Abstract
Introduction:
Hemolysis is still the most common reason for rejecting samples, while
reobtaining a new sample is an important problem. The aim of this study
was to investigate the effects of hemolysis in different hemolysis
levels for mostly used biochemical parameters to prevent unnecessary
rejections.
Materials and methods:
Sixteen healthy volunteers were enrolled in the study. Four hemolysis
levels were constituted according to hemoglobin concentrations and they
were divided into five groups: Group I: 0-0.10 g/L, Group II: 0.10-0.50
g/L, Group III: 0.51-1.00 g/L, Group IV: 1.01-2.50 g/L, Group V:
2.51-4.50 g/L. Lysis was achieved by mechanical trauma.
Results:
Hemolysis interference affected lactate dehydrogenase (LD) and aspartate
aminotransferase (AST) almost at undetectable hemolysis by visual
inspection (plasma hemoglobin < 0.5 g/L). Clinically meaningful
variations of potassium and total bilirubin were observed in moderately
hemolyzed samples (hemoglobin > 1 g/L). Alanine aminotransferase
(ALT), cholesterol, gamma glutamyltransferase (GGT), and inorganic
phosphate (P) concentrations were not interfered up to severely
hemolyzed levels (hemoglobin: 2.5-4.5 g/L). Albumin, alkaline
phosphatase (ALP), amylase, chloride, HDL-cholesterol, creatine kinase
(CK), glucose, magnesium, total protein, triglycerides, unsaturated iron
binding capacity (UIBC) and uric acid differences were statistically
significant, but remained within the CLIA limits.
Conclusion:
To avoid preanalytical visual inspection for hemolysis detection,
improper sample rejection, and/or rerun because of hemolysis, it is
recommended in this study that, routine determination of plasma or serum
free hemoglobin concentrations is important. For the analytes
interfered with hemolysis, new samples have to be requested.
Key words: hemolysis; preanalytical errors; interference; analytes.
Introduction
Hemolysis is the
most common preanalytical source of error in clinical laboratories and
responsible for nearly 60% of rejected samples (1,2). It may occur both in vivo or in vitro. The ratio of in vivo
hemolysis is only 3.2% of all the hemolyzed specimens (3). In vitro
hemolysis occurs more often and it is caused by improper sample drawing,
handling or centrifugation. Especially hardly collected samples, or
stored and/or transported, have increased risks for hemolysis.
Most of the
hemolyzed samples are being rejected on pre-analysis stage according to
the visual detection of serum interferences, even if the requested tests
may not be interfered with hemolysis. Besides, according to the
reports, visual assessment of sample hemolysis showed little agreement
with the actual concentration of hemoglobin interferent (4-7).
Conversely, even
if the hemolysis is not visible, there is also a discharge of the cell
constituents into serum or plasma (8). So invisible hemolysis is an
important cause of false results and has to be detected before the
investigation procedure. Increased number of biochemical tests, number
of samples and increased automation obligated laboratory staff to study
harder, give results faster, so pre-analytical determination of
hemolyzed sample and determination of interfered analytes before
analysis became more important.
Since the
knowledge of possible effects is important for correct interpretation of
the results, the aim of the present work was to evaluate the effects of
hemolysis interferences in different levels on routine commonly used
biochemistry tests. Results of this study may help to prevent
unnecessary rejections of samples.
Materials and methods
The venous blood
samples, collected from 16 healthy volunteers, were taken into 5
different heparinized tubes (Vacuette ®, Greiner Labortechnik, Germany)
to study the effect of in vitro hemolysis. Four samples were drawn
through the needles of 5 mL syringe (1.5 inch, 21 gauge) speedily for 2,
4, 6 and 8 times respectively to lyze the cells by mechanical trauma to
obtain slightly, mildly, moderately and severely hemolyzed samples.
They were all centrifuged at 1000 x g for 15 minutes. This method of
cell lysis was chosen because blood transferring into a tube by pushing
forcely down on the syringe plunger is analogous to the mechanical
disruption of erythrocytes that frequently occurs during blood
collection. In this method there is no standardization way of the force
applied while transferring the blood by syringe. Besides every patients’
fragility of red blood cell is different, so free Hb concentrations of
all samples were not correlated with the force.
After measuring
free Hb concentrations of the hemolyzed samples, the samples were
grouped according to their free Hb concentrations. The exclusion
criteria of a hemolyzed sample was the concentration of its free Hb
concentration discordant with the degree of hemolysis determined for
each group, so the number of samples were not equal at each group.
Free Hb of the samples were measured spectrophotometrically (Shimadzu Corporation; Kyoto, Japan) with Na2CO3
solution (10 mg/100 mL) as a reagent (9). Absorbances were measured at
415, 450 and 700 nm for all hemolyzed and diluted plasma.
Total plasma hemoglobin was calculated according to the formula:
Hb = 154.7 × (A425) – 130.7 × (A450) – 123.9 × (A700) (9).
(Reference ranges: 0-0.1 g/L for plasma free Hb).
Specimens were categorized according to the Hb concentrations into 5 groups:
- Group I (0-0.1 g/L), nonhemolyzed (N = 15);
- Group II (0.10-0.50 g/L) slightly hemolyzed (N = 12);
- Group III (0.51-1.00 g/L) mildly hemolyzed (N = 10);
- Group IV (1.01-2.50 g/L) moderately hemolyzed) (N = 15);
- Group V (2.51-4.5 g/L) severely hemolyzed (N = 12).
The results were
analyzed to determine if slight, mild, moderate or severe hemolysis had a
significant impact on the analyte concentrations.
All analytes were
measured with Olympus analyzer with original reagents (Olympus AU2700
system reagent, Olympus Diagnostica GmbH, Lismeehan, O’Callaghan’s
Mills, Co. Clare, Ireland).
Plasma
concentrations of albumin, alkaline phosphatase (ALP), alanine
aminotransferase (ALT), α-Amylase, aspartate aminotransferase (AST),
total bilirubin, calcium, sodium, potassium, chloride, total cholesterol
(TC), creatine kinase (CK), creatinine, gamma glutamyltransferase
(GGT), glucose, high density lipoprotein cholesterol (HDL-C), iron (Fe),
lactate dehydrogenase (LD), magnesium, inorganic phosphate, total
protein, triglyceride (TG), unsaturated iron binding capacity (UIBC),
urea, uric acid were analyzed in all groups.
The effects of
the hemolysis were evaluated according to the total allowable error
recommendations of Clinical Laboratory Improvement Amendments (CLIA’88)
(10) (Table 1). CLIA’ 88 regulations have established fixed limits for
assessing method and laboratory performance for specific regulated
analytes. In practice, the total allowable error for a given analytical
method must be less than the respective CLIA fixed limits for the
analyte in question.
Statistical analysis
To compare the concentrations of the hemolyzed samples with nonhemolyzed samples, bias percentage was calculated by the formula:
[(CX – C1)/ C1)] x 100.
C1: concentration of nonhemolyzed sample,
CX: concentration of hemolyzed sample.
All analyses were
performed using Statistical Package for Social Sciences statistical
package (SPSS, version 15.0 for Windows XP). Wilcoxon signed-rank test
analysis was used; P values < 0.05 were considered statistically
significant.
Results
As a result of
the mechanical trauma, the median free Hb concentrations for groups I,
II, III, IV and V were measured as 0.02, 0.27, 0.75, 1.27 and 3.34 g/L,
respectively. At free hemoglobin concentration 0.5 g/L, hemolysis was
visible by the red color of the plasma.The results of the investigations
were all presented in Table 1 and shown in Figure 1. Lactate
dehydrogenase appeared to be most sensitive to hemolysis; the increase
of č 1000 U of lactate dehydrogenase per liter resulted in a 4.5-fold
higher enzyme activity at 4.5 g of hemoglobin per liter of plasma than
at 0.27 g/L. A considerable increase of 30 U/L in aspartate
aminotransferase activity was a 2.5 fold increase in this enzyme’s
activity in severely hemolyzed plasma (hemoglobin < 4.5 g/L).
In electrolytes,
potassium values were most affected, in direct proportion to the
increased plasma free hemoglobin concentration, the average increase in
potassium concentration being 1.48 mmol/L for hemoglobin concentrations
up to 4.5 g/L, or about 1.4 fold (Figure 1).
Figure 1. Interferogram for hemoglobin and measured parameters: LD, AST, potassium and total bilirubin.
Table
1 Methods, median values of the analytes and free hemoglobin
concentrations for each group, % bias of analyte concentrations in
comparison to the nonhemolyzed group, and ± acceptable limits of CLIA’
88, ± desirable bias. Results with higher than CLIA limits and
statistically significant differences are marked in bold.
Table 1
summarizes the influence of hemolysis on routine biochemistry testing.
Differences are given as median values and percentage relative bias from
the baseline specimens (no lysis). As expected, LD, AST and potassium
values showed significant increases approximately linearly dependent on
the free Hb concentrations in hemolyzed plasma. There was a decrease
(100%) in total bilirubin concentration (Figure 1).
Values of
inorganic phosphorus were significantly increased linearly with the
increased free hemoglobin concentrations comparing with the baseline
specimens. Clinically meaningful (approximately 1.2 fold) increases in
enzyme activities were observed for GGT and ALT at 4.5 g of hemoglobin
per liter of plasma.
Of the analytes
evaluated, the bias values recorded for albumin, alkaline phosphatase,
amylase, calcium, chloride, HDL-cholesterol, creatine kinase,
creatinine, glucose, iron, magnesium, sodium, total protein,
triglycerides, unsaturated iron binding capacity, urea and uric acid
were lower than the CLIA allowable limits even in specimens containing
up to 4.5 g/L of plasma hemoglobin although for some analytes variations
were statistically significant (P < 0.05) (Table 1).
Although there
was no given acceptable limit for GGT in CLIA’ 88 considerations, we
assumed the limit as 20% like most of the other enzymes. For the
unsaturated iron binding capacity the evaluation was done according to
some studies which was determined as ±10% (11,12).
Discussion
Analytic
hemolysis interference occurs when the constituents of the plasma are at
lower concentrations than the constituents in erithrocytes. The release
of erythrocytic constituents can result in increased values for serum.
Dilution is another possible effect especially for gross hemolysis, and
may result in decreased values. While Hb absorbance peaks occur at č417,
540, and 575 nm and at 415 nm (Soret wave) absorbs light very strongly,
therefore at these wavelengths, spectrophotometric interference occurs
due to an increase in the optical absorbance or a change in the blank
value (8,11-13).
Cell contents at
higher concentrations of potassium, phosphate, AST and LD enter the
surrounding plasma when lysis of erythrocytes occurs (8,14). As expected
in the current study AST, LD, and potassium values showed clinically
meaningful increases like previous investigations (8,11-17,19). So we
can say that even if macroscopically invisible hemolysis occurs, AST and
LD measurements may contribute to high values.
Free hemoglobin
with its pseudo-peroxidase activity interferes in the bilirubin
procedure by inhibiting the diazonium color formation. In our study, we
found statistically low values for all groups as in literature (11) and
biochemical testing has to be done for this assay with a new sample at
hemoglobin concentrations higher than 1 g/L.
For cholesterol,
increase in concentration was significant at 2.5 g/L of Hb levels.
According to a study, the interference may result from Hb decomposition
of hydrogen peroxide. This effect can be compensated for by using an
appropriate sample blank (15). For HDL-C, the interference was not
clinically significant up to 10 g/L of hemoglobin concentration with the
same assay method (20).
Although Hb is
the major source of iron, hemolysis has a very little effect on the
serum iron assay which may be caused by spectral overlap and by a
chemical reaction between hemolysate and reaction components like ALP,
GGT, amylase and iron assays (11,12,21).
We found a slight
decrease in glucose and uric acid. This effect may become from a
premature decomposition of hydrogen peroxide by Hb (15). Dilutional
effect caused by the leakage of intracellular components into the
surrounding fluid especially in severe hemolysis may cause lower values
for glucose, sodium and calcium (14,19). In this study no lysate or any
other solution was added in specimens which may also has a dilutional
effect on the analysis.
Although CK is
not a constituent of erythrocytes, intracellular adenylate kinase has
been attributed to the interference in the CK assay (14,19). Correction
can be done by adding inhibitors such as adenosine monophosphate and
diadenosine pentaphosphate, or by substracting the activity measured in
the absence of creatine phosphate (21).
Nowadays serum
hemolysis index is a popular solution for interference detection
preanalytically. Manufacturers give the list of test-specific serum
indices for hemolysis, lipemia and bilirubin interferences. This can
help laboratory staff to be aware of interference, study or reject the
sample, and add comments to the results. But the standardization problem
of the various analytical systems and different decision thresholds for
various serum indices requires more effort (22).
Conclusion
We conclude that
hemolysis affects plasma concentration of a whole range of biochemical
parameters, whereas the most prominent effect of hemolysis is observed
for AST, LD, potassium and total bilirubin. For other analytes; albumin,
ALP, amylase, chloride, CK, HDL-cholesterol, glucose, magnesium, total
protein, triglycerides, UIBC and uric acid, differences were
statistically significant, but remained within the CLIA limits. We
therefore recommend routine free Hb level determination in serum or
plasma, or any other automated detection of the degree of hemolysis.
Only for those analyses which are affected by the estimated degree of
hemolysis, new samples have to be requested. Although time consuming,
this procedure is highly useful, since it may help to avoid unnecessary
rerun the samples and reduce the costs.
References : http://www.biochemia-medica.com/2011/21/79
1. Bonini P, Plebani M, Ceriotti F, Rubboli F. Errors in laboratory medicine. Clin Chem 2002;48:691-8.
2. Plebani M, Carraro P. Mistakes in a stat laboratory: types and frequency. Clin Chem 1997;43:1348-51.
3.
Carraro P, Servidio P, Plebani M. Haemolyzed specimens: a reason for
rejection or clinical challenge? Clin Chem 2000;46:306-7.
4. Hinckley C. Defining the best quality-control systems by design and inspection. Clin Chem 1997;43:873-9.
5.
Glick M, Ryder K, Jackson S. Graphical comparisons of interferences in
clinical chemistry instrumentation. Clin Chem 1986;32:470-5.
6. Plebani M. Laboratory errors: How to improve pre-and post-analytical phases? Biochem Med 2007;17:5-9.
7.
Simundic AM, Nikolac N, Ivankovic V, Ferenec-Ruzic D, Magdic B,
Kvaternik M, Topic E. Comparison of visual vs.automated detection of
lipemic, icteric and hemolyzed specimens: can we rely on a human eye?
Clin Chem Lab Med 2009;47:1361-5.
8. Thomas L. Haemolysis as influence and interference factor. eJIFCC vol 13 no 4: Available at: http://www.ifcc.org/ejifcc/vol13no4/130401002.htm. Acessed: September 25, 2010.
9.
Fairbanks VF, Klee GG. Biochemical aspects of hematology. In: Burtis
CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry. 3rd ed.
Philadelphia: WB Saunders Company; 1999. p.1673-6.
10. Clinical Laboratory Improvements Amendments of 1988. Final Rule. Laboratory Requirements. Federal Register 1992;57:7002-288.
11. Sonntag O. Haemolysis as an interference factor in clinical chemistry. J Clin Chem Clin Biochem 1986;24:127-39.
12.
Jay DW, Provasek D. Characterization and mathematical correction of
hemolysis interference in selected Hitachi 717 assays. Clin Chem
1993;39:1804-10.
13.
Grafmeyer D, Bondon M, Manchon M, Levillain P. The influence of
bilirubin, haemolysis and turbidity on 20 analytical tests performed on
automatic analysers. Results of an interlaboratory study. Eur J Clin
Chem Clin Biochem 1995;33:31-52.
14.
Lippi G, Salvagno LG, Montagnana M, Brocco G, Guidi GC. Influence of
hemolysis on routine clinical chemistry testing. Clin Chem Lab Med
2006;44:311-6.
15. Yucel D, Dalva C. Effect of in vitro hemolysis on 25 common biochemical tests. Clin Chem 1992;38:575-7.
16.
Scott MG, Heusel JW, Le Grys VA, Siggaard AO. In: Burtis CA, Ashwood
ER, eds. Tietz Textbook of Clinical Chemistry. 4th ed. Philadelphia: WB
Saunders Company; 2006.p.1058-60.
17.
Randall AG, Garcia-Webb P, Beilby JB. Interference by haemolysis,
icterus and lipaemia in assays on the Beckman Synchron CX5 and methods
for correction. Ann Clin Biochem 1990;27:345-52.
18.
Ricos C, Alvarez V, Cava F, Garcia –Lario JV, Hernandez A, Jimenez CV,
et al. Current databases on biologic variation: pros, cons and progress.
Scan J Clin Lab Invest 1999;59:491-500.
19.
Frank JJ, Bermes EW, Bickel MJ, Watkins BF. Effect of in vitro
hemolysis on chemical values for serum. Clin Chem 1978;24: 1966-70.
20.
Nauck M, März W, Wieland H. New immunoseparation-based homogeneous
assay for HDL-cholesterol compared with three homogeneous and two
heterogeneous methods for HDL-cholesterol. Clin Chem 1998;44:1443-51.
21.
Szasz G, Gerhardt W, Gruber W, Bernt E.Creatine kinase in serum:2.
Interference of adenylate kinase with the assay. Clin Chem
1976;2:1806-11.
22.
Simundic AM, Topic E, Nikolac N, Lippi G. Hemolysis detection and
management of hemolysed specimens. Biochemia Medica 2010;20:154-9.
