Practices for Identifying and Rejecting Hemolyzed
Specimens Are Highly Variable in Clinical Laboratories
Peter J. Howanitz, MD; Christopher M. Lehman, MD; Bruce A. Jones, MD; Frederick A. Meier, MD; Gary L. Horowitz, MD
Context.—Hemolysis is an important clinical laboratory
quality attribute that influences result reliability.
Objective.—To determine hemolysis identification and
rejection practices occurring in clinical laboratories.
Design.—We used the College of American Pathologists
Survey program to distribute a Q-Probes–type questionnaire about hemolysis practices to Chemistry Survey
participants.
Results.—Of 3495 participants sent the questionnaire,
846 (24%) responded. In 71% of 772 laboratories, the
hemolysis rate was less than 3.0%, whereas in 5%, it was
6.0% or greater. A visual scale, an instrument scale, and
combination of visual and instrument scales were used to
identify hemolysis in 48%, 11%, and 41% of laboratories,
respectively. A picture of the hemolysis level was used as
an aid to technologists’ visual interpretation of hemolysis
levels in 40% of laboratories. In 7.0% of laboratories, all
hemolyzed specimens were rejected; in 4% of laborato-
ries, no hemolyzed specimens were rejected; and in 88%
of laboratories, some specimens were rejected depending
on hemolysis levels. Participants used 69 different terms to
describe hemolysis scales, with 21 terms used in more than
10 laboratories. Slight and moderate were the terms used
most commonly. Of 16 different cutoffs used to reject
hemolyzed specimens, moderate was the most common,
occurring in 30% of laboratories. For whole blood
electrolyte measurements performed in 86 laboratories,
57% did not evaluate the presence of hemolysis, but for
those that did, the most common practice in 21 laboratories (24%) was centrifuging and visually determining the
presence of hemolysis in all specimens.
Conclusions.—Hemolysis practices vary widely. Standard assessment and consistent reporting are the first steps
in reducing interlaboratory variability among results.
(Arch Pathol Lab Med. 2015;139:1014–1019; doi:
10.5858/arpa.2014-0161-CP)
H
interferences or be accurate and consistent to identify and
deal with interfering substances so that clinicians can
provide appropriate treatments without concern for patient
safety. Because hemolysis is a major test interferent and the
most frequent cause of specimen rejection, accurate
laboratory results require that specimens be free of
hemolysis or account for the effects of hemolysis.1–3
Even though laboratorians have tried to improve the
quality of their procedures, in many institutions, blood
collection decentralization from laboratory personnel to
nonlaboratory personnel has resulted in increasing rates of
hemolysis.4,5 In addition, laboratorians have not taken full
advantage of automation to estimate accurately the degree
of hemolysis nor taken appropriate, consistent actions when
communicating the presence of hemolysis to clinicians.
Appropriate, consistent communication in this setting can
have often-overlooked patient safety consequences. Not all
hemolyzed specimens can be attributed to poor specimencollection techniques because more than 50 medical
conditions can cause in vivo hemolysis.6 In vivo hemolysis
can be masked by the occurrence of hemolysis from the use
of improper collecting and processing methods for specimens. In these circumstances, specimens hemolyzed because of a medical condition may be rejected because
hemolysis had been incorrectly ascribed to improper
specimen phlebotomy, without considering whether the
patient could be critically ill from a disease or condition
causing in vivo hemolysis.
emolysis is the disruption of the blood cell membranes,
with the release of the cell contents and membrane
fragments into the surrounding fluid. Unfortunately, in most
clinical laboratories, hemolysis interferes with a variety of
measurements made on analytes occurring in patient
specimens, making it difficult to interpret results correctly.
Clinical laboratory personnel modify patient results with
quantitative estimates of hemolysis as an aid to physicians in
interpreting patient results, but in some situations, such as
whole blood measurements, the occurrence of hemolysis is
not immediately obvious and cannot be estimated.
With increased emphasis on laboratory testing, clinical
laboratory procedures must be either less susceptible to
Accepted for publication November 4, 2014.
From the Department of Pathology, SUNY Downstate Medical
Center, Brooklyn, New York (Dr Howanitz); the Department of
Pathology, University of Utah, Salt Lake City (Dr Lehman); the
Department of Pathology and Laboratory Medicine, Henry Ford
Health System, Detroit, Michigan (Drs Jones and Meier); and the
Department of Pathology, Beth Israel Deaconess Medical Center,
Boston, Massachusetts (Dr Horowitz).
This manuscript was developed as a joint project by members of
the Quality Practices and the Chemistry Resource Committees of the
College of American Pathologists.
The authors have no relevant financial interest in the products or
companies described in this article.
Reprints: Peter J. Howanitz, MD, Department of Pathology, MS25,
SUNY Downstate Medical Center, 450 Clarkson Ave, Brooklyn, NY
11203 (e-mail: [email protected]).
1014 Arch Pathol Lab Med—Vol 139, August 2015
Variable Hemolysis Practices—Howanitz et al
surveys. One reminder notice was sent to nonrespondents
encouraging participants to complete their questionnaires and
return them to the Q-Probes CAP committee staff, who then
tabulated the results according to previously established Q-Probes
procedures.7 The questionnaire contained 28 multiple-choice
questions, with ‘‘other, please list’’ as a choice in 7 questions. If
participants did not answer specific questions, they were excluded
from the data only for the question(s) they did not answer.
Figure 1. Percentage of hemolyzed specimens received by clinical
laboratories (N ¼ 772 laboratories).
In this study, we sought to quantify the frequency of
hemolyzed specimens, to identify the methods for detecting
the presence and amount of hemolysis, to characterize the
current responses of laboratory personnel to the appearance
of hemolysis, and to suggest practice improvements.
MATERIALS AND METHODS
Members of the College of American Pathologists (CAP) Quality
Practices Committee developed an online questionnaire, in the
usual CAP Q-Probes format, which was sent to participants in the
2011 CAP C1, C3, C3X, CZ, CZX, CZ2, and CZ2X chemistry
RESULTS
Of the 3495 CAP customers enrolled in the Chemistry
Surveys, 846 (24%) responded. Seen in Figure 1 are
hemolysis rates for 772 laboratories. In 276 laboratories
(36%), the overall hemolysis rate was less than 1%; in 267
laboratories (35%), it was 1% to 2.9%; and in 8 laboratories
(1%), it was 15.0% or greater. In 71 laboratories (9%),
personnel were unsure of the overall hemolysis rate.
A visual scale of evaluating hemolysis was used in 369 of
779 (48%) of the responding clinical laboratories, an
instrument scale was used in 87 laboratories (11%), and
both visual and an instrument scales were used by 323
(41%) of responders. As shown in the Table, when visually
grading a specimen for hemolysis, a picture of the degree of
hemolysis was used to classify each specimen in 271 of 680
laboratories (40%); however, the visual grading of specimens for hemolysis was included as part of the clinical
laboratories’ competency assessment program in 120 of 678
laboratories (18%). In 64 of 756 laboratories (8%), a manual
procedure, such as placing a drop of specimen on gauze,
followed by visual inspection, was used to differentiate
hemolysis from icterus in difficult cases. Of 748 participants,
269 (36%) forwarded automated hemolysis flags that
accompany hemolyzed results from their major chemistry
instrument to the patients’ medical record. For all analytes
measured on their primary chemistry analyzers in 777
laboratories, 632 laboratories (81%) used the same hemolysis scales, 495 of 672 (74%) used the same hemolysis scales
Procedures Used in Identification of Hemolysis
Participants
Parameter
When visually grading a specimen for hemolysis, is a picture of
the degree of hemolysis used to classify each specimen?
Is the visual grading of specimens for hemolysis included as part of
your laboratory’s competency assessment program?
Are any manual procedures used to identify the presence of
hemolysis in difficult cases (ie, place a drop of specimen on
gauze, inspect the color and differentiate between hemolysis
and icterus [bilirubin])?
Is your laboratory able to forward automated hemolysis flags that
accompany hemolyzed results recorded on your major chemistry
instrument to the patients’ medical records?
Does your laboratory use automated result verification for the
primary chemistry analyzer?
Does your laboratory use the same hemolysis scale for all analytes
measured on your primary chemistry analyzer?
Does your laboratory use the same hemolysis scale for all analytes
measured on your primary chemistry and immunoassay
analyzers?
Does your laboratory use the same hemolysis scale for all analytes
measured on your primary chemistry analyzer and your backup
chemistry analyzer?
Does your laboratory systematically and regularly monitor the
percentage of hemolyzed specimens as part of your laboratory’s
performance improvement plan?
Arch Pathol Lab Med—Vol 139, August 2015
Answered Yes, Answered No, Answered Unsure,
Total, No.
No. (%)
No. (%)
No. (%)
680
271 (40)
389 (57)
20 (3)
678
120 (18)
494 (73)
64 (9)
756
64 (8)
658 (87)
34 (4)
748
269 (36)
378 (50)
101 (14)
830
266 (32)
547 (66)
17 (2)
777
632 (81)
105 (14)
40 (5)
723
503 (70)
165 (23)
55 (8)
672
495 (74)
130 (19)
47 (7)
692
325 (47)
321 (46)
46 (7)
Variable Hemolysis Practices—Howanitz et al 1015
Figure 2. Percentage of 707 laboratories
using each of the 21 most common of the
69 numeric and descriptive terms used to
classify specimens for hemolysis. Continuous
includes instruments that provide quantitative
hemolysis results on a unique scale, such as 0
to 1400.
on their primary and secondary chemistry analyzers, and
503 of 723 (70%) used the same hemolysis scales on the
primary chemistry analyzer and immunoassay analyzers. In
a few laboratories (325 of 692; 47%), hemolysis was
systematically and regularly monitored as part of the
laboratory’s performance improvement plan.
In most laboratories (630 of 710; 88%), specimens were
rejected depending on the level of hemolysis. However in 50
clinical laboratories (7.0%), all hemolyzed specimens were
rejected, and in 25 clinical laboratories (4%), no hemolyzed
specimens were rejected.
Sixty-nine specific terms were used to describe hemolysis:
21 terms were used commonly with each term used in at
least 10 participating laboratories to classify hemolysis.
Slight, moderate, gross, and marked were used in 389, 389,
326, and 161 of 707 laboratories (55%, 55%, 46%, and 23%),
respectively (Figure 2). Of the responders, 127 (18%)
indicated they used all 3 modifiers, slight, moderate, and
gross in their descriptive scales. The most commonly used
numeric terms were 1þ, 2þ, 3þ, and 4þ used in 195, 188,187,
and 170 laboratories (28%, 27%, 26%, and 24%), respectively. In the remaining 56 laboratories (8%), 48 additional
words, numbers, or phrases described hemolysis.
Figure 3 provides a list and the percentages for 20 cutoff
values used to describe specimens rejected for hemolysis in
597 laboratories. In those laboratories, 215 (36%) used a
numeric level, and 334 (56%) used a descriptive word to
describe the cutoff for specimen rejection. The most
common cutoffs that resulted in rejection were moderate
hemolysis, used in 182 laboratories (30%), followed by gross
hemolysis, which was used in 66 laboratories (11%). The
category other was used in 43 additional laboratories (7.2%),
where a unique term or additional modifier(s) was added to
one of the 20 other terms described in Figure 3. No
laboratories used 6þ for rejection, although one laboratory
used ‘‘greater than 6.’’
Figure 4 summarizes practices to detect hemolysis in
whole blood specimens in 91 clinical laboratories that use
whole blood analyzers not at the point-of-care. Approximately one-half of those using whole blood analyzers
measured glucose, potassium, sodium, and chloride, whereas about one-third of those with whole blood systems
measured calcium as well as other analytes. Of the 86
laboratories that used whole blood electrolyte analyzers, 48
(56%) did not determine whether specimens were hemolyzed. The most common method, used by 18 (21%) of these
Figure 3. Frequency of use for 20 numeric
and descriptive cutoff values at which 597
laboratories began to reject specimens for
hemolysis. Continuous includes instruments
that provide quantitative hemolysis results on
a unique scale, such as 0 to 1400.
1016 Arch Pathol Lab Med—Vol 139, August 2015
Variable Hemolysis Practices—Howanitz et al
Figure 4. Procedures used to detect whole blood hemolysis (N ¼ 86).
respondents to determine hemolysis, was to centrifuge all
whole blood specimens after measurement and then
visually determine whether hemolysis was present. In a
few clinical laboratories (9 of 86; 10%), personnel stored and
then visually inspected all specimens within an hour after
measurement to determine whether hemolysis was present.
In 5 (6%) of the clinical laboratories, only when potassium
was elevated were specimens either centrifuged (3 or 3%) or
stored for up to an hour and then visually inspected for
hemolysis (2 or 2%). When hemolysis was detected in any
of these circumstances, then a comment was added to the
quantitative result indicating the specimen was hemolyzed.
COMMENT
The overall hemolysis rate in 772 laboratories varied
greatly, ranging from less than 1% in 36% of laboratories (n
¼ 276) to 15% or more in 1% of laboratories (n ¼ 8). In more
than 70% of laboratories (n ¼ 543), the hemolysis rate was
less than 3%, and 9% (n ¼ 71) were unsure of their overall
hemolysis rates. This contrasts with a 1997 Q-Probes report1
in which 0.2% of more than 10 million specimens in 453
clinical laboratories were rejected for hemolysis. A number
of participants commented that the hemolysis rates were
highest in specimens from their emergency departments;
similar findings were described in a number of studies
reported in the literature, with hemolysis rates of 3.4%,8
12.8%,9 12.9%,4 and 19.8%.5 It is not surprising to us that in
9% of clinical laboratories (71 of 772), personnel did not
know what their overall hemolysis rates were, as common
practice is only to monitor and reduce the rates at specific
patient locations such as emergency departments where
rates are highest.
Some laboratories rejected all hemolyzed specimens. This
policy potentially places certain patients at risk such as the
case reported by Ismail and colleagues,10 who described a
patient with renal failure and unrecognized intravascular
hemolysis who died from the presumed arrhythmia of
hyperkalemia because their clinical laboratory had a policy
of not measuring potassium values when specimens were
hemolyzed. Although the World Health Organization
currently recommends that potassium results not be
reported when specimens are hemolyzed,11 we strongly
suggest that the laboratory community revisit this issue with
consideration of reporting results qualified by noting the
Arch Pathol Lab Med—Vol 139, August 2015
presence and degree of hemolysis and an assessment of the
effect it may have on the test result, allowing clinicians
responsible for the patient’s care to integrate this information into the differential diagnosis and subsequent treatment
or to repeat the analysis on another specimen obtained by
techniques minimizing in vitro hemolysis.
Most laboratories still used visual scales to grade
hemolysis, despite most chemistry analyzers used in central
hospital laboratories now measuring hemolysis. Visual
estimations of hemolysis are unreliable,6,12,13 and automated
methods are preferable. In addition, charts that are used for
visual estimation of hemolysis fade, leading to erroneous
interpretations. In most laboratories, the description of
hemolysis cannot be sent automatically to the patient’s
medical record, thereby underestimating the frequency of
reporting because technologists working in fast-paced
chemistry laboratories could easily forget to perform
nonautomated tasks, such as adding a modifier to a patient’s
medical record result. Because these hemolysis results
cannot be sent automatically to the laboratory information
system, these laboratories are probably prevented from
using an automated verification system. There is a second
consideration regarding nonautomated assessment of hemolysis. Although personnel performing laboratory tests in
the United States are required to be checked for color
blindness, only 120 of 678 laboratories (18%) evaluated
clinical laboratory employees for competency in visually
evaluating hemolysis.
Within laboratories, practices for identifying and reporting
hemolysis also appeared to vary greatly. Fourteen percent of
laboratories (105 of 777) did not use the same hemolysis
scale on their primary and secondary chemistry analyzer,
and in 23% of clinical laboratories (165 of 723), different
hemolysis scales were used on the primary chemistry
analyzer and the primary immunoassay analyzer. Different
scales emanating from the same laboratory are bound to
confuse clinicians. Other examples of variability in hemolysis procedures within the same laboratory included specific
reporting procedures that changed according to the analyte
measured.
Explanations of hemolysis in some textbooks suggest that
quantitative measurements be used to describe hemolysis,
and in the United States, these results frequently are
reported in milligrams per decaliter.14 However, in the
classic manuscript, Glick et al13 used 6 numeric, semiquantitative categories, each followed by a plus sign: 0, contains
no or trace interferent and 1þ, 2þ, 3þ, 4þ, or 5þ. Other workers
used descriptive terms that approximately correspond with 7
reportable, numeric categories, such as none (0), none to
slight (1), slight (2), slight to moderate (3), moderate (4),
moderate to gross (5), and gross (6).15 Some publications
relate descriptive terms to quantitative measurements, such
as the use of a scale such as mild (1 g/L), moderate (2.5 g/L),
and severe (5 g/L) by Hawkins15 or the use of slight (50 mg/
dL), moderate (100 mg/dL), and gross (200 mg/dL) by
Spencer and Roger.16 Our sampling confirmed the varied
classification with 69 different terms, numbers, or combinations of numbers and terms used. Some newer major
chemistry analyzers allow users to decide to implement a
feature of a quantitative, continuous scale throughout the
measurement range to describe hemolysis.
In our experience, we have found that laboratorians try to
simplify nonautomated processes by having the same rule
for all measurements performed on the same specimen type.
Hence, it was not surprising to us that most laboratories
Variable Hemolysis Practices—Howanitz et al 1017
(74%; 495 of 672) used one hemolysis level to reject or
accept specimens for all measurements. We found in
approximately 30% of laboratories (182 of 597), specimens
were rejected when hemolysis reached moderate levels, and
11% (66 of 597) when hemolysis levels were gross. For those
laboratories using a numeric scale, the most common
discrimination level for rejection or acceptance was 2þ, but
that level was used only in 9% (54 of 597) of the laboratories.
Whole blood measurements have gained favor when
speedy analyses are required, but because red blood cells are
distributed throughout these specimens, it is impossible to
detect whether some of the red color in whole blood
specimens is from hemolysis. Hawkins15 studied the
hemolysis frequency in whole blood gas analysis in his
clinical laboratory and found that hemolysis was more than
5 times higher in whole blood used for blood gases
compared with serum and that 42% of potassium results
in these specimens were downgraded from normokalemic
to hypokalemic or from hyperkalemic to normokalemia or
hypokalemic. The Hawkins17 data provide support in critical
whole blood potassium measurements for the need to
determine whether hemolysis is present.
There is no quick and reliable way to determine whether
hemolysis is present in whole blood specimens. In most
laboratories (56%; 48 of 86) in our study using whole blood
analyzers, laboratory personnel did not take additional steps
to detect whether whole blood specimens were hemolyzed.
Of the few who evaluated hemolysis in whole blood, the
most common practice among 21% of responders was to
centrifuge all whole blood specimens and then visually
examine whether the supernatant was red. The second most
common practice (10%) was to store the whole blood
specimen for up to an hour allowing the red blood cells to
settle and then visually noting whether hemolysis was
present. When potassium was elevated, some laboratories
identified hemolysis in whole blood specimens by storing
the whole blood specimen upright for up to an hour and
then visually examined the specimens for hemolysis (3%), or
inspecting other specimens obtained at the same time for
hemolysis (3%).
One obvious disadvantage of identifying hemolysis in
whole blood specimens is the time required. Visual
inspection required up to an hour before red blood cells
fully settle or required 10 minutes when separation was
hastened by centrifugation. Waiting for these procedures to
occur before visual inspection defeats the main objective of
rapid whole blood testing result reporting.
A second issue with identifying hemolysis in whole blood
specimens is that visual estimation of the amount of
hemolysis has been unreliable because experienced technologists cannot accurately distinguish between various
concentrations of hemolysis.6,12,13 A third issue is whether or
not hemolysis affects whole blood assays other than
potassium results. Whole blood measurements that are
commonly performed include sodium, calcium, magnesium,
and bilirubin. Effect on these measurements in hemolyzed
whole blood would not always be detected if clinical
laboratory procedures require evaluation of hemolysis only
when potassium was elevated. A potential solution alerting
physicians to hemolysis in a timely manner is to automatically quantitate hemolysis or free hemoglobin in a whole
blood specimen at, or before the time of, measurement of
the whole blood specimen. At this time, there are no US
Food and Drug Administration–approved methods or
instruments available that can make such a measurement.
1018 Arch Pathol Lab Med—Vol 139, August 2015
Widespread use of multianalyte, whole blood point-ofcare devices, such as the Abbott iSTAT (Abbott Laboratories, Abbott Park, Illinois) and the Alere epoc (Alere Inc,
Waltham, Massachusetts) blood gas analyzers, as well as
single-test whole blood devices, including glucose meters,
are subject to hemolysis interferences, but based on their
use at locations where no centrifuges are located, procedures used in clinical laboratories to identify hemolysis are
not applicable. The only useful procedure is that if a result is
unexpected and it could have been influenced by hemolysis,
a serum specimen should be sent to the clinical laboratory
for remeasurement.
Quantitative hemoglobin measurements are some of the
most common and accurate measurements made in hospital
clinical laboratories because hemoglobin routinely is measured by arterial blood gas instruments, complete blood cell
count analyzers, co-oximeters, pulse oximeters, and many
point-of-care devices. There is even a fourth edition of
reference methods for automated hemoglobin measurements
in human blood.18 Despite these resources, laboratorians
continue to use inaccurate visual methods to estimate
hemolysis and report hemolysis using poorly reproducible
terms that confuse clinicians. Finally, most laboratories
continue to accept or reject specimens for measurement of
various analytes using unscientific principles.
We certainly agree with van Rheenen and de Moor19 that
one place where visual estimates of hemoglobin may be the
best that can be achieved is in resource-poor countries
where no automated analyzers are available. In developed
countries, where clinical laboratory instruments that quantify hemoglobin accurately are in the same clinical
laboratory, it is time for laboratorians to standardize policies
and procedures for measurement and reporting of hemolysis. These policies and procedures should include quantification of hemoglobin on a method-by-method basis.
Until standard assessment and reporting occur, variability
among results from identical hemolyzed patient specimens
measured in different clinical laboratories will continue.
Besides standard reporting and consistent assessment of
interferences, we also recommend that instrument manufacturers consider developing whole blood analyzers that
simultaneously measure hemolysis.
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