CHAPTER 3

COLLECTING AND PROCESSING BLOOD AND BODY

FLUID SPECIMENS

QUALITYASSESSMENT THE PATIENT

Patient Care Partnership
Pediatric Patients
Adolescent Patients
Adult Patients
Geriatric Patients
INFECTION CONTROL
Isolation as Safety System
Standard and Additional Precautions
SPECIMEN COLLECTION
Blood Collection Variables
Blood Collection Procedures
Layersof Normal Anticoagulated Blood
Additives and Anticoagulants
Venipuncture Procedure

VENOUS BLOOD COLLECTION ( PHLEBOTOMY )

Supplies and Equipment

Initiation of the Procedure
Selection of an Appropriate Site
Preparation of the Venipuncuture Site
Performing the Venipuncture
Termination of the Procedure
Phlebotomy Problems
Phlebotomy Complications
Blood Culture Collection
Capillary or Peripheral Blood Collection by Skin Puncture
CAPILLARY BLOOD COLLECTION
Supplies and Equipment
Selection of an Appropriate Site
Preparation of the Site
Puncturing the Skin
Special Capillary Blood Collection
Capillary Blood for Slides
Collecting Microspecimens
Laser Equipment

SPECIMENS: GENERAL PREPARATION

Processing Blood Specimens

Unacceptable Specimens
Drug Effect on Specimens
Logging and Reporting Processes
Preserving and Storing Specimens
URINE
Types of Urine Specimens
Containers for Urine Collection
Collecting Urine Specimens
Preservation of Urine Specimens
Labeling and Processing of Urine Specimens
BODY FLUIDS
Cerebrospinal Fluid
Synovial Fluid
Pericardial, Pleural, and Peritoneal Fluids
Seminal Fluid
SWABS FOR CULTURE
Throat Culture Collection
Feces
Sweat
Saliva

CHAIN-OF-CUSTODY SPECIMEN INFORMATION

LEARNING OBJECTIVES
AT the conclusion of this chapter, the student will be able to:

Identify seven factors that should be monitored by quality assessment methods.

Demonstrate and describe the skills needed to interact with patients in the

collection of specimens.
Explain the Patient Care Partnership and its importance.
Describe the principles and applications of Standard Precautions.
Describe the equipment used for venous blood collection.
Explain and demonstrate the proper collection technique for venous blood.
Identify the color codes of evacuated tubes with the additives contained in the

tubes.
Compare common anticoagulants and additives used to preserve blood

specimens and the general use of each type of anticoagulant.

Describe the mode of action of EDTA and heparin.
Identify the major potential type of error in specimen collection.
List and explain five specific situations that could complicate venipuncture site

selection.
Identify eight typical phlebotomy problems, and describe the solution for each

pronlem.
Explain some techniques for obtaining blood from small or difficult veins.

Describe special considerations for pediatric and geriatric patients in the

collection of blood.
List the six categories of phlebotomy complications, and describe the

symptoms and treatment for each type of complication.

Demonstrate and describe the proper technique for the collection of a capillary

blood specimen.
Describe the purpose and use of the Unopette system.
Identify and compare various urine specimen requirements for a routine
urinalysis, including preservation and storage requirements.

Discuss the differences between various types of urine collection, including

midstream clean-catch, quantitative, and timed specimens.

Briefly explain collection procedures for other body fluid specimens, including

cerebrospinal fluid, pleural fluid, and synovial fluid.

Collect and explain the proper procedure for collecting a throat swab specimen

for culture.
Collect feces for an occult blood test and other tests.
Describe relevant medical-legal issues related to specimen collection.

QUALITY ASSESSMENT
The accuracy of laboratory testing begins with the quality of the specimen
received by the laboratory. This quality depends on how a specimen was
collected, transported, and processed. A laboratory assay will be no better
than the specimen on which it is performed. If the specimen is improperly
collected or stored or is mishandled in any way, the most perfect analysis is
invalid and cannot be used by the physician in diagnosis or treatment.
The term quality assessment or the older term, quality assurance, is
used to describe management of the treatment of the whole patient ( see
Chapter 8). As it applies to the clinical laboratory, quality assessment
requires establishing policies that maintain and control processes involving
the patient and laboratory analysis of specimens.
Quality assessment includes monitoring the following specimen collection
measures:

Preparation of patients for any specimens to be collected.

Collection of valid samples
Proper specimen transpor
Performance of the requested laboratory analyses
Validation of test results
Recording and reporting the assay results
Transmitting test results to the patients medical record.

Documentation, manintenance, and availability of records describing quality

assesment practices and quality control measures.

THE PATIENT
The role of the phlebotomist has never been more important to the patient and
the laboratory. More than two thirds of laboratory errors are caused by mistakes
before testing, or preanalytical errors. Most of these mistakes are related to
specimen collection and handling. Phlebotomists can reduce these mistake by
being well trained and constantly alert to sources of eror. In addition, the
phlebotomist is frequently the only laboratory staff member who a patient sees.
This means that the professional image of the laboratory is solely represented by
the phlebotomist.
The phlebotomist is expected to deliver unexcelled customer satisfaction. It is
important to understand and know the patients expectations, manage unrealistic
expectations through patient education, and be diplomatic with patient complaints.
If a patient is unhappy, the phlebotomist should listen with interest, express
genuine concern, and make an attempt to resolve the issue of concern. If the
phlebotomist is directly at fault, an apology would be appropriate.

Patient Care Partnership

The delivery of health care involves a partnership between patients and
physicians and other health care professionals. When collecting nlood specimens,
it is important that the phlebotomist consider the rights of the patient at all times.
The American Hospita! Association has developed the Patient Care Partnership
document, which replaces the former Patients Bill of Rights. This document
stresses the following:

High-quality hospital care

A clean and safe environment

Involvement by patients in their care
Protection of patients privacy
Help for patients when leaving the hospital
Help for patients with billing claims

Patient themselves, or another person chosen by the patient, can exercise these
patient rights. A proxy decision maker can act on the patients behalf if the patient
lacks decision-making ability, is legally incompetent, or is a minor.
The partnership nature of health care requires that patients, or their families or
surrogates, take part in their care. As such, patient are responsible for providing an
accurate medical history and any written advance directives, following the
hospital rules and regulations, and complying with activities that contribute to a
healthy lifestyle.

Pediatric Patients
When working with children, it is important to be gentle and compassionate.
Attempt to interact with the pediatric patient, realizing that both the patient and
the parent ( if present) may have anxiety about the procedure and may be
unfamiliar with the new settings. Acknowledge both the parent and the child.
Dont hurry; allow enough time for the procedure. It is important to take extra
time to gain a childs confidence before proceeding with the specimen collection.
In working with pediatric patients, it is important to bolster their morale as much
as possible. Ask for help in restraining a very small or uncooperative child. Older
children may be more responsive when permitted to :help (e.g.,by holding the
gauze).
In the nursery, each hospital will have its own rules, but a few general
precautions apply. After working with an infant in a crib, the crib sides must be
returned to the precollection position. If an infant is in an incubator, the portholes
should be closed as much as possible. When oxygen is in use, do not forget to

close the openings when the collection process is completed. Dispose of all waste
materials properly.

Adult Patients
Adult patients must be told briefly what is expected of them and what the
involves. Complete honesty is important. The patient should be greeted in a
friendly an tactful manner; without becoming overly familiar, a conversation can
be started in a quiet, pleasant, and calm manner. The patient should be told about
the purpose of the blood collection. Any personal information revealed by the
patient is told in confidence. The patients religious beliefs should be respected,
laboratory resports kept confidential, and any personal information also kept in
confidence. Information about other patients or physicians is always kept
confidentrial. If the same patient is seen frequently, the phlebotomist may become
familiar with the patients interests, hobbies, or family and use these as topics of
conversation. Many patients in the hospital are lonely and need a friend.
Occasionally, especially with the extremely ill patient, the person will not want to
talk at all, and this should be respected. It is important to be honest, but also to
attempt to boost the patients morale as much as possible.
Even if the patient is disagreeable, the phlebotomist should remain pleasant.
A smile can often work miracles. It is important to be firm when the patient is
unpleasant, to remain cheerful, and to express confidence in the work to be done.
In a hospital setting, before leaving the patients room the area should be
checked to see that everything is in place in the laboratory tray and that the room
has been left as it was found. The tray holding the blood collection supplies and
equipment should always be kept out of reach of the patient. All supplies should
be disposed of properly.

Geriatric Patients
It is extremely important to treat geriatric patients with dignity and respect.
Do not demean the patient. It is best to address the patient with a more formal
title, such as Mrs.,or Mr.,rather than by his or her first name. As with patients in
general, older patients may enjoy a short conversation. Keep a flexible agenda so
that enough time is allowed for the patient. If a patient appears to be having
difficulty hearing, speak slightly slower and louder.

INFECTION CONTROL
Isolation as Safety System
Isolation was previously defined as the separation of a scriously ill patient to
stop the spread of infection to others or to protect the patient from irritating
factors. The term isolation has changed from meaning a special set of precautions
performed by a few health care providers for a select few patients to a safety
system that is practiced by everyone in the course of routine patient care. Isolation
precautions are now a routine part of the everday work process.
Modern isolation techniques incorporate a broad-based theory that addresses the
needs of both patients and employees to ensure that the safest possible
environment is maintained throughout the health care facility. Current guidelines
use a two-tiered strategy to create this safety system.
Standard and Additional Precautions
The concept of Standard Precautions forces health care professionals to
change the way they view infection control. A two-tiered system has been
developed. The goal of this two-tiered system is to minimize the risk of infection
and to maximize the safery level within the health care facilitys environment.

The first tier of infection control is the practice of standard Precautions ( see
Chapter 2). Standard Precautions theory recognizes the need to reduce the risk of
microbial transmission, including HIV, from both identified and unidentified
sources of infection. These precautions require that protective protocols be
followed whenever contact is made with blood and body fluids.
A second tier of an infection control system was developed to provide additional
precautions to control the transmission of infectious agents under special
circumstances when Standard Precautions alone may not be enough.
Transmission-based precautions are divided into three basic categories: contact,
airborne, and droplet.
Contact Precautions
Contact precautions are designed to stop the spread of microorganisms via
direct contact, such as skin-to-skin contact and indirect contact, which is usually
the result of a person making contact with a contaminated inanimate object.
Contact precautions include wearing gloves when making contact with the
patients skin or with inanimate objects that have been in direct contact with the
patient. The use of gowns may be mandated when the health care workers
clothing is likely to come in contact with the patient or items in the patients room.
Airborne Precautions
Airborne precautions are designed to provide protection from extremely tiny
airborne bacteria or dust particles, which may be suspended in the air for an
extended period. Guidelines include the use of respiratory protection and the use
of special air-handling systems to control the airborne bacterial.
Droplet Precautions
Droplet precautions protect health care workers, visitors, and other patients
from droplets, which may be expelled during coughing, sneezing, or talking.
Guidelines include using a mask when working close to the patient. Guidelines for

10

patient placement, from the use of a private room to using a room with special airhadling capabilities, should bi implemented as well. Specific guidelines for the
transport and placement of patients and the environmental management of
equipment should be implemented according to each categorys requirements.

SPECIMEN COLLECTION
Blood is the type of specimen most frequently analyzed in the clinical
laboratory. Urine specimens and body fluids are also frequently analyzed. Fecal
specimens and other miscellaneous specimens, such as throat cultures and swabs
from wound abscesses, are sent to the microbiology laboratory for study.
Knowledge of proper collection, preservation, and processing of specimens is
essential. A properly collected blood specimen is crucial to quality performance in
the laboratory. Strict adherence to the rules of specimen collection is critical to the
accuracy of any test. Preanalytical errors, such as identification, are major
potential sources of error.
Blood specimens may be collected by health care personnel with several different
educational backgrounds, depending on the facility. In some institutions, blood
specimen collection is done by the clinical laboratory scientist/medical
technologist or the clinical laboratory technician/medical laboratory technician. In
other institutions, specially trained individuals, or phlebotomists, perform blood
collections. In addition to specimen procurement, related areas of specimen
transportation, handling, and processing must also be fully understood by anyone
who collects or handles blood specimens.
Blood Collection Variables
Most clinical laboratory determinations are done on whole blood, plasma, or
serum. Blood specimens may be drawn from fasting or non fasting patients. The

11

fasting state is defined as having no food or liquid other than water for 8 to 12
hours before blood collection. Fasting specimens are not necessary for most
laboratory determinations. Blood from fasting patients is usually drawn in the
morning before breakfast. Food intake, medication, activity, and time of day can
all influence the laboratory results for blood specimens.
Blood collected directly after a meal is described as a postprandial specimen. In
the case of blood glucose, a sample may be collected 2 hours postprandial. After 2
hours, blood glucose levels should return to almost fasting levels in patients who
are not diabetic. Blood should not be collected while intravenous solutions are
being administered, if possible.
Other controllable biological variations in blood include the following:

Posture ( whether the patient is lying in bed or standing up)

Immobilization (e.g.,resulting from prolonged bed rest)
Exercise
Circadian/diurnal variations ( cyclical variations throughout the day)
Recent food ingestion (e.g.,caffeine effect)
Smoking ( nicotine effect)
Alcohol ingestion
Administration of drugs

Blood Collection Procedures

There are two general sources of blood for clinical laboratory tests; peripheral
(or capillary) blood and venous blood. The Clinical and Laboratory Standards
Institute ( CLSI), formerly the National Committee for Clinical Laboratory
Standards (NCCLS), has set standards for the collection of venous blood
( venipuncture, or phlebotomy) and capillary blood ( skin puncture). Arterial
blood may be needed to perform specific procedures, such as blood gas analysis.
Layers of Normal Anticoagulated Blood
In vivo ( in the body) the blood is in a liquid form,but in vitro ( outside the
body) it will clot in a few minutes. Blood that is freshly drawn into a glass tube

12

appears as a translucent, dark-red fluid. In minutes it will start to clot, or

coagulate, forming a semisolid, jelly-like mass. If left undisturbed in the tube, this
mass will begin to shrink, or retract, in about I hour. Complete retraction normally
takes place within 24 hours.
When coagulation occurs, a pale-yellow fluid called serum separates from the
clot and appears in the upper portion of the tube. During the process of
coagulation, certain factors present in the original blood sample are depleted, or
used up ( see also Chapter 13). Fibrinogen is one important substance found in the
circulating blood (in the plasma portion) that is necssary for coagulation to occur.
Fibrinogen is converted to fibrin when clotting occurs, and the fibrin lends
structure to the clot in the form of fine threads in which the red blood cells
(RBCs, erythrocytes) and the white blood cells (WBCs, leukocytes) are
embedded. To assist in obataining serum, collection tube with a separator gel
additive are used. Serum is used extensively for chemical , serologic, and other
laboratory testing and can be obtained from the tube of clotted blood by
centrifuging.
When fresh whole blood is mixed with substances that prevent blood clotting,
called anticoagulants, the blood can be separated into plasma, a straw-colored
fluid, and the cellular components: erythrocytes, leukocytes, and platelets
( thrombocytes). Whole blood that is allowed to clot normally produces the strawcolored serum.
When an anticoagulated blood specimen is allowed to stand for a time, the
components will settle into three distinct layers( figure 3-1), as follows:

13

Additives and Anticoagulats

Some frequently used anticoagulants are dipotassium ethylenediaminetetraacetic
acid (KEDTA), sodium citrate, and heparin. Each of the anticoagulant types
prevents the coagulation of whole blood in a specific manner. The proper
proportion of anticoagulant to whole blood is important to avoid the introduction
of errors into test results. The specific type of anticoagulant needed for a
procedure should be stated in the laboratory procedure manual.

Dipotassium EDTA
The salts of the chelating (calcium-binding) agent K EDTA are
recommended by the International Council for Standardization in Haematology
( ICSH) and CLSI as the anticoagulant of choice for blood cell counting and
sizing because they produce less shrinkage of RBCs and less of an increase in cell
volume on standing. EDTA is spray-dried on the interior surface of evacuated
plastic tubes. The proper ratio of EDTA to whole blood is important because some
test results will be altered if the ratio is incorrect. Excessive EDTA produces

14

shrinkage of erythrocytes, thus affecting tests such as the manually performed

packed cell volume or microhematocrit.
Sodium Citrate
Sodium citrate in the concentration of a 3.2% solution has been adopted as
the appropriate concentration by the ICSH and the International Society for
Thrombosis and Hemostasis for coagulation studies. The College af American
Pathologists ( CAP) also recommends the use of 3.2% sodium citrate.
Sodium citrate is also used as an anticoagulant for activated prothrombin time
(aPTT) and prothrombin time (PT) testing and for the Westergren erythrocyte
sedimentation rate(ESR). The correct ratio of one part anticoagulant to nine parts
of whole blood in blood collection tubes is critical. An excess of anticoagulant can
alter the expected dilution of blood and produce errors in the results. Because of
the dilution of anticoagulant to blood, sodium citrate is generally unacceptable for
most other hematology tests.
Heparin
Heparin is used as an in vitro and in vivo anticoagulant. It acts as a substance
that inactivates the blood-clotting factor thrombin.

Heparin is a common

anticoagulant used in chemistry and special chemistry testing. Heparin is the only
anticoagulant device for the determination of pH, blood gases, electrolytes, and
ionized calcium. Heparin should not be used for coagulation or hematology
testing. It is the recommended anticoagulant for many determinations using whole
blood or plasma specimens because of its minimal chelating properties, minimal
effects on water shifts, and relatively low cation concentration.
Heparin is available as sodium, lithium, and ammonium salts. Lithium heparin is
the recommended form of heparin for use because it is least likely to interfere
when performining tests for other ions. Lithium heparin is essentially free of
extraneous ions. It should not be used for collection of blood for lithium levels.
Only a small amount of heparin is needed, so simply coating the insides of tubes

15

or syringes is often enough to give a good anticoagulant effect. Tubes containing

heparin should be inverted 8 to 10 times after collection to ensure thorough
mixing of the additive with the blood and thus complete anticoagulation of the
sample.
Sodium Fluoride
A dry additive and weak anticoagulant, sodium fluoride is used primarily for
preservation of blood glucose specimens to prevent glycolysis or destruction of
glucose (see Chapter 11).
Adverse Effects of Additives
The additives chosen for specific determinations must not alter the blood
components or affect the laboratory tests to be done. The following are some
adverse effects of using an improper additive or using the wrong amount of
additive:

Interference with the assay. The additive may contain a substance that is the
same, or reacts in the same way, as the substance being measured. An
example would be the use of sodium oxalate as the anticoagulant for a sodium

determination.
Removal of constituents. The additive may remove the constituent to be
measured. An example would be the use of an oxalate anticoagulant for a calcium
determination; oxalate removes calcium from the body by forming an insoluble

salt, calcium oxalate.

Effect on enzyme action. The additive may affect enzyme reactions. An example
would be the use of sodium fluoride as an anticoagulant in an enzyme

determination; sodium fluoride destroys many enzymes.

Alteration of cellular constituents. An additive may alter cellular constituents. An
example would be the use of an older anticoagulant additive, oxalate, in
hematology. Oxalate distorts the cell morphology; RBCs become crenated,
vacuoles apper in the granulocytes, and bizarre forms of lymphocytes and
monocytes appear rapidly when oxalate is used as the anticoagulant. Another

16

example is the use of heparin as an anticoagulant for blood to be used in the

preparation of blood films that will be stained with Wrights stain. Unless the films

are stained within 2 hours, heparin gives a blue background with Wrights stain.
Incorrect amount of anaticoagulant. If too little additive is used, partial clotting of
whole blood will occur. This interferes with cell counts. By comparison, if too
much liquid anticoagulant is used, it dilutes the blood sample and thus interferes
with certain quantitative measurements.

Venipuncture Procedure
Safe Blood Collection: Equipment and Supplies
An increased emphasis on sefety has led to new product development by
various companies. Newer designs of this equipment are reducing the incidence of
postphlebotomy needlesticks. Sarstedt ( Newton, NC) and the BD Company
( Franklin Lakies, N) are among the manufacturers who offer an extrensive variety
of safety-engineered, blood collection products.
The BD blood collection products include the following:
1. BD Vacutainer Eclipse Blood Collection Needle is a safety-engineered,
multisample blood collection needle that reduces the risk of needlestick
injuries.
2. BD Blood Transfer Device is an easy-to-use, latex-free device used to
facilitate safe and simple specimen transfers.
3. BD Vacutainer Safety-Lok Blood Collection Set is a safety-engineered
winged device indicated for both infusion and blood collection. These sets
feature a translucent, intergrated protectiv shield that provides one-handed

17

activation immediately after use to minimise the risk of needlestick

injuries and that allows for clear visibility of blood flashback.
4. BD Vacutainer Plasyic Tubes offer a safe method for blood collection that
reduces the risk of tube breakage and specimen spillage.
5. BD Genie Safety Lancets are safetyengineered, single-use capillary blood
sampling devices with a permanently retractable blade or needle feature
that minimizes the risk of injury or reuse.
6. BD Quikheel Safety Lancet is a safety-engineered product designed for
heelsticks on infants and premature babies. It features a sweeping gurgical
blade that permanently retracts after creating an incision.
Other new products manufactured by Ram Scientific ( Needham, Mass) (
www.rsleads.com) include preassembled capillary microtubes that are coated
with various anticoagulant additives and gel separators, the Haemolance Plus
Lockout Safety Lancet, and CapiJect, a series of silicone-coated, capillary
blood collection tubes with color-coded caps.
The standard needle for blood collection with asyringe or evacuated blood
collection tubes is a 21- gauge needle. Butterfly needle are being used more
frequently as the acuity of patients increases. The collecting needle is double
pointed; the longer end is for insertion into the patients vein, and the shorter
end pierces the rubber stopper of the collection tube. Sterile needles that fit a
standard holder are used. Various needle sizes are available. In addition to
length, needle are classified by gauge size: the higher the gauge number, the
smaller the inner diameter, or bore. The specially desingned, single-use needle
holder is used to secure the needle. It is no longer acceptable to wash and
reuse this plastic needle holder device. The BD Vacutainer Standard Yellow
Needle Holders ( reference numbers 364888 and 364983 ) have been
discontinued and are no loger available as a stand-alone holder for use with
blood collection devices. They are still used as a component for other safety
products, such as the BD Vacutainer Blood Transfer Device, BD Vacutainer
Direct Draw Adapter, and BD Vacutainer Luer-Lok Access Device.

18

The BD Vacutainer One Use Holder is a clear plastic needle holder

prominently marked with the words Do Not Reuse and Single Use Only.
Once a venipuncuture is completed, the entire needle and holder assembly is
disposed in a sharps container. The needle should not be removed from the
holder. This new needle holder is similar in feel to the reusable BD Vacutainer
Standard Yellow Needle Holder. No change in venipuncture techique is
required when transitioning to a single-use holder policy. The BD Vacutainer
One Use Holder is easy to implement into venipuncture procedures; it is
compatible with the entire BD Vacutainer Venous Blood Collection System,
including BD Vacutainer Eclipse Needles, BD Vacutainer Safety-Lok Blood
Collection Sets, and BD Vacutainer Multiple Sample Luer Adapters.
On October 15,2003, the U.S.Occupational Safety and Health Administration (
OSHA) posted a Saferty and Health Information Bulletin ( SHIB)
(www.oshe.gov) to clarify the OSHA position on reusing tube holders during
blood collection procedures, a clarification of the OSHA Bloodborne
Pathogens Standard [29 CFR 1910.1030 (d) (2) (vii) (A)]. The standaed
prohibits the removal of a contaminated needle

from a medical device.

Prohibition of needlremoval from any device is addressed in the 1991 and

2001 standards, the OSHA compliance directive ( CPL 2-2.69), and in a 2002
letter of interpretation. The purpose of the SHIB was to reiterate OSHAs
earlier statement (there is no grace period) that the best practice to prevent
needlestick injuries after phlebotomy procedures is the use of a sharp with
engineered sharps injury protection (SESIP) (e.g., safety neeedle) attached
to the blood tube holder and the immediate disposal of the entire unit after
each patients blood is drawn.
According to the OSHA SHIB and OSHAs 2001 Compliance Directive (CPL
2-2.69), certain situations may necessitate using a syringe to draw blood. The
blood collected into the syringe would then need to be transferred into a tube
before disposing of the contaminted syringe. In these situations, a syringe with
an engineered sharps injury prevention featur and work practices should be

19

used whenever possible. Transfer of the blood from the syringe to the tube
must be done using a needleless blood transfer device (e.g., BD Vacutainer).
As with any OSHA rule or regulation, non-compliance may result in the
issuance of citations by an OSHA compliance officer after the completion of a
site inspection. It is the responsibility of each facility to evaluate their work
pratices, implement appropriate engineering controls, and institute all other
applicable elements of exposure control to achieve compliance with current
OSHA rules and regulations. The OSHA SHIB provides a step-by-step
Evalution Toolbox for a facility to follow ( Box 3-1).
BOX31
OSHA Safety and Health Information Bulletin ( SHIB): Evaluation
Toolbox
1. Employers must firs evaluate, select, and use appropriate engineering
controls (e.g. sharps with engineered sharps injury protection), which
includes singe-use blood tube holders with sharps with engineered
sharps injury protection (SESIP) attached.
2. The use of engineering and work practice controls provide the highest
degree of control in order to eliminate potential injuries after
performing blood draws. Disposing of blood tube holders with
contaminated needles attached after the activation of the safety feature
affords the greatest hazard control.
3. In very rare situations, needle removal is acceptable.
If the employer can demonstrate that no feasible alternative to
needle removal is available ( e.g., inability to purchase single-use

blood tube holders because of asupply shortage of these devices).

If the removal is necessary for a specific medical or dental

procedure.
In these rare cases, the employer must ensure that the contaminated
needle is protected by a SESIP before disposal. In addition, the
employer must ensure that a proper sharps disposal container is
located in the immediate area of sharps use and is easily accessible

20

to employees. This information must be clearly detailed and

document in the employers Exposure Control Plan.
4. If it is necessary to draw blood with a syringe, a syringe with
engineered sharps injury protection must be used, in which the
protected needle is removed using safe work protected needle is
removed using safe work practices, and transfer of blood from the
syringe to the tube must be done using a needleless blood transfer
device.

Evacuated Blood Collection Tubes

Evacuated tubes are the most extensively used system for collecting venous
blood samples. An evacuated blood collection system consists of a collection
needle, a non-reusable needle holder, and a tube containing enough vacuum to
draw a specific amount of blood ( figure 3-2). Evacuated tubes come in various
(mL) sizes, including pediatric sizes, with color-coded stoppers. The stopper color
denotes the type of anticoagulant or the presence of a gel separator. BD
recommends that storage temperature for all BD Vacutainer blood collection tubes
not excees 25C or 77F. If plastic tubes reach higher temperatures, the tubes may
lose their vacuum or implode. Evacuted tubes are intended for one-time use.
When collecting multiple tubes of blood ,a specified order of draw protocol
needs to be followed to diminish the possibility of cross-contamination between
tubes caused by the presence of different additives ( Table 3-1). Errors in the order

21

of

draw

can

affect

laboratory

test

results.

Serum separator devices ( SSDs) assist in the processing of clotted whole blood.
To obtain the serum, special serum separator collection tubes are available. An
evacuated glass tube serves as the single system for both collection and processing
of the blood. Serum separator tubes are of two major types, those used during
centrifugation and those used after centrifugation. The tubes used during
centrifugation may be either intergrated gel tube systems or devices inserted into
the collection tube just before centrifugation. The integrated gel tubes contain a
special silicone gel layer, which, because of its viscosity and density, moves to
form a barrier between cells and serum during centrifugation.
Blood is forced into the gel layer during centrifugation, causing a temporary
change in viscosity. The gel starts at the bottom of the collection tube. Blood is
added to the tube, and the clot is allowed to form for a minimum of 30 minutes.
After clot formation, the tubes are centrifuged. The gel rises and lodges between

22

the packed RBCs and the top layer of serum. The gel hardens and forms an inert
barrier. These tubes do not need to have stoppers removed before centrifugation,
thus eliminating aerosol production and possible evaporation. The serum separator
tubes also give a higher yield of serum as well as a shorter processing time
because only a single centrifugation step is needed.
A new, specialized vacuum tube for collection, transport, and retention of whole
blood, the Cyto-ChexBCT, is designed for immunophenotyping by flow
cytometry. The preservative contained in this tube maintains the integrity of WBC
cluster of differentiation ( CD) markers for up to 7 days.Specimens are stable at
room temperature during transport and storinge.
Syringe Technique
Disposable plastic syringes are used for special cases of venous blood collection.
If a patient has particularly difficult veins, or if other special circumstances exist,
the syringe technique may be used. Some facilities recommend an order of draw
with a syringe that varies from the evacuated-tibe protocol.
General Protocal
1.Phlebotomist shiuld pleasantly introduce themselves to the patient and clearly
explain the procedure that is to be performed.
2. Patient identification is the critical first step in blood collection. It is necessary
both to ask the patients name and to check the identification band that is
physically attached to the patient. When the patient is unable to give his or her
name, or when identification is attached to the bed or is missing, nursing
personnel should be asked to identify the patient physically. Any variations in
protocal should on the test requisition.
3.Test requisitions should be checked and the appropriate evacute tubes asembled.
All specimens should be properly labeled immediately after the specimen is
drawn. Prelabeling is unacceptable.

23

4.The patients name, unique identificatin number and room number or clinic, and
date and time of collection are usually found on the label. In some cases, labels
must include the time of collection of the specimen and the type of specimen. A
properly completed request form should accompany all specimes sent to the
laboratory. Note: Capillary blood collection is performed with a sterile, disposble
lancet. These lancets should be properly discraded in a puncture proof container
after a single use.

Labels
Quality assesment policies are implemented in the clinical laboratory to
protect the patient from any adverse consequences of errors resulting from an
improprely handled specimen, beginning with the collection of that specimen.
Laboratory quality assessment and accreditation require that specimens be
properly labeled at the time of collection. All specimen containers must be labeled
by the person doing the collection to ensure that the specimen is actually collected
from the patient whose identification is on the label.
An unlabeled container or one labeled improperly should not be accepted by the
laboratory. Specimens are considered improperly labeled when there is
incomplete or on patient identification on the tube or container holding the
specimen. Many specimen containers are transported in leakproof plastic bags. It
is not acceptable practice for only the plastic bags to be labeled; the container
actually holding te specimen must be labeled as well. If the identification is
illegible, the specimen is unacceptable. A specimen is also unacceptable if the
specimen container identification does not match exactly the identification on the
request form for that specimen.
In many laboratories, labels are computer generated, which helps to ensure that
the proper identification information is included for each patient. Bar-coded labels
facilitate this process. One automated computer system, BD.id Patient

24

Identification System (http://www.bd.com) eliminares mislabeling because the

system reads the patients bar-coded wristband. The sofware indicates the tests,
appropriate tubes, and quantity of tubes required for the patient, then generates
bar-coded laboratory labels for tube identification at the patients bedside.
Each laboratory has a specific protocol for the hndling of mislabeled or
unacceotable specimens.

VENOUS BLOOD COLLECTION (PHLEBOTOMY)

Supplies and Equipment

Test requisition
Torniquet and disposble gloves
Sterile disposable needles and needle holder
Various evacuated blood tubes
Alcohol (70%) and gauze square or alcohol wipes
Any special equipment
Adhesive plastic strips

Initiaton of the Procedure

1. Properly identify the patient

25

2. Assemble all necessary equipment and evacuated tubes at the patients bedside
3. Put on disposable gloves
4. The plastic shield on a needle is to remain on the needle until immediately
Before the venipuncture. The evacuated tube is placed into the holder and
gently pushed until the top of the stopper reaches the guideline on the holder. Do
not push the tube all the way into the holder, or a loss of vacum wiil result.

Selection of an Appopriate Site

Obtaning a blood specimen from an intravenous(IV) line should be avoided
because it increases the risk of mixing the fluid with the blood sample and
producing test results.
1. Visually inspect both arms. Choose a site that has not been repeatedly
used for phelebotomy. In the arm, three veins are typically used for
venipuncture: the cephalic, basilic, and median cubital (Figure 3-3).
2. Apply the tourniquet (Figure3-3). Do not leave the tourniquet on for more
than 2 minutes. Prolonged on for more than 2 minutes. Prolonged
tourniquet application can elevate certain blood chemistry analytes,
including albumin, aspartate aminotransferase (AST), calcium, cholesterol,
iron, lipids, total bilirubin, and total protein.
3. To make the veins more prominent, ask the patient to make a fist. With the
index finger, palpate (feel) for an appropriate vein (see Figure3-3). The
ideal site is generally near or slighty below the bend in the arm. Palpation
is important for identifying the vein, which has a resilient feeling

26

compared with the surrounding tissues. Large veins are not always a good
choice because they tend to roll as you attempt the venipuncture.
Superficial and small veins should also be avoided. If no appropriate veins
are found in one arm, examine the other arm by applying the tourniquet
and palpating the arm. Veins in other areas, such as the wrist, hands, and
feet, can only be used by experienced phlebotomists.

Special Site Selection Situations

Five specific situations may result in a difficult venipuncture or may be
sources of preanalytical error.
INTRAVENOUS LINES
A limb with an IV line running should not be used for venipuncture
because of contamination to the specimen. The patients other arm or an
alternate site should be selected.
EDEMA
Edema is the abnormal acumulation of fluid in the intracellular spaces of
the tissue.

27

SCARRING OR BURN PATIENTS

Veins are very diffcult to palpate in areas with extensive scarring or burns.
Alternate sites or capilary blood collection should be used.
DIALYSIS PATIENTS
Blood should never be drawn from a vein in an arm with a cannula (a
permanent surgical fusion of a vein and an artery). A trained staff member
can draw blood from a cannula. The preferred venipuncture site is a hand
vein or a vein away from the fistula on the underside of the arm.

MASTECTOMY PATIENS
If a mastectomy patient has had lymph nodes adjacent to the breast removed,
venipuncture should not be performed on the breast removed, venipunctute should
not be performed on the same side as the mastectomy.
Preparation of the Venipuncture Site
1. After an appropriate site has been chosen, release the tourniquet
2. Using an alcohol pad saturated with 70% alcohol, cleanse the skin in the
area of the venipuncture site. Using a circular motion, clean the area from
the center and move outward. Do not go back over any area of the skin
once it has been cleansed.
3. Allow the site to air dry
Performing the Venipuncture
Avoid touching the cleansed venipuncture site.
1. Use one hand to hold the evacuated tube assembly. Position the patients
arm in a slightly downward position. Use one or more fingers of the other

28

hand to secure the skin area of the forearm below the intended
venipuncture site. This will tughten the skin and secure the veiin
2. Hold the needle with attached holder about 1 to 2 inches below and in a
straight line with the intended venipuncture site. Position the blooddrawing unit at an angle of about 20 degrees. The bevel of the needle
should be upward.
3. Insert the needle through the skin and into the vein. This insertion motion
should be smooth. One hand should steady the needle holder unit while the
other hand pushes the tube to the end of the plastic holder. It is important
to hold the needle steady during the phlebotomy to avoid interrupting the
flow of blood. Multiple samples can be drawn by inserting each additional
tube as soon as the tube attached to the needle holder has filled. (See
Table 3-1 for the order of drawing multiple evacuated tubes.)
Termination of the Procedure
1. The tourniquet can be released as soon as the blood begins to flow into
the evacuated tube or syringe or immediately before the final amount of
blood is drawn.
2. Ask the patient to open the hand.
3. Withdraw the blood collecting unit with one hand and immediately press
down on the gauze pad with the other hand. After the desired amount of
blood has been drawn.
4. If possible, have the patient elevate the entre arm and press on the gauze
pad with the opp or osite hand. If the patient is unable to do this, apply
pressure until bleeding ceases.
5. Place a nonallergenic adhesive spot or strip over the venipuncture site.
Failure to apply sufficient preassure to the venipuncture site could result in
a hematoma (a collection of blood under the skin that produces a bruise).
6. Mix tubes with anticoagulant by inverting the tubes several times. Do not
shake the tubes. Discard the used equipment into an appropriate punctureproof container.
7. Label all test tubes as required by the laboratory.
8. Clean up supplies from the work area, remove gloves, and wash hands. If
the patient is an outpatient, wait a few minutes after the venipuncture is

29

complete, and check to be sure that the patient does not feel dizzy or
nauseated before discharge.
Phlebotomy Problems
Occsionally a enipuncture is unsuccessful. Do not attempt to perform the
venipuncture more than two times. If two attemps are unsuccessful, notifty the
phlebotomy supervisor. Problems encontered in phlebotomy can include the
following:
1. Refusal by the patient to have blood drawn
2. Difficulty in obtaining a specimen because the bore the needle is against
the wall of the vein or going through the vein
3. Movement of the vein
4. Sudden movement by the patient or phlebotomist that causes the needle to
come out of the arm prematurely
5. Improper anticoagulant
6. An inadequate amount of blood in an evacuated tube
7. Fainting or illnes susequent to venipuncture
Phlebotomy Complications
Patients can experience complications resulting from a phlebotomy
procedure. These complications can be divided into six major categoris, as
follows:
1. Vascular complications. Bleeding from the site of the nipuncture and
hematoma formation are the most common vascular complications
2. Infections. The second most common complication of venipuncture is
infection.
3. Anemia. Latorgenic anemia is also known as nasocomialanemia,
physician-induced anemia, or anemia resulting from blood loss for testing.
This can be a particular problem with pediatric patients.
4. Neurological complications. Postphlebotomy patients can exhibit some
neurological complications, including seizure or pain.
5. Cardiovascular complications. Cardiovascular complications include
orthostatic hypotension, syncope, shock, and cardiac arrest.

30

6. Dermatological complications. The most common dermatological

consequence of phlebotomy is an allergic reaction to iodine in the case of
blood donors.

Blood Culture Collection

Eleveted false-positive rates are common and are associated with
substabtial health care costs. To ensure that the blood collected for culture
is free from contamination (from the patient, the phlebotomist, or other
personel), extra precautions are taken for cleaning the skin and the
collection tube before the actual collection.
The skin is cleaned three times with a povidone-iodine solution or a
chlorhexidine gluconate preparition. By use of a scrub applicator, the
povidone-iodine solution must be applied to the puncture site in a
concentric outward-moving circle, beginning at the site. This step is
repeated three times. After the triple cleaning, the povidone-iodine may be
removed with an alcohol pad if the color of the solution makes it difficult
to locate the vein. If the vein must be touched before the actual
venipuncture, the phlebotomists gloved finger must be triple-cleaned with
povidone-iodine. Perform the venipunctute using a sterile syringe and
needle, or collect directly into culture bottles using an evacuated (vacuum)
system.
Each culture bottle top must be cleaned with an alcohol pad before
injection of the required amount of blood sample inti the bottle. Culture
bottles are labeled and brought to the laboratory.
Capillary or Peripheral Blood Collection by Skin Puncture
Capillary blood can be used for a variety of laboratory assays. Capillary blood is
ofter used for point-of-care tests (POCTs ). A common POCT is bedside testing
for glucose using one of several available reading devices and the accompanying

31

reagent strips; as done at home by diabetic patients ( see following POCT

discussion).
Blood Spot Collection for Neonatal Screening Programs
Most states heve passed laws requiring that newborns be screened for certain
diseases that can result in serious abnormalities, including mental retardation, if
they are not diagnosed and treated early. These diseases include phenylketonuria
( PKU), galactosemia, hypothyroidism, and hemoglobinopathies. CLSI has set
standards for filter paper collection, or blood spot collection, of blood for these
screening programs. Blood should be collected 1 to3 days after birth, before the
infant is discharged from the hospital, and at least 24 hours after birth and after
ingestion of food for a valid PKU test. There is an increased chance of missing a
positive test result when an infant is tested for PKU before 24 hours of age. When
infants are discharged early, however, may physicians prefer to take a sample
early rather than risk no sample at all.
In most neonatal screening programs the specimen is collected on filter paper and
then sent to the approved testing laboratory for analysis. Special collection cards
with a filter paper portion are supplied by the testing laboratory; these are kept in
the hospital nursery or central laboratory. There is an information section on these
cards, and allrequested information must be provided and should be treated as any
other request form.The filter paper section of the card contains cirles designed to
identify the portion of the paper onto which the specimen should be placed, where
the filter paper will properly absorb the amount of blood necessary for the test.
Collection is usually done by heel puncture, following the accepted procedure for
the institution. When a drop of blood is present, the circle on the filter paper is
touched against the drop until the circle is completely filled. A sufficiently large
drop should be done in only one step. The filter paper is allowed to air-dry and
then is transported to the testing laboratory in a plastic transprt bag or other
acceptable container. The procedure established by the testing laboratory should
be followed for the collection step.

32

Capillary Blood for at the Bedside (Point-of-Care Testing)

Capillary blood samples for glucose testing and for other assays are used
frequently in many health care facilities for bedside testing, or point-of-care
testing (POCT). Quantitative daterminations for glucose are made available within
1 or 2 minutes, depending on the system employed. CLSI has set guidelines for
these tests because they are performed in acute care and long-term care facilities.
(POCT is also discussed in Chapter 1, under Alternate Sites of Testing)
POCT for glucose is also performed at home by many diabetic outpatients, using
their own blood and one of several glucose measuring devices. It is important for
diabetic patients, especially those with insulin-dependent diabetes melitus, to
monitor their own blood glucose levels several times a day and to be able to adjust
their dosage of insulin accrodingly to maintain good glucose control.
For the diabetic inpatient, POCT is also a valuable tool for diabetes management.
The blood glucose level is often unstable in these patients, a situation that may
necessitate frequent adjustments of insulin dosage. POCT provides results that are
immediate, so dosages can be adjusted more quickly. Ordering and collecting
venous blood specimens for glucose tests done by a central laboratory, with be
necessary frequenc and rapidty of reporting repuired, are often impractical,
thereby making POCT much more useful. Good quality control programs must be
used, however, to ascettain the realibility ofthe POCT results. Whole blood
samples should be collected by puncture from the heel (for infants only), finger, or
flushed heparinized line, using policies for Standard Precautions. Arterial or
venous blood should not be used, unles the directions from the manufacturer of
the POCT device specify the appropriateness of these alternative blood specimens.
The POCT instrument should be calibrated and the test performed accroding to
the manufacturers directions. Results should be recorded permanently in the
patients medical record in a manner that distinguishes between bedside text
results and central laboratory test results.

33

It is critical to understand and consider the specific limitations of each POCT

detection system, as desrcibed by the manufacturer, so that reliable results arc
obtained. The use of a quality assesment of these procedures. The use of POCT,
whether bedside testing or self-testing for glucose, is intended for management of
diabetic patients and not for an initial diagonis. POCT is not used to reolace the
standard laboratory tests for glucose, but only as a supplement.
Several commercial instruments are available, and with each product a meter
provides quantitative determination of glucosa present

when used with an

accompanying reagent strip. A drop of capillary blood is touched to the reagent

strip pad and, accroding to the specific procedure, read in the meter. The
instrumen provides an accurate and standardized reading when used according to
the manufacturers directions. The reagent strips must be handled with care and
used within their proper shelf life. The strips are specific are only for glucose. The
meters are packaged in convenient carrying cases and are small enough to be
pleaced in a pocket or briefcase.

CAPILLARY BLOOD COLLECTION

Supplies and Equipment

Alcohol (70%) and gauze squares or alcohol wipes

Disposable gloves and sterile small gauze squares
Sterile disposable blood lancets
Equipment specific to the test ordered (e.g.glass slides for blood smears,
micropipette and diluent for CBCs, microhematocrit tubes)

Selection of an Appropriate Site

1. Usually the fingertip of the third or fourth finger, heel, and big toe are
appropriate sites for the collection of small quantities of capillary blood.
The earlobe may be used as a site of last resort in adults. Do not puncture
the skin through previous sites, which may be infected. The plantar surface

34

(sole) of the heel or big toe is an appropriate site in infants or in special

cases such as burn victims. The ideal site in infants is the medial or lateral
plantar surface of the heel, with a puncture no deeper than 2.0 mm beneath
the plantar heel-skin surface and no more than half this distance at the
posterior curve of the heel (Figure 3-5, A). CLSI recommendations sre not
use fingers of infants. The back of the heel should never be used because
of the danger of injuring the heel bone, cartilage, and nerves in this area.
2. The site of blood collection must be warm to ensure the free flow of blood.

Preparation of the Site

1. Hold the area to be punctured with the thumb and index finger of a gloved
hand.
2. Wipe the area with a 70% alcohol pad and allow to air-dry.
3. Wipe the area with a dry gauze square. If the area is not dry, the blood will
not form a rounded drop and will be defficult to collect.

Puncturing the Skin

1. Use a disposable sterile lancet once, and discard it properly in a punctureproof container.
2. Securely hold the area, and puncture once with a firm motion (Figure35,B).
3. Wipe away the first drop of blood because the first drop of blood is mixed
with lymphatic fluid and possibly alcohol.
4. Apply gentle pressure to the area to obtain a suitable specimen.

Special Capillary Blood Collection Unopette

The Unopette system is a microsample collection system for use in certain
manual or automated procedures (Figure 3-6).

35

Although various diluents and sample sizes differ from test to test, the blood
collection procedures are basically similar. Standard Precautions should be
practiced as required during specimen processing and the disposel of supplies.
Each system consists of a capillary pipette and a reservoir containing a
premeasured amount of specific diluent.

Collection and Dilution Procedure

1. Place the reservoir on a flat surface. Hold the reservoir with one hand.
With the other hand, take the micropipette, covered with a pipette shield,
and firmly push the tip of the pipette shield unit through the neck of the
reservoir.
2. Remove the pipette and shield from the neck of the reservoir. Remove the
sheild from the pipette.
3. Collect the free-flowing capillary blood into the pipette section using the
technique described in the procedure for collection of capillary blood.
When the pipette has filled up to the end of the capillary bore in the neck
of the pipette, it will not draw more blood.
4. Wipe any excess blood from the outside of the pipette, being careful not to
touch the blood sample in the capillary tube.
5. While squeezing the reservoir slightly with one hand, cover the flagged
end of the pipette with the index finger of the other handm and insert
capillary pipette with the index finger of the other hand, and insert the
capillary pipette into the reservoir.
6. Simultaneously, release the pressuare on the reservoir and the index finger
from the pipette. This action will draw the blood into the diluent.
7. Rinse the bore of the pipette by squezzing and releasing the reservoir
( repeat steps 5 and 6 ) two or theree times. This will throughly rinse the
blood from the capillary pipette.
8. Place the index finger over the top of the inverted pipette, and gently tilt
entire unit up and down several times to mix

36

9. To transport the specimen, remove the pipette from the reservoir and place
the flagged end info the reservoir. Label the container with the patients
name and other appropriate identification.
10. To use this unit for testing, invert the specimen to mix it, and expel several
drops of the dilution. The unit can then be used as a pipette to perform
such procedures as loading a hemocytometer.

Capillary Blood for Slides

A finger or heel puncture is made, and after the first drop is wiped away, the glass
slide is touched to the second drop formed. The slide is placed on a flat surface
and a spreader slide used to prepare the smear ( see Chapter 12). The slide is
allowed to airdry, is properly labeled, and then is transported to the laboratory for
examination.
Collecting Microspecimens
At times, only small amounts of capillary blood can be collected, and many
laboratory determinations have been devised for testing small amounts of sample.
In general, the same procedure is followed as for any other drawing of capillary
blood. For chemistry procedures, blood can be collected in a capillary tube or
microcontainer by touching the tip of the tube to a large drop of blood while the
tube is held in a slightly downward position. The blood enters the collection unit
by capillary action. Several tubes can be filled from a single skin puncture, if
needed. Tubes are capped and brought to the laboratory for testing. Careful

37

centrifugation technique must be used, if serum is needed. Microcontainers are

available with various additives, including serum separator gels.
Capillary tubes may be heparinized or plain. For spccial tests, a 100 or 200
lambda micropipette may be used. Box 3-2 indicates the order of draw for
capillary specimens.
Laser Equipment
Laser techology is the first radical change in phlebotomy in more than 100 years.
Revolutionary devices approval from the food and Drug Administration (FDA)in
1997. The Lasette ( Cell Robotics, Albuquerque, NM) and the Laser Lancet
( Transmedica International, Little Rock, Ark) can draw blood without the use of
sharp objects.
A laser device emits a pulse of light energy that lasts a fration of a second. The
laser concentrates on a very small portion of skin, literally vaporizing the tissue
about 1 to 2 mm to the capillary bed. The device can draw a 100 uL blood sample,
a sufficient amount for certain tests. The laser process is less painful and heals
faster than when blood is drawn with traditional lancets. The patient feels a
sensation similar to heat rather than the prick of a sharp object.
BOX 3-2
Order of Draw for Capillary Specimens

1.
2.
3.
4.
5.

Blood gases
Slides/smears
EDTA tubes
Other additive minicontainers
Serum containers,minicontainers

SPECIMENS: GENERAL PREPARATION

Accurate chemical analysis of biological fluids depends on proper collection,
preservation, and preparation of the sample, in addition to the technique and

38

method of analysis used. The most quantitatively perfect determination is of no

use if the specimen is not properly handled in the initial steps of the procedure.
Processing Blood Specimens
Blood specimens must be properly handled after collection. CLSI has publiched
standards for handling blood specimen after collection by venipuncture. If no
anticoagulant is used, the blood will clot and serum is obtained. After being
placed in a plain tube with no additives, the blood is allowed to clot. The serum is
then removed from the clot by centrifugation. To prevent excessive handling of
biological fluids, many laboratory instrumentation system can now use the serum
directly from the centrifuged tube, without another sepaation step and without
removing the stopper.
It is important to remove the plasma or serum from the remaining blood cells, or
clot, as soon as possible. Because biological specimens are being handled, the
need for certain safety precautions is stressed. The Standard Precautions policy
should be used because all blood specimen should be considered infectious and
must be handled with gloves. The outside of the tubes may be bloody, and initial
laboratory handling of all specimens necessitates direct contact with the tubes.
When stoppers must be removed from the tubes, they must be removed carefully
and not popped off, because this could cause infection by inhalation or by contact
of the infectious aerosol with mucous membranes. Stoppers should be twisted
gently while being covered with protective gauze to minimize the risk from
aerosol. This processing step can be done using a protective plastic sheild to
prevent direct splashes. To separate the serum and plasma from the remaining
blood cells, the tube must be centrifuged.
It is generally best to test specimens as quickly as possible. Specimens should be
processed to the point where they can be properly stored so that the constituents to
be measured will not be altered. Specimens collected at collection station away
from the testing laboratory need to guarantee that delivery will be made in less

39

than 2 hours from collection and that specimens have been stored properly,
including refrigeration, or freezing if necessary.
If the centrifuged serum or plasma must be removed into a separate tube or vial,
pipette the serum or plasma by using mechanical suction and a disposable pipette;
use a protective plastic shield to prevent direct splashes. All serum and plasma
tubes, as will as the original blood tubes, should be discaeded properly in
biohazard containers when they are no longer needed for the determination.
Use of Serum Separator Tubes
With many automanted methods, processing, the blood often takes longer than the
actual analysis.A fast, efficient way of separating serum from cells is needed. As
discussed earlier, special serum separator tubes. Which resemble ordinary vacuum
tubes but contain inert silicone gel, can be used. The gel is displaced up inside the
tube during centrifugation and from a barrier between the serum and the cells. The
serum can easily be removed to the appropriate container or can be aspirated
directly into the analyzer used for testing. By directly aspirating the centrifuged
specimen in the primary collection tube into the analyzer, one step is saved, and
the risk of transmitting biohazardous material is reduced. Direct use of the
primary tube in testing also reduces the risk of mislabeling the specimen during its
transfer to an additional tube. Special specimen collection products are designed
to save time and to provide a safer mechanism for processing blood specimens.
Centrifuging the Specimens
After clotting has taken place, the tube is centrifuged with its cap on. It is
important to remind staff who handle blood specimens in all steps of the
laboratory analysis to use Standard Precautions. Standard Precaution require all
persons handling specimens to wear gloves. When necessary, stoppers must be
carefully removed from blood collection tubes to prevent aerosolization of the
specimen. Centrifuges must be covered and placed in a shielded area. When
serum or plasma samples must be removed from the blood cells or clot,

40

mechanical suction is used for pipetting, and all specimen tubes and supplies must
be discarded properly in biohazard containers.
The use of automated analyzers often allows the use of the primary collection
tube for the analysis itself. In these cases the primary blood tube is centrifuged
with its cap on and the serum aspirated directly into the analyzer.
Unacceptable Specimens
Various conditions render a blood speecimen unsuitable for testing. Clotted
specimens are not suitable for cell counts because the cells are trapped in the clot
and are therefore not counted. A cell count on a clotted sample will be falsely low.
Hemolyzed Specimens
Hemolysis in specimens is perhaps the most common cause of an abnormal
appearance. Hemolyzed serum or plasma is unfit as a specimen for several
chemistry determinations.
A specimen that is hemolyzed appears red, usually clear red, because the RBCs
have been lysed and the hemoglobin has been released into the liquid portion of
the blood. Often the cause of hemolysis in specimens is the technique used for
venipuncture. A poor venipuncture, with excessive trauma to the blood vessel, can
result in a hemolyzed specimen. Inappropriate needle bore size and contact with
alcohol on the skin are other causes. Hemolysis of blood can also result from
freezing, prolonged exposure to warmth, or allowing the serum or plasma to
remain too long on the cells before testing or removal to another tube. A
determination of whether the hemolysis is in vitro or in vivo is also useful.
Although relatively rare, in vivo hemolysis is a clinically significant finding.
Hemolyzed serum or plasma is unsuitable for several chemistry determinations
because substances usually present within cells (e.g., K +) can be released into the
serum or plasma if serum is left on the cels for a prolonged period. In addition,
several other constituents, including the enzymes, acid phospates, lactate

41

dehydrogenase (LDH), and aspartate aminotransferase (AST, GOT), are present in

large amounts in RBCs, so hemolysis of red cells will significantly elevate the
value obtained for these substances in serum. Hemoglobin is released during
hemolysis and may directly interfere with a reaction, or its color may interfere
with photometric analysis of the specimen. The procedure to be done should
always be checked to determine whether abnormal looking specimens can be
used.
Icteric Specimens
Icteric (yellow) serum or plasma is another specimen with an abnormal
appearance. When serum or plasma takes on an abnormal brownish yellow color,
there has most likely been an increase in bile pigments, namely, bilirubin.
Excessive intravlarascular desctruction of RBCs, obstruction of the bile duct, or
impairment of the liver leads to an accumulation of bile pigments in the blood,
and the skin becomes yellow. Those performing clinical laboratory determinations
should note any abnormal appearance of serum or plasma and record it on the
report from. The abnormal color of the serum can interfere with photometric
measurements.
Lipemic specimens
Lipemic plasma or serum takes on a milky-white color. The presence of lipid, in
serum or plasma can cause this abnormal appearance. Often, the lipemia results
from collecting the blood from the patient too soon after a meal. Use of a lipemic
serum specimen does not interfere with some chemical determination but may
interfere with others (e.g., triglyceride assay).
Drug Effect on Specimens
Blood drawn from patients taking certain types of medication can give invalid
chemistry results for some constituents. Drugs can alter several chemical
reactions.drugs can affect laboratory resukts in two general ways: some action of

42

the drug or its metabolite can cause an alteration (in vivo) in the concentration of
the substance being measured, or some phsycal or chemical property of the drug
can alter the analysis directly (in vitro). The number of drugs that affect laboratory
measurements is increasing.
Logging and Reporting Processes
As part of the processing and handling of laboratory specimens, a careful,
accurate logging and recording process must be in place in the laboratory,
regardless if the size of the facility. A log shet and a printed report from are vital
to the operation of any laboratory. The log sheet documents on a daily basis the
various patient specimens received in the laboratory.log sheets and result reports
are generated by laboratory information systems, when used (see Chapter 10).
Items to be listed on the log sheet are the patients name, identifiction number,
type of specimen collected (description of the specimen and its source), date and
time of specimen collection, and laboratory tests to be done. The log sheet should
also indicate the time when the specimen arrived in the laboratory. The log sheet
can also include a column for test results and the date when the tests are
completed. Results can be documented by hand, by use of laboratory instrumentprinted reports, or by computer printouts. The log sheet data are part of the
permanent record of the laboratory and must be stored and available for future
reference.
A printed report is often sent to the physician with the vital data pertaining to the
test results. Result reports are also available electronically in many facilities. The
following information should be included in the report: patients name,
identification number, date and time of specimen collection, description and
source of specimen, the initials of the person who collected the specimen, tests
requested, the name of the physician requesting the tests, the test results and the
initials or signature of the person who performed the test. Much of this
documentation of data is being done with the use of laboratory computerized

43

information systems. Copies of this laboratory report may be sent to the medical
records departement and to the accounting office for patient billing purposes.
Preserving and Storing Specimens
Some chemical constituents change rapidly after the blood is removed from the
vein. The best policy is to perform tests on fresh specimens. When the specimen
must be preserved until the test can be done, there are ways to impede alteration.
because it prevents glycolysis.
With few exceptions, the lower the temperature, the greater is the stability of the
chemical constituents. Furthermore, the growth of bacteria is considerably
inhibited by refrigeration and is completely inhibited by freezing. Room
temperature is generally cinsidered to be 18o to 30oC, the refrigerator temperature
about 4oC, and freezing about -5oC or less. Refrigeration is a simple and reliable
means of impeding alterations, including bacteriology action and glycolysis,
although some changes still take place. Refrigerated specimens must be brought to
room temperature before chemical analysis. Removing cells from plasma and
serum is another of preventing some changes. Some specimens needed for certain
assays, such as bilirubin, must be shielded from the light or tested immediately.
Bilirubin is a light sensitive substance.
Serum or plasma may be preserved by freezing. Whole blood cannor be frozen
satisfactorily because freezing ruptures the RBCs (hemolysis). Freezing preserves
enzyme activities in serum and plasma. Serum and plasma freeze in layers with
different concetrations, and therefore these specimens must be well mixed before
they are used in a chemical determination.
Every preceaution must be taken to preserve the chemical constituents in the
specimen from the time of collection to the time of testing in the laboratory, if the
results are to be meaningful. In general, tubes for collecting blood for chemical
determinations do not have to be sterile, but they should be chemically clean.
Serum is usually preferred to whole blood or plasma when the constituents to be

44

measured are relatively evenly distributed between the intracelluler and extra
celluler portions of the blood.
Storage of Processed Specimens
The processing of individual serum or plasma tubes will depend on the analysis to
be done and the time that will elapse before analysis. Serum or plasma may be
kept at room temperature, refrigerated frozen, or protected from light, depending
on the circumstances and the determination to be done. Some specimens must be
analyzed immediately after they reach the laboratory, such as specimens for blood
gas and pH analyses. Blood specimens for hematology can be stored in the
refrigerator for 2 hours being used in testing. After storage, anticoagulated blood,
serum, or plasma must be thoroughly mixed after it has reached room
temperature.
Plasma and serum often can be frozen and preserved satisfactorily until a
determination can be done. Whole blood cannot be frozen because RBCs rupture
on freezing. Freezing preserves most chemical constituents in serum and plasma
provides a methoad of sample preservation for the laboratory. In general,
refrigerating specimens impedes alterations of many constituents. With all
biological specimens, however, preservation should be the exception rather than
the rule. A laboratory determination is best done on a fresh specimen.
Removing Interfering Substances
Biological fluids are very complex in their composition. There are hundreds of
detectable substances in urine and blood; chemical analysis would be impossible
if it were necessary to isolate each substance completely before it could be
measured. An optimal method is one that can test for a specific substance while
the other substances remain. A test is said to be specific when none of the other
substances interfere. In chemical analysis, however,almost all determinations are
subject to some interference. Sometimes the interference is small enough or
constant enough that it does not significantly alter the accuracy or precision of the

45

test results. Sometimes the interference does affect the results, and the specimen
must be specially treated before the analysis can take place. That is, the substances
causing the interference must be isolated, or removed, from the specimen.
URINE
Urine yields a great amount of valuable information quickly and economically.
Clinical information obtained from a urine specimen is influenced by the
collection method, timing, and handling.
Various types of collection and transport containers for urine specimens are
available. A specimen must be carefully collected, preserved, and processed
before analysis in order for the reported results to be reliable. If urine testing
cannot be performed within 2 hours of collection, the specimen should be stored
4 C as soon as possible after collection. Specimens can be stored under
refrigeration for 6 to 8 hours with no gross alteration in constituents.
Types of Urine Specimens
Random Specimen
A random urine specimen is the most common type for analysis. Random
specimens, or specimens collected at any time, can give an inaccurate view of a
patients health because the specimen is too diluted and analyte values are
artificially lowered. Although there are no specific guidelines on how the
collection should be conducted, avoiding the introduction of contaminants into the
specimen is recommended. This requires explicit instructions to patients so that
they do not touch the inside of the cup or cup lid with their body.
First Morning Specimen
The first urine voided in the morning is the specimen of choice for urinalysis and
microscopic examination. This urine is generally more concentrated because of
the length of time the urine is allowed to remain in the bladder overnight. The

46

specimen contains relatively high levels of cellular elements and analytes ( e.g.,
glucose, protein ). Any urine that is voided from the bladder during the 8- hour
( typically overnight) collection period should be prooled and refrigerated so that
a true 8 hour sample is obtained. To test for the presence of urine sugar, the best
specimen to use is one voided 2 to 3 hours after a meal. This is the one exception
to the recommended use of the first morning specimen.
Midstream Clean-Catch Specimen
Midstream, clean-catch urine is the preferred type of specimen for culture and
sensitivity testing because of the reduced incidence of cellular and microbial
contamination. Patients are required first to cleanse the urethral area and then to
void the first portion of the urine stream info the toilet. These first steps
significantly reduce the incidence of contamination of the urine specimen. A
midstream sample of urine is then collected into a clean container. This method of
collection can be conducted at any time of day or night.
24-Hour or Timed Specimen
The most common tests requiring the 24-hour urine specimen include those
measuring creatinine, urine urea nitrogen, glucosa, sodium, potassium, and
substances ( e.g., catecholamines,17- hydroxysteroids) that are affected by diurnal
variations. The bladder is emptied before beginnig the timed collection. Then, for
the duration of the designated 24 hour period, all urine is collected and pooled
into a collection container, with the final collection at the end of the period.
Usually the specimen is refrigerated. Accurate iming is critical to determining the
concentration of various analytes and calculated ratios.
Catheter Collection Specimen
This assisted procedure is conducted when a patient is confined to bed or cannot
urinate independently. A health care provider can use an existing catheter or can

47

insert a Foley catheter into the bladder through the urethra to collect the urine
specimen.
Suprapubic Aspiration Specimen
This method is used when a bedridden patient cannot be catheterized or a sterile
specimen is required. The urine specimen is collected by needle aspiration
through the abdominal wall into the bladder.
Pediatric Specimen
For infants and small children, a special urine collection bag is adhered to the skin
surrounding the urethral area. Once the collection is completed, the urine is
poured into a collection cup or transferred directly into an evacuated tube with a
transfer straw. Urine collected from a diaper is not recommended for laboratory
testing because contamination from the diaper material may affect test results. If a
24-hour pediatric specimen is required, a special tube can be attached to the bag,
which in turn is connected to a collection bottle.
Containers for Urine Collection
It is essential that the containers used to collect the urine specimen be clean, dry,
and free of particles or interfering substances. Containers should not be reused.
Several types of containers are sultable for this purpose. Disposable, inert, plastic
containers with leak-resistant lids, plastic bags or jars are most often used.
Any bedpans that are used to collect voided urine must be scrupulously clean and
free of cleaning agents or bleach. Labels must remain fixed to the urine specimen
container at all times and must be on the container, not on the lid.
Urine Collection Cups
Urine collection container cups come in a variety of shapes and sizes, with either
snap-on or screw-on lids. CLSI guidelines for urine ( GP-16A2) recommend the
use of a primary collection container that houlds at least 50 mL, has a wide base,

48

and has an opening of at least 4 cm. The wide base prevents spillage, and a 4 cm
opening is an adequate target for urine collection.
Leak-resistant cups should be used to protect health care personnel from exposure
to the specimen and protect the specimen from exposure to contaminants. Some
urine transport cup closures have special access ports that allow closed- system
transfer of urine directly from the collection device to the tube.
Urinalysis Tubes
Evacuated tubes, similarto those used in blood collection, are filled through a
straw device from cups with integrated transfer devices built into their lid, or from
direct sampling devices, and are used to access catheter sampling ports.
For testing purpose, conical bottom test tubes provide the best sediment collection
for microscopic analysis. Some tubes are specially designed to be used with a
pipetter that allows for standardized sampling. Fill volumes of urinalysis tubes
usually range from 8 to 15 mL.
BD manufactures a plastic urine preservative tube.This tube contains
chlorhexidine, ethylparaben, and sodium propionate and maintains sample
integrity for up to 72 hours without refigeration.
24-Hour Collection Containers
Urine collection containers for 24-hour specimens should hold up to 3 L and may
be colored to protect light-sensitive analytes ( e.g., porphyrins, urobilinogen) from
degradation.
If a preservative is required, the least hazardous type should be selected and added
to the collection container before the urine collection begins. Common 24-hour
preservatives are hydrochloric acid, boric acid, acetic acid, and toluene. Warning
labels should be placed on the container. A corresponding material safety data

49

sheet (MSDS) should be givento the patient, and the health care provider should
explain any potential hazards.
Urine Culture Containers
CLSI guidelines recommed sterile collection containers for microbiology
specimens. These containers should have secure closures to prevent specimen loss
and to protect the specimen from contamination.
Urine Transport Tubes
Transport tubes should be compatible with automated system and instruments
used by the laboratory. Collection containers and transport tubes should be
compatible with the pneumatic tube system if one is used for urine specimen
transort in the facility. A leakproof device in this situation is critical.

Collecting Urine Specimens

Collection of Routine specimens
A specimen for urinalysis should be collected in a clean, dry container, and the
specimen should be fresh. For routine screening, a freshly voided, random,
preferably midstream ( freely flowing) urine specimen is usually suitable. For
most routine urinalysis, including protein content and urinary sediment
constituents, the concentrated first morning specimen is the most satisfactory one
to use.
Occasionally a catheterized specimen may be needed.This type of specimen is
obtained by a physician or designee and is obtained by introducing a catheter into
the bladder, through the urethra, for the withdrawal for urine. Catheterization may
be required under special circumstances or for obtaining a sterile urine specimen
for bacteriologic examination. The risk of introducing infection is always present
when an invasive procedure such as catheterization is performed. Under most

50

conditions, a free-flowing ( midstream) voided specimen is satisfactory for

bacteriologic cultures.

Patient Collection Instructions for Midstream, Clean-Catch Urine Specimen

for Culture
1. Wash your hands thoroughly with soap and water.
2. Open the lid of the urine container provided. Be careful no to touch the
inside.
3. Cleanse your genital area using the following procedure:
Man
a. If you are uncircumcised, draw back the foreskin before cleansing
b. Clean the tip of your penis using a sterile cleansing towelette,
beginning at the tip and moving toward the base. Repeat the cleansing
process using a second towelette.
Woman
a. Squat over the toilet, and use the fingers of one hand to separate and
hold open the folds of the skin in your genital area.
b. Clean the urinary opening and surrouding area with a sterile cleansing
towelette, moving from front to back. Repeat the cleansing process
using a second towelette.
4. Discard the towelettes in a trash receptacle ( not in the toilet).
5. both
Begin
urinating intoculture
the toilet
Afterurinalysis
the urineare
hasneeded
flowedonfor
When
a bacteriologic
andbowl.
a routine
theseveral
same
seconds
into the
toilet,
catchbethe
midportion
urinetests,
flow
in the
specimen,
the culture
should
always
done
first, then of
the the
routine
to avoid
collection
When culturing
sufficient
urine has media.
been Procedure
collected
contamination
of thecontainer.
specimen before
on bacteriologic
( approximately
halfoffull),
urinating
into
toilet.
3-1 desribes
the collection
urinecontinue
specimens
suitable
forthe
culture.
6. Tightly screw the cap on the specimen container.
7. Wash your hands thoroughly with soap and water.
Collection
of Timed
Specimens
8. Promptly
give Urine
the specimen
container to the nurse or laboratory personnel,
or leave in the place specified.
9. Ensure that the specimen label contains your proper identification.

51

The patient is carefully instructed about details of the urine collection process, if
the collection will be done on an outpatient basis. The bladder is emptied at the
starting time (e.g., 8 AM) and this time is noted on the collection container. The
first urine voided at the beginning of the collection is always discarded. All
subsequent voidings are collected and put into the container, up to and including
the urine voided at 8 AM the following day. This last urine specimen will
complete the 24 hour collection.
For timed collection of other than 24 hours, the sample collection principle
applies. These timed collection specimens are preserved by refrigeration between
collections, with the appropriate chemical preservative added to the container
before the beginning of the collection process.
The total volume of the timed collection sample is measured and recorded, and the
sample well mixed, before a measured aliquot is with drawn for analysis.
Collection of Urine for Culture
A clean-catch, midstream urine specimen is desirable for culture ( see Procedure
3-1). It is important that the glans penis in the male and the urethral orifice in the
female be thoroughly cleaned with a mild antiseptic solution by means of sterile
gauze or cotton balls. The patient should be instructed to urinate focibly and to
allow the intial stream of urine to pass into the toilet or bedpan. Throughout the
urination process for the female, the labia ahould be separated so that no
contamination results. The midstream specimen should be collected in a sterile
container, and no portion of the perineum ( female) should come in contact with
the collection container. After the specimen has been collected, the remaining
urine is discarded.
Preservation of Urine Specimens
If a fresh specimen of urine is letf at room temperature for a period, the urine
rapidly undergoes changes. Decomposition of urine begins within 30 minutes after
collection. Specimens left at room temperature will soon begin to decompose,

52

primarily because of the action of urea-splitting bacteria, which produces

ammonia. On combining with hydrogen ions, ammonia forms ammonium ions,
causing an increase in urine pH, which will contribute to the decomposition of
casts and certain cells, if present in the urine. The various laboratory tests planned
for a urine specimen should be performend promptly after collection. No longer
that 1 or 2 hours should elapse before the tests are done, unless the urine is
preserved in some way.
The best method of preservation is immediate refrigeration during and after
collection. The specimen may be kept 6 to 8 hours under refrigeration, with no
chemical preservative added, with o gross alterations. Specimen can be frozen (at
-24 to -166 C) after collection. Several chemical preservatives are available as
additives for routine urine specimens. Preservatives have different roles but
usually are addedto reduce bacterial action or chemical decomposition or to
solubilizc consituents that might otherwise precipitate from the solution.
Specimen for some types of analysis should not have preservatives added because
of the possibility of interference with analytical methods. Generally, the length of
preservation capacity ranges from 24 to 72 hours.
In addition to refrigeration or freezing, common chemical preservatives are
hydrochloric acid , boric acid and acetic acid. Boric acid allows urine to be kept at
room temperature while still providing results comparable to those of refrigerated
urine. Other preservatives include the following:

Toluene, a solution lighter that urine or water, prevents the growth of

bacterial by excluding contact of urine with air. A thin layer of toluene is
added, just enough to cover thee surface of the urine. The toluene should
be skimmed off or the urine pipetted from beneath it when the urinr is
examined. Toluene ( toluol) is the best all-around preservative because it

does not interfere with the various tests done in the routine urinalysis.
Formaldehyde ( formalin), a liquid preservative, acts by fixing the formed
elements in the urinary sediment, including bacteria. It may interfere with
the reduction tests for urine sugar, however, and may from a precipitate

53

with urea that interferes with the microscopic exammination of the

sediment. Preservative tablets that produce frmaldehyde are commercially
available. The tablets are more convenient to use that the liquid formalin

and do not interfere with the usual chemical and microscopic examination.
Thymol, a crystalline substance, works to prevent the growth of bacteria.

Thymol may interfere with tests for urine protein and bilirubin.
The BD Vacutainer Plus Plastic UA Preservative Tube contains a
proprietary additive ( chlorhexidine, ethyl paraben, sodim propionate) that
maintains sample integrity of up to 72 hours without refrigeration.When a
specimen is directly transferred from a collection cup into a preservative
tube, it rovides a stable environment for the specimen until testing can be
conducted and reduces the risk of bacterial overgrowth or specimen
decomposition.

Specialized additives include nitric acid for mercury analysis, sodium bicarbonate
and EDTA for porphyrins, and sodium bicarbonate for urobilinogen analysis.
The most common preservative of urine for culture and sensitivity ( C&S) testing
is boric acid, which comes in tablet, powder, or lyophilized form. Clinical
evidence suggests that nonbuffered borin acid may be harmful to certain
organisme and that buffered boric acid preservatives can reduce the harmful
effects of the preservative on the organisms. C&S preservatives are designed to
maintain the specimen in a state equivalent to refrigeration by deterring the
proliferation of organisms that could result in a false-positive culture or bacterial
overgrowth.
Preserved urine specimens can be stored at room temperature until time of testing.
Product claims regarding duration of preservative potency should be obtained
from the particular manufacturer.
Specimen Preservation Guidelines

54

1. CLSI guidelines for microbiological urine testing recommend refrigeration of

specimens at 2 to 8 C or the use of chemical preservatives if the specimen
cannot be processed within 2 hours of collection.
2. Chemical preservatives should be nonmercuric and environmentally friendly.
The American Hospital Association and the Environmental Protection Agency
issued a Memorandum of Understanding for the virtual elimination of
mercury containing waste from the health care industry waste stream by
2005 (http://www.epa.gov/mercury).
3. The proper specimen to additive ratio must be maintained when using a
chemical preservative to ensure accurate test results. Maintaining the correct
ratio is especially important when transferring fill lines on the tube are used to
ensure proper fill.
4. An evacuated tube system is designed to achieve proper fill volume to ensure
the proper specimen to additive ratio and proper preservative function.
Evacuated systems also reduce the potential exposere of the health care
worker to the specimen.
Labeling and Processing of Urine Specimens
As with any type of laboratory specimen, certain criteria need to be met for proper
collection and transportation of urine specimens.
Labels
Include the patient name and identification information on labels. Make sure that
the information on the container label and the requistion match. If the collection
container is used for transport, the label should be placed on the container, not on
the lid, because the lid can be mistakenly placed on a different container. Ensure
that the labels used on the containers are adherent under refrigerated conditions.
Collection Date and Time
Include the date and time of the urine collection on the specimen label. This will
confirm that the collection was done correctly. For timed specimens, verify start

55

and stop times of collection. Document the time at which the specimen was
received in the laboratory for verification of proper handling and transport after
collection.
Collection Method
The method of collection should be checked when the specimen is received in the
laboratory to ensure the type of specimen submitted meets the needs of the test
ordered.
Proper Preservation
Check if there is a chemical preservative present or if the specimen has not been
refrigerated for longer than 2 hours after collection. Verify taht the method of
preservation used is appropriate for the selected test.
Light Protection
Verify that specimens submitted for testing of light sensitiye analytes are collected
in containers that protect the specimen from light.
BODY FLUIDS
Sterile body fluid can be found in various body cavities under normal conditions.
In various disorders and diseases, the quantity of these fluids can increase
significantly. Fluid specimens aspirated from different anatomical sites ( Tabel 32) can be analyzed for the total number of red and white blood cells,
differentiation of white blood cell types, chemical composition, and
microorganisms. Standard Precautions must be practiced when handling all types
of body fluids.
The type of examination performed on the body fluid depends on the source of the
specimen. The specimen must be fresh. Cell counts cannot be done on a clotted
specimen; anticoagulants must be used to prevent coagulation of the specimen
when a cell count is needed.

56

Cerebrospinal Fluid
Cerebrospinal fluid (CSF) acts as a shock absorber for the brain and spinal cord,
circulates nutrients, lubricates the central nervous system (CNS), and may
contribute to the nourishment of brain tissue.
TABEL 3.2
Body Fluids
Fluid

Synonyms

Bronchoalveolar lavage

Bronchial washings

Cerebrospinal fluid

Spinal fluid
Lumbar puncture fluid
Ventricular fluid
Meningeal fluid

Peritoneal fluid

Dialysate fluid
Paracentesis fluid
Ascitic fluid

Perincardial fluid

Fluid from around the heard

Pericardiocentesis fluid

Plcural fluid

Chest fluid
Thoracic fluid
Thoracentesis fluid

Seminal fluid

Semen

Synovial fluid

Joint fluid

57

CSF is found inside all the ventricles, in the central canal of the spinal cord, and in
the central canal of the spinal cord, and in the subarachnoid space around both the
brain and the spinal cord.
The total maximum volume of CSF is about 150 mL in adults and approximately
60 mL in neonates. In the laboratory, a specimen of CSF is examined visually and
microscopically. Clinically the examination of spinal fluid is useful in diagnosing
a variety of disorders, including subarachnoid hemorhage, meningeal infection
(meningitis), multiple sclerosis, and neoplasms.
Normal CSF is clear and colorless. Any presence of color should be noted. A
yellow coloring of a specimen of the supernatant of a centrifuged specimen is
refered to as xanthochromia, a condition caused by the release of hemoglobin
from hemolyzed erythrocytes (RBCs) in the CSF. Groos blood may also be
observed in traumatic tap specimens or in cases of photological bleeding caused
by spontaneous subarachnoid hemorrhage or intracerebal hemorrhage. Normal
CSF has the viscosity of water. Clotting can result from increased protein. Gel
formation on standing is caused by an increased fibrinogen content.
CSF specimens must be immediately delivered to the laboratory for examination.
The four or five collection tubes must be handled using Standard Precautions.
Tubes are designated for routine testing in hematology, microbiology, clinical
chemistry, and immunology/serology.
Synovial Fluid
Synovial fluid is the fluid that is contained in the joint spaces. Artrocentesis
constitutes a liqued biopsy of the joint. Normal joints have very little synovial
fluid. Aspiration of this fluid from the joints by arthrocentesis provides
information about joint diseases. A veriety of disordes (e.g.,rheumatoid arthritis,
gout) produce changes in the number and types of cells, the chemical

58

composition, and crystals in the fluid. In addition, arthocentesis may alleviate

elevated intraarticuler may alleviate elevated intraarticular pressure.
Synovial fluid differs from other body cavity fluids because of the importance of
finding crystals in the specimen and because it is normally very viscous. Ideally
the specimen should be collected into three tubes: (1) a sterile tube for culture; (2)
a tube with either sodium heparin or liquid EDTA anticoagulant, preferably
heparin, for cell counts, crystal identification, and prepared smears; and (3) a plain
tube without additive anticoagulant (and not in serum seperator gel tube) to
observe for groos apperance, crystal analysis, and fibrinogen clots and for
chemistry or immunologic tests. Sodium heparin or liquid EDTA is the addtive of
choice. To test for clot formation, the fluid must be collected in a plain tube
without anticoagulant.
Pericardial, Pleural, and Peritoneal Fluids
The fluids of the pericardial, pleural, and pertoneal cavitesare called serous
fluids. They normaly are formed continuosualy in the body cavities are
reabsorbed, leaving only very small volumes. The normal apperance of these
fluids is pale and straw colored. The fluid becomes more turbid as the total cell
count rises, an indication of inflammation. Increases in the amounts of these body
cavity fluids formed are seen in infammation and when the serum protein level
falls.
Serous fluids are aspirated by a physician if they are mechanically inhibiting the
function of the associated organs, as well as for diagnostic purposes. The
specimen is collected into various containers, depending on the laboratory testing
to be done. An EDTA tube is used for cell counts and smear evaluation, sterile
tubes are used for cultures and oxalate or fluoride tubes for protein, glucose, or
other chemistry tests. If a large volume of fluid is aspirated, it is collected in a
container with an appropriate addtive to prevent clotting. If the fluid clots, it is
useles for many analyses.

59

Seminar Fluid
The main function of seminal fluid is to transport sperm to female cervical mucus.
After deposition in the female reproductive tract, sperm remain in seminal plasma
for a short time while sttempting to enter the mucus. Each of the male
reproductive structures contributes specific components to seminal fluid. In
addition to spermatozoa, which constitue only a small part of the total volume of
seminal fluid, this fluid has a highly varied composition.
Seminal fluid is examined physically, chemically, and microscopically physically,
chemically, and microscopically. These procedures are performed to determine the
phsical and chemical properties, to quantitate the number of spern cells, and to
examine cellular motility and morpology. Seminal fluid can be analyzed for a
number of reasons, including infertility studies, artifical insemination protocols,
postvasectomy assesment, and evaluation of probable sexual assault.
Seminal Fluid
The main function of seminal fluid is to transport sperm to female cervical mucus.
After deposition in the female cervical mucus. After deposition in the female
reproductive tract, sperm remain in seminal plasma for a short time while
attempting to enter the mucus. Each of the male reproductive structures fluid. In
addition to spermatozoa, which constitute only a small part of the total volume of
seminal fluid, this fluid has a highly varied composition.
Seminal fluid is examined physically, chemically, and microscopically. These
procedures are performed to determine the phsycal and chemical properties, to
quantitate the number of sperm cells, and to examine celluler motility and
morphology. Seminal fluid can be analyzed for a number of reasons, including
infertility studies, artificial insemination protocols, postvasectomy assesment, and
evaluation of probable sexual assault.

60

A fresh specimen is needed. The specimen may be collected in a clean, sterile,

glass or plastic container. Ideally, seminal fluid should be analyzed within
30minutes of collection. It is mandatory that the specimen be kept at 37 o C and
examined within 1 to 2 of collection. After 60 minutes of stronge in a plastic
container, sperm motility is
Collecting and Processing Laboratory Specimens
for Throat Culture
1. Ask the patient to open his or her mouth.
2. Using a sterile tongue blade to hold the tongue down and a sterile swab to collect the
specimen, take the specimen directly from the back of the throat, being careful not to
touch the teeth, ceeks, gums, or tongue when inseting or removing the swab (see
Figure 3-7).
3. The tonsillar fauces and rear pharyngeal wall should be wabbed, not just gently
touched, in order to remove organisms adhering to the membranes. White patches of
exuadate in the tonsillar area are especially productive for isolating the streptococcal
organisms.
4. The swab containing the specimen can be placed in a special container with transport
media. Commercial collection sets containing both swabs and transport media are
availabe. Stretococci survice on dry swabs for up to 2 to 3 hours and on swabs in
transport (holding) media at 4o C for 24 to 28 hours.
5. The specimen container must be labeled with be mecessary patient identification.

Significantly reduced. Most laboratoris examine two specimens collected a few

days apart. Collection, proper transport, and prompt examination are critical
factors in the analysis of seminal fluid. Standard precautions should be adhered to
when handling semen, blood, and other body fluids.
It is recommended that a 3 to 5 day period of sexual abstinence be observed
before specimen collection. Two days may be sufficient; the period should not
excedd 5 days. Condoms treated with spermicide or lubricants with spermicidal
properties must be avoided during specimen collection. In additon. Patient must
be advised to keep the specimen warn if collected at home and to deliver it
promptly to the laboratory.
In medicolegal cases, identification and security are paramount, and the
procedural protocal is determined by local juridicition. In cases of alleged rape or
suspected sexual assault, vaginal smears may be submitted for evaluation of the

61

presence of sperm. Sperm can be detected in the vagina for 24 to 27 hours after
intercourse, but the absence of sperm does not mean that intercourse has not mean
that intercourse has not taken place.
SWABS FOR CULTURE
Swabs with samples of specimens from wounds, abscesses, throats, and other sites
are brought to the laboratory in a sterile transport tube for culture. These swabs
are potentialy from infecticulture. These swabs are potentially from infectious ares
and should be treated carefully in the laboratory. Again, the container with the
swab in it must be properly labeled and the culture done immediately (see Chapter
16). Most bacteria will die if stored on a dry swab, so if the culture cannot be done
immediately, a transport medium should be used, with some means to keep the
swab moist and cool. Most organisme can live for many hours if stored prioperly;
however, immediate culture is still best. Proper technique for disposal of
contaminated material must be used.
Throat Culture Collection
Throat swab specimens are used for detection of group A hemolytic stretococci
causing pharyngits be used for the classic culture on sheep blood media or for one
of the rapid direct tests utilizing extraction of the cell well polysaccharide antigen
and its gained popularity, especially inphyisicians offices, because results are
available within minutes instead of hours (see Chapter 16).

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Feces
Feces, or stool specimens, should be collected in a clean plastic container. An
aliquot of excreted stool is preferabel to faces obtained from a glove as part
labeled properly, including the time of collection (for a timed specimens) and the
laboratory tests desired.
Small amounts of fecal material are frequently analyzed for the presence of
occualt, or hidden, blood. The presence of occult blood is recognized as an
important sign in sreening for colon cancer.
GAMBAR

Outpatients are often asked to recover small amounts of their own faces and apply
them directly to the cardboard filter paper supplied by the physician. The labeled
specimens are than mailed back to the physician or laboratory for testing. In
adults, certain metabolic balance studies and measurement of fecal nitrogen and
far require a 3-day (72-hour) fecal collection.

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Faces from childern can be screened can be for trypsin activity to detect cytic
fibrosis. The fecal samples are usually obtained from a soiled diaper.
Sweat
Sweat testing for detection of increased electrolyte concentration is used to
confrim the diagnosis of cystic fibrosis (see Chapter 11).
Salivia
Salivia, a clear, alkaline and viscous fluid secreted by mucous glands of the
mouth, can be used for various analyses. Microbial studies of viruses and bacteria
and chemical testing of hormones, therapeutic drugs, and drugs of abuse can be
performed on salivia. The most common way of collecting a specimen is to have a
patient chew on wax or absorbent dental cotton for several minutes and then
collect the salivia.
CHAIN-OF-CUSTODY SPECIMEN INFORMATION
When specimens are involved in possible medicolegal situation, certain specimenhandling policies are require that any data pertaining to the specimen in question
be determined in such a court of law. Processing steps for such specimens,
including the initial collection, transportation, storage, and anylitical testing, must
be documented by careful record keeping. Documentation ensures that there has
been no tampering with the specimen by any interested parties, that the specimen
has been collected from the appropriate person, and that the results reported are
accurate. Each step of the collection, handling, proccesing, testing, and reporting
processes must be documented; this is called the chain of custody.
Chain-of-custody documentation must be signed by every person who has handled
the specimens involved in the case in wuestion. The specimens involved in the
case in question. The actual process may very in different health care facilities,
but the general purpose of this process is to make certain that any data obtained by
the clinical laboratory will be admisible on a court of law and that all steps have
been taken to ensure the integrity of the information produced.

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REFERENCES
1. American Hospital Association: Patient Care Partenrship, www.aha.org
(retrieved August 2005).
2. Clinical and Laboratory Standars Institute: Procedures and devices for the
collection of diagnostic capillary bllod specimens: approved standard, ed
5, Wayne, Pa, 2004,H-4-A5
3. Clinical and Laboratory Standars Institute: Tubes and additives for venous
blood specimen collection: approved standard, ed5, wayne, Pa, 2003, H1A5.
4. Clinical and Laboratory Standars Institute: Blood collection on filter paper
for newborn sreening programs: approved standard, ed4, Wayne, Pa,
2003, LA4-A3.
5. Clinical and Laboratory Standars Institue: Point-of-care blood glucose
testing in acure and chronic care facilites: approved guideline, ed 2,
Wayne, Pa, 2002, C30-A2.
6. Clinical and Laboratory Standars Institute: Procedures for the collection of
diagnostic blood specimens by venipuncture: approved standard, ed5,
Wayne, Pa, 2003, H3-H5.
7. Clinical and Laboratory Standars Institute: Routine urinalysis and
collection, transportation, and preservation of urine specimens: approved
guideline, ed 2, Wayne, Pa, 2001, GP16-A2.
8. Skobe C: The basics of specimen collection and handling of urine testing,
Lab Notes 14(2), 2004
(www.bd.com/vacutainer/labnotesVolume14Number2/, retrieved May
2005)

65

BIBLIOGRAPHY
Arzoumanian L: Tech Talk 1(2), May 2002 (www.bd.com).
Burtis CA Ashwood ER, editors: Tietz fundamentals of clinical chemistry, ed5,
Philadelpia, 2001, Sunders.
Busn V: Why doesnt my heparinized plasma specimen remain
anticoagulated? Lab Notes 13, 2003 (www.bd.com, retrieved July 2003)
Bush V, Mangan L: The hemolyzed specimen: cause, effects and reduction,
Lab Notes 13 (1),2003 ( www.bd.com)
Dale JC: Phlebotomy complications. Paper presented at Mayo Laboratorys
Phlebotomy Conference, August 1996, Boston.
Ernst D, Calam R: NCCLS simplifres the order of draw: a brief history, Med
Lab Obsever MLO 36 (5): 26, 2004
Fabel V: Phlebotomy and the aging patient, Adv Med Lab Prof 29 (1): 24,
1998
Forbes BA, Sahm DF, Weissfeld A: Bailzy & Scotts diagnostic microbiology,
ed 11,St Louis 2002, Mosby.
Foubister V: Quick on the draw: coagulation tube response, Cap To day 16
(10): 38,2002
Gerberding JL: Occupational exposure to HIV in health care settings, N Engl J
Med 348 (9): 826,2003
Haraden L: Pediatric phlebotomy: great expectations, Adv Med Lab Prof 28
(11): 12,1997
Hurley TR: Considerations for the pediatric and geriatric patient. Paper
presented at Mayo Laboratorys Phlebotomy Conference, Auhust 1996,
Boston.
Latshaw J: Laser takes sting out of phlebotomy, Adv Med Lab Prof 28(12): 40,
1997

66

Lee F, Lind N: Isolation guidelines, Infection Control Today

(www.infectioncontroltoday. Com/articles/051col15.html.retrieved May 2005)
Magee LS: Preanalytical variable in the chemistry laboratory, Lab Notes 15,
2005.
Marrinan D: Tech Talk 1 (1),2002
Norbeg A et al: Contamination raset of blood cultures obtained by dedicated
phlebotomy vs intravenous catheter, JAMA 289:726,2003
Occupational Safety and Health Administration, US Department of Labor:
Disposal of contaminated needles and blood tube holders used in phlebotomy,
Safery
and
Health
Information
Bulletin
(www.osha.gov/dts/shib/shib101503.html,retieved May 2005).
Occupational Safery and Health Administration, US Department of Labor:
Best practice:OSHAs position on the reuse of blood collection tube holders,
Safety
and
Health
Information
Bulletin
(www.osha.gov/dts/shib/shib101503.html, retrieved May 2005).
Ogden-Grable H, Gill GW; Preventing phlebotomy errors potential for
harming your patients, Labmedicine 36(7): 430,2005.
Roark J: HICPAC revises isolation and TB guidelines, Infection Control
Today (www.infectioncontroltoday.com/articles/531Clinical.html, retrieved
May 2005).
Turgeon M: Clinical hematology: theory and procedures, ed4, Philadelphia,
2005, Lippincott-Williams & Wilkins, p 18.
Tyndall L, Smith S: Tios for urine analysis, Tech Talk 2(1), 2003
(www.bd.com)
Understanding
additives:
hepatin,
Lab
Notes
14(1),
2004
(www.bd.com/vacutainer/labnotes/2004winterspring/additeves_heparin.asp,
retrieved May 2005).

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