California Code of Regulations › Title 8 - Industrial Relations › Division 1 - Department of Industrial Relations › Chapter 4 - Division of Industrial Safety › Subchapter 7 - General Industry Safety Orders › Group 16 - Control of Hazardous Substances › Article 110 - Regulated Carcinogens › Appendix F - Nonmandatory Protocol for Biological Monitoring

Current through Register 2024 Notice Reg. No. 28, July 12, 2024
1.0 INTRODUCTION
Under the final OSHA cadmium rule, monitoring of
						  biological specimens and several periodic medical examinations are required for
						  eligible employees. These medical examinations are to be conducted regularly,
						  and medical monitoring is to include the periodic analysis of cadmium in blood
						  (CDB), cadmium in urine (CDU) and beta-2-microglobulin in urine (B2MU). As CDU
						  and B2MU are to be normalized to the concentration of creatinine in urine
						  (CRTU), then CRTU must be analyzed in conjunction with CDU and B2MU
						  analyses.
The purpose of this protocol is to provide procedures
						  for establishing and maintaining the quality of the results obtained from the
						  analyses of CDB, CDU and B2MU by commercial laboratories. Laboratories
						  conforming to the provisions of this nonmandatory protocol shall be known as
						  "participating laboratories." The biological monitoring data from these
						  laboratories will be evaluated by physicians responsible for biological
						  monitoring to determine the conditions under which employees may continue to
						  work in locations exhibiting airborne- cadmium concentrations at or above
						  defined actions levels (see paragraphs (1)(3) and (1)(4) of the final rule).
						  These results also may be used to support a decision to remove workers from
						  such locations.
Under the medical monitoring program for cadmium, blood
						  and urine samples must be collected at defined intervals from workers by
						  physicians responsible for medical monitoring; these samples are sent to
						  commerical laboratories that perform the required analyses and report results
						  of these analyses to the responsible physicians. To ensure the accuracy and
						  reliability of these laboratory analyses, the laboratories to which samples are
						  submitted should participate in an ongoing and efficacious proficiency testing
						  program. Availability of proficiency testing programs may vary with the
						  analyses performed.
To test proficiency in the analysis of CDB, CDU and
						  B2MU, a laboratory should participate either in the interlaboratory comparison
						  program operated by the Centre de Toxicologie du Quebec (CTQ) or an equivalent
						  program. (Currently, no laboratory in the U.S. performs proficiency testing on
						  CDB, CDU or B2MU.) Under this program, CTQ sends participating laboratories 18
						  samples of each analyte (CDB, CDU and/or B2MU) annually for analysis.
						  Participating laboratories must return the results of these analyses to CTQ
						  within four to five weeks after receiving the samples.
The CTQ program pools analytical results from many
						  participating laboratories to derive consensus mean values for each of the
						  samples distributed. Results reported by each laboratory then are compared
						  against these consensus means for the analyzed samples to determine the
						  relative performance of each laboratory. The proficiency of a participating
						  laboratory is a function of the extent of agreement between results submitted
						  by the participating laboratory and the consensus values for the set of samples
						  analyzed.
Proficiency testing for CRTU analysis (which should be
						  performed with CDU and B2MU analyses to evaluate the results properly) also is
						  recommended. In the U.S., only the College of American Patholgists (CAP)
						  currently conducts CRTU proficiency testing; participating laboratories should
						  be accredited for CRTU analysis by the CAP.
Results of the proficiency evaluations will be
						  forwarded to the participating laboratory by the proficiency-testing
						  laboratory, as well as to physicians designated by the participating laboratory
						  to receive this information. In addition, the participating laboratory should,
						  on request, submit the results of their internal Quality Assurance/Quality
						  Control (QA/QC) program for each analytic procedure (i.e., CDB, CDU and/or
						  B2MU) to physicians designated to receive the proficiency results. For
						  participating laboratories offering CDU and/or B2MU analyses, QA/QC
						  documentation also should be provided for CRTU analysis. (Laboratories should
						  provide QA/QC information regarding CRTU analysis directly to the requesting
						  physician if they perform the analysis in-house; if CRTU analysis is performed
						  by another laboratory under contract, this information should be provided to
						  the physician by the contract laboratory.)
QA/QC information, along with the actual biological
						  specimen measurements, should be provided to the responsible physician using
						  standard formats. These physicians then may collate the QA/QC information with
						  proficiency test results to compare the relative performance of laboratories,
						  as well as to facilitate evaluation of the worker monitoring data. This
						  information supports discretionary decisions made by the physician with regard
						  to the biological monitoring program, and for mandating medical removal.
This protocol describes procedures that may be used by
						  the responsible physicians to identify laboratories most likely to be
						  proficient in the analysis of samples used in the biological monitoring of
						  cadmium; also provided are procedures for record keeping and reporting by
						  laboratories participating in proficiency testing programs, and recommendations
						  to assist these physicians in interpreting analytical results determined by
						  participating laboratories. As the collection and handling of samples affects
						  the quality of the data, recommendations are made for these tasks.
						  Specifications for analytical methods to be used in the medical monitoring
						  program are included in this protocol as well.
In conclusion, this document is intended as a
						  supplement to characterize and maintain the quality of medical monitoring data
						  collected under the final cadmium rule promulgated by OSHA. OSHA has been
						  granted authority under the Occupational Safety and Health Act of 1970 to
						  protect workers from the effects of exposure to hazardous substances in the
						  work place and to mandate adequate monitoring of workers to determine when
						  adverse health effects may be occurring. This nonmandatory protocol is intended
						  to provide guidelines and recommendations to improve the accuracy and
						  reliability of the procedures used to analyze the biological samples collected
						  as part of the medical monitoring program for cadmium.
2.0 DEFINITIONS
When the terms below appear in this protocol, use the
						  following definitions.
Accuracy:
A measure of the bias of a data set. Bias is a
						  systematic error that is either inherent in a method or caused by some artifact
						  or idiosyncrasy of the measurement system. Bias is characterized by a
						  consistent deviation (positive or negative) in the results from an accepted
						  reference value.
Arithmetic Mean:
The sum of measurements in a set divided by the number
						  of measurements in a set.
Blind Samples:
A quality control procedure in which the concentration
						  of analyte in the samples should be unknown to the analyst at the time that the
						  analysis is performed.
Coefficient of Variation:
The ratio of the standard deviation of a set of
						  measurements to the mean (arithmetic or geometric) of the measurements.
Compliance Samples:
Samples from exposed workers sent to a participating
						  laboratory for analysis.
Control Charts:
Graphic representations of the results for quality
						  control samples being analyzed by a participating laboratory.
Control Limits:
Statistical limits which define when an analytic
						  procedure exceeds acceptable parameters; control limits provide a method of
						  assessing the accuracy of analysts, laboratories, and discrete analytic
						  runs.
Control Samples:
Quality control samples.
F/T:
The measured amount of an analyte divided by the
						  theoretical value (defined below) for that analyte in the sample analyzed; this
						  ratio is a measure of the recovery for a quality control sample.
Geometric Mean:
The natural antilog of the mean of a set of natural
						  log- transformed data.
Geometric Standard Deviation:
The antilog of the standard deviation of a set of
						  natural log-transformed data.
Limit of Detection:
Using a predifined level of confidence, this is the
						  lowest measured value at which some of the measured material is likely to have
						  come from the sample.
Mean:
A central tendency of a set of data; in this protocol,
						  this mean is defined as the
arithmetic
mean (see definition of
						  arithmetic mean above) unless stated otherwise.
Performance:
A measure of the overall quality of data reported by a
						  laboratory.
Pools:
Groups of quality-control samples to be established for
						  each target value (defined below) of an analyte. For the protocol provided in
						  attachment 3, for example, the theoretical value of the quality control samples
						  of the pool must be within a range defined as plus or minus (±) 50% of
						  the target value. Within each analyte pool, there must be quality control
						  samples of at least 4 theoretical values.
Precision:
The extent of agreement between repeated, independent
						  measurements of the same quantity of an analyte.
Proficiency:
The ability to satisfy a specified level of analyte
						  performance.
Proficiency Samples:
Specimens, the values of which are unknown to anyone at
						  a participating laboratory, and which are submitted by a participating
						  laboratory for proficiency testing.
Quality or Data Quality:
A measure of the confidence in the measurement
						  value.
Quality Control (QC) Samples:
Specimens, the value of which is unknown to the
						  analyst, but is known to the appropriate QA/QC personnel of a participating
						  laboratory; when used as part of a laboratory QA/QC program, the theoretical
						  values of these samples should not be known to the analyst until the analyses
						  are complete. QC samples are to be run in sets consisting of one QC sample from
						  each pool (see definition of "pools" above).
Sensitivity:
For the purposes of this protocol, the limit of
						  detection.
Standard Deviation:
A measure of the distribution or spread of a data set
						  about the mean; the standard deviation is equal to the positive square root of
						  the variance, and is expressed in the same units as the original measurements
						  in the data set.
Standards:
Samples with values known by the analyst and used to
						  calibrate equipment and to check calibration throughout an analytic run. In a
						  laboratory QA/QC program, the values of the standards must exceed the values
						  obtained for compliance samples such that the lowest standard value is near the
						  limit of detection and the highest standard is higher than the highest
						  compliance sample or QC sample. Standards of at least three different values
						  are to be used for calibration, and should be constructed from at least 2
						  different sources.
Target Value:
Those values of CDB, CDU or B2MU which trigger some
						  action as prescribed in the medical surveillance section of the regulatory text
						  of the final cadmium rule. For CDB, the target values are 5, 7, 10 and 15
						  µg/l. For CDU, the target values are 3, 5, 10 and 15 µg/g CRTU. For
						  B2MU, the target value are 300, 500, 1000 and 1500 µg/g CRTU. (Note that
						  target values may vary as a function of time.)
Theoretical Value (or Theoretical Amount):
The reported concentration of a quality-control sample
						  (or calibration standard) derived from prior characterizations of the
						  sample.
Value or Measurement Value:
The numerical result of a measurement.
Variance:
A measure of the distribution or spread of a data set
						  about the mean; the variance is the sum of the squares of the differences
						  between the mean and each discrete measurement divided by one less than the
						  number of measurements in the data set.
3.0 PROTOCOL
This protocol provides procedures for characterizing
						  and maintaining the quality of analytic results derived for the medical
						  monitoring program mandated for workers under the final cadmium rule.
3.1 Overview
The goal of this protocol is to assure that medical
						  monitoring data are of sufficient quality to facilitate proper interpretation.
						  The data quality objectives (DQOs) defined for the medical monitoring program
						  are summarized in Table 1. Based on available information, the DQOs presented
						  in Table 1 should be achievable by the majority of laboratories offering the
						  required analyses commercially; OSHA recommends that only laboratories meeting
						  these DQOs be used for the analysis of biological samples collected for
						  monitoring cadmium exposure.
TABLE 1
RECOMMENDED DATA QUALITY
									 OBJECTIVES (DQOs) FOR THE CADMIUM MEDICAL MONITORING PROGRAM
Analyte/Concentration
									 Pool
Limit of
									 Detection
Precision
									 (CV)
Accuracy
Cadmium in Blood
0.5 µg/l
40
%±1 µg/l or 15 %
[LESS THAN EQUAL TO]2
								  µg/l
20 %
of the mean
>2 µg/1
Cadmium in Urine
0.5 µg/g
40 %
±1 µg/l or 15
								  %
[LESS THAN EQUAL TO]2
								  µg/l
creatinine
20 %
of the mean
creatinine
>2 µg/l e
creatinin
ß-2-Microglobulin in
								  Urine
100 µg/g
5 %
±15 % of the
								  mean
100
								  µg/g
creatinine
creatinine
To satisfy the DQOs presented in Table 1, OSHA provides
						  the following guidelines:
1. Procedures for the collection and handling of blood
						  and urine are specified (Section
						  3.4.1 of this protocol);
2. preferred analytic methods for the analysis of CDB,
						  CDU and B2MU are defined (and a method for the determination of CRTU also is
						  specified since CDU and B2MU results are to be normalized to the level of
						  CRTU).
3. procedures are described for identifying
						  laboratories likely to provide the required analyses in an accurate and
						  reliable manner;
4. these guidelines (Sections
						  3.2.1 to
						  3.2.3, and Section
						  3.3) include recommendations
						  regarding internal QA/QC programs for participating laboratories, as well as
						  levels of proficiency through participation in an interlaboratory proficiency
						  program;
5. procedures for QA/QC record keeping (Section
						  3.3.2), and for reporting QC/QA
						  results are described (Section
						  3.3.3); and,
6. procedures for interpreting medical monitoring
						  results are specified (Section
						  3.4.3).
Methods recommended for the biological monitoring of
						  eligible workers are:
1. The method of Stoeppler and Brandt (1980) for CDB
						  determinations (limit of detection: 0.5 µg/l);
2. the method of Pruszkowska et al. (1983) for CDU
						  determinations (limit of detection: 0.5 µg/l of urine); and,
3. the Pharmacia Delphia test kit (Pharmacia 1990) for
						  the determination of B2MU (limit of detection: 100 µg/l urine).
Because both CDU and B2MU should be reported in
						  ±
												µg/g CRTU, an independent determination of CRTU is recommended.
						  Thus, both the OSHA Salt Lake City Technical Center (OSLTC) method (OSHA, no
						  date) and the Jaffe method (Du Pont, no date) for the determination of CRTU are
						  specified under this protocol (i.e., either of these 2 methods may be used).
						  Note that although detection limits are not reported for either of these CRTU
						  methods, the range of measurements expected for CRTU (0.9-1.7 µg/l) are
						  well above the likely limit of detection for either of these methods (Harrison,
						  1987).
Laboratories using alternate methods should submit
						  sufficient data to the responsible physicians demonstrating that the alternate
						  method is capable of satisfying the defined data quality objectives of the
						  program. Such laboratories also should submit a QA/QC plan that documents the
						  performance of the alternate method in a manner entirely equivalent to the
						  QA/QC plans proposed in Section
						  3.3.1.
3.2 Duties of the Responsible Physician
The responsible physician will evaluate biological
						  monitoring results provided by participating laboratories to determine whether
						  such laboratories are proficient and have satisfied the QA/QC recommendations.
						  A requirement of the medical monitoring program mandated under the cadmium rule
						  is that responsible physicians have the duty to employ laboratories to perform
						  the required CDB, CDU and B2MU analyses of biological samples collected from
						  eligible workers (paragraph (
l
)(1)(iv) of the final rule). In
						  determining which laboratories to employ for this purpose, these physicians
						  should review proficiency and QA/QC data submitted to them by the participating
						  laboratories.
Participating laboratories should demonstrate
						  proficiency for each analyte (CDU, CDB and B2MU) sampled under the biological
						  monitoring program. Participating laboratories involved in analyzing CDU and
						  B2MU also should demonstrate proficiency for CRTU analysis, or provide evidence
						  of a contract with a laboratory proficient in CRTU analysis.
3.2.1 Recommendations for Selecting Among Existing
						  Laboratories
OSHA recommends that existing laboratories providing
						  commercial analyses for CDB, CDU and/or B2MU for the medical monitoring program
						  satisfy the following criteria:
1. Should have performed commercial analyses for the
						  appropriate analyte (CDB, CDU and/or B2MU) on a regular basis over the last 2
						  years;
2. should provide the responsible physician with an
						  internal QA/QC plan;
3. if performing CDU or B2MU analyses, the
						  participating laboratory should be accredited by the CAP for CRTU analysis, and
						  should be enrolled in the corresponding CAP survey (note that alternate
						  credentials may be acceptable, but acceptability is to be determined by the
						  responsible physician); and,
4. should have enrolled in the CTQ interlaboratory
						  comparison program for the appropriate analyte (CDB, CDU and/or B2MU).
Participating laboratories should submit appropriate
						  documentation demonstrating compliance with the above criteria to the
						  responsible physician. To demonstrate compliance with the first of the above
						  criteria, participating laboratories should submit the following documentation
						  for each analyte they plan to analyze (note that each document should cover a
						  period of at least 8 consecutive quarters, and that the period designated by
						  the term "regular analyses" is at least once a quarter):
1. Copies of laboratory reports providing results from
						  regular analyses of the appropriate analyte (CDB, CDU and/or B2MU);
2. copies of 1 or more signed and executed contracts
						  for the provision of regular analyses of the appropriate analyte (CDB, CDU
						  and/or B2MU); or,
3. copies of invoices sent to 1 or more clients
						  requesting payment for the provision of regular analyses of the appropriate
						  analyte (CDB, CDU and/or B2MU).
Whatever the form of documentation submitted, the
						  specific analytic procedures conducted should be identified directly. The forms
						  that are copied for submission to the responsible physician also should
						  identify the laboratory which provided these analyses.
To demonstrate compliance with the second of the above
						  criteria, a laboratory should submit to the responsible physician an internal
						  QA/QC plan detailing the standard operating procedures to be adopted for
						  satisfying the recommended QA/QC procedures for the analysis of each specific
						  analyte (CDB, CDU and/or B2MU). Procedures for internal QA/QC programs are
						  detailed in Section
						  3.3.1 below.
To satisfy the third of the above criteria,
						  laboratories analyzing for CDU or B2MU also should submit a QA/QC plan for
						  creatinine analysis (CRTU); the QA/QC plan and characterization analyses for
						  CRTU must come from the laboratory performing the CRTU analysis, even if the
						  CRTU analysis is being performed by a contract laboratory.
Laboratories enrolling in the CTQ program (to satisfy
						  the last of the above criteria) must remit, with the enrollment application, an
						  initial fee of approximately $100 per analyte. (Note that this fee is only an
						  estimate, and is subject to revision without notice.) Laboratories should
						  indicate on the application that they agree to have proficiency test results
						  sent by the CTQ directly to the physicians designated by participating
						  laboratories.
Once a laboratory's application is processed by the
						  CTQ, the laboratory will be assigned a code number which will be provided to
						  the laboratory on the initial confirmation form, along with identification of
						  the specific analytes for which the laboratory is participating. Confirmation
						  of participation will be sent by the CTQ to physicians designated by the
						  applicant laboratory.
3.2.2 Recommended Review of Laboratories Selected to
						  Perform Analyses
Six months after being selected initially to perform
						  analyte determinations, the status of participating laboratories should be
						  reviewed by the responsible physicians. Such reviews should then be repeated
						  every 6 months or whenever additional proficiency or QA/QC documentation is
						  received (whichever occurs first).
As soon as the responsible physician has received the
						  CTQ results from the first 3 rounds of proficiency testing (i.e., 3 sets of 3
						  samples each for CDB, CDU and/or B2MU) for a participating laboratory, the
						  status of the laboratory's continued participation should be reviewed. Over the
						  same initial 6-month period, participating laboratories also should provide
						  responsible physicians the results of their internal QA/QC monitoring program
						  used to assess performance for each analyte (CDB, CDU and/or B2MU) for which
						  the laboratory performs determinations. This information should be submitted
						  using appropriate forms and documentation.
The status of each participating laboratory should be
						  determined for each analyte (i.e., whether the laboratory satisfies minimum
						  proficiency guidelines based on the proficiency samples sent by the CTQ and the
						  results of the laboratory's internal QA/QC program). To maintain competency for
						  analysis of CDB, CDU and/or B2MU during the first review, the laboratory should
						  satisfy performance requirements for at least 2 of the 3 proficiency samples
						  provided in each of the 3 rounds completed over the 6-month period. Proficiency
						  should be maintained for the analyte(s) for which the laboratory conducts
						  determinations.
To continue participation for CDU and/or B2MU analyse,
						  laboratories also should either maintain accreditation for CRTU analysis in the
						  CAP program and participate in the CAP surveys, or they should contract the CDU
						  and B2MU analyses to a laboratory which satisfies these requirements (or which
						  can provide documentation of accreditation/participation in an equivalent
						  program).
The performance requirement for CDB analysis is defined
						  as an analytical result within ±
												µg/l blood or 15% of the
						  consensus mean (whichever is greater). For samples exhibiting a consensus mean
						  less than 1 µg/l, the performance requirement is defined as a
						  concentration between the detection limit of the analysis and a maximum of 2
						  [MICRO]g/l. The purpose for redefining the acceptable interval for low CDB
						  values is to encourage proper reporting of the actual values obtained during
						  measurement; laboratories, therefore, will not be penalized (in terms of a
						  narrow range of acceptability) for reporting measured concentrations smaller
						  than 1 µg/l.
The performance requirement for CDU analysis is defined
						  as an analytical result within ±1 µg/l urine or 15% of the
						  consensus mean (whichever is greater). For samples exhibiting a consensus mean
						  less than 1Ã¦g/l urine, the performance requirement is defined as
						  a concentration between the detection limit of the analysis and a maximum of 2
						  µg/l urine. Laboratories also should demonstrate proficiency in
						  creatinine analysis as defined by the CAP. Note that reporting CDU results,
						  other than for the CTQ proficiency samples (i.e., compliance samples), should
						  be accompanied with results of analyses for CRTU, and these 2 sets of results
						  should be combined to provide a measure of CDU in units of µg/g
						  CRTU.
The performance requirement for B2MU is defined as
						  analytical results within ±15% of the consensus mean. Note that
						  reporting B2MU results, other than for CTQ proficiency samples (i.e.,
						  compliance samples), should be accompanied with results of analyses for CRTU,
						  and these 2 sets of results should be combined to provide a measure of B2MU in
						  units of µg/g CRTU.
There are no recommended performance checks for CRTU
						  analyses. As stated previously, laboratories performing CRTU analysis in
						  support of CDU or B2MU analyses should be accredited by the CAP, and
						  participating in the CAP's survey for CRTU.
Following the first review, the status of each
						  participating laboratory should be reevaluated at regular intervals (i.e.,
						  corresponding to receipt of results from each succeeding round of proficiency
						  testing and submission of reports from a participating laboratory's internal
						  QA/QC program).
After a year of collecting proficiency test results,
						  the following proficiency criterion should be added to the set of criteria used
						  to determine the participating laboratory's status (for analyzing CDB, CDU
						  and/or B2MU): A participating laboratory should not fail performance
						  requirements for more than 4 samples from the 6 most recent consecutive rounds
						  used to assess proficiency for CDB, CDU and/or B2MU separately (i.e., a total
						  of 18 discrete proficiency samples for each analyte). Note that this
						  requirement does
not
replace, but supplements, the
						  recommendation that a laboratory should satisfy the performance criteria for at
						  least 2 of the 3 samples tested for each round of the program.
3.2.3 Recommendations for Selecting Among Newly-Formed
						  Laboratories (or Laboratories that Previously Failed to Meet the Protocol
						  Guidelines)
OSHA recommends that laboratories that have not
						  previously provided commercial analyses of CDB, CDU and/or B2MU (or have done
						  so for a period less than 2 years), or which have provided these analyses for 2
						  or more years but have not conformed previously with these protocol guidelines,
						  should satisfy the following provisions for each analyte for which
						  determinations are to be made prior to being selected to analyze biological
						  samples under the medical monitoring program:
1. Submit to the responsible physician an internal
						  QA/QC plan detailing the standard operating procedures to be adopted for
						  satisfying the QA/QC guidelines (guidelines for internal QA/QC programs are
						  detailed in Section
						  3.3.1;
2. submit to the responsible physician the results of
						  the initial characterization analyses for each analyte for which determinations
						  are to be made;
3. submit to the responsible physician the results, for
						  the initial 6-month period, of the internal QA/QC program for each analyte for
						  which determinations are to be made (if no commercial analyses have been
						  conducted previously, a minimum of 2 mock standardization trials for each
						  analyte should be completed per month for a 6-month period;
4. enroll in the CTQ program for the appropriate
						  analyte for which determinations are to be made, and arrange to have the CTQ
						  program submit the initial confirmation of participation and proficiency test
						  results directly to the designated physicians. Note that the designated
						  physician should receive results from 3 completed rounds from the CTQ program
						  before approving a laboratory for participation in the biological monitoring
						  program;
5. laboratories seeking participation for CDU and/or
						  B2MU analyses should submit to the responsible physician documentation of
						  accreditation by the CAP for CRTU analyses performed in conjunction with CDU
						  and/or B2MU determinations (if CRTU analyses are conducted by a contract
						  laboratory, this laboratory should submit proof of CAP accreditation to the
						  responsible physician); and,
6. documentation should be submitted on an appropriate
						  form.
To participate in CDB, CDU and/or B2MU analyses, the
						  laboratory should satisfy the above criteria for a minimum of 2 of the 3
						  proficiency samples provided in each of the 3 rounds of the CTQ program over a
						  6-month period; this procedure should be completed for each appropriate
						  analyte. Proficiency should be maintained for each analyte to continue
						  participation. Note that laboratories seeking participation for CDU or B2MU
						  also should address the performance requirements for CRTU, which involves
						  providing evidence of accreditation by the CAP and participation in the CAP
						  surveys (or an equivalent program).
The performance requirement for CDB analysis is defined
						  as an analytical result within ±1 µg/l or 15% of the consensus
						  mean (whichever is greater). For samples exhibiting a consensus mean less than
						  1 µg/l, the performance requirement is defined as a concentration between
						  the detection limit of the analysis and a maximum of 2 µg/l. The purpose
						  of redefining the acceptable interval for low CDB values is to encourage proper
						  reporting of the actual values obtained during measurement; laboratories,
						  therefore, will not be penalized (in terms of a narrow range of acceptability)
						  for reporting measured concentrations less than 1 µg/l.
The performance requirement for CDU analysis is defined
						  as an analytical result within ±1 µg/l urine or 15% of the
						  consensus mean (whichever is greater). For samples exhibiting a consensus mean
						  less than 1 µg/l urine, the performance requirement is defined as a
						  concentration that falls between the detection limit of the analysis and a
						  maximum of 2 µg/l urine. Performance requirements for the companion CRTU
						  analysis (defined by the CAP) also should be met. Note that reporting CDU
						  results, other than for CTQ proficiency testing (i.e., compliance samples),
						  should be accompanied with results of CRTU analyses, and these 2 sets of
						  results should be combined to provide a measure of CDU in units of µg/g
						  CRTU.
The performance requirement for B2MU is defined as an
						  analytical result within ±15% of the consensus mean. Note that reporting
						  B2MU results, other than for CTQ proficiency testing (i.e., compliance
						  samples), should be accompanied with results of CRTU analysis, these 2 sets of
						  results should be combined to provide a measure of B2MU in units of µg/g
						  CRTU.
Once a new laboratory has been approved by the
						  responsible physician for conducting analyte determinations, the status of this
						  approval should be reviewed periodically by the responsible physician as per
						  the criteria presented under Section
						  3.2.2.
Laboratories which have failed previously to gain
						  approval of the responsible physician for conducting determinations of 1 or
						  more analytes due to lack of compliance with the criteria defined above for
						  existing laboratories (Section
						  3.2.1), may obtain approval by
						  satisfying the criteria for newly-formed laboratories defined under this
						  section; for these laboratories, the second of the above criteria may be
						  satisfied by submitting a new set of characterization analyses for each analyte
						  for which determinations are to be made.
Reevaluation of these laboratories is discretionary on
						  the part of the responsible physician.
Reevaluation, which normally takes about 6 months, may
						  be expedited if the laboratory can achieve 100% compliance with the proficiency
						  test criteria using the 6 samples of each analyte submitted to the CTQ program
						  during the first 2 rounds of proficiency testing.
For laboratories seeking reevaluation for CDU or B2MU
						  analysis, the guidelines for CRTU analyses also should be satisfied, including
						  accreditation for CRTU analysis by the CAP, and participation in the CAP survey
						  program (or accreditation/participation in an equivalent program).
3.2.4 Future Modifications to the Protocol
						  Guidelines
As participating laboratories gain experience with
						  analyses for CDB, CDU and B2MU, it is anticipated that the performance
						  achievable by the majority of laboratories should improve until it approaches
						  that reported by the research groups which developed each method. OSHA,
						  therefore, may choose to recommend stricter performance guidelines in the
						  future as the overall performance of participating laboratories
						  improves.
3.3 Guidelines for Record Keeping and Reporting
To comply with these guidelines, participating
						  laboratories should satisfy the above-stated performance and proficiency
						  recommendations, as well as the following internal QA/QC, record keeping, and
						  reporting provisions.
If a participating laboratory fails to meet the
						  provisions of these guidelines, it is recommended that the responsible
						  physician disapprove further analyses of biological samples by that laboratory
						  until it demonstrates compliance with these guidelines. On disapproval,
						  biological samples should be sent to a laboratory that can demonstrate
						  compliance with these guidelines, at least until the former laboratory is
						  reevaluated by the responsible physician and found to be in compliance.
The following record keeping and reporting procedures
						  should be practiced by participating laboratories.
3.3.1 Internal Quality Assurance/Quality Control
						  Procedures
Laboratories participating in the cadmium monitoring
						  program should develop and maintain an internal quality assurance/quality
						  control (QA/QC) program that incorporates procedures for establishing and
						  maintaining control for each of the analytic procedures (determinations of CDB,
						  CDU and/or B2MU) for which the laboratory is seeking participation. For
						  laboratories analyzing CDU and/or B2MU, a QA/QC program for CRTU also should be
						  established.
Written documentation of QA/QC procedures should be
						  described in a formal QA/QC plan; this plan should contain the following
						  information: Sample acceptance and handling procedures (i.e.,
						  chain-of-custody); sample preparation procedures; instrument parameters;
						  calibration procedures; and, calculations.
Documentation of QA/QC procedures should be sufficient
						  to identify analytical problems, define criteria under which analysis of
						  compliance samples will be suspended, and describe procedures for corrective
						  actions.
3.3.1.1 QA/QC procedures for establishing control of
						  CDB and CDU analyses
The QA/QC program for CDB and CDU should address, at a
						  minimum, procedures involved in calibration, establishment of control limits,
						  internal QC analyses and maintaining control, and corrective-action protocols.
						  Participating laboratory should develop and maintain procedures to assure that
						  analyses of compliance samples are within control limits, and that these
						  procedures are documented thoroughly in a QA/QC plan.
A nonmandatory QA/QC protocol is presented in
						  Attachment 1. This attachment is illustrative of the procedures that should be
						  addressed in a proper QA/QC program.
Calibration. Before any analytic runs are conducted,
						  the analytic instrument should be calibrated. Calibration should be performed
						  at the beginning of each day on which QC and/or compliance samples are run.
						  Once calibration is established, QC or compliance samples may be run.
						  Regardless of the type of samples run, about every fifth sample should serve as
						  a standard to assure that calibration is being maintained.
Calibration is being maintained if the standard is
						  within ±15% of its theoretical value. If a standard is more than
						  ±15% of its theoretical value, the run has exceeded control limits due
						  to calibration error; the entire set of samples then should be reanalyzed after
						  recalibrating or the results should be recalculated based on a statistical
						  curve derived from that set of standards.
It is essential that the value of the highest standard
						  analyzed be higher than the highest sample analyzed; it may be necessary,
						  therefore, to run a high standard at the end of the run, which has been
						  selected based on results obtained over the course of the run (i.e., higher
						  than any standard analyzed to that point).
Standards should be kept fresh; as samples age, they
						  should be compared with new standards and replaced if necessary.
Internal Quality Control Analyses. Internal QC samples
						  should be determined interspersed with analyses of compliance samples. At a
						  minimum, these samples should be run at a rate of 5% of the compliance samples
						  or 2 samples per analytic run, whichever is greater. If only 2 samples are run,
						  they should contain different levels of cadmium.
Internal QC samples may be obtained as commercially-
						  available reference materials and/or they may be internally prepared.
						  Internally-prepared samples should be well characterized and traced, or
						  compared to a reference material for which a consensus value is
						  available.
Levels of cadmium contained in QC samples should not be
						  known to the analyst prior to reporting the results of the analysis.
Internal QC results should be plotted or charted in a
						  manner which describes sample recovery and laboratory control limits.
Internal Control Limits. The laboratory protocol for
						  evaluating internal QC analyses per control limits should be clearly defined.
						  Limits may be based on statistical methods (e.g., as 2[SIGMA] from the
						  laboratory mean recovery), or on proficiency testing limits (e.g., ±2
						  µg or 15% of the mean, whichever is greater).
Statistical limits that exceed ±40% should be
						  reevaluated to determine the source error in the analysis.
When laboratory limits are exceeded, analytic work
						  should terminate until the source of error is determined and corrected;
						  compliance samples affected by the error should be reanalyzed. In addition, the
						  laboratory protocol should address any unusual trends that develop which may be
						  biasing the results. Numerous, consecutive results above or below laboratory
						  mean recoveries, or outside laboratory statistical limits, indicate that
						  problems may have developed.
Corrective Actions. The QA/QC plan should document in
						  detail specific actions taken if control limits are exceeded or unusual trends
						  develop. Corrective actions should be noted on an appropriate form, accompanied
						  by supporting documentation.
In addition to these actions, laboratories should
						  include whatever additional actions are necessary to assure that accurate data
						  are reported to the responsible physicians.
Reference Materials. The following reference materials
						  may be available:
Cadmium in Blood (CDB)
1. Centre de Toxicologie du Quebec, Le Centre
						  Hospitalier de l'Universite Laval, 2705 boul. Laurier, Quebec, Que., Canada G1V
						  4G2. (Prepared 6 times per year at 1-15 µg Cd/l.)
2. H. Marchandise, Community Bureau of Reference-BCR,
						  Directorate General XII, Commission of the European Communities, 200, rue de la
						  Loi, B-1049, Brussels, Belgium. (Prepared as Bl CBM-1 at 5.37 µg Cd/l,
						  and Bl CBM-2 at 12.38 µg Cd/l.)
3. Kaulson Laboratories Inc., 691 Bloomfield Ave.,
						  Caldwell, NJ 07006; tel: (201) 226-9494, FAX (201) 226-3244. (Prepared as #0141
						  [As, Cd, Hg, Pb] at 2 levels.)
Cadmium in Urine (CDU)
1. Centre de Toxicologie du Quebec, Le Centre
						  Hospitalier de l'Universite Laval, 2705 boul. Laurier, Quebec, Que., Canada G1V
						  4G2. (Prepared 6 times per year.)
2. National Institute of Standards and Technology
						  (NIST), Dept. of Commerce, Gaithersburg, MD; tel: (301) 975-6776. (Prepared as
						  SRM 2670 freeze-dried urine [metals]; set includes normal and elevated levels
						  of metals; cadmium is certified for elevated level of 88.0 µg/l in
						  reconstituted urine.)
3. Kaulson Laboratories Inc., 691 Bloomfield Ave.,
						  Caldwell, NJ 07006; tel: (201) 226-9494, FAX (201) 226-3244. (Prepared as #0140
						  [As, Cd, Hg, Pb] at 2 levels.)
3.3.1.2 QA/QC procedures for establishing control of
						  B2MU
A written, detailed QA/QC plan for B2MU analysis should
						  be developed. The QA/QC plan should contain a protocol similar to those
						  protocols developed for the CDB/CDU analyses. Differences in analyses may
						  warrant some differences in the QA/QC protocol, but procedures to ensure
						  analytical integrity should be developed and followed.
Examples of performance summaries that can be provided
						  include measurements of accuracy (i.e., the means of measured values verses
						  target values for the control samples) and precision (i.e., based on duplicate
						  analyses). It is recommended that the accuracy and precision measurements be
						  compared to those reported as achievable by the Pharmacia Delphia kit
						  (Pharmacia 1990) to determine if and when unsatisfactory analyses have arisen.
						  If the measurement error of 1 or more of the control samples is more than 15%,
						  the run exceeds control limits. Similarly, this decision is warranted when the
						  average CV for duplicate samples is greater than 5%.
3.3.2 Procedures for Record Keeping
To satisfy reporting requirements for commercial
						  analyses of CDB, CDU and/or B2MU performed for the medical monitoring program
						  mandated under the cadmium rule, participating laboratories should maintain the
						  following documentation for each analyte:
1. For each analytic instrument on which analyte
						  determinations are made, records relating to the most recent calibration and QC
						  sample analyses;
2. for these instruments, a tabulated record for each
						  analyte of those determinations found to be within and outside of control
						  limits over the past 2 years;
3. results for the previous 2 years of the QC sample
						  analyses conducted under the internal QA/QC program (this information should
						  be: Provided for each analyte for which determinations are made and for each
						  analytic instrument used for this purpose, sufficient to demonstrate that
						  internal QA/QC programs are being executed properly, and consistent with data
						  sent to responsible physicians.
4. duplicate copies of monitoring results for each
						  analyte sent to clients during the previous 5 years, as well as associated
						  information; supporting material such as chain-of- custody forms also should be
						  retained; and,
5. proficiency test results and related materials
						  received while participating in the CTQ interlaboratory program over the past 2
						  years; results also should be tabulated to provide a serial record of relative
						  error (derived per Section
						  3.3.3 below).
3.3.3 Reporting Procedures
Participating laboratories should maintain these
						  documents: QA/QC program plans; QA/QC status reports; CTQ proficiency program
						  reports; and, analytical data reports. The information that should be included
						  in these reports is summarized in Table 2; a copy of each report should be sent
						  to the responsible physician.
TABLE 2
REPORTING PROCEDURES FOR
									 LABORATORIES PARTICIPATING IN THE CADMIUM MEDICAL MONITORING
									 PROGRAM
Report
Frequency (Time
									 Frame)
Contents
1
QA/QC Program Plan
Once
								  (initially)
A detailed description of the QA/QC protocol to be
								  established by the laboratory to maintain control of analyte
								  determinations.
2
QA/QC Status Report
Every 2
								  months
Results of the QC samples incorporated into regular runs for
								  each instrument (over the period since the last report).
3
Proficiency Report
Attached to
								  every data report
Results from the last full year of proficiency
								  samples submitted to the CTQ program.
Results of the 100
								  most recent QC samples incorporated into regular runs for each instrument.
4
Analytical Data Report
For all
								  reports of data results
Date the sample was received.
Date the sample was
								  analyzed.
Appropriate
								  chain-of-custody information.
Types of analyses
								  performed.
Results of the
								  requested analyses.
Copy of the most
								  current proficiency report.
As noted in Section
						  3.3.1, a
QA/QC program
						  plan
should be developed that documents internal QA/QC procedures
						  (defined under Section
						  3.3.1) to be implemented by the
						  participating laboratory for each analyte; this plan should provide a list
						  identifying each instrument used in making analyte determinations.
A
QA/QC status report
should be
						  written bimonthly for each analyte. In this report, the results of the QC
						  program during the reporting period should be reported for each analyte in the
						  following manner: The number (N) of QC samples analyzed during the period; a
						  table of the target levels defined for each sample and the corresponding
						  measured values; the mean of F/T value (as defined below) for the set of QC
						  samples run during the period; and, use of X ± 2[SIGMA] (as defined
						  below) for the set of QC samples run during the period as a measure of
						  precision.
As noted in Section
						  2, an F/T value for a QC sample is
						  the ratio of the measured concentration of analyte to the established (i.e.,
						  reference) concentration of analyte for that QC sample. The equation below
						  describes the derivation of the mean for F/T values, X:
Click
						  here to view image
The standard deviation, [SIGMA], for these measurements
						  is derived using the following equation (note that 2[SIGMA] is twice this
						  value):
Click
						  here to view image
The nonmandatory QA/QC protocol (see Attachment 3)
						  indicates that QC samples should be divided into several discrete pools, and a
						  separate estimate of precision for each pools then should be derived. Several
						  precision estimates should be provided for concentrations which differ in
						  average value. These precision measures may be used to document improvements in
						  performance with regard to the combined pool.
Participating laboratories should use the CTQ
						  proficiency program for each analyte. Results of the this program will be sent
						  by CTQ directly to physicians designated by the participating laboratories.
						  Proficiency results from the CTQ program are used to establish the accuracy of
						  results from each participating laboratory, and should be provided to
						  responsible physicians for use in trend analysis. A
proficiency
						  report
consisting of these proficiency results should accompany data
						  reports as an attachment.
For each analyte, the proficiency report should include
						  the results from the 6 previous proficiency rounds in the following
						  format:
1. Number (N) of samples analyzed;
2. mean of the target levels,
						  (1/N)[SIGMA]Ti, with Ti being a
						  consensus mean for the sample;
3. mean of the measurements,
						  (1/N)[SIGMA]Mi, with Mi being a
						  sample measurement;
4. a measure of error defined by:
(1/N) [SIGMA]
						  (Ti-Mi)2
Analytical data reports should be submitted to
						  responsible physicians directly. For each sample, report the following
						  information: The date the sample was received; the date the sample was
						  analyzed; appropriate chain-of-custody information; the type(s) of analyses
						  performed; and, the results of the analyses. This information should be
						  reported on a form similar to the form provided an appropriate form. The most
						  recent proficiency program report should accompany the analytical data reports
						  (as an attachment).
Confidence intervals for the analytical results should
						  be reported as X ± 2[SIGMA], with X being the measured value and
						  2[SIGMA] the standard deviation calculated as described above.
For CDU or B2MU results, which are combined with CRTU
						  measurements for proper reporting, the 95% confidence limits are derived from
						  the limits for CDU or B2MU, (p), and the limits for CRTU, (q), as
						  follows:
Click
						  here to view image
For these calculations, X ± p is the measurement
						  and confidence limits for CDU or B2MU, and Y ± q is the measurement and
						  confidence limit for CRTU.
Participating laboratories should notify responsible
						  physicians as soon as they receive information indicating a change in their
						  accreditation status with the CTQ or the CAP. These physicians should not be
						  expected to wait until formal notice of a status change has been received from
						  the CTQ or the CAP.
3.4 Instructions to Physicians
Physicians responsible for the medical monitoring of
						  cadmium-exposed workers must collect the biological samples from workers; they
						  then should select laboratories to perform the required analyses, and should
						  interpret the analytic results.
3.4.1 Sample Collection and Holding Procedures
Blood Samples. The following procedures are recommended
						  for the collection, shipment and storage of blood samples for CDB analysis to
						  reduce analytical variablility; these recommendations were obtained primarily
						  through personal communications with J.P. Weber of the CTQ (1991), and from
						  reports by the Centers for Disease Control (CDC, 1986) and Stoeppler and Brandt
						  (1980).
To the extent possible, blood samples should be
						  collected from workers at the same time of day. Workers should shower or
						  thoroughly wash their hands and arms before blood samples are drawn. The
						  following materials are needed for blood sample collection: Alcohol wipes;
						  sterile gauze sponges; band-aids; 20- gauge, 1.5-in. stainless steel needles
						  (sterile); preprinted labels; tourniquets; vacutainer holders; 3-ml "metal
						  free" vacutainer tubes (i.e., dark-blue caps), with EDTA as an anti- coagulant;
						  and, styrofoam vacutainer shipping containers.
Whole blood samples are taken by venipuncture. Each
						  blue- capped tube should be labeled or coded for the worker and company before
						  the sample is drawn. (Blue-capped tubes are recommended instead of red-capped
						  tubes because the latter may consist of red coloring pigment containing
						  cadmium, which could contaminate the samples.) Immediately after sampling, the
						  vacutainer tubes must be thoroughly mixed by inverting the tubes at least 10
						  times manually or mechanically using a Vortex device (for 15 sec). Samples
						  should be refrigerated immediately or stored on ice until they can be packed
						  for shipment to the participating laboratory for analysis.
The CDC recommends that blood samples be shipped with a
						  "cool pak" to keep the samples cold during shipment. However, the CTQ routinely
						  ships and receives blood samples for cadmium analysis that have not been kept
						  cool during shipment. The CTQ has found no deterioration of cadmium in
						  biological fluids that were shipped via parcel post without a cooling agent,
						  even though these deliveries often take 2 weeks to reach their
						  destination.
Urine Samples. The following are recommended procedures
						  for the collection, shipment and storage of urine for CDU and B2MU analyses,
						  and were obtained primarily through personal communications with J.P. Weber of
						  the CTQ (1991), and from reports by the CDC (1986) and Stoeppler and Brandt
						  (1980).
Single "spot" samples are recommended. As B2M can
						  degrade in the bladder, workers should first empty their bladder and then drink
						  a large glass of water at the start of the visit. Urine samples then should be
						  collected within 1 hour. Separate samples should be collected for CDU and B2MU
						  using the following materials: Sterile urine collection cups (250 ml); small
						  sealable plastic bags; preprinted labels; 15-ml polypropylene or polyethylene
						  screw-cap tubes; lab gloves ("metal free"); and, preservatives (as
						  indicated).
The sealed collection cup should be kept in the plastic
						  bag until collection time. The workers should wash their hands with soap and
						  water before receiving the collection cup. The collection cup should not be
						  opened until just before voiding and the cup should be sealed immediately after
						  filling. It is important that the inside of the container and cap are not
						  touched by, or come into contact with, the body, clothing or other
						  surfaces.
For CDU analyzes, the cup is swirled gently to
						  resuspend any solids, and the 15-ml tube is filled with 10-12 ml urine. The CDC
						  recommends the addition of 100 µl concentrated
						  HNO3 as a preservative before sealing the tube and then
						  freezing the sample. The CTQ recommends minimal handling and does not acidify
						  their interlaboratory urine reference materials prior to shipment, nor do they
						  freeze the sample for shipment. At the CTQ, if the urine sample has much
						  sediment, the sample is acidified in the lab to free any cadmium in the
						  precipitate.
For B2M, the urine sample should be collected directly
						  into a polyethylene bottle previously washed with dilute nitric acid. The pH of
						  the urine should be measured and adjusted to 8.0 with 0.1 N NaOH immediately
						  following collection. Samples should be frozen and stored at -20ØC until
						  testing is performed. The B2M in the samples should be stable for 2 days when
						  stored at 2-8ØC, and for at least 2 months at -20ØC. Repeated
						  freezing and thawing should be avoided to prevent denaturing the B2M (Pharmacia
						  1990).
3.4.2 Recommendations for Evaluating
						  Laboratories
Using standard error data and the results of
						  proficiency testing obtained from CTQ, responsible physicians can make an
						  informed choice of which laboratory to select to analyze biological samples. In
						  general, laboratories with small standard errors and little disparity between
						  target and measured values tend to make precise and accurate sample
						  determinations. Estimates of precision provided to the physicians with each set
						  of monitoring results can be compared to previously-reported proficiency and
						  precision estimates. The latest precision estimates should be at least as small
						  as the standard error reported previously by the laboratory. Moreover, there
						  should be no indication that precision is deteriorating (i.e., increasing
						  values for the precision estimates). If precision is deteriorating, physicians
						  may decide to use another laboratory for these analyses. QA/QC information
						  provided by the participating laboratories to physicians can, therefore, assist
						  physicians in evaluating laboratory performance.
3.4.3 Use and Interpretation of Results
When the responsible physician has received the CDB,
						  CDU and/or B2MU results, these results must be compared to the action levels
						  discussed in the final rule for cadmium. The comparison of the sample results
						  to action levels is straightforward. The measured value reported from the
						  laboratory can be compared directly to the action levels; if the reported value
						  exceeds an action level, the required actions must be initiated.
4.0 BACKGROUND
Cadmium is a naturally-occurring environmental
						  contaminant to which humans are continually exposed in food, water, and air.
						  The average daily intake of cadmium by the U.S. population is estimated to ben
						  10-20 µg/day. Most of this intake is via ingestion, for which absorption
						  is estimated at 4-7% (Kowal et al. 1979). An additional nonoccupational source
						  of cadmium is smoking tobacco; smoking a pack of cigarettes a day adds an
						  additional 2-4 µg cadmium to the daily intake, assuming absorption via
						  inhalation of 25-35% (Nordberg and Nordberg 1988; Friberg and Elinder 1988;
						  Travis and Haddock 1980).
Exposure to cadmium fumes and dusts in an occupational
						  setting where air concentrations are 20-50
						  µg/m3 results in an additional daily intake of
						  several hundred micrograms (Friberg and Elinder 1988, p. 563). In such a
						  setting, occupational exposure to cadmium occurs primarily via inhalation,
						  although additional exposure may occur through the ingestion of material via
						  contaminated hands if workers eat or smoke without first washing. Some of the
						  particles that are inhaled initially may be ingested when the material is
						  deposited in the upper respiratory tract, where it may be cleared by
						  mucociliary transport and subsequently swallowed.
Cadmium introduced into the body through inhalation or
						  ingestion is transported by the albumin fraction of the blood plasma to the
						  liver, where it accumulates and is stored principally as a bound form complexed
						  with the protein metallothionein. Metallothionein-bound cadmium is the main
						  form of cadmium subsequently transported to the kidney; it is these 2 organs,
						  the liver and kidney, in which the majority of the cadmium body burden
						  accumulates. As much as one half of the total body burden of cadmium may be
						  found in the kidneys (Nordberg and Nordberg 1988).
Once cadmium has entered the body, elimination is slow;
						  about 0.02% of the body burden is excreted per day via urinary/fecal
						  elimination. The whole-body half-life of cadmium is 10-35 years, decreasing
						  slightly with increasing age (Travis and Haddock 1980).
The continual accumulation of cadmium is the basis for
						  its chronic noncarcinogenic toxicity. This accumulation makes the kidney the
						  target organ in which cadmium toxicity usually is first observed (Piscator
						  1964). Renal damage may occur when cadmium levels in the kidney cortex approach
						  200 µg/g wet tissue-weight (Travis and Haddock 1980).
The kinetics and internal distribution of cadmium in
						  the body are complex, and depend on whether occupational exposure to cadmium is
						  ongoing or has terminated. In general, cadmium in blood is related principally
						  to recent cadmium exposure, while cadmium in urine reflects cumulative exposure
						  (i.e., total body burden)(Lauwerys et al. 1976; Friberg and Elinder
						  1988).
4.1 Health Effects
Studies of workers in a variety of industries indicate
						  that chronic exposure to cadmium may be linked to several adverse health
						  effects including kidney dysfunction, reduced pulmonary function, chronic lung
						  disease and cancer (Federal Register 1990). The primary sites for
						  cadmium-associated cancer appear to be the lung and the prostate.
Cancer. Evidence for an association between cancer and
						  cadmium exposure comes from both epidemiological studies and animal
						  experiments. Pott (1965) found a statistically significant elevation in the
						  incidence of prostate cancer among a cohort of cadmium workers. Other
						  epidemiology studies also report an elevated incidence of prostate cancer;
						  however, the increases observed in these other studies were not statistically
						  significant (Meridian Research, Inc. 1989).
One study (Thun et al. 1985) contains sufficiently
						  quantitative estimates of cadmium exposure to allow evaluation of dose-response
						  relationships between cadmium exposure and lung cancer. A statistically
						  significant excess of lung cancer attributed to cadmium exposure was found in
						  this study, even after accounting for confounding variables such as coexposure
						  to arsenic and smoking habits (Meridian Research, Inc. 1989).
Evidence for quantifying a link between lung cancer and
						  cadmium exposure comes from a single study (Takenaka et al. 1983). In this
						  study, dose-response relationships developed from animal data were extrapolated
						  to humans using a variety of models. OSHA chose the multistage risk model for
						  estimating the risk of cancer for humans using these animal data. Animal
						  injection studies also suggest an association between cadmium exposure and
						  cancer, particularly observations of an increased incidence of tumors at sites
						  remote from the point of injection. The International Agency for Research on
						  Cancer (IARC) (Supplement 7, 1987) indicates that this, and related, evidence
						  is sufficient to classify cadmium as an animal carcinogen. However, the results
						  of these injection studies cannot be used to quantify risks attendant to human
						  occupational exposures due to differences in routes of exposure (Meridian
						  Research, Inc. 1989).
Based on the above-cited studies, the U.S.
						  Environmental Protection Agency (EPA) classifies cadmium as "B1," a probable
						  human carcinogen (USEPA 1985). IARC in 1987 recommended that cadmium be listed
						  as a probable human carcinogen.
Kidney Dysfunction. The most prevalent nonmalignant
						  effect observed among workers chronically exposed to cadmium is kidney
						  dysfunction. Initially, such dysfunction is manifested by proteinuria (Meridian
						  Research, Inc. 1989; Roth Associates, Inc. 1989). Proteinuria associated with
						  cadmium exposure is most commonly characterized by excretion of low-molecular
						  weight proteins (15,000-40,000 MW), accompanied by loss of electrolytes, uric
						  acid, calcium, amino acids, and phosphate. Proteins commonly excreted include
						  ß-2-microglobulin (B2M), retinol-binding protein (RBP), immunoglobulin
						  light chains, and lysozyme. Excretion of low molecular weight proteins is
						  characteristic of damage to the proximal tubules of the kidney (Iwao et al.
						  1980).
Exposure to cadmium also may lead to urinary excretion
						  of high-molecular weight proteins such as albumin, immunoglobulin G, and
						  glycoproteins (Meridian Research, Inc. 1989; Roth Associates, Inc. 1989).
						  Excretion of high-molecular weight proteins is indicative of damage to the
						  glomeruli of the kidney. Bernard et al. (1979) suggest that cadmium-associated
						  damage to the glomeruli and damage to the proximal tubules of the kidney
						  develop independently of each other, but may occur in the same
						  individual.
Several studies indicate that the onset of
						  low-molecular weight proteinuria is a sign of irreversible kidney damage
						  (Friberg et al. 1974; Roels et al. 1982; Piscator 1984; Elinder et al. 1985;
						  Smith et al. 1986). For many workers, once sufficiently elevated levels of B2M
						  are observed in association with cadmium exposure, such levels do not appear to
						  return to normal even when cadmium exposure is eliminated by removal of the
						  worker from the cadmium-contaminated work environment (Friberg, exhibit 29,
						  1990).
Some studies indicate that cadmium-induced proteinuria
						  may be progressive; levels of B2MU increase even after cadmium exposure has
						  ceased (Elinder et al. 1985). Other researchers have reached similar
						  conclusions (Frieburg testimony, OSHA docket exhibit 29, Elinder testimony,
						  OSHA docket exhibit 55, and OSHA docket exhibits 8-86B). Such observations are
						  not universal, however (Smith et al. 1986; Tsuchiya 1976). Studies in which
						  proteinuria has not been observed, however, may have initiated the reassessment
						  too early (Meridian Research, Inc.1989; Roth Associates, Inc. 1989; Roels
						  1989).
A quantitative assessment of the risks of developing
						  kidney dysfunction as a result of cadmium exposure was performed using the data
						  from Ellis et al. (1984) and Falck et al. (1983). Meridian Research, Inc.
						  (1989) and Roth Associates, Inc. (1989) employed several mathematical models to
						  evaluate the data from the 2 studies, and the results indicate that cumulative
						  cadmium exposure levels between 5 and 100
						  µg-years/m3 correspond with a
						  one-in-a-thousand probability of developing kidney dysfunction.
When cadmium exposure continues past the onset of early
						  kidney damage (manifested as proteinuria), chronic nephrotoxicity may occur
						  (Meridian Research, Inc. 1989; Roth Associates, Inc. 1989). Uremia, which is
						  the loss of the glomerulus' ability to adequately filter blood, may result.
						  This condition leads to severe disturbance of electrolyte concentrations, which
						  may result in various clinical complications including atherosclerosis,
						  hypertension, pericarditis, anemia, hemorrhagic tendencies, deficient cellular
						  immunity, bone changes, and other problems. Progression of the disease may
						  require dialysis or a kidney transplant.
Studies in which animals are chronically exposed to
						  cadmium confirm the renal effects observed in humans (Friberg et al. 1986).
						  Animal studies also confirm cadmium-related problems with calcium metabolism
						  and associated skeletal effects, which also have been observed among humans.
						  Other effects commonly reported in chronic animal studies include anemia,
						  changes in liver morphology, immunosuppression and hypertension. Some of these
						  effects may be associated with cofactors; hypertension, for example, appears to
						  be associated with diet, as well as with cadmium exposure. Animals injected
						  with cadmium also have shown testicular necrosis.
4.2 Objectives for Medical Monitoring
In keeping with the observation that renal disease
						  tends to be the earliest clinical manifestation of cadmium toxicity, the final
						  cadmium standard mandates that eligible workers must be medically monitored to
						  prevent this condition (as well as cadmium-induced cancer). The objectives of
						  medical-monitoring, therefore, are to: Identify workers at significant risk of
						  adverse health effects from excess, chronic exposure to cadmium; prevent future
						  cases of cadmium-induced disease; detect and minimize existing cadmium-induced
						  disease; and, identify workers most in need of medical intervention.
The overall goal of the medical monitoring program is
						  to protect workers who may be exposed continuously to cadmium over a 45-year
						  occupational lifespan. Consistent with this goal, the medical monitoring
						  program should assure that:
1. Current exposure levels remain sufficiently low to
						  prevent the accumulation of cadmium body burdens sufficient to cause disease in
						  the future by monitoring CDB as an indicator of recent cadmium exposure;
2. cumulative body burdens, especially among workers
						  with undefined historical exposures, remain below levels potentially capable of
						  leading to damage and disease by assessing CDU as an indicator of cumulative
						  exposure to cadmium; and,
3. health effects are not occurring among exposed
						  workers by determining B2MU as an early indicator of the onset of cadmium-
						  induced kidney disease.
4.3 Indicators of Cadmium Exposure and Disease
Cadmium is present in whole blood bound to albumin, in
						  erythrocytes, and as a metallothionein-cadmium complex. The
						  metallothionein-cadmium complex that represents the primary transport mechanism
						  for cadmium delivery to the kidney. CDB concentrations in the general,
						  nonexposed population average 1 µg Cd/l whole blood, with smokers
						  exhibiting higher levels (see Section
						  5.1.6). Data presented in Section
						  5.1.6 shows that 95% of the general
						  population not occupationally exposed to cadmium have CDB levels less than 5
						  µg Cd/l.
If total body burdens of cadmium remain low, CDB
						  concentrations indicate recent exposure (i.e., daily intake). This conclusion
						  is based on data showing that cigarette smokers exhibit CDB concentrations of
						  2-7 µg/l depending on the number of cigarettes smoked per day (Nordberg
						  and Nordberg 1988), while CDB levels for those who quit smoking return to
						  general population values (approximately 1 µg/l) within several weeks
						  (Lauwerys et al. 1976). Based on these observations, Lauwerys et al. (1976)
						  concluded that CDB has a biological half-life of a few weeks to less than 3
						  months. As indicated in Section
						  3.1.6, the upper 95th percentile
						  for CDB levels observed among those who are not occupationally exposed to
						  cadmium is 5 µg/l, which suggests that the absolute upper limit to the
						  range reported for smokers by Nordberg and Nordberg may have been affected by
						  an extreme value (i.e., beyond 2[SIGMA] above the mean).
Among occupationally-exposed workers, the occupational
						  history of exposure to cadmium must be evaluated to interpret CDB levels. New
						  workers, or workers with low exposures to cadmium, exhibit CDB levels that are
						  representative of recent exposures, similar to the general population. However,
						  for workers with a history of chronic exposure to cadmium, who have accumulated
						  significant stores of cadmium in the kidneys/liver, part of the CDB
						  concentrations appear to indicate body burden. If such workers are removed from
						  cadmium exposure, their CDB levels remain elevated, possibly for years,
						  reflecting prior long-term accumulation of cadmium in body tissues. This
						  condition tends to occur, however, only beyond some threshold exposure value,
						  and possibly indicates the capacity of body tissues to accumulate cadmium which
						  cannot be excreted readily (Friberg and Elinder 1988; Nordberg and Nordberg
						  1988).
CDU is widely used as an indicator of cadmium body
						  burdens (Nordberg and Nordberg 1988). CDU is the major route of elimination
						  and, when CDU is measured, it is commonly expressed either as µg Cd/l
						  urine (unadjusted), µg Cd/l urine (adjusted for specific gravity), or
						  µg Cd/g CRTU (see Section
						  5.2.1). The metabolic model for CDU
						  is less complicated than CDB, since CDU is dependent in large part on the body
						  (i.e., kidney) burden of cadmium. However, a small proportion of CDU still be
						  attributed to recent cadmium exposure, particularly if exposure to high
						  airborne concentrations of cadmium occurred. Note that CDU is subject to larger
						  interindividual and day-to-day variations than CDB, so repeated measurements
						  are recommended for CDU evaluations.
CDU is bound principally to metallothionein, regardless
						  of whether the cadmium originates from metallothionein in plasma or from the
						  cadmium pool accumulated in the renal tubules. Therefore, measurement of
						  metallothionein in urine may provide information similar to CDU, while avoiding
						  the contamination problems that may occur during collection and handling urine
						  for cadmium analysis (Nordberg and Nordberg 1988). However, a commercial method
						  for the determination of metallothionein at the sensitivity levels required
						  under the final cadmium rule is not currently available; therefore, analysis of
						  CDU is recommended.
Among the general population not occupationally exposed
						  to cadmium, CDU levels average less than 1 µg/l (see Section
						  5.2.7). Normalized for creatinine
						  (CRTU), the average CDU concentration of the general population is less than 1
						  µg/g CRTU. As cadmium accumulates over the lifespan, CDU increases with
						  age. Also, cigarette smokers may eventually accumulate twice the cadmium body
						  burden of nonsmokers, CDU is slightly higher in smokers than in nonsmokers,
						  even several years after smoking cessation (Nordberg and Nordberg 1988).
						  Despite variations due to age and smoking habits, 95% of those not
						  occupationally exposed to cadmium exhibit levels of CDU less than 3 µg/g
						  CRTU (based on the data presented in Section
						  5.2.7).
About 0.02% of the cadmium body burden is excreted
						  daily in urine. When the critical cadmium concentration (about 200 ppm) in the
						  kidney is reached, or if there is sufficient cadmium-induced kidney
						  dysfunction, dramatic increases in CDU are observed (Nordberg and Nordberg
						  1988). Above 200 ppm, therefore, CDU concentrations cease to be an indicator of
						  cadmium body burden, and are instead an index of kidney failure.
Proteinuria is an index of kidney dysfunction, and is
						  defined by OSHA to be a material impairment. Several small proteins may be
						  monitored as markers for proteinuria. Below levels indicative of proteinuria,
						  these small proteins may be early indicators of increased risk of
						  cadmium-induced renal tubular disease. Analytes useful for monitoring
						  cadmium-induced renal tubular damage include:
1. ß-2-Microglobulin (B2M), currently the most
						  widely used assay for detecting kidney dysfunction, is the best characterized
						  analyte available (Iwao et al. 1980; Chia et al. 1989);
2. Retinol Binding Protein (RBP) is more stable than
						  B2M in acidic urine (i.e., B2M breakdown occurs if urinary pH is less than 5.5;
						  such breakdown may result in false [i.e., low] B2M values [Bernard and
						  Lauwerys, 1990]);
3. N-Acetyl-B-Glucosaminidase (NAG) is the analyte of
						  an assay that is simple, inexpensive, reliable, and correlates with cadmium
						  levels under 10 µg/g CRTU, but the assay is less sensitive than RBP or
						  B2M (Kawada et al. 1989);
4. Metallothionein (MT) correlates with cadmium and B2M
						  levels, and may be a better predictor of cadmium exposure than CDU and B2M
						  (Kawada et al. 1989);
5. Tamm-Horsfall Glycoprotein (THG) increases slightly
						  with elevated cadmium levels, but this elevation is small compared to increases
						  in urinary albumin, RBP, or B2M (Bernard and Lauwerys 1990);
6. Albumin (ALB), determined by the biuret method, is
						  not sufficiently sensitive to serve as an early indicator of the onset of renal
						  disease (Piscator 1962);
7. Albumin (ALB), determined by the Amido Black method,
						  is sensitive and reproducible, but involves a time-consuming procedure
						  (Piscator 1962);
8. Glycosaminoglycan (GAG) increases among cadmium
						  workers, but the significance of this effect is unknown because no relationship
						  has been found between elevated GAG and other indices of tubular damage
						  (Bernard and Lauwerys 1990);
9. Trehalase seems to increase earlier than B2M during
						  cadmium exposure, but the procedure for analysis is complicated and unreliable
						  (Iwata et al. 1988); and,
10. Kallikrein is observed at lower concentrations
						  among cadmium-exposed workers than among normal controls (Roels et al.
						  1990).
Of the above analytes, B2M appears to be the most
						  widely used and best characterized analyte to evaluate the presence/absence, as
						  well as the extent of, cadmium-induced renal tubular damage (Kawada, Koyama,
						  and Suzuki 1989; Shaikh and Smith 1984; Nogawa 1984). However, it is important
						  that samples be collected and handled so as to minimize B2M degradation under
						  acidic urine conditions.
The threshold value of B2MU commonly used to indicate
						  the presence of kidney damage 300 µg/g CRTU (Kjellstrom et al. 1977a;
						  Buchet et al. 1980; and Kowal and Zirkes 1983). This value represents the upper
						  95th or 97.5th percentile level of urinary excretion observed among those
						  without tubular dysfunction (Elinder, exbt L-140-45, OSHA docket H057A). In
						  agreement with these conclusions, the data presented in Section
						  5.3.7 of this protocol generally
						  indicate that the level of 300 µg/g CRTU appears to define the boundary
						  for kidney dysfuncion. It is not clear, however, that this level represents the
						  upper 95th percentile of values observed among those who fail to demonstrate
						  proteinuria effects.
Although elevated B2MU levels appear to be a fairly
						  specific indicator of disease associated with cadmium exposure, other
						  conditions that may lead to elevated B2MU levels include high fevers from
						  influenza, extensive physical exercise, renal disease unrelated to cadmium
						  exposure, lymphomas, and AIDS (Iwao et al. 1980; Schardun and van Epps 1987).
						  Elevated B2M levels observed in association with high fevers from influenza or
						  from extensive physical exercise are transient, and will return to normal
						  levels once the fever has abated or metabolic rates return to baseline values
						  following exercise. The other conditions linked to elevated B2M levels can be
						  diagnosed as part of a properly- designed medical examination. Consequently,
						  monitoring B2M, when accompanied by regular medical examinations and CDB and
						  CDU determinations (as indicators of present and past cadmium exposure), may
						  serve as a specific, early indicator of cadmium- induced kidney damage.
4.4 Criteria for Medical Monitoring of Cadmium
						  Workers
Medical monitoring mandated by the final cadmium rule
						  includes a combination of regular medical examinations and periodic monitoring
						  of 3 analytes: CDB, CDU and B2MU. As indicated above, CDB is monitored as an
						  indicator of current cadmium exposure, while CDU serves as an indicator of the
						  cadmium body burden; B2MU) is assessed as an early marker of irreversible
						  kidney damage and disease.
The final cadmium rule defines a series of action
						  levels that have been developed for each of the 3 analytes to be monitored.
						  These action levels serve to guide the responsible physician through a
						  decision-making process. For each action level that is exceeded, a specific
						  response is mandated. The sequence of action levels, and the attendant actions,
						  are described in detail in the final cadmium rule.
Other criteria used in the medical decision-making
						  process relate to tests performed during the medical examination (including a
						  determination of the ability of a worker to wear a respirator). These criteria,
						  however, are not affected by the results of the analyte determinations
						  addressed in the above paragraphs and, consequently, will not be considered
						  further in these guidelines.
4.5 Defining to Quality and Proficiency of the Analyte
						  Determinations
As noted above in Sections
						  2 and
						  3, the
quality
of
						  a measurement should be defined along with its
value
to
						  properly interpret the results. Generally, it is necessary to know the
accuracy
and the
precision
of a measurement
						  before it can be properly evaluated. The precision of the data from a specific
						  laboratory indicates the extent to which the repeated measurements of the same
						  sample vary within that laboratory. The accuracy of the data provides an
						  indication of the extent to which these results deviate from average results
						  determined from many laboratories performing the same measurement (i.e., in the
						  absence of an independent determination of the true value of a measurement).
						  Note that terms are defined operationally relative to the manner in which they
						  will be used in this protocol. Formal definitions for the terms in italics used
						  in this section can be found in the list of definitions (Section
						  2).
Another data quality criterion required to properly
						  evaluate measurement results is the
limit of detection
of that
						  measurement. For measurements to be useful, the range of the measurement which
						  is of interest for biological monitoring purposes must lie entirely above the
						  limit of detection defined for that measurement.
The overall quality of a laboratory's results is termed
						  the
performance
of that laboratory. The degree to which a
						  laboratory satisfies a minimum performance level is referred to as the
proficiency
of the laboratory. A successful medical monitoring
						  program, therefore, should include procedures developed for monitoring and
						  recording laboratory performance; these procedures can be used to identify the
						  most proficient laboratories.
5.0 Overview of Medical Monitoring Tests for CDB, CDU,
						  B2MU and CRTU
To evaluate whether available methods for assessing
						  CDB, CDU, B2MU and CRTU are adequate for determining the parameters defined by
						  the proposed action levels, it is necessary to review procedures available for
						  sample collection, preparation and analysis. A variety of techniques for these
						  purposes have been used historically for the determination of cadmium in
						  biological matrices (including CDB and CDU), and for the determination of
						  specific proteins in biological matrices (including B2MU). However, only the
						  most recent techniques are capable of satisfying the required accuracy,
						  precision and sensitivity (i.e., limit of detection) for monitoring at the
						  levels mandated in the final cadmium rule, while still facilitating automated
						  analysis and rapid processing.
5.1 Measuring Cadmium in Blood (CDB)
Analysis of biological samples for cadmium requires
						  strict analytical discipline regarding collection and handling of samples. In
						  addition to occupational settings, where cadmium contamination would be
						  apparent, cadmium is a ubiquitous environmental contaminant, and much care
						  should be exercised to ensure that samples are not contaminated during
						  collection, preparation or analysis. Many common chemical reagents are
						  contaminated with cadmium at concentrations that will interfere with cadmium
						  analysis; because of the widespread use of cadmium compounds as colored
						  pigments in plastics and coatings, the analyst should continually monitor each
						  manufacturer's chemical reagents and collection containers to prevent
						  contamination of samples.
Guarding against cadmium contamination of biological
						  samples is particularly important when analyzing blood samples because cadmium
						  concentrations in blood samples from nonexposed populations are generally less
						  than 2 µg/l (2 ng/ml), while occupationally-exposed workers can be at
						  medical risk to cadmium toxicity if blood concentrations exceed 5 µg/l
						  (ACGIH 1991 and 1992). This narrow margin between exposed and unexposed samples
						  requires that exceptional care be used in performing analytic determinations
						  for biological monitoring for occupational cadmium exposure.
Methods for quantifying cadmium in blood have improved
						  over the last 40 years primarily because of improvements in analytical
						  instrumentation. Also, due to improvements in analytical techniques, there is
						  less need to perform extensive multi-step sample preparations prior to
						  analysis. Complex sample preparation was previously required to enhance method
						  sensitivity (for cadmium), and to reduce interference by other metals or
						  components of the sample.
5.1.1 Analytical Techniques Used to Monitor Cadmium in
						  Biological Matrices
Click
						  here to view image
A number of analytical techniques have been used for
						  determining cadmium concentrations in biological materials. A summary of the
						  characteristics of the most widely employed techniques is presented in Table 3.
						  The technique most suitable for medical monitoring for cadmium is atomic
						  absorption spectroscopy (AAS).
To obtain a measurement using AAS, a light source
						  (i.e., hollow cathode or lectrode-free discharge lamp) containing the element
						  of interest as the cathode, is energized and the lamp emits a spectrum that is
						  unique for that element. This light source is focused through a sample cell,
						  and a selected wavelength is monitored by a monochrometer and photodetector
						  cell. Any ground state atoms in the sample that match those of the lamp element
						  and are in the path of the emitted light may absorb some of the light and
						  decrease the amount of light that reaches the photodetector cell. The amount of
						  light absorbed at each characteristic wavelength is proportional to the number
						  of ground state atoms of the corresponding element that are in the pathway of
						  the light between the source and detector.
To determine the amount of a specific metallic element
						  in a sample using AAS, the sample is dissolved in a solvent and aspirated into
						  a high-temperature flame as an aerosol. At high temperatures, the solvent is
						  rapidly evaporated or decomposed and the solute is initially solidified; the
						  majority of the sample elements then are transformed into an atomic vapor.
						  Next, a light beam is focused above the flame and the amount of metal in the
						  sample can be determined by measuring the degree of absorbance of the atoms of
						  the target element released by the flame at a characteristic wavelength.
A more refined atomic absorption technique, flameless
						  AAS, substitutes an electrothermal, graphite furnace for the flame. An aliquot
						  (10-100 µl) of the sample is pipetted into the cold furnace, which is
						  then heated rapidly to generate an atomic vapor of the element.
AAS is a sensitive and specific method for the
						  elemental analysis of metals; its main drawback is nonspecific background
						  absorbtion and scattering of the light beam by particles of the sample as it
						  decomposes at high temperatures; nonspecific absorbance reduces the sensitivity
						  of the analytical method. The problem of nonspecific absorbance and scattering
						  can be reduced by extensive sample pretreatment, such as ashing and/or acid
						  digestion of the sample to reduce its organic content.
Current AAS instruments employ background correction
						  devices to adjust electronically for background absorbtion and scattering. A
						  common method to correct for background effects is to use a deuterium arc lamp
						  as a second light source. A continuum light source, such as the deuterium lamp,
						  emits a broad spectrum of wavelengths instead of specific wavelengths
						  characteristic of a particular element, as with the hollow cathode tube. With
						  this system, light from the primary source and the continuum source are passed
						  alternately through the sample cell. The target element effectively absorbs
						  light only from the primary source (which is much brighter than the continuum
						  source at the characteristic wavelengths), while the background matrix absorbs
						  and scatters light from both sources equally. Therefore, when the ratio of the
						  two beams is measured electronically, the effect of nonspecific background
						  absorption and scattering is eliminated. A less common, but more sophisticated,
						  background correction system is based on the Zeeman effect, which uses a
						  magnetically- activated light polarizer to compensate electronically for
						  nonspecific absorbtion and scattering.
Atomic emission spectroscopy with inductively-coupled
						  argon plasma (AES-ICAP) is widely used to analyze for metals. With this
						  instrument, the sample is aspirated into an extremely hot argon plasma flame,
						  which excites the metal atoms; emission spectra specific for the sample element
						  then are generated. The quanta of emitted light passing through a monochrometer
						  are amplified by photomultiplier tubes and measured by a photodetector to
						  determine the amount of metal in the sample. An advantage of AES- ICAP over AAS
						  is that multi-elemental analyses of a sample can be performed by simultaneously
						  measuring specific elemental emission energies. However, AES-ICAP lacks the
						  sensitivity of AAS, exhibiting a limit of detection which is higher than the
						  limit of detection for graphite-furnace AAS (Table 3).
Neutron activation (NA) analysis and isotope dilution
						  mass spectrometry (IDMS) are 2 additional, but highly specialized, methods that
						  have been used for cadmium determinations. These methods are expensive because
						  they require elaborate and sophisticated instrumentation.
NA analysis has the distinct advantage over other
						  analytical methods of being able to determine cadmium body burdens in specific
						  organs (e.g., liver, kidney)
in vivo
(Ellis et al. 1983).
						  Neutron bombardment of the target transforms cadmium- 13 to cadmium-114, which
						  promptly decays ( < 10-14 sec) to its ground
						  state, emitting gamma rays that are measured using large gamma detectors;
						  appropriate shielding and instrumentation are required when using this
						  method.
IDMS analysis, a definitive but laborious method, is
						  based on the change in the ratio of 2 isotopes of cadmium (cadmium 111 and 112)
						  that occurs when a known amount of the element (with an artificially altered
						  ratio of the same isotopes [i.e., a cadmium 111 "spike"] is added to a weighed
						  aliquot of the sample (Michiels and De Bievre 1986).
5.1.2 Methods Developed for CDB Determinations
A variety of methods have been used for preparing and
						  analyzing CDB samples; most of these methods rely on one of the analytical
						  techniques described above. Among the earliest reports, Princi (1947) and Smith
						  et al. (1955) employed a colorimetric procedure to analyze for CDB and CDU.
						  Samples were dried and digested through several cycles with concentrated
						  mineral acids (HNO3 and
						  H2SO4) and hydrogen peroxide
						  (H2O2). The digest was
						  neutralized, and the cadmium was complexed with diphenylthiocarbazone and
						  extracted with chloroform. The dithizone-cadmium complex then was quantified
						  using a spectrometer.
Colorimetric procedures for cadmium analyses were
						  replaced by methods based on atomic absorption spectroscopy (AAS) in the early
						  1960s, but many of the complex sample preparation procedures were retained.
						  Kjellstrom (1979) reports that in Japanese, American and Swedish laboratories
						  during the early 1970s, blood samples were wet ashed with mineral acids or
						  ashed at high temperature and wetted with nitric acid. The cadmium in the
						  digest was complexed with metal chelators including diethyl dithiocarbamate
						  (DDTC), ammonium pyrrolidine dithiocarbamate (APDC) or diphenylthiocarbazone
						  (dithizone) in ammonia-citrate buffer and extracted with methyl isobutyl ketone
						  (MIBK). The resulting solution then was analyzed by flame AAS or graphite-
						  furnace AAS for cadmium determinations using deuterium-lamp background
						  correction.
In the late 1970s, researchers began developing simpler
						  preparation procedures. Roels et al. (1978) and Roberts and Clark (1986)
						  developed simplified digestion procedures. Using the Roberts and Clark method,
						  a 0.5 ml aliquot of blood is collected and transferred to a digestion tube
						  containing 1 ml concentrated HNO3. The blood is then
						  digested at 110°C for 4 hours. The sample is reduced in volume by continued
						  heating, and 0.5 ml 30% H2O2 is
						  added as the sample dries. The residue is dissolved in 5 ml dilute (1%)
						  HNO3, and 20 µl of sample is then analyzed by
						  graphite-furnace AAS with deuterium-background correction.
The current trend in the preparation of blood samples
						  is to dilute the sample and add matrix modifiers to reduce background
						  interference, rather than digesting the sample to reduce organic content. The
						  method of Stoeppler and Brandt (1980), and the abbreviated procedure published
						  in the American Public Health Association's (APHA) Methods for Biological
						  Monitoring (1988), are straightforward and are nearly identical. For the APHA
						  method, a small aliquot (50-300 µl) of whole blood that has been
						  stabilized with ethylenediaminetetraacetate (EDTA) is added to 1.0 ml 1M
						  HNO3, vigorously shaken and centrifuged. Aliquots (10-25
						  µl) of the supernatant then are then analyzed by graphite-furnace AAS
						  with appropriate background correction.
Using the method of Stoeppler and Brandt (1980),
						  aliquots (50-200 µl) of whole blood that have been stabilized with EDTA
						  are pipetted into clean polystyrene tubes and mixed with 150-600 µl of 1
						  M HNO3. After vigorous shaking, the solution is
						  centrifuged and a 10-25 µl aliquot of the supernatant then is analyzed by
						  graphite-furnace AAS with appropriate background correction.
Claeys-Thoreau (1982) and DeBenzo et al. (1990) diluted
						  blood samples at a ratio of 1:10 with a matrix modifier (0.2% Triton X-100, a
						  wetting agent) for direct determinations of CDB. DeBenzo et al. also
						  demonstrated that aqueous standards of cadmium, instead of spiked, whole-blood
						  samples, could be used to establish calibration curves if standards and samples
						  are treated with additional small volumes of matrix modifiers (i.e., 1%
						  HNO3, 0.2% ammonium hydrogenphosphate and 1 mg/ml
						  magnesium salts.)
These direct dilution procedures for CDB analysis are
						  simple and rapid. Laboratories can process more than 100 samples a day using a
						  dedicated graphite-furnace AAS, an auto-sampler, and either a Zeeman- or a
						  deuterium-background correction system. Several authors emphasize using optimum
						  settings for graphite-furnace temperatures during the drying, charring, and
						  atomization processes associated with the flameless AAS method, and the need to
						  run frequent QC samples when performing automated analysis.
5.1.3 Sample Collection and Handling
Sample collection procedures are addressed primarily to
						  identify ways to minimize the degree of variability that may be introduced by
						  sample collection during medical monitoring. It is unclear at this point the
						  extent to which collection procedures contribute to variability among CDB
						  samples. Sources of variation that may result from sampling procedures include
						  time-of-day effects and introduction of external contamination during the
						  collection process. To minimize these sources, strict adherence to a sample
						  collection protocol is recommended. Such a protocol must include provisions for
						  thorough cleaning of the site from which blood will be extracted; also, every
						  effort should be made to collect samples near the same time of day. It is also
						  important to recognize that under the recent OSHA blood-borne pathogens
						  standard (29 CFR
						  1910.1030), blood samples and certain body
						  fluids must be handled and treated as if they are infectious.
5.1.4 Best Achievable Performance
The best achievable performance using a particular
						  method for CDB determinations is assumed to be equivalent to the performance
						  reported by research laboratories in which the method was developed.
For their method, Roberts and Clark (1986) demonstrated
						  a limit of detection of 0.4 µg Cd/l in whole blood, with a linear
						  response curve from 0.4 to 16.0 µg Cd/l. They report a coefficient of
						  variation (CV) of 6.7% at 8.0 µg/l.
The APHA (1988) reports a range of 1.0-25 µg/l,
						  with a CV of 7.3% (concentration not stated). Insufficient documentation was
						  available to critique this method.
Stoeppler and Brandt (1980) achieved a detection limit
						  of 0.2 µg Cd/l whole blood, with a linear range of 0.4-12.0 µg
						  Cd/l, and a CV of 15-30%, for samples at 1.0Ã¦g µl.
						  Improved precision (CV of 3.8%) was reported for CDB concentrations at 9.3
						  µg/l.
5.1.5 General Method Performance
For any particular method, the performance expected
						  from commercial laboratories may be somewhat lower than that reported by the
						  research laboratory in which the method was developed. With participation in
						  appropriate proficiency programs and use of a proper in-house QA/QC program
						  incorporating provisions for regular corrective actions, the performance of
						  commercial laboratories is expected to approach that reported by research
						  laboratories. Also, the results reported for existing proficiency programs
						  serve as a gauge of the likely level of performance that currently can be
						  expected from commercial laboratories offering these analyses.
Weber (1988) reports on the results of the proficiency
						  program run by the Centre de Toxicologie du Quebec (CTQ). As indicated
						  previously, participants in that program receive 18 blood samples per year
						  having cadmium concentrations ranging from 0.2-20 µg/l. Currently, 76
						  laboratories are participating in this program. The program is established for
						  several analytes in addition to cadmium, and not all of these laboratories
						  participate in the cadmium proficiency-testing program.
Under the CTQ program, cadmium results from individual
						  laboratories are compared against the consensus mean derived for each sample.
						  Results indicate that after receiving 60 samples (i.e., after participation for
						  approximately three years), 60% of the laboratories in the program are able to
						  report results that fall within ± 1 µg/l or 15% of the mean,
						  whichever is greater. (For this procedure, the 15% criterion was applied to
						  concentrations exceeding 7 µg/l.) On any single sample of the last 20
						  samples, the percentage of laboratories falling within the specified range is
						  between 55 and 80%.
The CTQ also evaluates the performance of participating
						  laboratories against a less severe standard: ± 2 µg/l or 15% of
						  the mean, whichever is greater (Weber 1988); 90% of participating laboratories
						  are able to satisfy this standard after approximately 3 years in the program.
						  (The 15% criterion is used for concentrations in excess of 13 µg/l.) On
						  any single sample of the last 15 samples, the percentage of laboratories
						  falling within the specified range is between 80 and 95% (except for a single
						  test for which only 60% of the laboratories achieved the desired
						  performance).
Based on the data presented in Weber (1988), the CV for
						  analysis of CDB is nearly constant at 20% for cadmium concentrations exceeding
						  5 µg/l, and increases for cadmium concentrations below 5 µg/l. At 2
						  µg/l, the reported CV rises to approximately 40%. At 1 µg/l, the
						  reported CV is approximately 60%.
Participating laboratories also tend to overestimate
						  concentrations for samples exhibiting concentrations less than 2 µg/l
						  (see Figure 11 of Weber 1988). This problem is due in part to the proficiency
						  evaluation criterion that allows reporting a minimum ± 2.0 µg/l
						  for evaluated CDB samples. There is currently little economic or regulatory
						  incentive for laboratories participating in the CTQ program to achieve greater
						  accuracy for CDB samples containing cadmium at concentrations less than 2.0
						  µg/l, even if the laboratory has the experience and competency to
						  distinguish among lower concentrations in the samples obtained from the
						  CTQ.
The collective experience of international agencies and
						  investigators demonstrate the need for a vigorous QC program to ensure that CDB
						  values reported by participating laboratories are indeed reasonably accurate.
						  As Friberg (1988) stated:
"Information about the quality of published data has
						  often been lacking. This is of concern as assessment of metals in trace
						  concentrations in biological media are fraught with difficulties from the
						  collection, handling, and storage of samples to the chemical analyses. This has
						  been proven over and over again from the results of interlaboratory testing and
						  quality control exercises. Large variations in results were reported even from
						  "experienced" laboratories."
The UNEP/WHO global study of cadmium biological
						  monitoring set a limit for CDB accuracy using the maximum allowable deviation
						  method at Y=X±(0.1X+1) for a targeted concentration of 10 µg Cd/l
						  (Friberg and Vahter 1983). The performance of participating laboratories over a
						  concentration range of 1.5-12 µg/l was reported by Lind et al. (1987). Of
						  the 3 QC runs conducted during 1982 and 1983, 1 or 2 of the 6 laboratories
						  failed each run. For the years 1983 and 1985, between zero and 2 laboratories
						  failed each of the consecutive QC runs.
In another study (Vahter and Friberg 1988), QC samples
						  consisting of both external (unknown) and internal (stated) concentrations were
						  distributed to laboratories participating in the epidemiology research. In this
						  study, the maximum acceptable deviation between the regression analysis of
						  reported results and reference values was set at Y=X±(0.05X+0.2) for a
						  concentration range of 0.3-5.0 µg Cd/l. It is reported that only 2 of 5
						  laboratories had acceptable data after the first QC set, and only 1 of 5
						  laboratories had acceptable data after the second QC set. By the fourth QC set,
						  however, all 5 laboratories were judged proficient.
The need for high quality CDB monitoring is apparent
						  when the toxicological and biological characteristics of this metal are
						  considered; an increase in CDB from 2 to 4 µg/l could cause a doubling of
						  the cadmium accumulation in the kidney, a critical target tissue for selective
						  cadmium accumulation (Nordberg and Nordberg 1988).
Historically, the CDC's internal QC program for CDB
						  cadmium monitoring program has found achievable accuracy to be ± 10% of
						  the true value at CDB concentrations > 5.0 µg/l (Paschal 1990). Data
						  on the performance of laboratories participating in this program currently are
						  not available.
5.1.6 Observed CDB Concentrations
As stated in Section
						  4.3, CDB concentrations are
						  representative of ongoing levels of exposure to cadmium. Among those who have
						  been exposed chronically to cadmium for extended periods, however, CDB may
						  contain a component attributable to the general cadmium body burden.
5.1.6.1 CDB concentrations among unexposed
						  samples
Numerous studies have been conducted examining CDB
						  concentrations in the general population, and in control groups used for
						  comparison with cadmium-exposed workers. A number of reports have been
						  published that present erroneously high values of CDB (Nordberg and Nordberg
						  1988). This problem was due to contamination of samples during sampling and
						  analysis, and to errors in analysis. Early AAS methods were not sufficiently
						  sensitive to accurately estimate CDB concentrations.
Table 4 presents results of recent studies reporting
						  CDB levels for the general U.S. population not exposed occupationally to
						  cadmium. Other surveys of tissue cadmium using U.S. samples and conducted as
						  part of a cooperative effort among Japan, Sweden and the U.S., did not collect
						  CDB data because standard analytical methodologies were unavailable, and
						  because of analytic problems (Kjellstrom 1979; SWRI 1978).
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Arithmetic and/or geometric means and standard
						  deviations are provided in Table 4 for measurements among the populations
						  defined in each study listed. The range of reported measurements and/or the 95%
						  upper and lower confidence intervals for the means are presented when this
						  information was reported in a study. For studies reporting either an arithmetic
						  or geometric standard deviation along with a mean, the lower and upper 95th
						  percentile for the distribution also were derived and reported in the
						  table.
The data provided in Table 4 from Kowal et al. (1979)
						  are from studies conducted between 1974 and 1976 evaluating CDB levels for the
						  general population in Chicago, and are considered to be representative of the
						  U.S. population. These studies indicate that the average CDB concentration
						  among those not occupationally exposed to cadmium is approximately 1
						  µg/l.
In several other studies presented in Table 4,
						  measurements are reported separately for males and females, and for smokers and
						  nonsmokers. The data in this table indicate that similar CDB levels are
						  observed among males and females in the general population, but that smokers
						  tend to exhibit higher CDB levels than nonsmokers. Based on the Kowal et al.
						  (1979) study, smokers not occupationally exposed to cadmium exhibit an average
						  CDB level of 1.4 µg/l.
In general, nonsmokers tend to exhibit levels ranging
						  to 2 µg/l, while levels observed among smokers range to
						  5Ã¦g/l. Based on the data presented in Table 4, 95% of those not
						  occupationally exposed to cadmium exhibit CDB levels less than 5
						  µg/l.
5.1.6.2 CDB concentrations among exposed
						  workers.
Table 5 is a summary of results from studies reporting
						  CDB levels among workers exposed to cadmium in the work place. As in Table 4,
						  arithmetic and/or geometric means and standard deviations are provided if
						  reported in the listed studies. The absolute range, or the 95% confidence
						  interval around the mean, of the data in each study are provided when reported.
						  In addition, the lower and upper 95th percentile of the distribution are
						  presented for each study i which a mean and corresponding standard deviation
						  were reported. Table 5 also provides estimates of the duration, and level, of
						  exposure to cadmium in the work place if these data were reported in the listed
						  studies. The data presented in Table 5 suggest that CDB levels are dose
						  related. Sukuri et al. (1983) show that higher CDB levels are observed among
						  workers experiencing higher work place exposure. This trend appears to be true
						  of every the studies listed in the table.
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CDB levels reported in Table 5 are higher among those
						  showing signs of cadmium-related kidney damage than those showing no such
						  damage. Lauwerys et al. (1976) report CDB levels among workers with kidney
						  lesions that generally are above the levels reported for workers without kidney
						  lesions. Ellis et al. (1983) report a similar observation comparing workers
						  with and without renal dysfunction, although they found more overlap between
						  the 2 groups than Lauwerys et al.
The data in Table 5 also indicate that CDB levels are
						  higher among those experiencing current occupational exposure than those who
						  have been removed from such exposure. Roels et al. (1982) indicate that CDB
						  levels observed among workers experiencing ongoing exposure in the work place
						  are almost entirely above levels observed among workers removed from such
						  exposure. This finding suggests that CDB levels decrease once cadmium exposure
						  has ceased.
A comparison of the data presented in Tables 4 and 5
						  indicates that CDB levels observed among cadmium-exposed workers is
						  significantly higher than levels observed among the unexposed groups. With the
						  exception of 2 studies presented in Table 5 (1 of which includes former workers
						  in the sample group tested), the lower 95th percentile for CDB levels among
						  exposed workers are greater than 5 µg/l, which is the value of the upper
						  95th percentile for CDB levels observed among those who are not occupationally
						  exposed. Therefore, a CDB level of 5 µg/l represents a threshold above
						  which significant work place exposure to cadmium may be occurring.
5.1.7 Conclusions and Recommendations for CDB
Based on the above evaluation, the following
						  recommendations are made for a CDB proficiency program.
5.1.7.1 Recommended method
The method of Stoeppler and Brandt (1980) should be
						  adopted for analyzing CDB. This method was selected over other methods for its
						  straightforward sample-preparation procedures, and because limitations of the
						  method were described adequately. It also is the method used by a plurality of
						  laboratories currently participating in the CTQ proficiency program. In a
						  recent CTQ interlaboratory comparison report (CTQ 1991), analysis of the
						  methods used by laboratories to measure CDB indicates that 46% (11 of 24) of
						  the participating laboratories used the Stoeppler and Brandt methodology
						  (HNO3 deproteinization of blood followed by analysis of
						  the supernatant by GF-AAS). Other CDB methods employed by participating
						  laboratories identified in the CTQ report include dilution of blood (29%), acid
						  digestion (12%) and miscellaneous methods (12%).
Laboratories may adopt alternate methods, but it is the
						  responsibility of the laboratory to demonstrate that the alternate methods meet
						  the data quality objectives defined for the Stoeppler and Brandt method (see
						  Section 5.1.7.2 below).
5.1.7.2 Data quality objectives
Based on the above evaluation, the following data
						  quality objectives (DQOs) should facilitate interpretation of analytical
						  results.
Limit of Detection. 0.5 µg/l should be achievable
						  using the Stoeppler and Brandt method. Stoeppler and Brandt (1980) report a
						  limit of detection equivalent to <0.2 µg/l in whole blood using 2
						  µl aliquots of deproteinized, diluted blood samples.
Accuracy. Initially, some of the laboratories
						  performing CDB measurements may be expected to satisfy criteria similar to the
						  less severe criteria specified by the CTQ program, i.e., measurements within 2
						  µg/l or 15% (whichever is greater) of the target value. About 60% of the
						  laboratories enrolled in the CTQ program could meet this criterion on the first
						  proficiency test (Weber 1988).
Currently, approximately 12 laboratories in the CTQ
						  program are achieving an accuracy for CDB analysis within the more severe
						  constraints of ±1 µg/l or 15% (whichever is greater). Later, as
						  laboratories gain experience, they should achieve the level of accuracy
						  exhibited by these 12 laboratories. The experience in the CTQ program has shown
						  that, even without incentives, laboratories benefit from the feedback of the
						  program; after they have analyzed 40-50 control samples from the program,
						  performance improves to the point where about 60% of the laboratories can meet
						  the stricter criterion of ±1 µg/l or 15% (Weber 1988). Thus, this
						  stricter target accuracy is a reasonable DQO.
Precision. Although Stoeppler and Brandt (1980) suggest
						  that a coefficient of variation (CV) near 1.3% (for a 10 µg/l
						  concentration) is achievable for within-run reproducibility, it is recognized
						  that other factors affecting within- and between- run comparability will
						  increase the achievable CV. Stoeppler and Brandt (1980) observed CVs that were
						  as high as 30% for low concentrations (0.4 µg/l), and CVs of less than 5%
						  for higher concentrations.
For internal QC samples (see Section
						  3.3.1), laboratories should to
						  attain an overall precision near 25%. For CDB samples with concentrations less
						  than 2 µg/l, a target precision of 40% is reasonable, while precisions of
						  20% should be achievable for concentrations greater than 2 µg/l. Although
						  these values are more strict than values observed in the CTQ interlaboratory
						  program reported by Webber (1988), they are within the achievable limits
						  reported by Stoeppler and Brandt (1980).
5.1.7.3 Quality assurance/quality control
Commercial laboratories providing measurement of CDB
						  should adopt an internal QA/QC program that incorporates the following
						  components: Strict adherence to the selected method, including all calibration
						  requirements; regular incorporation of QC samples during actual runs; a
						  protocol for corrective actions, and documentation of these actions; and,
						  participation in an interlaboratory proficiency program. Note that the
						  nonmandatory QA/QC program presented in Attachment 3 is based on the Stoeppler
						  and Brandt method for CDB analysis. Should an alternate method be adopted, the
						  laboratory should develop a QA/QC program satisfying the provisions of Section
						  3.3.1.
5.2 Measuring Cadmium in Urine (CDU)
As in the case of CDB measurement, proper determination
						  of CDU requires strict analytical discipline regarding collection and handling
						  of samples. Because cadmium is both ubiquitous in the environment and employed
						  widely in coloring agents for industrial products that may be used during
						  sample collection, preparation and analysis, care should be exercised to ensure
						  that samples are not contaminated during the sampling procedure.
Methods for CDU determination share many of the same
						  features as those employed for the determination of CDB. Thus, changes and
						  improvements to methods for measuring CDU over the past 40 years parallel those
						  used to monitor CDB. The direction of development has largely been toward the
						  simplification of sample preparation techniques made possible because of
						  improvements in analytic techniques.
5.2.1 Units of CDU Measurement
Procedures adopted for reporting CDU concentrations are
						  not uniform. In fact, the situation for reporting CDU is more complicated than
						  for CDB, where concentrations are normalized against a unit volume of whole
						  blood.
Concentrations of solutes in urine vary with several
						  biological factors (including the time since last voiding and the volume of
						  liquid consumed over the last few hours); as a result, solute concentrations
						  should be normalized against another characteristic of urine that represents
						  changes in solute concentrations. The 2 most common techniques are either to
						  standardize solute concentrations against the concentration of creatinine, or
						  to standardize solute concentrations against the specific gravity of the urine.
						  Thus, CDU concentrations have been reported in the literature as "uncorrected"
						  concentrations of cadmium per volume of urine (i.e., µg Cd/l urine),
						  "corrected" concentrations of cadmium per volume of urine at a standard
						  specific gravity (i.e., µg Cd/l urine at a specific gravity of 1.020), or
						  "corrected" mass concentration per unit mass of creatinine (i.e., µg Cd/g
						  creatinine). (CDU concentrations [whether uncorrected or corrected for specific
						  gravity, or normalized to creatinine] occasionally are reported in nanomoles
						  [i.e., nmoles] of cadmium per unit mass or volume. In this protocol, these
						  values are converted to µg of cadmium per unit mass or volume using 89
						  nmoles of cadmium=10 µg.)
While it is agreed generally that urine values of
						  analytes should be normalized for reporting purposes, some debate exists over
						  what correction method should be used. The medical community has long favored
						  normalization based on creatinine concentration, a common urinary constituent.
						  Creatinine is a normal product of tissue catabolism, is excreted at a uniform
						  rate, and the total amount excreted per day is constant on a day-to-day basis
						  (NIOSH 1984b). While this correction method is accepted widely in Europe, and
						  within some occupational health circles, Kowals (1983) argues that the use of
						  specific gravity (i.e., total solids per unit volume) is more straightforward
						  and practical (than creatinine) in adjusting CDU values for populations that
						  vary by age or gender.
Kowals (1983) found that urinary creatinine (CRTU) is
						  lower in females than males, and also varies with age. Creatinine excretion is
						  highest in younger males (20-30 years old), decreases at middle age (50-60
						  years), and may rise slightly in later years. Thus, cadmium concentrations may
						  be underestimated for some workers with high CRTU levels.
Within a single void urine collection, urine
						  concentration of any analyte will be affected by recent consumption of large
						  volumes of liquids, and by heavy physical labor in hot environments. The
						  absolute amount of analyte excreted may be identical, but concentrations will
						  vary widely so that urine must be corrected for specific gravity (i.e., to
						  normalize concentrations to the quantity of total solute) using a fixed value
						  (e.g., 1.020 or 1.024). However, since heavy-metal exposure may increase
						  urinary protein excretion, there is a tendency to underestimate cadmium
						  concentrations in samples with high specific gravities when specific-gravity
						  corrections are applied.
Despite some shortcomings, reporting solute
						  concentrations as a function of creatinine concentration is accepted generally;
						  OSHA therefore recommends that CDU levels be reported as the mass of cadmium
						  per unit mass of creatinine ( µg/g CTRU).
Reporting CDU as µg/g CRTU requires an additional
						  analytical process beyond the analysis of cadmium: Samples must be analyzed
						  independently for creatinine so that results may be reported as the ratio of
						  cadmium to creatinine concentrations found in the urine sample. Consequently,
						  the overall quality of the analysis depends on the combined performance by a
						  laboratory on these 2 determinations. The analysis used for CDU determinations
						  is addressed below in terms of µg Cd/l, with analysis of creatinine
						  addressed separately. Techniques for assessing creatinine are discussed in
						  Section 5.4.
Techniques for deriving cadmium as a ratio of CRTU, and
						  the confidence limits for independent measurements of cadmium and CRTU, are
						  provided in Section
						  3.3.3.
5.2.2 Analytical Techniques Used to Monitor CDU
Analytical techniques used for CDU determinations are
						  similar to those employed for CDB determinations; these techniques are
						  summarized in Table 3. As with CDB monitoring, the technique most suitable for
						  CDU determinations is atomic absorption spectroscopy (AAS). AAS methods used
						  for CDU determinations typically employ a graphite furnace, with background
						  correction made using either the deuterium-lamp or Zeeman techniques; Section
						  5.1.1 provides a detailed
						  description of AAS methods.
5.2.3 Methods Developed for CDU Determinations
Princi (1947), Smith et al. (1955), Smith and Kench
						  (1957), and Tsuchiya (1967) used colorimetric procedures similar to those
						  described in the CDB section above to estimate CDU concentrations. In these
						  methods, urine (50 ml) is reduced to dryness by heating in a sand bath and
						  digested (wet ashed) with mineral acids. Cadmium then is complexed with
						  dithiazone, extracted with chloroform and quantified by spectrophotometry.
						  These early studies typically report reagent blank values equivalent to 0.3
						  µg Cd/l, and CDU concentrations among nonexposed control groups at
						  maximum levels of 10 µg Cd/l--erroneously high values when compared to
						  more recent surveys of cadmium concentrations in the general population.
By the mid-1970s, most analytical procedures for CDU
						  analysis used either wet ashing (mineral acid) or high temperatures (
						  >400°C) to digest the organic matrix of urine, followed by cadmium
						  chelation with APDC or DDTC solutions and extraction with MIBK. The resulting
						  aliquots were analyzed by flame or graphite-furnace AAS (Kjellstrom
						  1979).
Improvements in control over temperature parameters
						  with electrothermal heating devices used in conjunction with flameless AAS
						  techniques, and optimization of temperature programs for controlling the
						  drying, charring, and atomization processes in sample analyses, led to improved
						  analytical detection of diluted urine samples without the need for sample
						  digestion or ashing. Roels et al. (1978) successfully used a simple sample
						  preparation, dilution of 1.0 ml aliquots of urine with 0.1 N
						  HNO3, to achieve accurate low-level determinations of
						  CDU.
In the method described by Pruszkowska et al. (1983),
						  which has become the preferred method for CDU analysis, urine samples were
						  diluted at a ratio of 1:5 with water; diammonium hydrogenphosphate in dilute
						  HNO3 was used as a matrix modifier. The matrix modifier
						  allows for a higher charring temperature without loss of cadmium through
						  volatilization during pre-atomization. This procedure also employs a stabilized
						  temperature platform in a graphite furnace, while nonspecific background
						  absorbtion is corrected using the Zeeman technique. This method allows for an
						  absolute detection limit of approximately 0.04 µg Cd/l urine.
5.2.4 Sample Collection and Handling
Sample collection procedures for CDU may contribute to
						  variability observed among CDU measurements. Sources of variation attendant to
						  sampling include time-of-day, the interval since ingestion of liquids, and the
						  introduction of external contamination during the collection process.
						  Therefore, to minimize contributions from these variables, strict adherence to
						  a sample-collection protocol is recommended. This a protocol should include
						  provisions for normalizing the conditions under which urine is collected. Every
						  effort also should be made to collect samples during the same time of
						  day.
Collection of urine samples from an industrial work
						  force for biological monitoring purposes usually is performed using "spot"
						  (i.e., single-void) urine with the pH of the sample determined immediately.
						  Logistic and sample-integrity problems arise when efforts are made to collect
						  urine over long periods (e.g., 24 hrs). Unless single-void urines are used,
						  there are numerous opportunities for measurement error because of poor control
						  over sample collection, storage and environmental contamination.
To minimize the interval during which sample urine
						  resides in the bladder, the following adaption to the "spot" collection
						  procedure is recommended: The bladder should first be emptied, and then a large
						  glass of water should be consumed; the sample may be collected within an hour
						  after the water is consumed.
5.2.5 Best Achievable Performance
Performance using a particular method for CDU
						  determinations is assumed to be equivalent to the performance reported by the
						  research laboratories in which the method was developed. Pruszkowska et al.
						  (1983) report a detection limit of 0.04 µg/l CDU, with a CV of
						  <4%between0-5 µg/l. The CDC reports a minimum CDU detection limit of
						  0.07 µg/l using a modified method based on Pruszkowska et al. (1983). No
						  CV is stated in this protocol; the protocol contains only rejection criteria
						  for internal QC parameters used during accuracy determinations with known
						  standards (Attachment 8 of exhibit 106 of OSHA docket H057A). Stoeppler and
						  Brandt (1980) report a CDU detection limit of 0.2 µg/l for their
						  methodology.
5.2.6 General Method Performance
For any particular method, the expected initial
						  performance from commercial laboratories may be somewhat lower than that
						  reported by the research laboratory in which the method was developed. With
						  participation in appropriate proficiency programs, and use of a proper in-house
						  QA/QC program incorporating provisions for regular corrective actions, the
						  performance of commercial laboratories may be expected to improve and approach
						  that reported by a research laboratories. The results reported for existing
						  proficiency programs serve to specify the initial level of performance that
						  likely can be expected from commercial laboratories offering analysis using a
						  particular method.
Weber (1988) reports on the results of the CTQ
						  proficiency program, which includes CDU results for laboratories participating
						  in the program. Results indicate that after receiving 60 samples (i.e., after
						  participating in the program for approximately 3 years), approximately 80% of
						  the participating laboratories report CDU results ranging between ± 2
						  µg/l or 15% of the consensus mean, whichever is greater. On any single
						  sample of the last 15 samples, the proportion of laboratories falling within
						  the specified range is between 75 and 95%, except for a single test for which
						  only 60% of the laboratories reported acceptable results. For each of the last
						  15 samples, approximately 60% of the laboratories reported results within
						  ± 1 µg or 15% of the mean, whichever is greater. The range of
						  concentrations included in this set of samples was not reported.
Another report from the CTQ (1991) summarizes
						  preliminary CDU results from their 1991 interlaboratory program. According to
						  the report, for 3 CDU samples with values of 9.0, 16.8, 31.5 µg/l,
						  acceptable results (target ± 2 µg/l) were achieved by only 44-52%
						  of the 34 laboratories participating in the CDU program. The overall CVs for
						  these 3 CDU samples among the 34 participating laboratories were 31%, 25%, and
						  49%, respectively. The reason for this poor performance has not been
						  determined.
A more recent report from the CTQ (Weber, private
						  communication) indicates that 36% of the laboratories in the program have been
						  able to achieve the target of ± 1 µg/l or 15% for more than 75% of
						  the samples analyzed over the last 5 years, while 45% of participating
						  laboratories achieved a target of ± 2 µg/l or 15% for more than
						  75% of the samples analyzed over the same period.
Note that results reported in the interlaboratory
						  programs are in terms of µg Cd/l of urine, unadjusted for creatinine. The
						  performance indicated, therefore, is a measure of the performance of the
						  cadmium portion of the analyses, and does not include variation that may be
						  introduced during the analysis of CRTU.
5.2.7 Observed CDU Concentrations
Prior to the onset of renal dysfunction, CDU
						  concentrations provide a general indication of the exposure history (i.e., body
						  burden) (see Section
						  4.3). Once renal dysfunction
						  occurs, CDU levels appear to increase and are no longer indicative solely of
						  cadmium body burden (Friberg and Elinder 1988).
5.2.7.1 Range of CDU concentrations observed among
						  unexposed samples
Surveys of CDU concentrations in the general population
						  were first reported from cooperative studies among industrial countries (i.e.,
						  Japan, U.S. and Sweden) conducted in the mid-1970s. In summarizing these data,
						  Kjellstrom (1979) reported that CDU concentrations among Dallas, Texas men (age
						  range: 9-59 years; smokers and nonsmokers) varied from 0.11-1.12 µg/l
						  (uncorrected for creatinine or specific gravity). These CDU concentrations are
						  intermediate between population values found in Sweden (range: 0.11-0.80
						  µg/l) and Japan (range: 0.14-2.32 µg/l).
Kowal and Zirkes (1983) reported CDU concentrations for
						  almost 1,000 samples collected during 1978-79 from the general U.S. adult
						  population (i.e., nine states; both genders; ages 20-74 years). They report
						  that CDU concentrations are lognormally distributed; low levels predominated,
						  but a small proportion of the population exhibited high levels. These
						  investigators transformed the CDU concentrations values, and reported the same
						  data 3 different ways: µg/l urine (unadjusted), µg/l (specific
						  gravity adjusted to 1.020), and µg/g CRTU. These data are summarized in
						  Tables 6 and 7.
Based on further statistical examination of these data,
						  including the lifestyle characteristics of this group, Kowal (1988) suggested
						  increased cadmium absorption (i.e., body burden) was correlated with low
						  dietary intakes of calcium and iron, as well as cigarette smoking.
CDU levels presented in Table 6 are adjusted for age
						  and gender. Results suggest that CDU levels may be slightly different among men
						  and women (i.e., higher among men when values are unadjusted, but lower among
						  men when the values are adjusted, for specific gravity or CRTU). Mean
						  differences among men and women are small compared to the standard deviations,
						  and therefore may not be significant. Levels of CDU also appear increase with
						  age. The data in Table 6 suggest as well that reporting CDU levels adjusted for
						  specific gravity or as a function of CRTU results in reduced
						  variability.
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The data in the Table 6 indicate the geometric mean of
						  CDU levels observed among the general population is 0.52 µg Cd/l urine
						  (unadjusted), with a geometric standard deviation of 3.0. Normalized for
						  creatinine, the geometric mean for the population is 0.66 µg/g CRTU, with
						  a geometric standard deviation of 2.7. Table 7 provides the distributions of
						  CDU concentrations for the general population studied by Kowal and Zirkes. The
						  data in this table indicate that 95% of the CDU levels observed among those not
						  occupationally exposed to cadmium are below 3 µg/g CRTU.
5.2.7.2 Range of CDU concentrations observed among
						  exposed workers
Table 8 is a summary of results from available studies
						  of CDU concentrations observed among cadmium-exposed workers. In this table,
						  arithmetic and/or geometric means and standard deviations are provided if
						  reported in these studies. The absolute range for the data in each study, or
						  the 95% confidence interval around the mean of each study, also are provided
						  when reported. The lower and upper 95th percentile of the distribution are
						  presented for each study in which a mean and corresponding standard deviation
						  were reported. Table 8 also provides estimates of the years of exposure, and
						  the levels of exposure, to cadmium in the work place if reported in these
						  studies. Concentrations reported in this table are in µg/g CRTU, unless
						  otherwise stated.
Data in Table 8 from Lauwerys et al. (1976) and Ellis
						  et al. (1983) indicate that CDU concentrations are higher among those
						  exhibiting kidney lesions or dysfunction than among those lacking these
						  symptoms. Data from the study by Roels et al. (1982) indicate that CDU levels
						  decrease among workers removed from occupational exposure to cadmium in
						  comparison to workers experiencing ongoing exposure. In both cases, however,
						  the distinction between the 2 groups is not as clear as with CDB; there is more
						  overlap in CDU levels observed among each of the paired populations than is
						  true for corresponding CDB levels. As with CDB levels, the data in Table 8
						  suggest increased CDU concentrations among workers who experienced increased
						  overall exposure.
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Although a few occupationally-exposed workers in the
						  studies presented in Table 8 exhibit CDU levels below 3 µg/g CRTU, most
						  of those workers exposed to cadmium levels in excess of the PEL defined in the
						  final cadmium rule exhibit CDU levels above 3 µg/g CRTU; this level
						  represents the upper 95th percentile of the CDU distribution observed among
						  those who are not occupationally exposed to cadmium (Table 7).
The mean CDU levels reported in Table 8 among
						  occupationally-exposed groups studied (except 2) exceed 3 µg/g CRTU.
						  Correspondingly, the level of exposure reported in these studies (with 1
						  exception) are significantly higher than what workers will experience under the
						  final cadmium rule. The 2 exceptions are from the studies by Mueller et al.
						  (1989) and Kawada et al. (1990); these studies indicate that workers exposed to
						  cadmium during pigment manufacture do not exhibit CDU levels as high as those
						  levels observed among workers exposed to cadmium in other occupations. Exposure
						  levels, however, were lower in the pigment manufacturing plants studied.
						  Significantly, workers removed from occupational cadmium exposure for an
						  average of 4 years still exhibited CDU levels in excess of 3 µg/g CRTU
						  (Roels et al. 1982). In the single-exception study with a reported level of
						  cadmium exposure lower than levels proposed in the final rule (i.e., the study
						  of a pigment manufacturing plant by Kawada et al. 1990), most of the workers
						  exhibited CDU levels less than 3 µg/g CRTU (i.e., the mean value was only
						  1.3 µg/g CRTU). CDU levels among workers with such limited cadmium
						  exposure are expected to be significantly lower than levels reported on Table
						  8.
Based on the above data, a CDU level of 3 µg/g
						  CRTU appear to represent a threshold above which significant work place
						  exposure to cadmium occurs over the work span of those being monitored. Note
						  that this threshold is not as distinct as the corresponding threshold described
						  for CDB. In general, the variability associated with CDU measurements among
						  exposed workers appears to be higher than the variability associated with CDB
						  measurements among similar workers.
5.2.8 Conclusions and Recommendations for CDU
The above evaluation supports the following
						  recommendations for a CDU proficiency program. These recommendations address
						  only sampling and analysis procedures for CDU determinations specifically,
						  which are to be reported as an unadjusted µ/g Cd/l urine. Normalizing
						  this result to creatinine requires a second analysis for CRTU so that the ratio
						  of the 2 measurements can be obtained. Creatinine analysis is addressed in
						  Section 5.4. Formal procedures for
						  combining the 2 measurements to derive a value and a confidence limit for CDU
						  in µg/g CRTU are provided in Section
						  3.3.3.
5.2.8.1 Recommended method
The method of Pruszkowska et al. (1983) should be
						  adopted for CDU analysis. This method is recommended because it is simple,
						  straightforward and reliable (i.e., small variations in experimental conditions
						  do not affect the analytical results).
A synopsis of the methods used by laboratories to
						  determine CDU under the interlaboratory program administered by the CTQ (1991)
						  indicates that more than 78% (24 of 31) of the participating laboratories use a
						  dilution method to prepare urine samples for CDU analysis. Laboratories may
						  adopt alternate methods, but it is the responsibility of the laboratory to
						  demonstrate that the alternate methods provide results of comparable quality to
						  the Pruszkowska method.
5.2.8.2 Data quality objectives
The following data quality objectives should facilitate
						  interpretation of analytical results, and are achievable based on the above
						  evaluation.
Limit of Detection. A level of 0.5 µg/l (i.e.,
						  corresponding to a detection limit of 0.5 µg/g CRTU, assuming 1 g CRT/l
						  urine) should be achievable. Pruszkowska et al. (1983) achieved a limit of
						  detection of 0.04 µg/l for CDU based on the slope the the curve for their
						  working standards (0.35 pg Cd/0.0044, A signal=1% absorbance using
						  GF-AAS).
The CDC reports a minimum detection limit for CDU of
						  0.07 µg/l using a modified Pruszkowska method. This limit of detection
						  was defined as 3 times the standard deviation calculated from 10 repeated
						  measurements of a "low level" CDU test sample (Attachment 8 of exhibit 106 of
						  OSHA docket H057A).
Stoeppler and Brandt (1980) report a limit of detection
						  for CDU of 0.2 µg/l using an aqueous dilution (1:2) of the urine
						  samples.
Accuracy. A recent report from the CTQ (Weber, private
						  communication) indicates that 36% of the laboratories in the program achieve
						  the target of ±1 µg/l or 15% for more than 75% of the samples
						  analyzed over the last 5 years, while 45% of participating laboratories achieve
						  a target of ±2 µg/l or 15% for more than 75% of the samples
						  analyzed over the same period. With time and a strong incentive for
						  improvement, it is expected that the proportion of laboratories successfully
						  achieving the stricter level of accuracy should increase. It should be noted,
						  however, these indices of performance do not include variations resulting from
						  the ancillary measurement of CRTU (which is recommended for the proper
						  recording of results). The low cadmium levels expected to be measured indicate
						  that the analysis of creatinine will contribute relatively little to the
						  overall variability observed among creatinine-normalized CDU levels (see
						  Section 5.4). The initial target value for
						  reporting CDU under this program, therefore, is set at ±1 µg/g
						  CRTU or 15% (whichever is greater).
Precision. For internal QC samples (which are
						  recommended as part of an internal QA/QC program, Section
						  3.3.1), laboratories should attain
						  an overall precision of 25%. For CDB samples with concentrations less than 2
						  µg/l, a target precision of 40% is, while precisions of 20% should be
						  achievable for CDU concentrations greater than 2 µg/l. Although these
						  values are more stringent than those observed in the CTQ interlaboratory
						  program reported by Webber (1988), they are well within limits expected to be
						  achievable for the method as reported by Stoeppler and Brandt (1980).
5.2.8.3 Quality assurance/quality control
Commercial laboratories providing CDU determinations
						  should adopt an internal QA/QC program that incorporates the following
						  components: Strict adherence to the selected method, including calibration
						  requirements; regular incorporation of QC samples during actual runs; a
						  protocol for corrective actions, and documentation of such actions; and,
						  participation in an interlaboratory proficiency program. Note that the
						  nonmandatory program presented in Attachment 1 as an example of an acceptable
						  QA/QC program, is based on using the Pruszkowska method for CDU analysis.
						  Should an alternate method be adopted by a laboratory, the laboratory should
						  develop a QA/QC program equivalent to the nonmandatory program, and which
						  satisfies the provisions of Section
						  3.3.1.
5.3 Monitoring l2-Microglobulin in Urine (B2MU)
As indicated in Section
						  4.3, B2MU appears to be the best of
						  several small proteins that may be monitored as early indicators of
						  cadmium-induced renal damage. Several analytic techniques are available for
						  measuring B2M.
5.3.1 Units of B2MU Measurement
Procedures adopted for reporting B2MU levels are not
						  uniform. In these guidelines, OSHA recommends that B2MU levels be reported as
						  µg/g CRTU, similar to reporting CDU concentrations. Reporting B2MU
						  normalized to the concentration of CRTU requires an additional analytical
						  process beyond the analysis of B2M: Independent analysis for creatinine so that
						  results may be reported as a ratio of the B2M and creatinine concentrations
						  found in the urine sample. Consequently, the overall quality of the analysis
						  depends on the combined performance on these 2 analyses. The analysis used for
						  B2MU determinations is described in terms of µg B2M/l urine, with
						  analysis of creatinine addressed separately. Techniques used to measure
						  creatinine are provided in Section
						  5.4. Note that Section
						  3.3.3 provides techniques for
						  deriving the value of B2M as function of CRTU, and the confidence limits for
						  independent measurements of B2M and CRTU.
5.3.2 Analytical Techniques Used to Monitor B2MU
One of the earliest tests used to measure B2MU was the
						  radial immunodiffusion technique. This technique is a simple and specific
						  method for identification and quantitation of a number of proteins found in
						  human serum and other body fluids when the protein is not readily
						  differentiated by standard electrophoretic procedures. A quantitative
						  relationship exists between the concentration of a protein deposited in a well
						  that is cut into a thin agarose layer containing the corresponding monospecific
						  antiserum, and the distance that the resultant complex diffuses. The wells are
						  filled with an unknown serum and the standard (or control), and incubated in a
						  moist environment at room temperature. After the optimal point of diffusion has
						  been reached, the diameters of the resulting precipition rings are measured.
						  The diameter of a ring is related to the concentration of the constituent
						  substance. For B2MU determinations required in the medical monitoring program,
						  this method requires a process that may be insufficient to concentrate the
						  protein to levels that are required for detection.
Radioimmunoassay (RIA) techniques are used widely in
						  immunologic assays to measure the concentration of antigen or antibody in
						  body-fluid samples. RIA procedures are based on competitive-binding techniques.
						  If antigen concentration is being measured, the principle underlying the
						  procedure is that radioactive-labeled antigen competes with the sample's
						  unlabeled antigen for binding sites on a known amount of immobile antibody.
						  When these 3 components are present in the system, an equilibrium exists. This
						  equilibrium is followed by a separation of the free and bound forms of the
						  antigen. Either free or bound radioactive-labeled antigen can be assessed to
						  determine the amount of antigen in the sample. The analysis is performed by
						  measuring the level of radiation emitted either by the bound complex following
						  removal of the solution containing the free antigen, or by the isolated
						  solution containing the residual-free antigen. The main advantage of the RIA
						  method is the extreme sensitivity of detection for emitted radiation and the
						  corresponding ability to detect trace amounts of antigen. Additionally, large
						  numbers of tests can be performed rapidly.
The enzyme-linked immunosorbent assay (ELISA)
						  techniques are similar to RIA techniques except that nonradioactive labels are
						  employed. This technique is safe, specific and rapid, and is nearly as
						  sensitive as RIA techniques. An enzyme-labeled antigen is used in the
						  immunologic assay; the labeled antigen detects the presence and quantity of
						  unlabeled antigen in the sample. In a representative ELISA test, a plastic
						  plate is coated with antibody (e.g., antibody to B2M). The antibody reacts with
						  antigen (B2M) in the urine and forms an antigen-antibody complex on the plate.
						  A second anti-B2M antibody (i.e., labeled with an enzyme) is added to the
						  mixture and forms an antibody-antigen- antibody complex. Enzyme activity is
						  measured spectrophotometrically after the addition of a specific chromogenic
						  substrate which is activated by the bound enzyme. The results of a typical test
						  are calculated by comparing the spectrophotometric reading of a serum sample to
						  that of a control or reference serum. In general, these procedures are faster
						  and require less laboratory work than other methods.
In a fluorescent ELISA technique (such as the one
						  employed in the Pharmacia Delphia test for B2M), the labeled enzyme is bound to
						  a strong fluorescent dye. In the Pharmacia Delphia test, an antigen bound to a
						  fluorescent dye competes with unlabeled antigen in the sample for a
						  predetermined amount of specific, immobile antibody. Once equilibrium is
						  reached, the immobile phase is removed from the labeled antigen in the sample
						  solution and washed; an enhancement solution then is added that liberates the
						  fluorescent dye from the bound antigen-antibody complex. The enhancement
						  solution also contains a chelate that complexes with the fluorescent dye in
						  solution; this complex increases the fluorescent properties of the dye so that
						  it is easier to detect.
To determine the quantity of B2M in a sample using the
						  Pharmacia Delphia test, the intensity of the fluorescence of the enhancement
						  solution is measured. This intensity is proportional to the concentration of
						  labeled antigen that bound to the immobile antibody phase during the initial
						  competition with unlabeled antigen from the sample. Consequently, the intensity
						  of the fluorescence is an inverse function of the concentration of antigen
						  (B2M) in the original sample. The relationship between the fluorescence level
						  and the B2M concentration in the sample is determined using a series of graded
						  standards, and extrapolating these standards to find the concentration of the
						  unknown sample.
5.3.3 Methods Developed for B2MU Determinations
B2MU usually is measured by radioimmunoassay (RIA) or
						  enzyme-linked immunosorbent assay (ELISA); however, other methods (including
						  gel electrophoresis, radial immunodiffusion, and nephelometric assays) also
						  have been described (Schardun and van Epps 1987). RIA and ELISA methods are
						  preferred because they are sensitive at concentrations as low as micrograms per
						  liter, require no concentration processes, are highly reliable and use only a
						  small sample volume.
Based on a survey of the literature, the ELISA
						  technique is recommended for monitoring B2MU. While RIAs provide greater
						  sensitivity (typically about 1 µg/l, Evrin et al. 1971), they depend on
						  the use of radioisotopes; use of radioisotopes requires adherence to rules and
						  regulations established by the Atomic Energy Commission, and necessitates an
						  expensive radioactivity counter for testing. Radioisotopes also have a
						  relatively short half-life, which corresponds to a reduced shelf life, thereby
						  increasing the cost and complexity of testing. In contrast, ELISA testing can
						  be performed on routine laboratory spectrophotometers, do not necessitate
						  adherence to additional rules and regulations governing the handling of
						  radioactive substances, and the test kits have long shelf lives. Further, the
						  range of sensitivity commonly achieved by the recommended ELISA test (i.e., the
						  Pharmacia Delphia test) is approximately 100 µg/l (Pharmacia 1990), which
						  is sufficient for monitoring B2MU levels resulting from cadmium exposure. Based
						  on the studies listed in Table 7 (Section
						  5.3.7), the average range of B2M
						  concentrations among the general, nonexposed population falls between 60 and
						  300 µg/g CRTU. The upper 95th percentile of distributions, derived from
						  studies in Table 9 which reported standard deviations, range between 180 and
						  1,140 µg/g CRTU. Also, the Pharmacia Delphia test currently is the most
						  widely used test for assessing B2MU.
5.3.4 Sample Collection and Handling
As with CDB or CDU, sample collection procedures are
						  addressed primarily to identify ways to minimize the degree of variability
						  introduced by sample collection during medical monitoring. It is unclear the
						  extent to which sample collection contributes to B2MU variability. Sources of
						  variation include time-of-day effects, the interval since consuming liquids and
						  the quantity of liquids consumed, and the introduction of external
						  contamination during the collection process. A special problem unique to B2M
						  sampling is the sensitivity of this protein to degradation under acid
						  conditions commonly found in the bladder. To minimize this problem, strict
						  adherence to a sampling protocol is recommended. The protocol should include
						  provisions for normalizing the conditions under which the urine is collected.
						  Clearly, it is important to minimize the interval urine spends in the bladder.
						  It also is recommended that every effort be made to collect samples during the
						  same time of day.
Collection of urine samples for biological monitoring
						  usually is performed using "spot" (i.e., single-void) urine. Logistics and
						  sample integrity become problems when efforts are made to collect urine over
						  extended periods (e.g., 24 hrs). Unless single-void urines are used, numerous
						  opportunities exist for measurement error because of poor control over sample
						  collection, storage and environmental contamination.
To minimize the interval that sample urine resides in
						  the bladder, the following adaption to the "spot" collection procedure is
						  recommended: The bladder should be emptied and then a large glass of water
						  should be consumed; the sample then should be collected within an hour after
						  the the water is consumed.
5.3.5 Best Achievable Performance
The best achievable performance is assumed to be
						  equivalent to the performance reported by the manufacturers of the Pharmacia
						  Delphia test kits (Pharmacia 1990). According to the insert that comes with
						  these kits, QC results should be within ± 2 SDs of the mean for each
						  control sample tested; a CV of less than or equal to 5.2% should be maintained.
						  The total CV reported for test kits is less than or equal to 7.2%.
5.3.6 General Method Performance
Unlike analyses for CDB and CDU, the Pharmacia Delphia
						  test is standardized in a commercial kit that controls for many sources of
						  variation. In the absence of data to the contrary, it is assumed that the
						  achievable performance reported by the manufacturer of this test kit will serve
						  as an achievable performance objective. The CTQ proficiency testing program for
						  B2MU analysis is expected to use the performance parameters defined by the test
						  kit manufacturer as the basis of the B2MU proficiency testing program.
Note that results reported for the test kit are
						  expressed in terms of µg B2M/l of urine, and have not been adjusted for
						  creatinine. The indicated performance, therefore, is a measure of the
						  performance of the B2M portion of the analyses only, and does not include
						  variation that may have been introduced during the analysis of
						  creatinine.
5.3.7 Observed B2MU Concentrations
As indicated in Section
						  4.3, the concentration of B2MU may
						  serve as an early indicator of the onset of kidney damage associated with
						  cadmium exposure.
5.3.7.1 Range of B2MU concentrations among unexposed
						  samples
Most of the studies listed in Table 9 report B2MU
						  levels for those who were not occupationally exposed to cadmium. Studies noted
						  in the second column of this table (which contain the footnote "d") reported
						  B2MU concentrations among cadmium-exposed workers who, nonetheless, showed no
						  signs of proteinuria. These latter studies are included in this table because,
						  as indicated in Section
						  4.3, monitoring B2MU is intended to
						  provide advanced warning of the onset of kidney dysfunction associated with
						  cadmium exposure, rather than to distinguish relative exposure. This table,
						  therefore, indicates the range of B2MU levels observed among those who had no
						  symptoms of renal dysfunction (including workers with none of these
						  symptoms).
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To the extent possible, the studies listed in Table 9
						  provide geometric means and geometric standard deviations for measurements
						  among the groups defined in each study. For studies reporting a geometric
						  standard deviation along with a mean, the lower and upper 95th percentile for
						  these distributions were derived and reported in the table.
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The data provided from 15 of the 19 studies listed in
						  Table 9 indicate that the geometric mean concentration of B2M observed among
						  those who were not occupationally exposed to cadmium is 70-170 µg/g CRTU.
						  Data from the 4 remaining studies indicate that exposed workers who exhibit no
						  signs of proteinuria show mean B2MU levels of 60-300 µg/g CRTU. B2MU
						  values in the study by Thun et al. (1989), however, appear high in comparison
						  to the other 3 studies. If this study is removed, B2MU levels for those who are
						  not occupationally exposed to cadmium are similar to B2MU levels found among
						  cadmium-exposed workers who exhibit no signs of kidney dysfunction. Although
						  the mean is high in the study by Thun et al., the range of measurements
						  reported in this study is within the ranges reported for the other
						  studies.
Determining a reasonable upper limit from the range of
						  B2M concentrations observed among those who do not exhibit signs of proteinuria
						  is problematic. Elevated B2MU levels are among the signs used to define the
						  onset of kidney dysfunction. Without access to the raw data from the studies
						  listed in Table 9, it is necessary to rely on reported standard deviations to
						  estimate an upper limit for normal B2MU concentrations (i.e., the upper 95th
						  percentile for the distributions measured). For the 8 studies reporting a
						  geometric standard deviation, the upper 95th percentiles for the distributions
						  are 180-1140 µg/g CRTU. These values are in general agreement with the
						  upper 95th percentile for the distribution (i.e., 631 µg/g CRTU) reported
						  by Buchet et al. (1980). These upper limits also appear to be in general
						  agreement with B2MU values (i.e., 100-690 µg/g CRTU) reported as the
						  normal upper limit by Iwao et al. (1980), Kawada et al. (1989), Wibowo et al.
						  (1982), and Schardun and van Epps (1987). These values must be compared to
						  levels reported among those exhibiting kidney dysfunction to define a threshold
						  level for kidney dysfunction related to cadmium exposure.
5.3.7.2 Range of B2MU concentrations among exposed
						  workers
Table 10 presents results from studies reporting B2MU
						  determinations among those occupationally exposed to cadmium in the work place;
						  in some of these studies, kidney dysfunction was observed among exposed
						  workers, while other studies did not make an effort to distinguish among
						  exposed workers based on kidney dysfunction. As with Table 9, this table
						  provides geometric means and geometric standard deviations for the groups
						  defined in each study if available. For studies reporting a geometric standard
						  deviation along with a mean, the lower and upper 95th percentiles for the
						  distributions are derived and reported in the table.
The data provided in Table 10 indicate that the mean
						  B2MU concentration observed among workers experiencing occupational exposure to
						  cadmium (but with undefined levels of proteinuria) is 160-7400 µg/g CRTU.
						  One of these studies reports geometric means lower than this range (i.e., as
						  low as 71 µg/g CRTU); an explanation for this wide spread in average
						  concentrations is not available.
Seven of the studies listed in Table 10 report a range
						  of B2MU levels among those diagnosed as having renal dysfunction. As indicated
						  in this table, renal dysfunction (proteinuria) is defined in several of these
						  studies by B2MU levels in excess of 300 µg/g CRTU (see footnote "c" of
						  Table 10); therefore, the range of B2MU levels observed in these studies is a
						  function of the operational definition used to identify those with renal
						  dysfunction. Nevertheless, a B2MU level of 300 µg/g CRTU appears to be a
						  meaningful threshold for identifying those having early signs of kidney damage.
						  While levels much higher than 300 µg/g CRTU have been observed among
						  those with renal dysfunction, the vast majority of those not occupationally
						  exposed to cadmium exhibit much lower B2MU concentrations (see Table 9).
						  Similarly, the vast majority of workers
not
exhibiting renal
						  dysfunction are found to have levels below 300 µg/g CRTU (Table
						  9).
The 300 µg/g CRTU level for B2MU proposed in the
						  above paragraph has support among researchers as the threshold level that
						  distinguishes between cadmium-exposed workers with and without kidney
						  dysfunction. For example, in the guide for physicians who must evaluate
						  cadmium-exposed workers written for the Cadmium Council by Dr. Lauwerys, levels
						  of B2M greater than 200-300 µg/g CRTU are considered to require
						  additional medical evaluation for kidney dysfunction (exhibit 8-447, OSHA
						  docket H057A). The most widely used test for measuring B2M (i.e., the Pharmacia
						  Delphia test) defines B2MU levels above 300 µg/l as abnormal (exhibit
						  L-140-1, OSHA docket H057A).
Dr. Elinder, chairman of the Department of Nephrology
						  at the Karolinska Institute, testified at the hearings on the proposed cadmium
						  rule. According to Dr. Elinder (exhibit L-140-45, OSHA docket H057A), the
						  normal concentration of B2MU has been well documented (Evrin and Wibell 1972;
						  Kjellstrom et al. 1977a; Elinder et al. 1978, 1983; Buchet et al. 1980; Jawaid
						  et al. 1983; Kowal and Zirkes, 1983). Elinder stated that the upper 95 or 97.5
						  percentiles for B2MU among those without tubular dysfunction is below 300
						  µg/g CRTU (Kjellstrom et al. 1977a; Buchet et al. 1980; Kowal and Zirkes,
						  1983). Elinder defined levels of B2M above 300Ã¦g/g CRTU as
						  "slight" proteinuria.
5.3.8 Conclusions and Recommendations for B2MU
Based on the above evaluation, the following
						  recommendations are made for a B2MU proficiency testing program. Note that the
						  following discussion addresses only sampling and analysis for B2MU
						  determinations (i.e., to be reported as an unadjusted µg B2M/l urine).
						  Normalizing this result to creatinine requires a second analysis for CRTU (see
						  Section 5.4) so that the ratio of the 2
						  measurements can be obtained.
5.3.8.1 Recommended method
The Pharmacia Delphia method (Pharmacia 1990) should be
						  adopted as the standard method for B2MU determinations. Laboratories may adopt
						  alternate methods, but it is the responsibility of the laboratory to
						  demonstrate that alternate methods provide results of comparable quality to the
						  Pharmacia Delphia method.
5.3.8.2 Data quality objectives
The following data quality objectives should facilitate
						  interpretation of analytical results, and should be achievable based on the
						  above evaluation.
Limit of Detection. A limit of 100 µg/l urine
						  should be achievable, although the insert to the test kit (Pharmacia 1990)
						  cites a detection limit of 150 µg/l; private conversations with
						  representatives of Pharmacia, however, indicate that the lower limit of 100
						  µg/l should be achievable provided an additional standard of 100
						  µg/l B2M is run with the other standards to derive the calibration curve
						  (Section 3.3.1.1). The lower detection limit
						  is desirable due to the proximity of this detection limit to B2MU values
						  defined for the cadmium medical monitoring program.
Accuracy. Because results from an interlaboratory
						  proficiency testing program are not available currently, it is difficult to
						  define an achievable level of accuracy. Given the general performance
						  parameters defined by the insert to the test kits, however, an accuracy of
						  ± 15% of the target value appears achievable.
Due to the low levels of B2MU to be measured generally,
						  it is anticipated that the analysis of creatinine will contribute relatively
						  little to the overall variability observed among creatinine-normalized B2MU
						  levels (see Section
						  5.4). The initial level of accuracy
						  for reporting B2MU levels under this program should be set at ±
						  15%.
Precision. Based on precision data reported by
						  Pharmacia (1990), a precision value (i.e., CV) of 5% should be achievable over
						  the defined range of the analyte. For internal QC samples (i.e., recommended as
						  part of an internal QA/QC program, Section
						  3.3.1), laboratories should attain
						  precision near 5% over the range of concentrations measured.
5.3.8.3 Quality assurance/quality control
Commercial laboratories providing measurement of B2MU
						  should adopt an internal QA/QC program that incorporates the following
						  components: Strict adherence to the Pharmacia Delphiad method, including
						  calibration requirements; regular use of QC samples during routine runs; a
						  protocol for corrective actions, and documentation of these actions; and,
						  participation in an interlaboratory proficiency program. Procedures that may be
						  used to address internal QC requirements are presented in Attachment 1. Due to
						  differences between analyses for B2MU and CDB/CDU, specific values presented in
						  Attachment 1 may have to be modified. Other components of the program
						  (including characterization runs), however, can be adapted to a program for
						  B2MU.
5.4 Monitoring Creatinine in Urine (CRTU)
Because CDU and B2MU should be reported relative to
						  concentrations of CRTU, these concentrations should be determined in addition
						  CDU and B2MU determinations.
5.4.1 Units of CRTU Measurement
CDU should be reported as µg Cd/g CRTU, while
						  B2MU should be reported as µg B2M/g CRTU. To derive the ratio of cadmium
						  or B2M to creatinine, CRTU should be reported in units of g crtn/l of urine.
						  Depending on the analytical method, it may be necessary to convert results of
						  creatinine determinations accordingly.
5.4.2 Analytical Techniques Used to Monitor CRTU
Of the techniques available for CRTU determinations, an
						  absorbance spectrophotometric technique and a high-performance liquid
						  chromatography (HPLC) technique are identified as acceptable in this
						  protocol.
5.4.3 Methods Developed for CRTU Determinations
CRTU analysis performed in support of either CDU or
						  B2MU determinations should be performed using either of the following 2
						  methods:
1. The Du Pont method (i.e., Jaffe method), in which
						  creatinine in a sample reacts with picrate under alkaline conditions, and the
						  resulting red chromofore is monitored (at 510 nm) for a fixed interval to
						  determine the rate of the reaction; this reaction rate is proportional to the
						  concentration of creatinine present in the sample (a copy of this method is
						  provided in Attachment 2 of this protocol); or,
2. the OSHA SLC Technical Center (OSLTC) method, in
						  which creatinine in an aliquot of sample is separated using an HPLC column
						  equipped with a UV detector; the resulting peak is quantified using an
						  electrical integrator (a copy of this method is provided in Attachment 3 of
						  this protocol).
5.4.4 Sample Collection and Handling
CRTU samples should be segregated from samples
						  collected for CDU or B2MU analysis. Sample-collection techniques have been
						  described under Section
						  5.2.4. Samples should be preserved
						  either to stabilize CDU (with HNO3) or B2MU (with NaOH).
						  Neither of these procedures should adversely affect CRTU analysis (see
						  Attachment 3).
5.4.5 General Method Performance
Data from the OSLTC indicate that a CV of 5% should be
						  achievable using the OSLTC method (Septon, L private communication). The
						  achievable accuracy of this method has not been determined.
Results reported in surveys conducted by the CAP (CAP
						  1991a, 1991b and 1992) indicate that a CV of 5% is achievable. The accuracy
						  achievable for CRTU determinations has not been reported.
Laboratories performing creatinine analysis under this
						  protocol should be CAP accredited and should be active participants in the CAP
						  surveys.
5.4.6 Observed CRTU Concentrations
Published data suggest the range of CRTU concentrations
						  is 1.0-1.6 g in 24-hour urine samples (Harrison 1987). These values are
						  equivalent to about 1 g/l urine.
5.4.7 Conclusions and Recommendations for CRTU
5.4.7.1 Recommended method
Use either the Jaffe method (Attachment 2) or the OSLTC
						  method (Attachment 3). Alternate methods may be acceptable provided adequate
						  performance is demonstrated in the CAP program.
5.4.7.2 Data quality objectives
Limit of Detection. This value has not been formally
						  defined; however, a value of 0.1 g/l urine should be readily achievable.
Accuracy. This value has not been defined formally;
						  accuracy should be sufficient to retain accreditation from the CAP. Precision.
						  A CV of 5% should be achievable using the recommended methods.
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ATTACHMENT 1: NONMANDATORY PROTOCOL FOR AN INTERNAL
						  QUALITY ASSURANCE/QUALITY CONTROL PROGRAM
The following is an example of the type of internal
						  quality assurance/quality control program that assures adequate control to
						  satisfy mandatory OSHA requirements under this protocol. However, other
						  approaches may also be acceptable.
As indicated in Section
						  3.3.1 of the protocol, the
						  mandatory QA/QC program for CDB and CDU should address, at a minimum, the
						  following:
* calibration;
* establishment of control limits;
* internal QC analyses and maintaining control;
						  and
* corrective action protocols.
This illustrative program includes both initial
						  characterization runs to establish the performance of the method and ongoing
						  analysis of quality control samples intermixed with compliance samples to
						  maintain control.
CALIBRATION
Before any analytical runs are conducted, the analytic
						  instrument must be calibrated. This is to be done at the beginning of each day
						  on which quality control samples and/or compliance samples are run. Once
						  calibration is established, quality control samples or compliance samples may
						  be run. Regardless of the type of samples run, every fifth sample must be a
						  standard to assure that the calibration is holding.
Calibration is defined as holding if every standard is
						  within plus or minus (±) 15% of its theoretical value. If a standard is
						  more than plus or minus 15% of its theoretical value, then the run is out of
						  control due to calibration error and the entire set of samples must either be
						  reanalyzed after recalibrating or results should be recalculated based on a
						  statistical curve derived from the measurement of all standards.
It is essential that the highest standard run is higher
						  than the highest sample run. To assure that this is the case, it may be
						  necessary to run a high standard at the end of the run, which is selected based
						  on the results obtained over the course of the run.
All standards should be kept fresh, and as they get
						  old, they should be compared with new standards and replaced.
INITIAL CHARACTERIZATION RUNS AND ESTABLISHING
						  CONTROL
A participating laboratory should establish four pools
						  of quality control samples for each of the analytes for which it wishes to be
						  accredited. The concentrations of quality control samples within each pool are
						  to be centered around each of the four target levels for the particular analyte
						  identified in Section
						  4.4 of the protocol.
Within each pool, at least 4 quality control samples
						  need to be established with varying concentrations ranging between plus or
						  minus 50% of the target value of that pool. Thus for the medium-high cadmium in
						  blood pool, the theoretical values of the quality control samples may range
						  from 5 to 15 µg/l, (the target value is µ 10 g/l). At least 4
						  unique theoretical values must be represented in this pool.
The range of theoretical values of plus or minus 50% of
						  the target value of a pool means that there will be overlap of the pools. For
						  example, the range of values for the medium-low pool for cadmium in blood is
						  3.5 to 10.5 µg/l while the range of values for the medium-high pool is 5
						  to 15 µg/l. Therefore, it is possible for a quality control sample from
						  the medium-low pool to have a higher concentration of cadmium than a quality
						  control sample from the medium-high pool.
Quality control samples may be obtained as commercially
						  available reference materials, internally prepared, or both. Internally
						  prepared samples should be well characterized and traced or compared to a
						  reference material for which a consensus value for concentration is available.
						  Levels of analyte in the quality control samples must be concealed from the
						  analyst prior to the reporting of analytical results. Potential sources of
						  materials that may be used to construct quality control samples are listed in
						  Section 3.3.1 of the protocol.
Before any compliance samples are analyzed, control
						  limits must be established. Control limits should be calculated for every pool
						  of each analyte for which a laboratory seeks accreditation, and control charts
						  should be kept for each pool of each analyte. A separate set of control charts
						  and control limits should be established for
each
analytical
						  instrument in a laboratory that will be used for analysis of compliance
						  samples.
At the beginning of this QA/QC program, control limits
						  should be based on the results of the analysis of 20 quality control samples
						  from each pool of each analyte. For any given pool, the 20 quality control
						  samples should be run on 20 different days. Although no more than one sample
						  should be run from any single pool on a particular day, a laboratory may run
						  quality control samples from different pools on the same day. This constitutes
						  a set of initial characterization runs.
For each quality control sample analyzed, the value F/T
						  (defined in the glossary) should be calculated. To calculate the control limits
						  for a pool of an analyte, it is first necessary to calculate the mean, X, of
						  the F/T values for each quality control sample in a pool and then to calculate
						  its standard deviation ^[SIGMA]. Thus, for the control limit for a pool, X is
						  calculated as:
Click
						  here to view image
and ^ [SIGMA] is calculated as
Click
						  here to view image
where N is the number of quality control samples run
						  for a pool.
The control limit for a particular pool is then given
						  by the mean plus
or minus 3 standard deviations (X
						  ±3^[SIGMA]).
The control limits may be no greater than 40% of the
						  mean F/T value. If three standard deviations are greater than 40% of the mean
						  F/T value, then analysis of compliance samples may not
						  begin1. Instead, an investigation into the causes of
						  the large standard deviation should begin, and the inadequacies must be
						  remedied. Then, control limits must be reestablished which will mean repeating
						  the running 20 quality control samples from each pool over 20 days.
__________
1 Note that the value, "40%"
						  may change over time as experience is gained with the program.
INTERNAL QUALITY CONTROL ANALYSES AND MAINTAINING
						  CONTROL
Once control limits have been established for each pool
						  of an analyte, analysis of compliance samples may begin. During any run of
						  compliance samples, quality control samples are to be interspersed at a rate of
						  no less than 5% of the compliance sample workload. When quality control samples
						  are run, however, they should be run in
sets
consisting of one
						  quality control sample from each pool. Therefore, it may be necessary, at
						  times, to intersperse quality control samples at a rate greater than 5%.
There should be at least one
set
of
						  quality control samples run with any analysis of compliance samples. At a
						  minimum, for example, 4 quality control samples should be run even if only 1
						  compliance sample is run. Generally, the number of quality control samples that
						  should be run are a multiple of four with the minimum equal to the smallest
						  multiple of four that is greater than 5% of the total number of samples to be
						  run. For example, if 300 compliance samples of an analyte are run, then at
						  least 16 quality control samples should be run (16 is the smallest multiple of
						  four that is greater than 15, which is 5% of 300).
Control charts for each pool of an analyte (and for
						  each instrument in the laboratory to be used for analysis of compliance
						  samples) should be established by plotting F/T versus date as the quality
						  control sample results are reported. On the graph there should be lines
						  representing the control limits for the pool, the mean F/T limits for the pool,
						  and the theoretical F/T of 1.000. Lines representing plus or minus (±)
						  2[SIGMA] should also be represented on the charts. A theoretical example of a
						  control chart is presented in Figure 1
Click
						  here to view image
All quality control samples should be plotted on the
						  chart, and the charts should be checked for visual trends. If a quality control
						  sample falls above or below the control limits for its pool, then corrective
						  steps must be taken (see the section on corrective actions below). Once a
						  laboratory's program has been established, control limits should be updated
						  every 2 months, prior to sending OSHA the updated data required to maintain
						  accreditation.
The updated control limits should be calculated from
						  the results of the last 100 quality control samples run for each pool. If 100
						  quality control samples from a pool have not been run at the time of the
						  update, then the limits should be based on as many as have been run provided at
						  least 20 quality control samples from each pool have been run over 20 different
						  days.
The trends that should be looked for on the control
						  charts are:
1. 10 consecutive quality control samples falling above
						  or below the mean;
2. 3 consecutive quality control samples falling more
						  than 2[SIGMA] from the mean (above or below the 2[SIGMA] lines of the chart);
						  or
3. the mean calculated to update the control limits
						  falls more than 10% above or below the theoretical mean of 1.000.
If any of these trends is observed, then all analysis
						  must be stopped, and an investigation into the causes of the errors must begin.
						  Before the analysis of compliance samples may resume, the inadequacies must be
						  remedied and the control limits must be reestablished for that pool of an
						  analyte. Reestablishment of control limits will entail running 20 sets of
						  quality control samples over 20 days.
Note that alternative procedures for defining internal
						  quality control limits may also be acceptable. Limits may be based, for
						  example, on proficiency testing, such as ± 1 µg or 15% of the mean
						  (whichever is greater). These should be clearly defined.
CORRECTIVE ACTIONS
Corrective action is the term used to describe the
						  identification and remediation of errors occurring within an analysis.
						  Corrective action is necessary whenever the result of the analysis of any
						  quality control sample falls outside of the established control limits. The
						  steps involved may include simple things like checking calculations of basic
						  instrument maintenance, or it may involve more complicated actions like major
						  instrument repair. Whatever the source of error, it must be identified and
						  corrected (and a Corrective Action Report (CAR) must be completed and sent to
						  OSHA with the next reaccreditation).
To maintain accreditation, laboratories must report
						  corrective actions to the OSHA Salt Lake Technical Center. A form to be used
						  for reporting corrective actions is provided in Attachment 5 of the
						  protocol.
Attachment 2:
CREATININE IN URINE (JAFFE PROCEDURE)
INTENDED USE: The CREA pack is used in the Du Pont
						  ACA® discrete clinical analyzer to quantitatively measure creatinine in
						  serum and urine.
SUMMARY: The CREA method employs a modification of the
						  kinetic Jaffe reaction reported by Larsen. This method has been reported to be
						  less susceptible than conventional methods to interference from non-creatinine,
						  Jaffe-positive compounds.1
A split sample comparison between the CREA method and a
						  conventional Jaffe procedure on Autoanalyzer® showed a good correlation.
						  (See SPECIFIC PERFORMANCE CHARACTERISTICS).
Autoanalyzer,® is a registered trademark of
						  Technicon Corp., Tarrytown, NY.
PRINCIPLES OF PROCEDURE: In the presence of a strong
						  base such as NaOH, picrate reacts with creatinine to form a red chromophore.
						  The rate of increasing absorbance at 510 nm due to the formation of this
						  chromophore during a 17.07-second measurement period is directly proportional
						  to the creatinine concentration in the sample.
NaOH
Creatinine +
								  Picrate [RIGHTWARDS ARROW]
Red chromophore
(absorbs at 510
								  nm)
REAGENTS:
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
______________________________
Compartmenta
Form
Ingredient
Quantityb
#2,3, &
								  4
Liquid
Picrate
0.11 mmol
#6
Liquid
NaOH (for pH
								  adjustment)c
__________
a. Compartments are numbered 1-7, with compartment #7
						  located closest to pack fill position #2.
b. Nominal value at manufacture.
c. See PRECAUTIONS.
PRECAUTIONS: COMPARTMENT #6 CONTAINS 75 µL OF 10
						  N NaOH; AVOID CONTACT; SKIN IRRITANT; RINSE CONTACTED AREA WITH WATER.
USED PACKS CONTAIN HUMAN BODY FLUIDS; HANDLE WITH
						  APPROPRIATE CARE.
FOR IN VITRO DIAGNOSTIC USE.
MIXING & DILUTING: Mixing and diluting are
						  automatically performed by the ACA® discrete clinical analyzer. The sample
						  cup must contain sufficient quantity to accommodate the sample volume plus the
						  "dead volume"; precise cup filling is not required.
Sample Cup Volumes
									 (µL)
Standard
Microsystem
Analyzer
Dead
Total
Dead
Total
II, III
120
3000
10
500
IV, SX
120
3000
30
500
V
90
3000
10
500
STORAGE OF UNPROCESSED PACKS: Store at 2-8°C. Do
						  not freeze. Do not expose to temperatures above 35° C or to direct
						  sunlight.
EXPIRATION: Refer to EXPIRATION DATE on the tray
						  label.
SPECIMEN COLLECTION: Serum or urine can be collected
						  and stored by normal procedures.2
KNOWN INTERFERING
						  SUBSTANCES3: Serum Protein Influence. Serum protein
						  levels exert a direct influence on the CREA assay. The following should be
						  taken into account when this method is used for urine samples and when it is
						  calibrated:
Aqueous creatinine standards or urine specimens will
						  give CREA results depressed by approximately 0.7 mg/dL [62 µmol/L]d and
						  will be less precise than samples containing more than 3 g/dL [30 g/L]
						  protein.
All urine specimens should be diluted with an albumin
						  solution to give a final protein concentration of at least 3 g/dL [30 g/L]. Du
						  Pont Enzyme Diluent (Cat. #790035-901) may be used for this purpose.
* High concentration of endrogenous bilirubin ( >20
						  mg/dL [ >342 µmol/L]) will give depressed CREA results (average
						  depression 0.8 mg/dL [71 µmol/L]).4
* Grossly hemolyzed (hemoglobin100 mg/dL [ >62
						  mol/L]) or visibly lipemic specimens may cause falsely elevated CREA
						  results.5,6
* The following cephalosporin antibiotics do not
						  interfere with the CREA method when present at the concentrations indicated.
						  Systematic inaccuracies (bias) due to these substances are less than or equal
						  to 0.1 mg/dL [8.84 µmol/L] at CREA concentrations of approximately 1 mg/
						  dL [88 µmol/L].
Peak
Drug
Serum
									 Level7,8,9
Concentration
Antibiotic
mg/dL
[mmol/L]
mg/dL
[mmol/L]
Cephaloridine
1.4
0.3
25
6.0
Cephalexin
0.6-2.0
0.2-0.6
25
7.2
Cephamandole
1.3-2.5
0.3-0.5
25
4.9
Cephapirin
2.0
0.4
25
5.6
Cephradine
1.5-2.0
0.4-0.6
25
7.1
Cefazolin
2.5-5.0
0.55-1.1
50
11.0
* The following cephalosporin antibiotics have been
						  shown to affect CREA results when present at the indicated concentrations.
						  System inaccuracies (bias) due to these substances are greater that 0.1 mg/dL
						  [8.84 mol/L] at CREA concentrations of:
Peak
Drug
Serum
								  Level7,8,9 >
Concentration
Effect
Antibiotic
mg/dL
[mmol/L]
mg/dL
[mmol/L]
Cephalothin
1-6
0.2-1.5
100
25.2
â20-25%
Cephoxitin
2.0
0.5
5.0
1.2
â35-40%
* The single wavelength measurement used in this method
						  eliminates interference from chromophores whose 510 nm absorbance is constant
						  throughout the measurement period.
d. Systeme International d'unites (S.I. Units) are in
						  brackets.
* Each laboratory should determine the acceptability of
						  its own blood collection tubes and serum separation products. Variations in
						  these products may exist between manufacturers and, at times, from lot to
						  lot.
PROCEDURE:
TEST MATERIALS
Item
II,III Du
									 Pont
IV,SX V Du Pont Cat.
									 $
Du Pont Du Pont Cat.
									 #
ACA® CREA Analytical Test
								  Pack
701976901
701976901
701976901
Sample System
								  Kit
710642901
710642901
713697901
or
Micro Sample System
								  Kit
702694901
710356901
NA
and
Micro Sample System
								  Holders
702785000
NA
NA
DYLUX® Photosensitive Printer
								  Paper
700036000
NA
NA
Thermal Printer
								  Paper
NA
710639901
713645901
Du Pont Purified
								  Water
704209901
710615901
710815901
Cell Wash
								  Solution
701864901
710664901
710864901
TEST STEPS
The operator need only load the sample kit and
						  appropriate test pack(s) into a properly prepared ACA® discrete clinical
						  analyzer. It automatically advances the pack(s) through the test steps and
						  prints a result(s). See the Instrument Manual of the ACA® analyzer for
						  details of mechanical travel of the test pack(s).
* Preset Creatinine (CREA) Test Conditions
* Sample Volume: 200 µL
* Diluent: Purified Water
* Temperature: 37.0±0.1°C
* Reaction Period: 29 seconds
* Type of Measurement: Rate
* Measurement Period: 17.07 seconds
* Wavelength: 510 nm
* Units: mg/dL [µmol/L]
CALIBRATION
The general calibration procedure is described in the
						  Calibration/Verification chapter of the Manuals. The following information
						  should be considered when calibrating the CREA method.
* Assay Range: 0-20 mg/mL[0-1768
						  µmol/L]e
* Reference Material: Protein containing primary
						  standardsf or secondary calibrators such as Du Pont
						  Elevated Chemistry Control (Cat. #790035903) and Normal Chemistry Control (Cat.
						  #790035905)g
* Suggested Calibration levels: 1,5,20, mg/mL [88, 442,
						  1768 µmol/L]
* Calibration Scheme: 3 levels, 3 packs per
						  level
* Frequency: Each new pack lot. Every 3 months for any
						  one pack lot.
e. For the results in S.I. units [µmol/L] the
						  conversion factory is 88.4.
f. Refer to the Creatinine Standard Preparation and
						  Calibration Procedure available on request from a Du Pont
						  Representative.
g. If the Du Pont Chemistry Controls are being used,
						  prepare them according to the instructions on the product insert sheets.
Preset Creatinine (CREA) Test
						  Conditions
Item
ACA
®
II Analyzer
ACA
®
III, IV, SX, V Analyzer
Count by
One(1)
NA
[Five (5)]
Decimal
Point
0.0 mg/dL
000.0 mg/dL
Location
[000.
								  µmol/L]
[000 µmol/L]
Assigned
Starting
999.8
-1.000 E1
Point or Offset
								  C0
[9823.]
[-8.840 E2]
Scale Factor
or
0.2000
2.004 E-1h
Assigned
mg/dL/counth
Linear Term
C1h
[0.3536
[1.772E1]
µmol/L/count]
__________
h. The preset scale
						  factor(linear term) was derived from the molar absorptivity of the indicator
						  and is based on an absorbance to activity relationship (sensitivity) of 0.596
						  (mA/min)/(U/L). Due to small differences in filters and electronic components
						  between instruments, the actual scale factor (linear term) may differ slightly
						  from that given above.
QUALITY CONTROL
Two types of quality control procedures are
						  recommended:
* General Instrument Check. Refer to the Filter Balance
						  Procedure and the Absorbance Test Method described in the Instrument Manual.
						  Refer also to the ABS Test Methodology literature.
* Creatinine Method Check. At least once daily run a
						  CREA test on a solution of known creatinine activity such as an assayed control
						  or calibration standard other than that used to calibrate the CREA method. For
						  further details review the Quality Assurance Section of the Chemistry Manual.
						  The result obtained should fall within acceptable limits defined by the
						  day-to-day variability of the system as measured in the user's laboratory. (See
						  SPECIFIC PERFORMANCE CHARACTERISTICS for guidance.) If the result falls outside
						  the laboratory's acceptable limits, follow the procedure outlined in the
						  Chemistry Troubleshooting Section of the Chemistry Manual.
A possible system malfunction is indicated when
						  analysis of a sample with five consecutive test packs gives the following
						  results:
Level
SD
1 mg/dL
>0.15 mg/dL
[88 µmol/L]
[ >13
								  µmol/L]
20 mg/dL
>0.68 mg/dL
[1768 µmol/L]
[ >60
								  µmol/L]
Refer to the procedure outlined in the Trouble Shooting
						  Section of the Manual.
RESULTS
The ACA® analyzer automatically calculates and
						  prints the CREA result in mg/dL [µmol/L].
LIMITATION OF PROCEDURE
Results >20 mg/dL [1768 µmol/L]:
* Dilute with suitable protein base diluent. Reassay.
						  Correct for diluting before reporting.
The reporting system contains error messages to warn
						  the operator of specific malfunctions. Any report slip containing a letter code
						  or word immediately following the numerical value should not be reported. Refer
						  to the Manual for the definition of error codes.
REFERENCE INTERVAL
SERUM:11,i Males - 0.8-1.3
						  md/dL [71-115 µmol/L] Females - 0.6-1.0 md/dL [53-88 µmol/L]
URINE12
Males - 0.6-2.5 g/24 hr [53-221 mmol/24 hr]Females -
						  0.6-1.5 g/24 hr [53-133 mmol/24 hr]
Each laboratory should establish its own reference
						  intervals for CREA as performed on the analyzer.
i. Reference interval data obtained from 200 apparently
						  healthy individuals (71 males, 129 females) between the ages of 19 and
						  72.
SPECIFIC PERFORMANCE CHARACTERISTICS
						  REPRODUCIBILITYK
Standard
								  Deviation (% CV)
Material
Mean
Within-Run
Between-Day
Lyophilized
1.3
0.05
								  (3.7)
0.05 (3.7)
Control
[115]
[4.4]
[4.4]
Lyophilized
20.6
0.12
								  (0.6)
0.37 (1.8)
Control
[1821]
[10.6]
[32.7]
CORRELATION
Regression
						  Statistics1
Comparative
Correlation
Method
Slope
Intercept
Coefficient
n
Autoanalyzer®
1.03
0.03[2.7]
0.997
260
j. ALL SPECIFIC PERFORMANCE CHARACTERISTICS tests were
						  run after normal recommended equipment quality control checks were performed
						  (see Instrument Manual).
k. Specimens at each level were analyzed in duplicate
						  for twenty days. The within-run and between-day standard deviations were
						  calculated by the analysis of variance method.
l. Model equation for regression statistics is: Result
						  of ACA® Analyzer = Slope Comparative method result) + intercept
ASSAY RANGEm
0.0-20.0 mg/dl [0-1768 µmol]
m. See REPRODUCIBILITY for method performance within
						  the assay range.
ANALYTICAL SPECIFICITY
See KNOWN INTERFERING SUBSTANCES section for
						  details.
BIBLIOGRAPHY:
1 Larsen, K,
Clin
						  Chem Acta
41, 209 (1972).
2 Tietz, NW,
Fundamentals of Clinical Chemistry
, W. B. Saunders Co.,
						  Philadelphia, PA, 1976, pp 47-52, 1211.
3 Supplementary information
						  pertaining to the effects of various drugs and patient conditions on in vivo or
						  in vitro diagnostic levels can be found in "Drug Interferences with Clinical
						  Laboratory Tests,"
Clin. Chem
21 (5) (1975), and "Effects of
						  Disease on Clinical Laboratory Tests,"
Clin Chem
, 26 (4)
						  1D-476D (1980).
4 Watkins, R. Fieldkamp, SC,
						  Thibert, RJ, and Zak, B,
Clin Chem
, 21, 1002 (1975).
5 Kawas, EE, Richards, AH,
						  and Bigger, R,
An Evaluation of a Kinetic Creatinine Test for the Du
						  Pont ACA
, Du Pont Company, Willmington, DE (February 1973).n
6 Westgard, JO,
Effects of Hemolysis and Lipemia
on ACA
Creatinine
Method
,0.200 µL,
Sample Size
, Du Pont Company, Wilmington, DE (October
						  1972).
7
Physicians' Desk
						  Reference
, Medical Economics Company, 33 Edition, 1979.
8 Henry, JB,
Clinical Diagnosis and Management by Laboratory Methods
, W.B.
						  Saunders Co., Philadelphia, PA 1979, Vol. III.
9 Krupp, MA, Tierney, LM
						  Jr., Jawetz, E, Roe, Rl, Camargo, CA,
Physicians Handbook
,
						  Lange Medical Publications, Los Altos, CA, 1982 pp 635-636.
10 Sarah, AJ, Koch, TR,
						  Drusano, GL, Celoxitin Falsely Elevates Creatinine Levels, JAMA 247, 205-206
						  (1982).
11 Gadsden, RH, and Phelps,
						  CA,
A Normal Range Study of Amylasein Urine and Serum on the Du Pont
						  ACA
, Du Pont Company, Wilmington, DE (March
						  1978)n
12 Dicht, JJ, Reference
						  Intervals for Serum Amylase and Urinary Creatinine on the Du Pont ACA®
						  Discrete Clinical Analyzer, Du Pont Company, Wilimgton, DE (November
						  1984).
n. Reprints available from
						  Du Pont Company, Diagonostic Systems.
Attachment 3
Analysis of Creatinine for the Normalization of Cadmium
						  and Beta-2-Microglobulin Concentrations in Urine
Matrix: Urine
Target Concentration: 1.1 g/L (this amount is
						  representative of creatinine concentrations found in urine).
Procedure: A 1.0 mL aliquot of urine is passed through
						  a C18 SEP-PAK (Waters Associates). Approximately 30 mL of HPLC (high
						  performance liquid chromatography) grade water is then run through the SEP-PAK.
						  The resulting solution is diluted to volume in a 100-mL volumetric flask and
						  analyzed by HPLC using an ultraviolet (UV) detector.
Special Requirements: After collection, samples should
						  be appropriately stabilized for cadmium (Cd) analysis by using 10% high purity
						  (with low Cd background levels) nitric acid (exactly 1.0 mL of 10% nitric acid
						  per 10 mL of urine) or stabilized for Beta-2-Microglobulin (B2M) by taking to
						  pH 7 with dilute NaOH (exactly 1.0 mL of 0.11 N NaOH per 10 mL of urine). If
						  not immediately analyzed, the samples should be frozen and shipped by overnight
						  mail in an insulated container.
Date: January 1992.
Chemists: David B. Armitage, Duane Lee, Organic Service
						  Branch II, OSHA Technical Center, Salt Lake City, Utah.
1. General Discussion
1.1. Background
1.1.1. History or procedure
Creatinine has been analyzed by several methods in the
						  past. The earliest methods were of the wet chemical type. As an example,
						  creatinine reacts with sodium picrate in basic solution to form a red complex,
						  which is then analyzed colorimetrically (Refs. 5.1 and 5.2).
Since industrial hygiene laboratories will be analyzing
						  for Cd and B2M in urine, they will be normalizing those concentrations to the
						  concentration of creatinine in urine. A literature search reveled several HPLC
						  methods (Refs. 5.3., 5.4., 5.5. and 5.6) for creatinine in urine and because
						  manu industrial hygiene laboratories have HPLC equipment, it was desirable to
						  develop an industrial hygiene HPLC method for creatinine in urine. The method
						  of Hausen, Fuchs, and Wachter was chosen as the starting point for method
						  development, SEP-PAKs were used for sample clarification and cleanup in this
						  method to protect the analytical column. The urine aliquot which has been
						  passed through the SEP-PAK is then analyzed by reverse-phase HPLC using
						  ion-pair techniques.
This method is very similar to that of Ogata and
						  Taguchi (Ref. 5.6.) except they used centrifugation for sample clean-up. It is
						  also of note that they did a comparison of their HPLC results to those of the
						  Jaffe method (a picric acid method commonly used in the health care industry)
						  and found a linear relationship of close to 1:1. This indicates that either
						  HPLC or colorimetric methods may be used to measure creatinine concentrations
						  in urine.
1.1.2. Physical properties (Ref 5.7.)
Molecular weight: 113.12
Molecular formula:
						  C4-H7-N3-O
Chemical name:
						  2-amino-1.5-dihydro-1-methyl-4H-imidazol-4-one
CAS#: 60-27-5
Melting point: 300 >C (decomposes)
Appearance: white powder
Solubility: soluble in water; slightly soluble in
						  alcohol; practically insoluble in acetone, ether, and chloroform
Synonyms: 1-methylglycocyamidine, 1-methylhydation-2-
						  imide
Structure: see Figure #1
Click
						  here to view image
Figure #1
1.2. Advantages
1.2.1. This method offers a simple, straightforward,
						  and specific alternative method to the Jaffe method.
1.2.2. HPLC instrumentation is commonly found in many
						  industrial hygiene laboratories.
2. Sample Stabilization Procedure
2.1. Apparatus Metal-free plastic container for urine
						  sample.
2.2. Reagents
2.2.1. Stabilizing Solution-1) Nitric acid (10%, high
						  purity with low Cd background levels) for stabilizing urine for Cd analysis or
						  2) NaOH, 0.11 N, for stabilizing urine for B2M analysis.
2.2.2. HPLC grade water
2.3. Technique
2.3.1. Stabilizing solution is added to the urine
						  sample (see section
						  2.2.1.) The stabilizing solution
						  should be such that for each 10 mL of urine, add exactly 1.0 mL of stabilizer
						  solution. (Never add water or urine to acid or base. Always add acid or base to
						  water or urine.) Exactly 1.0 mL of 0.11 N NaOH added to 10 mL of urine should
						  result in a pH of 7. Or add 1.0 mL of 10% nitric acid to 10 mL of urine.
2.3.2. After sample collection seal the plastic bottle
						  securely and wrap it with an appropriate seal. Urine samples should be frozen
						  and then shipped by overnight mail (if shipping is necessary) in an insulted
						  container. (Do not fill plastic bottle too full. This will allow for expansion
						  of contents during the freezing process.)
2.4. The Effect of Preparation and Stabilization
						  Techniques on Creatinine Concentrations
Three urine samples were prepared by making one sample
						  acidic, not treating a second sample, and adjusting a third sample to pH 7. The
						  samples were analyzed in duplicate by two different procedures. For the first
						  procedure a 1.0 mL aliquot of urine was put in a 100-mL volumetric flask,
						  diluted to volume with HPLC grade water, and then analyzed directly on an HPLC.
						  The other procedure used SEP-PAKs. The SEP-PAK was rinsed with approximately 5
						  mL of methanol followed by approximately 10 mL of HPLC grade water and both
						  rinses were discarded. Then 1.0 mL of the urine sample was put through the
						  SEP-PAK, followed by 30 mL of HPLC grade water. The urine and water were
						  transferred to a 100-mL volumetric flask, diluted to volume with HPLC grade
						  water and analyzed by HPLC. These three urine samples were analyzed on the day
						  they were obtained and then frozen. The results show that whether the urine is
						  acidic, untreated or adjusted to pH 7, the resulting answer for creatinine is
						  essentially unchanged. The purpose of stabilizing the urine by making it acidic
						  or neutral is for the analysis of Cd or B2M respectively.
COMPARISON OF PREPARATION AND STABILIZATION
						  TECHNIQUES
Sample
w/o SEP-PAK (g/L
									 creatinine)
with SEP-PAK (g/L
									 creatinine)
Acid
1.10
1.10
Acid
1.11
1.10
Untreated
1.12
1.11
Untreated
1.11
1.12
pH 7
1.08
1.02
pH 7
1.11
1.08
2.5. Storage After 4 days and 54 days of storage in a
						  freezer, the samples were thawed, brought to room temperature and analyzed
						  using the same procedures as in section
						  2.4. The results of several days of
						  storage show that the resulting answer for creatinine is essentially
						  unchanged.
Storage Data
4 days
54 days
Sample
w/o SEP-PAK (g/L
									 creatinine)
with SEP-PAK (g/L
									 creatinine)
w/o SEP-PAK (g/L
									 creatinine)
with SEP-PAK (g/L
									 creatinine)
Acid..........................
1.09
1.09
1.08
1.09
Acid..........................
1.10
1.10
1.09
1.10
Acid..........................
..........
..........
1.09
1.09
Untreated..........................
1.13
1.14
1.09
1.11
Untreated..........................
1.15
1.14
1.10
1.10
Untreated..........................
..........
..........
1.09
1.10
pH
								  7..........................
1.14
1.13
1.12
1.12
pH 7..........................
1.14
1.13
1.12
1.12
pH 7..........................
..........
..........
1.12
1.12
2.6. Interferences: None.
2.7. Safety precautions
2.7.1. Make sure samples are properly sealed and frozen
						  before shipment to avoid leakage.
2.7.2. Follow the appropriate shipping
						  procedures.
The following modified special safety precautions are
						  based on those recommended by the Centers for Disease Control (CDC)
						  (Ref.5.8).
2.7.3. Wear gloves, lab coat, and safety glasses while
						  handling all human urine products. Disposable plastic, glass, and paper (pipet
						  tips, gloves, etc.) that contact urine should be placed in a biohazard
						  autoclave bag. These bags should be kept in appropriate containers until sealed
						  and autoclave. Wipe down all work surfaces with 10% sodium hypochlorite
						  solution when work is finished.
2.7.4. Dispose of all biological samples and diluted
						  specimens in a biohazard autoclave bag at the end of the analytical run.
2.7.5. Special care should be taken when handling and
						  dispensing nitric acid. Always remember to add acid to water (or urine). Nitric
						  acid is a corrosive chemical capable of severe eye and skin damage. Wear
						  metal-free gloves, a lab coat, and safety glass. If the nitric acid comes in
						  contact with any part of the body, quickly wash with copious quanties of water
						  for at least 15 minutes.
2.7.6. Special care should be taken when handling and
						  dispensing NaOH. Always remember to add base to water (or urine). NaOH can
						  cause severe eye and skin damage. Always wear the appropriate gloves, a lab
						  coat, and safety glasses. If the NaOH comes in contact with any part of the
						  body, quickly wash with copius quantities of water for at least 15
						  minutes.
3. Analytical Procedure
3.1. Apparatus
3.1.1. A high performance liquid chromatograph equipped
						  with pump, sample injector and UV dector.
3.1.2. A C18 HPLC column; 25 cm X 4.6 mm I.D.
3.1.3. An electronic integrator, or some other suitable
						  means of determining analyte response.
3.1.4. Stripchart recorder.
3.1.5. C18 SEP-PAKs (Waters Associates) or
						  equivalent.
3.1.6. Luer-lock syringe for sample preparation (5 mL
						  or 10 mL).
3.1.7. Volumetric pipettes and flasks for standard and
						  sample preparation.
3.1.8. Vacuum system to aid sample preparation
						  (optional).
3.2. Reagents
3.2.1. Water, HPLC grade.
3.2.2. Methanol, HPLC grade.
3.2.3. PIC B-7 (Waters Associates) in small
						  vials.
3.2.4. Creatinine, anhydrous, Sigma Chemical Corp.,
						  purity not listed.
3.2.5. 1-Heptanesulfonic acid, sodium salt
						  monohydrate.
3.2.6. Phosphoric acid.
3.2.7. Mobile phase. It can be prepared by mixing one
						  vial of PIC B-7 into a 1 L solution of 50% methanol and 50% water. The mobil
						  phase can also be made by preparing a solution that is 50% methanol and 50%
						  water with 0.005M heptanesulfonic acid and adjusting the pH of the solution to
						  3.5 with phosphoric acid.
3.3. Standard preparation
3.3.1. Stock standards were prepared by weighing 10 to
						  15 mg of creatinine. This is transferred to a 25-mL volumetric flask and
						  diluted to volume with HPLC grade water.
3.3.2. Dilutions to a working range of 3 to 35 g/mL are
						  made in either HPLC grade water or HPLC mobil phase (standards give the same
						  detector response in either solution).
3.4. Sample preparation
3.4.1. The C18 SEP-PAK is connected to a Luer-lock
						  syringe. It is rinsed with 5 mL HPLC grade methanol and then 10 mL of HPLC
						  grade water. These rinses are discarded.
3.4.2. Exactly 1.0 mL of urine is pipetted into the
						  syringe. The urine is put through the SEP-PAK into a suitable container using a
						  vacuum system.
3.4.3. The walls of the syringe are rinsed in several
						  stages with a total of approximately 30 mL of HPLC grade water. These rinses
						  are put through the SEP-PAK into the same container. The resulting solution is
						  transferred to a 100-mL volumetric flask and then brought to volume with HPLC
						  grade water.
3.5. Analysis (conditions and hardware are those used
						  in this evaluation.)
3.5.1. Instrument conditions
Column..........................
Zorbax' ODS, 5-6 µm particle size; 25 cm X 4.6 mm I.D.
Mobile phase..........................
See Section
								  3.2.7.
Detector..........................
Dual wavelength UV; 229 nm (primary) 254 nm (secondary),
Flow rate..........................
0.7 mL/minute.
Retention
								  time..........................
7.2 minutes.
Sensitivity..........................
0.05 AUFS.
Injection
								  volume..........................
20 µL.
3.5.2. Chromatogram (See Figure #2).
Click
						  here to view image
3.6. Interferences
3.6.1. Any compound that has the same retention time as
						  creatinine and absorbs at 229 nm is an interference.
3.6.2. HPLC conditions may be varied to circumvent
						  interferences. In addition, analysis at another UV wavelength (i.e. 254 nm)
						  would allow a comparison of the ratio of response of a standard to that of a
						  sample. Any deviations would indicate an interference.
3.7. Calculations
3.7.1. A calibration curve is constructed by plotting
						  dector response versus standard concentration (See Figure #3).
3.7.2. The concentration of creatinine in a sample is
						  determined by finding the concentration corresponding to its detector response.
						  (See Figure #3).
Click
						  here to view image
3.7.3. The µg/mL creatinine from section
						  3.7.2. is then multiplied by 100
						  (the dilution factor). This value is equivalent to the micrograms of creatinine
						  in the 1.0 mL stabilized urine aliquot or the milligrams of creatinine per
						  liter of urine. The desire unit, g/L, is determined by the following
						  relationship:
µg/mL
mg/L
g/L=
___________________________
=
___________________________
1000
1000
3.7.4. The resulting value for creatinine is used to
						  normalize the urinary concentration of the desired analyte (A) (Cd or B2M) by
						  using the following formula.
µg A/g creatinine=
µg A/L
								  (experimental)
g/L creatinine
Where A is the desired analyte. The protocol of
						  reporting such normalized results is µg A/g creatinine.
3.8. Safety precautions. See section
						  2.7
4. Conclusions
The determination of creatinine in urine by HPLC is a
						  good alternative to the Jaffe method for industrial hygiene laboratories.
						  Sample clarification with SEP-PAKs did not change the amount of creatinine
						  found in urine samples. However, it does protect the analytical column. The
						  results of this creatinine in urine procedure are unaffected by the pH of the
						  urine sample under the conditions tested by this procedure. Therefore, no
						  special measure are required for creatinine analysis whether the urine sample
						  has been stabilized with 10% nitric acid for the Cd analysis or brought to a pH
						  of 7 with 0.11 NaOH for the B2M analysis.
5. References
5.1. Clark, L.C.; Thompson, H.L. Anal. Chem. 1949, 21,
						  1218.
5.2. Peters, J.H; J. Biol. Chem. 1942, 146, 176.
5.3. Hausen. V.A.; Fuchs, D; Wachter, H.;J. Clin. Chem.
						  Clin. Biochem. 1981, 19,373-378.
5.4. Clark, P.M.S.; Kricka, L.J.; Patel, A.; J. Liq.
						  Chrom. 1980, 3(7), 1031-1046.
5.5. Ballerini, R.; Chinol, M.; Cambi, A; J. Chrom.
						  1979, 179, 365-369.
5.6. Ogata, M.; Taguchi, T.; Industrial Health 1987,
						  25, 225-228.
5.7. "Merck Index", 11th ed.; Windholz, Martha Ed.;
						  Merck: Rahway, N.J., 1989; p. 403.
5.8. Kimberly, M.; "Determination of Cadmium in Urineby
						  Graphite Furance Atomic Absorption Spectrometry with Zeeman Background
						  Correction." Centers for Disease Control, Atlanta, Georgia, unpublished, update
						  1990.
1.
						  Editorial correction moving NOTE and HISTORIES 1-5 to precede Appendix A to
						  section 5207 (Register 99, No. 28). For
						  prior history of Appendices, see section
						  5207.