EXERCISE 13:
SPECIMEN PROCESSING
Skills: 25 points
Objectives:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Explain how a centrifuge works.
Name five types of centrifuges and tell what each is used for.
State the correct operation of a clinical centrifuge.
Distinguish between serum and plasma and state the different types of plasma which may be used for
testing.
Define and recognize hemolysis, icterus, and lipemia and explain their effects on laboratory tests.
List 14 reasons that a specimen would be rejected by the laboratory.
State the action which must be taken when a sample is rejected by the laboratory.
Explain the importance of verifying the blood specimen identification with the requisition slip.
Demonstrate proper accessioning of laboratory specimens by correctly filling in the accessioning log
sheet.
Demonstrate appropriate specimen processing by comparing requisitions with specimens and noting
any discrepancies, problems with specimen suitability, and/or missing specimens.
State the resolution of any problems noted on the Specimen Accession Log.
State the information which must be present on a tube labeled for an aliqot.
Correctly label a transfer tube for serum or plasma aliquot.
Safely and accurately separate serum or plasma from cells using appropriate PPE.
Discussion
Serum versus Plasma
Whole blood is composed of all cellular elements (red blood cells or RBC, white blood cells or WBC, and
platelets or PLT) and the liquid component, which is either serum or plasma. Adult blood has about 40%
cellular elements and 60% serum or plasma.
Serum is the liquid expressed from clotted blood (blood drawn into a tube with no additive). Blood is allowed
to clot and fibrinogen, along with some of the other coagulation factors, is used up in the formation of the clot.
Serum, therefore, does not contain fibrinogen or other coagulation factors. Serum is the preferred specimen
for most chemistry, blood bank and serology tests because fibrinogen may cause interference in the test
procedure performed, removing it prevents this problem. Some additives may also interfere with these
analyses and it is best that they not be present.
Plasma is the liquid portion of the blood present in anticoagulated specimens. Plasma contains all the
coagulation factors except calcium. Calcium is not present in plasma as most anticoagulants prevent
coagulation by binding (chelating) the calcium. Heparin is a naturally occurring anticoagulant which acts as
an anti-thrombin. Thrombin is another essential component of the coagulation mechanism. The effects of
heparin are short lived. Blood drawn in heparin will begin to clot in approximately 48 hours. Plasma tends to
be somewhat hazier than serum.
Exercise 13: Specimen Processing (Revised 12/10/2013)
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Centrifuges
Centrifuges are instruments used to spin specimens of blood or other body fluids at high rates of speed, forcing
the heavier particles to the bottom of the container. The most frequent laboratory use of the centrifuge is to
separate the cellular components of blood from the liquid so that the liquid may be used for testing.
Centrifuges vary in size, capacity, and speed capability. Clinical centrifuge is the name given to tabletop
models which can be used for urinalysis or serum separation. These usually have a speed capacity of up to
3000 rpms (revolutions per minute), and will hold tubes ranging from 5 to 50 mL sizes, depending on the
adapters. A serofuge is a small centrifuge used in blood banking a serology to spin serological tubes.
Microcentrifuges or microfuges are also becoming widely used. These will spin special microtubes 1.5 cm
capacity at high speeds, usually about 12,000 rpm. The microhematocrit centrifuge is a variation of the
microfuge; it spins capillary tubes at high speeds so that hematocrits can be measured.
Other types of centrifuges include high-speed centrifuges which rotate at speeds up to 20,000 rpm and
ultracentrifuges which rotate at speeds over 50,000 rpm. These centrifuges are specially equipped so that
specimens may be kept cool while being centrifuged. Centrifuges such as these are typically used only in
research laboratories and are not required for routine clinical testing.
Operating the Centrifuge
The manufacturer's instructions must always be followed when using a centrifuge or any other type of
equipment. Some general rules to follow in the operation of all centrifuges are:
1.
2.
3.
4.
5.
Do not operate centrifuges with the lids open.
Balance the contents of the centrifuge before operating. For example, if there is only one specimen to
be centrifuged a tube identical in size and volume of solution contained must be placed in the rotor
opposite the specimen tube. The rotor is the part of the centrifuge which holds the tubes and rotates
during the operation of the centrifuge. For every specimen placed in the rotor, there must be a
balancing specimen placed directly opposite.
Do not open the centrifuge lid until the rotor has stopped spinning.
Spin specimens with lids on to avoid creating aerosols.
Use only tubes that are specified as appropriate for that particular centrifuge.
Preventive Maintenance
Centrifuges should be checked and have maintenance performed on a regular basis. The routine checks of
revolutions per minute and time of centrifugation using either a tachometer or a stroboscope are absolutely
essential for consistent results that meet quality control standards.
Exercise 13: Specimen Processing (Revised 12/10/2013)
2
Aliquoting a Serum or Plasma Specimen
Many laboratory tests require only the serum or plasma. Proper and accurate aliquoting of blood specimens is
an essential skill. Clotted or anticoagulated specimens must be centrifuged and the serum or plasma separated
from the cells so that it can be used in various analyses. It is critical that the serum or plasma not be
contaminated with red blood cells as this could cause falsely increased or decreased results.
The transfer tube in which the aliquot of serum or plasma is placed must be labeled BEFORE the aliquot is
added to the tube. The label MUST include the following:
1. Two patient identifiers: Name plus some other identifier such as date of birth, lab accession number,
medical record number, etc.
2. Date and time of collection.
3. Initials of the individual aliquoting the specimen.
4. Specimen type: EDTA plasma, citrate plasma, heparinized plasma, sodium fluoride plasma, serum,
etc.
5. Special information such as trough or peak drug level.
Take great care not to put the wrong specimen in the wrong tube. This could have devastating consequences
since these results are used to diagnose and treat patients.
Serum or plasma can be separated from the cells in two ways:
1.
The tube is centrifuged and the serum or plasma is carefully pipetted off into another clean, properly
labeled test tube with a clean pipette. Care must be taken not to contaminate the serum with red
blood cells. Red cells present in a serum or plasma sample will alter the results of many laboratory
tests.
2.
Serum separator tubes (SST) and Plasma separator tubes (PST) have a polymer gel. The blood is drawn
into the tube which is then centrifuged. During centrifugation the gel moves above the clot to form a
physical barrier between the serum or plasma and the clot or red cell layer. The serum or plasma can
then be poured off without contamination by red blood cells.
Specimen Appearance
Once the serum or plasma is transferred to an appropriately labeled tube the appearance should be evaluated.
Observe the serum or plasma - it should be clear to hazy and pale yellow in color. There are several different
appearances one should be aware of and which may require notation on the laboratory requisition or rejection
of the specimen:
1.
Hemolysis is a red or reddish color in the serum or plasma which will appear as a result of red
blood cells rupturing and releasing the hemoglobin molecules. Hemolysis is usually due to a
traumatic venipuncture (i.e., vein collapses due to excessive pressure exerted with a syringe,
"digging" for veins, or negative pressure damages innately fragile cells. Gross hemolysis (serum or
plasma is bright red) affects most lab tests performed and the specimen should be recollected.
Check the specific lab test to see what effect hemolysis has on it. Slight hemolysis (serum or
plasma is lightly red) affects some tests, especially serum potassium and LDH (lactate
dehydrogenase). Red blood cells contain large amounts of both of these substances and hemolysis
will falsely elevate their measurements to a great extent. Laboratories must set criteria for the level
of hemolysis which is considered acceptable. Most hemolyzed samples are rejected and must be
recollected.
Exercise 13: Specimen Processing (Revised 12/10/2013)
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2.
3.
Icterus. Serum or plasma can be bright yellow or even brownish due to either liver disease or damage
or excessive red cell breakdown inside the body. Icterus can, like hemolysis, affect many lab tests, but
unfortunately, recollection is not an option since the coloration of the serum or plasma is due to the
patient's disease state. However, appearance should be noted on the lab report as "serum icteric".
Handle icteric serum with extreme caution due to the possibility that the patient may have hepatitis.
Lipemia. Rarely serum or plasma may appear milky. Slight milkiness may be caused when the
specimen is drawn from a non-fasting patient who has eaten a heavy meal. A thick milky appearance
occurs in rare cases of hereditary lipemia. As with icterus, the appearance should be noted on the lab
report as "serum lipemic".
Specimen Storage
If the specimen is to be stored in the refrigerator or freezer, the tube should be tightly capped or tightly sealed
with parafilm. If the specimen is to be frozen, the ideal freezer is a true laboratory freezer which maintains
constant temperature. Regular kitchen refrigerator freezers are generally frost free and go through freeze and
thaw cycles, which may adversely affect the specimen.
Accessioning Specimens
Every lab has a procedure to accession specimens in the lab. Patient names and identification numbers are
logged in on a computer or on a log sheet or book along with the test(s) to be performed. This accessioning
serves as a record of tests done and is a convenient way to check if the specimen has actually arrived in the lab
for testing in the event that the physician or nurse calls to check on the results. It is extremely important that
all patient information is correctly transcribed, that includes spelling of the patient's first and last names and
the correct identification numbers. Remember, most lab errors are clerical in nature; someone misspelled a
name or transposed names or numbers. Be careful! You will be graded down if you make clerical errors.
Specimen Rejection
Each laboratory will have policies in place which will determine the acceptability of specimens for laboratory
testing and the protocols to follow for specimen rejection. An essential component of quality control in the
laboratory is the careful evaluation of the suitability of every specimen by every individual involved in the
handling and testing. Documentation of specimen rejection and the reason the specimen was found unsuitable
are important elements of the quality management program. The records are carefully reviewed to identify the
cause of the error and develop an action plan to reduce the number of specimen rejections.
It is critical to identify whether a single individual or department has a consistently high specimen rejection
rate. Corrective action must be initiated. Communication between the laboratory and the individual or
department involved is crucial. The cause of the error may be determined by asking specific questions related
to techniques which may have lead to the specimen rejection. Follow-up training should be provided and
documented. Additional measures may need to be taken if the problems continue.
The following are causes of specimen rejection and will require that the specimen be recollected:
Exercise 13: Specimen Processing (Revised 12/10/2013)
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1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Labeling errors:
a. Name misspelled or wrong name.
b. Identification number does not match to number on requisition.
c. Date of collection, time of collection and/or phlebotomist’s initials are missing.
Unlabeled tubes.
Wrong tube collected.
Fasting specimen not collected on fasting patient.
Timed specimen not collected at correct time.
“Quantity not sufficient” (QNS) - inadequate volume.
Specimen hemolyzed.
Anticoagulated specimen clotted or has clots in it.
Improper transport (time, temperature, light exposure).
Outdated (expired) supplies.
Contaminated specimens.
Stand your ground and reject improperly labeled and/or collected specimens.
Exercise 13: Specimen Processing (Revised 12/10/2013)
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EXERCISE 13:
SPECIMEN PROCESSING
Procedure
1. The instructor will give each student 3 different patient requisition slips and specimens.
2. Fill in the date and time columns with today’s date and the current time.
a. Compare the names and identification numbers on EACH tube with the names and
identification numbers on the requisition slip. After each tube is evaluated place upright in a
test tube rack.
b. If any errors are identified, note the problem on your "Specimen Accession Log"
c. Describe the action which must be taken to resolve the problem.
3. Write the patient's name and identification number in the appropriate column on your form.
4. List each test ordered on the requisition and the tubes received in the appropriate columns. If a
problem is identified list the problem and the resolution.
a. Make sure that you have the correct specimen for each test ordered.
b. Note on the log if specimens are missing or inappropriate specimens were received.
c. Describe the action which must be taken to resolve the problem, i.e. patient must be redrawn.
5. There will be one specimen which requires aliquoting.
6. If a centrifuge is available in your lab you may be required to centrifuge your specimen.
a. Make sure each tube has one directly opposite from it with identical size and blood volume. If
no match is found create a balance tube.
b. Once the centrifuge is full spin for 7 - 10 minutes.
7. Label a test tube for each specimen to be separated with the following information
a. patient's name
b. identification number or DOB
c. Specimen type (refer to procedure)
d. Date and time of collection
e. Your initials
f. Special labeling, i.e., peak, trough, etc.
8. Carefully separate the serum or plasma from the original tube into the labeled tube with a clean pipette
making sure not to contaminate the serum or plasma with red blood cells. If the red blood cells are
resuspended the tube must be respun.
9. Parafilm or cap the top of each tube.
Name ____________________________________
Points________/25
Date ______________________
EXERCISE 13: SPECIMEN PROCESSING
SPECIMEN ACCESSION LOG
Read the instructions on the previous page CAREFULLY before beginning!!
Date
Received
Time
Received
Patient Name
Patient ID #
Exercise 13: Specimen Processing (Revised 12/10/2013)
Test
Ordered
Tube
Received
Problem
Resolution
Initials
6
Instructor Use ONLY
25 Points
1.
2.
3.
4.
5.
6.
7.
8.
9.
Date and time – 0.5 each (3 total)
Correct spelling of name, last name first – 1 each (3 points)
Correct patient ID # - 1 each (3 points)
Lists test ordered on requisition - deduct 0.5 for each test NOT listed (1 point each patient – 3 total)
Tube Received – deduct 0.5 if tube is received but not recorded OR is not received but is recorded (1 point each patient – 3 total)
Problem – deduct 1 point if problem is present but student did not document OR 0.5 if student lists problem that is NOT a problem. (2
total)
Resolution – deduct 1 point if resolution of a problem is not listed (2 total)
Initials – (3 points)
Aliquot label MUST have: Name (0.5), number(0.5), specimen type (1 Point), date/time of collection(0.5) and initials (0.5) - (3 points)
EXERCISE 13:
SPECIMEN PROCESSING - STUDY QUESTIONS
Name________________________
1.
Compare and contrast serum and plasma including whether or not coagulation factors are present. (2
points).
a.
Serum
b.
2.
Date__________________ Points __________/25
Plasma
List 5 rules to observe when operating a centrifuge (5 points).
a.
Exercise 13: Specimen Processing (Revised 12/10/2013)
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b.
c.
d.
e.
3.
Explain how most anticoagulants prevent coagulation of the blood specimen (1 point).
4.
Explain how heparin works as an anticoagulant (1 point).
5.
What is a serofuge used for? What is a microhematocrit centrifuge used for? (2 points)
a. Serofuge
b.
6.
Microhematocrit centrifuge
Define and explain the clinical significance of each of the following terms (3 points):
a.
Hemolysis
Define:
Clinical Significance:
b.
Icterus
Define:
Clinical Significance:
Exercise 13: Specimen Processing (Revised 12/10/2013)
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c.
Lipemia
Define:
Clinical Significance:
7.
List 3 labeling errors that would cause a specimen to be rejected by the laboratory. (1. 5 points)
a.
b.
c.
8.
List 4 reasons that a blood specimen may be rejected by the laboratory. (2 points)
a.
b.
c.
d.
9.
What action must be taken if a discrepancy exists between the information on the laboratory specimen
and that found on the requisition slip? (1 point)
10.
Why is a regular kitchen refrigerator not the most desirable one to use to freeze a clinical specimen?
(1.5 point)
11.
State the reason that plasma from an EDTA tube cannot be used for a plasma calcium level. (1 point)
12.
List 4 items of information which MUST be transcribed on to the tube into which an aliquot will be
Exercise 13: Specimen Processing (Revised 12/10/2013)
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placed? (2 points)
a.
b.
c.
d.
13.
State the two methods frequently used for separating serum or plasma from the cells. (2 points)
a.
b.
Exercise 13: Specimen Processing (Revised 12/10/2013)
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