INTERNATIONAL JOU RNAL OF LABO RATO RY HEMATO LOGY

Spurious counts and spurious results on haematology

analysers: a review. Part II: white blood cells, red blood
cells, haemoglobin, red cell indices and reticulocytes
M. ZANDECKI, F. GENEVIEVE, J. GERARD, A. GODON

Haematology Laboratory, SUMMARY

University Hospital of Angers,
Angers, France
Correspondence:
M. Zandecki, Haematology Labor-
atory, University Hospital of Angers,
4, rue Larrey, 49000 Angers,
France. Tel.: +33 241 35 53 53; Fax:
+33 2 41 35 55 99; E-mail:
mazandecki@chu-angers.fr
doi:10.1111/j.1365-2257.2006.00871.x
Received 30 January 2006;
accepted for publication 30 July
2006
Keywords
Haematology analysers, automated
count, cell blood count, spurious
count, white blood cells, haemo-
globin, red blood cells, mean cell
volume
Haematology analysers provide quick and accurate results in most
situations. However, spurious results, related either to platelets (part I
of this report) or to other parameters from the cell blood count (CBC)
may be observed in several instances. Spuriously low white blood cell
(WBC) counts may be observed because of agglutination in the
presence of ethylenediamine tetra-acetic acid (EDTA). Cryoglobulins,
lipids, insufficiently lysed red blood cells (RBC), erythroblasts and
platelet aggregates are common situations increasing WBC counts. In
most of these instances flagging and/or an abnormal WBC differential
scattergram will alert the operator. Several situations lead to abnormal
haemoglobin measurement or to abnormal RBC count, including
lipids, agglutinins, cryoglobulins and elevated WBC counts. Mean (red)
cell volume (MCV) may be also subject to spurious determination,
because of agglutinins, excess of glucose or salts and technological
considerations. In turn, abnormality related to one measured param-
eter will lead to abnormal calculated RBC indices: mean cell haemo-
globin content (MCHC) is certainly the most important RBC indices to
consider, as it is as important as flags generated by the haematology
analysers (HA) in alerting the user to a spurious result. In many
circumstances, several of the measured parameters from CBC may be
altered, and the discovery of a spurious change on one parameter
frequently means that the validity of other parameters should be
considered. Sensitive flags now allow the identification of several
spurious counts, but only the most sophisticated HA have optimal
flagging and more simple HA, especially those without a WBC
differential scattergram, do not possess the same sensitivity for
detecting anomalous results. Reticulocytes are integrated now into
the CBC in many HA, and several situations may lead to abnormal
counts.

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22 M. ZANDECKI ET AL. SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS
INTRODUCTION
Many haematology analysers (HA) that enumerate
WBC generate a WBC differential scattergram, which
is the basis for the automated WBC differential. Such
WBC scattergrams are also pivotal for the generation
of flags or alarms, detecting that the WBC count (and
the WBC differential) is possibly erroneous and giving
some information about the possible origin of the
anomaly. As mentioned in part I of this report, the
WBC differential scattergram is also of crucial import-
ance to identify anomalies of platelet (PLT) counting,
PLT clumps being the most obvious example.
Red blood cell (RBC) counts, haemoglobin (Hb),
and mean (Red) cell volume (MCV) are other
parameters measured by HA, which are also subject
to the spurious values in several situations. Red cell
(Wintrobe) indices other than MCV and packed cell
volume (haematocrit; Hct) are usually calculated
from these measured parameters, and in most cir-
cumstances erroneous directly measured RBC param-
eters will subsequently cause in turn erroneous
calculated RBC indices. These indices can be used as
a flag for abnormal counts. Reticulocytes may be
considered now as a part of the CBC, and many HA
have integrated reticulocyte counts under a fully
automatized method.
WHITE BLOOD CELLS
For blood cell analysis, each large sized particle
(greater than the size of a PLT) that is not destroyed
by haemolytic agents will be identified as a WBC on
most HA. After enumeration, and according to the
type, impedance with low- and high-frequency elec-
tromagnetic or direct current, laser light scattering (at
one or at various angles), or peroxidase staining
intensity are used, either individually or together, to
generate a five-, six-, or even seven- part differential
(for review, see Bain & Bates, 2001). It is not in the
scope of this report to study how the various WBC
are classified but it must be kept in mind that scatter-
grams generated by the HA to display the WBC differ-
ential must be fully understood by operators (Bain &
Bates, 2001). In many instances, WBC scattergrams
allow the detection of abnormalities related to spur-
ious counts and/or help to explain them. As a rule,
instruments that do not generate WBC differential
Journal compilation  2007 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2007, 29, 21–41
scattergrams will be most prone to overlook several
factitious counts.
Spuriously low WBC counts
Polymorphonuclear neutrophil aggregates
Polymorphonuclear neutrophil (PMN) aggregates may
be observed on blood samples drawn into EDTA. The
incidence is low but certainly underestimated, corre-
sponding to 2/65 000 full blood counts in USA
(Epstein & Kruskall, 1988), 1/9500 in Italy (Bizzaro,
1993), 1/7500 in France (Lesesve et al., 2000). Both
male and female show the anomaly. Although no
pathology or no specific disease is associated with
clustering of PMN, an acute or chronic inflammatory
context (Luke, Koepke & Siegel, 1971; Savage, 1989;
Kahlil, 1991; Robbins, Conly & Oettinger, 1991;
Imbing et al., 1996; Lesesve et al., 2000), liver diseases
(Epstein & Kruskall, 1988; Savage, 1989; Kobayashi
et al., 1991; Vinatier et al., 1994; Imbing et al., 1996),
or circumstances associated with the generation of
cold agglutinins (Guibaud, Plumet-Leger & Frobert,
1983; Epstein & Kruskall, 1988; Robbins, Conly &
Oettinger, 1991; Deol, Hernandez & Pierre, 1995;
Imbing et al., 1996; Lesesve et al., 2000) have been
reported in many instances. The phenomenon may or
may not be transitory (Antonsen & Beyer, 1989; Bizz-
aro, 1993; Schinella, Kojikara & Curci, 1995).The
decrease may be either moderate or clinically signifi-
cant, leading at times to suspect agranulocytosis and
to generate unneeded investigations (bone marrow
aspiration or biopsy) or therapy (antibiotics; Epstein &
Kruskall, 1988; Vinatier et al., 1994; Schinella, Kojikara
& Curci, 1995). There is no relationship between the
phenomenon described here, which is a pure in vitro
anomaly related to sampling in EDTA anticoagulant,
and the in vivo anomaly occurring in diseases such as
adult respiratory distress syndrome or leukostasis, in
which PMN tend to aggregate as a consequence of
membrane interactions with complement (Jacob et al.,
1980).
The mechanism leading to agglutination is not
fully elucidated (Carr et al., 1996). It is always an in
vitro phenomenon, mainly EDTA dependent (Epstein
& Kruskall, 1988), although agglutination after the
use of sodium citrate or heparin as anticoagulants
has also been reported in some instances (Rohr &
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 M. ZANDECKI ET AL.
Rivers, 1990; Robbins, Conly & Oettinger, 1991; Vin-
atier et al., 1994; Deol, Hernandez & Pierre, 1995;
Carr et al., 1996). Using transfer experiments, either
plasma from the affected patients in contact with
WBC from normal patients or normal plasma in con-
tact with PMN from the affected patients, the anom-
aly has proved to be related to a plasma component,
and incubation of plasma with an anti-IgM antibody
or with dithiothreitol abolished or quantitatively
decreased PMN aggregation (Bizzaro, 1993). Using
flow cytometry, IgM was found on the PMN surface
in a patient (Carr et al., 1996). Some authors have
reported that the size of the aggregates was larger at
low temperature and that they disappeared at 37 C
(Bizzaro, 1993; Carr et al., 1996). Several other
authors reported that WBC agglutination in EDTA-
anticoagulated blood was not corrected by warming
the EDTA blood sample, at least in some cases, min-
imizing the implication of a cold agglutinin in the
genesis of the clusters (Robbins, Conly & Oettinger,
1991; Vinatier et al., 1994; Deol, Hernandez & Pierre,
1996). A high level of integrin expression (CD11b–
CD18) on PMN membrane was proposed to be rela-
ted to the generation of PMN clumps (Galifi et al.,
1993).
White blood cell differential scattergrams (using
either size and complexity or size and peroxidase con-
tent) may demonstrate anomalies, but it must be kept
in mind that the largest aggregates are overlooked,
and only the smallest will trigger flagging. On imped-
ance-type HA, the anomaly is suspected when events
are present above the area of PMN (top of the WBC
graph), or when it is difficult to separate WBC classes
(Galifi et al., 1993), and the most frequent flags gener-
ated using such HA are ‘immature granulocytes’ or
‘band cells’ (Lippi et al., 1994). On Bayer-Technicon
HA, the smallest clusters may appear as a band of dots
in the upper right of the WBC differential scattergram:
as these aggregates contain many PMN they are
reported as peroxidase-rich particles, and an alarm
corresponding to high peroxidase content is gener-
ated. On some HA, analysis of WBC nuclei is per-
formed on a dedicated channel after the use of a
drastic solution ensuring lysis of WBC membranes:
number of free nuclei is used as a control for WBC
count. Discrepancy between WBC count obtained
from that channel and that from the fluorescence or
peroxidase channel is observed (Bayer, Tarrytown,
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SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 23
Figure 1. Aggregate of polymorphonuclear neutroph-
ils observed in an infected patient.
NY, USA; Abbott, Abbott Park, IL, USA). White blood
cell aggregates are destroyed after drastic lysis of WBC
membranes (Galifi et al., 1993). Blood films may show
small (up to five cells), moderate (up to 50 cells), or
large (>100 cells) clusters of PMN (Figure 1). Careful
morphological examination of these aggregates shows
that a few lymphocytes or monocytes may be at times
entrapped within the aggregates (Hillyer, Knopf &
Berkman, 1990). Immature granulocytes (myelocytes,
metamyelocytes) and band cells are not infrequently
reported as a part of the clustered cells, and it was
proposed that aggregates might develop around
myelocytes, whereas PMN alone failed to cluster
together (Deol, Hernandez & Pierre, 1995). Aggregates
of PMN are devoid of PLT, in contrast to PLT–PMN
aggregates (discussed in part I). Eventually, as aggre-
gation of PMN in the presence of EDTA leads to a
reduction in their number, HA may generate flags
corresponding to spuriously abnormal WBC differen-
tials, e.g. spurious agranulocytosis or spurious lympho-
cytosis (Epstein & Kruskall, 1988; Vinatier et al., 1994;
Schinella, Kojikara & Curci, 1995).
Home-made anticoagulants were proposed to over-
come the agglutination (Schinella, Kojikara & Curci,
1995). As mentioned above, warming the sample at
37 C may reduce both size and number of clusters in
some instances, but full disappearance is far from
being a consistent finding, and that method cannot be
proposed to overcome the anomaly. Finger prick and
immediate dilution of the blood sample prevents the
agglutination.
24 M. ZANDECKI ET AL. SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS
Aggregation of WBC other than polymorphs
in the presence of EDTA
Clusters of normal (nonmalignant) lymphocytes were
reported in a patient with urinary tract infection, in a
patient with a B-cell lymphoma without bloodstream
involvement, in a patient with escarres, and in
another one with chronic myelomonocytic leukaemia
(Deol, Hernandez & Pierre, 1995; Lesesve et al., 2001).
Lymphocyte aggregation was also reported to occur in
chronic lymphocytic leukaemia patients, spontane-
ously when lymphocyte counts are extremely high
(>400 · 109/l; O’Flaherty, Kreutzer & Ward, 1978), or
not (Bizzaro & Piazza, 1991). Aggregates of three to
50 lymphoma cells were observed in two cases of
Splenic Lymphoma with Villous Lymphocytes (SLVL;
Juneja et al., 1992; Imbing et al., 1995) and in one
case to non-Hodgkin lymphoma mimicking SLVL
(Shelton & Frank, 2000). In these situations, the lar-
gest aggregates are overlooked by the HA and WBC
count is spuriously but variably low. The smallest
aggregates may disturb WBC differential and may or
may not generate a flag, depending on the type of HA
used (Shelton & Frank, 2000). In all cases reported so
far EDTA was implicated, although small clusters of
cells were also observed on heparinized samples
drawn as controls (Deol, Hernandez & Pierre, 1996)
and sodium citrate did not appreciably change the
clustering tendency of the lymphocytes in one case
(Shelton & Frank, 2000). Fingerpricking and immedi-
ate dilution of blood seems the best way to avoid
aggregates (Lesesve et al., 2001). Heating (37 C) was
reported to reduce clumping either partly (Shelton &
Frank, 2000) or had no significant effect on the size
of clumps (Lesesve et al., 2001). As the number of
cases reported is low, only hypotheses on the mech-
anism(s) leading to lymphocyte agglutination have
been proposed, implicating various molecules such as
adrenalin (epinephrin), arachidonic acid, or leukotrien
B4 (Villa et al., 1984; Shelton & Frank, 2000). In one
instance aggregates involving all WBC classes was
reported, in a patient with a long-standing history of
alcohol abuse and alcoholic cirrhosis (Savage, 1989).
Nature and amount of anticoagulant
A decrease in WBC count not related to agglutination
was reported in situations corresponding to samples
Journal compilation  2007 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2007, 29, 21–41
containing excess of K3-EDTA (but not K2-EDTA)
anticoagulant, resulting from insufficient blood drawn
after vein puncture (Goossens, Van Duppen & Verwil-
ghen, 1991).
Spuriously high WBC counts
PLT aggregates and large platelets
Pseudoleukocytosis may be secondary to PLT clumps
large enough to mimic WBC size (Solanki & Blackburn,
1983; Cornbleet, 1983; Savage, 1984; Payne & Pierre,
1984; Lombarts & de Kieviet, 1988; Schrezenmeier
et al., 1995). All modern HA that analyse WBC sub-
populations detect this anomaly: PLT clumps are
localized as a rocket shaped area of dots at the lower
left hand corner of the WBC differential scattergram,
and a flag corresponding to inability to discriminate
among the WBC categories, namely lymphocytes,
is generated (see Figure 2, part I). Several authors
have reported that HA without WBC differential
scattergrams are unable to detect such anomaly
(Payne & Pierre, 1984; Lombarts & de Kieviet, 1988).
Some very large PLT may be encountered in myelo-
proliferative and in myelodysplastic disorders, whose
size and volume may reach those of WBC and which
may be enumerated as WBC: flags are usually gener-
ated, that mention the presence of pathological parti-
cles that cannot be classified as WBC (usually ‘giant
PLT’, or ‘PLT aggregates’, depending on the HA).
Nucleated red blood cells (NRBC)
They may be found in the blood stream in physiologi-
cal (newborns) and in pathological circumstances, and
at times they may be much more numerous than
WBC. Using HA, these NRBC are in contact with lysis
agents that destroy their membrane, leaving nuclei
free, the latter being responsible for anomalies. Free
NRBC nuclei are usually particles <40–50 fl, a size
that is superimposable to that of PLT clumps or large
PLT. Unsurprisingly, NRBC and PLT clumps disturb
WBC counts identically and both artifacts generate
equally the corresponding flags (Figure 2). According
to the HA, NRBC are either included within the WBC
count (instruments that do not generate any WBC dif-
ferential scattergram, but also some that do), or not
enumerated as WBC, or counted separately. On
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 M. ZANDECKI ET AL.
Figure 2. Nucleated red blood cells (NRBC ¼ 85 · 109/l) in a neonate (haemolytic anaemia): the WBC scattergram
(right) shows a large number of dots, corresponding to NRBC nuclei, as compared to a normal WBC scattergram
(left; Bayer Advia 120).
several recent and sophisticated HA, NRBC are specif-
ically identified and may be enumerated using fluor-
escence technology (Kim et al., 1998; Wang et al.,
2003).
Red blood cells resistant to lysis
Red blood cell may not be destroyed by lysis reagents
in various circumstances, either physiological (neo-
nates) or pathological (abnormal haemoglobins, liver
disease, uraemia, chemotherapy): they are considered
as WBC and falsely increase the WBC count (Luke,
Koepke & Siegel, 1971; Cornbleet, 1983; Griswold &
Champagne, 1985; Vinatier et al., 1994; Mellors &
McArdle, 1995; Elghetany & Hudnall, 1996). White
blood cell differential scattergrams demonstrate dots of
small size located near or within the lymphocyte win-
dow, and inability to perform a WBC differential leads
to the generation of a flag. The picture is more or less
superimposable to that observed with the presence of
NRBC. This phenomenon seems to be a consistent
finding in haemoglobin C – containing RBC (either
CC, Cbthal, or CS) but not in b thalassemic patients
(either trait or major) or in haemoglobin S disease
(Booth & Mead, 1983). HA without WBC differential
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SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 25
scattergrams may fail to detect the anomaly, and in
patients undergoing chemotherapy it may be of cru-
cial importance, as it leads to normal or even elevated
WBC counts in otherwise leukopenic patients: com-
parison with previous results (delta check) and exami-
nation of blood smear are necessary in such instances.
Some HA generate a message « RBC resistant to
lysis » and give access to an extended lysis mode
which lengthens contact of blood cells with the lysis
reagent (Abbott CellDyn 4000). On HA using a dedi-
cated channel to enumerate WBC nuclei, discrepancy
between full WBC count and WBC nuclei count is
obvious: the channel analysing WBC nuclei uses lysis
agents strong enough to destroy all membranes (WBC
and RBC) and gives the accurate WBC count.
Cryoglobulins
The presence of cryoglobulin was first reported as
causing erroneous WBC counts (Emori, Bluestone &
Goldsberg, 1973; Taft et al., 1973; Haeney, 1976; Gul-
liani, Hyun & Gabaldon, 1977), but it also leads in
many instances to spuriously elevated PLT counts,
and at times to altered RBC counts or Hb measure-
ments (cf., see Fohlen-Walter et al., 2002). Abnormal-
26 M. ZANDECKI ET AL. SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS

Figure 3. Several morphological aspects of cryoglobulins may be observed on peripheral blood samples, and two
among them are shown here: small lucent precipitates scattered around RBC (left), or invisible precipitates that
change morphology of RBC into a ‘moth-eaten’ aspect (right).

ities that occur on HA are neither consistent nor rela-
ted to the amount of cryoglobulin or to its nature
(each type may be involved in spurious counts). How-
ever, anomalies generated must be considered because
they have some peculiar characteristics, and their
recognition may be the first clue leading to the diag-
nosis of cryoglobulinaemia (Zandecki et al., 1989; Foh-
len-Walter et al., 2002). Cryoprecipitates may be
observed in some instances on stained blood smears
(Figure 3), and in nearly all cases after examination of
fresh blood sample using a phase contrast microscope
(more obvious if performed at low temperature). Var-
ious morphological aspects were reported, including
dense amorphous clusters or flake-like particles, nee-
dle-shaped crystals, and pinkish globules (cf., see Zan-
decki et al., 1989). As cryoglobulins are
immunoglobulins that precipitate at temperature
lower than 37 C, abnormal counts are observed
mainly on HA that both use reagents and perform
analysis at room temperature. However, HA that use
prewarmed reagents, even with low pH reagents that
normally avoid precipitation of cryoglobulins, are not
free of anomalies and spurious counts are also
observed (Fohlen-Walter et al., 2002). According to
their size, cryoprecipitates may only affect PLT counts,
leading to spuriously elevated ones, up to eight times
(Fohlen-Walter et al., 2002): histograms of PLT vol-
umes show large number of small-sized particles, and
Journal compilation  2007 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2007, 29, 21–41
an alarm is usually generated (see Figure 8, part I). If
cryoglobulin particles cluster together or if their size is
larger, reaching that of leukocytes, WBC count is spu-
riously increased: a cloud of small dots may be
observed on the WBC differential scattergram, near
the lymphocytes or above the threshold for PLT, or at
the top of the PMN cloud, depending on the HA used
(Fohlen-Walter et al., 2002). When samples are
warmed to 37 C for at least 30 min and reanalysed
promptly anomalies disappear, but it is not a fully
consistent finding and not infrequently a new sample
must be taken and kept at 37 C until analysis to
obtain accurate CBC counts (Zandecki et al., 1989;
Infanti et al., 1998; Figure 4). Hb determination and
RBC counts may at times be altered in the presence of
cryoglobulins (see later). In some situations cryoglob-
ulins may form a gel, leading to inaccurate aspiration,
and an alarm may be generated on some HA (insuffi-
cient sampling, or related).
Cryofibrinogen or related
Particles related to cryofibrinogen or to fibrin have
been reported to elevate WBC counts and at times
PLT counts and, according to the reports, PLT and
WBC counts may be found up to twice and up to 16
times the accurate value, respectively (Gulliani, Hyun
& Gabaldon, 1977; Griswold & Champagne, 1985;
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 M. ZANDECKI ET AL. SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 27

Corberand et al., 1991). Careful examination of the

corresponding blood smears shows thin fibrils, related
to fibrin strands after electron microscopic study
(Figure 5). These fibrils might correspond either to
cryofibrinogen, although biochemical research may be
negative in some instances (Griswold & Champagne,
1985), or to a mixture of cryoglobulin and cryofibri-
nogen (Gulliani, Hyun & Gabaldon, 1977). However,
analysis of new EDTA blood samples from the patients
frequently fail to reproduce the phenomenon,
hypothesizing that such particles might be the result
of fibrin polymerization after a difficult venepuncture,
initiating coagulation in vitro before contact of the
blood with EDTA (Corberand et al., 1991). After
warming the samples at 37 C abnormal counts are
either less pronounced or fully disappear.

Figure 4. Low grade B-cell lymphoma and cryoglobu-
lin type I (Ig M Kappa). Analysis at room tempera-
ture (20 C; left) shows that WBC count was not
performed and that PLT count was inaccurate, and
several flags were generated. After warming the
same sample at 37 C and prompt analysis all abnor-
malities disappear (37 C; right; STKS II Coulter).
Lipids
As previously discussed (part 1), lipids may generate
droplets large enough to disturb PLT counts, but also
WBC counts, together with or independently of PLT
count. Abnormal WBC differential scattergrams are
generated, displaying various changes, at times more
or less superimposable to those outing from the
presence of cryoglobulins (Figure 6). Comments on
lipids disturbing Hb determination will be discussed
later.

Figure 5. In some instances (see text), fibrin strands may be observed on peripheral blood smears (MGG staining;
left); according to their size they may be enumerated as PLT or/and as WBC. Electron microscopic analysis (right;
a) shows such strands intermixed with PLT and WBC (arrows), and inset (right; b) shows peculiar periodic ultra-
structure corresponding to fibrin strands.

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28 M. ZANDECKI ET AL. SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS

Figure 6. Lipids in a patient with liver disease. When compared with a normal scattergram (left), lipids generated
here a strand of dots elongating PLT region (arrow; Sysmex XE2100).

Microorganisms Overfilling blood collection vacuum tubes

Bacteria or fungi may be observed on peripheral blood
smears and, as previously discussed (part I), may lead
to spuriously elevated PLT counts. They were also
reported as disturbing WBC differentials if they
clumped together, and in vitro studies adding large
number of such microorganisms were shown to
increase WBC count (Marshall et al., 1990).
Haemozoin contained in PMN and monocytes in
malarial cases may cause depolarization of the laser
light and subsequently misclassification of WBC types
on some HA (Sysmex, Kobe, Japan), although full
WBC count does not clearly change (Huh et al., 2005)
Adipose tissue
Total WBC count was reported as artefactually eleva-
ted, caused by contamination with subcutaneous adi-
pose tissue of the blood sample obtained by traumatic
femoral vein puncture: peculiar scatter plots also dem-
onstrated an abnormal WBC differential (Whiteway &
Bain, 1999).
As previously mentioned together with spurious PLT
counts, overfilling the collection tube leads to inade-
quate sample mixing and setting of cellular contents:
all the parameters are altered (Pewarchuk, Vander-
Boom & Blajchman, 1992).
Concluding remarks on spurious WBC counts
As for spurious PLT counts, it is not always possible to
find or to identify the phenomenon that leads to spur-
ious WBC counts, even if something is visible on per-
ipheral blood smears (Savage, 1989). The WBC
scattergram from HA is useful and must be interpreted
carefully; examination of stained films must also be
considered.
HAEMOGLOBIN, RBC COUNT AND RBC
PARAMETERS
HA measure several RBC parameters, including RBC
count and MCV both determined on the same chan-

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 M. ZANDECKI ET AL.
nel(s), and Hb concentration which is measured on
another channel(s). Haematocrit (Hct), mean cellular
Hb (MCH), and mean corpuscular Hb concentration
(MCHC) are usually calculated from the measured
parameters. Circumstances leading to erroneous meas-
ured RBC parameters will subsequently generate
abnormal calculated RBC indices. So, an anomaly of
at least one of the RBC indices, beside its pathophysi-
ological significance, must also be considered as a flag
for the detection of an error of at least one among the
red cell parameters measured (RBC, MCV, Hb). As
well as the classical Wintrobe RBC indices, other
parameters derived from the technology have been
introduced, which differ according to the HA, but
their use may also be helpful for the detection of
abnormal counts or measurements. One example is
the assessment of Hb concentration of individual RBC,
also named cellular haemoglobin concentration mean
(CHCM), which is subsequently compared with the
calculated MCHC, generating a flag for discordant data
(Abbott, Bayer).
Haemoglobin
Hb concentration is measured using a colorimetric
(spectrophotometric) method, either by a modification
of the manual cyanmethaemoglobin method or by
addition of various reagents, such as sodium lauryl
sulphate or imidazole. According to the manufactur-
ers, various nonionic detergents are included to insure
rapid RBC lysis and to reduce turbidity because of cell
membranes and plasma lipids.
Lipids and hyperchylomicronemia
Lipemia may cause erroneous PLT (see part I) and
WBC counts (see above), but may also induce inter-
ferences by turbidity (Creer & Ladenson, 1983; Sand-
berg, Sonstabo & Christensen, 1989). An abnormally
high MCHC (>36 g/dl) corresponding to an errone-
ously high Hb has been reported for patients with
severe constitutional or acquired hypertryglyceridemia
(Gagne et al., 1977; Mayan et al., 1996), and for
patients receiving intravenous administration of fat
emulsions (Nosanchuck, Roark & Wanser, 1974;
Shah, Patel & Rao, 1975; Nicholls, 1975, 1977; Creer
& Ladenson, 1983; Artiss & Zak, 1987; Sandberg,
Sonstabo & Christensen, 1989; Cantero, Conejo &
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SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 29
Jimenez, 1996). After the study of various types of
hyperlipoproteinemias, it was observed that erroneous
Hb and MCHC >36 g/dl were observed in patients
with at least 20 g/l of triglycerids, corresponding to
type I and part of type V hyperlipoproteinemias (rich
in chylomicrons) but not to type IV hyperlipoprote-
inemias (rich in very low density lipoproteins; Gagne
et al., 1977). Samples taken after a meal may at times
demonstrate superimposable spurious Hb measure-
ment. Even the most recent HA are sensitive to
hyperlipemia, although to a variable extent, as for
example Abbott Cell Dyn 4000 that is defined as giv-
ing true Hb values for up to 13 and 9 g/l of triglycerid
and cholesterol levels, respectively (M.C. Chrieten,
personal communication). Although excess of lipids
usually spuriously increases Hb, a spurious fall of Hb
was also reported once (Savage, 1989). Similar to the
presence of cold agglutinins, anomaly related to lipids
is suspected when MCHC is >36 g/dl, or when WBC
scattergrams demonstrate high number of particles of
low to moderate size (Figure 6). For laser-beam HA
that measure Hb level within each RBC and generate
the so-called « measured MCHC » or CHCM, a differ-
ence between the CHCM and the calculated MCHC is
observed. Various methods have been proposed to
obviate the abnormality on the relevant sample,
including isovolumetrical replacement of hyperlipemic
plasma with isoosmotic diluent, or ether extraction of
lipids. As previously mentioned, such methods may in
turn lead to erroneous PLT and WBC counts.
High WBC counts
White blood cell may induce excessive turbidity and
disturb Hb measurement if their number is sufficiently
high. There is no clear-cut threshold for WBC counts
associated with spuriously elevated Hb, but one must
be careful with all samples with WBC count over 50
or 100 · 109/l (Cornbleet, 1983; Sandberg, Sonstabo
& Christensen, 1989), although some HA define
threshold at 250 · 109/l or even seem to be fully
insensitive to WBC count because of entire WBC lysis
performed before Hb measurement (Sysmex). As for
lipid disturbance, CHCM determined on some HA
may help to find the true Hb value (McVeigh, Faim &
van der Weyden, 1989). However, RBC count may be
also disturbed by high WBC counts (see later), and
one must pay attention to the limits in recalculating
30 M. ZANDECKI ET AL. SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS
RBC indices, and a packed cell volume (centrifuged
Hct) should be considered.
Immunoglobulins
They have been reported to interfere with a number
of clinical laboratory tests (cf., see Roberts, Fontenot
& Lehman, 2000). False elevation of Hb measurement
by automated methods was observed for several
patients with Waldenström’s macroglobulinemia and
monoclonal IgM, or with multiple myeloma and
monoclonal IgA or IgG (Wallis & Ford, 1987; McMul-
lin, Wilkin & Elder, 1995; Roberts et al., 2000). This
anomaly is related to high levels of Ig that interact
with reagents of the lysis solution. For IgM, this phe-
nomenon has been related to the amount of mono-
meric component within the circulating paraprotein
(Goodrick et al., 1993). HA employing Hb conversion
to cyanmethaemoglobin seem to be more affected
than others, because of addition of surfactants for
cyanide-free methods. Red blood cell count and MCV
are unaffected in this situation but, as Hb is overesti-
mated, MCHC usually exceeds 36 g/dl. In order to
obtain more accurate results in this instance, it was
proposed to determine ‘plasma Hb’ after centrifugation
of the sample (which gives turbidity because of the
paraprotein) and to withdraw it from Hb of the whole
blood (Roberts et al., 2000). For Sysmex instruments,
it seems that if the sample is half diluted by the oper-
ator before analysis on the HA, the phenomenon does
not occur (Roberts et al., 2000). For laser-beam HA
that determine the measured MCHC (CHCM) a clear-
cut difference with the calculated MCHC generates an
alarm, and accurate Hb from the sample may be
obtained from measured MCHC.
Cryoglobulins
Spurious Hb values have been reported in some
instances, and several mechanisms are proposed to
explain these abnormal findings. In some cases spuri-
ously high Hb values were related to a mechanism
similar to that described above for immunoglobulins
(Taft et al., 1973; Cornbleet, 1983) or to the distur-
bance of light transmittance, whereas in other cases a
slight decrease of both Hb measurement and RBC
count was related to a flow anomaly (Taft et al., 1973;
Bremmelgaard & Nygard, 1991; Fohlen-Walter et al.,
Journal compilation  2007 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2007, 29, 21–41
2002). However, spurious Hb measurement is far from
being a consistent finding in the presence of cryoglob-
ulins (Fohlen-Walter et al., 2002).
Haemolysis
Hb free within plasma is measured together with that
from the RBC, but its amount ranges from 10 to
40 mg/l in normal conditions and does not affect total
Hb measurement. However, in situations related to
major intravascular haemolysis, including chemicals,
mechanical haemolysis associated with heart valves,
and haemolytic anaemias associated with blood trans-
fusion, free plasma Hb may be elevated enough to
affect total Hb measurement. MCHC may be >36 g/dl.
Centrifuged Hct shows a pink or red plasma tinge,
namely if free plasma Hb is >200 mg/l, and is, in some
instances, the only reliable RBC parameter. Some
laser-beam HA directly determine Hb within each
RBC, the Cellular Haemoglobin Concentration Mean
(CHCM), which allows the accurate Hb value to be
calculated. A short time lapse between venepuncture
and analysis is of crucial importance because haemo-
lysis may continue in vitro, leading to a spurious
decrease of RBC count and total Hb with a spurious
increase of free plasma Hb.
Chemical structure of haemoglobin and bilirubin
In physiological situations, Hb is more or less coupled
to oxygen or to carbon-di-oxide and, according to
which molecule is coupled to Hb, the peak of optimal
light absorbance differs slightly. The addition of
several reagents leave Hb free from coupled molecules
and changes it into one stable molecule (cyanmethae-
moglobin is an example), the latter demonstrating a
narrow peak of light absorbance, which allows accu-
rate determination of Hb concentration. However,
high amounts of carbon monoxide coupled to Hb
may not be fully transformed, and in such situations
a spuriously high Hb concentration is reported
(Cornbleet, 1983; Vinatier & Flandrin, 1993). In con-
trast, sulfhaemoglobin in high amount has been
reported as lowering Hb measurement (Cornbleet,
1983). Bilirubin: although one must pay attention to
very high amounts of bilirubin within the plasma,
most HA do not presently demonstrate any interfer-
ence with bilirubin, at least for concentrations up to
 2007 The Authors
 M. ZANDECKI ET AL.
250 mg/l. Above theses values, however, attention is
needed.
Spurious RBC counts and red cell parameters
According to the technology used, spurious results
may or may not be observed using some HA, and
some knowledge about the methods analysing blood
parameters is necessary. On impedance – type HA an
aliquot of the blood sample is diluted isoosmotically
and the number and height of electric pulses gener-
ated by the electrical resistance of RBC that pass
through a small orifice allow the determination of
both the RBC count and the MCV. Using laser-beam
methods, scattering at least at two angles allows the
determination of RBC count and MCV. In order to
improve accuracy, pretreatment of RBC with a specific
reagent that changes them isovolumetrically from dis-
coid to a sphere is performed on some HA. Setting dis-
crimination thresholds is an important consideration:
discriminating the smallest RBC from the largest PLT
is at times a challenge, already discussed (part I). On
the contrary, even in extreme pathological situations,
MCV does not exceed 150–160 fl and, as there is no
particle above that size in the blood stream in health
or in disease, HA do not analyse any particle above
200–300 fl in volume.
Spuriously elevated RBC counts
High WBC counts
Most HA enumerate RBC and WBC together within
the same channel(s), and the RBC count reported is
the sum of both RBC and WBC counts. In physiologi-
cal conditions, it is not of any importance, as it corres-
ponds to overestimate RBC count by 0.1% (if we
consider a WBC count of 5 · 109/l and a RBC count
of 5 · 1012/l). However, high WBC counts
(>100 · 109/l) may lead to a significant change in the
RBC count, particularly if the patient is also anaemic
(Bain & Bates, 2001). Moreover, in the latter
instances the MCV reported corresponds to the mean
volume of RBC and of WBC from the sample and
may be spurious, variably according to the nature and
the number of WBC from the relevant sample. So,
high WBC counts may induce several abnormal find-
ings, including Hb (discussed above), RBC count,
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Journal compilation  2007 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2007, 29, 21–41
SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 31
MCV, and subsequently lead to abnormal calculated
MCHC (discussed later).
Giant platelets
High number of giant PLT may lead to spuriously low
PLT count (see part 1) but, as they are enumerated as
RBC, they may also affect RBC count in a way similar
to that for WBC; however, RBC count is usually only
slightly affected in this instance (Cornbleet, 1983;
Bain & Bates, 2001).
Spuriously decreased RBC counts
Cold agglutinins
Cold agglutinins aggregate RBC when the temperature
is lower than 37 C. Unsurprisingly, peculiar anomal-
ies of RBC parameters in the presence of cold aggluti-
nins were reported first on counters acting at room
temperature (Hattersley et al., 1971; Petrucci, Duanne
& Chapman, 1971; Bessman & Banks, 1980). Accord-
ing to the HA, the upper threshold that may consider
particles as RBC into the RBC channel(s) is located
between 200 and 300 fl. So, only particles correspond-
ing either to isolated RBC or to small RBC clumps
(two or three RBC), are analysed, whereas large RBC
clumps are fully neglected by the HA. This leads to
spuriously low RBC counts and to abnormally high
MCV (each small RBC clump is considered as one sin-
gle particle). Haematocrit (RBC · MCV) is erroneous
and spuriously low, contrasting with Hb that is meas-
ured after RBC lysis and is unaffected by agglutinins.
As a rule, the MCHC is spurious, usually >36 g/dl. The
peculiar association of low RBC count, high MCV and
MCHC >36 g/dl is almost pathognomonic of the cold
induced artifact on instruments that work at laborat-
ory temperature, and not infrequently helps to diag-
nose the cold agglutinin in the patient analysed
(Petrucci, Duanne & Chapman, 1971). HA working
with reagents at temperatures near 37 C are not fully
insensitive to cold agglutinins, however, but changes
are less obvious and may remain undiscovered in some
instances (Solanki & Blackburn, 1985). HA that mea-
sure directly the amount of Hb within each RBC (mea-
sured MCHC, or CHCM) usually show discrepancy
between the measured MCHC and the calculated one.
Whatever the HA used, the common finding is that Hb
32 M. ZANDECKI ET AL. SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS
value is unaffected and that anomalies disappear when
the sample is warmed at 37 C and analysed promptly
afterwards. Amplification of the anomalies after the
sample has been cooled at 4 C for 1 or 2 h reinforces
the diagnosis. As viscosity of the sample may be high,
leading to inaccurate aspiration, an alarm may be
generated on some HA (insufficient sampling, or
related). Co-existence of RBC agglutination with
EDTA-dependant thrombocytopenia was reported but
antibodies directed against RBC and those directed
against PLT differed (Bizzaro & Fiorin, 1999).
Warm autoimmune haemolytic anaemia
In some instances warm autoantibodies were also
reported as inducing RBC agglutination, leading to
spurious MCV and RBC count, in a situation superim-
posable to that observed for cold agglutinins, but not
reversible by warming (Weiss & Bessman, 1984).
Very small RBC and discrimination with PLT
As discussed in part 1, PLT counts may be disturbed
by microcytic cells, namely if RBC volume is <36 fl:
some HA consider particles over this threshold as
RBC, and in severe microcytic anaemias PLT count
may be altered. Underestimation of RBC counts may
be observed in turn, because of the elimination of the
smallest erythrocytes from the RBC count. In such
instances up to 6% lowered RBC counts were repor-
ted (Savage et al., 1985).
Cryoglobulins
As previously mentioned, a slight decrease of both Hb
measurement and RBC count was reported in a few
instances, related to a flow anomaly (Taft et al., 1973;
Cornbleet, 1983; Bremmelgaard & Nygard, 1991; Foh-
len-Walter et al., 2002).
Considerations related to the quality of the sample.
Clotting, or abnormal mixing secondary to overfilling
of the sample (Pewarchuk et al., 1992), may lead to
an aspirated aliquot unrepresentative of the blood
sample. Cryoglobulins and cold agglutinins may lead
to high viscosity of the sample, leading to inadequate
aspiration.
Journal compilation  2007 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2007, 29, 21–41
In vitro haemolysis
As mentioned above, in some situations, namely hae-
molysis related to chemicals or blood transfusion, RBC
may continue to lyse within the sample, leading to
spuriously low RBC count, together with abnormal
Hb measurement and abnormal red cell indices (see
Hb measurement).
Mean cell volume and haematocrit
Some situations leading to spurious MCV (see Table 1)
are already discussed above [high WBC counts, cold
(warm) agglutinins]. Spurious Hct is a frequent find-
ing, related to abnormal MCV or/and to abnormal
RBC count as it is a parameter that is usually calcula-
ted by the HA: situations leading to abnormal Hct,
either increased or decreased, will not be discussed in
details as they may be deducted from MCV changes
(Table 1).
MCV and the technology used for measurement
Using impedance-type HA, disc-shaped RBC become
elongated into a cigar shape as they pass through the
aperture. Elongation is more pronounced for particles
flowing at the periphery of the orifice than at the cen-
tral part, and also when Hb content is low within the
RBC. Mean (red) cell volume is spuriously lowered
and MCH is spuriously elevated, mainly in hypochro-
mic disorders, and for MCV of 55 fl a spurious
decrease of up to 7 fl may be observed (Mohandas
et al., 1980; Paterakis et al., 1994; Bain & Bates, 2001).
Hydrodynamic focusing, a technical change that
directs the flow of particles at the centre of the aper-
ture, improves MCV measurement. For laser-beam
HA, the discoid shape of RBC was a major drawback
for peer analysis of RBC until the isovolumetric spher-
isation was applied, allowing current instruments to
generate accurate MCV (Mohandas et al., 1986). Some
impedance-type HA use both technical improvements
to analyse RBC (Abbott), whereas others measure the
Hct and calculate MCV using the HCT and RBC (Sys-
mex; ABX, Kyoto, Japan; some Beckman instruments,
Miami, FL, USA). In that latter instance however,
the presence of cold agglutinins, which disturbs RBC
count (see above) leads to erroneous MCV (Sysmex
XE-2100 operator’s manual – revised April 2004).
 2007 The Authors
 M. ZANDECKI ET AL. SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 33

Table 1. Situations leading to erroneous results on haematology analysers

Other parameters altered

WBC: spurious decrease

Agglutination of PMN (EDTA - related)
Agglutination of WBC other than PMN
(lymphocytes, lymphoma cells, leukaemic blasts)
Excess amount of K3-EDTA anticoagulant Shrinkage of RBC: MCV and Hctfl
Coagulation within the sample All parameters
WBC: spurious increase
PLT aggregates PLTfl
(Very) large PLT PLTfl
Nucleated red blood cells
RBC resistant to lysis (newborns, abnormal Hb,
chemotherapy, uraemia, liver disease…)
Cryoglobulin, cryofibrinogen, immunoglobulins PLT›
Lipids PLT›, Hb›, MCH›
Microorganisms (bacterial aggregates) PLT›
Others (adipose tissue, overfilling vacuum tubes) All parameters
RBC: spurious decrease
Cold agglutinins, warm agglutinins MCV›, MCH›
Very small RBC PLT›
Cryoglobulins (fl flow, inadequate aspiration) Hbfl, WBCfl, PLTfl
In vitro haemolysis Hb›, MCH›
Coagulation All parameters
RBC: spurious increase
High WBC counts MCV›, Hct›, MCH›
Giant PLT PLTfl
Haemoglobin: spurious increase
Lipids WBC›, Hb›, MCH›
High WBC counts MCH›, RBC›
Immunoglobulins (and cryglobulins) WBC›, PLT›, MCH›,
In vitro haemolysis MCH›
Carboxyhaemoglobin (high amount)
Bilirubin (>250–300 mg/l) MCH›
Haemoglobin: spurious decrease
Coagulation within the sample All parameters
Overfilling vaccum tube All parameters
Veinipuncture near a drip MCV› (glucose drip)
Sulfhaemoglobin
MCV
Cold agglutinins, warm agglutinins: › MCH›, RBCfl, PLTfl
High WBC counts: › RBC›
Hyperglycemia: › MCH›
K2 EDTA in excess: › MCHfl
Hyper- or hyponatremia: › or fl MCHfl or ›
Technology: impedance without hydrodynamic focusing MCH›
(MCVfl in hypochromic anaemias)
MCH > 36 g/dl (not related to spurious counts in some disorders: spherocytosis, xerocytosis, abnormal Hb: see text)
Cold agglutinins, warm agglutinins RBC, MCV›
Lipids WBC›, Hb›
Immunoglobulins Hb›
In vivo and in vitro haemolysis Hb altered, Hct
Carboxyhaemoglobin (>10–20%)
Bilirubin (>300 mg/l, at times less)
Immunosuppressive drugs (see also spuriously high Hb, spuriously low MCV)
MCH <32 g/dl
Hyperglycemia (see also spuriously high MCV, spuriously low Hb) MCV›

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Journal compilation  2007 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2007, 29, 21–41
34 M. ZANDECKI ET AL. SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS

RBC RBC

50 100 200 300 fL 50 100 200 300 fL

12 12
RBC = 1. 99 10 /l Hb = 6.4 g/dl RBC = 3.78 10 /l Hb = 12.5 g/dl
MCV = 141.5 fl MCH = 22.9 g/dl MCV = 97.1 fl MCH = 34.1 g/dl

Figure 7. Venepuncture performed near a glucose infusion. Blood sample from that patient was diluted (Hb low)
and excess of glucose led to a swelling of RBC: MCV was spuriously high and in turn MCHC spuriously low. Sam-
ple drawn correctly by the next morning showed normal values (no transfusion had been performed). RBC histo-
gram on diluted sample (left) and on new sample (right; Beckman Coulter STKS II).

present (blood drawn near a glucose drip). Studies

MCV and high WBC counts
As mentioned above, WBC are counted together with
RBC, and MCV reported is the mean of both RBC and
WBC volumes. In situations related to high WBC
counts and low RBC counts (e.g. acute leukaemias),
MCV is altered. Up to a 15–20 fl increase may be
observed in some instances. HA which calculate MCV
using the HCT and RBC (Sysmex, ABX, some Beck-
man instruments) are also sensitive to leukocytosis
over 100 · 109/l (Sysmex XE-2100 operator’s manual
– revised april 2004).
Severe hyperglycemia
Mean (red) cell volume is reported as spuriously high
in blood samples containing high levels of glucose,
either related to severe hyperglycemia in the relevant
patient or to a sample, which has been drawn near an
intravenous glucose infusion (Morse et al., 1981;
Strauchen et al., 1981; Holt, DeWandler & Arvan,
1982; Evan-Wong & Davidson, 1983; Savage & Hoff-
man, 1983; Planas, Van Voolen & Kelly, 1985; Van
Duijnhoven & Treskes, 1996). In the latter situation,
the sample is diluted leading at times to a severe spur-
ious macrocytic and hypochromic anaemia (Figure 7).
The calculated Hct is spuriously high and the MCH is
spuriously low: RBC count and Hb are unaffected in
diabetic patients and variably decreased if samples are
diluted because of glucose infusion (Figure 7). This
phenomenon is related to plasma glucose concentra-
tion, which balances that of intraerythrocytic, either
in the body or in the sample if an excess of glucose is
Journal compilation  2007 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2007, 29, 21–41
performed on RBC at various concentrations of glu-
cose have shown that within a few seconds after dilu-
tion into the HA (diluents from the manufacturers)
water penetrates within RBC rich in glucose, inducing
swelling: return to a normal size is progressively
observed and is complete after up to 5 min. According
to the time spent between dilution and MCV meas-
urement, the anomaly will be more or less conspicu-
ous (Holt, DeWandler & Arvan, 1982). The anomaly
is more obvious if RBC are isovolumetrically sphered,
because stabilization of RBC volume occurs within a
few seconds after the dilution of the sample (Van
Duijnhoven & Treskes, 1996). For the above-men-
tioned reasons HA are more or less sensitive to this
phenomenon, but glucose concentrations over
20 mmol/l may begin to increase MCV, and blood
glucose >35 mmol/l may overestimate MCV up to
50 fl (Holt, DeWandler & Arvan, 1982; Planas, Van
Voolen & Kelly, 1985; Van Duijnhoven & Treskes,
1996; Figure 7). Increased MCV leads to an increase
in the calculated Hct and to a spuriously low MCHC.
CHCM measured on some HA is also altered. CBC
demonstrating macrocytic hypochromia may be con-
sidered first as related to hyperglycemic sample.
Considerations related to the anticoagulant
The use of either K2- or K3-EDTA salt does not
induce any difference in the CBC, in optimal condi-
tions of sampling. However, when concentration of
anticoagulant is increased, because of insufficient
volume of blood drawn after venepuncture (or in
 2007 The Authors
 M. ZANDECKI ET AL.
neonates), some changes may be observed. Decrease
of centrifuged Hct resulting from shrinking of RBC
was reported in conditions related mainly to excess of
K3 salt (Goossens, Van Duppen & Verwilghen, 1991;
Hinchliffe, Bellamy & Lilleyman, 1992), in contrast
with data obtained on HA: no influence of K3-EDTA
concentration was observed on MCV, while K2-EDTA
at high concentrations resulted in a slight increase
in MCV, and such phenomenon could be observed
using several different HA (Goossens, Van Duppen &
Verwilghen, 1991).
Hyper- and hypo-natremia
Macrocytic and hypochromic RBC changes were
observed on blood samples in conditions related to
hypernatremia, whereas hyponatremia generated a
tendency towards microcytic and hyperchromic RBC
(Cornbleet, 1983). Such situations were also reported
in animals (Boisvert, Tvedten & Scott, 1999). Hyper-
and hypo-natremia are both situations also reported
as leading to spurious centrifuged Hct (Cornbleet,
1983).
Storage of the sample and MCV
EDTA used as anticoagulant allows the accurate deter-
mination of the CBC up to 24 h after the sample has
been drawn. However, after that time, MCV may
increase, namely if the sample is stored at room tem-
perature. This coupled with low haemoglobin could
cause the operator to suspect that a slightly microcytic
anaemia is normocytic and that a normocytic could be
mistaken for a macrocytic anaemia (Cohle, Saleem &
Makkaoui, 1981).
Mean cell haemoglobin content (MCHC)
Measured parameters allow the calculation of mean
cell haemoglobin of individual RBC [MCH as
expressed in pg ¼ Hb (g/l)/RBC (1012/l)] and MCHC
[MCHC in g/dl ¼ Hb (g/l) · 100/MCV (fl) · RBC
(1012/l)]. Some HA measure Hb concentration directly
within RBC, named cellular Hb concentration mean
(CHCM): discordant values (usually difference over
1.5 g/dl) between MCHC and CHCM allow in many
instances the detection of anomalies related to one of
the measured RBC parameters.
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Journal compilation  2007 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2007, 29, 21–41
SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 35
MCHC >36 g/dl is infrequent on most impedance-
type HA, whereas it may be occasionally observed on
laser-beam HA, in several constitutive disorders in
which RBC are ‘dehydrated’, including hereditary
spherocytosis, various haemoglobin disorders (CC, SC,
Cb thal), and some rare RBC disorders (xerocytosis).
Some acquired conditions mimic constitutive ones,
namely acquired immune haemolytic anaemias
caused by warm agglutinins, in which RBC coated
with warm antibodies may transform gradually into
spheres in vivo or in vitro after venepuncture, leading
to more or less dehydrated and spherised cells. Many
situations corresponding to abnormally high Hb val-
ues, and/or abnormally low RBC, and/or spuriously
low MCV, also lead to increase MCHC (see the corres-
ponding paragraphs and Table 1). Although the mech-
anism is unknown, some immunosuppressive drugs
may slightly increase MCHC, usually not above
37.5 g/dl (Cornbleet 1983).
MCHC <32 g/dl. As mentioned above, hyperglyce-
mia leads to artefactual hypochromic macrocytosis.
Storage of blood samples has been reported to cause a
facticious increase in percentage of hypochromic cells,
and become conspicuous after 24 h of storage
(Murphy, Spaven & Casey 2002). Although changes
are not major, the number of hypochromic cells may
be an important element to consider, as for example
in iron deficiency anaemias or during erythropoietin
therapy (Richardson, Bartlett & Will, 2001).
RETICULOCYTES
Enumeration of peripheral blood reticulocytes is
essential in the diagnosis and management of anaemic
patients and may be considered now as a part of the
CBC. If manual counting by light microscopy remains
the standard of reticulocyte enumeration, automated
methods developed during the past two decades are
now more accurate, precise, and cost-effective than
manual counting and, in addition, provide a variety of
reticulocyte-related parameters, such as volume, hae-
moglobin concentration, and maturity, which are
unavailable with light microscopy (Cavill, 1993; Van
Petegem et al., 1993; Davis et al., 1994; Koepke, 1999;
Brugnara, 2000; Siekmeier, Bierlich & Jaross, 2000;
Riley et al., 2001; Bain & Bates, 2001; Pierre, 2002;
Riley et al., 2002). Automated reticulocyte counts can
be performed using general purpose flow cytometers,
36 M. ZANDECKI ET AL. SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS
dedicated flow cytometers, or integrated with the
other parameters from the CBC under a fully automa-
tized method. RNA from reticulocytes is stained using
the classical new methylene blue (part of Abbott and
Beckman Coulter instruments), oxazine (Bayer), or
one among various fluorescent dyes, including thiaz-
ole orange (Horiba ABX), auramine O (Sysmex),
CD4K530 (Abbott), or coriphosphine O (Beckman
Coulter). According to the HA and to the reagent
used, flow cytometric analysis is performed, using
either the measurement of volume, conductivity, and
light scatter (VCS) or the fluorescence detection (for
review see Riley et al., 2002).
Careful gating of reticulocytes is crucial in deter-
mining accurate counts, because dyes stain also RNA
from PLT and WBC, and may also combine with the
DNA of nucleated cells. Red blood cells and reticulo-
cytes being larger than PLTs and smaller than WBC,
both the former are discriminated from both the latter
according to the size. Unsurprisingly, gating may be
difficult in the presence of blood components of
abnormal size and containing stained RNA/DNA.
Giant PLT, PLT clumps, abnormal WBC, abnormal
number of WBC, and WBC fragments, are situations
reported as a potential source of interference with
automated methods of reticulocyte analysis
(Oyamatsu et al., 1989; Pappas, Owens & Flick, 1992;
Riley et al., 2001). NRBC have been reported as dis-
turbing reticulocyte counts (Pappas, Owens & Flick,
1992; Riley et al., 2001), but, as NRBC themselves are
not counted as reticulocytes in the HA, it has been
hypothesized that abnormal reticulocyte count
observed in that instance could be due to increased
number of very young reticulocytes which frequently
appeared with NRBC (Oyamatsu et al., 1989).
After gating of RBCs, reticulocytes are identified
within the RBC population according to the coloured
particles or fluorescent material they contain. Cyto-
plasmic particles other than RNA that can be stained
by supravital dyes, including Howell–Jolly bodies,
Pappenheimer bodies, or basophilic stippling, may be
confused with reticulum granules using automated
techniques in HA, as well as they interfere in manual
techniques (Pappas, Owens & Flick, 1992; Ghevaert
et al., 1997; Riley et al., 2001), although such interfer-
ences have been found to be more or less conspicuous
according to the machines used (Oyamatsu et al.,
1989; Lofsness, Kohnke & Geier, 1994). Heinz bodies
Journal compilation  2007 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2007, 29, 21–41
observed in severe haemolytic anaemias, thalassaemia
major, congenital Heinz-body anaemia, or post-sple-
nectomy disorders, are also situations reported as dis-
turbing automated reticulocyte counts (Hinchliffe,
1993; Espanol, Pedro & Remacha, 1999; listed in Riley
et al., 2001), as well as sickle cells (Pappas, Owens &
Flick, 1992; Ghevaert et al., 1997), spherocytes
(Ghevaert et al., 1997), and haemoglobin H inclusion
bodies (Riley et al., 2001).
Although they are only faintly stained using fluor-
escent dyes, intraerythrocytic parasites (malaria, babe-
sia) may interfere with automated reticulocyte count,
and value six times higher than the manual count
was reported in a patient demonstrating 70% of RBC
infected with plasmodium falciparum (Laurencet,
Martinez & Beris, 1997).
Intensity of intracellular staining or fluorescence of
reticulocytes is assessed in most HA, the brightest reti-
culocytes being the youngest, leading to peculiar
indices such as the immature reticulocyte fraction
(IRF; for review see Brugnara, 2000). White blood
cells displaying intense staining were reported as lead-
ing to an erroneous estimation of reticulocyte matur-
ation index, the error being directly correlated with
the WBC count (Villamor et al., 1996). Very mature
reticulocytes contain only a few coloured dots and
may be insufficiently detected in neonates using flow
cytometry, possibly due to the low concentration of
the colouring matter used in some HA (Bayer; Wieg-
and et al., 2004). Although reticulocyte counts are s
after storing blood samples for 72 h at 4 C and 24–
48 h at room temperature (Brugnara et al., 1994;
Rudensky, 1997; Lacombe et al., 1999; Bain & Bates,
2001), immature reticulocyte fraction was observed as
stable for only 8 h at 4 C and 6 h at room tempera-
ture (Lacombe et al., 1999).
Various other situations are mentioned as gener-
ating occasionally spurious reticulocyte counts, inclu-
ding agglutinated cells in the presence of cold
agglutinins (Oyamatsu et al., 1989; Riley et al., 2001),
autofluorescence of RBC (porphyria, drugs) and diag-
nostic intravenous fluorescent dyes (Riley et al.,
2001), high amount of paraproteins, and haemolysis
(Riley et al., 2001).
Although depending on the software gate correc-
tions, in most of the above described situations the
HA show abnormal flags, prompting the technologist
to perform a manual count. However, exceptions to
 2007 The Authors
 M. ZANDECKI ET AL.
Table 2. Situations leading to possible interference
with automated methods for reticulocyte analysis
Inaccurate gating of RBCs: giant PLTs, PLT clumps,
abnormal WBCs, abnormal number of WBCs, WBC
fragments, nucleated red blood cells
Intraerythrocytic particles: Howell-Jolly bodies,
Pappenheimer bodies, basophilic stippling, Heinz bodies,
sickle cells, spherocytes, Haemoglobin H inclusions,
plasmodium, babesia
Others: cold agglutinin disease, autofluorescence of RBCs
(drugs, porphyria), paraproteins, haemolysis, diagnostic
intravenous fluorescent dyes
flagging have been reported, at least in cold agglutinin
disease (Oyamatsu et al., 1989) and in beta-thalas-
saemia major (Ghevaert et al., 1997; see Table 2).
CONCLUDING REMARKS
The widespread use of HA has led to major improve-
ment of cellular haematology, because of quick and
accurate results found in most instances, and now
preanalytical and analytical variables should be con-
sidered first within the laboratory when spurious
results from the HA are found (see also concluding
remarks from part I). In most situations, HA generate
flags or peculiar scattergrams in response to clinical or
artifactual parameters, independent of the technology
used. Inadequate blood sample may be responsible for
various anomalies, including initiation of coagulation
within the tube because of difficult venepuncture or
to low blood flow, excess of EDTA salt result of insuf-
ficient filling of the tube, which generate PLT and/or
WBC aggregates, fibrin precipitates, or others. Over-
filling of blood sample and time elapsed from samp-
ling to analysis must also be considered at times. In
several instances the disease itself generates changes
that disturb measurements performed by HA (RBC
agglutination, cryoprecipitates). Some anomalies,
however, are intrinsic to the technology used for ana-
lysis, and have led manufacturers to develop technical
changes in analysing blood cells, one example corres-
ponding to the use of hydrodynamic focusing, which
dramatically improved measurement of MCV in
impedance-type HA. Some protocols for measurement
of blood cells have been discarded, such as PLT counts
 2007 The Authors
Journal compilation  2007 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2007, 29, 21–41
SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS 37
performed after RBC lysis, leaving intraglobular com-
ponents free and enumerated together with PLT.
Improvements in analysis of PLT and RBC parameters
has led to an increase in the accuracy of results and
has also led to the generation of several flags in
abnormal situations. The enumeration of PLT in
thrombocytopenias, the identification of NRBC leading
to spurious PLT and WBC counts, and other situa-
tions, have led manufacturers to optimise computer-
ized analysis for the detection and analysis of every
blood cell, and moreover to develop further methods
for their specific identification and their specific enu-
meration on the same sample and on the same HA.
The improvement of WBC analysis and the careful
study of WBC differential scattergrams have led to
another major improvement in blood cell analysis,
allowing WBC differential to be performed automatic-
ally but also to exhibit and to give explanation for
several anomalous CBC results. Several peculiar WBC
scattergrams generated were progressively identified,
and related to spurious counts. As discussed all along
both reports, inaccurate identification, analysis, or
enumeration of one or several components from the
CBC leads in many instances to an abnormal WBC
differential scattergram. By the rule the WBC differen-
tial is flagged or invalidated, so a blood smear and an
optical count are often needed. Automated abnormal
WBC differentials related to spurious counts must be
included together with the various but insufficiently
reported situations related to the inability of the HA
to perform WBC differential or to identify specifically
one or several cells from the WBC differential. All
these situations should certainly need a specific
report.
So, if some spurious counts were found to be
numerous enough to generate technical improve-
ments to identify them clearly and create other meth-
ods for measurement, several other spurious counts
either do not generate specific flags to identify the
anomaly or even do not generate any flag at all.
Moreover, if the most recent and powerful HA are
able to generate several flags related to at least a part
of spurious counts, it is stressed that simpler HA,
namely those without any WBC differential scatter-
gram, will not. So, if acquiring a new HA is a personal
choice, each haematologist must know how his
machine will react when (at least) the situations
reported here occur.
38 M. ZANDECKI ET AL. SPURIOUS COUNTS ON HAEMATOLOGY ANALYSERS
ACKNOWLEDGEMENTS
We are indebted to Mathilde LINARD for kindly
reviewing English manuscript.
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