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newSchoolWorks.com › VoTechWorks › Health & Personal Services › Medical Assisting › Phlebotomy: Venipuncture, Capillary Collection, and Specimen Handling › Lesson 9
HLT-MEDI38-03 · Lesson 9 · 13 clock hours · lab

Specimen Processing: Centrifugation, Aliquoting, and Preservation

Objective. Students correctly process a blood specimen through centrifugation, identify serum versus plasma, aliquot into labeled secondary tubes, and apply correct preservation or refrigeration based on the test ordered.

⚠ Pending professional review

This lesson, its worked examples and its practice questions has not yet been verified by a licensed clinician in the field or by a certification or approval body. It is a study draft and may contain errors. Do not use it to make decisions about any patient or medication. Always follow your instructor, your supervising professional, and current official references and regulations.

VoTechWorks program approval and accreditation are still in progress. Credentials are issued only by the certifying bodies named in each program, after their own exams.

Why Specimen Processing Matters

Drawing blood correctly is only the first step. What happens to a tube after it leaves the patient's arm determines whether the laboratory receives a usable specimen or one that must be recollected. Improper centrifugation, mislabeled aliquots, or wrong storage conditions can cause patient harm through delayed or incorrect results. As a medical assistant, you are responsible for every step that occurs between collection and transport.

Centrifugation Basics

A centrifuge spins tubes at high speed to separate blood components by density. The heavier formed elements (red cells, white cells, platelets) settle to the bottom; the lighter liquid portion rises to the top.

Serum is the liquid layer above the clot in a tube that contains no anticoagulant, such as a serum separator tube (SST). Serum lacks clotting factors because they were consumed during clotting.

Plasma is the liquid layer above the cell pellet in a tube that contains an anticoagulant (EDTA, heparin, or citrate). Plasma retains clotting factors.

Knowing which you are working with matters because some tests require serum and others require plasma; substituting one for the other invalidates the result.

Balancing the Centrifuge

An unbalanced centrifuge vibrates excessively, can walk off the bench, damages the instrument, and may break tubes — creating a bloodborne pathogen hazard. Always place tubes directly opposite one another. Tubes must be matched by volume, not just by count. If you have an odd number of tubes, balance with a water-filled tube of the same type and volume. Never run a centrifuge unless every position opposite a sample is occupied by a counterweight of equal mass.

Operating the Centrifuge

1. Confirm tubes are capped and correctly balanced.

2. Select the speed (RPM) and time specified by your laboratory's procedure manual — SSTs typically require a specific spin time after the clot has fully formed (allow the manufacturer-recommended clot time before spinning).

3. Close and latch the lid before starting.

4. Do not open the lid until the rotor has come to a complete stop — never brake manually.

5. After the run, inspect tubes for hemolysis (pink or red color in the supernatant) and note any cracks or breakage before removing.

Reading the Tube After Centrifugation

An SST contains a gel separator that migrates between the clot and the serum layer during centrifugation, creating a physical barrier. When you look at a spun SST:

Measure the volume of the supernatant layer visually or with a transfer pipette. A normal 5 mL SST typically yields approximately 2–2.5 mL of serum, though yield varies with hematocrit.

Aliquoting

Aliquoting means transferring a measured portion of the supernatant into a smaller, labeled secondary tube so multiple tests can run from one draw, or so a portion can be sent to a reference laboratory.

Steps for safe aliquoting:

1. Put on gloves (and eye protection if splashing is possible).

2. Confirm the primary tube identity matches the requisition before touching anything.

3. Use a clean transfer pipette for each aliquot to prevent cross-contamination.

4. Draw up the specified volume gently — avoid disturbing the cell pellet or gel layer.

5. Dispense into the pre-labeled secondary microtube without introducing bubbles.

6. Cap immediately and verify the label again.

7. Discard the pipette into a sharps container or biohazard waste per your facility protocol.

Labeling Secondary Aliquots

Every aliquot tube must carry five label fields at minimum:

1. Patient full name

2. Date of birth (or second patient identifier per facility policy)

3. Collection date and time

4. Specimen type (serum, plasma — and anticoagulant if plasma)

5. Test or aliquot number

A missing or incorrect field is a critical error. The competency check for this lesson requires all five fields to be correct.

Specimen Preservation and Storage

Different analytes degrade at different rates and under different conditions. Always consult the laboratory directory or procedure manual for the specific test. Common principles:

| Condition | Examples |

|---|---|

| Room temperature, immediate transport | Blood cultures (never refrigerate — keeps organisms viable for detection in the bottle, transport promptly) |

| Refrigerate (2–8 °C) | Potassium, lipid panel, urinalysis (chemical), urine culture (if not immediately plated) |

| Freeze (−20 °C or lower) | Cortisol and many hormone assays when delayed; check lab manual |

| Protect from light | Bilirubin, some vitamin assays |

| Immediate transport / no delay | Ammonia (unstable — place on ice slurry and transport within minutes); CBC should be analyzed promptly or refrigerated briefly |

Ammonia is among the most time-sensitive routine tests; even short delays at room temperature cause falsely elevated results. Blood cultures must be kept at or near body temperature until incubated — do not refrigerate them.

Documentation

Complete the specimen processing log immediately after each action, not from memory at the end of the shift. Record: patient ID, tube type, spin time and speed, time of aliquot, volume transferred, storage condition applied, and your initials. Accurate documentation protects the patient and protects you.

Safety Reminders

Key terms8

SerumThe liquid portion of blood remaining after clotting has occurred and the clot has been removed; it does not contain clotting factors.
PlasmaThe liquid portion of blood in a tube containing anticoagulant; it retains clotting factors and other proteins removed during clot formation.
CentrifugationThe process of spinning tubes at controlled speed and time to separate blood components by density.
AliquotA measured portion of a specimen transferred from the primary collection tube into a secondary container for testing or storage.
Gel separatorA thixotropic gel present in SST tubes that migrates to form a physical barrier between the cell pellet and the serum layer during centrifugation.
HemolysisThe rupture of red blood cells releasing hemoglobin into the specimen, recognized as a pink or red tinge in the supernatant; can interfere with many laboratory tests.
CounterbalanceA tube of equal volume and mass placed directly opposite a sample tube in the centrifuge rotor to ensure balanced spinning.
Chain of custody / specimen integrityThe documented, unbroken record of a specimen's handling from collection through result reporting, essential for accuracy and legal defensibility.

Worked examples4

1. You need to spin five SST tubes (each 5 mL) in a six-position rotor. How do you arrange them to achieve balance?
  1. Note that five tubes in a six-position rotor leaves one empty position — you cannot simply place them sequentially because the rotor will be unbalanced.
  2. Fill a sixth tube with 5 mL of water and cap it to serve as a counterweight.
  3. Place tubes in positions so that each tube sits directly opposite an equal-mass tube: positions 1 & 4, 2 & 5, 3 & 6 (on a standard six-position rotor numbered around the ring).
  4. Verify by looking across each diameter — every tube has an equal counterpart directly across from it.
Answer: Use a water-filled tube of the same size (5 mL) as the sixth position, then arrange all six tubes symmetrically so each is directly opposite its counterpart.
2. You have a spun SST tube and need to transfer 1.0 mL of serum into a labeled microtube. Using a graduated transfer pipette marked in 0.5 mL increments, describe the measurement.
  1. Hold the pipette vertically and squeeze the bulb before inserting the tip into the serum layer — keep the tip above the gel plug.
  2. Release the bulb slowly to draw serum up to the 1.0 mL mark on the pipette.
  3. Confirm the meniscus bottom sits at the 1.0 mL line — neither above nor below.
  4. Dispense gently into the microtube by slowly squeezing the bulb; read the delivered volume as 1.0 mL.
Answer: Draw serum to exactly the 1.0 mL graduation mark and dispense fully — the aliquot volume is 1.0 mL.
3. A 4-position rotor holds tubes of varying volumes: Position 1 = 10 mL tube, Position 3 (opposite) = 10 mL tube. Position 2 = 3 mL tube. What must go in Position 4?
  1. In a four-position rotor the positions opposite each other are: 1 & 3, and 2 & 4.
  2. Position 1 (10 mL) is balanced by Position 3 (10 mL) — correct.
  3. Position 2 holds a 3 mL tube, so Position 4 must hold an equal counterweight of 3 mL.
  4. Place a 3 mL tube (sample or water-filled) in Position 4.
Answer: A 3 mL tube (or water-filled tube of the same volume) must be placed in Position 4 to balance the 3 mL tube in Position 2.
4. A spun 4 mL EDTA tube yields a plasma layer measuring approximately 2 mL. The requisition asks for two aliquots: one of 0.5 mL and one of 1.0 mL. Is there sufficient plasma, and how much remains after aliquoting?
  1. Total plasma available: 2.0 mL.
  2. Volume needed: 0.5 mL + 1.0 mL = 1.5 mL.
  3. Remaining after aliquoting: 2.0 mL − 1.5 mL = 0.5 mL.
  4. 0.5 mL remains in the primary tube; both aliquots can be filled as ordered.
Answer: Yes, there is sufficient plasma. After transferring both aliquots (1.5 mL total), approximately 0.5 mL remains in the primary tube.
In class: Load a balanced centrifuge with six tubes (including one intentionally unbalanced setup for demonstration); instructor shows the effect of imbalance on the instrument and students practice calculating and arranging balanced loads for all common tube volumes. · After centrifugation of an SST tube, students identify and measure the serum layer, use a transfer pipette to aliquot a specified volume into a labeled secondary microtube, and document the process on a specimen processing log. · Complete a specimen preservation matching exercise: given eight laboratory test orders (potassium, cortisol, ammonia, urinalysis, CBC, lipid panel, blood culture, urine culture), students select storage condition (room temperature, refrigerate, freeze, protect from light, immediate transport) and time limit before degradation.
Materials: Centrifuge (tabletop clinical model), evacuated tubes (SST, EDTA, heparin), transfer pipettes, labeled microtube aliquot vials, specimen processing log forms, permanent markers, biohazard bags, cool-pack transport box, phlebotomy textbook chapter on processing
Check: Students complete a full processing sequence (centrifuge, read, aliquot, label, document) on one tube set; instructor reviews the specimen log for accuracy and inspects the aliquot for correct volume and labeling — 100% correct on all five label fields required to pass.

Practice check8

Answer each question, then check it. Nothing is recorded; this is your own practice.

Phlebotomy / Specimen Processing
1. A medical assistant places five 7 mL SST tubes in a six-position centrifuge rotor without adding a counterweight. What is the most likely result?
Clinical Laboratory / Specimen Processing
2. Which of the following best distinguishes serum from plasma?
Phlebotomy / Specimen Integrity
3. A student notices a pink tinge in the serum layer of a freshly spun SST tube. This finding most likely indicates:
Phlebotomy / Specimen Handling
4. An ammonia level is ordered. After collection, the medical assistant should:
Safety / Infection Control
5. Which action should be taken if a tube breaks inside the centrifuge during a run?
Phlebotomy / Specimen Preservation
6. A blood culture bottle has been drawn and the laboratory is not immediately available. The medical assistant should:
Medical Assisting / Administrative and Clinical
7. When labeling a secondary aliquot tube, which combination of fields meets minimum requirements?
Clinical Laboratory / Specimen Preservation
8. A cortisol specimen cannot be analyzed on the same day it is collected. According to general specimen preservation principles, the medical assistant should:
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