Red Blood Cell-Cholinesterase Testing and Quality Assurance

16 Pages·2001·0.61 MB·English

by  Department of the Army

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 OF THE A R M Y
30 NOVEMBER 2001
MED 590
TECHNICABLU LLETIN
DEPARTMENT OF THE ARMY
No. MED 590 WASHINGTON7 DC, 30 NOVEMBER 2001
RED BLOOD CELL-CHOLINESTERASE TESTING AND QUALITY ASSURANCE
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APPROVED FOR PUBLIC RELEASE;
DISTRIBUTION IS UNLIMITED
Page
CHAPTER1 . INTRODUCTION
1-1 1-1
1-2 1-1
1-3 1-1
1-4 1-1
1-5 1-1
1-6 1-2
2-1 2-1
2-2 2-1
2-3 2-1
2-4 2-1
2-5 2-2
2-6 2-2
Reporting test results 2-7 2-2
Program procedures 2-8 2-2
CHAPTER3. PRIMARY ChE TESTING
Authority 3-1 3-1
Background 3-2 3-1
Program participation 3-3 3-1
Submitting primary sp to CRL 3-4 3-1
Reporting test results 3-5 3-1
4. PROCEDURES FOR PERFORMING RBC-ChE TESTING
Purpose 4-1 4-1
Safety and 4-2 4-1
Blood specimen collection 4-3 4-1
Reagent preparation 4-4 4-1
Blood specimen analysis 4-5 4-1
Instruments and equipment req 4-6 4-1
Reference range 4-7 4-2
Baseline determination 4-8 4-2
APPENDIXA . REFERENCES A-1
Glossary Glossary-1
"This bulletin supersedes TB MED 292, 30 May 1975.
TB MED 590
CHAPTER 1
INTRODUCTION
1-1. Purpose 1-2. References
This bulletin-
Appendix A lists the references used in this bul-
a. Describes the methodology for measuring red letin.
blood cell cholinesterase (RBC-ChE) activity in the
Department of Defense (DOD) Cholinesterase
1-3. Abbreviations and terms
Monitoring Program.
b. Provides information to- The glossary provides a list of abbreviations and
terms used in this bulletin.
(1) Installation commanders who have a ChE-
inhibiting substance mission.
(2) Health care providers who are responsible 1-4. Background
for evaluating RBC-ChE depressions and for
a. Cholinesterase testing is an essential element
maintaining the RBC-ChE test records. (See DA
in the medical surveillance of chemical agent and
Pam 40-8.)
pesticide workers. The enzyme ChE hydrolyzes
(3) Laboratory personnel responsible for con-
acetylcholine resulting in the release of acetic acid
ducting RBC-ChE assays.
and choline. This mechanism is important for the
c. Describes the controls and standards included breakdown of the neurotransmitter acetylcholine
in the DOD laboratory test method. to retain normal function of nervous, muscle, and
d. Describes the procedures for- gland tissues. Acetylcholine inhibiting compounds,
such as the nerve agents GA, GB, GD, VX and
(1) Requesting RBC-ChE assay analysis.
organophosphorous insecticides, bind to ChE, ren-
(2) Collecting and processing blood specimens.
dering the enzyme inactive. This inhibition will re-
(3) Completing test results.
sult in concentration dependent compromise of
(4) Reporting test results. nervous system function, which can eventually
e. Describes the procedures used by the Cholin- lead to death.
esterase Reference Laboratory (CRL) at the U.S. b. The RBC-ChE is a representative of the true
Army Center for Health Promotion and Preventive ChE of the nervous system. Exposure to ChE-in-
Medicine (USACHPPM) to- hibiting compounds, even at sub-clinical levels, can
(1) Support standardized testing require- be detected by monitoring RBC-ChE activity. Ex-
ments. posure to inhibitors is detected by comparing an
(2) Provide limited primary testing. individual's RBC-ChE activity level to a pre-
viously established baseline level and checking for
(3) Establish and maintain quality control
a depression in enzyme activity.
(QC) and quality assurance (QA) procedures.
c. The DOD has adopted the use of a single
(4) Maintain assay performance standardiza-
standardized method for RBC-ChE testing: the
tion by-
Manual 17-Minute Change in pH Method. A QA
(a)S pecifying procedures, reagents, and in-
program has been established to ensure-
strumentation.
(1) The accuracy and precision of the meas-
(b) Providing technical training and consulta-
urements.
tion.
(2) That the results obtained by all testing
(c) laboratory technicians.
sites are comparable.
(5) Maintain transferability of test results
throughout the program by performing- 1-5. Functions
(a)T est result validation by representative a. The USACHPPM CRL-
specimen retesting. (1) Performs RBC-ChE QA oversight for
(b) Statistical analysis of test-retest dif- nerve agent stockpile and demilitarization sites.
ferences. (2) Provides limited primary RBC-ChE test-
(6) Conduct onsite laboratory audits at least ing services to DOD organizations and DOD con-
annually. tractors.
1-1
TB MED 590
(3) Evaluates and approves testing site lab- (4) Maintain test equipment on loan from the
oratories’ requests for RBC-ChE testing. CRL.
(4) Provides training to testing site laboratory (5) Submit RBC-ChE specimens with appro-
staff. priate patient information to the CRL in a timely
(5) Provides select testing equipment to test- manner.
ing sites on a loan basis. c. Primary sites submit RBC-ChE specimens
(6) Provides standing operating procedures with appropriate patient information to the CRL
pertinent to RBC-ChE testing. in a timely manner.
b. RBC-ChE testing sites-
1-6. Technical assistance
(1) Request admission to the program
from the CRL. To contact the CRL on safety or other issues, write
to Commander, U.S. Army Center for Health Pro-
(2) Provide qualified technicians to the CRL
for training. motion and Preventive Medicine, 5158 Blackhawk
Road, ATTN: MCHB-TS-LRD, Aberdeen Proving
(3) Dedicate adequate testing facilities and
Ground, MD 21010-5403.
supplies.
1-2
TB 590
CHAPTER 2
RBC-ChE QUALITY ASSURANCE PROGRAM
2-1. Authority However, services provided to military laboratories
are generally reimbursable if not covered by cen-
DA Pam 40-8 authorizes the QA program for
tral funding.
RBC-ChE testing at nerve agent stockpile and de-
militarization.
2-4. CRL services
2-2. Background The CRL will provide the following services:
a. Training and certification in the Manual 17-
To ensure a consistent and reliable ChE testing
Minute Change in pH Method. The initial techni-
environment within DOD, the CRL provides QA
cian training consists of a 40-hour training block
services and oversight for the RBC-ChE testing
at the CRL, which includes hands-on experience in
program. This program verifies performance for
ChE determination, operation and maintenance of
each testing site.
ChE testing equipment, and QC procedures. Upon
successful completion of this training, technicians
2-3. Program participation
will receive a certificate. Annual technician recer-
Eligible laboratories engaged in RBC-ChE testing tification will be given based on ChE testing per-
within the DOD RBC-ChE surveillance mission, formance during the previous year and perform-
must participate in the QA program. To join the ance assessed during site visits.
RBC-ChE testing program, testing site labora- b. Quarterly proficiency surveys of participating
tories should contact the CRL. The following re- laboratories. Blind specimens will be sent to test-
quirements must be fulfilled before new labora- ing sites on a quarterly basis. Results from all the
tories can be accepted as RBC-ChE testing sites. participating laboratories will be compiled, and in-
a. Laboratory personnel must meet the stand- dividual sites must stay within 2 standard devi-
ards for high complexity testing set forth by the ations of the population mean.
DOD Clinical Laboratory Improvement Program c. RBC-ChE testing performance monitoring.
(DOD CLIP) in the Armed Forces Institute of Pa- (1) After completion of onsite testing, the test-
thology (AFIP) Pamphlet 40-24. ing sites will send all specimens to the CRL. To
b. Laboratories performing RBC-ChE testing ensure continued satisfactory testing performance,
must be CLIP or Clinical Laboratory Improvement 20 to 30 percent of specimens from testing sites
Amendments (CLIA) registered and accredited by will be retested at the CRL. The results from test-
either Commission on Office Laboratory Accredita- ing sites must fall within 0.05 pH units, when
tion (COLA), College of American Pathologists compared to the CRL results obtained on the same
(CAP), or Joint Commission on Accreditation of specimen, to meet acceptability criteria.
Healthcare Organizations (JCAHO). (2) The CRL will provide the testing site with
c. New laboratories should submit a written jus- a written report of the comparative test. If results
tification to USACHPPM CRL explaining the ex- do not meet acceptability criteria, the CRL staff
pected duration of RBC-ChE testing needed and will remotely aid (that is, telephone and or email)
an approximate number of blood specimens ex- the testing sites in troubleshooting. If remote trou-
pected to be processed. bleshooting does not solve the problem, the CRL
d. The CRL will assign an account number to will conduct an onsite visit. The expenses for this
each laboratory that participates in the DOD visit are the responsibility of the testing site.
RBC-ChE monitoring program. This account num- d. Onsite laboratory audits. The CRL will con-
ber must accompany each specimen submitted to duct annual audits to assure testing site oper-
the CRL to ensure proper processing of specimens. ations are in compliance with DOD RBC-ChE pro-
The program manager for the submitting labora- gram standards. If laboratories show poor perform-
tory will be responsible for providing the CRL with ance, the CRL may conduct audits more fre-
the account information. Reimbursement for CRL quently. The CRL will furnish the laboratories an
services will be determined on an individual basis. assessment report summarizing any deficiencies
2-1
TB MED 590
found during the audits. Laboratories must correct g. Patient’s baseline (if available) and category.
the deficiencies and submit a written report of cor- h. Date and time specimen was taken.
rective actions taken to the CRL within 60 days
i. Address where test results are to be reported.
after the audit.
e. QC specimens. The CRL will provide QC. Test- 2-6. Submitting specimens for
ing sites must keep QC charts for control results, analysis to CRL
and all corrective actions taken for controls that do
Specimens will be submitted to the CRL per
not meet acceptability criteria will be logged on
USACHPPM SOP CRL 40-10.
QC charts.
f: Calibration of equipment. All equipment used 2-7. Reporting test results
for quantitative analysis or the purpose of col- To ensure effective ChE health monitoring, testing
lecting quantitative data must be calibrated in ac- laboratories will-
cordance with CRL or local SOPs. If available, Na- a. Provide a written report of the results for pri-
tional Institute of Standards and Testing (NIST)
mary RBC-ChE determinations within 72 hours of
traceable reference materials must be used for
specimen receipt for category A personnel and
calibration.
within 1 week of specimen receipt for all other cat-
egories (B, C, and D).
2-5. Requesting RBC-ChE analysis
b. Inform the referring health care provider or
installation medical authority (IMA) immediately
Blood specimens submitted for RBC-ChE’ testing
if RBC-ChE depressions equal to or greater than
should be accompanied by a request memorandum
25 percent of baseline are detected.
containing the following information:
a. Patient name. c. Refer variations of greater than 10 percent
from baseline value (low or high) to the IMA for
b. Age.
further analysis.
c. SSN.
d. Type of test ordered. 2-8. Program procedures
e. Name and signature of physician ordering the Detailed procedures for the QA program may be
test. found in USACHPPM SOPs CRL 40-9, 40-13, and
f: Date test ordered. 40-15.
2-2
TB MED 590
CHAPTER 3
PRIMARY ChE TESTING
3-1. Authority for providing the CRL with the account informa-
DA Pam 40-8 authorizes limited primary ChE tion. In general, services provided to civilian con-
tractors are reimbursable.
testing for DOD agencies and contractors.
3-2. Background Submitting primary specimens to
CRL
To provide ChE testing capability for Government
sites and Government contractors without onsite Specimens should be submitted to the CRL per
testing facilities, the CRL performs ChE deter- USACHPPM SOP CRL 40-10. Specimens must be
minations for specimens submitted to the CRL on submitted with the following information:
a limited basis. a. Patient name.
b. Age.
3-3. Program participation
c. SSN.
a. New customers need to provide the CRL with
the following information: d. Type of test ordered.
(1) Written justification of the service needed e. Name and signature of physician ordering the
and the expected duration of this service. test.
(2) A point of contact for administrative Date test ordered.
issues. g. Patient’s baseline (if available) and category.
(3)A complete mailing address for test results. h. Date and time specimen was taken.
b. The CRL will assign an account number to all i. Address where test results are to be reported.
customers participating in the primary testing pro-
3-5. Reporting test results
gram. This account number must accompany all
primary specimens submitted to the CRL to en- The CRL will furnish a written report with the
sure proper processing of specimens. The program ChE test results to the submitting site within 3
manager for the submitting site will be responsible days of receipt of specimen.
3-1
TB MED 590
CHAPTER 4
PROCEDURES FOR PERFORMING RBC-ChE TESTING
4-1. Purpose (3) Sodium barbital (0.01 M) -phosphate
(0.002 M) -saponin (0.015 % weight per volume)
This chapter provides and explains the procedures
working buffer, pH 8.05-8.10.
for-
a. Collecting and processing blood specimens. 4-5. Blood specimen analysis
b. Preparing reagents. a. The RBC-ChE assay currently in use is based
c. Analyzing blood specimens. on a modification (R.I. Ellin, et al., 1973) of a
potentiometric assay method developed by H.O.
d. Using equipment and materials in the man-
Michel (1949). The enzyme activity is expressed in
ual method.
units of change in pH per hour
e. Using the reference range.
b. To set up the standard equipment and per-
Determining the initial RBC-ChE activity form the enzyme assay-
baseline value.
(1) Prepare the water bath, pH meter, and
4-2. Safety and administrative proce- dual syringe dilutor per USACHPPM SOPs CRL
dures 40-5, CRL 40-6, and CRL 40-3.
(2) Perform a balance verification on the ana-
Follow all standard laboratory safety and adminis-
lytical balance. Refer to USACHPPM SOP CRL
trative procedures, to include-
40-9 and the operator’s manual for this procedure.
a. SOPs recommended by the CAP, COLA, and
(3) Equilibrate the blood specimens and re-
agents. Allow all specimens, reagents, and controls
b. Local bloodborne pathogens exposure control to equilibrate to room temperature.
(4) Prepare the working buffer to be used in
this method.
(5) Prepare the RBC-ChE data runsheet.
e. Part 1910.1030, title 29, Code of Federal Reg- (6) Prepare specimens per USACHPPM SOP
ulations (CFR), 1991. CRL 40-2.
Volume 56, Federal Register, p. 235. (7) Perform the enzyme assay per
g. Part 1910.1200, title 29, Code of Federal Reg- USACHPPM SOP CRL 40-2.
ulations. (8) Calculate the RBC-ChE activity in units
of per USACHPPM SOP CRL 40-2.
4-3. Blood specimen collection
4-6. Instruments and equipment re-
a. Collect blood specimens per USACHPPM SOP
quired to perform RBC-ChE assays
CRL 40-10 and local SOPs on phlebotomy.
To ensure accurate results and comparability of re-
b. Centrifuge the specimen to separate the RBCs
sults between laboratories, equipment for RBC-
from the plasma and remove the plasma within 4
ChE testing must be standardized.
hours of collection.
a. The CRL, on a loan basis, will provide the fol-
Reagent preparation
lowing equipment:
a. All reagents will be prepared per USACHPPM (1) pH meter.
SOP CRL 40-2. All chemicals and solutions must (2) Dual syringe diluter.
be labeled with dates received or prepared, date
(3) Constant temperature water bath.
opened and expiration date, and must be. stored
b. This equipment will remain the property of
properly.
the CRL and will not be transferred to any testing
b. The following solutions are required for RBC-
facility property book. The equipment will be tem-
ChE testing:
porarily loaned to testing sites on DA Form 2062
(1) Acetylcholine bromide solution for enzyme (Hand Number).
substrate, 0.11 moles per liter c. Each testing site will receive two sets of the
(2) Sodium barbital (0.01 M) equipment items mentioned in paragraph a above.
(0.002 M) stock buffer, pH 8.19-8.25. One equipment item will be in use and the other
4-1
TB MED 590
will be as a back up. In the event of oratory is responsible for purchasing and main-
equipment failure, the testing facility must coordi- taining these items.
nate with the CRL promptly to arrange for repair
or replacement. Only user maintenance is author- 4-7. Reference range
ized. All other repairs must be coordinated and ap-
The reference range for RBC-ChE activities is
proved by the CRL.
0.63-0.89 This reference range should only
d. Costs for the repair and replacement of equip-
be used in an emergency in the absence of patient
ment will be paid by the CRL. Shipping costs are
baseline values.
the responsibility of the testing facility.
e. The CRL will provide operator’s manuals and
4-8. Baseline determination
for each item.
A list of recommended equipment and RBC-ChE baseline levels will be determined per
consumables is available from the CRL. Each lab- DA Pam 40-8.
4-2
TB MED 590
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,A TTN:
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
USACHPPM, 5158 Blackhawk Road, Aberdeen Proving Ground, MD 21010-5403)
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
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 Method for Measuring Red Blood Cell
Cholinesterase Activity. Arch. Environ. Health 27:48-49
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
r 29 CFR 1910, Occupational Safety and Health Standards
A-5. Referenced forms
DA Form 2062
Hand Receipt/Annex Number
A-1