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HLT-PHLE88-04 · Lesson 6 · 6 clock hours · lab

Serum and Plasma Separation and Aliquoting

Objective. Students will separate serum or plasma from centrifuged specimens and aliquot measured volumes into labeled transfer tubes using correct technique and without contaminating the sample.

⚠ 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 Separation and Aliquoting Matter

After a blood specimen is centrifuged, the liquid portion must be carefully removed and distributed into smaller tubes called aliquots before it is sent to different laboratory departments. An aliquot is simply a measured portion of the original specimen. If this step is done carelessly—picking up red cell debris, disturbing the buffy coat, or measuring the wrong volume—test results can be falsely elevated or lowered, and the patient may receive an incorrect diagnosis or treatment. Accuracy and clean technique at this stage are as critical as the venipuncture itself.

What Centrifugation Produces

When a tube is centrifuged, the contents separate into distinct layers based on density.

In a gel separator tube (SST or PST), a thixotropic gel migrates during centrifugation and forms a physical barrier between the cells and the liquid layer above, which makes aspiration somewhat easier but does not eliminate the need for careful technique.

Serum comes from tubes with no anticoagulant (red-top or gold/SST tubes). The blood has clotted, so the liquid above the clot is serum, which contains no clotting factors.

Plasma comes from anticoagulated tubes (such as lavender-top EDTA, light-blue citrate, or green-top heparin). Because clotting was prevented, clotting factors remain in the plasma.

Never mix serum and plasma, and never transfer liquid from one tube type into an aliquot tube intended for a different additive type. Cross-contamination invalidates results.

Personal Protective Equipment and Safety

All separation and aliquoting steps are performed in a biological safety cabinet or at a designated processing area. Before touching any centrifuged tube:

1. Put on gloves, lab coat, and eye protection (safety glasses or a face shield).

2. Inspect the tube for cracks before opening. Do not use a cracked tube.

3. Open tubes away from your face. Some labs require an additional splash shield.

4. Never mouth-pipette under any circumstances.

5. Discard tips and consumables into a biohazard waste container immediately after use.

6. Remove and dispose of gloves before leaving the processing area; wash hands.

Aspiration Technique Step by Step

1. Confirm the Tube

Check the tube label: patient name (or training tube ID), tube type, and date/time of collection. Confirm this matches your aliquot log before opening anything.

2. Inspect the Separation

Look at the tube while it is still closed and upright. Identify the clear supernatant layer and the gel barrier or buffy coat below it. If the layers are not fully separated, the specimen may need re-centrifugation.

3. Aspirate the Supernatant

Insert a calibrated disposable transfer pipette into the liquid layer by gently piercing or removing the stopper as directed by your instructor. Draw up the liquid slowly and steadily. Watch the tip as you lower it toward the interface:

4. Dispense into the Labeled Aliquot Tube

Hold the aliquot tube upright and dispense gently down the inside wall—do not splash. Splashing creates aerosols, which are a biohazard. Check the volume against the reference line on the tube or read the calibration markings on the pipette.

5. Cap and Label

Immediately cap the aliquot tube. Verify that the label shows tube type, volume, destination department, technician initials, and time of aliquoting.

6. Document on the Aliquot Log

Record each aliquot on the log form as you go, not afterward. Logging from memory introduces errors.

Volume Accuracy

Many laboratory tests require specific minimum volumes. Sending too little may result in a "quantity not sufficient" (QNS) rejection. Sending too much wastes specimen that could be needed for add-on tests. When using a calibrated disposable pipette, draw up slightly more than the target volume, then express the excess back into the primary tube before dispensing into the aliquot tube. This ensures the dispensed volume is as close to target as possible.

For this course, your target accuracy is within ±0.1 mL of the ordered volume.

Common Errors to Avoid

| Error | Consequence |

|---|---|

| Aspirating into buffy coat | Cell contamination; falsified results |

| Incorrect volume | QNS rejection or wasted specimen |

| Mislabeled aliquot tube | Wrong patient result—critical safety event |

| Incomplete aliquot log | Chain of custody broken; regulatory issue |

| Re-using a pipette tip | Cross-contamination between specimens |

Connecting to Certification

The NHA, AMT, and ASCP Certified Phlebotomy Technician exams all include questions on specimen handling and processing, including centrifugation, separation, and proper labeling. Mastering this skill in the lab ensures you can answer those questions from direct experience, not just memorization.

Key terms9

AliquotA measured portion of a larger specimen transferred into a separate tube for distribution to a specific laboratory department or test.
SerumThe liquid portion of blood remaining after the blood has been allowed to clot and the clot and cells have been removed by centrifugation; it contains no clotting factors.
PlasmaThe liquid portion of anticoagulated blood remaining after cells are removed by centrifugation; it retains clotting factors because clotting was prevented by the tube additive.
Buffy coatThe thin pale layer between the red blood cell layer and the serum/plasma layer after centrifugation, consisting of white blood cells and platelets; must not be aspirated into a serum or plasma aliquot.
Thixotropic gelA synthetic gel used in separator tubes (SST, PST) that migrates during centrifugation to form a physical barrier between the cellular layer and the serum or plasma layer above.
SupernatantThe liquid layer that rises to the top after centrifugation; in phlebotomy processing this refers to the serum or plasma to be aliquoted.
QNS (Quantity Not Sufficient)A specimen rejection code indicating that the volume of sample received by the laboratory is too small to perform the ordered test.
Calibrated transfer pipetteA disposable pipette marked with volume gradations that allow the user to aspirate and dispense a precise, measurable volume of liquid.
AerosolTiny droplets of liquid suspended in air, created by splashing or improper pipetting technique; a biohazard risk in specimen processing.

Worked examples4

1. You need to aliquot 0.5 mL of serum into each of two labeled tubes from a centrifuged SST. The calibrated pipette holds a maximum of 2.0 mL and has markings at every 0.1 mL. How much total serum must you successfully aspirate without contamination, and what is the minimum serum volume the primary tube must contain?
  1. Identify the total volume needed: 2 aliquots × 0.5 mL = 1.0 mL.
  2. Add a small overage to allow precise dispensing and account for any residual in the pipette tip: plan to aspirate approximately 1.1–1.2 mL total.
  3. Confirm the primary tube must have at least 1.0 mL of clear serum above the gel barrier to meet the order; a tube with less should be flagged as potentially QNS before aliquoting.
  4. Aspirate to the 1.1 mL mark on the pipette, express back 0.1 mL into the primary tube, then dispense exactly 0.5 mL into the first aliquot tube, verify against the reference line, then dispense the remaining 0.5 mL into the second aliquot tube.
Answer: You need to aspirate at least 1.0 mL (plus a small overage of ~0.1 mL) from the primary tube. Each aliquot receives 0.5 mL, verified at the reference mark, for a total dispensed volume of 1.0 mL.
2. Your target volume for an aliquot is 1.0 mL. After dispensing, you compare the fill level to the reference mark and estimate you have dispensed 0.85 mL. Is this within the acceptable ±0.1 mL tolerance, and what should you do?
  1. Calculate the deviation: |1.0 mL − 0.85 mL| = 0.15 mL.
  2. Compare to the tolerance limit: 0.15 mL > 0.10 mL, so this is outside the acceptable range.
  3. Do not attempt to add more specimen by returning to the primary tube with the same pipette tip (cross-contamination risk). Instead, notify the instructor.
  4. In a real laboratory, the aliquot would be returned to the primary tube if policy allows, the tube re-mixed gently by inversion (for plasma), and re-aliquoted using a fresh pipette to the correct volume, or flagged for supervisor review.
Answer: 0.85 mL is 0.15 mL below target, which exceeds the ±0.1 mL tolerance. The aliquot does not pass; corrective action is required using a fresh pipette.
3. You are aliquoting three specimens: Specimen A requires 1.0 mL to Chemistry, Specimen B requires 0.5 mL to Hematology, and Specimen C requires 1.5 mL to Immunology. What is the total volume of supernatant you must successfully aspirate across all three specimens?
  1. List each required volume: Specimen A = 1.0 mL, Specimen B = 0.5 mL, Specimen C = 1.5 mL.
  2. Add them: 1.0 + 0.5 + 1.5 = 3.0 mL total.
  3. Remember this is the minimum dispensed volume; aspirated volume will be slightly higher to allow for pipette overage and precise delivery.
  4. Each specimen is handled separately with its own pipette tip to prevent cross-contamination.
Answer: A minimum of 3.0 mL of supernatant must be successfully aspirated and dispensed across the three specimens (plus small overages per pipette to ensure accurate delivery).
4. You are filling out the aliquot log for Specimen B above (0.5 mL to Hematology). You aliquot at 10:47 AM. List every field required on the log and what you would enter for each.
  1. Tube ID / Specimen identifier: enter the label ID exactly as it appears on Specimen B's primary tube.
  2. Tube type: enter the tube additive type (e.g., EDTA lavender-top if this is a plasma specimen going to Hematology).
  3. Volume transferred: 0.5 mL.
  4. Destination department: Hematology.
  5. Time of aliquoting: 10:47 AM.
  6. Technician initials: your initials as trained by your program.
Answer: A complete log entry includes: specimen ID, tube type, volume transferred (0.5 mL), destination department (Hematology), time (10:47 AM), and technician initials. All fields must be filled in at the time of aliquoting, not reconstructed from memory later.

The math in this lesson1

Learners total aliquot volumes and plan overage before pipetting serum.
6.RP.3
In class: Using centrifuged training tubes containing colored water to simulate separated specimens, students practice aspirating the supernatant layer with a transfer pipette, stopping before the buffy coat or gel barrier, and dispensing into a labeled aliquot tube. · Students measure and dispense specific volumes (e.g., 0.5 mL, 1.0 mL) using calibrated transfer pipettes, then verify volume against a reference mark on the aliquot tube; instructor checks for accuracy and technique. · Students complete a three-specimen aliquoting exercise documenting tube type, volume transferred, destination department, and time on an aliquot log; the log is reviewed against a completed example for accuracy.
Materials: Centrifuged training tubes (colored water, gel separator tubes), labeled aliquot tubes, calibrated disposable transfer pipettes, aliquot log form, marker pens, biohazard bags, gloves, lab coat, eye protection
Check: Instructor observes technique: aliquot must not contain visible pellet or buffy coat contamination, volume within ±0.1 mL of target, and log completed fully—all three criteria must be met to pass.

Practice check8

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

Specimen Handling and Processing
1. When aspirating serum from a centrifuged gel separator tube, the phlebotomy technician should stop drawing when the pipette tip is:
Specimen Handling and Processing
2. A phlebotomy student dispenses 0.92 mL into an aliquot tube when the target volume was 1.0 mL. According to the ±0.1 mL accuracy standard used in this course, this aliquot:
Specimen Handling and Processing
3. Which of the following best describes the buffy coat?
Specimen Handling and Processing
4. A laboratory returns a specimen with the notation 'QNS.' This means:
Safety and Compliance
5. Which personal protective equipment is required when performing serum or plasma aliquoting?
Specimen Handling and Processing
6. What is the key difference between serum and plasma relevant to aliquoting?
Safety and Compliance
7. While aliquoting, a student notices the primary tube has a small crack near the base. The correct action is to:
Specimen Handling and Processing
8. A student completes three aliquots and fills in the aliquot log after leaving the processing area, relying on memory. What is the primary risk of this practice?
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