• All specimens (regardless of physiological source) must be labeled to positively identify the individual or animal from whom it was collected; at a minimum, the individual's full name, unique personal identification number (social security number, when possible), military unit and location, and date and time of collection should be written on the label of the specimen container.
• All specimens are collected using aseptic techniques. All specimens are packaged, handled, and transported in a manner that ensures they arrive at the final destination laboratory in a testable condition. Personal protection guidelines must be adhered to when collecting or processing specimens; at a minimum, this includes gloves and a mask. In the laboratory, a gown or other protective items may also need to be used. In the field, under suspect NBC conditions, collectors should be in MOPP Level 4 or inside NBC-protected vehicles. Common sense and the clinical and/or tactical situation will determine the extent of personal protection necessary.
• Preservation of specimens, either chemically or mechanically (cooling), will be necessary to minimize the amount of analyte degradation that occurs after removing the specimen from its physiological microenvironment. The optimal preservation technique will vary with different laboratory tests and must be confirmed for each requested assay. While freezing may preserve some serum constituents, freeze-thawing cycles may denature others. Freezing may also completely destroy certain microorganisms. This caution also applies to tissue specimens since "fixing" tissue with a standard 10 percent formalin solution will preserve tissue for special staining techniques; however, it renders the specimens completely useless for microbiological culture. Always verify specimen preservation requirements for storage and transport with the supporting medical laboratory before processing the specimen. Ideally, confirmation of the correct handling conditions should be coordinated before collection.
• The importance of coordinating sample/specimen collection with the supporting laboratory facility cannot be overstated. Contact the receiving laboratory for instructions when doubt exists about the appropriate source, collection technique, storage and preservation conditions (such as, aerobic or anaerobic environment), and transportation requirements for samples/specimens. Extremely small volumes of samples/specimens, properly collected and handled, can yield a tremendous amount of information to assist in making medical, tactical, and strategic decisions. Conversely, very large quantities of poorly collected and insufficiently preserved samples/specimens are essentially worthless for most analytical techniques.
• Analysis beyond intratheater capabilities will be coordinated by the supporting laboratory, when deployed, or through medical channels in the absence of an in-theater supporting laboratory.

b. Post mortem and Forensic Specimens. The analysis of specimens from deceased humans and animals can provide valuable information about the disease, organisms, injuries, or environmental conditions at the time of death. This information can greatly enhance the treatment of others affected by the same, or physiologically similar, process. Specimen collection for post mortem or forensic examination is very important; the techniques involved reflect a significant degree of training, experience, and skill. Most specimens will be of the same type and size as for ante mortem specimens, but types and amounts of specimens will be determined by the collector.

(1) The collection of specimens from remains should be conducted exclusively by a pathologist, or other personnel specifically trained in forensic collection techniques. An exception is when Special Operations Forces (SOF) personnel are operating under radio silence conditions; the most qualified medical person with the operation collects, preserves, and transports or coordinates transport of specimens for evaluation. The same chain of custody requirements applies to specimens collected by SOF personnel, as with all other specimens.

(2) A large amount of support information can be gained by analyzing the site of injury and subsequent death. This "site scene" investigation requires a tremendous attention to detail and a trained observer. If forensic personnel cannot be contacted, or will be unduly delayed in arriving at the scene, then photographs of the victim and the immediate surroundings should be made. The scope and extent of the photographs should be composed to reflect as much detail as possible to assist forensic personnel in reviewing the scene retrospectively. In the event that photography is not feasible, detailed sketches of the scene should be made to assist the forensic investigation.

(3) Techniques such as cardiac or bladder puncture, needle biopsy of organs, spinal tap, or exploratory laparotomy will not be performed by untrained personnel unless specifically requested and directed by forensic investigators.

Table B-1. Specimen Collection for Suspect Biological Warfare Agents

c. Water Sample Collection.

(1) Water samples for identification or verification of biological agent contamination are collected by PVNTMED personnel. The supporting laboratory should provide guidance on sampling procedures and collecting kits for use in collecting the samples. In the absence of guidance, a technique for use of the Sep-Pak^TM is described in FM 3-19.

(2) When sampling kits are not available, samples may be collected in other available sterile containers. The best containers for use are the 100-ml glass bottles used for collecting routine water samples. All water samples must be collected and placed in a cooler or refrigerator until the sample is transported to its destination. During transportation the samples must be maintained at a temperature between 1°C and 4°C.

d. Food Samples. Veterinary personnel must collect suspect biologically contaminated food samples for submission to the supporting laboratory for in-theater verification of contamination. All food samples must be collected and placed in sterile containers. Place the samples in a cooler or refrigerator until the sample is transported to its destination. During transportation the samples must be maintained at a temperature between 1°C and 4°C.

e. Animal Specimens. Veterinary personnel collect specimens from suspect biologically contaminated/diseased animals. The same types and amounts of specimens are prepared and shipped in the same manner as are human specimens.

f. Environmental Samples. Environmental samples are collected as directed in the operators' manual or other publications for operating collection systems. Example: The Biological Integrated Detection System (BIDS) collects an environmental sample using a single liquid sample collector. The collector is a high-volume aerosol sampling and collection device. On demand it samples ambient air through a two-stage virtual impactor that concentrates aerosol particles in the 2 to 10 micrometer diameter-size range. The concentrate particle stream is directed through a wet collector containing a buffer solution and, over a 45-minute period, a 40 to 50 ml sample is collected. On order or when test results indicate a suspected agent, the sample and associated documentation are packaged and transported IAW FM 3-101-4.

B-4. Chain of Custody

• When was the sample/specimen collected?
• Who has maintained custody of the sample/specimen?
• What has been done with the sample/specimen at each change of custody?

b. The samples/specimens must be appropriately packaged, labeled, and evacuated to the designated medical laboratory for confirmation of a biological attack. The standard chain of custody for the evacuation would be as follows:

• Sampling unit.
• Unit S2/security office or medical operations officer.
• Technical escort unit or other command-designated escort personnel.
• In-theater supporting medical laboratory.
• Designated CONUS laboratory.

Section II. SAMPLING TECHNIQUES AND PROCEDURES

B-5. General

Table B-2. Example NBC Collection and Shipping Equipment List

B-6. Expended Material

The NBC recon units collect samples under various circumstances. For example, a recon unit may collect samples in an area free of hostile forces. The Special Forces NBC Reconnaissance 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 material used to collect the samples should be turned in to the laboratory with the samples. This material 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-3 compares different types of samples.

B-7. Environmental Samples

Control or background samples that are collected from clean samples must be identical to the samples collected from the areas near the attack areas as baseline data. The contaminated samples must be 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-4, page B-20).

Table B-3. Comparison of Sample Types

B-8. Collection of Air and Vapor Samples

B-9. Collection of Water Samples

• Step 1. Ensure that the lever on the stopcock is turned sideways with the 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 the bottom or sides of the body of water.
• Step 3. Draw 60 cc of water into the syringe.
• Step 4. Turn the stopcock lever to the off position by positioning the lever to point toward the stopcock.
• Step 5. Push the 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 a plastic tubing from the Sep-Pak^TM, and discard it as contaminated waste.
• Step 8. Detach Sep-Pak^TM from 3-way stopcock; place into sample container; seal with pressure-sensitive tape; and mark for Identification.

• Step 9. Dispose of the syringe and stopcock as contaminated waste.
• Step 10. Insert the sample container in a cooler or refrigerator until the sample is transported to its destination.

g. For samples to be representative of the overall contaminated area, the collection point should be carefully selected. Collect samples from—

• Drains and slow-moving streams, since contamination and dilution from other sources are minimized.
• 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.

h. Collect the sample without the Sep-Pak^TM by immersing a capped or stoppered container to the desired depth, removing the cap or stopper, letting the container fill, and then capping the container. An alternate method for deeper water is to use a plastic, pump-operated siphon to pump water from a specific depth.

i. The best time to collect a sample of water from a location 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.

B-10. Collection of Soil Samples

a. Collect the soil samples by using a knife, spoon, spatula, or similar item to scrape a square of topsoil (2×5×1 centimeters) from areas that appear to have been contaminated in to a collection container. If chunks or clods of earth are collected, select those that are no larger than 10×5×1 centimeters (see Table B-4). Also, collect a control sample of soil of the same type and texture from a nearby uncontaminated area.

b. Use a glass bottle, jar, or Teflon^TM jar as the container when available. When these containers are not available, Mylar bags may be used. When using a glass bottle, jar, or Teflon^TM jar, seal the cap with either pressure-sensitive or Teflon^TM tape, and mark for identification. When using Mylar bags, place each sample in a separate bag, push excess air out, and seal by folding the open end over two to three times and wrapping the bag with tape. Insert the first bag into a second bag, seal, tape, and mark for identification. If possible, place the samples in a piglette.

c. Collect samples as soon as possible when directed, upon detection of a suspect substance, or after the alleged incident.

B-11. Collection of Contaminated Vegetation

B-12. Medical Specimens

b. Selection of humans for control sampling is somewhat more complicated than selection of environmental control samples. This is because ethnic diets, racial differences, physiological makeup, and actual living conditions of persons who are outwardly similar may introduce potentially large deviations. Each of these factors must be accurately considered before selecting subjects as controls.

c. 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 in the ground and extracting the product little by little over several months.

d. 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.

e. Physiological makeup is critical because of the differences in hormone balance and tissue composition in males, females, adults, and juveniles. For this reason, medical control specimens should be drawn from individuals of the same gender and approximate age as specimens from exposed personnel, if possible.

f. 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.

g. The bottom line in selecting subjects for medical control sampling is that they be as similar in all aspects as possible.

B-13. Collection of Medical Specimens

c. Collect blood specimens using either a standard 10 cc disposable syringe with a 1- to 1½-inch needle (20 to 22 gauge), or by using a Vacutainer^TM system. When using a Vacutainer^TM system, ensure that multiple specimen 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 specimens, collectors need to check specimen containers for paper labels IAW guidelines for labeling medical specimens. Collect blood specimens using the following materials equipment:

• Gloves.
• 10 cc sterile, disposable syringe.
• 1- to 1.5-inch sterile needle (20 to 22 gauge).
• Vacutainer^TM device (adapter with needle).
• Constricting band.
• Disinfectant pads, Betadine, or alcohol.
• Sterile 2×2-inch gauze pads.
• Labels.