HaÈmatologie
Preanalytical Issues in Hematology
PraÈanalytik in der HaÈmatologie
Sheshadri Narayanan
Summary: Physiological variables such as age, gen-
der, circadian rhythm and effects of lifestyle such as ex-
ercise, smoking, and alcohol influence hematological
measurements. Specimen collection and processing var-
iables including analyte stability affect hematological
test results. Time-dependent morphological changes oc-
cur in cells, which are accentuated by changes in speci-
men-anticoagulant blood ratio and the temperature of
storage of specimen, thus impacting on the stability of
cell populations. The pH of specimen anticoagulant can
influence the size of the erythrocyte. Homogeneity of
sample prior to analysis is a prerequisite. In vitro circu-
lating antibody-specimen anticoagulant interactions can
introduce changes in platelets and leukocytes. Increase
in the concentration of some chemical analytes can af-
fect hematological test results.
Keywords: preanalytical; hematology variables;
physiological; specimen collection; anticoagulants;
processing.
Zusammenfassung: Physiologische Variablen wie
Alter, Geschlecht, circadiane Rhythmik und Effekte des
Lebensstils wie koÈrperliche AktivitaÈt, Rauchen und Al-
koholkonsum beeinflussen haÈmatologische Untersu-
chungen. Die Bedingungen der Specimensammlung
und -verarbeitung einschlieûlich AnalytenstabilitaÈt be-
einflussen Testergebnisse. Die Zellen erfahren zeitab-
haÈngig morphologische VeraÈnderungen, die durch die
relative Antikoagulanzienkonzentration und die Tempe-
ratur der Probenlagerung verstaÈrkt werden und die Sta-
bilitaÈt der Zellpopulationen beeinflussen. Der pH des
Antikoagulanz im Specimen beeinflusst die GroÈûe des
Erythrozyten. Die ProbenhomogeneitaÈt ist essentiell. In
vitro, koÈnnen Interaktionen von AntikoÈrpern und Anti-
koagulanzien PlaÈttchen und Leukozyten veraÈndern. Ver-
aÈnderte Konzentrationen mancher chemischer Analyte
kann haÈmatologische Untersuchungsergebnisse beein-
flussen.
Department of Pathology and Laboratory Medicine, Weill Medical
College of Cornell University, USA
Correspondence: Sheshadri Narayanan, Department of Pathology
and Laboratory Medicine, F 715, Box 79, Weill Medical College of
Cornell University, New York, USA.
Fax: +1 646 414 72 91
E-mail: Narayan_med_edu@msn.com
ã 2003 Blackwell Verlag, Berlin
Redaktion: Silke Heller
SchluÈsselwoÈrter: PraÈanalytik; HaÈmatologie; Pro-
benentnahme und -lagerung; Antikoagulanzien.
A wide range of physiological variables influence the
results obtained on hematological measurements.
Specimen collection and processing variables as well as
stability of cellular constituents affect hematological
results. Abnormal concentrations of selected biochemi-
cal analytes can impact on the quality of hematological
results.
Physiological Variables
Age
Hemoglobin (Hb) and Erythrocyte or Red blood cell
(RBC) counts are at a maximum in neonates (1 to 3
days old) [1, 2]. Thus the mean Hb concentration in a
neonate may approximate 185 g/L (115 mmol/L), while
the mean RBC level may approach 5.3 ´ 1012/L. Both
the Mean Corpuscular Volume (MCV) and the hema-
tocrit are also increased in the new born. At 2 weeks of
life the Hb concentration can drop by as much as
20 g/L (12.4 mmol/L) and the RBC count by approxi-
mately 0.4 ´ 1012/L, with a slight decrease in both hem-
atocrit and MCV. At 2 months of life, the Hb and RBC
count exhibit a sharp decrease of approximately 50 g/L
(31 mmol/L) for Hb and 0.9 ´ 1012/L for RBC count
compared to a 2-week-old baby. The hematocrit and
MCV are also at lower levels compared to a 2-week-old
baby. By age 18 years, the Hb, hematocrit, RBC count,
and MCV normalize to adult reference intervals. The
increase in Hb in the neonate has been ascribed to in-
creased arterial oxygen content leading to the destruc-
tion of RBC and the consequent increase in the Hb con-
centration [2]. Table 1 illustrates the progression of
reference intervals from newborn to adult values for
Hb, RBC, and MCV. Similar age-related changes are
seen for leukocyte or white blood cell (WBC) counts
and neutrophil and lymphocyte counts. The highest
mean WBC counts approximating 22.8 ´ 109/L are seen
12 hours after birth. In the 24 hours after birth, they
drop by approximately 3.9 ´ 109/L, and during the 1st
week after birth drop by as much as 10.6 ´ 109/L com-
pared to the value seen at 24 hours. The WBC counts
thereafter drop progressively until they normalize at
age 21 to adult reference intervals [3]. The total number
of neutrophils also reach the highest levels at 12 hours
J Lab Med 2003; 27 (7/8): 243±248 243
S. Narayanan: In Preanalytical Issues in Hematology

Table 1 Progression of reference intervals from newborn

Circadian rhythm
to adult values The WBC count is higher in the evening (P.M.) when
compared to counts in the morning (A.M.). This in-
#s given reflect mean values. Adapted from reference (3) crease is primarily due to an increase in circulating T-
Age Hb RBC MCV
lymphocytes, especially the CD4+ T helper cells whose
12
levels are higher in the P.M. hours as compared to A.M.
(mmol/L) (x10 /L) (fl) [1, 7]. In contrast to plasma cortisol rhythm with high-
1±3 days 115 5.3 108 est values in the early morning and lowest values at
2 weeks 103 4.9 105 midnight, the number of circulating lymphocytes peak
2 months 72 4.0 96
at 7:00 a. m., reaches the lowest values at noon (12:00
p. m.), begins to increase in the afternoon and reaches
2±6 years 78 4.6 81 highest values during sleep [7].
6±12 years 84 4.6 86
12±18 years
Male 90 4.9 88
Effects of Lifestyle
Female 86 4.6 90 Exercise
Note: fl=femtoliter. During or after exercise, there is an increase in the
WBC count [8]. Strenuous exercise can also result in
intravascular hemolysis and the depletion of haptoglo-
bin as it binds with free hemoglobin to clear the com-
after birth declining sharply at 24 hours. Thereafter, plex [1, 9].
they drop progressively before normalizing to adult
reference intervals at age 21 [1, 3, 4]. The lymphocyte Altitude
count is also increased at birth reaching a maximum at High altitude of 1,400 m has an effect of raising the Hb
6 months of age, at which time the neutrophil: lympho- and hematocrit up to 8 %. Sports exercise at high alti-
cyte ratio is reversed [1, 3, 4]. The monocyte count is tude can have a similar effect [1, 2, 8]. The effect of hy-
increased for up to 2 weeks after birth, while the eosi- poxia at high altitude results in an increase in erythro-
nophil count remains elevated for up to 1 week after poietin leading to an increase in hemoglobin and
birth [1, 3±5]. In contrast, the basophil count is in- hematocrit. In this context, it should be noted that high
creased only transiently for up to 1 day after birth [1]. altitude climbers train either at high altitude or by
Table 2 illustrates the progression of reference inter- undergoing treatment with erythropoietin. Approxi-
vals from newborn to adult values for WBC, neutro- mately 3 weeks are required for adaptation to high alti-
phils and lymphocytes. tude and for alteration of biochemical analytes.
Climbers returning to ambient altitude require 3 weeks
Gender for adjustment of the affected laboratory tests to nor-
RBC count, hematocrit and Hb values are slightly lower mal.
in females when compared to males [6].
Smoking
Long-term smoking has an impact on hematological
constituents in blood. Since cigarette smoke contains
carbon monoxide, which has a much greater affinity for
Table 2 Range of WBC, neutrophils and lymphocyte
hemoglobin than oxygen, carbon monoxide levels in
counts: newborn to adult blood are increased. There is also an increase in hemo-
globin, WBC, and RBC counts. The MCV is also in-
#s reflect mean values. creased since the RBCs become larger. Long-term
Adapted from references (3 and 4) healthy smokers who smoked 1 pack of cigarettes a day
Age WBC neutrophils lymphocytes for 1 year were shown to have WBC count of approxi-
9 mately 1000 cells/mL (1x109/L), and those who smoked
(x10 /L) (x109/L) (x109/L) 2 packs a day had approximately 2000 cells/mL
12 hours 22.8 15.5 5.5 (2 ´ 109/L) higher when compared to healthy non smok-
24 hours 18.9 11.5 5.8 ers [1, 9].
1st week 12.2 5.5 5.0
Alcohol
6 months 11.9 3.8 7.3 Chronic alcoholism results in an increase in MCV
2 years 10.6 3.5 6.3 which may be due either to a direct toxic effect of etha-
6 years 8.5 4.3 3.5 nol on erythropoiesis or due to folate deficiency [10].
21 years 7.4 4.4 2.5

244 J Lab Med 2003; 27 (7/8): 243±248


Specimen collection and
processing variables
Anticoagulant used for specimen collection
Ethylene diamine tetraacetic acid (EDTA) is the most
commonly used anticoagulant for routine hematological
determinations. The pH of EDTA varies depending on
the salt that is used. The disodium (Na2) and dipotassi-
um (K2) EDTA salts have a lower pH compared to tri-
potassium (K3) EDTA whose pH is 7.5 ± 1.0. K2 EDTA,
in contrast, has a pH of 4.8 ± 1.0. While all the salts of
EDTA are hyperosmolar, causing the water to leave the
cells and the cells to shrink, the effect of low pH of Na2
EDTA and K2 EDTA is to actually cause the cells to
swell, thus compensating for the osmotically induced
shrinkage. Thus, the microhematocrit (centrifuged hem-
atocrit) is lowered with K3 EDTA and not with Na2 and
K2 EDTA [1]. Because of the swelling effect, it has
been reported that increasing the concentration of K2
EDTA beyond the nominal value of 1.5 g/L can result
in an increase in MCV [11]. The low pH of K2 EDTA
can be accentuated further in patients with metabolic
acidosis. It has been demonstrated that MCV can be
spuriously elevated in hemodialysis patients when
blood is collected in K2 EDTA as compared to results
obtained with K3 EDTA [12].
Stability of cellular constituents:
Time-dependent morphological changes occur in neu-
trophils upon collection of blood in EDTA. Both the
time between blood collection and the preparation of a
peripheral blood smear and the concentration of EDTA
influence morphological changes. Even at an optimal
EDTA concentration of 1.5 g/L of blood, minor changes
such as swelling of neutrophils and loss of structure in
the neutrophil lobes begin to occur. These changes are
followed by loss of granulation in the cytoplasm and
formation of vacuoles in the nucleus or cytoplasm of
cells [1, 13]. The nucleus swells further, with a cross-
over of nuclear chromatin when peripheral blood
smears are prepared between 1 and 3 hours after speci-
men collection at an optimal EDTA concentration.
Preparation of peripheral blood smears beyond 3 hours
after blood collection, even at an optimal EDTA con-
centration, further distorts the morphological features
of neutrophils due to loss of bridges between the nu-
clear lobules and loss of cytoplasmic boundary. If a pe-
ripheral blood smear is prepared with an increased
EDTA concentration of 2.5 g/L of blood, approximating
to a blood collection tube filled to one half of its nomi-
nal volume, even immediately after blood collection,
the morphological features of the neutrophils are se-
verely distorted due to disintegration of cellular struc-
ture with a severe crossover of nuclear and cytoplasmic
boundaries [13].
The mononuclear cells undergo similar time-related
and concentration-related changes, but to a lesser extent
than the neutrophils. Platelets undergo changes such as
swelling, which can give rise to giant platelets that may
S. Narayanan: In Preanalytical Issues in Hematology
eventually disintegrate yielding fragments in the same
size range as the platelets. Platelet counts are spuriously
increased if these fragments are counted as platelets [1].
Mean platelet volume (MPV) measurements in EDTA
are unreliable since the platelets rapidly change their
shape from discoid to spherical upon blood collection
[14].
Because of time-dependent cellular changes in blood
collected in EDTA, the stability of WBC differential
count, especially the neutrophils, is limited after blood
collection for up to 6 hours of storage at room temper-
ature in most of the 5-part differential analyzers [1, 14].
The stability at room temperature even of bone mar-
row aspirates collected in EDTA decreases with time,
thereby inducing spurious dyserythropoietic changes
that could lead to an incorrect diagnosis of myelodys-
plastic syndrome. Thus, upon storage for 1 day at room
temperature, the mean percentage of erythroblasts with
cytoplasmic vacuoles increased by 22.2 % compared to
analysis at initial time and nuclear shape changes in-
creased by 6.21 %. However, these changes were signif-
icantly reduced when the bone marrow aspirates col-
lected in EDTA were maintained under refrigerated
conditions [15].
With some of the limitations inherent in EDTA, the
question has been raised whether EDTA is still the ad-
equate anticoagulant for specimen collection intended
for routine hematology determinations [14].
An alternative anticoagulant suitable for MPV meas-
urements has been proposed [16]. This anticoagulant
formulation consists of a mixture of trisodium citrate
(17 mmol), pyridoxal phosphate (11.3 mmol) and Tris
(hydroxymethyl) amino methane (24.6 mmol). In con-
trast to EDTA, with this anticoagulant mixture, MPV
was stable at room temperature (25° C) for 24 hours.
There was no evidence of platelet clumping since pyri-
doxal phosphate is an effective antiaggregant and disag-
gregant. Results of other routine hematology tests, such
as cell counts and indices, obtained on a healthy popu-
lation with this anticoagulant mixture were comparable
to that obtained with EDTA [16].
In addition to EDTA, other anticoagulants, such as
Acid citrate dextrose (ACD) and heparin have found ap-
plication for the separation of mononuclear cells (lym-
phocytes and monocytes). However, beyond 24 hours of
specimen collection, with all these three anticoagulants,
the lymphocyte fraction became contaminated with
granulocytes [17]. Lymphocyte separation is apparently
compromised with time in EDTA blood to the extent that
9 RBCs were present for every lymphocyte that was sep-
arated using 2-day-old EDTA blood [18].
ACD and heparin are reported to be superior to
EDTA for maintaining viable RBCs overnight in speci-
mens intended for flow cytometric analysis [19]. How-
ever, the viability of WBCs is comparable in EDTA,
ACD and heparin in specimens analyzed the same day.
It is only at and beyond 24 hours that granulocyte via-
bility can decrease substantially in blood specimens
collected in EDTA [19].
J Lab Med 2003; 27 (7/8): 243±248 245
S. Narayanan: In Preanalytical Issues in Hematology
The anticoagulant used for blood collection is re-
ported to influence in vitro studies of platelet, mono-
cyte, and neutrophil activation [20]. Thus, when EDTA,
citrate, heparin and hirudin were compared as anticoa-
gulants for blood collection, both monocyte activation
as measured by release of tissue factor and tumor ne-
crosis factor, and neutrophil activation as measured by
lipopolysaccharide-induced release of lactoferrin were
found to be lowest with EDTA [20].
The comparison of the above-mentioned four antico-
agulants in platelet activation studies by the measure-
ment of the platelet alpha-granule marker, platelet fac-
tor 4 (PF4) indicated that EDTA apparently suppresses
platelet activation. Thus PF4 levels were lowest with
EDTA-collected platelet-poor plasma (217 mg/L), when
compared to unfractionated heparin (1,180 mg/L) which
activated platelets significantly. While the PF4 levels
obtained on both platelet-poor plasma derived from cit-
rate (440 mg/L) and hirudin (469 mg/L) were compara-
ble, they were higher than that obtained with EDTA and
significantly lower than that obtained with unfractio-
nated heparin [20].
Homogeneity of sample prior to analysis
Proper mixing of blood sample immediately prior to
analysis to achieve homogeneity is a prerequisite for
hematological analysis. Overfilling of a blood collec-
tion tube can eliminate headspace or bubble required to
effect proper mixing especially when a rocking-type
mixer as opposed to a rotary-type mixer is used. Inex-
plicable results were reported in one study in which the
hemoglobin value had doubled compared to a value ob-
tained a week before on the same patient, while the
WBC and platelet counts were drastically reduced.
However, after the analysis was repeated on the same
specimen 4 times, results on hemoglobin, WBC and
platelets were comparable to results obtained on the pa-
tient's specimen a week before. Apparently, the remov-
al of aliquots of blood during repeated analysis created
sufficient head space for the air bubble to move to
achieve thorough mixing on the rocking-type mixer
[21].
The importance of achieving thorough homogeniza-
tion of blood specimen immediately prior to analysis
was highlighted in a study comparing hemoglobin
measurements performed by trained nurses and labora-
tory personnel on a point-of-care (POC) analyzer.
When the results obtained by nurses and laboratory per-
sonnel were compared to results obtained on a mecha-
nized analyzer in the laboratory, the correlation
achieved in the hands of the laboratory personnel was
excellent (N = 103, r = 0.99, slope = 0.98 and y inter-
cept = 0.11). However, the correlation in the hands of
the nurses thoroughly trained to operate the POC ana-
lyzer was poor (N = 235, r = 0.61, slope = 0.81, y inter-
cept = 2.83). The discrepancy between the results ob-
tained by nurses and the laboratory personnel was
246 J Lab Med 2003; 27 (7/8): 243±248
attributed to the fact that the samples in the laboratory
were mixed thoroughly on a rotary-type mixer, whereas
at the nurse's station, the POC samples were mixed
manually by rotating tubes by hand , apparently result-
ing in non-uniform and improper mixing [22].
Antibody-specimen anticoagulant
interactions
EDTA-dependent pseudothrombocytopenia is an in vi-
tro phenomenon of platelet agglutination that is encoun-
tered in blood collected in EDTA due to the presence of
antibodies in blood that react with platelets [1]. These
antibodies target antigens such as the glycoprotein IIb/
IIIa complex sequestered within the platelet membrane.
These antigens become exposed when EDTA chelates
calcium. At low (4° C) and room (25° C) temperature
the exposed platelet antigen further becomes modified
to permit platelet-derived antibodies to agglutinate pla-
telets. This EDTA-induced phenomenon of pseudo-
thrombocytopenia is, however, abolished when the
blood specimen is brought to 37° C, as the glycoprotein
IIb/IIIa complex is dissociated at this temperature. In
addition to spuriously decreasing platelet count, EDTA-
induced pseudothrombocytopenia can also spuriously
increase the WBC count, especially if the platelet ag-
gregates are in the same size range as WBCs, and, as
such, are included in the WBC count. In contrast, if the
platelet aggregates clump neutrophils and the size of
the large neutrophil-platelet clumps exceed the size
range for counting WBCs by the instrument, both pseu-
dothrombocytopenia and pseudoleukopenia are ob-
served in EDTA anticoagulated blood stored at room
temperature (25° C) which is abolished when the blood
is warmed to 37° C [23].
The mechanism for EDTA-induced pseudothrombo-
cytopenia is apparently due to EDTA-dependent IgG
antibodies that are directed to an epitope on platelet
membrane glycoprotein IIb [24].
EDTA-dependent IgG autoantibodies that are direc-
ted to the glycoprotein IIb/IIIa complex in the platelet
membrane as well as to the neutrophil Fc-gamma recep-
torIII (CD16) cause an in vitro phenomena called plate-
let satellitism, so named since the platelets virtually sur-
round the neutrophils [25]. This phenomenon which is
observed at room temperature (25° C) is abolished at
37° C. However, peripheral blood smears prepared im-
mediately from freshly collected EDTA anticoagulated
or capillary blood do not evidence this phenomenon. In
addition to EDTA, occasionally platelet satellitism has
also been observed in both citrated and heparinized
blood [26]. Severe platelet satellitism can cause spuri-
ous pseudothrombocytopenia.
In addition to EDTA-induced IgG antibodies, spuri-
ously low automated WBC count has been observed
with an EDTA-dependent IgM antibody of low titer,
and most active at room temperature (25° C). The spuri-
ous WBC count was related to the antibody-induced

leukocyte aggregation noticeable on a peripheral blood
smear, with the aggregates falling outside the size range
for counting WBCs. This phenomenon was not ob-
served in the same patient's blood specimen collected
in either citrate or heparin [27].
Effect of chemical analytes:
A transient increase in MCV is observed when glucose
concentration exceeds 33.3 mmol/L (6 g/L). This in-
crease is observed when blood specimens are diluted
automatically by the instrument with an isotonic diluent
and analyzed immediately. The osmotic effect of glu-
cose causes water to enter the RBC as the cells are sus-
pended in an isotonic diluent leading to a spurious in-
crease in the MCV. However, if the sample is analyzed
5-minutes after microdilution, an accurate MCV result
is obtained as glucose within the cell slowly equili-
brates leading to an efflux of water along with glucose
from the RBC [28].
Spuriously high hemoglobin values are obtained in
hyperlipidemic specimens with a triglyceride concen-
tration exceeding 11.29 mmol/L (10 g/L). The effect of
lipemia can be overcome by either resuspending the
RBCs in saline prior to analysis or by making correc-
tions with a plasma hemoglobin blank [28].
Spurious increases in WBC count and hemoglobin
have been observed in the presence of monoclonal pro-
teins in myeloma patients or in macroglobulinemia due
to the turbidity resulting from the precipitation of these
proteins by the lysis reagents that are used to lyse RBC
by the instrument [28].
RBC agglutination or rouleaux formation can be in-
duced due to a variety of conditions such as for exam-
ple hyperfibrinogenemia, and when patients are receiv-
ing infusions of dextran or polyvinyl-pyrolidon [2].
These RBC aggregates could be counted as single cells
thus resulting in a spurious decrease in the RBC count.
These RBC aggregates can lead to an overestimation of
MCV and an underestimation of hematocrit since the
MCV of the aggregate is less than the volume of RBC
in the aggregate. The presence of cryoproteins, cryoglo-
bulins and cryofibrinogen can lead to spurious increases
in WBC and platelet counts since the cryoprotein aggre-
gates are counted as WBC, while the cryoglobulin crys-
tals are counted as platelets. This phenomenon is abol-
ished in blood warmed to 370 C [29].
Conclusion
A variety of preanalytical variables can influence hema-
tological measurements. Optimization of anticoagulant
for specimen collection can considerably minimize
the component of controllable preanalytical error.
Although, EDTA is the widely used anticoagulant for
routine hematological measurements, recommending
one EDTA salt (K2EDTA) over another (K3EDTA) is
S. Narayanan: In Preanalytical Issues in Hematology
not entirely without pitfalls, since MCV can be spuri-
ously elevated in hemodialysis patients in specimens
collected in K2EDTA.
Indeed the time is ripe for an alternative anticoagu-
lant that can replace EDTA and also improve specimen
stability. Finally, while a variety of anticoagulants have
been used for specialized hematological studies, what is
lacking is an evidence-based approach towards evaluat-
ing the efficacy of alternative anticoagulants for their
application not only in routine hematological analysis,
but also for specialized studies on platelets, mononu-
clear cells and neutrophils. Clearly there is a need for
such an endeavor.
References
1. Narayanan S. The preanalytic phase. An important component
of laboratory medicine. Am J Clin Pathol 2000; 113:429±52.
2. Guder WG, Narayanan S, Wisser H, Zawta B. Samples: From
the patient to the laboratory: The impact of preanalytical variables
on the quality of laboratory results. 2 ed. Darmstadt, Germany: GIT
VERLAG, 2001:165pp.
3. Perkins SL. Normal blood and bone marrow values in humans.
In: Lee GR, Foerster J, Lukens J, Paraskevas F, Greer JP, Rodgers
GM, eds. Wintrobe's Clinical Hematology. 10th edition, Vol. 2,
Baltimore: Williams and Wilkins 1999:2738±48.
4. Monroe BL, Weinberg AG, Rosenfeld CR, Browne R. The neo-
natal blood count in health and disease, I: Neutrophilic cells. J
Pediatr 1979;95:89±98.
5. Weinberg AG, Rosenfeld CR, Monroe BL, Browne R. Neonatal
blood cell count in health and disease, II: Values for lymphocytes,
monocytes and eosinophils. J Pediatr 1985;106:462±6.
6. Kratz A, Lewandrowski KB. Normal reference laboratory
values. N Engl J Med 1998;339:1063±72.
7. Miyawaki T, Taga K, Nagaoki T, Seki H, Suzuki Y, Taniguchi N.
Circadian changes of T lymphocyte subsets in human peripheral
blood. Clin Exp Immunol 1984;55:618±22.
8. Young DS. Effects of preanalytical variables on clinical labora-
tory tests. 2 ed. Washington D.C.: AACC press, 1997:1283 pp.
9. Statland BE, Winkel P. Effect of preanalytical factors on the in-
tra-individual variation of analytes in the blood of healthy subjects:
Consideration of preparation of the subject and time of venipunc-
ture. Crit Rev Clin Lab Sci 1977;9:105±44.
10. Rico H. Alcohol and bone disease. Alcohol Alcoholism 1990;
25:345±52.
11. Goosens W, Van Duppen V, Verwilghen RL. K2 or K3EDTA: the
anticoagulant of choice in routine hematology? Clin Lab Haematol
1991;13:291±5.
12. Asanuma M, Taguchi C, Kumagai T, Uesaka H, Hosokawa H,
Kuriya SI. The hydrogen ion concentration (pH) in blood samples
with K2EDTA and K3EDTA affects mean corpuscular volume values
in Hemodialysis patients. Lab Hematol 2000;6:67±72.
13. Sacker IS. Specimen collection: In. Lewis SM, Coster JF, edi-
tors. Quality control in Hematology. Symposium of the international
committee for standardization in Hematology. New York: Academic
press, 1975:211±29.
14. Reardon DM, Warner B, Trowbridge EA. EDTA, the traditional
anticoagulant of hematology: with increased automation is it time
for a review? Med Lab Sci 1991;48:72±5.
15. Wang LJ, Glasser L. Spurious dyserythropoesis. Am J Clin
Pathol 2002;117:57±9.
16. Lippi U, Schiniella M, Modena N, Nicoli M, Lippi G. Advan-
tages of a new anticoagulant in routine hematology on the Coulter
Counter S-Plus STKR analyzer. Am J Clin Pathol 1990;93:760±4.
J Lab Med 2003; 27 (7/8): 243±248 247
S. Narayanan: In Preanalytical Issues in Hematology
17. Narayanan S. Effect of anticoagulants used for blood collection
on laboratory tests. Jpn J Clin Pathol 1996;103:73±80.
18. Nicholson JKA, Jones BM, Cross D, McDougal JS. Comparison
of T-and B-cell analysis on fresh and aged blood. J Immunol
Methods 1984;73:29±40.
19. Carter PH, Resto-Ruiz S, Washington GC, Ethridge S, Palini A,
Vogt R, et al. Flow cytometric analysis of whole blood lysis, three
anticoagulants, and five cell preparations. Cytometry 1992;13:68±
74.
20. Engstad CS, Gutteberg TJ, Osterud B. Modulation of blood cell
activation by four commonly used anticoagulants. Thromb Haemost
1997;77:690±6.
21. Pewarchuk W, Vanderbroom J, Blajchman MA. Pseudopolycy-
themia, pseudothrombocytopenia, and pseudoleukopenia due to
overfilling of blood collection vacuum tubes. Arch Pathol Lab Med
1992;116:90±2.
22. Neville RG. Evaluation of portable haemoglobinometer in gen-
eral practice. BMJ 1987;294:1263±7.
248 J Lab Med 2003; 27 (7/8): 243±248
23. Moraglio D, Banfi G, Arnelli A. Association of pseudothrombo-
cytopenia and pseudoleukopenia. Scand J Clin Lab Invest 1994;54:
257±65.
24. Fiorin F, Steffan A, Pradella P, Bizzaro N, Potenza R, DeAngelis
V. IgG platelet antibodies in EDTA-dependent pseudothrombocyto-
penia bind to platelet membrane glycoprotein IIb. Am J Clin Pathol
1998;110:178±83.
25. Bizzaro N, Goldschmeding R, van dem Borne AE. Platelet satel-
litism is Fc gamma R III (CD16) receptor mediated. Am J Clin Path-
ol 1995;103:740±4.
26. Peters MJ, Heyderman RS, Hatch DJ, Klein NJ. Investigation of
platelet-neutrophil interactions in whole blood by flow cytometry.
J Immunol Methods 1997;209:125±35.
27. Hillyer CD, Knopf AN, Berkman EM. EDTA-dependent leu-
koagglutination. Am J Clin Pathol 1990;94:458±61.
28. Cornbleet J. Spurious results from automated hematology cell
counters. Lab Med 1983;14:509±14.
29. Bridgen ML, Dalal BI. CE update-Spurious and artifactual test
results I. Cell counter-related abnormalities. LAB MED 1999;30:
325±34.