Clinical Biochemistry 50 (2017) 1317–1322

Contents lists available at ScienceDirect

Clinical Biochemistry
journal homepage: www.elsevier.com/locate/clinbiochem

Review

Causes, consequences and management of sample hemolysis in the clinical T

laboratory
Laura Heiremana,⁎, Pieter Van Geelb, Lorenz Musgera, Evelien Heylena, Wim Uyttenbroecka,
Boris Mahieua
a
Department of Laboratory Medicine, Ziekenhuis Netwerk Antwerpen, Antwerp, Belgium
b
Department of Orthopedic Surgery, Ziekenhuis Netwerk Antwerpen, Antwerp, Belgium

A R T I C L E I N F O A B S T R A C T

Keywords: Preanalytical hemolysis of blood samples is a common problem in medical practice, especially in emergency
Preanalytical hemolysis departments. Several potential influences on sample hemolysis have been investigated, including sampling
Causes of hemolysis techniques, centrifugation and sample transport. In particular, the use of intravenous catheters and the vacuum
Affected laboratory parameters sampling technique have often been demonstrated to provoke hemolysis. Other factors playing a role include the
Management of hemolysis
use of inappropriate puncture sites, complicated blood sampling, prolonged tourniquet application, underfilling
of tubes and excessive shaking of specimens. Training of phlebotomists can play a pivotal role in overcoming
these issues. A sample may also undergo hemolysis at the point of centrifugation, more specifically when cen-
trifugation lasts too long or is done repeatedly. Pneumatic tube system (PTS)-transported samples tend to be
more strongly affected by hemolysis compared to hand-carried ones, though whether this difference is clinically
relevant remains questionable. The velocity at which the sample moves, the distance it covers and the shock
forces it sustains all determine to what extent hemolysis occurs during PTS transport. The use of cushion inserts
in the carrier to stabilize the samples and the presence of a gel separator in the transported serum tubes may
prevent PTS-induced hemolysis. Finally, there is considerable variation between patients in the extent to which
samples are prone to hemolysis. Sample hemolysis leads to unreliable laboratory results, delayed diagnosis and
patients suffering avoidable discomfort. Specifically, hemolysis may interfere with laboratory results due to
release of intracellular components, dilution effects, proteolysis and interference with analytical techniques.
There is ongoing debate about how laboratories should deal with results altered by hemolysis. Laboratory
specialists should clearly communicate with the ordering clinicians in order to make an informed decision about
how to interpret hemolysis-affected analytical results. This review looks into current evidence concerning the
causes and consequences of in vitro hemolysis, and aims to explain how to deal with it.

1. Introduction of 12 h must be respected, any exercise 72 h before blood sampling

The preanalytical phase is the phase of the total analytical process in
laboratory medicine that includes all the procedures before the start of
sample analysis and involves different healthcare professionals [1].
Preanalytical processes are responsible for most medical errors that
occur in laboratory diagnostics, particularly when they involve a lot of
manual handling or when they are poorly standardized [2,3]. Blood
collection and handling are important sources of variability in labora-
tory results [1]. These variations may be related to patient preparation,
time of tourniquet application, order of blood draw, mixing of blood
tubes and labelling of primary blood tubes [1]. First of all, a fasting time
must be avoided and a minimum of 15–20 min of resting must be ob-
served since food intake, physical exercise and postural change during
blood collection are major sources of bias in routine clinical chemistry,
hematology and coagulation testing [1,4]. Applying tourniquets for
longer than 60 s causes prolonged venous stasis which may influence
laboratory results and should therefore be avoided [4]. The order of
draw as recommended by EFLM (blood culture, citrate, serum, heparin,
EDTA, glycolysis inhibitors) should be respected since contamination of
anticoagulants or additives from one collection tube to another during
blood sampling may introduce errors [2]. Though it is often re-
commended to gently mix blood tubes by inverting them 5–10 times

Abbreviations: PTS, pneumatic tube system; HI, hemolysis index; Hb, hemoglobin; K, potassium; LDH, lactate dehydrogenase; AST, aspartate aminotransferase; NSE, neuron-specific
enolase; ED, emergency department; LIS, laboratory information system
⁎
Corresponding author at: Department of Laboratory Medicine, Ziekenhuis Netwerk Antwerpen, Lindendreef 1, 2020 Antwerp, Belgium.
E-mail address: heireman.laura@gmail.com (L. Heireman).

http://dx.doi.org/10.1016/j.clinbiochem.2017.09.013
Received 2 July 2017; Received in revised form 13 September 2017; Accepted 18 September 2017
Available online 22 September 2017
0009-9120/ © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
L. Heireman et al.
after filling, this does not apply for every type of additive-containing
tube [1,4]. Mislabelling of blood tubes, a frequent cause of erroneous
patient identification, can be avoided by applying them at bedside [1].
Other issues arising from inappropriate sample collection or handling
include insufficient sample volume, inappropriate clotting, con-
tamination from infusion routes and, importantly, sample hemolysis
[2]. The latter accounts for up to 40–70% of all unsuitable specimens,
making them the main reason for sample rejection [2,5].
Hemolysis is defined as the rupture of erythrocytes resulting in the
release of its intracellular components in plasma or serum [6]. Hemo-
lysis may occur either in vitro or in vivo, with in vivo hemolysis ac-
counting for < 2% of cases [5]. This type of hemolysis can be dis-
tinguished from in vitro hemolysis by the decrease in haptoglobin
concentration, increase in unconjugated bilirubin concentration and
reticulocyte count [7,8]. Haptoglobin tends to bind Hb intravascularly
and allows effective clearance by the reticuloendothelial system, pre-
venting oxidative damage [7,8]. In vitro hemolysis results from in-
adequate specimen collection or handling and poses challenging pro-
blems in hospitals [5]. Only after centrifugation sample hemolysis can
be recognized [5,9]. Macroscopic evaluation is an unreliable measure of
sample hemolysis resulting in incorrect estimates of its prevalence
[10,11]. Also, macroscopic evaluation tends to vary with different
sample types [10,11]. Modern chemistry analysers are equipped with
spectrophotometric systems that automatically measure the level of
hemolysis, expressed as the so-called hemolysis index (HI) [5]. A HI of
500 corresponds to a serum hemoglobin (Hb) concentration of ap-
proximately 500 mg/dL [5]. The advantages of spectrophotometry over
visual inspection include increased reproducibility, better sensitivity
and improved reliability [12]. Objective and standardized systems for
detection of unsuitable specimens need to be adopted by each labora-
tory [11].
This (non-systematic) review looks into current evidence concerning
the causes and consequences of in vitro hemolysis, and aims to explain
how to deal with it.
2. Factors associated with increased sample hemolysis
2.1. Blood collection as a source of hemolysis
Preanalytical hemolysis often occurs during blood sampling with
intravenous catheters being particularly notorious for inducing red cell
damage. Indeed, catheters are associated with significantly more he-
molysis than the standard venipuncture using a straight needle
[12–19]. Partial obstruction of intravenous catheters in particular may
promote hemolysis of blood samples [20]. Straszewski et al. also de-
monstrated a significant reduction in the frequency of hemolysis when
using 21 gauge butterfly needles instead of 21 gauge intravenous ca-
theters [21]. Wollowitz et al. too reported a significant reduction in
hemolysis rate using butterfly needles (21 or 23 gauge) rather than
intravenous catheters (16, 18, 20 or 22 gauge) [13]. A study of Lippi
et al. failed to show a significant difference in rate of sample hemolysis
between the use of 21-gauge straight needles and 21-gauge butterfly
needles [22]. They stated that using butterfly needles provides a reli-
able alternative to the standard needle [22]. However, in another study
of Lippi et al. the use of small butterfly needles, especially those > 23
gauge, was associated with the high prevalence of sample hemolysis in
the pediatric department [18].
Lippi et al. also investigated the frequency of in vitro hemolysis in
samples obtained via intravenous catheters by manual aspiration com-
pared to standard vacuum tubes [23]. The mean levels of K, LDH and
free Hb were significantly lower when the sample was aspirated
manually as compared to the vacuum technique [23]. A subsequent
study demonstrated that the introduction of Sarstedt S-monovette®
blood tubes reduced the rate of hemolysis in the emergency department
(ED) compared to the previously used BD Vacutainer® SST II Plus plastic
serum tubes [24]. This observation is explained by the difference in
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Clinical Biochemistry 50 (2017) 1317–1322
shear stress between both techniques [23]. S-monovette® tubes are
fitted with a plunger, allowing manual aspiration of blood much like a
classic syringe [24]. The importance of this feature was stressed when
Heiligers-Duckers et al. found stronger sample hemolysis with vacuum
collection compared to manual aspiration from IV catheters [25].
Mrazek et al. demonstrated increasing hemolysis rates when using va-
cuum tubes (8 mL Greiner BioOne Vacuette® lithium heparin gel tube,
5 mL Greiner BioOne Vacuette® low-vacuum tube, 4.5 mL Greiner
BioOne Vacuette® lithium heparin tube without gel and 3 mL BD Va-
cutainer® PST II lithium heparin gel tube) compared to the Sarstedt
aspiration system, except for the 3 mL BD Barricor® tube [26]. Grant
reported that combining intravenous catheters with classic syringes
instead of BD Vacutainer® tubes lowered the extent of hemolysis [17]. A
study of Strubi-Vuillaume et al. showed that using lithium heparin
tubes (BD Vacutainer®) in aspiration mode ameliorated accuracy of LDH
determination compared to using them in vacuum mode [27].
Fernandez et al. found a lower rate of hemolyzed sample rejection
for smaller tubes (3.5 mL and 4 mL) compared to bigger tubes (8 mL,
8.5 mL and 9 mL) for BD Vacutainer® and Greiner BioOne Vacuette®
serum tubes [28]. Giavarina, looking at Greiner BioOne Vacuette® li-
thium heparin collection tubes, also demonstrated a significant reduc-
tion in hemolysis using 2.5 mL instead of 5 mL tube volume [29].
Mrazek et al. found a convincing correlation between the amount of
negative pressure in vacuum tubes and hemolysis levels [26]. They
noticed that hemolysis rates can be lowered to levels comparable to
aspiration systems when low vacuum tubes are used [26]. Lippi et al.
observed no reduction in hemolysis in ED samples using low-volume
tubes [30]. In contrast, an increase in sample hemolysis was seen using
3.5 mL instead of 5.0 mL BD Vacutainer® SST II Plus plastic serum tubes
[30].
Blood tubes that are less than semi-filled are more often associated
with hemolysis than those that are filled more adequately [7,13].
Munnix et al. reported that hemolysis occurred primarily in the first
collected sample and less so in those collected subsequently [12]. Dis-
carding the first tube can be applied in practice in cases of difficult
punctures [12]. However, Heiligers-Duckers et al. showed blood
drawing with discard tubes and low vacuum tubes can potentially re-
duce hemolysis compared to using standard vacuum tubes, but to a
lesser extent than the use of the manual aspiration technique [25].
Regarding the type of blood tube, Ryan et al. found that using BD Va-
cutainer® rapid serum tubes instead of BD Vacutainer® plasma tubes
failed to change the rate of clinically significant alterations in hemolysis
markers [31].
Hemolysis occurs more frequently when blood is drawn from other
locations than the antecubital fossa [13,15,32]. In the antecubital re-
gion, sample collection from the basilic vein is associated with a much
higher amount of hemolyzed samples than median cephalic vein,
median basilic vein, median anterobrachial vein or cephalic vein [1].
Intravenous catheters inserted via the hand are more often associated
with sample hemolysis [12,15,32]. The risk of hemolysis increases with
difficulty in venous access, unsatisfactory attempts, fragile veins and
forcing blood into the collection tube [8,12,13,18,32]. Theoretically,
aspiration of non-vaporized alcohol in the needle during venepuncture
may cause spurious hemolysis [1]. However, Salvagno et al. observed
no significant differences in sample hemolysis between patients in
whom blood was drawn with or without letting the alcoholic disin-
fectant dry [33].
Tightening the tourniquet around the arm for over 1 min during
blood collection is also associated with hemolysis as a result of the long-
lasting constriction of the blood vessels [13,34]. However, the CLSI
H03-A6 document does not caution against unsuitable tourniquet ap-
plication time [35]. Lima-Oliveira therefore proposed changes in this
protocol and recommended to cleanse the venepuncture site before
applying the tourniquet and to remove the tourniquet immediately
when the first tube starts to fill [36]. This resulted in a mean reduction
of 88 s in tourniquet application time [36].
L. Heireman et al.
Several manufacturers recommend that samples be mixed by gently
inverting them 5–10 times [37]. Whether vigorous shaking of blood
tubes after collection influences hemolysis has not been unequivocally
demonstrated. Lippi et al. observed that excessive shaking the blood
tube after collection may induce hemolysis [18]. However, Lima-Oli-
veira et al. observed that the (apparently incorrect) vigorous mixing of
blood tubes does not induce variability in laboratory results (with
gentle mixing as reference) [39]. Parenmark et al. and Lima-Oliveira
et al. studied three types of mixing procedures: instant mixing, mixing
after 5 min of rest and no mixing [37,40]. Parenmark et al. only showed
a small but significant increase in HI and LDH measured in samples that
were immediately mixed through a horizontal mixing tray compared to
no mixing [37]. Lima-Oliveira only noted a significant difference in HI
between mixing and no mixing when mixing occurred after 5 min rest
[40]. Further, in the study of Parenmark et al., activated partial trom-
boplastin time was seriously affected in one of twenty non-mixed tube
with citrate buffer [37]. Lima-Oliveira et al. only observed significant
differences for Na between instant mixing and mixing after 5 min/no
mixing [40]. The authors stated that mixing primary blood samples
after venepuncture is not mandatory for all types of tubes since the
blood turbulence generated by the vacuum pressure inside the tubes
may be sufficient for solubilization, mixing and stabilization of ad-
ditives and blood [37,40].
Standardized training as well as reduction of the number of people
involved in performing venipuncture are claimed to improve sample
quality [41]. Cadamuro et al. showed that sample hemolysis was less
likely when blood was drawn by nursing staff who received specific
training compared to untrained staff [41]. Since the number of un-
trained staff was comparatively small, the amount of people involved in
blood collection seems to play a minor role. Introduction of an educa-
tional program in the ED was able to significantly reduce rates of
sample hemolysis [14]. Changes brought about by the educational in-
tervention included increased use of a syringe rather than Vacutainer®,
increased use of venipuncture instead of catheters for blood sampling,
reduced arterial sampling, increased sample volume and reduced time
interval from sampling to analysis [14]. Only the first two changes were
associated with a significant reduction in hemolysis [14].
Trying to determine whether teaching phlebotomists the procedures
recommended by CLSI/NCCLS might improve the quality of the pro-
cess, Lima-Oliveira et al. found that as a result of this training, un-
necessary rubbing of skin, filling tubes in an incorrect order and mixing
tubes inadequately had all been eliminated [35]. The usefulness of
education has also been demonstrated in a study by Bölenius et al. [42].
An educational intervention program led to significant improvements in
patient identification, patient rest, test request management and test
tube labelling [42]. Giavarina et al. recommended that the training
should include education about sampling devices, sampling techniques,
procedures for patient management and safety information for phle-
botomists [2]. The use of checklists seemed to be effective for reducing
errors in healthcare settings, especially in surgical departments [2].
Hemolysis is observed more often in samples received from the ED
than in those originating from other hospital departments, affecting as
much as 10–30% of ED samples [13]. A possible explanation is the
common use of intravenous catheters for blood collection in the ED
[18,43]. Their use, however, is indispensable in this department since
these catheters carry the advantage of offering optimal patient comfort
as they have to be introduced only once [9,13]. This makes it also a
time-efficient technique [9]. Lippi et al. demonstrated that the ex-
penditure generated by specimen recollection due to spurious hemo-
lysis can be as high as 22.8% of the overall cost of serum sample col-
lection in the ED [44]. Moreover, they showed that up to 99% of the
total cost of sample recollection is attributable to hemolysis in speci-
mens drawn from catheters [44]. They stated that continuous educa-
tion, increased use of straight needle venepuncture and introduction of
blood collection devices especially suited for drawing blood from in-
travenous catheters may overcome this problem [44]. A study by Berg
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Clinical Biochemistry 50 (2017) 1317–1322
et al. demonstrated that phlebotomy techniques in the ED often do not
conform to the manufacturer's recommendations [45]. This may help to
explain the strikingly high number of hemolyzed samples coming from
the ED [45]. It is worth pointing out, however, that this study did not
take into account blood sampling techniques used in other hospital
departments.
2.2. Centrifugation as a source of hemolysis
Long-lasting and excessive centrifugation forces have been shown to
cause in vitro hemolysis [5,12]. In the case of serum tubes, hemolysis
tends to occur when the collected blood is centrifuged before the coa-
gulation process is complete [5,20]. This is particularly troublesome in
case the patient is taking anticoagulant medication [5,20]. In addition,
incomplete formation of the separator barrier and repeated cen-
trifugation of tubes with gel separators may induce sample hemolysis
[5].
2.3. Sample transport as a source of hemolysis
Transport of blood samples from the location of blood collection to
the laboratory may also be associated with hemolysis. Fernandes et al.
reported a significantly lower percentage of troublesome hemolysis
when centrifugation took place at the site of collection rather than in
the laboratory [46]. Exposure of blood samples to extreme tempera-
tures during transport and long transport duration may cause hemolysis
[5,46]. Fernandes et al. observed that transport times of > 2 h led to
sample rejection more frequently compared to transport times < 15
min [46].
Another possible source of in vitro hemolysis related to sample
transport is the pneumatic tube system (PTS). PTSs are widely used in
modern hospitals and allow rapid transport of patient specimens, di-
minishing laboratory turnaround time [47]. PTSs may contribute to
hemolysis due to the kinetic forces they impose on samples with large
acceleration changes (g-forces) being notably problematic [38,48].
Studies that have investigated the influence of PTSs on hemolysis report
conflicting results, possibly due to the different specifications of the
PTSs that were used (configuration, speed, distance, intensity of g-
forces). Pupek et al. compared human courier transport and PTS
transport and showed no impact of PTS transport on sample hemolysis
[49]. Also, Fernandes et al. observed no significant difference in he-
molysis rate of ED serum samples between PTS and human courier
transport [46]. However, Koessler et al. demonstrated significant in-
creases for LDH concentration in serum samples with gel barrier of
healthy volunteers transported via PTS compared to hand-carried
transport [50]. Sodi et al. compared the HI measured in different pa-
tient sample types transported via PTS to hand-carried transport [51].
Hemolysis occurred more frequently in serum gel samples (diameter
16 mm, Sarstedt S-monovettes®) when transported by PTS instead of
courier. This effect was even more pronounced with plain serum tubes,
suggesting that gel contained in serum tubes may offer some protection
against hemolysis [51]. Böckel-Frohnhöfer et al. reported a significantly
higher HI in patient samples contained in lithium heparin tubes with gel
separator than in serum tubes with gel separator, both transported via
PTS [52]. Pasqualetti et al. demonstrated a significantly lower HI of
lithium heparin plasma samples collected from ED patients before
compared to after PTS transport [53]. For serum samples with gel
barrier, the opposite effect was noticed [53]. It thus seems that serum
samples are less susceptible to hemolysis than plasma samples when
transported via PTS and so that blood samples in tubes with clot acti-
vator and without anticoagulant may be more resistant to PTS-induced
hemolysis [53].
Acceleration forces are expressed as multiples of gravitational force
or g-force (9.81 m/s2). Monitoring of g-forces during PTS transport can
be performed using a data logger [54]. Acceleration forces measured
during PTS transport are small compared to those occurring during
L. Heireman et al.
centrifugation, the latter ranging from 1500 to 3000 g [55]. However,
not the high accelerations alone, but more likely large and rapid ac-
celeration changes induce hemolysis [48]. During centrifugation, ac-
celerations and decelerations are highly controlled. Streichert et al.
measured temperature, humidity, pressure and 3-axis accelerations
during PTS transport by sending a mini-data logger along with blood
samples of healthy volunteers [54]. They showed a direct positive
correlation between the area under the vector sum acceleration dis-
tribution with cut-off 2 g and PTS speed [54]. The peak vector sum
accelerations during PTS transport amounted to 15 g for 2.5 m/s, 14 g
for 2 m/s, 13 g for 1.5 m/s and 9 g for hand-carried transport [54]. The
measured area under the curve was 500 arbitrary units for 1.5 m/s,
1000 arbitrary units for 2 m/s and 1500 arbitrary units for 2.5 m/s
[54]. Further, a clearly positive correlation between measured serum K
concentrations, LDH activity, AST activity, and area under the curve
was noted [54]. At a speed of 2.5 m/s, relevant alterations were ob-
served for K, phosphate, AST and LDH concentrations and for the latter
two parameters also at 1.5 m/s compared to hand-carried transport
[54]. Also, Gomez-Rioja et al. showed a linear relation between total
sum of sudden acceleration changes and HI, K, LDH and AST in healthy
volunteers [56]. They also found significant interindividual differences
in correlation of hemolysis parameters (HI, K, LDH and AST) and total
sum of sudden acceleration forces during PTS transport [56]. Kapoula
et al. stated that the statistically significant effect of PTS on K, LDH and
AST may lead to the conclusion that the PTS is associated with a mild
degree of sample haemolysis [57]. They noticed, however, that this
difference may not have a significant clinical effect on laboratory re-
sults [57].
A simpler way to measure acceleration forces are provided by
smartphones equipped with a data logger app (for example Sensor
Kinetics Pro and VibSensor) [48,58]. Smartphones are readily acces-
sible, tend to be small enough to fit in a PTS carrier and have been
shown to generate data with satisfactory reproducibility [48,58]. One
disadvantage, however, is that acceleration forces over 8 g cannot be
registered [48]. Using a smartphone, Mullins et al. were able to de-
monstrate that the number of shock forces > 3 g experienced by sam-
ples during PTS transport showed a positive linear relationship both
with HI and plasma LDH measured in lithium heparin plasma from
healthy volunteers [48]. Blood samples were photographed during PTS
transport, demonstrating turbulence as well as a foamy appearance of
the blood specimens during and after transport [48]. It is hypothesized
that such formation of air bubbles in blood samples may provoke he-
molysis due to the hydrodynamic pressure they exert on red blood cells
[48]. In a subsequent study, Mullins et al. demonstrated that sample
haemolysis occurs during PTS transport as bubbles accumulate [59].
They showed that prevention of bubble formation by removal of sample
air space protects blood from hemolysis during PTS transport [59]. A
combination of high speed and long distance of the PTS may also play a
major role in hemolysis [60]. A study of Tiwari et al. showed that
sending serum samples via PTS at a velocity of 3 m/s significantly in-
creased measured LDH, supernatant Hb and K compared to courier
transport [60]. This difference could not be demonstrated in samples
transported via PTS at a velocity of 2 m/s [60].
It has been suggested that samples be prevented from moving within
the carrier during transport. In one study, packing samples in bubble
wrap before PTS transport allowed for a more accurate measurement of
LDH concentration [27]. Cakirca et al. observed a significant difference
in hemolysis rate between hand-carried transport and PTS only when
no cushion inserts were used [61]. Amukele et al. measured hemolysis
in serum gel separator tubes transported by drones and failed to identify
any impact of the flight on hemolysis [62]. The authors suggest the
foam scaffold used for transport may have prevented the samples from
being subjected to excessive kinetic forces, thus precluding undue he-
molysis [62]. Suchsland et al. evaluated an innovative PTS designed to
transport one sample at a time without the use of cartridges [55]. No
significant differences in K, LDH or AST levels, measured in lithium
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Clinical Biochemistry 50 (2017) 1317–1322
heparin plasma samples, were observed when comparing this in-
novative PTS to hand-carried transport [55]. In addition, innovative
PTS was shown to reduce sample transport time compared to courier
transport [55]. Importantly, defects in PTSs may increase hemolysis
rates [63]. Ellis reported a case of abnormal sample hemolysis caused
by a dislodged tube support that had damaged the so-called O-rings of
the PTS [63]. These are rings that slow down the sample carrier as it
approaches the arrival station, thus softening the landing [63]. Damage
to such O-rings may lead to excessive deceleration of the carriers, in-
creasing hemolysis levels [63]. It is recommendable that every la-
boratory validate its own PTS as the degree of sample hemolysis seems
to depend on the specifications of each single PTS [57].
2.4. Interindividual susceptibility to sample hemolysis
Shear stress, blood composition and materials the blood comes into
contact with may all play a role in damaging red blood cells [64]. Age
and gender may also be determining factors in sample hemolysis. Sö-
derberg et al. reported significantly more hemolyzed samples collected
from men compared to women and in patients above the median age
(63 years) compared to younger patients in health care centres, but not
in the ED or nursing homes [43]. The association between age and
hemolysis may be due to greater difficulties in obtaining venous access
in the elderly [43]. The authors failed to explain the differences in
hemolysis between men and women, however [43]. Contrary to this
finding, Fang et al. observed no significant correlation between gender
or age of patients and the risk of hemolysis [15]. Stark et al. observed a
significantly greater proportion of hemolysis-related specimen rejection
for African Americans compared to Caucasians in the ED [65]. A study
of Kavsak et al. showed that samples obtained from patients with he-
matological/oncological diseases are more susceptible to PTS-related
hemolysis than those of patients admitted to the intensive care unit
(56% and 13% respectively) [66].
3. Consequences of hemolysis
Preanalytical hemolysis often necessitates additional blood sam-
pling, thereby prolonging turnaround time [12]. Repeating blood col-
lection results in additional patient discomfort, delayed treatment and
increased health care costs [6,12]. Laboratory results of hemolyzed
samples that do get accepted for analysis may be incorrect, leading to
incorrect clinical decision-making [6].
Any substance with a plasma or serum concentration > 10 times as
low as in red blood cells are particularly susceptible to increase in case
of hemolysis [7]. Accordingly, plasma levels of potassium (K), lactate
dehydrogenase (LDH), aspartate aminotransferase (AST), alanine ami-
notransferase, magnesium, phosphate, folate and urea concentrations
may be falsely increased [5,8]. Variations in K results are particularly
important since they may incorrectly suggest a life-threatening situa-
tion and so lead physicians to make inappropriate treatment changes
[53,67]. Red blood cells also contain high amounts of neuron-specific
enolase (NSE), resulting in falsely elevated NSE concentrations mea-
sured in hemolyzed serum or cerebrospinal fluid and complicating the
diagnosis of small-cell lung cancer and neuroblastoma [68]. Levels of
blood components with a mainly extracellular localisation may be fal-
sely decreased in case of sample hemolysis as a result of plasma or
serum dilution due to release of intraerythrocytic fluid [5,8]. This is the
case for glucose, sodium, chloride, bilirubin, gamma-glutamyl-
transferase, alkaline phosphatase and albumin [5,8].
Release of proteolytic enzymes from the red blood cells causes de-
gradation of insulin, glucagon, calcitonin, parathyroid hormone, adre-
nocorticotropic hormone and gastrin [5]. Therefore, the measured
concentration of these hormones may be decreased [5]. Hb released
from red blood cells absorbs visible light at wavelengths of mainly
415 nm, 540 nm and 570 nm, causing interference with spectro-
photometric measurements at these wavelengths [5]. Levels of alkaline
L. Heireman et al.
phosphatase, gamma-glutamyltransferase, and bilirubin may be falsely
decreased when measured spectrophotometrically, whilst lipase and
iron levels may be falsely increased [5,8]. Adenylate kinase, when re-
leased from erythrocytes, causes an increase in measured creatine ki-
nase concentration [5,8]. The release of Hb from red blood cells also
causes a positive interference in the troponin I assay, whilst it induces
false negative results for troponin T along with the release of proteolytic
enzymes [5]. Interference of haptoglobin-Hb complexes and free Hb
with serum protein electrophoresis is possible [5]. Finally, antibodies
used in immunological tests can cross-react with components released
from the red blood cells [5]. The unpredictable response of several
blood components to hemolysis precludes the implementation of usable
correction factors [8].
4. Management of hemolysis
There is ongoing debate about how laboratories ought to deal with
samples affected by hemolysis [11]. In the absence of guidelines and
clinical data, there is substantial heterogeneity in the way laboratories
manage preanalytical altered results [8,69]. One option would be to
repeat sample collection. Another would be to perform sample analysis
anyway, yet urge restraint in clinical interpretation of the values ob-
tained by providing a disclaimer. Finally, results could be adjusted post
factum according to the estimated degree of sample hemolysis [11].
Both correction of results and reporting result with a disclaimer might
be useful in making an early diagnosis [11]. However, mathematical
correction of results may introduce bias and lead to inaccurate and
misleading results [8,11]. Disclaimers may not be interpreted properly
or even be overlooked by the clinician [11]. In addition, erroneous
results may be misleading anyway and lead to wrong clinical decision-
making in monitoring or treatment [11,70].
The best option, it seems, is to request another sample, though re-
peating sample collection is not always possible [11]. If a new sample
cannot be obtained, Simundic et al. recommend the laboratory spe-
cialist should communicate with the physician and seek a solution
tailored to the individual patient [11]. In some cases not reporting re-
sults might delay diagnoses that otherwise could have been made using
hemolysis-affected yet indicative laboratory results [70]. This is true in
particular when samples are difficult to obtain or when the patient's
condition is critical [69,70]. Cadamuro et al. advice laboratory spe-
cialists to ask the clinician why sample analysis was requested, whether
the patient is in a critical condition, how feasible it is to obtain a new
sample and how accurate the results need to be for medical decision-
making [70]. This way, a risk assessment is done by weighing the
probability of inducing harm by not reporting a result against the
probability of inflicting harm based on reporting a result with above-
normal uncertainty [69]. The authors also suggest the clinician should
be provided with information about the likely influence of hemolysis on
the test results in order to help decide whether to make use of them or
not [70].
A disadvantage is that consulting the ordering physican is rather
time-consuming [70]. Therefore, Cadamuro et al. came up with a pro-
posal that is easy to implement and highly automatable in every la-
boratory information system (LIS) capable of defining algorithms,
thereby saving all human resources [70]. They suggest mentioning the
result only in a separated comment box [70]. In this comment section,
additional information should be provided on the possible deviation of
the laboratory result [70]. This way, the clinician is forced to read the
information necessary for interpreting the laboratory report [70]. The
authors defined an algorithm in their LIS by which these comments are
generated automatically based on the HI [70].
5. Conclusion
Proper training and knowledge of the factors that may induce he-
molysis as well as implementation of standardized collection
1321
Clinical Biochemistry 50 (2017) 1317–1322
procedures are essential to minimize hemolysis rates and improve the
reliability of laboratory results. Decisions regarding whether and how
to report results should be tailored to the specific situation the patient is
in. It is therefore mandatory that laboratory specialists communicate
clearly with clinicians who order the tests in order for them to make an
informed decision about how to interpret hemolysis-affected analytical
results.
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