Sampling Techniques and Procedures
The collection of environmental, biomedical, and background (control) samples is an integral part of investigating allegations pertaining to the first use of chemical or biological agents. The types of samples taken and the collection methods primarily depend upon the circumstances encountered by the collector. During all chemical and biological sampling operations, the commander establishes the required protective equipment to fit the situation. This appendix ends with a recommended list of equipment for use during chemical and biological sampling operations.
NBC recon units collect samples under various circumstances. For example, a recon unit may collect samples in an area free of hostile forces. The LB team may collect samples within the enemy area of operations or deep into the enemy's rear area. Samples include toxic agent munitions, chemical products, air, water, soil, and vegetation. In addition, all expended equipment used to collect the samples should be turned in to the laboratory. This equipment includes items such as expended M256A1 kits, M8 and M9 paper. These items should be recovered, packaged, and shipped with the suspected samples for analysis. Different information may be derived from each type of sample. Table B-1 compares different types of samples.
Control or background samples are collected from clean areas near the attack areas as baseline data. The control samples must be identical to the samples collected from the contaminated areas. The contaminated samples are compared to the baseline data (control samples). This is especially true if unknown or nonstandard chemical and/or suspected biological agents were employed. The analysis center uses the control samples to compare with a contaminated one. The recon unit collects control samples of soil, water, and vegetation from areas about 500 meters upwind of an alleged attack area. Control samples generically are the same as those collected in an alleged attack area. For example, if leaves from an apple tree in an attack area were collected as a suspected contaminated sample, the recon team should collect leaves (as a control sample) from an apple tree outside of the contaminated area. If water from a pond in the attack area is collected, the recon unit should collect control samples of water from a pond (not a moving stream) in a nearby clean area. The size of an environmental control sample should be about the same as the suspected contaminated sample collected from the attack area (see Table B-2).
Collection of Air and Vapor Samples
Air is a suitable sample, because it is well-mixed. A sample of air contains a freed amount of contaminants. The amount of contaminants is determined by several factors:
- Wind speed.
- Rate the contaminant flowed into the environment.
- Physical state of the contaminant.
- Contour of the terrain.
How to Collect Air Samples
Two general guidelines apply to collecting air samples:
1. The recon team collects samples of air, using devices that draw air through a filter material that selectively removes certain compounds from the air.
2. To avoid contamination, persons conducting air sampling should not use cologne, perfume, insect repellent, medical creams, or strong soaps before taking a sample. The fragrances from these products are volatile organic compounds that may be absorbed on the filter and skew analytical results. Smoke also severely interferes with air sampling--therefore, avoid cigarette and vehicle exhaust smoke.
Detailed Procedures for Collecting Air Samples. The primary method for collecting air samples is with the PAS 1000 automatic air sampler in conjunction with a Tenex tube for a total of three to four minutes when possible. Upon completion of sampling, place the Tenex tube in a 2¼-inch piglette. Seal the piglette around the cap with either pressure sensitive or Teflon® tape. Once sealed place the piglette into a Mylar easy-close or "ziplock" bag. Fold the bag around the piglette in a circular motion, then apply another bag and fold again. Once the bag is folded around the piglette, use any type tape to secure the bag around the piglette. Place the piglette into a refrigerator or cooler until the sample is transported to its destination.
Where to Collect Air Sample. When chemicals are permitted into the atmosphere from a facility, the best places to obtain samples are close to the emission source where the concentration of the chemical is not diluted. The further from the original point of release, the more diluted the sample becomes from mixing with air, water, or environmental pollutants.
Natural and man-made terrain features such as hills, valleys, and rows of buildings, sometimes aid the collector by channeling emissions. When these features are associated with a particular facility, their downwind side is a suitable place to collect a sample because the emission remains more concentrated further from the release point.
For collection in a possibly contaminated location, and if the situation permits, initially use a detection kit such as the M18A2/M256A1 to determine if a possible vapor hazard exists from known chemical agents. Also, use the kit when personnel are required to examine possible toxic agent munitions. In any case, collect air samples with the white band tubes and save for identification and analysis.
Small air samplers also enable the collector to obtain vapor samples from alleged toxic agent munitions at a safe distance while explosive ordnance disposal (EOD) operations are performed. If EOD personnel are not on the scene, the air sampler can be activated, and the collector can stand at a safe distance while the sampler is operating.
When to Sample. Perform sampling operations as soon as possible when directed by a higher headquarters or after suspected chemical or biological contamination is encountered.
Collection of Water Samples
Water sampling is a matter of collecting enough water to get acceptable information about the contaminants. The collector should provide the analysis center with one C18 and one silica cartridge when using the Sep-Pak technique.
How to Collect Water Sample. General guidelines: If it is believed that the threat has used standard chemical agents during an attack, use the M272 chemical agent water test kit for initial screening and sampling.
Detailed procedures for collecting water samples. The following items are required along with the Sep-Pak C18 cartridge:
- One 60cc syringe without needle.
- One 3-way sterile, plastic, stop cock with protective covers.
- One piece of plastic tubing (3/16" ID x 6" long minimum).
- Sterile water or methanol.
- One clean container, such as a Teflon® cup or glass jar.
Step 1. Attach Sep-Pak directly to 60cc syringe.
Step 2. Pour small amount of sterile water or methanol into container.
Step 3. Insert tip of Sep-Pak into container.
Step 4. Withdraw at least 40cc of solution.
Step 5. Detach Sep-Pak from syringe and discard solution from syringe.
Step 6. Repeat steps 3 through 5 using the same syringe.
After priming is complete, assemble components in the following configuration:
1. Attach 3-way stop cock to 60cc syringe.
2. Attach Sep-Pak to opposite end of stop cock.
3. Attach plastic tubing to open end of Sep-Pak.
Use the following procedures to collect samples with Sep-Pak:
Step 1. Assure lever on stop cock is turned sideways with off arrow pointed toward the large outlet port.
Step 2. Place the open end of the plastic tubing into the water near the bottom, without touching bottom or sides of the body of water.
Step 3. Draw 60cc of water into syringe.
Step 4. Turn stop cock lever to off position by positioning lever to point toward stop cock.
Step 5. Push plunger all the way in, discharging the water from the syringe through the outlet port.
Step 6. Repeat steps 1 through 5.
Step 7. Detach plastic tubing from Sep-Pak, and discard as contaminated waste.
Step 8. Detach Sep-Pak from 3-way stop cock; place into sample container; seal with pressure sensitive tape; and mark for identification.
Note: You should take a minimum of four (4) samples: three (3) of suspected contamination and one (1) control sample from a nearby unaffected (none contaminated) area for reference.
Step 9. Dispose of syringe and stop cock as contaminated waste.
Step 10. Insert sample container in cooler or refrigerator until the sample is transported to its destination.
Where To Collect Water Samples. Drains are ideal sites, since contamination and dilution from other sources are minimized.
The recon team collects water samples from the slow moving parts of the stream because the turbulence and speed of rapidly flowing water tend to dilute chemical concentration. If an oil-stain-like fan, globules of organic materials, or an unnatural appearing powder-like material is visible on the water's surface, collect a surface sample of the material. If not, collect the sample from near the bottom of the stream. The upper layers of water may have lesser amounts of contaminants, due to higher temperatures that promote decomposition. Since most chemicals of interest are more dense than water, contaminants usually sink to lower levels.
To collect a sample at a given depth, the collector simply immerses a capped or stoppered container to the desired depth, removes the cap or stopper, lets the container fill, and then caps the container. An alternate method for deeper water is to use a plastic, pump operated siphon to pump water from a specific depth.
The recon team may collect samples from stagnant pools of water if the pools of water are part of chemical waste areas, such as a landfill or chemical disposal area. Chemicals may percolate into stagnant pools or sumps close to the site.
When To Collect Water Samples. The best time to collect a sample of water from a facility is when intelligence or local reports indicate that a process of possible interest is ongoing. In the absence of reliable reporting, this may be indicated by increased activity, higher than normal amounts of security, or increased flow from facility chimneys or water discharge pipes.
In field areas where a toxic agent has been sprayed or disseminated over a land area, the best time to collect water samples is just after the start of a rainstorm when runoff is beginning. Natural surface drainage will concentrate any remnants of toxic compounds in depressions, streams, or ditches.
Collection of Soil Samples
Soil is a suitable place to collect samples for toxic organic compounds. A critical point, however, is that the precise site of the agent deposition must be sampled for best results.
How To Collect Soil Samples. Soil samples are collected by scraping into a collection container 2 to 5 centimeters of top soil from areas that appear to have been contaminated. If chunks or clods of earth are collected, select those that are no larger than 10 by 5 by 1 centimeters (see Table B-2). Also, collect a control sample of soil of the same type and texture from a nearby uncontaminated area.
Detailed procedures for collecting soil samples. Using a knife, spoon, spatula, or similar item, scrape 2 to 5 centimeters of suspect top soil into a collection container. Use a glass bottle, jar, or Teflon® jar as a container when available. Mylar bags also may be used. When using a identification. When using Mylar bags, place each sample in one bag, push excess air out, and seal by folding the open end over two to three times and wrapping with tape. Insert the first bag into a second bag, seal, tape, and mark for identification. If possible, place samples in a piglette.
Where To Collect Samples. Contamination is often recognized by discoloration or apparent deposition of material on the soil's surface.
If discoloration or deposits of material are evident, use something such as a garden trowel or wooden tongue depressor to carefully scrape up the soil. Collect only discolored soil or deposited materials, if possible.
Avoid direct contact with the sample (MOPP4 is required).
When To Collect Samples. Sample as soon as possible when directed or after the alleged incident.
Collection of Contaminated Vegetation
Before collecting samples of vegetation from an alleged attack area, the collector makes a visual survey of the area, dons protective equipment (MOPP4), and then enters the area from upwind.
Collect samples of vegetation that appear to be different from normal. Select leaves that have wilted or appear to have been chemically burned. Collect samples of vegetation that appear to have liquid or solid substances deposited on their surfaces (this may be noticed as a shiny or moist area).
Detailed Procedures for Collecting Vegetation. Collect samples of vegetation at several locations within the suspected contaminated area. Using a cutting tool or any sharp object, cut several affected leaves and/or a handful of grass whenever possible. Do not crush the sample. Place the sample into a Mylar or "ziplock" bag. Squeeze excess air out of the bag and seal. Fold open end of the bag over two to three times, and wrap with tape. The minimum size for a sample is three leaves or three handfuls of grass. One leaf is of little value, but is better than nothing. Bark is acceptable but not preferred. Mark the bag for identification. Take a control sample of similar material from an unaffected (uncontaminated) area. Fold, seal, tape, and mark the control sample in the same manner as the actual sample.
Where To Collect Vegetation Samples. When it is possible to determine a probable center of attack in an area, collect vegetation samples near the center of the area, about 100 meters upwind of the area, and in several 100-meter increments downwind of the area. If the collector can discern a contamination pattern in the area, this should be reported.
Just as blood and urine samples are taken from humans who were allegedly exposed in an attack, also collect samples from individuals who claim not to be affected by a toxic agent and are from the same group as exposed personnel. The purpose is the same as collecting environmental control samples; that is, to determine if a toxic substance is present in the individuals' natural environment or if it has been artificially introduced.
Selection of humans for control sampling is somewhat more complicated than selection of environmental control samples. This is because there are potentially large deviations introduced by ethnic diets, racial differences, physiological makeup, and actual living conditions of persons who are outwardly similar. Each of these factors must be accurately considered before selecting subjects as controls.
Consideration of ethnic diets is important because of unique foods or methods of food preparation that may exist. As an example, individuals in settled areas may purchase beer that has been carefully filtered and sterilized, while individuals in a nearby unsettled area may ferment their own beer by burying home crafted jugs for fermenting beer in the ground and extracting the product little by little over several months.
Racial differences can account for differences in mortality and morbidity rates in specific populations. One example of this could be the high rate of hemophilia in a population versus the rarity of the disease in another.
Physiological makeup is critical because of the differences in hormone balance and tissue composition in males, females, adults, and juveniles. For this reason, biomedical controls should be drawn from individuals of the same gender and approximate age as samples from exposed personnel, if possible.
Differences in the actual living conditions of people also require a close look. The point here is that conditions in remote, semicivilized camps are seldom the same as those in a well established camp that has access to modern amenities.
The bottom line in selecting subjects for biomedical control sampling is that they be as similar in all aspects as possible.
Trained medical technicians or physicians should collect biomedical samples (human or animal); however, LB team personnel are trained to do this procedure. Remember, the collector must have express permission (authority) to collect biomedical samples from the dead, because of religious beliefs in many cultures. To obtain such samples without permission may result in unnecessary mission complications. Ensure all personnel handling or collecting biomedical samples have received proper immunizations for their own protection. They must be inoculated IAW the surgeon general's guidance.
Biomedical samples collected during an investigation include blood, urine, and tissue samples from living victims and blood and urine samples from unexposed persons (background control samples).
Collect samples using either a standard 10 cc disposable syringe with a 1- to 1½-inch needle (20 to 22 gauge), or by using a vacutainer system. When using a vacutainer system, ensure that multiple sample. needles and "red top" vacuum tubes are used. Ten cc of blood is sufficient for analytical testing. Do not take more than 5 cc from small, malnourished children. After blood is collected, it should be transferred to a polypropylene-type container and sealed with parafilm before transporting. All body fluids should be collected without violating antiseptic techniques. Also, prior to transporting samples, collectors need to check specimen containers for paper labels IAW guidelines for labeling biomedical samples.
Note: Gloves should be worn for self-protection from transmission of contaminants whenever handling bodily ids. Do not freeze liquid blood and urine samples (ideal cooling temperature is between 35 and 40 degrees Fahrenheit, 2-4 degrees Celsius.)
Collect blood samples using the following materials and equipment:
- 10cc sterile, disposable syringe.
- 1- to 1.5-inch sterile needle (20 to 22 gauge).
- Vacutainer device (adapter with needle).
- Constricting band.
- Disinfectant pads.
- Sterile 2x2-inch gauze pads.
- Betadine or alcohol.
Collect urine samples using either a standard urine cup or by a urine catheter. When using a urine cup, the person must urinate into the cup until sufficient fluid is collected (40 cc of urine is preferable, although 10 cc can support analytical testing). When the person is unable to urinate, the catheterization technique is preferable. The catheterization technique is best performed in a clinical environment. As with other body fluids collected, urine must be kept cod. Do not freeze.
Note: For correct procedures on catheterization refer to STP 8-91B25-SMTG.
When alleged victims have unidentified skin lesions, photographs of the lesion(s) and overall photos of the extent of the lesion(s) should be taken, using color film before biopsy. A sample of the lesion should be obtained. This is done by surgically removing a portion of the skin with a sterile pair of scissors and forceps.
Place tissue samples in a Teflon® container filled 1/4 inch from the bottom with a preservative, (formalin 10%) for preservation of the sample until it reaches its proper destination. Seal the container and lid with parafilm. As with any other biomedical sample, tissue samples are refrigerated prior to shipment; but, do not freeze tissue samples.
Postmortem Examination. If a collector is able to collect samples from dead humans or animals, the following samples are collected:
- Blood. Use a 50-60cc sterile syringe with an 18-gauge, 5-inch (large bore) needle to collect blood from the heart, and urine directly from the bladder. Use a spinal needle to collect cerebral spinal fluids. Three of each samples must be collected.
- Lungs. A biopsy needle is needed to properly collect lung tissue samples. After collecting samples from the lungs, place sample in a plastic or Teflon® container filled with 10% formalin (preservative) and seal the container for transporting to its destination.
- Liver. If possible collect liver core samples, using a large-gauge needle (18-gauge, 5-inch long) via intercostal or abdominal puncture. Or, if the family consents, perform a mini laparotomy and obtain one or two 2x2x2cm sections of liver. Store and package the sample as directed for tissue samples.
Note: Before attempting any of the following procedures, collector must be certified by a qualified person (medical doctor) on the correct procedures to collect samples from cadavers.
Reporting, Packaging, and Shipment
Although a sample collected from an alleged attack area can be significant, it can become useless if proper steps are not talent to record critical information about its collection or if it is improperly packed and breaks during shipment to an analysis center, This section discusses the information needed when acquiring samples and the preferred methods for handling and packing samples for shipment.
A complete history of the circumstances about each sample's acquisition must be provided to the agency analyzing the sample.
Critical information includes--
- Circumstances of Acquisition. How the sample was obtained, where it was found, and how it was collected.
- Physical Description. The physical state (solid, liquid, powder, apparent viscosity), color, approximate size, identity of the specimen (such as military nomenclature), dirt, leaves, or so forth.
- Circumstances of Agent Deposition. The type of delivery system, a description of how the weapon functioned, how the agent acted on release, sounds heard during dissemination, a description of any craters or shrapnel found associated with a burst, and colors of smoke, flames, or mist that may be associated with the attack.
- Agent Effects on Vegetation. A description of the general area (jungle, mountain, grassland) and changes in the vegetation after agent deposition (such as color change, wilting, drying, dead) in the main attack and fringe areas.
- Agent Effects on Humans. How the agent affected personnel in the main attack area versus fringe areas; the duration of agent effects; peculiar odors that may have been noticed in the area prior to, during, and/or after an attack; measures taken that alleviated or deteriorated the effects; and the approximate number of victims and survivors, to include their ages and genders.
- Agent Effects on Animals. The types of animals that were or were not affected by an attack and a description of how they were affected.
Handling and Packaging Materials
Materials used for packaging samples primarily consist of Mylar collection bags, Teflon® specimen jars and tubes, pigs and piglettes, ice chests, sealing materials, and wrapping and cushioning supplies.
Use the Mylar bag as the initial container for such samples as protective masks and filter canisters, individual antidote and decon kits, munition fragments, and other items too large to place in a specimen jar. Use it also to package sample containers to ensure a vapor barrier in case the container is broken in transit. The bag acts as an initial or secondary vapor barrier to prevent air from leaking inward and toxic material outward. Follow the procedures below when using the bag.
If packaging a specimen container or non-environmental sample, first, verify it has a sample number. Carefully place the sample in a bottom corner of the Mylar bag.
Squeeze all the air out of the bag and seal it by removing the adhesive's protective strip, and pressing the two sides together.
Place a piece of 2-inch-wide fiber or cloth tape across the end of the bag that you just sealed to reseal the Mylar bag on the outside. This serves as extra insurance in case the internal seal is broken.
With the bag lying in front of you and the seal at the top, fold the bag across its width to as small a size as possible without damaging the sample. At this point, use tape to hold the fold. Next, fold the bag from the top down to the bottom of the bag to as small a size as possible. The sealing of the bag is the most critical step during the packaging process.
At this point, turn the bag over and use a marker or file label to put the sample number on the outside of the bag so that analysis center personnel can identify the sample.
Place the folded Mylar bag in a clear plastic ziplock bag, if available. Following the same steps you used for the Mylar bag, fold and seal the plastic bag. When this has been completed, again mark the sample number on the exterior of the bag.
Glass Specimen Jars and Polypropylene Tubes
Use glass containers to hold small environmental samples or autopsy specimens. Use polypropylene tubes to hold biomedical samples such as blood or urine. Polypropylene containers may be used for autopsy samples if required; however, glass containers are preferred. The use of glass rather than plastic containers is preferred for environmental samples because toxic agents may leach chemicals from plastics into a sample, introducing contamination and confusing the analysis efforts.
If the container has a screw-on lid, place Teflon® plumber's tape (NSN 8030-00-889-3535; Tape, Anti-Seize) on the threads of the container before putting on the lid. This helps to limit the leakage of liquids and vapor from the container and to assure the lid will not fall off while in transit. If the lid has a cardboard liner, remove the liner and replace it with one or two layers of parafilm (a laboratory sealant film).
Once the lid is on, stretch parafilm around the outside of the container at the junction of the lid and the glass. Two wraps of the film is enough to provide a leakage barrier and more assurance that the lid cannot fall off.
At this point, ensure the sample number is on the outside of the container. Use a diamond etching pencil or an adhesive label to put the sample number on the exterior of the container.
Six-Pound Metal Can
Use metal cans as the external container for packaging small items that have been sealed in Mylar bags, specimen jars, and polypropylene tubes containing biomedical samples. The metal can helps absorb shock from rough handling during shipment and eliminates the spread of contamination if a specimen container is broken, The six-pound metal can is capable of holding more than one sample (depending upon size of samples).
Before placing samples in the can for shipping, ensure a sample number is assigned and is visible on each item.
Place about 1 to 2 inches of packing material in the bottom of the can.
Wrap jars and tubes in plastic bubble wrap or 1/8- to 1/4-inch-thick foam rubber sheeting, secure the wrap with tape or a rubber band, and place the wrapped item in the can.
If bubble wrap or foam rubber is not available, use newspaper. The guiding principle is that the sample containers should fit snugly and not be able to move in the can.
Standard polyethylene or metal ice chests are the most easily procured items used for transworld shipment of CB samples. The most easily used size is about 24 inches long by 18 inches high by 15 inches deep. This size permits the sender to ship two or three 6-pound metal cans in each chest with sufficient dry ice to maintain freezing temperatures for about four days. Also, each chest remains at a weight that one individual can handle.
The best coolant available in most areas is dry ice. It maintains low temperatures for several days and is easy to handle. Blue ice, a plastic containerized refrigerant, is acceptable and used if available, but will not maintain freezing temperatures for as long as dry ice. Standard ice should only be used as a last resort, because of its rapid melting rate and the possibility that melted ice may contaminate samples.
While a commercial ice chest provides good insulation of both the samples and the coolant, it is best to place extra insulation and cushioning around the metal cans inside the chest.
Newspapers, plastic bubble wrap, and foam rubber may all be used with almost equally good results except newspapers and standard ice do not mix well.
The collector must provide a formatted message for transmission as soon as possible to report acquisition and shipment of samples, During special operations in a theater in which a special forces group (SFG) is deployed, the message is transmitted by the fastest means through the fewest channels to the chemical-biological sampling control element (CBSCE). If a CBSCE has not been deployed to the area of operations, as in a low-sample volume peacetime CB sampling operation, the message is transmitted by the fastest means through the fewest channels to the message addressees below. In addition, a written report accompanies each sample or batch of samples. The collector ensures that the acquisition message has been properly classified.
The acquisition report includes at least the following addressees:
DIR AFMIC FT DETRICK MD//AFMIC-CR/AFMIC-SA//
CMDT USACMLS FT MccLELLAN AL//ATZN-CM-CU//
CDR CBDA APG
CDR FSTC CHARLOTTESVILLE VA//AIAST-RA-ID2//
An acquisition message contains the following information:
- The sample identification number is part of the subject line if only a single sample is referred to in the text. Otherwise, refer to the sample number within the message body with its background information.
- The shipment date, mode of transportation, courier identification, air bill of lading number, flight number destination, and estimated time of arrival are included if the sample is to be shipped immediately. Also, the material courier receipt form (DD Form 1911) should be used to maintain chain of custody.
- Background information on the sample. Questionable circumstances surrounding acquisition of a sample. The name of another country or agency that acquired a sample from the same event or area and is not shown on the message address.
- A recommended priority and rationale for analysis to guide the analysis center on the assessment of the potential value of the sample.
- All details relating to the acquisition of the sample, regardless of how insignificant they may seem to the collector.
Dispose of samples according to the physical category of each.
Ship all samples by the fastest, safest means--preferably by a technical escort unit (TEU)--to the theater CBSCE or to a location the CBSCE designates. If there is no CBSCE in the theater, send the samples IAW preplanned instructions from the Chemical-Biological Sampling Control Center (CBSCC) at CBDA, Aberdeen, Maryland. The CBSCC uses the following criteria to determine the final destination of each sample:
- Is the sample chemical or biological in content?
- Is the sample content completely unknown?
- Is the sample a possible combination of chemical and biological material?
In any case, the CBSCC must be notified in advance of shipment of the sample so additional instructions or deviations from standard instructions can be given. Figure B-1 shows an example of a shipping notification message. The CBSCC will direct, in advance, that samples be sent to one or more of the following locations, depending on the category of the samples.
Prior to shipment, contact must be made with--
Technical Escort Unit
Aberdeen Proving Ground, MD 21010
|DSN--||584-4381 (Duty hours)|
|584-2773 (After duty hours)|
This unit controls the transport of samples to their final destination(s). Do not ship suspected toxic samples or munition systems to CONUS technical centers or intelligence agencies without coordination and prior approval by the recipient.
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