Attach_24_USAPHC_Red_Blood_Cell-Cholinesterase_Testing_and_Quality_Assurance.pdf

Medical Support Services

Center for Domestic Preparedness (CDP)

Anniston, AL

Attachment 24

USAPHC Red Blood Cell-Cholinesterase

Testing and Quality Assurance

TECHNICAL N

OCCUPATIONAL AND ENVIRONMENTAL

HEALTH

RED BLOOD CELL-CHOLINESTERASE TESTING

AND QUALITY ASSURANCE

APPROVED FOR PUBLIC RELEASE; DISTRIBUTION IS UNLIMITED

H E A D Q U A R T E R S , D E P A R T M E N T O F T H E A R M Y

30 NOVEMBER 2001

MED 590

TECHNICAL BULLETIN

No. MED 590

DEPARTMENT OF THE ARMY

WASHINGTON7 DC, 30 NOVEMBER 2001

RED BLOOD CELL-CHOLINESTERASE TESTING AND QUALITY ASSURANCE

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I I

APPROVED FOR PUBLIC RELEASE;

DISTRIBUTION IS UNLIMITED

CHAPTER 1. INTRODUCTION

Reporting test results Program procedures

Authority Background Program participation Submitting primary sp to CRL Reporting test results PROCEDURES FOR PERFORMING RBC-ChE TESTING Purpose Safety and Blood specimen collection Reagent preparation Blood specimen analysis Instruments and equipment req Reference range Baseline determination

CHAPTER 3. PRIMARY ChE TESTING

4.

APPENDIX A. REFERENCES

Glossary

"This bulletin supersedes TB MED 292, 30 May 1975.

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Glossary-1

TB MED 590

CHAPTER 1

INTRODUCTION

1-1. Purpose This bulletin-

a. Describes the methodology for measuring red blood cell cholinesterase (RBC-ChE) activity in the Department of Defense (DOD) Cholinesterase Monitoring Program.

b. Provides information to-

(1) Installation commanders who have a ChE-inhibiting substance mission.

(2) Health care providers who are responsible for evaluating RBC-ChE depressions and for maintaining the RBC-ChE test records. (See DA Pam 40-8.)

(3) Laboratory personnel responsible for con-ducting RBC-ChE assays.

c. Describes the controls and standards included in the DOD laboratory test method.

d. Describes the procedures for-

(1) Requesting RBC-ChE assay analysis.

(2) Collecting and processing blood specimens.

(3) Completing test results.

(4) Reporting test results.

e. Describes the procedures used by the Cholin-esterase Reference Laboratory (CRL) at the U.S.

Army Center for Health Promotion and Preventive Medicine (USACHPPM) to-

(1) Support standardized testing require-ments.

(2) Provide limited primary testing.

(3) Establish and maintain quality control

(QC) and quality assurance (QA) procedures.

(4) Maintain assay performance standardiza-tion by-

(a) Specifying procedures, reagents, and in-strumentation.

( b ) Providing technical training and consulta-tion.

( c ) laboratory technicians.

( 5 ) Maintain transferability of test results throughout the program by performing-

(a) Test result validation by representative specimen retesting.

( b ) Statistical analysis of test-retest dif-ferences.

(6) Conduct onsite laboratory audits at least annually.

1-2. References Appendix A lists the references used in this bul-letin.

1-3. Abbreviations and terms The glossary provides a list of abbreviations and terms used in this bulletin.

1-4. Background

a. Cholinesterase testing is an essential element in the medical surveillance of chemical agent and pesticide workers. The enzyme ChE hydrolyzes acetylcholine resulting in the release of acetic acid and choline. This mechanism is important for the breakdown of the neurotransmitter acetylcholine to retain normal function of nervous, muscle, and gland tissues. Acetylcholine inhibiting compounds, such as the nerve agents GA, GB, GD, VX and organophosphorous insecticides, bind to ChE, ren-dering the enzyme inactive. This inhibition will re-sult in concentration dependent compromise of nervous system function, which can eventually lead to death.

b. The RBC-ChE is a representative of the true ChE of the nervous system. Exposure to ChE-in-hibiting compounds, even at sub-clinical levels, can be detected by monitoring RBC-ChE activity. Ex-posure to inhibitors is detected by comparing an individual's RBC-ChE activity level to a pre-viously established baseline level and checking for a depression in enzyme activity.

c. The DOD has adopted the use of a single standardized method for RBC-ChE testing: the Manual 17-Minute Change in pH Method. A QA program has been established to ensure-

(1) The accuracy and precision of the meas-urements.

(2) That the results obtained by all testing sites are comparable.

1-5. Functions

a. The USACHPPM CRL-

(1) Performs RBC-ChE QA oversight for nerve agent stockpile and demilitarization sites.

(2) Provides limited primary RBC-ChE test-ing services to DOD organizations and DOD con-tractors.

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( 3 ) Evaluates and approves testing site lab-

(4) Provides training to testing site laboratory

( 5 ) Provides select testing equipment to test-

(6) Provides standing operating procedures oratories’ requests for RBC-ChE testing.

staff.

ing sites on a loan basis.

pertinent to RBC-ChE testing.

b. RBC-ChE testing sites-

(1) Request admission to the program

(2) Provide qualified technicians to the CRL

(3) Dedicate adequate testing facilities and from the CRL.

for training.

supplies.

(4) Maintain test equipment on loan from the

CRL.

( 5 ) Submit RBC-ChE specimens with appro-priate patient information to the CRL in a timely manner.

c. Primary sites submit RBC-ChE specimens with appropriate patient information to the CRL in a timely manner.

1-6. Technical assistance To contact the CRL on safety or other issues, write to Commander, U.S. Army Center for Health Pro-motion and Preventive Medicine, 5158 Blackhawk Road, ATTN: MCHB-TS-LRD, Aberdeen Proving Ground, MD 21010-5403.

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TB 590

CHAPTER 2

RBC-ChE QUALITY ASSURANCE PROGRAM

2-1. Authority DA Pam 40-8 authorizes the QA program for RBC-ChE testing at nerve agent stockpile and de-militarization.

2-2. Background To ensure a consistent and reliable ChE testing environment within DOD, the CRL provides QA services and oversight for the RBC-ChE testing program. This program verifies performance for each testing site.

2-3. Program participation Eligible laboratories engaged in RBC-ChE testing within the DOD RBC-ChE surveillance mission, must participate in the QA program. To join the RBC-ChE testing program, testing site labora-tories should contact the CRL. The following re-quirements must be fulfilled before new labora-tories can be accepted as RBC-ChE testing sites.

a. Laboratory personnel must meet the stand-ards for high complexity testing set forth by the DOD Clinical Laboratory Improvement Program (DOD CLIP) in the Armed Forces Institute of Pa-thology (AFIP) Pamphlet 40-24.

b. Laboratories performing RBC-ChE testing must be CLIP or Clinical Laboratory Improvement Amendments (CLIA) registered and accredited by either Commission on Office Laboratory Accredita-tion (COLA), College of American Pathologists (CAP), or Joint Commission on Accreditation of Healthcare Organizations (JCAHO).

c. New laboratories should submit a written jus-tification to USACHPPM CRL explaining the ex-pected duration of RBC-ChE testing needed and an approximate number of blood specimens ex-pected to be processed.

d. The CRL will assign an account number to each laboratory that participates in the DOD RBC-ChE monitoring program. This account num-ber must accompany each specimen submitted to the CRL to ensure proper processing of specimens.

The program manager for the submitting labora-tory will be responsible for providing the CRL with the account information. Reimbursement for CRL services will be determined on an individual basis.

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However, services provided to military laboratories are generally reimbursable if not covered by cen-tral funding.

2-4. CRL services The CRL will provide the following services:

a. Training and certification i n the Manual 17- Minute Change in p H Method. The initial techni-cian training consists of a 40-hour training block at the CRL, which includes hands-on experience in ChE determination, operation and maintenance of ChE testing equipment, and QC procedures. Upon successful completion of this training, technicians will receive a certificate. Annual technician recer-tification will be given based on ChE testing per-formance during the previous year and perform-ance assessed during site visits.

b. Quarterly proficiency surveys of participating laboratories. Blind specimens will be sent to test-ing sites on a quarterly basis. Results from all the participating laboratories will be compiled, and in-dividual sites must stay within 2 standard devi-ations of the population mean.

c. RBC-ChE testing performance monitoring.

(1) After completion of onsite testing, the test-ing sites will send all specimens to the CRL. To ensure continued satisfactory testing performance, 20 to 30 percent of specimens from testing sites will be retested at the CRL. The results from test-ing sites must fall within 0.05 pH units, when compared to the CRL results obtained on the same specimen, to meet acceptability criteria.

(2) The CRL will provide the testing site with a written report of the comparative test. If results do not meet acceptability criteria, the CRL staff will remotely aid (that is, telephone and or email) the testing sites in troubleshooting. If remote trou-bleshooting does not solve the problem, the CRL will conduct an onsite visit. The expenses for this visit are the responsibility of the testing site.

d. Onsite laboratory audits. The CRL will con-duct annual audits to assure testing site oper-ations are in compliance with DOD RBC-ChE pro-gram standards. If laboratories show poor perform-ance, the CRL may conduct audits more fre-quently. The CRL will furnish the laboratories an assessment report summarizing any deficiencies found during the audits. Laboratories must correct the deficiencies and submit a written report of cor-rective actions taken to the CRL within 60 days after the audit.

e. QC specimens. The CRL will provide QC. Test-ing sites must keep QC charts for control results, and all corrective actions taken for controls that do not meet acceptability criteria will be logged on QC charts.

f: Calibration of equipment. All equipment used for quantitative analysis or the purpose of col-lecting quantitative data must be calibrated in ac-cordance with CRL or local SOPs. If available, Na-tional Institute of Standards and Testing (NIST) traceable reference materials must be used for calibration.

2-5. Requesting RBC-ChE analysis Blood specimens submitted for RBC-ChE’ testing should be accompanied by a request memorandum containing the following information:

a. Patient name.

b. Age.

c. SSN.

d. Type of test ordered.

e. Name and signature of physician ordering the f: Date test ordered.

test.

g. Patient’s baseline (if available) and category.

h. Date and time specimen was taken.

i. Address where test results are to be reported.

2-6. Submitting specimens for analysis to CRL Specimens will be submitted to the CRL per

USACHPPM SOP CRL 40-10.

2-7. Reporting test results To ensure effective ChE health monitoring, testing laboratories will-

a. Provide a written report of the results for pri-mary RBC-ChE determinations within 72 hours of specimen receipt for category A personnel and within 1 week of specimen receipt for all other cat-egories (B, C, and D).

b. Inform the referring health care provider or installation medical authority (IMA) immediately if RBC-ChE depressions equal to or greater than 25 percent of baseline are detected.

c. Refer variations of greater than 10 percent from baseline value (low or high) to the IMA for further analysis.

2-8. Program procedures Detailed procedures for the QA program may be found in USACHPPM SOPs CRL 40-9, 40-13, and 40-15.

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CHAPTER 3

PRIMARY ChE TESTING

3-1. Authority DA Pam 40-8 authorizes limited primary ChE testing for DOD agencies and contractors.

3-2. Background To provide ChE testing capability for Government sites and Government contractors without onsite testing facilities, the CRL performs ChE deter-minations for specimens submitted to the CRL on a limited basis.

3-3. Program participation

a. New customers need to provide the CRL with

(1) Written justification of the service needed

(2) A point of contact for administrative

(3) A complete mailing address for test results.

b. The CRL will assign an account number to all customers participating in the primary testing pro-gram. This account number must accompany all primary specimens submitted to the CRL to en-sure proper processing of specimens. The program manager for the submitting site will be responsible the following information:

and the expected duration of this service.

issues.

for providing the CRL with the account informa-tion. In general, services provided to civilian con-tractors are reimbursable.

Submitting primary specimens to

CRL

Specimens should be submitted t o the CRL per USACHPPM SOP CRL 40-10. Specimens must be submitted with the following information:

a. Patient name.

b. Age.

c. SSN.

d. Type of test ordered.

e. Name and signature of physician ordering the

Date test ordered.

g. Patient’s baseline (if available) and category.

h. Date and time specimen was taken.

i. Address where test results are to be reported.

test.

3-5. Reporting test results The CRL will furnish a written report with the ChE test results to the submitting site within 3 days of receipt of specimen.

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CHAPTER 4

PROCEDURES FOR PERFORMING RBC-ChE TESTING

4-1. Purpose This chapter provides and explains the procedures for-

a. Collecting and processing blood specimens.

b. Preparing reagents.

c. Analyzing blood specimens.

d. Using equipment and materials in the man-

e. Using the reference range.

Determining the initial RBC-ChE activity baseline value.

4-2. Safety and administrative proce-dures Follow all standard laboratory safety and adminis-trative procedures, to include-

a. SOPs recommended by the CAP, COLA, and

b. Local bloodborne pathogens exposure control ual method.

e. Part 1910.1030, title 29, Code of Federal Reg-

Volume 56, Federal Register, p. 235.

g. Part 1910.1200, title 29, Code of Federal Reg-ulations (CFR), 1991.

ulations.

4-3. Blood specimen collection

a. Collect blood specimens per USACHPPM SOP

CRL 40-10 and local SOPs on phlebotomy.

b. Centrifuge the specimen t o separate the RBCs from the plasma and remove the plasma within 4 hours of collection.

Reagent preparation

a. All reagents will be prepared per USACHPPM

SOP CRL 40-2. All chemicals and solutions must be labeled with dates received or prepared, date opened and expiration date, and must be. stored properly.

b. The following solutions are required for RBC- ChE testing:

(1) Acetylcholine bromide solution for enzyme

(2) Sodium barbital (0.01 M) substrate, 0.11 moles per liter

(0.002 M) stock buffer, pH 8.19-8.25.

(3) Sodium barbital (0.01 M) -phosphate (0.002 M) -saponin (0.015 % weight per volume) working buffer, pH 8.05-8.10.

4-5. Blood specimen analysis

a. The RBC-ChE assay currently in use is based on a modification (R.I. Ellin, et al., 1973) of a potentiometric assay method developed by H.O.

Michel (1949). The enzyme activity is expressed in units of change in pH per hour

b. To set up the standard equipment and per-form the enzyme assay-

(1) Prepare the water bath, pH meter, and dual syringe dilutor per USACHPPM SOPs CRL 40-5, CRL 40-6, and CRL 40-3.

(2) Perform a balance verification on the ana-lytical balance. Refer to USACHPPM SOP CRL 40-9 and the operator’s manual for this procedure.

(3) Equilibrate the blood specimens and re-agents. Allow all specimens, reagents, and controls to equilibrate t o room temperature.

(4) Prepare the working buffer t o be used in this method.

(5) Prepare the RBC-ChE data runsheet.

(6) Prepare specimens per USACHPPM SOP

(7) Perform the enzyme assay per

(8) Calculate the RBC-ChE activity in units

CRL 40-2.

USACHPPM SOP CRL 40-2.

of per USACHPPM SOP CRL 40-2.

4-6. Instruments and equipment re-quired to perform RBC-ChE assays To ensure accurate results and comparability of re-sults between laboratories, equipment for RBC- ChE testing must be standardized.

a. The CRL, on a loan basis, will provide the fol-lowing equipment:

(1) pH meter.

(2) Dual syringe diluter.

(3) Constant temperature water bath.

b. This equipment will remain the property of the CRL and will not be transferred t o any testing facility property book. The equipment will be tem-porarily loaned to testing sites on DA Form 2062 (Hand Number).

c. Each testing site will receive two sets of the equipment items mentioned in paragraph a above.

One equipment item will be in use and the other

4-1 will be as a back up. In the event of equipment failure, the testing facility must coordi-nate with the CRL promptly to arrange for repair or replacement. Only user maintenance is author-ized. All other repairs must be coordinated and ap-proved by the CRL.

d. Costs for the repair and replacement of equip-ment will be paid by the CRL. Shipping costs are the responsibility of the testing facility.

e. The CRL will provide operator’s manuals and for each item.

A list of recommended equipment and consumables is available from the CRL. Each lab-oratory is responsible for purchasing and main-taining these items.

4-7. Reference range The reference range for RBC-ChE activities is 0.63-0.89 This reference range should only be used in an emergency in the absence of patient baseline values.

4-8. Baseline determination RBC-ChE baseline levels will be determined per DA Pam 40-8.

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APPENDIX A

REFERENCES

A-1. Department of Defense Publications

AFIP Pamphlet 40-24, Technical Instructions for the Department of Defense Clinical Laboratory Improve-ment Program. (Available from Armed Forces Institute of Pathology, 6825 16th Street NW, ATTN:

CLIP Office, Washington, DC 20306-6000.)

A-2. Department of the Army Publications

DA Pam 40-8, Occupational Health Guidelines for the Evaluation and Control of Occupational Exposure to Nerve Agents GA, GB, GD, and VX

A-3. Cholinesterase Reference Laboratory Standing Operating Procedures (Available from

CRL 40-2, 17-Minute RBC Acetylcholinesterase Procedure CRL 40-3, Operation of the Dual Syringe Diluter CRL 40-4, Calibration of the Substrate Repipet CRL 40-5, Operation of Precision Circulating Water Bath CRL 40-6, Operation of Models 345 and 445 pH Meters CRL 40-8, Operation of the Virtis Unitop Model 600SL Freeze Dryer CRL 40-9, Quality Assurance Program

USACHPPM, 5158 Blackhawk Road, Aberdeen Proving Ground, M D 21010-5403)

CRL 40-10, Sample Collection, Processing, Shipping and Receiving CRL 40-11, Bloodborne Pathogens Exposure Control CRL 40-13, Preparation of Samples CRL 40-14, Data Entry CRL 40-15, Proficiency Testing

Other publications

Ellin R.I., Burkhardt B.H., and Hart R.D. (1973), A Cholinesterase Activity. Arch. Environ. Health 27:48-49

Method for Measuring Red Blood Cell

Michel, H.O. (1949), An Electrometric Method for the Determination of Red Blood Cell and Plasma Cholin-esterase Activity. J. Lab. Clinic. Med. 34: 1564-1568

29 CFR 1910, Occupational Safety and Health Standards r

A-5. Referenced forms

DA Form 2062 Hand Receipt/Annex Number

A-1

GLOSSARY

Section ABBREVIATIONS

AFIP

Armed Forces Institute of Pathology

CAP

College of American Pathologists

CAS

Chemical Abstract Service

CFR

Code of Federal Regulations

ChE cholinesterase

CLIA

Clinical Laboratory Improvement Amendments

CLIP

Clinical Laboratory Improvement Program

COLA

Commission on Office Laboratory Accreditation

CRL

Cholinesterase Reference Laboratory

DOD

Department of Defense

FR

Federal Register

IMA

installation medical authority

JCAHO

Joint Commission on Accreditation of Healthcare Organizations

M moles per liter

MTF

Military Treatment Facility

Glossary-1

NIST

National Institute of Standards and Testing change in pH per hour

QA

quality assurance

QC

quality control

RBC

red blood cell

SF

standard form

SOP

standing operating procedure

USACHPPM

U.S. Army Center for Health Promotion and Preventive Medicine

Section II. TERMS

Agent GA The chemical dimethylphosphoramido-cyanidate, Chemical Abstracts Service (CAS) registry number 77-

81-6, in pure form and in the various impure forms which may be found in storage as well as in indus-trial, depot, or laboratory operations.

Agent GB The chemical isopropyl registry number in pure form and in the various impure forms which may be found in storage as well as in industrial, depot, or laboratory operations.

Agent GD The chemical phosphonofluoridic acid, methyl-1, 2, 2-trimethylpropyl ester, CAS registry number 96-64-

0, in pure form and in the various impure forms which may be found in storage as well as in indus-trial, depot, or laboratory operations.

Agent VX The chemical phosphonothioic acid, methyl-, S- (2- (bis amino) ethyl) O-ethyl ester, CAS registry number 50782-69-9, in pure form and in the various impure forms which may be found in storage as well as in industrial, depot, or laboratory operations.

Category A personnel Personnel with a high risk of potential exposure due to the nature of the agent operations being conducted.

(See DA Pam 40-8, para 4-2.)

Category B personnel Personnel with a low risk or infrequent potential exposure t o nerve agents in routine industrial, labora-tory, or security operations and job requirements involving the prolonged wearing of protective ensem-bles during training and emergency responses. (See DA Pam 40-8, para 4-2.)

Category C personnel Personnel with minimal probability of exposure to nerve agents even under accident conditions, but whose activities may place them in close proximity to agent areas. (See DA Pam 40-8, para 4-2.)

Category D personnel Transient visitors to agent areas where there is a potential for exposure and who are not included in the medical surveillance program for nerve agents at the visited installation. (See DA Pam 40-8, para 4- 2.)

Data runsheet Worksheet that lists all the specimens analyzed during a single assay run that is used for entering raw data and computing RBC-ChE results.

Primary site Any site submitting an RBC-ChE specimen to the CRL for determination of RBC-ChE enzyme activity and which does not have onsite RBC-ChE testing capability.

Testing site Any RBC-ChE testing location that is currently certified by the RBC-ChE program and is partici-pating in the RBC-ChE QA program.

Glossary3

By Order of the Secretary of the Army:

Official:

JOEL B. HUDSON

Administrative Assistant to the

Secretary of the Army

TB MED 590

ERIC K. SHINSEKI

General, United States Army

Chief of Staff

Distribution:

MED 590.

To be distributed in accordance with Initial Distribution Number (IDN) 341762, requirements for TB r

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