Hemolyzed Blood Samples and Specimen Rejection
Why a sample turns pink, which results it distorts, how the laboratory detects and grades it, the other reasons specimens get rejected, and the collection and transport steps that stop the redraw.
Hemolysis is the rupture of red blood cells. When it happens in a collection tube, hemoglobin and everything else the cells were holding leaks into the serum or plasma, the sample turns pink or red, and a set of results move away from the patient's true values. A hemolyzed sample is the single most common reason a laboratory rejects a specimen: one review put it at 40% to 70% of all unsuitable specimens, and in a landmark study of laboratory errors 61.9% occurred in the pre-analytical phase, before the sample ever reached an instrument.
Two things are worth separating at the start. Almost all hemolysis seen in the laboratory is in vitro, produced by the draw, the handling or the transport. In vivo hemolysis, where cells are breaking down inside the patient, accounts for only a small minority of hemolyzed samples, around 2% to 3% in the studies that have looked, and carries its own signature. This guide is about the first kind: how it happens, what it does to results, how it is detected, and what stops it.
“My test was hemolyzed.” What that means
It means the sample was damaged, not that you are. Red cells in the tube broke open and changed some of the numbers, so the laboratory could not trust them and will not report them. The tests that are sensitive to hemolysis, potassium above all, have to be repeated on a new sample. Tests that are not sensitive to it are usually reported from the same tube.
A redraw is an inconvenience, not a warning sign. It becomes worth a conversation with a clinician only if careful repeat draws keep hemolyzing, because that pattern, together with other markers, is how in vivo hemolysis is picked up.
How the laboratory knows, and why the eye is not enough
After centrifugation, hemolysis shows as pink to red serum or plasma where straw-colored is expected. The color becomes visible at a free hemoglobin concentration of roughly 0.3 grams per liter. Below that, hemolysis can be present and invisible, and above it, two people looking at the same tube frequently grade it differently. A 2009 comparison of visual and automated detection concluded that the human eye cannot be relied on for the job.
Modern chemistry analyzers therefore measure a hemolysis index from the sample's light absorbance, alongside indices for icterus and lipemia, and compare each against the interference limit the assay manufacturer has published. Above the limit, the result is suppressed or flagged rather than reported. CLSI's guideline on HIL indices describes how those limits are established. Where grading is still done by eye, for example at a collection site deciding whether to redraw before the courier leaves, a printed color reference makes the grades mean the same thing to everyone. The CDC's hemolysis reference palette, for example, runs seven tabs from minimal to 1,000 mg/dL of hemoglobin and marks 100 mg/dL and darker as unfit for its serology testing.
Which results hemolysis changes, and in which direction
Red cells and plasma hold very different concentrations of the same substances. Rupture the cells and the plasma inherits what was inside.
| Analyte | Effect | Why |
|---|---|---|
| Potassium | Falsely high | Red cells hold roughly 23 times the potassium of plasma; even mild hemolysis lifts the result, which is the mechanism behind pseudohyperkalemia. |
| Lactate dehydrogenase (LDH) | Falsely high, markedly | About 160 times more concentrated inside red cells than outside; the most hemolysis-sensitive routine analyte. |
| AST and ALT | Falsely high | AST is roughly 40 times more concentrated in red cells; ALT rises less but still rises. |
| Magnesium, phosphate, iron | Falsely high | All are intracellular-rich and leak with the hemoglobin. |
| Folate | Falsely high | Red cell folate is many times the serum level. |
| Haptoglobin | Falsely low | Free hemoglobin binds haptoglobin; in vitro hemolysis mimics the in vivo pattern on this one analyte. |
| Bilirubin, troponin, some immunoassays | Assay-dependent | Free hemoglobin absorbs light near the wavelengths several methods read, and interferes chemically with others; each manufacturer publishes its own hemolysis limit. |
| Coagulation tests (PT, aPTT) | Unreliable | Hemolysis signals cell damage and activation that can shorten or distort clotting times; most laboratories reject grossly hemolyzed citrate tubes outright. |
The concentration ratios are long-established values (Caraway 1962; Kroll and Elin 1994). Interference limits are assay-specific and published by each manufacturer; the laboratory applies them, not the collector. Mild hemolysis often leaves most of a panel reportable; potassium, LDH and AST are the results voided first.
Where hemolysis is made
Almost always mechanical, and almost always before the laboratory sees the tube. The draw, the tube and the transport each contribute.
Before the needle
- Tourniquet left on longer than one minute, or the patient pumping a fist
- Alcohol not allowed to dry before puncture
- Drawing through an intravenous catheter rather than by straight venipuncture
- A small-gauge needle (25G) on a large-volume draw
During the draw
- Probing for a vein or partial occlusion of the bevel
- Pulling hard on a syringe plunger, or forcing blood from a syringe through a needle into a tube
- Underfilling a vacuum tube, so the remaining vacuum shears cells
- Drawing above an IV site or from a hematoma
After the draw
- Vigorous shaking instead of gentle inversion
- Whole blood chilled to freezing, or left in a hot vehicle
- Delayed separation of serum or plasma from cells
- Pneumatic tube transport or rough courier handling
- Centrifuging too fast, too long or twice
The best-evidenced single cause is the intravenous catheter. A laboratory medicine best practices systematic review of emergency department studies found that drawing by straight-needle venipuncture rather than through an IV catheter reduces hemolysis, and recommended it as a practice; the benchmark that literature works to is a hemolysis rate of 2% or less. Emergency departments, where catheter draws are routine and staff are hurried, run far above it; dedicated phlebotomists run far below it. The difference is technique, not luck.
Transport adds its own share. Whole blood that freezes hemolyzes completely; blood left in a hot vehicle hemolyzes partially; a pneumatic tube system subjects tubes to acceleration and vibration. Meta-analyses consistently find higher LDH in tube-transported samples than in hand-carried ones, most clearly at speeds of 6 m/s and above or runs of 250 m and more, while the effect on potassium and on the overall hemolysis rate is small or inconsistent. None of this is visible until the tube is spun, which is why a transport record with time and temperature is the only way to tell a draw problem from a delivery problem. The order of draw guide covers the tube sequence; the cold chain guide covers the temperature bands.
The other reasons a specimen is rejected
Hemolysis leads the list, but a specimen acceptability policy covers all of these, and most of them are decided before the sample leaves the collection site.
Lipemic
Turbid plasma from triglyceride-rich lipoproteins, visible above roughly 300 mg/dL. Usually a non-fasting sample; sometimes a disorder or lipid infusion. Interferes with light-based methods and displaces plasma water.
Icteric
Yellow to brown plasma from elevated bilirubin. Interferes spectrally and chemically with several assays, and it is a property of the patient rather than the draw, so a redraw does not fix it.
Clotted
Fibrin strands or a clot in an anticoagulant tube, from delayed or inadequate inversion. Blocks hematology analyzers and invalidates coagulation and plasma chemistry results.
Quantity not sufficient
Too little sample for the tests ordered, or a citrate tube filled outside the 9:1 blood-to-additive ratio it needs.
Wrong tube or additive
EDTA in a chemistry request, heparin in a coagulation request, a serum tube where plasma was required. The order of draw exists to prevent the carryover version of this.
Unlabeled, mislabeled or unmatched
A specimen whose identity cannot be confirmed against the requisition with two identifiers. Rejected regardless of quality, because the alternative is a result in the wrong chart.
Out of time or temperature
Transported outside the analyte's stability window or its required temperature band, or received with no record of either.
Expired or damaged collection device
Tubes past their expiration date lose vacuum and additive performance; leaking or cracked containers are rejected on receipt.
Rejection criteria, and the record the laboratory has to keep
Specimen rejection is not a judgment call made tube by tube. Under CLIA, a laboratory must have written policies covering specimen acceptability and rejection (42 CFR 493.1242), must record the condition and disposition of any specimen that fails them (493.1283), and must say so on the report (493.1291). The criteria are the laboratory's own, informed by the assay manufacturers' interference limits and the accreditor's checklist, which is why the same degree of hemolysis can be accepted for one test and rejected for another.
The rates are lower than the attention suggests and higher than anyone wants. A College of American Pathologists Q-Probes study across 453 laboratories found an overall chemistry specimen rejection rate of about 0.35%, with hemolysis the leading reason. At the volume of a busy outreach program that fraction is a recollection every few hundred draws, each one a delayed result, a second appointment or home visit, a second courier leg and a second accession. The cost sits with the program long before it reaches the laboratory. Programs that get ahead of it track the hemolysis rate as a quality indicator, by site, by collector and by collection method, against the 2% benchmark, and feed the numbers back to the people holding the needle.
What stops the redraw
Three places to design hemolysis out: the technique, the kit and the transport. The collector controls the first; the program controls the other two.
At the draw
- Tourniquet on for no more than a minute, released as blood begins to flow
- Straight-needle venipuncture with a 21G or 22G needle where the vein allows; avoid drawing through IV catheters
- Let the alcohol dry; do not probe
- If a syringe is unavoidable, use a transfer device and let the vacuum fill the tube
- Fill to the line, then invert gently the number of times the tube manufacturer states
In the kit
- The right tube, additive and volume for each test, specified once and printed on the requisition
- Fill lines, order-of-draw prompts and inversion counts on the instructions the collector actually reads
- Absorbent, secondary containment and a packout matched to the temperature band, so transport does not undo the draw
In transit and receipt
- Separate serum and plasma from cells within the window the assay requires when the lab is more than a couple of hours away
- Hold whole blood at the band the analyte needs, never frozen
- A tracking record with time and temperature, so a hemolyzed arrival can be traced to a cause instead of a guess
- Pneumatic tube policies that exclude hemolysis-sensitive tests, or validated settings
Specimen quality as a designed outcome
DS2 works on all three places at once. Its mobile phlebotomists draw to the CLSI venipuncture standard, which is where the 2% benchmark is met. Its collection kits put the correct tube, fill line, inversion count and packout in the collector's hands, printed on the requisition and label they actually read. And its specimen transport keeps whole blood inside its temperature band with a time and temperature record, so a hemolyzed arrival can be traced to its cause and the program can fix the cause rather than the symptom.
Hemolysis and specimen rejection questions
What does hemolyzed mean on a blood test?
It means red blood cells in the sample broke open and released hemoglobin and other cell contents into the serum or plasma, turning it pink or red and changing some results. In almost every case this happened in the tube, at the draw or in transit, not in the body. It is a problem with the sample, not a diagnosis, and the usual outcome is a redraw for the affected tests.
If my sample was hemolyzed, is something wrong with me?
Very unlikely. The large majority of hemolyzed samples are hemolyzed in vitro, meaning after the blood left the vein. True in vivo hemolysis, where cells are breaking down inside the body, is uncommon and shows a different laboratory pattern, including low haptoglobin and raised bilirubin, that a clinician will recognize. If repeat samples drawn carefully keep coming back hemolyzed, that is when the question is worth asking.
How can you tell if a blood sample is hemolyzed?
After centrifugation the serum or plasma is pink to red instead of straw-colored. The eye picks this up at a free hemoglobin concentration of roughly 0.3 grams per liter and above, but visual grading is unreliable between observers, so most laboratory analyzers now measure a hemolysis index automatically and apply the interference limit for each test.
What is the hemolysis index?
A semi-quantitative measurement of free hemoglobin in serum or plasma made by the chemistry analyzer from the sample's light absorbance, reported alongside icterus and lipemia indices (the HIL indices). Each assay has a published hemolysis limit; when the index exceeds it, the result is suppressed or flagged. CLSI's guideline on HIL indices describes how the limits are set and used.
Which blood tests are most affected by hemolysis?
Potassium, lactate dehydrogenase (LDH), AST, magnesium, phosphate, iron and folate read falsely high because red cells hold far more of them than plasma does. Haptoglobin reads falsely low. Bilirubin, troponin and several immunoassays are affected to a degree that depends on the method, and grossly hemolyzed coagulation samples are usually rejected outright.
What is pseudohyperkalemia?
An artificially high potassium result that does not reflect the potassium in the patient's blood. Classically it is defined as serum potassium exceeding plasma potassium by more than 0.4 mmol/L in samples kept at room temperature and tested within an hour of collection. Hemolysis is the most common cause; prolonged tourniquet time, fist clenching, very high platelet or white cell counts and delayed separation are others.
How do you prevent hemolysis during a blood draw?
Keep the tourniquet on for under a minute, use straight-needle venipuncture with a 21G or 22G needle rather than drawing through an IV catheter, let the alcohol dry, avoid probing, let the tube's vacuum do the filling, fill to the line and invert gently rather than shaking. After the draw, keep whole blood out of the freezer and the hot car, separate serum or plasma within the required window, and transport with a time and temperature record.
What happens when a laboratory rejects a specimen?
The laboratory records the condition and disposition of the specimen, reports that it did not meet acceptability criteria, and requests a recollection for the affected tests. CLIA requires written acceptability and rejection criteria, records of what was rejected and why, and a report that says so. For the patient it means another draw; for the program it means a delayed result and a cost that is almost always cheaper to prevent than to repeat.
References
- 1.Lippi G, Blanckaert N, Bonini P, et al. Haemolysis: an overview of the leading cause of unsuitable specimens in clinical laboratories. Clin Chem Lab Med. 2008;46(6):764-772.
- 2.Carraro P, Plebani M. Errors in a stat laboratory: types and frequencies 10 years later. Clin Chem. 2007;53(7):1338-1342.
- 3.Heyer NJ, Derzon JH, Winges L, et al. Effectiveness of practices to reduce blood sample hemolysis in EDs: a laboratory medicine best practices systematic review and meta-analysis. Clin Biochem. 2012;45(13-14):1012-1032.
- 4.Carraro P, Servidio G, Plebani M. Hemolyzed specimens: a reason for rejection or a clinical challenge? Clin Chem. 2000;46(2):306-307: in vivo hemolysis accounted for about 3.2% of hemolyzed specimens.
- 5.Caraway WT. Chemical and diagnostic specificity of laboratory tests. Am J Clin Pathol. 1962;37:445-464; Kroll MH, Elin RJ. Interference with clinical laboratory analyses. Clin Chem. 1994;40(11):1996-2005: red cell to plasma concentration ratios for potassium, AST and LDH.
- 6.Simundic AM, Nikolac N, Ivankovic V, et al. Comparison of visual vs. automated detection of lipemic, icteric and hemolyzed specimens: can we rely on a human eye? Clin Chem Lab Med. 2009;47(11):1361-1365.
- 7.Ding Y, et al. Pneumatic tube system transport and blood sample hemolysis: meta-analysis. Scand J Clin Lab Invest. 2021: LDH higher in pneumatic-tube samples, most clearly at 6 m/s and above or 250 m and more; no consistent difference in hemolysis rate or potassium.
- 8.CLSI C56-A, Hemolysis, Icterus, and Lipemia/Turbidity Indices as Indicators of Interference in Clinical Laboratory Analysis.
- 9.CLSI PRE02, Collection of Diagnostic Venous Blood Specimens, 8th edition (formerly GP41): tourniquet time, needle selection, tube filling and mixing.
- 10.Jones BA, Calam RR, Howanitz PJ. Chemistry specimen acceptability: a College of American Pathologists Q-Probes study of 453 laboratories. Arch Pathol Lab Med. 1997;121(1):19-26.
- 11.Nikolac N. Lipemia: causes, interference mechanisms, detection and management. Biochem Med (Zagreb). 2014;24(1):57-67.
- 12.CDC, Reference Tool to Determine Hemolysis Status (hemolysis reference palette): seven color tabs from minimal to 1,000 mg/dL hemoglobin; 100 mg/dL and darker not suitable for serology.
- 13.42 CFR 493.1242(a)(7), 493.1283(a)(3) and 493.1291(c)(7) (CLIA): written specimen acceptability and rejection criteria, records of the condition and disposition of specimens that do not meet them, and reporting of that condition.
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