Chapter XV: 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 Vacutainer^{TM} tube collection system. Blood collected with
a syringe and needle should be transferred to an appropriate
Vacutainer^{TM} 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
Vacutainer^{TM}); two 7 to 10 ml tubes with potassium or
sodium ethylenediaminetetraacetate (EDTA) (lavender-stopper
Vacutainer^{TM}).

· 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_

========================================================================
EARLY POSTEXPOSURE CLINICAL
CONVALESCENT/TERMINAL/POSTMORTEM
------------------------------------------------------------------------
=ANTHRAX=
_0 TO 24 HOURS._ _24 TO 72 HOURS._
NASAL AND THROAT SWABS, AND SERUM (TT OR RT) FOR TOXIN ASSAYS.
INDUCED RESPIRATORY SECRETIONS BLOOD (E, C, H) FOR PCR.
FOR CULTURE, FA, AND PCR. BLOOD (BC OR C) FOR CULTURES.
_3 TO 10 DAYS._
SERUM (TT OR RT) FOR TOXIN ASSAYS.
BLOOD (BC OR C) FOR CULTURE.
PATHOLOGY SPECIMENS.
------------------------------------------------------------------------
=PLAGUE=
_0 TO 24 HOURS._ _24 TO 72 HOURS._
NASAL SWABS, SPUTUM, AND BLOOD (BC AND C) FOR CULTURE AND
INDUCED RESPIRATORY SECRETIONS BLOODY SPUTUM (C) FOR FA. SERUM
FOR CULTURE, FA, (TT OR RT) FOR F-1 ANTIGEN ASSAYS.
AND PCR. BLOOD (E, C, OR H) FOR PCR.
_>6 DAYS._
SERUM (TT OR RT) FOR IgM,
LATER FOR IgG.
PATHOLOGY SPECIMENS.
------------------------------------------------------------------------
=TULAREMIA=
_0 TO 24 HOURS._ _24 TO 72 HOURS._
NASAL SWABS, SPUTUM, AND BLOOD (BC OR C) FOR CULTURE.
INDUCED RESPIRATORY BLOOD (E, C, OR H) FOR PCR.
SECRETIONS FOR CULTURE, FA, SPUTUM FOR FA AND PCR.
AND PCR. _>6 DAYS._
SERUM (TT OR RT) FOR IgM AND
LATER IgG, AGGLUTINATION TITERS.
PATHOLOGY SPECIMENS.
------------------------------------------------------------------------
=MELIOIDOSIS/GLANDERS=
_0 TO 24 HOURS._ _24 TO 72 HOURS._
NASAL SWABS, SPUTUM, AND BLOOD (BC OR C) FOR CULTURE.
INDUCED RESPIRATORY BLOOD (E, C, OR H) FOR PCR.
SECRETIONS FOR CULTURE AND SPUTUM AND DRAINAGE FROM SKIN
PCR. LESIONS FOR PCR AND CULTURE.
_>6 DAYS._
BLOOD (BC OR C) AND TISSUE FOR
CULTURE.
SERUM (TT OR RT) FOR
IMMUNOASSAYS.
PATHOLOGY SPECIMENS.
------------------------------------------------------------------------
=BRUCELLOSIS=
_0 TO 24 HOURS._ _24 TO 72 HOURS._
NASAL SWABS, SPUTUM, AND BLOOD (BC OR C) FOR CULTURE.
INDUCED RESPIRATORY BLOOD (E, C, AND H) FOR PCR.
SECRETIONS FOR CULTURE AND _>6 DAYS._
PCR. BLOOD (BC OR C) AND TISSUE FOR
CULTURE.
SERUM (TT OR RT) FOR
IMMUNOASSAYS.
PATHOLOGY SPECIMENS.
------------------------------------------------------------------------
=Q FEVER=
_0 TO 24 HOURS._ _2 TO 5 DAYS._
NASAL SWABS, SPUTUM, AND BLOOD (BC OR C) FOR CULTURE IN EGGS
INDUCED RESPIRATORY OR MOUSE INOCULATION.
SECRETIONS FOR CULTURE AND BLOOD (E, C, AND H) FOR PCR.
PCR. _>6 DAYS._
BLOOD (BC OR C) FOR CULTURE IN
EGGS OR MOUSE INOCULATION.
PATHOLOGY SPECIMENS.
------------------------------------------------------------------------
=BOTULISM=
_0 TO 24 HOURS._ _24 TO 72 HOURS._
NASAL SWABS AND INDUCED NASAL SWABS AND RESPIRATORY
RESPIRATORY SECRETIONS FOR SECRETIONS FOR PCR (CONTAMINATING
PCR (CONTAMINATING BACTERIAL DNA) AND TOXIN ASSAYS.
BACTERIAL DNA) AND TOXIN _>6 DAYS._
ASSAYS. USUALLY NO IgM OR IgG.
SERUM (TT OR RT) FOR TOXIN PATHOLOGY SPECIMENS (LIVER AND
ASSAYS. SPLEEN FOR TOXIN DETECTION).
------------------------------------------------------------------------
=RICIN INTOXICATION=
_0 TO 24 HOURS._ _36 TO 48 HOURS._
NASAL SWABS AND INDUCED SERUM (TT OR RT) FOR TOXIN
RESPIRATORY SECRETIONS FOR ASSAY.
PCR (CONTAMINATING CASTOR TISSUE FOR IMMUNOHISTOLOGICAL
BEAN DNA) AND TOXIN ASSAYS. STAINING.
SERUM (TT OR RT) FOR TOXIN PATHOLOGY SPECIMENS.
ASSAYS. _>6 DAYS._
SERUM (TT OR RT) FOR IgM AND IgG
IN SURVIVORS.
------------------------------------------------------------------------
=STAPH ENTEROTOXICOSIS=
_0 TO 3 HOURS._ _2 TO 6 HOURS._
NASAL SWABS AND INDUCED URINE FOR IMMUNOASSAYS.
RESPIRATORY SECRETIONS FOR NASAL SWABS AND INDUCED
PCR (CONTAMINATING BACTERIAL RESPIRATORY SECRETIONS FOR
DNA) AND TOXIN ASSAYS. PCR (CONTAMINATING BACTERIAL
SERUM (TT OR RT) FOR TOXIN DNA) AND TOXIN ASSAYS.
ASSAYS. SERUM (TT OR RT) FOR TOXIN
ASSAYS.
_>6 DAYS._
SERUM FOR IgM AND IgG.
------------------------------------------------------------------------
=T-2 TOXICOSIS=
_0 TO 24 HOURS POSTEXPOSURE_ _1 TO 5 DAYS._
NASAL AND THROAT SWABS AND SERUM (TT OR RT) AND TISSUE FOR
INDUCED RESPIRATORY TOXIN DETECTION.
SECRETIONS FOR IMMUNOASSAYS, _>6 DAYS POSTEXPOSURE._
HPLC/MASS SPECTROMETRY. URINE FOR DETECTION OF TOXIN
METABOLITES.
------------------------------------------------------------------------
=EQUINE ENCEPHALOMYELITIS=
(VEE, EEE, AND WEE VIRUSES) _24 TO 72 HOURS._
_0 TO 24 HOURS._ SERUM (TT OR RT) AND THROAT FOR
NASAL SWABS AND INDUCED CULTURE.
RESPIRATORY SECRETIONS FOR SERUM (E, C, H, TT, OR RT) FOR
RT-PCR AND VIRAL CULTURE. RT-PCR. THROAT SWABS UP TO 5 DAYS
FOR CULTURE THEN CSF.
SERUM (TT OR RT) FOR ANTIGEN
ELISA.
_>6 DAYS._
SERUM (TT OR RT) FOR IgM.
PATHOLOGY SPECIMENS PLUS BRAIN.
------------------------------------------------------------------------
=POX=
(SMALLPOX AND MONKEYPOX) _2 TO 5 DAYS._
_0 TO 24 HOURS._ SERUM (TT OR RT) FOR VIRAL
NASAL SWABS AND INDUCED CULTURE
RESPIRATORY SECRETIONS FOR _>6 DAYS._
PCR AND VIRAL CULTURE. SERUM (TT OR RT) FOR VIRAL CULTURE
DRAINAGE FROM SKIN LESIONS/
SCRAPINGS FOR MICROSCOPY, EM,
VIRAL CULTURE, AND PCR.
PATHOLOGY SPECIMENS.
------------------------------------------------------------------------
=EBOLA=
_0 TO 24 HOURS._ _2 TO 5 DAYS._
NASAL SWABS AND INDUCED SERUM (TT OR RT) FOR VIRAL
RESPIRATORY SECRETIONS FOR CULTURE.
RT-PCR AND VIRAL CULTURE. _>6 DAYS._
SERUM (TT OR RT) FOR VIRAL CULTURE
PATHOLOGY SPECIMENS PLUS ADRENAL
GLAND.
------------------------------------------------------------------------
=LEGEND:=

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-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

_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.