Dr.
Patrick Parsons, Dr. Susan Cummins, Ms. M. Chaudhary-Webb, Dr. Wayne
Matson, Dr. D.K. Saxena, Dr. Robert Parr, Dr. Sheila Rajarathanam,
and Dr. Jude Vaz
Blood
Lead Sample
Collection Issues
Dr. Madhu Chaudhary-Webb
Correct sample collection
procedures and techniques are paramount to ensure the quality of analytical
outcomes. Proper pre- and post-collection procedures ensure that the
samples collected are of high quality and free from contamination, coagulation,
over-dilution, or breakage during shipping. These procedures include
the use of proper supplies, universal precautions, blood collection
technique, and sample handling after collection, and storage and shipping
procedures.
The first step
in any venous or capillary blood collection process is the use of correctly
screened or certified "lead-free" blood collection supplies.
All supplies that come in contact with the blood specimen should be
tested for the presence of lead. These supplies include but are not
limited to needles, syringes, lancets, and blood collection tubes or
vials. CDC has developed screening guidelines, which suggest that each
"lot" of supplies needs to have at least 42 units from that
"lot" screened. For example, out of a specific batch number
of lancets, 42 of the lancets are selected for screening. A "lot"
is deemed to be "lead-free" if the lead contribution in each
individual unit (from the 42 units) has no more than 5% of a normal
expected blood lead level.
Universal precautions
must be practiced at all times when dealing with patients or blood materials.
This protects both the analyst and the patient against infection from
biological materials. Gloves, safety glasses, and lab coats should be
worn at all times when collecting blood or handling specimens. Gloves
must also be changed between each patient. Additionally, the gloves
used must be powder free to avoid potential lead contamination from
the powder. All supplies used in the collection process must be one-use
only and must be disposed of after use in biohazard disposal containers
for incineration or sterilization. Lancets, needles, and syringes with
attached needles should be placed in biohazard "Sharps" disposal
containers.
The preferable
technique for setting up a blood collection work area is to establish
a clean workspace with fresh supplies laid out within reach of the blood
collector. A disposable plastic backed absorbent pad ("chuck")
should be laid out with a needle and syringe or lancet, gauze, alcohol
swabs, bandage, labeling pen or labels, blood collection vial(s) or
tube(s) and disposable biohazard containers within reach. Once an appropriate
work area has been created, the patient must be instructed to thoroughly
wash the site of blood collection with soap and water. The washed site
must not come in contact with any surface until after blood collection
is complete in order to avoid lead contamination from the surfaces.
It is recommended that only trained phlebotomists or medical personnel
collect venous samples. However, if capillary bloods are to be collected,
most laboratorians and/or health care providers can be trained to safely
collect the capillary specimen. If a venous sample is obtained, the
vessel should contain nearly the full capacity of blood. This is necessary
to avoid potential dilution from the liquid anticoagulant contained
in some types of blood collection tubes. Additionally, certain technologies
require properly filled tubes for both venous and capillary samples
to avoid the problem of competitive binding which can potentially cause
low lead results. Most capillary specimens can be collected from a fingerstick
into various micro vials or tubes, but for children under 6 months of
age, a heel stick is preferred. After washing the blood collection site,
the site is wiped with a clean alcohol swab, then dried with a clean
gauze pad. The third or fourth finger is punctured on the distal side
with a lancet. In the case of heelsticks, the puncture should be done
on either side of the heel (never in the center of the heel). The first
drop of blood is wiped away, then the next few drops of blood are collected.
The finger should not be "milked," as this could cause dilution
of the blood from the surrounding tissue fluids resulting in falsely
low blood lead results. Constant pressure at the puncture site is appropriate.
The vial or tube used should be filled to the specified collection volume
of that vial or tube. After collection, the patient’s puncture site
should be bandaged. Immediately after collecting the blood, it should
be thoroughly mixed with the anticoagulant in the tube or vial before
it is stored. Once the blood collection is complete, all the used supplies
and gloves can be rolled up in the disposable "chuck" and
disposed of very conveniently in an appropriate biohazard disposable
container. A new blood collection work area should be set up with a
new "chuck" and new blood collection supplies, and a new set
of gloves.
Depending upon
the analytical procedure or technology selected for the survey or study,
sample storage requirements will vary. Ideally, fresh blood should be
stored at 4 ° C for short terms or frozen at or less than -20 ° C for
long terms. In accordance with the National Committee for Clinical Laboratory
Standards (NCCLS) guidelines, it is preferable in most situations to
ship blood samples in coolers with "cool-packs" to maintain
the temperature around 4 ° C, especially during periods of hot weather.
This ensures the integrity of the specimens. However, many labs do accept
blood specimens shipped at ambient temperature. Regardless of the shipping
temperature, care should be taken to properly package the specimens
to protect the specimens from breakage. In the event of breakage, packing
should be capable of absorbing the spill. There should be three layers
of protection between the sample and the exterior. The first layer being
the tube in which the blood is contained, the second layer being a bag
or box in which the tube is placed, and the third layer being the actual
shipping container. Within the shipping container, there should be numerous
layers of absorbent materials such as paper towels, "chucks",
or newspaper that can soak up the blood should there be breakage during
shipping.
In conclusion,
proper blood lead sample collection and handling is a multifaceted process.
All supplies used for the collection process must be "lead-free"
as determined by testing or certification. Universal precautions must
be routinely practiced in order to protect both the patient and the
analyst from infectious disease. This includes wearing protective gear
and using fresh single use disposable supplies per patient. For the
actual blood collection event, a clean work space should be created
by placing a disposable "chuck" along with the blood collection
supplies and disposal containers within reach. The chuck is deposed
of along with all the used collection supplies, and a fresh workspace
is created for each new patient. And finally, care should be taken in
the packaging and shipping process to ensure the integrity of the specimens.
The packing should be able to protect the specimens from breakage, to
soak up any spills, and keep the specimens cool.
References
1. Schlenker TL,
Fritz CJ, Mark D, Laude M, Linke G, Murphy A, Matte T. Screening for
Pediatric Lead Poisoning: Comparability of Simultaneously Drawn Capillary
and Venous Blood Samples. JAMA 1994;271:1346-1348.
2. Schonfeld DJ,
Cullen MR, Rainey PM, Berg AT, Brown DR, Hogan JC Jr, Turk DS, Rude
CS, Cicchetti DV. Screening for Lead Poisoning in an Urban Pediatric
Clinic Using Samples Obtained by Fingerstick. Pediatrics 1994;94(2):174-179.
3. Parsons PJ, Reilly
AA, Esernio-Jenssen D. Screening Children Exposed to Lead: An Assessment
of the Capillary Blood Lead Fingerstick Test. Clin Chem 1997;43(2):302-311.
4. NCCLS. Proposed
Guideline C40-P. Analytical Procedures for the Determination of Lead
in Blood and Urine. Wayne, PA: National Committee for Clinical Laboratory
Standards, 1998;8(4):1-51.
Measurement
of Lead in Humans with Anodic Stripping Voltammetry in the Laboratory
and in the Field
Dr. Wayne R.
Matson
Introduction
Anodic stripping
voltammetry (ASV) was one of the first micro blood lead techniques introduced
specifically for screening children for lead poisoning in the late 1960’s.
During the early 1970’s the first versions of the technology were used
by the Centers for Disease Control and ESA Inc. to describe the extent
of pediatric lead poisoning in thirty cities around the United States.
Subsequently more rapid and portable versions of the technology have
been used in field studies to screen children for lead in locations
around the world, most recently in extensive initial studies, to define
the extent of lead poisoning in India. The ASV devices have also been
used in a number of central clinical laboratories for routine lead testing.
Recently in
1998 ASV technology has been adapted to a hand held device that allows
complete portability in field-testing. This portable device has been
used to define the extent of lead poisoning in children in various studies
in Russia, China, Ecuador, and other countries. And used in conjunction
with laboratory based ASV devices for initiating screening programs.
ASV techniques
have been shown in a number of studies and long term proficiency tests
to be highly accurate. Both alone, and in conjunction with other techniques
of assessing lead burden they offer some unique capabilities in the
structuring of effective programs for reduction of lead damage to children.
The Technology
of ASV
There are many
confusing and erudite ways of explaining the arcane arts of electrochemistry,
however, anodic stripping voltammetry is fundamentally quite simple.
To perform an ASV test one starts with a sample that has a small amount
of positive lead ions and a lot of other stuff in it. One puts an electrode
in the sample and makes its voltage negative. This brings the positive
lead ions to the electrode and lets them accumulate there leaving the
other stuff behind. After the lead has accumulated onto the electrode
one makes the voltage on the electrode go positive (Anodic). The accumulated
lead is stripped (Stripping) off the electrode and the electrons that
it gives when the voltage changes are counted (Voltammetry). If one
controls the times and voltages and chemistries with a modicum of skill
the number of electrons that are counted equal the amount of lead in
the sample.
Types of ASV
There are two
kinds of ASV that have been routinely used to screen children in the
field and in clinical laboratories. The first uses a composite mercury
graphite electrode (a dispersion of a small millionth of a gram amount
of mercury on graphite) to accumulate the lead from a sample. With this
device a measured 100 (l sample of blood (about three drops) is placed
in a prepackaged tube of reagent containing acidic chromium and other
materials to make the lead in the blood accessible. The tube is then
placed on the electrode assembly and the process of ASV is initiated.
The result of the blood lead analysis is reported in about 80 seconds.
This instrument is designed to work with both fingerstick and stored
venous blood samples, and is suitable for small clinical laboratories
or satellite screening centers performing ca 100-250 tests a day. It
has also been used as a semi portable device for screening centers that
are set up for at least a day or more.
The second type
of ASV uses a screen-printed disposable gold electrode with a protective
coating. With this device (which is about the size of a pocket calculator)
a 50 (l blood sample is placed in a prepackaged tube of reagent and
a drop of the mixture is then placed on the disposable slide. The ASV
process is initiated and the results of the blood lead analysis are
reported in three minutes. This device is particularly useful in a structured
program for identifying " hot spots" or regions where there
is a specific high incidence of lead insult, and for use in a physician’s
office or a remote location where instant results are needed.
Both types of
ASV are unique in that a central laboratory largely performs the quality
control of standards, reagents and procedures. This allows a high level
of control of results in situations where extensive laboratory infrastructure
is not widely available.
ASV and the Issue
of Micro-Fingerstick Samples
Both types of
ASV technology were specifically designed to be applied to fingerstick
blood samples taken from children. The total system of analysis of sample
acquisition, preparation and analysis was integrated for this specific
purpose. There is a recurrent question of how well a fingerstick sample
can be taken without contamination or dilution artifacts.
We have run
screening programs for children in some pretty unlikely places; a private
home in Beijing, a university office in Moscow, store front clinics
and gymnasiums around the USA, and once in a Caribbean bar in St. Thomas.
The blood lead data obtained was confirmed to be as good as that of
the best central laboratories. It can be argued that the people taking
the samples were highly trained and extraordinarily good at getting
clean accurate samples from a child. (The author would like to think
so.).
However, we
also in the 1970’s, provided analytical support for twenty-five small
cities with over a hundred volunteers taking duplicate fingerstick samples.
What we found was that initially there was a high incidence of contaminated
samples. But each volunteer was coded, and his or her personal results
were broken out and reported to him or her. After three to five sampling
events with this feed back they almost all were able to draw duplicates
with minimal contamination or volume error. The critical thing is that
a field compatible technique like ASV provides immediate feedback, not
only to the parent of the child, but also to the technician or volunteer
drawing the sample. This immediate feed back is important in learning
the techniques of accurate sample acquisition.
ASV and the Economics
of Screening
By far the greatest
cost of lead poisoning prevention is in the infrastructure of the program,
finding the children, getting them sampled, following up on the results
of the samples and abating the sources of insult. Having the result
of a blood lead test immediately reduces follow up costs, and eliminates
the frequent problem of not being able to locate an elevated child again.
There is also a more subtle effect. If the caregivers are right there
when a high result is obtained, it is an optimum time to start the educational
and awareness training for beginning to address the problem.
When we were
screening children in an urban Beijing school last fall one of the teachers
noticed as the data was being obtained, that the boys were more affected
than the girls. An ad hoc environmental assessment indicated that the
boys played preferentially in a dusty yard near the highway. A quick
adaptation of the equipment indicated high levels in the dust, and plans
were immediately made to change play habits and reduce dust levels.
Clearly this
one incident is not a global solution to environmental lead pollution,
but equally clearly it illustrates the utility of a technology that
gives immediate results which can be immediately acted upon.
Summary
ASV is one of
many assessment tools for measuring lead insult in children. It has
unique operating capabilities that are useful in structuring a large
scale screening program. It can be used either as a primary screening
tool, or in conjunction with other methodologies to provide an overall
analytical capability for supporting the elimination of lead damage
to children.
Blood
Lead Measurements Using Electrothermal Atomization Atomic Absorption
Spectrometry (ETAAS)
Dr. Patrick J.
Parsons
Background
Historically,
atomic absorption spectrometry (AAS) has proven very successful for
measuring lead in blood, urine and other body tissues. The Delves-cup
AAS arrangement enabled quite small blood volumes (<100 µL) to be
analyzed, and led to establishment of mass screening programs in North
America, Europe and elsewhere. Delves-cup AAS is still used today, and
with very good success, by some laboratories. For more than 25 years,
electrothermal atomization AAS, which is also known as graphite furnace
AAS, has been used for routine blood lead measurements. Early methods
and instrumentation were prone to interferences, and protein separation
was considered mandatory. However, modern furnace instrumentation is
now much more successful, and ETAAS has largely replaced Delves-cup
AAS as the method of choice in most clinical laboratories.
Method Summary
A simple but
robust ETAAS method for blood lead is now available based on a concept
called the Stabilized Temperature Platform Furnace (STPF). This approach
requires the use of: platform atomization to achieve a more isothermal
atomization environment; integrated absorbance signals for improved
precision; chemical modifiers to stabilize the analyte during pyrolysis;
stopped gas-flow and maximum power heating rates during atomization;
and several other conditions. Thus, a consensus method was adopted by
the National Committee for Clinical Laboratory Standards (NCCLS) in
the United States because it appears to be easily transferable between
instruments from different manufacturers.
Sample preparation: 1+9
dilution of 50-µL whole blood with modifier off-line.
Modifier solution: 0.5%
(w/v) NH4H2PO4 + 0.5% (v/v) Triton
X-100 + 0.2% (v/v) HNO3.
Calibration: 3
points + a blank either aqueous Pb in modifier or blood matrix-matched.
Quality
controls: Tri-level QC samples low (10 µg/dL), medium (20-30
µg/dL) and high (40-60 &g/dL).
Instrumentation: l
= 283.3 nm; 0.5-0.7 nm slit width; 12-µL deposited on the platform.
Method Validation
This method
has been validated against certified reference materials from NIST,
CEC BCR, CDC, and NYS DOH. It has proven successful in several proficiency
testing programs for blood lead in the US (CAP, NYS DOH, WSLH and PA
DOH) as well as several external quality assessment (EQAS) schemes (Quebec
CHUL, UK EQAS, and Robens). The method, which was developed for the
Perkin-Elmer 4100ZL (THGA) bench, has been shown to be directly transferable
to instrumentation from Varian Optical Instruments (SpectrAA 400Z, SpectrAA
400P), Thermo Jarrell Ash (AtomSpec GF) and Hitachi (Z-8100, Z-9000),.
It has even proven transferable to very low-cost ETAAS instrumentation
using continuum correction, as well as Zeeman and Smith-Hieftje background
correction systems. Typical analytical run times (excluding preparation)
are of the order of one minute per cycle. The method has also been validated
for measuring lead in urine.
Recommendations
The success
of childhood lead poisoning prevention programs will depend to a large
extent on the quality of data generated by the analytical laboratory.
This is as true of clinical laboratories that must confirm positive
capillary blood screening results with a venous blood lead test as it
is of environmental laboratories that identify the source(s) of lead
exposure. Lead screening programs need access to local specialized laboratories
with the demonstrated skills and experience to report reliable blood
lead measurements for diagnostic purposes and environmental remediation
purposes. These local analytical laboratories should be operated under
the guidance of a centralized reference laboratory(ies) that would be
responsible for operating a proficiency testing program, producing reference
materials certified for lead, and for training and educating laboratory
technicians and supervisors. Given the large numbers of blood specimens
involved, ETAAS is the obvious choice because modern instrumentation
is designed to be run unattended. The key to success will be good training
and adherence to established standards and practices.
Quality
Control and Quality Assurance for Blood Lead Measurements; Role of Certified
Reference Materials; Proficiency Testing Programs
Robert M. Parr
Introduction
Analytical quality
assurance is an essential component of any kind of meaningful program
of environmental monitoring and screening. Appropriate QA/QC procedures
are needed in order to assure (1) comparability of measurements conducted
by participating laboratories for different populations groups and/or
geographical areas, (2) comparability with measurements reported for
similar population groups in other countries and/or for other types
of exposure, and (3) comparability over time, e.g. for monitoring the
effects of interventions.
Quality Systems
Guidelines for
the establishment of quality systems are available from various sources,
particularly the International Organization for Standardization (ISO).
The most relevant guides are ISO-25 and ISO-9000. An internationally
harmonized protocol for proficiency testing prepared by IUPAC, ISO and
AOAC provides similar guidelines for external quality control testing.
Other relevant international guidelines have been proposed within the
framework of the Global Environmental Monitoring System (GEMS) of the
World Health Organization.
Certified Reference
Materials
More than 500
certified reference materials (CRMs) with biological and/or environmental
matrices are available from various international suppliers containing
reference concentration values for lead. Eighteen of these have been
made from human or bovine blood. No CRMs for lead are currently available
from Indian producers, though several are in preparation (human blood,
cattle milk, soil, fly ash, and seawater) and are expected to be available
shortly.
Recommendations
All analytical
laboratories that are participating in a national or regional program
for lead monitoring should have an appropriate quality system in place
to give assurance of their analytical competence. Such quality systems
should conform to the general requirements of international recommendations
on this subject, such as ISO-25 and ISO-9000. Accreditation by a certifying
body is desirable, but not absolutely essential if proficiency-testing
results are satisfactory.
All such participating
laboratories should be able demonstrate their analytical competence
using appropriate CRMs. For method validation, at least two CRMs (preferably
more) should be chosen which cover both the low end and the high end
of the working concentration range that is of interest.
Appropriate in-house
QC materials should be included in every analytical run, and control
charts should be maintained. A Central Reference Laboratory should be
appointed to conduct external QC tests (proficiency tests) on a recurring
basis using "blind" QC materials. The results should be evaluated and
reported (z-scores) in accordance with the relevant IUPAC/ISO/AOAC protocol.
Indian CRMs and testing materials should be used to the extent that
they are available. Otherwise, CRMs should be obtained from other producers
(NIST, BCR, AMI, etc.).
Although the main
emphasis of a program on lead screening will involve the analysis of
whole blood as a surrogate measure of dose, other kinds of samples (air,
water, food, dust, and soil) may also need to be studied to assess different
sources of exposure. The quality system should be able to take care
of such matrices as well.
Conducting
Surveillance and Screening Projects for Childhood Lead Poisoning: Epidemiologic
and Program Issues
Dr. Susan Cummins
Lead exposure is
the most common environmental hazard facing children in the United States
and worldwide. The widespread nature of this persistent environmental
hazard has been characterized by numerous epidemiologic surveys. This
summary reviewed key considerations in the design and conduct of childhood
lead poisoning screening surveys.
Use exposure
Source and Pathway Information to Design the Survey
Any survey for childhood
lead poisoning should focus first on the subgroup of children who are
most likely to be lead exposed. There are two major issues to consider
when selecting an appropriate study population for childhood lead poisoning
screening projects. These are the likely exposure sources and pathways
and the feasible methods for identifying and recruiting subjects. To
illustrate these points, several previous screening projects were reviewed.
Exposure sources
and pathways predict important characteristics of the exposed population.
For example, the most common remaining source of lead poisoning in the
United States is lead-contaminated dust from deteriorating lead containing
paint and leaded gasoline emissions. Young children between the ages
of 6 months and three years are most likely exposed because they engage
in normal hand-to-mouth behavior that brings them in contact with large
amounts of dust. When these behaviors naturally cease, usually at the
age of three years, lead poisoning declines. Additionally, these young
children have a very high metabolic and respiratory rate, and so take
in and absorb more lead dust than do older children. When lead dust
is the most common source of lead exposure, young children must be the
focus of study.
Contrast this example
with a food-borne source of exposure. In California, we recently identified
a cluster of childhood lead poisoning among school-aged children and
teens from consumption of lead contaminated candy imported from Mexico.
Infants and young children did not consume the candy, so did not become
poisoned. In this exposure situation, a study focussing on young children
alone would have missed this important childhood lead poisoning outbreak.
Ascertaining
the population
Sample ascertainment
requires a balance between practical considerations and research needs.
It is usually easier and less expensive to find participants through
a central location where subjects gather, such as a clinic or community
center. However, this approach may miss those who do not use the clinic
or facility, who may be at highest risk for lead exposure. The optimal
research sample is a randomly selected one, and moreover, developing
a listing of the study population is often a barrier and the study sample
will be harder to find and test.
Selecting an
Approach for Measuring Blood Lead Levels
Again, selecting
a blood collection and lead measurement method require balancing practical
and research considerations. Using a fingerstick sample and analyzing
it with the hand held analyzer is the simplest and least expensive method
for obtaining an accurate measurement of individual blood lead levels.
Additionally, participants may receive their results immediately, which
provides an opportunity to provide real benefit to them. However, there
is a great risk of environmental contamination of field samples, which
would result in false elevations in the measurement. The gold standard
method is a venous sample analyzed in a laboratory, However, this method
is more expensive, and requires collection of blood in lead free tubes,
transport, and other logistic difficulties.
Using Survey
Data to Advance Policy
Surveys that identify
a high prevalence of lead poisoning in a community or population provide
powerful information for mobilizing lead poisoning prevention efforts.
A well-designed survey can identify those in the community who are most
affected and the main sources of their poisoning. More importantly,
these data provide the scientific basis for developing programs to reduce
and eliminate childhood lead poisoning over time.
