9.5 Hemolysis & Hemoconcentration
Key Takeaways
Hemolysis is the rupture of red cells, releasing intracellular contents into the plasma or serum.
Potassium, LDH, AST, magnesium, and phosphorus are the analytes most falsely elevated by hemolysis.
Common causes include a needle gauge that is too small, puncturing through wet alcohol, vigorous shaking, and forceful syringe transfer.
Hemoconcentration results from prolonged tourniquet time and falsely elevates protein-bound and cellular analytes.
Visible pink to red serum or plasma is the bedside indicator of hemolysis; the specimen should be recollected.
9.5 Hemolysis & Hemoconcentration
Quick Answer: Pre-analytical errors account for over 70% of all laboratory diagnostic errors. Hemolysis (erythrocyte rupture) is caused by small needle gauges (<23G), forceful syringe aspiration, vigorous tube shaking, drawing from hematomas, and wet alcohol; it causes catastrophic pseudohyperkalemia (potassium is 20–25x higher inside RBCs) and falsely elevates LDH, AST, magnesium, iron, and phosphorus. Hemoconcentration (loss of plasma water into tissues) results from leaving a tourniquet on for >1 minute or vigorous fist pumping, falsely elevating total protein, albumin, calcium, cholesterol, and hematocrit. When collecting from a patient with an active IV line, always use the opposite arm; if unavoidable, draw distal to (below) the IV site after a nurse stops the infusion for at least 2 minutes, apply the tourniquet below the IV, discard a 5 mL discard tube, and document the site on the requisition.
The diagnostic accuracy of clinical chemistry, hematology, and coagulation testing depends directly on pre-analytical specimen integrity. Phlebotomists serve as the primary guardians of specimen quality; collecting compromised specimens leads to erroneous diagnoses, inappropriate medical treatments, and compromised patient care.
Hemolysis: Mechanisms, Causes & Altered Analytes
Hemolysis is defined as the disruption and rupture of red blood cell (erythrocyte) membranes, leading to the release of free hemoglobin, intracellular enzymes, and electrolytes into surrounding serum or plasma. In the laboratory, hemolyzed serum or plasma ranges from light pink (mild hemolysis) to cherry red or dark ruby (gross hemolysis).
EIGHT PRIMARY CAUSES OF IN VITRO HEMOLYSIS
┌────────────────────────────────────────────────────────────────────────┐
│ 1. Needle Gauge Too Small: Using <23G needles (e.g., 25G) generates │
│ intense hydrodynamic shear stress that shears erythrocyte membranes.│
├────────────────────────────────────────────────────────────────────────┤
│ 2. Forceful Syringe Plunger Aspiration: Pulling a syringe plunger too │
│ rapidly creates high negative suction pressure, bursting RBCs. │
├────────────────────────────────────────────────────────────────────────┤
│ 3. Drawing Blood From or Adjacent to a Hematoma: Aspirates already │
│ lysed, clotted, and degraded red blood cells into the tube. │
├────────────────────────────────────────────────────────────────────────┤
│ 4. Vigorous Shaking or Agitation of Tubes: Shaking tubes violently │
│ instead of gentle 180° inversions causes mechanical impact lysis. │
├────────────────────────────────────────────────────────────────────────┤
│ 5. Incomplete Drying of 70% Isopropyl Alcohol: Puncturing wet alcohol │
│ introduces alcohol into the stream, causing chemical RBC lysis. │
├────────────────────────────────────────────────────────────────────────┤
│ 6. Thermal Extremes: Exposing blood to excessive heat (leaving in a hot│
│ vehicle) or freezing un-separated whole blood (ice crystals lyse). │
├────────────────────────────────────────────────────────────────────────┤
│ 7. Centrifuging Incompletely Clotted Serum: Centrifuging before full │
│ clotting (30–60 min) causes fibrin strands to shear red cells. │
├────────────────────────────────────────────────────────────────────────┤
│ 8. Prolonged Contact With Un-Separated Clot: Leaving whole blood at │
│ room temperature for >2 hours allows progressive metabolic leakage. │
└────────────────────────────────────────────────────────────────────────┘
Analytes Falsely Elevated by Hemolysis
Because erythrocytes contain high concentrations of specific intracellular components, cell rupture releases these molecules directly into the specimen, causing severe false elevations:
| Analyte Altered | Normal Intracellular Concentration vs. Plasma | Clinical Consequence of Hemolytic Error |
|---|---|---|
| Potassium () | 20 to 25 times higher inside RBCs (~140 mEq/L vs. ~4.0 mEq/L). | Pseudohyperkalemia: Even mild, imperceptible hemolysis falsely spikes potassium, potentially prompting erroneous diagnoses of hyperkalemic emergency, unnecessary emergency dialysis, or dangerous anti-hyperkalemic treatments. |
| Lactate Dehydrogenase (LDH) | 100 times higher inside RBCs than in normal serum. | Falsely elevates cardiac, liver, and oncological diagnostic markers, mimicking tissue necrosis or myocardial infarction. |
| Aspartate Aminotransferase (AST) | Significantly elevated inside erythrocytes. | Falsely elevates liver function tests and cardiac enzyme panels. |
| Magnesium () | Abundant intracellular divalent cation. | Falsely elevates serum magnesium, masking true hypomagnesemia. |
| Phosphorus / Phosphate | High intracellular organic phosphate concentration. | Falsely elevates serum phosphate readings. |
| Serum Iron () | Iron released from intracellular hemoglobin heme groups. | Falsely elevates serum iron and total iron-binding capacity calculations. |
Analytes Falsely Decreased or Interferred by Hemolysis
- Intact Red Blood Cell (RBC) Count & Hematocrit (Hct): Lysed red blood cells cannot be counted by automated cell counters, producing falsely low RBC counts and falsely depressed hematocrit levels.
- Optical & Photometric Interference (Bilirubin & Troponin): Free hemoglobin strongly absorbs light at wavelengths between 400 nm and 580 nm, optically interfering with spectrophotometric and immunoassay methodologies used to measure Bilirubin, Cardiac Troponin, and other chemistry analytes.
Hemoconcentration: Venous Stasis & Plasma Water Loss
Hemoconcentration is a pre-analytical condition in which the concentration of large, non-diffusible molecules (proteins, enzymes, bound minerals) and cellular elements in circulating blood increases relative to plasma water.
PATHOPHYSIOLOGY OF HEMOCONCENTRATION
┌────────────────────────────────────────────────────────────────────────┐
│ Prolonged Tourniquet Application (> 1 Minute) / Vigorous Fist Pumping │
└───────────────────────────────────┬────────────────────────────────────┘
│
▼
┌────────────────────────────────────────────────────────────────────────┐
│ Localized Venous Stasis & Elevated Intravascular Hydrostatic Pressure │
└───────────────────────────────────┬────────────────────────────────────┘
│
▼
┌────────────────────────────────────────────────────────────────────────┐
│ Plasma Water & Small Filterable Electrolytes Forced Across Capillary │
│ Walls into Interstitial Tissue │
└───────────────────────────────────┬────────────────────────────────────┘
│
▼
┌────────────────────────────────────────────────────────────────────────┐
│ Non-Filterable Large Molecules & Cellular Elements Remain Trapped in │
│ the Intravascular Compartment │
│ (Falsely Elevated: Total Protein, Albumin, Calcium, Lipids, RBCs, Hct) │
└────────────────────────────────────────────────────────────────────────┘
Causes & Affected Analytes
- Causes: Leaving a tourniquet applied for longer than 1 minute (60 seconds), vigorous or repetitive fist pumping, severe patient dehydration, and occluded or sclerosed veins.
- Falsely Elevated Analytes:
- Total Protein & Albumin: Large molecular weight proteins cannot cross intact capillary walls.
- Total Calcium: Approximately 40% to 50% of circulating serum calcium is bound to albumin. When albumin concentrates due to venous stasis, total calcium levels falsely spike (pseudohypercalcemia).
- Cholesterol & Triglycerides: Macromolecular lipid-protein complexes concentrate rapidly.
- Cellular Elements: Red Blood Cells (RBCs), White Blood Cells (WBCs), Platelets, Hemoglobin, and Hematocrit.
- Serum Iron & Enzymes: Large enzymes and protein-bound iron.
- Prevention: Apply the tourniquet for a maximum of 1 minute. Once the vein is successfully accessed and blood begins flowing into the first tube, release the tourniquet immediately.
A clinical laboratory technician flags a chemistry specimen for severe hemolysis. Which of the following pre-analytical errors is the most common cause of in vitro erythrocyte hemolysis during blood collection?
Allowing 70% isopropyl alcohol to air-dry completely for 60 seconds before needle insertion.
Using an excessively small needle gauge, pulling a syringe plunger too forcefully, or vigorously shaking evacuated tubes after collection.
Maintaining the collection tube in an upright vertical position during specimen transport.
Inverting an anticoagulant tube gently 8 to 10 times immediately after filling.
Which serum or plasma electrolyte is most severely and falsely elevated as a result of even mild specimen hemolysis, and what is the underlying physiological reason?
Sodium, because sodium is the primary intracellular cation contained within erythrocytes.
Chloride, because hemoglobin binds chloride ions and releases them during centrifuge spinning.
Potassium, because the intracellular concentration of potassium inside erythrocytes is approximately twenty times higher than in normal plasma.
Bicarbonate, because cellular lysis converts carbonic acid into gaseous carbon dioxide.
A phlebotomist leaves a tourniquet tightly tied to a patient's upper arm for four minutes while palpating for a suitable antecubital vein. Which physiological phenomenon occurs, and how does it alter laboratory test results?
Hemodilution occurs, causing water to enter the vascular compartment and falsely lowering total protein and calcium levels.
Hemolysis occurs, causing rapid destruction of leukocytes and falsely elevating circulating blood glucose.
Petechiae formation occurs, causing all serum enzymes and coagulation factors to precipitate and read as zero.
Hemoconcentration occurs, causing plasma water to filter out of capillaries into interstitial tissue and falsely elevating large molecules such as total protein, albumin, calcium, and cellular counts.
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