EXERCISE 16:
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.
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Centrifuges
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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.
Aliquoting a Serum or Plasma Specimen
Many laboratory tests require only the serum or plasma.
Proper and accurate aliquoting of blood
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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.
consequences since these results are used to diagnose and treat patients.
This could have devastating
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.
2.
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
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3.
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:
1.
Labeling errors:
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2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
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.
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EXERCISE 16:
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.
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Name ____________________________________
Date ______________________
EXERCISE 16: SPECIMEN PROCESSING
SPECIMEN ACCESSION LOG
Read the instructions on the previous page CAREFULLY before beginning!!
Date
Received
Time
Received
Patient Name
Patient ID #
Test Ordered
Tube Received
Problem
Resolution
Initials
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)
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EXERCISE 16: SPECIMEN PROCESSING
STUDY QUESTIONS
Name_________________________________
Points: 25
1.
/25
Compare and contrast serum and plasma including whether or not coagulation factors are present. (2
points).
a.
Serum
b.
2.
Student Grade:
Date__________________
Plasma
List 5 rules to observe when operating a centrifuge (5 points).
a.
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).
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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:
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.
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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
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.
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