Prof. Dr. L. Thomas, Kirschbaumweg 8, 60489 Frankfurt, Deutschland
E-mail: th-books@t-online.de

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Introduction

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 neurone-specific enolase.

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 testing.

Haemolysis

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 mg/L.

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 process².

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.

Causes of Haemolysis

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.

In vitro-haemolysis¹

Several factors causing in vitro haemolysis are encountered during the venipuncture procedure (Table 1). Further possibilities for inaccuracies are presented in Table 2. 

In vivo-haemolysis

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 detection

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 mg/L.

Plasma/Serum changes through Haemolysis

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.

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 plasmacytoma⁸.

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 days¹⁰.

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 4.

Haemolysis as an interference factor

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.

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1. Wilke G, Rath W, Schutz E, Armstrong VW, Kuhn W. Haptoglobin as a sensitive marker of haemolysis in HELLP-syndrome. Int J Gynecol Obstet 1992, 39: 29-34

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1. Druml W, Grimm G, Laggner AN, Schneewei� B, Lenz K. Hyperlipaemia in acute haemolysis. Klein Wochenschr 1991; 69: 426-9.

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1. Glick MR, Ryder KW, Jackson SA. Graphical comparisons of interferences in clinical chemistry instru-mentation. Clin Chem 1986, 32:470-5

1. Grafmeyer D, Bondon M, Machon M, Levillain R, The influence of bilirubin, haemolysis and turbidity on 20 analytical tests performed on automatic analyzers, Eur J Clin Chem Clin Biochem 1995; 33:31-52