.)

(page 11)
"Collection and Storage of Test Areas" E. Retaining small items. iii. Place an item stored sticker on the packaging..."

The serologists no longer use stickers for any purpose in the lab. (The use of stickers arises in several locations in the SPM v1.0, none of which are followed.)

(page 2)
SOP: Onestep ABAcard p30 Test for the Identification of Semen B. p30 test procedures 7. Within each batch... positive control - a semen standard or p30 standard diluted to a final...

There is no explanation as to what the semen standard is (crude?), or what the p30 standard is (pure isolated p30?), or any reference to the QC procedures in Appendix I. Equally confusing, however, page 6 of Appendix 1 (page 6) lists the Semen Standard (SERI) and the p30 Standard (Sigma) to be used for QC procedures of the p30 ABAcards, while page 7 ("p30 Reagents") states that...
"...the semen/p30 standard may be crude semen containing p30 (SERI p30 electrophoresis standard), purified p30 (Sigma Chemical), or equivalent..."

Clarification on the differences of each of these reagents (and what "equivalent" is acceptable) is necessary. (That is, the p30 standard routinely used in the lab is seminal fluid (no spermatozoa) and comes from SERI. Is the "crude semen p30 electrophoresis standard" the same as what is normally used in the lab? Note: the ACP quantitation electrophoresis protocol, which uses the electrophoresis standard, is no longer used in the lab.)

This protocol also introduces the use of Worksheets generated from a computer. Are these worksheets "locked"? What worksheets should be "locked"? The SPM v1.0 should explain which worksheets are considered "editable by the analysts". Also introduced in this protocol is the electronic storage of digital images. Rational explanation for the removal/trash/empty trash procedure should be included within the SPM v1.0.

(page 18) Leucomalachite Green (LMG) Test for Blood I. LMG Reagent QC A. QC of the LMG Reagent... i. "...a positive control swab (1:10,000 human blood)..."

Out of convenience, the serologist have opted to periodically make batches (500+) of positive control swabs, storing them in the freezer. On the day of use, for QCing the LMG reagent prior to casework, a single swab is removed from the freezer and used for case work. However, there is no official protocol for the preparation and storage of batches of positive control swabs for the LMG test (this is exactly analogous to the batch preparation of positive control 1:1000 semen swabs...see above). Also, similar to the stock semen, the human blood used as a standard in the lab does not fall within the definition of a "standard" as set forth in the QMPQM v2.3/4 (analogous to the semen standard). However, the human blood used typically does have the donor's name associated with it (and sometimes the phlebotomist's name, too.) The lab performs no other analytical tests on the donor blood to confirm reliability of the peroxidase-like activity (turns the LMG blue-green in a positive test) of the stock human blood standard. Only recently has the Serology Lab begun logging in chemicals and reagents received and/or prepared (and/pr QCd) into a common binder along side the DNA reagents and materials. (This was done before with serology reagents and solutions prepared/QCd in a separate serology binder, but stock chemicals were typically not logged.) But there appears to be some confusion among the serologists as to what stock chemicals (typically used to make critical reagents) warrant the documentation of expiration dates and/or disposal those chemicals past the expiration date. This confusion originated from a situation approximately 3 months ago when the stock chemical sodium perborate 4hydrate (the major component of the critical reagent LMG) was discovered to be 3 YEARS past expiration date (05/05 according to the manufacturer's date printed on the bottle). Nowhere in the logbooks had this notation been made for each successive preparation of LMG. The discovery of the expired reagent was reported to the higher authorities with the declaration that the LMG reagent prepared (with the unknown expired chemical) was adequate to use provided that the LMG reagent passed QC (1:10,000 dilution of blood on a swab.) In a similar fashion, expired HemaTrace and p30 ABAcards can be re-QC'd, thereby extending their shelf-life another 2 months. However, the spray bottles containing the 10% bleach solution (or rather, the 1:10 dilution created

upon spraying) were disposed of because they were beyond the expiration date. (Bleach stored in a "normal" 4L jug is acceptable to use and, seemingly, never expires.) For clarification, the serologists should be informed as to which stock chemicals, reagents, and materials used routinely require monitoring and reporting of expiration dates (as well as other pertinent information).

(page 42)
1. "...Determine whether failure is due to the expiration of the control swab before preparing new reagent..."

There is no expiration date given for the control swabs made in batch, and there is no protocol (or previous in-house experimental study) for determining the expiration date of the swabs. (page 20)
'Species of Origin Determination using the Ouchterlony Double Diffusion Method"

The procedure states that the "...positive control to be used is human serum". However, serologists in the lab routinely use dilutions of whole human blood as positive controls, not human serum. Also, the protocol does not state the dilution of the positive control to be used (i.e. the limit of detection). In addition, the procedure as written requires 2 positive controls to be used per rosette. Arguably, only one positive control needs to be included per rosette because using 2 positive controls leaves open the possibility of a experimental result where one positive control gives an expected precipitin band (true positive) and the other positive control does not (a false negative reaction). These conflicting results can confuse the analyst into either believing the test is valid or invalid. Two positive controls may be used (per rosette) if they are of different dilutions or if they are different human blood sources. This should be explicitly stated in the protocol and/or examples should be given. (Another, weaker argument...if 2, why not 3 positive controls??) Also, there is no mention of a negative control per rosette. There is, however, a mention of a negative control per plate. Arguably, the experiment is the "rosette" (the anti-serum in the center vs. the outer wells containing the unknowns) and not the plate (which may have a number of rosettes, each with a different anti-serum in the center depending upon the experiment...see II.B.iv). Each anti-serum must demonstrate "non-reactivity" with the extract buffer (i.e. no precipitin band). Therefore, for simplicity, for each anti-serum used (i.e. rosette) there should be a single positive control and a single negative control.

(page 21)
III. Optional Procedure. Stain and destain.

The serologists in the lab do not perform this step largely because the procedures for "destaining" are not explained. (page 22)
VI. Cross-over procedure

This procedure is no longer in use per the Memo.

(page 32)
B. Designated communal storage areas must be locked at all times. Keys will be kept in a designated place within the laboratory, accessible only to Forensic Biology Personnel.

The communal locked cabinet is accessible by everyone who has access to the lab (key + magnacard). Also communal, but never locked, are the 4C fridge and the -20C freezers. The reasoning for locking some communal areas (that everyone has keys for) and not others is not clear to the serologists.

(page 36)
G. Autopsy kits. i. " Store all swabs regardless of the result of analysis..."

It is common practice in the lab to only store those swabs that test positive (analogous to Sexual Assault kits). The remaining swabs go back into the kit. There is no documentation of when the protocol changed. This was a verbally communicated change.

(page 36)
J. Condoms. "...Condoms should be stored intact (as submitted) in the FB freezer until analysis..."

There are no freezers in the Evidence Registration area, therefore, condoms can not be stored in freezer until analysis. Even when the condom is in possession by the analyst, it is not stored in the FB lab's freezer. Again, this was a verbally communicated change, without documentation.

Appendix 1. Reagents and Solutions: Preparation, Storage, and QC

(page 39)

The recipe for ACP Quantitation Assay Buffer and ACP Quantitation Assay Stop Solution are still listed. This procedure is no longer done in the lab per the Memo (page 43) The instructions for using P30 antiserum for cross-over electrophoresis. This procedure is no longer done in the lab per the Memo. (date??) Also listed is the recipe for P30 Assay buffer (page 44) The recipe for p-Nitrophenyl phosphate substrate is still listed. However, the ACP Quantitation assay is no longer performed in the lab per Memo.

(Appendix 1, no page number),

Quality Control Procedures for the Onestep ABA HemaTrace Blood Cards for the Identification of Human Blood.

The negative control stated in the protocol for the ABAcard HemaTrace procedure is deionized water. This is erroneous because the HemaTrace Buffer (not deionized water) is used for the extraction of blood from the test item. Therefore, the HemaTrace buffer should be used as the negative control. In addition, the positive control is whole blood diluted in HemaTrace Buffer (not blood diluted in deionized water.) Therefore, the HemaTrace Buffer is the correct negative control. If an alternative buffer is used to extract the blood (not the HemaTrace Buffer), the alternative buffer would be the correct negative control. Of greater concern is the fact that this page is a photocopy of a page from the SWIFS Validation Experiments for the use of Onestep ABA HemaTrace Blood Cards in case work (circa 1993). This fact should be further investigated to insure the correct buffers and protocols were used during the Validation Experiments. To confuse matters more, the CORRECT buffer to be used is stated in... (Chapter 10, no page number),
SOP:OneStep ABAcard HemaTrace for the Forensic Identification of Human Blood C. Evaluation of test results... b. To be able to report out results in a batch... " 1. The positive control should consist of a 1:1,000,000 dilution of human blood and the negative control should consist of the extraction buffer.

A contradiction of appropriate reagents within the same Manual can lead to misinterpretation of protocol by the analyst.

Worksheets (page 56) Acid Phosphatase Assay Worksheet...obsolete protocol still in SPM v1.0 (page 57) P30 Worksheet...obsolete protocol still in SPM v1.0 (page 58) Suspect Kit worksheet...no longer used??

Insofaras the update and revisions to the Serology Training Guide v1.1 (STG v1.1), many ideas and suggestions come to mind. An absolute criteria that must be incorporated into the STG are those criteria and standards used by the Supervisors to establish performance evaluations at the end of the 3 month and 6 month periods.
Dallas County Code DIVISION 7. PROBATIONARY PERIOD* Sec. 86-271. Performance evaluations; failure to complete probationary period. The immediate supervisor will periodically advise the employee of his progress and ensure that the employee receives any necessary training required in order for the employee to successfully perform the job duties. The supervisor shall complete performance evaluations after the first three and immediately preceding the completion of the six months of the probationary period. Failure of the employee to satisfactorily complete the probationary period will result in dismissal without right to appeal. (Admin. Policy Manual, A(2.07)) Secs. 86-272--86-290. Reserved.

A trainee has no basis of comparison when it comes to an "average" timeframe for completing those requirements necessary to becoming a "trained" serologist (and a "real" Dallas County employee). Therefore, a schedule containing detailed benchmarks and milestones should be presented to the trainee at the beginning of training.

Has this email been introduced to the QM Web Portal, and the appropriate notations made in the QMPQP v2.4?
Subject: Creation Date: From: Created By: Peer Technical Review sheets for oral rinse only kits 11/10/2008 9:56:35 AM Timothy Sliter TSliter@dallascounty.org

An issue has come to my attention while reviewing serology reports. For no analysis sexual assault kits with an oral rinse, no peer technical review form is being completed. The reports are considered administrative in nature because they relate to the receipt and release of evidence only, and because no analytical result is being reported. Because no analytical result is being reported, the reports are considered not to be subject to peer technical review under the Institute Quality Management Program (QMP 16.3. Technical/Peer Review of Case Records) I am going to change this policy effective immediately. Because we are performing (and billing for) technical processing of an evidence item (centrifugation of the oral rinse, transfer of the pellet to a swab, drying of the swab), these reports will be considered analytical in nature, and a peer technical review sheet will be completed.

Is there documentation supporting the receipt, reading, and understanding by those it directly involves?