Section I. INTRODUCTION
B-1. General
a. Critical elements for accuracy in analysis of NBC samples and physiological specimens are correct collecting, packaging, handling, and transporting techniques. The quality of any analytical evaluation is directly related to the quality of the sample/specimen and the degree of postcollection degradation that occurs prior to testing. Health service support personnel collect and submit specimens for suspect NBC hazards/agents involving humans and animals. Chemical corps and other nonmedical units collect and submit environmental (air, plant, and soil) samples for suspect NBC hazards/agents. Preventive medicine personnel collect and submit water and ice samples for suspect NBC hazards/agents. Veterinary personnel collect and submit food samples, such as fruits and vegetables, and specimens from animals for suspect NBC hazards/agents. Specimens collected from patients that are suspect of being exposed to a biological agent are forwarded to the supporting medical laboratory (such as the TAML, AML or US Navy Forward Deployed PVNTMED Unit) for analysis.
b. Essentially all military operations from war to stability operations and support operations may generate medical laboratory testing requirements. Each scenario, geographical region, population base, and suspect agent will impact on the type and amount of samples/specimens required and the collection process. During all operations, express permission is required before collecting specimens from civilians because of religious or sociological beliefs in many cultures. To obtain such specimens without permission could result in unnecessary mission complications.
NOTES
1. The term "sample" refers to nonhuman and nonanimal origin. The term "specimen" refers to human and animal origin.
2. Always consider that chemical agents may have been employed. Check for chemical agents before collecting a biological sample/specimen. Chemical agents can damage or destroy biological agents. Also, chemical agents not identified in the sample/specimen can pose a hazard to receiving laboratory personnel. Mark all samples that are potentially contaminated with chemical agents as such.
3. Precautions should be taken to protect the sample/specimen collector from potential BW agents; at a minimum, respiratory protection and rubber gloves must be worn. Additional care must be taken when collecting samples/specimens to prevent cross-contamination. Gloves must be changed or decontaminated between sample/specimen collections.
4. Samples will not be delivered to the clinical laboratory of an MTF for analysis. They must be delivered to the designated supporting medical laboratory for processing. This will prevent accidentally spreading a biological agent in the MTF.
c. Coordination for follow-on testing is absolutely critical to the sample/specimen collection process.
d. Coordination with the receiving laboratory should be made to establish sample requirements, preferred collection techniques, methods of preservation, and transportation conditions, when the tactical situation and/or mission permits.
e. The number of medical specimens that need to be collected varies with the type of analysis performed and the impact of the values determined. The number and types of "control" samples/specimens required to validate test information is determined by the supporting medical laboratory personnel. Random sampling, matched with control populations, or other techniques will be employed as the requirements are identified.
B-2. Sample/Specimen Background Information
a. A complete history of the circumstances about each sample's/specimen's acquisition must be provided to the agency conducting the analysis.
b. Critical information includes, but is not limited to—
- Meteorological conditions. Describe what the meteorological conditions were at the time of the alleged attack and at the time of the sampling.
- Attack to collection time. State the length of time after alleged attack when sample/specimen was taken.
- Circumstances of acquisition. Describe how the sample/specimen was obtained and the source of the sample/specimen.
- Physical description. Describe the physical state of the sample/specimen (solid, liquid, powder, apparent viscosity), color, approximate size, identity of the sample/specimen (that is, dirt, leaves, blood, tissue), and dose rate (if radiologically contaminated).
- Circumstances of the agent deposition. Describe 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 the burst, and colors of smoke, flames, or mists that may be associated with the attack.
- Agent effects on vegetation. Describe the general area (jungle, mountain, grassland) and changes in the vegetation after the agent deposition (that is, 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 before, during, or after an attack; measures taken that alleviated or worsened the effects; and the approximate number of victims and survivors (include age and gender).
- Agent effects on animals. Describe how they are affected.
- Grid coordinates or other descriptive information on sample collection location.
B-3. Sample/Specimen Collection and Preservation
a. Ante mortem Specimens. Physiological specimens from living human or animal patients can include just about any conceivable body source or excreted by-product. It must be noted that specimen types are seldom interchangeable; the exact type and amount of specimen required for a specific assay must be known before a collection procedure is initiated (see [Table B-1]).
- Patients seen in an MTF may be the first and in some cases the only source for sampling for suspect biological agent release. The primary medical care provider will determine the level of treatment for these patients and the specimens required for laboratory diagnosis. The MTF laboratory is not equipped to handle biological agents and, therefore, specimens generated will be forwarded to the supporting medical laboratory for analysis. Patient disposition will be based on evacuation policies, exposure, suspect agent, clinical symptoms, and required treatment/isolation.
- Blood specimens represent the most common analytical sample. Certain techniques and special care must be exercised to ensure an acceptable specimen is collected and to minimize an adverse affect to the patient or specimen collector. In general, phlebotomy requires the use of a 20 to 22-gauge needle to minimize mechanical hemolysis during aspiration using a syringe or VacutainerTM tube collection system. Blood collected with a syringe and needle should be transferred to an appropriate VacutainerTM tube immediately after collection. The type of tube, type of anticoagulant or preservative, and amount of blood collected will vary with the specific assay requested. Unless some special sample preparation step is required, the blood is best left in the original rubber-stopper tube for transport.
- Urine specimens are best collected using a clean-catch (midstream, if possible) technique in a sterile urine cup. The volume of sample required will vary depending on the specific assay requested; however, 25 to 50 ml is sufficient for most tests.
- Tissue specimens can originate from any body source accessible by scraping, swabbing, or minor excision. Tissue specimens are collected only by medical trained personnel. Specific techniques for collecting these specimens are not provided in this appendix.
- Sputum specimens are best collected using a sterile cup. The volume of specimen will normally be very small. However, a sufficient quantity must be collected to provide for in-theater testing and to provide for CONUS laboratory testing.
- Nasal swabs should be collected using sterile cotton-tipped swabs. The swabs with specimen from each person should be placed in a separate sterile container to prevent cross-contamination.
NOTE
In cases where the supporting laboratory cannot be contacted, as a minimum the following specimens should be collected: Urine—25 to 50 ml in a sterile container. Blood—two 7 to 10 ml tubes without anticoagulant (red-stopper VacutainerTM); two 7 to 10 ml tubes with potassium or sodium ethylenediaminetetraacetate (EDTA) (lavender-stopper VacutainerTM).
- 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
| BC | Blood culture |
| C | Citrated blood |
| CSF | cerebrospinal fluid |
| DNA | deoxyribonucleic acid |
| E | EDTA |
| EEE | eastern equine encephalitis |
| ELISA | enzyme-linked immunosorbent assay |
| EM | electron microscopy |
| F-1 | fraction-1 |
| FA | fluorescent antibody |
| H | Heparin |
| HPLC | high-pressure liquid chromatography |
| IgG | immunoglobulin class G |
| IgM | immunoglobulin class M |
| PCR | polymerase chain reaction |
| RT | Red Top, if TT is not available |
| RT-PCR | reverse transcriptase/ polymerase chain reaction |
| TT | Tiger top |
| VEE | Venezuelan equine encephalitis |
| WEE | western equine encephalitis |
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-PakTM 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
a. A strict chain of custody must be maintained for every sample/specimen collected. Use DD Form 1911 (Material Courier Receipt), or other document (such as DA Form 4137 [Evidence/Property Custody Document]) as directed for each sample/specimen collected. The chain of custody document must accompany the sample/specimen during transport from the point of collection to the final receiving laboratory. Each time the sample/specimen is transferred to another individual, the receiving person must sign the document to show that they received the sample/specimen and state what happened to the sample/specimen while in their custody. The document will provide the answer to the following questions:
- 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.