Prof. Dr. L.
Thomas, Kirschbaumweg 8, 60489 Frankfurt, Deutschland
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Haemolysis is an important interference factor that must be
considered when making laboratory measurements. Its influence
should not be ignored. Because of the automation of many analytical
processes, including increased automation of the pre-analytical
phase, screening test material for haemolysis is often lacking. In
particular samples collected for complete blood testing in
non-laboratory settings have to be transported over longer
distances because of the increasing consolidation of laboratories.
As a result there is an increased risk of haemolysis during storage
and/or transportation. Even if haemolysis is not visually
detectable, a discharge of intracellular constituents into the
plasma/serum can have occurred. If the tests are on patients with
the haemolytic syndrome (in-vivo haemolysis), then differentiating
it from in vitro haemolysis, mostly resulting from inappropriate
specimen collection, is rarely possible. Consequently, analytical
results are often false, fluctuating between either too high or too
low, or giving unexpected pathological findings, for such
measurements as potassium, LDH, AST, acid phosphatase, and/or
The objective of this report is to point out the extent of
haemolytic interferences in laboratory findings, and thereby to
decrease the number of inappropriate specimen collections and
Haemolysis is the release of intracellular
components from erythrocytes, thrombocytes and leukocytes into the
extracellular fluid, i.e. the plasma or serum1.
Haemolysis is visible as a red colouration of the plasma or serum
after centrifugation of the sample. Reports in the literature
on the concentration of free haemoglobin, which is visible as a red
coloration in the plasma or serum, varies between 100 and 300
Haemolysis can lead to changes of a specific
parameter in the test material. It is called a biological influence
factor if the release of the blood-cell constituents took place in
vivo. In vitro haemolysis is an interference factor if it occurs
after specimen collection and changes the results of the analytical
Thrombocytolysis and granulocytolysis can also influence test
results without visual haemolysis3. On examination of
the coagulation process it is evident that thrombocytolysis is
responsible for the higher concentration of a number of
intracellular components in serum compared to the plasma. An
intravascular destruction of leukocytes can lead to increased
lysozyme levels in myeloid and monocytic leukaemia�s.
Visual pre-analytical inspection of
centrifuged blood samples for haemolysis is the reason for
rejection in 60% of the rejected samples. In a medical
study4, 3.3 % of the specimens that were sent to the
laboratory for clinical chemical investigation were haemolysed.
When examining the reason, in only 3.2 % of haemolysed samples was
in vivo haemolysis the cause.
Several factors causing in vitro haemolysis
are encountered during the venipuncture procedure (Table 1).
Further possibilities for inaccuracies are presented in Table
Table 1: Causes of haemolysis during venipuncture
strong aspiration, particularly while puncturing superficial veins.
Aspiration using thin needles should cause less haemolysis than use
of large ones because the flow-rate, flow speed and turbulence is
less, and as a result haemolysis is reported to be
Table 2: Problems encountered after
In-vivo haemolysis is caused by antibodies, biochemically
through medications, by toxic substances, through hereditary
factors (e.g. haemoglobinopathies), through enzyme defects
(acholuric jaundice) or by infections (e.g. malaria). When
suspecting in vivo haemolysis the plasma should be checked to
exclude the possibility of additional in vitro haemolysis1 caused
by the coagulation process.
Haemolysis is visible in non-icteric samples as a red coloration
of serum and plasma. It is visible to the eye if the concentration
of free haemoglobin is above 300 mg/L. Quantitation of free
haemoglobin below this concentration is obtained
immuno-nephelometrically7 or spectrophotometrically
using a bichromatic method (e.g. ACA). The upper reference limit
for free haemoglobin in plasma is 20 mg/L; for serum it is 50
Released components of blood cells, whose intracellular
concentrations are more than 10x higher than their extracellular
concentrations, might increase the plasma/serum concentrations of
these components significantly through in vivo and in vitro
haemolysis. In particular potassium, LD and AST levels become
elevated. Measured components whose levels are higher in serum than
in plasma come from thrombocytes, i.e. potassium, neurone-specific
enolase and acid phosphatase. Indicators of haemolysis are
presented in Table 3.
Table 3: Indicators of haemolysis
unexpected increase in potassium, LD, AST, acid
phosphatase, neurone-specific enolase levels
Differentiation of in
vivo- and in vitro-haemolysis
Even if in vitro haemolysis occurs more
frequent than in vivo, the latter is of greater clinical importance
because of its pathological origin, and because the parameters
influenced by haemolysis are relevant for diagnosis, follow-up and
therapeutic monitoring of the diseases. In cases of suspected in
vivo haemolysis sample rejection is considered malpractice. For
this reason a differentiation (in vivo/in vitro) has to be carried
out on every haemolytic sample.
In vivo released haemoglobin is bound to
haptoglobin and transported into the reticuloendothelial system
(RES), mostly to the spleen. Free haemoglobin can only be measured
in plasma if the haptoglobin transport capacity is exceeded. Then
haptoglobin cannot be measured using immunonephelometric or
immunoturbidimetric methods. Haptoglobin concentrations in the
reference range can be measured if there is a simultaneous
acute-phase response (CRP increased) or in patients with
hypersplenism. Increase in haptoglobin concentration and free
haemoglobin has been reported in patients with
In patients having the HELLP-syndrome the
concentration of haptoglobin in plasma can be normal on diagnosis.
However, it will decrease within the following 10
When suspecting a haemolytic syndrome the
decrease of haptoglobin and the rise in LD, indirect bilirubin and
the reticulocyte index depend on the degree of haemolysis. In
extensive haemolysis, changes in LD, indirect bilirubin and
haptoglobin will occur within 24 hours. However, a rise in the
reticulocyte index occurs 2 days later, at the earliest. An
increase in indirect bilirubin is only measurable if the haemolysis
rate (usually 0,8 %) rises above 5 %. In intravasal haemolysis a
rise in LD (which causes a change in the LD electrophoresis pattern
particularly of LD1and LD2) can only be measured if the
reticulocyte index is above 10 %. In a subject with an LD activity
of 165 U/L and an in vitro haemolysis of 800 mg the serum
haemoglobin caused a 58 % increase in LD activity /10/. Massive
intravasal haemolysis, with a decrease in the haematocrit of
more than 25 % within 12 hours, can cause a hypertri-glyceridaemia.
Reduced triglyceride metabolism is caused by diminished micro
circulation and/or mobilization of free fatty acids, and their
re-esterification to triglycerides11. Criteria to
distinguish in vivo and in vitro haemolysis are shown in Table
Table 4: Different types of haemolysis
parallel increase of haemoglobin (red coloration
of plasma/serum), potassium, LD and AST respectively, but
haptoglobin and reticulocyte-index remains normal,
unforeseen increase in potassium, but no red
coloration of plasma/serum, LD in reference range, i.e. if whole
blood is stored for several days,
parallel increase in haemoglobin (red coloration
of plasma/serum) and LD but no parallel increase in potassium,
no red coloration of plasma/serum, but decrease
in haptoglobin and potential increase in LD, indirect bilirubin
and/or reticulocyte-index, respectively,
serum/plasma without red coloration, but
increase in LD, potassium and acid phosphatase. In plasma no
increase of these parameters (i.e. has been noticed in
Haemolysis as an
Constituents that have been released from blood cells and
that subsequently have been mixed with the extracellular fluid can
change the plasma/serum concentration or activity of certain
components for the following reasons:
- Increased or decreased values because of a concentration
gradient between the cells and the plasma.
- Constituents released from blood cells can interfere with the
chemical reactions used to analyse for plasma/serum components
(e.g. the peroxidase activity of haemoglobin affects bilirubin
measurement and released adenylkinase the measurement of CK).
- Haemoglobin in the sample can interfere with chemical reactions
because it changes the molar extinction coefficient of the
substrate or reaction product to be measured.
Hemoglobin absorbs light very strongly at 415 nm (Soret wave).
Haemolysis therefore increases absorption in this wavelength range
and causes an apparent increase in the concentration of
analytes measured in this range. Interferences of clinical chemical
tests through haemolysis have been pub-lished many times 12 �
14. A list of frequently interfering parameters is presented
in Table 5.
Interferences in parameters and methods
The activity of AST in erythrocytes is 40x higher than in plasma.
In patients with AST activities in the reference range haemolysis
with haemoglobin values of 1.5 g/L causes an elevated AST
False low concentrations are measured using the
Jendrassik-Gr�f-method, because the pseudoperoxidase activity of
haemoglobin inhibits the formation of the azo dye. The inhibition
can be observed if the free haemoglobin concentration in serum is
higher than 0,8 g/L12.
Released erythrocyte adenylkinase increases the enzymatically
measured CK- and CK-MB activities. Adenylatkinase added to the
chemical reaction mixture cannot be inhibited through AMP and
diadenosinpentaphospate. Consequently there is an increase in the
Potentially haemoglobin is a huge source for iron. However, the
additive iron effect is insignificant14, because the
iron-porphyrin binding is stronger than the iron-transferin binding
and methods for the determination of iron only measure iron
released from transferrin.
The additive effect of haemoglobin
on the total protein concentration is small, but significant.
Only high haemoglobin concentrations cause lower serum values. The
uricase-catalase method (Kageyama-reaction) is more susceptible to
interference than the uricase-peroxidase method.
The concentration of potassium in
red blood cells is approximately 25 x higher than in plasma. The
concentration of potassium is increased, even if the in vitro
haemolysis is not visible through red coloration. This can be
noticed if a whole blood sample with low glucose values is stored
several hours at room temperature.
Blood cells have a high phosphate level, but the major part
is organically bound. The addition of
organic phosphate esters to serum can produce a release of
inorganic phosphate that can falsely increased phosphate
concentrations. For this reason serum should be separated from
erythrocytes within 2 hours after specimen collection.
Haemoglobin-haptoglobin-complexes move between the a2- and
�-Globulin fractions. Free haemoglobin migrates as a diffuse
reddish band in the �-globulin fraction.
Immunoassays are evaluated by diagnostic kit manufacturers for
interferences to haemolysis in the same way as other clinical
chemistry tests. However, the manufacturers often only add
haemoglobin (mostly human methemoglobin is used) to samples. When
haemolysis interference factor of an immunoassay haemoglobin
should not be confused with haemolysis. Blood cells contain
components other than haemoglobin that can hamper immunoassays.
Therefore, it is very important to ask the product manufacturer how
haemolysis testing was performed.
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