48.0 Sample Processing


Title of Manual: Specimen Collection

Version No: 1.0

Title: Sample Processing

Effective Date: 12/20/10
Revised Date: 06/23/14

Document #: GPA.SPC.48.0


I. PURPOSE

Proper specimen handling assures the results reported reflects the concentration in which the analyte exists in the patient as precisely as technologically possible. Once outside the circulatory system, blood is subject to the effects of time, temperature, light and other natural processes that take place when the blood is no longer in circulation. The race against the effects of these processes begins the moment blood enters the collection tube and continues until the specimen is analyzed. Those factors that alter test results include clotting, glycolysis, and the transfer of ions between serum and red blood cells.

This applies to all personnel who are authorized and trained to perform phlebotomy.

II. MATERIALS

Reagents

Supplies

Equipment

● Centrifuge
● Transport Bags
(if Applicable)
● Plastic Pour Off Tubes
● Pipettes


III. PROCEDURE

1. Verification of Specimen Requirements
    a. Appropriate specimen verification should be performed by reviewing specimen requirements in the        Directory of Service, Outreach, or Lifepoint. If there is any questions, placing a call to the clinical lab is        appropriate.
    b. Although some samples may have to be separated for storage and transport at the appropriate         temperatures, the order will be placed as one order in the computer system. Do not split an order.
2. Preventing Clotting
    a. Clotting can be prevented by properly mixing tubes containing anticoagulants immediately
        upon collection.
3. Preventing Glycolysis and Ion Exchange
    a. Glycolysis refers to the metabolism of glucose.
    b. When serum or plasma remains in contact with red blood cells during specimen transport and storage,         glycolysis can result in falsely lower glucose results.
    c. To avoid glycolysis and ion exchange, collect specimens into a gel separator tube (SST) and centrifuge
        within 2 hours of collection to separate serum or plasma from the cells.
4. Centrifuge Use
    a. Centrifugation forces all the cellular components of the blood (red cells, white cells and platelets) to
        the bottom of the tube with the cell-free serum or plasma to be tested remaining above the cells.
        The liquid portion of whole blood is called Serum when no anticoagulant is in the tube. When an
        anticoagulant like EDTA (lavender top), Heparin (green top), and Citrate (blue top) has been added
        to the tube, the liquid supernate is termed Plasma. Serum or plasma that remains in contact with cells
        for a long period of time will cause changes in many test results.
    b. Serum collection tubes (red and SST) must be gently inverted 7-8 times to activate the clotting process
        and be allowed to clot for a minimum of 30 minutes before spinning. Serum tubes should be allowed to
        clot in an upright position in a test tube rack. A simple test to determine if a tube has clotted is to invert
        the tube. The sample will remain at the bottom of the tube in a completely clotted tube. The tube will
        be sufficiently clotted when a large fibrin clot can be seen when gently rocking the tube back and forth.
    c. The centrifuge must be completely balanced prior to use. See procedure PHL2010.05 for specific details
        on centrifuge operation.
    d. After spinning, serum separator tubes (SST) should be visually inspected to ensure there is a complete         gel barrier in-between the cells and serum. A completely spun down SST when inverted should show
        now red cells streaming past the gel barrier. Red cells having prolonged contact with serum can affect
        chemistry results, for example glucose levels will be lower and potassium levels will increase.
    e. When spinning blue top tubes for special coagulation test, the brake should not be used.
5. Platelet Poor Plasma Specimens
    a. When platelet poor plasma is the required specimen, blue top tubes for special coagulation tests,
        the brake should not be used.
    b. White cells and platelets are lighter than red cells so they form a whitish layer (buffy coat) on top of the
        red cells. When a centrifuge brake is used the very light platelets tend to re-suspend into the citrated
        plasma. These platelets will adversely affect the special coagulation test such as PTT, Factor VIII,
        Mixing Studies, Lupus Anticoagulant, Von Willebrand, etc. These special coag test require platelet
        poor plasma (platelet count <10 K/uL), which is why the plasma is removed and centrifuged a
        second time.
    c. Step by step process:
            i.Centrifuge capped citrate tube for 10 minutes.
            ii.Using a plastic transfer pipet, remove the top ¾ plasma. Transfer this plasma to a properly labeled
                pour off tube with cap.
            iii.Centrifuge the plasma (in the pour off tube) for another 10 minutes.
            iv.Using a plastic transfer pipet, transfer the top ¾ of the spun plasma into a properly labeled pour
                off tube (at least ½ of plasma per aliquot). And freeze immediately. Do not disturb the plasma in the

                bottom of the spun tube, where any residual platelets will be.
            v.Aliquots with visible red cells or hemolysis (pink plasma) are not acceptable.
6. Ambient specimens must remain at room temperature and should be bagged together for
    specimen integrity.
7. Effects of light.
    a. Some analytes deteriorate when exposed to light.
    b. These specimens can be shielded from light during handling, storage, and transport by wrapping
        the specimen in aluminum foil or encasing in a light tight transport container.
8. Temperature Verification
    a. Storage temperatures for refrigerators and freezers must be documented daily to ensure integrity.
        See Temperature Dependant Equipment PHL2010.28
9. Add on test
    b. The requesting physician’s office or IOP should call the clinical lab directly to request add on test when
        a sample has already been transported to the lab.
    c. If the phlebotomist is asked to add on a test per the physicians request this must be documented in the
        daily requisition manifest printed from outreach.


IV. QUALITY CONTROL

N/A

V. CALCULATIONS/CALIBRATION

N/A

VI. INTERPRETATIONS

N/A

VII. METHOD PERFORMANCE SPECIFICATIONS

N/A

VIII. REFERENCES

Collection, Transport, and Processing of Blood For Testing Plasma – Based Coagulation Assay;
Approved guideline – 4th edition, Vol. 23, H21 – A4, NCCLS, Wayne PA. 2003

IX. RELATED DOCUMENTS

N/A

X. DOCUMENT HISTORY

X - MINOR REVISION
(Laboratory Director’s Signature on Original Subsequent Document Attached)

MAJOR REVISION
(Requires Laboratory Director & Department Director Signature - where applicable)

Reason for Change

2/10/14 New Document Control Format

06/23/14 Moved to Specimen Collection Manual



Laboratory Director

(Signature)

Date

Natalie Depcik-Smith, MD
(Print)



Department Director

(Signature)

Date

Robert M. Gay, M.D.
(Print)



Designee

Date

(Print)

(Signature)

(Print)

(Signature)

(Print)

(Signature)

(Print)

(Signature)

(Print)

(Signature)



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