Appendix D

BIOLOGIC SAMPLE COLLECTION AND ANALYSIS PLANS:

Collection:

URINE

BLOOD

BUCCAL CELLS

Analysis:

METALS

NON-PERSISTENT AND PERSISTENT PESTICIDES

POLYCHLORINATED BIPHENYLS (PCBs)

VOLATILE ORGANIC COMPOUNDS (VOCs)

INFECTIOUS AGENTS

### Prepared in Support of:

CDC/NCEH Cross Sectional Assessment Study

Prepared by:

Centers for Disease Control and Prevention

National Center for Environmental Health

National Center for Infectious Diseases

## Cross-sectional Exposure Assessment of Environmental Contaminants in

## Churchill County, Nevada: Protocol for Collecting Urine Specimens

## 

***COLLECTION PROCEDURE***

Urine collection cups will be provided for each participant. Instruct
each person to do the following for urine collection:

\- Hands should be washed with soap and water.

\- Do not remove the cap from cup until ready to void.

\- Collect at least 30 mL urine in the cup.

\- Do not touch the inside of the cup or cap at anytime.

\- Recap the specimen and deliver to investigator.

\- Place a label for **[URINE CONTAINER]{.underline}** on cup.

One aliquot is needed. Any specific analyte can be prepared at CDC from
this container.

\- Pour 25 mL urine into a 1 oz glass bottle with the green screw cap.

\- Place the participant's ID label on each container.

***SHIPPING LIST***

A collection log is provided to record samples that are collected.
Please mark the appropriate spaces indicating which aliquots were
collected, date collected and any problems that were encountered in
collection, storage, or shipping.

***SHIPPING PROCEDURE***

1\. Pack the shipping box with the boxes of urine samples. Place each
box in the zip-lock bags before packing. Fill the shipper with dry ice,
cover with the Styrofoam lid and tape down the cardboard outer flaps.
Place a dry ice label on the outside of the container and write in the
amount of dry ice in the shipper.

2\. Ship to the following address:

Charles Dodson

Centers for Disease Control and Prevention

4770 Buford Highway NE

> Building 17 Loading Dock

Atlanta, GA 30341

##  3. Please call (770) 488-4305 on the day the shipment is made. Also, if any questions arise, please call the above number. 

## Cross-sectional Exposure Assessment of Environmental Contaminants in

## Churchill County, Nevada: Protocol for Collecting Blood Specimens

1\) Have the following items on hand and available:

-   Blue Absorbent Pad

-   Powder free gloves

-   Tourniquet

-   Alcohol disinfectant swabs (individually wrapped)

-   Gauze bandages (sterile, individually wrapped)

-   21g or 23g vacutainer butterfly

-   Vacutainer needle holder

-   7 mL PURPLE top tube with Hemagard cap

-   7 mL GREY top tube with Hemagard cap

-   7 mL PURPLE top tube with Hemagard cap

-   Bandage

-   Sharps disposal container for used needles and butterflies

2\) Select the appropriate size butterfly and attach to the Vacutainer
needle holder.

3\) Wipe the three tube caps with an alcohol wipe **immediately** before
collection.

4\) Tie the tourniquet onto the upper arm so that it can be quickly
released with one hand.

5\) Swab the venipuncture area with an alcohol pad.

6\) Wipe off excess alcohol with the gauze bandages. Allow to air dry
for 5 - 10 seconds.

7\) Puncture the vein with the butterfly needle.

8\) Insert the **first 7mL purple top tube** into the barrel of the
vacutainer needle holder and push until blood enters the tube. The tube
will draw only 6.5 mL of blood. When full, remove tube and invert 4-6
times to mix.

9\) Insert the **GREY top 7mL tube** into the barrel of the vacutainer
needle holder and push until blood enters the tube. When full, remove
tube and invert 4-6 times to mix.

10\) Insert the **second 7mL purple top tube** into the barrel of the
vacutainer needle holder and push until blood enters the tube.

11\) Release the tourniquet when the last tube has filled half way.
Allow tube to finish filling, remove from holder, invert 4-6 times to
mix, and then apply pressure with sterile gauze to venipuncture site as
you remove the needle.

12\) Carefully remove vacutainer needle or butterfly from holder and
dispose of it in the sharps container.

13\) Mix all of the blood tubes well by inverting 4-6 times upon removal
from the holder to ensure good distribution of the anticoagulant
throughout the blood.

14\) Immediately upon completion of venipuncture, while pressure is
being applied to site, pick up collected tubes and invert 6-10 more
times to mix.

15\) Place pressure on the venipuncture site for a few minutes with a
gauze pad. Cover the venipuncture site with a bandage.

16\) Place a bar-coded ID label on the vacutainer tubes. Make sure that
the label edge starts on the edge of the tube label and that there is a
"window" so that one can see the tube contents. Place the label on the
tube so that the barcode looks like a "ladder" when the tube is held
upright.

# 17) Record the sample number on the collection log indicating results of the collection and appropriate collection comments if difficulties were encountered.

18\) Place samples in the sample tube rack or box provided. Give the
blood and urine specimens to the CDC laboratory personnel.

*Notes:*

If unable to collect blood after two tries, contact the CDC laboratory
personnel.

If the CDC laboratory personnel are unable to collect a blood specimen
after two additional tries then consult the CDC MD.

## Cross-sectional Exposure Assessment of Environmental Contaminants in

## Churchill County, Nevada: Protocol for Collecting Buccal Cell Specimens

![](media/image1.png){width="2.3in" height="1.1444444444444444in"}

1)  Read and sign the enclosed consent forms.

2\) Twist it at the blue arrow to remove the cap. Take the brush out of
the tube. Be sure **NOT** to touch the brush end.

![](media/image2.png){width="2.609722222222222in"
height="2.0006944444444446in"}

3\) Place the brush inside the subject's mouth, against the inside of
the cheek. Rub the brush firmly up and down along the inside of the
cheek 30 times.

4\) Put the brush back into the tube with the brush end first. Cover the
tube with the cap. Do not re-use this brush on another person.

5\) Place the Barcode label on the tube and write the **date** the
sample was collected on the tube label.

6)![](media/image3.png){width="2.6in" height="1.3368055555555556in"}
Repeat steps 2-5 with the second tube. This time, use the brush on the
other cheek.

7)  Store the tubes on dry ice.

# METALS

**Laboratory Methods for the Elemental Analysis of Blood and Urine
Samples**

**Collected during the Churchill County Leukemia Investigation**

**Collection of Blood Samples**

The blood sampling was conducted by drawing from all study participants
by venipuncture whole blood samples into EDTA-anticoagulated tubes.
Blood specimens were refrigerated then aliquoted within two hours into
cryovials immediately frozen and shipped on dry ice. All blood samples
were stored frozen at or lower than -20ºC until analyzed. All blood
collection supplies including butterflies, syringes, needles, evacuated
tubes, transfer pipettes and cryovials were prescreened for metal
contamination before use.

**Analysis of Blood Samples**

[Lead and Cadmium]{.underline}

Lead was measured by graphite furnace atomic absorption spectrometry
(GFAAS). The GFAAS included simultaneous determination of lead and
cadmium utilizing Zeeman background correction and resulted in a blood
lead limit of detection for lead of 0.3 µg/dL and for Cadmium of 0.3
µg/L. The reported lead and cadmium results were the average of two
measurements. The blood lead/cadmium instrument was calibrated by using
calibrators prepared by using the National Institute of Standards (NIST)
Standard Reference Material (SRM) aqueous lead. Analytical quality
control was monitored by utilizing six quality control pools to assure
adherence to quality control standards.

[Mercury]{.underline}

Whole blood specimens were analyzed for total and inorganic mercury.
Specimens were analyzed using an automated cold vapor atomic absorption
spectrophotometry instrument (PerkinElmer Flow Injection Mercury System
\[FIMS\] 400 with an AS-91 autosampler). The detection limit was 0.14
parts per billion (ppb) for total mercury and 0.4 ppb for inorganic
mercury. Matrix matched calibration methods were used for both blood
measurements. The total blood mercury analysis utilized a Maxidigest MX
350 in-line microwave digester connected to the FIMS-400 system. The
inorganic mercury analysis utilized stannous chloride as the reducing
agent and the total mercury analysis utilized sodium borohydride as the
reducing agent. The blood mercury analysis requires 0.2 mL of blood for
the total and an additional 0.2 mL of blood for the inorganic analysis.
The reported mercury result was the average of two measurements. NIST
SRM 966 was used as a bench quality control material as well as 3 levels
of in-house blood pools traceable to NIST SRM 966 for daily QC.

**Collection of Plasma Samples**

The blood sampling was conducted by drawing from all participants by
venipuncture whole blood samples into EDTA-anticoagulated tubes. Samples
were spun down and plasma was aliquoted into cryovials within two hours
then immediately frozen and shipped on dry ice. All plasma samples were
stored frozen at or lower than -20ºC until analyzed. All blood
collection supplies utilized for plasma collections including
butterflies, syringes, needles, evacuated tubes, transfer pipettes and
cryovials were prescreened for selenium contamination before use.

**Analysis of Plasma Samples**

[Plasma Selenium]{.underline}

Plasma selenium concentrations were determined by using Inductively
Coupled Plasma Dynamic Reaction Cell Mass Spectrometry (ICP-DRC-MS).
This multi-element analytical technique is based on quadruple ICP-MS
technology and includes DRC™ technology, which minimizes or eliminates
many argon-based polyatomic interferences.

In this method, selenium was measured at mass 80 utilizing methane as
the reaction gas. Plasma samples were diluted 1+23 with 1% (v/v)
double-distilled nitric acid, 0.01% Triton-X-100, 10% ethanol containing
gallium for internal standardization. The detection limit for plasma
selenium was 3 µg/L. The ICP-MS was calibrated using NIST traceable
matrix matched calibrators. The reported selenium concentration was the
average of three measurements. Quality control was monitored by
utilizing three quality control pools to assure adherence to quality
control standards.

**Collection of Urine Samples**

Urine samples were collected from study participants by collecting a
midstream void into a sterile urine collection cup. Urine specimens were
then aliquoted into cryovials, frozen and shipped on dry ice. All urine
samples were stored frozen at or lower than -20º C until analyzed. All
urine collection supplies including urine cups, transfer pipettes, glass
bottles, and cryovials were prescreened for all metal contamination
before use. The mercury urine specimen was aliquoted into a cryovial
that included a preservative added for the stability of the mercury
specimen.

**Analysis of Urine Samples**

[Mercury]{.underline}

Samples were analyzed for mercury using an automated cold vapor atomic
absorption spectrophotometry instrument (PerkinElmer FIMS-400 with an
AS-91 autosampler). The detection limit for mercury was 0.14 µg/L. A
matrix matched calibration method was used. The urine mercury analysis
required 0.2 mL of urine. The reported mercury result was the average of
two measurements. Analytical quality control was monitored by utilizing
three quality control pools to assure adherence to quality control
standards.

[Chromium]{.underline}

Chromium in urine was measured by GFAAS. The GFAAS determination of
chromium utilizes Zeeman background correction and results in a limit of
detection for urine chromium of 0.3 µg/L. The reported chromium results
were the average of two measurements. The urine chromium instrument was
calibrated by using calibrators prepared by using the NIST SRM aqueous
SRM. Analytical quality control was monitored by utilizing four quality
control pools to assure adherence to quality control standards.

[Nickel]{.underline}

Nickel in urine was measured by GFAAS. The GFAAS determination of nickel
utilizes Zeeman background correction and results in a limit of
detection for urine nickel of 1.5 µg/L. The reported nickel results were
the average of two measurements. The urine nickel instrument was
calibrated by using calibrators prepared by using the NIST SRM aqueous
SRM. Analytical quality control was monitored by utilizing four quality
control pools to assure adherence to quality control standards.

[Arsenic (total)]{.underline}

Urine arsenic (total) concentrations are determined by using ICP-DRC-MS.
This multielement analytical technique is based on quadruple ICP-MS
technology and includes DRC™ technology, which minimizes or eliminates
many argon-based polyatomic interferences. In this method, arsenic
(isotope mass 75) and gallium (isotope mass 69) or tellurium (isotope
mass 126) are measured in urine by ICP-DRC-MS using argon/hydrogen
(90%/10%, respectively) as a reaction gas. Urine samples are diluted 1+9
with 2% (v/v) double-distilled nitric acid containing gallium or
tellurium for internal standardization. The detection limit for urine
arsenic is 0.3 µg/L. The ICP-MS was calibrated using NIST traceable
matrix matched calibrators. The reported urine arsenic concentration was
the average of three measurements. Quality control was monitored by
utilizing four quality control pools to assure adherence to quality
control standards.

[Manganese]{.underline}

Urine Manganese concentrations were determined by using Inductively
Coupled Plasma Dynamic Reaction Cell Mass Spectrometry (ICP-DRC-MS).
This multielement analytical technique is based on quadruple ICP-MS
technology and includes DRC™ technology, which minimizes or eliminates
many argon-based polyatomic interferences. In this method, manganese was
measured at mass 55 utilizing methane as the reaction gas. Plasma
samples were diluted 1+23 with 1% (v/v) double-distilled nitric acid,
0.01% Triton-X-100, 10% ethanol containing rhodium for internal
standardization. The detection limit for plasma selenium was 0.24 µg/L.
The ICP-MS was calibrated using NIST traceable matrix matched
calibrators. The reported manganese concentration was the average of
three measurements. Quality control was monitored by utilizing three
quality control pools to assure adherence to quality control standards.

[Multi-Element]{.underline}

Multiple elements in urine were measured by ICP-MS. This ICP-MS method
measured the following 12 elements in urine: Beryllium (Be), Cobalt
(Co), Molybdenum (Mo), Cadmium (Cd), Antimony (Sb), Cesium (Cs), Barium
(Ba), Tungsten (W), Platinum (Pt), Thallium (Tl), Lead (Pb), and Uranium
(U). Urine samples were diluted 1+9 with 2% (v/v), double-distilled,
concentrated nitric acid containing both iridium (Ir) and rhodium (Rh)
for multi-internal standardization. The detection limits for the urine
metals were: Be 0.14 µg/L, Co 0.10 µg/L, Mo 1.7 µg/L, Cd 0.09 µg/L, Sb
0.08 µg/L, Cs 0.22 µg/L, Ba 0.30 µg/L, W 0.04 µg/L, Pt 0.06 µg/L, Tl
0.03 µg/L, Pb 0.22 µg/L, and U 0.006 µg/L. The ICP-MS was calibrated
using NIST traceable matrix matched calibrators. The reported urine
metals concentration was the average of four measurements. Quality
control was monitored by utilizing four quality control pools to assure
adherence to quality control standards.

*PESTICIDES AND POLYCHLORINATED BIPHENYLS (PCBs)*

###### Laboratory Methods for the Analysis of Pesticides and Polychlorinated Biphenyls

**and/or their Metabolites in Blood and Urine Samples**

**Collected during the Churchill County Leukemia Investigation**

**Collection of Urine Samples**

Urine samples were collected from study participants by collecting a
midstream void into a sterile urine collection cup. Urine specimens were
then aliquoted into cryovials, frozen, and shipped on dry ice. All urine
samples were stored frozen at -70oC until analyzed.

**Analysis of Urine Samples**

[Class-Specific Metabolites of Organophosphate Pesticides]{.underline}

Samples (2 mL) were lyophilized then the metabolites were derivatized to
their respective chloropropyl phosphate esters. The esters were analyzed
using gas chromatography-tandem mass spectrometry. The detection limits
ranged from 0.1 to 0.6 ng/mL. A matrix-matched isotope dilution
calibration method was used. Analytical quality control was monitored by
utilizing two quality control pools to assure adherence to quality
control standards.

[Chlorinated Phenols, Chemical-Specific Organophosphate Pesticide
Metabolites, Carbamate Metabolites, Fungicides]{.underline}

Samples (2 mL) were extracted with n-butyl chloride/diethyl ether then
the metabolites were derivatized to their respective chloropropyl
ethers. The ethers were analyzed using gas chromatography-tandem mass
spectrometry. The detection limits ranged from 0.1 to 1.1 ng/mL. A
matrix-matched isotope dilution calibration method was used. Analytical
quality control was monitored by utilizing two quality control pools to
assure adherence to quality control standards.

[Herbicides, Pyrethroids and Repellents]{.underline}

Samples (2 mL) were extracted using solid phase extraction cartridges.
The extracts were analyzed using high-performance liquid
chromatography-atmospheric pressure chemical ionization-tandem mass
spectrometry. The detection limits ranged from 0.01 to 0.5 ng/mL. A
matrix-matched isotope dilution calibration method was used. Analytical
quality control was monitored by utilizing two quality control pools to
assure adherence to quality control standards.

**Collection of Plasma Samples**

The blood sampling was conducted by drawing from all participants by
venipuncture whole blood samples into EDTA-anticoagulated tubes. Samples
were spun down and plasma was aliquoted into cryovials within 2 hours
then immediately frozen and shipped on dry ice. All plasma samples were
stored at -70oC until analyzed.

**Analysis of Plasma Samples**

### 

[PCB/Organochlorine Pesticide Analysis]{.underline}

Samples (1 mL) were lyophilized on hydromatrix in an accelerated solvent
extraction cell. The cell was subsequently filled with florisil and
inverted. The sample was extracted using accelerated solvent extraction
with dichloromethane:hexane and received an in-line florisil cleanup
during the extraction process. The extracts were analyzed using gas
chromatography-high resolution mass spectrometry. The detection limit
for the PCBs and organochlorine pesticides were 5 and 20 pg/mL,
respectively. An isotope dilution calibration method was used.
Analytical quality control was monitored by utilizing two quality
control pools to assure adherence to quality control standards.

*VOLATILE ORGANIC COMPOUNDS (VOCs)*

###### Laboratory Methods for the Analysis of Volatile Organic Compounds

**in Blood Samples Collected during the Churchill County Leukemia
Investigation**

Volatile organic compounds (VOCs) are measured in whole blood by solid
phase microextraction/gas chromatography/isotope dilution mass
spectrometry based on the method described by Cardinali, et al. (1). The
analytes are in equilibrium between the whole blood matrix and the
headspace above the sample. A solid-phase microextraction fiber is
inserted into the headspace and the VOCs partition into the phase on the
outside of the fiber shaft. This fiber is then inserted into the heated
GC inlet where the VOCs rapidly desorb due to the increased temperature.
Extracted VOCs are focused at the head of the GC column using a
cryo-trap. Analytes are separated on a DB-VRX column and quantified
using selected ion monitoring mass spectrometry (unit mass resolution).
Comparison of relative response factors with known standard
concentrations yields individual analyte concentrations. The method is
applicable to the determination of 31 VOCs in 3 mL blood with detection
limits in the low parts per trillion range. Since non-occupationally
exposed individuals have blood VOC concentrations in this range, the
method is applicable for determining these quantities and investigating
cases of low-level exposure to VOCs.

*INFECTIOUS AGENTS*

**Laboratory Methods Used to Detect Viruses, Retroviruses and Retrovirus
Activity in**

##### Blood Samples Collected during the Churchill County Leukemia Investigation

+----------------------------------------+-----------------------------+
| # **Specific Virus Tested**            | ## Detection Method         |
+----------------------------------------+-----------------------------+
| **Retroviruses**                       |                             |
+----------------------------------------+-----------------------------+
| Reverse transcriptase activity ^a^     | AmpRT activity ^b^          |
+----------------------------------------+-----------------------------+
| Human T-cell lymphotropic virus types  | Serology ^c,\ d,\ e^        |
| 1 and 2                                |                             |
|                                        |                             |
| (HTLV-1/2)                             |                             |
+----------------------------------------+-----------------------------+
| Feline Leukemia Virus (FeLV)           | PCR ^f,\ g^                 |
+----------------------------------------+-----------------------------+
| Avian Leukosis Virus (ALV)             | Serology ^c,\ d^            |
+----------------------------------------+-----------------------------+
|                                        |                             |
+----------------------------------------+-----------------------------+
| **DNA viruses**                        |                             |
+----------------------------------------+-----------------------------+
| Epstein-Barr virus (EBV) ^h^           | Serology ^c,\ h^ by:        |
|                                        |                             |
| EBV viral capsid antigen (VCA)-IgG ^i^ | #### IFA ^k^                |
|                                        |                             |
| EBV nuclear antigen in Raji cells      | ACIF ^l^                    |
| (EBNA-Raji)                            |                             |
|                                        | ACIF ^l^ confirmed by       |
| EBV nuclear antigen 1 (EBNA-1)         | EIA^d^                      |
|                                        |                             |
| EBV viral capsid antigen (VCA)-IgM ^j^ | EIA^d^                      |
+----------------------------------------+-----------------------------+

^a^ Reverse transcriptase activity (also called 'ampRT') is a general
screen for the presence of living, active retroviruses in the blood
stream. It can be positive when someone has an active retroviral
infection. The retrovirus must be making new virus (replicating) above a
certain level for this test to be positive. Some viruses become dormant
or inactive. The laboratory test can be negative when a retroviral
infection is dormant (latent) or inactive or when levels of the product
of actively replicating virus are low in peripheral blood.

^b^ Heneine et al. The Journal of Infectious Diseases 1995; 171.

Garcia Lerma et al. The Journal of Infectious Diseases 1998; 177.

^c^ Serology detects the presence of blood stream antibodies to certain
organisms. In general, antibodies are proteins the body produces to
protect people against certain infections. Antibodies usually circulate
in the blood stream. Different tests are used to measure antibodies.

^d^ Enzyme-linked immunoassay (EIA), enzyme-linked immunosorbent assay
(ELISA), and western blot (WB) are types of antibody tests for serology.
Sometimes only one type of test is needed to detect infection. Sometimes
combinations of tests are needed to get the final test result. This
study used ELISA confirmed by WB (when indicated) to test for HTLV-1/2
serology. The study used WB testing for ALV serology.

^e^ Vironostilka HTLV-I/II Microelisa System, Organon Teknika
Corporation, Durham, NC; Genelabs Diagnostics HTLV BLOT 2.4 Western Blot
Assay, Zeptometrix Corporation, Buffalo, NY.

^f^ Polymerase chain reaction to detect DNA (deoxyribonucleic acid) is a
molecular test that detects nucleic acids, or parts of genes, within
certain limits. It does not tell whether the organism is alive or dead.

^g^ Butera et al. Journal of the American Veterinary Medical Association
2000; 217.

^h^ EBV testing was performed by a contractor. The combination of test
results from a four-part EBV test panel determines the infection status
of an individual. The parts of the panel are:

-   EBV viral capsid antigen (VCA)-IgG ^i^ appears at almost the same
    time as VCA-IgM ^j^, peaks 2-3 months after onset of infection, and
    usually remains at a stable, detectable level for the rest of a
    person's life.

-   EBV viral capsid antigen (VCA)-IgM is detectable 2-6 weeks after
    acute infection and disappears for life in the majority of
    individuals.

-   EBV nuclear antigen 1 (EBNA-1) is the dominant one of six proteins
    in the Epstein-Barr complex of nuclear antigens; antibodies to
    EBNA-1 first appear 10-12 weeks after acute EBV infection and so
    give a longer interval during which to detect acute infection than
    do IgM antibodies. Once the titer (level) of antibodies to EBNA-1 is
    ≥20 (about 3 months after onset of infection), \>95% of individuals
    no longer have detectable VCA-IgM antibody.

-   EBV nuclear antigen in Raji cells (EBNA-Raji) is the reference
    source of Epstein-Barr nuclear antigens.

^i^ Immunoglobulin G (IgG) is a type of antibody in the blood stream. It
usually indicates past infection and can be measured for years after an
infection. IgG antibodies can also be measured during some persistent
infections.

^j^ Immunoglobulin M (IgM) is a type of antibody in the blood stream. It
is usually detected during or shortly after acute infection then
disappears over time.

^k^ Indirect immunofluorescence assay (IFA) is a type of test used to
detect antibodies in the blood stream or serum.

^l^ Anticomplement immunofluorescence (ACIF) is a test used to detect
specific proteins produced by the body during certain infections.