aging stability of complete blood count and white blood cell differential parameters analyzed by...

Aging stability of complete blood count and white blood

cell differential parameters analyzed by Abbott CELL-DYN

Sapphire hematology analyzerP. HEDBERG, T. LEHTO

INTRODUCTION

The recent trends towards large centralized laborato-

ries, and changes in laboratory organizations, have

brought redistribution activity to a new perspective.

Laboratories now test specimens that have been dis-

patched over a long distance; as a result, testing is

often delayed by 12–24 h or more after venipuncture.

Although laboratories should still give reliable results,

excessive delays in processing might affect the reliabil-

ity, accuracy and imprecision analysis. Cellular ele-

ments are known to have limited stability in

ethylenediaminetetraacetic acid (EDTA)-anticoagulat-

ed blood (Buttarello, 2004). Also, the different behav-

ior of automated counters using impedance and

optical methods may have an effect, and this should

be taken into account (Wood et al., 1999).

Of the three EDTA salts used for the anticoagula-

tion of blood specimens for hematological testing,

potassium salts are the most readily soluble (England

et al. 1993). K3EDTA is dispensed as a liquid and thus

causes a slight dilution of the specimen. This salt also

affects the red blood cell size at increased concen-

trations and on storage than the dipotassium salt.

Department of Clinical Chemistry,

Laboratory, Oulu University

Hospital, University of Oulu, Oulu,

Finland

Correspondence:

Pirjo Hedberg, Laboratory, Oulu

University Hospital, PO Box 500,

FI-90029 OYS, Oulu, Finland.

Tel.: +358 8 3155453;

Fax: +358 8 3154409;

E-mail: [email protected]

doi:10.1111/j.1751-553X.2007.01009.x

Received 31 May 2007; accepted

for publication 8 October 2007

Keywords

Automated blood cell counts,

hematology, hematology analyzer,

stability, WBC differential, WBC

flagging

SUMMARY

This study presents the results of an aging stability study of complete

blood count (CBC) and leukocyte differential parameters using the

Abbott CELL-DYN Sapphire hematology analyzer. Stability studies

showed no substantial change in CBC parameters up to 24–48 h at

+23 ± 2 �C (room temperature), except for optical platelet count

(PLTo). For specimens aged over 24, the value of impedance platelet

count yielded more reliable results than the routine PLTo. White blood

cell (WBC) differential parameters, except eosinophils, were stable for

up to 48 h at +23 ± 2 �C. CBC parameters were stable for 72 h, except

mean platelet volume, which slightly increased between 48 and 72 h,

at +4 �C. WBC differentials were stable 48–72 h, with a slight decrease

observed in absolute neutrophils and lymphocytes at +4 �C.

ORIGINAL ARTICLE INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

� 2008 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2009, 31, 87–96 87

Therefore, in 1993, the International Council for

Standardization in Haematology recommended that

K2EDTA should be used as the anticoagulant of choice

in specimen collection for blood cell counting and

sizing (England et al. 1993). However, the cellular ele-

ments in hematology specimens have limited stability

in EDTA-anticoagulated blood during storage at room

temperature or at +4 �C. Of these two temperatures,

conservation of specimens at +4 �C is known to

improve the stability of some complete blood count

and white blood cell differential parameters, but there

have not been any systematic studies that take into

account all the modern instruments or that considered

normal specimens, as well as a wide variety of pathol-

ogies, where a delay in analysis might give different

results (Buttarello, 2004).

In order to decide whether to accept or reject the

aged specimen, laboratory staff needs to be familiar

with the changes known to occur in blood speci-

mens during storage. For reticulocyte counts, the

National Committee for Clinical Laboratory Stan-

dards (NCCLS) H44-A protocol (National Committee

for Clinical Laboratory Standards, 1997) recom-

mended that analyses should be performed within

6 h of collection if the specimen is kept at room

temperature; conserving specimens at +4 �C stabilizes

parameters for 72 h. Also, for the differential leuko-

cyte count, it was recommended that smears should

be prepared for microscopic analysis within 4 h

(National Committee for Clinical Laboratory Stan-

dards, 1996).

Several studies have been published during recent

years that include data concerning the stability of

complete blood count and automated differential

count using different analyzers (Warner & Reardon,

1991; Briggs, Harrison & Grant, 1999; Wood et al.,

1999; Walters & Garrity, 2000; Gulati et al., 2002).

Our study investigated the stability of complete

blood count (CBC), reticulocyte and white blood cell

(WBC) differential parameters in EDTA-anticoagulated

blood at room temperature (+23 ± 2 �C) and at +4 �C.

The WBC viability fraction (WVF) and leukocyte flag-

ging changes were also followed during these condi-

tions. The measurements were undertaken using an

Abbott CELL-DYN Sapphire hematology analyzer (Ab-

bott Laboratories, Diagnostic Division, Abbott Park, IL,

USA) at Oulu University Hospital Laboratory, Oulu,

Finland.

MATERIALS AND METHODS

Analytical methods

The Abbott CELL-DYN Sapphire Hematology System

uses multi-angle polarized scatter separation (MAPSS)

and focused-flow impedance technologies, combined

with three-color fluorescent flow cytometry. This sys-

tem has a fully automated reticulocyte analysis with

immature reticulocyte fraction (IRF), a five-part WBC

differential, fluorescent DNA staining of nucleated red

blood cell absolute count (NRBC), optical and imped-

ance platelet measurement and fully automated

monoclonal antibody testing for CD 3/4/8 and CD61.

A cyanide-free method is used to measure hemoglobin

(HGB) colorimetrically.

Samples

For these studies, residual fresh (<2 h) EDTA-antico-

agulated samples submitted for routine full blood cell

counts were used. All samples were drawn into Bec-

ton Dickinson Vacutainer K2EDTA tubes (Becton

Dickinson Vacutainer, Cat. No. 388010, Becton Dick-

inson Systems, Plymouth, UK). Sample selection crite-

ria were used only to ensure a representative balance

of normal and abnormal parameter ranges. Data

exclusions were only made when there was clear evi-

dence of incomplete or suboptimal sample aspiration

or processing faults. Samples were not selected accord-

ing to any special treatment ward. All samples were

processed anonymously.

Aging stability

Every sample was analyzed at time point of <2 h

(baseline measurement). Twenty-five different sam-

ples were maintained after the first measurement at

room temperature (+23 ± 2 �C) and were reanalyzed

after 6-, 24-, 48- and 72-h storage. The specimens

randomly collected from routine workflow for these

studies included hematologically abnormal samples.

Abnormal cells may have a different stability from

normal cells. Three of the samples had an abnormal

WBC, with immature granulocytes (IG) being flagged

at the baseline measurement. In the second study, 40

samples were divided into four aliquots and stored

in polystyrene tubes (75 · 12 mm, ref. 55.476.005


Journal compilation � 2008 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2009, 31, 87–96

88 P. HEDBERG AND T. LEHTO AGING STABILITY OF AUTOMATED HEMATOLOGY PARAMETERS

Sarstedt, D-51588) at +4 �C. One aliquot from each

sample was analyzed after 6, 24, 48 and 72 h. However,

it is not known how aliquoting into nonblood collec-

tion tubes could affect any analysis. It is difficult to

maintain specimens at +4 �C when using automated

sample loading. Samples stored at +4 �C were initially

cold, taken from the refrigerator and then reanalyzed

after a brief warming period (1–5 min) at 23 ± 2 �C. All

of the time measurement groups (6, 24, 48 and 72 h)

included pathologic specimens according to the data

attained from the first measurements. At baseline

measurements, six of the samples in the group stored

for 6 h at +4 �C were flagged as abnormal, with one

variant lymphocyte (VARLYM), three IG, one uniden-

tified fluorescent population (FP?) or NRBC and one

BLAST flags. Of the samples stored for 24 h, nine

were abnormal (five IG and four BAND flags) and

eight of the samples in the group stored for 48 h were

abnormal (five VARLYM, one IG, one FP? or NRBC

and one BAND flags). Of the samples stored for 72 h,

eight were abnormal (four IG, one FP? or NRBC and

three BLAST flags). In the third study, 20 different

samples (six were abnormal from the beginning; four

IG, one BLAST and one FP? or NRBC flags) were

stored at +4 �C in between the measurements (6 h,

24 h and 48 h) and warmed to room temperature

before analysis (altogether four cycles). Additionally,

the stability of the leukocyte flagging was studied. The

suspect population flags BAND, IG, BLAST, VARLYM

and FP? were followed up, as were any positive NRBC

results. Stability profiles were derived from longitudi-

nal comparisons of parameter changes.

Statistical analysis

Statistical processing was performed using personal

computer EXCEL software. The statistical significance of

the differences between the means was assessed by

Student’s t-test as appropriate. P < 0.05 was consid-

ered statistically significant.

RESULTS AND DISCUSSION

Aging stability at +23 ± 2 �C (room temperature)

Changes observed in the CBC results of blood speci-

mens stored at +23 ± 2 �C are summarized in Table 1.

At this temperature, the WBC, RBCi (impedance mea-

surement of red blood cells), RBCo (optical measure-

ment of RBC), HGB, MCH (mean corpuscular

hemoglobin), RDW (red cell distribution width) and

PLTi (impedance measurement of PLT) were found to

be stable over the time, with mean percentage

changes of less than ±10%. However, a statistically

significant change (P < 0.05) was found for RDW at

48- and 72-h storage. The MCV (mean corpuscular

volume), HCT (hematocrit) and MPV (mean platelet

volume) increased over time, producing mean per-

centage changes over 10% within 72 h (10.6%,

11.1% and 11.1%, respectively). The statistically sig-

nificant changes were found at 24-, 48- and 72-h stor-

age for MCV, at 48- and 72-h storage for HCT and at

72-h storage for MPV. The MCHC (mean corpuscular

hemoglobin concentration) trended downward over

time, with change of 13.1% within 48 h. Statistically

significant changes were seen at all storage time

points. There was a proportional 12.1% decrease at

24-h storage, 19.8% decrease at 48-h storage and

24.4% decrease at 72-h storage for PLTo (optical mea-

surement of PLT). The R% (reticulocyte percent) and

RETC (reticulocyte absolute count) and IRF (imma-

ture reticulocyte fraction) fell slightly at 72 h, but not

at earlier time points, at +23 ± 2 �C. A statistically

significant change (P < 0.05) was found for R% at

24- and 72-h storage.

The results of the automated differential count

for specimens stored at +23 ± 2 �C are summarized

in Table 1. Storage of specimens at +23 ± 2 �Cresulted in a fast decrease in WVF (leukocyte viabil-

ity fraction) (P < 0.05 at 6-, 24-, 48- and 72-h stor-

age). After 24 h, the change was already )29.4%

and tended to decrease further with time. Storage of

specimens for 6 h resulted in decreased absolute

basophil counts ()31.1%). In contrast, prolonged

storage up to 72 h showed an increase in basophils.

Basophils represented the smallest fraction of all the

WBC. The standard deviations of the percentage

changes of the absolute basophil counts were very

high. Also, there was a poor analytical day-to-day

precision of the basophil counting (data not shown).

Additionally, one of the samples in this study with

very low absolute basophil counts measured after

the 6-h storage differed from the first measurement

more than the other samples thus giving a high

overall bias%. If this sample was not included in

the analysis, the decrease in the absolute counts of



P. HEDBERG AND T. LEHTO AGING STABILITY OF AUTOMATED HEMATOLOGY PARAMETERS 89

basophils would be 2%. Eosinophil counts had

decreased by 78.8% during 24-h storage and

showed a progressive decline over the time. Statisti-

cally significant (P < 0.05) changes were found for

eosinophils at 24-, 48- and 72-h storage. Also the

changes over 10% were seen for basophils and

monocytes at 72 h.

The stability of NRBC values were assessed and

any NRBC value from the analyzer was considered

as a positive result with discrimination level of

>0%. None of the samples analyzed at baseline, at

6-h and 24-h time points had a positive NRBC value.

Four of the samples analyzed after 48- and 72-h

storage had positive NRBC values (48 h: 0.35, 0.25,

0.27, 0.25 · 109/l and 72 h: 0.54, 0.44, 0.23,

0.39 · 109/l).

Aging stability at +4 �C

The effects of +4 �C storage on the CBC and automated

WBC differential parameters are shown in Table 2 and

Figures 1 and 2. Prolonged storage of specimens for up

to 72 h at +4 �C revealed stability of the CBC parame-

ters, reticulocyte parameters and monocyte counts

within 10% of their original values. However, a statisti-

cally significant change (P < 0.05) was seen in the

MCHC at 48 and 72 h. A decrease in PLTo at +23 ±2 �Cwas corrected by storage at +4 �C. A small increase in

MPV was seen during storage (up to 11.8%). A statisti-

cally significant change (P < 0.05) was observed for

MPV at 24-, 48- and 72-h storage. A substantial

decrease in the absolute counts of eosinophils and in

WVF with storage at +23 ± 2 �C was not observed

Table 1. Changes of CBC parameter and WBC differential values induced by storage of blood at +23 ± 2 �C. Data are

presented as means of parameter values, changes (% from the original value, which is 100%) at different time points

and standard deviation (SD) of changes in parenthesis. Statistically significant changes (P < 0.05; Student’s t-test)between the means are marked with asterisks (*)

<2 h 6 h 24 h 48 h 72 h

Mean Mean (%/SD) Mean (%/SD) Mean (%/SD) Mean (%/SD)

WBC, ·109/l 9.67 9.61 (99.3/2.6) 9.52 (98.2/2.4) 9.42 (96.8/4.2) 9.47 (97.7/2.5)

RBCi, ·1012/l 3.96 3.96 (100.0/0.8) 3.95 (99.8/1.1) 3.98 (100.7/0.7) 3.99 (100.71.0)

RBCo, ·1012/l 4.06 4.05 (99.9/0.6) 4.04 (99.6/0.8) 4.05 (99.5/0.8) 4.06 (100.0/0.6)

Hemoglobin, g/l 121 120 (99.1/1.2) 120 (99.7/0.7) 121 (99.6/0.9) 121 (99.7/0.6)

Hematocrit, l/l 0.36 0.37 (101.2/0.9) 0.39 (105.6/1.1) 0.40 (109.6/1.1)* 0.41 (111.1/1.3)*

MCV, fl 92.2 93.4 (101.3/0.2) 97.8 (105.8/0.6)* 101.5 (109.1/0.8)* 103.1 (110.6/0.8)*

MCH, pg 30.8 30.5 (99.1/1.2) 30.7 (99.7/1.3) 30.4 (98.8/1.0) 30.4 (98.8/1.1)

MCHC, g/l 334 326 (97.7/1.4)* 314 (93.6/1.4)* 299 (88.7/1.5)* 295 (86.9/1.5)*

RDW, %CV 13.2 13.3 (101.1/0.9) 13.8 (104.8/1.1) 14.1 (106.7/1.4)* 14.3 (107.9/1.8)*

PLTo, ·109/l 259 249 (95.8/2.2) 248 (95.8/2.2) 203 (80.2/6.4) 208 (75.6/8.4)

PLTi, ·109/l 252 262 (103.2/8.1) 262 (103.2/8.1) 268 (106.0/8.7) 268 (105.8/10.3)

MPV, fl 8.4 9.0 (102.1/2.5) 8.5 (100.6/3.0) 9.2 (104.6/10.9) 9.7 (111.1/9.1)*

RETC, ·109/l 75.6 74.7 (98.0/3.3) 68.2 (90.6/5.5) 72.2 (97.7/8.0) 65.6 (86.1/9.3)

R% 1.8 1.8 (97.8/3.4) 1.6 (90.4/5.9)* 1.7 (97.3/7.7) 1.6 (85.2/8.8)*

IRF 0.35 0.34 (95.1/11.0) 0.34 (94.1/9.3) 0.33 (91.9/10.2) 0.30 (79.0/17.3)

WVF 0.992 0.987 (98.8/1.0)* 0.789 (70.6/18.9)* 0.568 (13.8/39.4)* 0.441 ()41.6/54.3)*

Neutrophils, ·109/l 6.60 6.67 (101.5/3.3) 6.77 (103.2/3.7) 6.68 (101.4/5.5) 6.74 (102.5/4.8)

Lymphocytes, ·109/l 1.94 1.85 (93.4/7.6) 1.80 (91.3/9.4) 1.81 (92.0/10.5) 1.92 (97.9/10.9)

Monocytes, ·109/l 0.80 0.77 (96.1/9.2) 0.72 (91.2/13.2) 0.79 (91.8/21.9) 0.69 (64.0/41.3)

Eosinophils, ·109/l 0.28 0.28 (95.7/16.6) 0.17 (21.2/66.7)* 0.10 ()188.2/228.1)* 0.07 ()440.5/454.1)*

Basophils, ·109/l 0.04 0.05 (66.3/87.9) 0.05 (91.1/77.4) 0.05 (98.4/64.7) 0.05 (110.2/53.8)

WBC, white blood cell; RBCi, impedance measurement of red blood cells; RBCo, optical measurement of red blood

cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCHC, mean cell hemoglobin concentra-

tion; RDW, red cell distribution width; PLTo, optical measurement of platelets; PLTi, impedance measurement of plate-

lets; MPV, mean platelet volume; WVF, white blood cell viability fraction, RETC, reticulocyte count; R%, percentage

reticulocytes; IRF, immature reticulocyte fraction.




Tabl

e2.

Ch

an

ges

of

CB

Cpara

mete

ran

dW

BC

dif

fere

nti

alvalu

es

indu

ced

by

stora

ge

ofblo

od

at

+4

�C.D

ata

are

pre

sen

ted

as

mean

sofpara

mete

rvalu

es,

chan

ges

(%fr

om

the

ori

gin

al

valu

e,

wh

ich

is100%

)at

dif

fere

nt

tim

epoin

tsan

dSD

sof

chan

ges

inpare

nth

esi

s.Th

est

ati

stic

all

ysi

gn

ifica

nt

chan

ges

(P<

0.05;Student’s

t-test)betweenthemeansare

marked

withasterisks(*)

Batc

h1

Batc

h2

Batc

h3

Batc

h4

<2

h6

h<

2h

24

h<

2h

48

h<

2h

72

h

Mean

Mean

(%/S

D)

Mean

Mean

(%/S

D)

Mean

Mean

(%/S

D)

Mean

Mean

(%/S

D)

n=

40

(n=

20

inR

ETC

,R

%an

dIR

F)

WB

C,

·10

9/l

9.8

59.8

7(1

00.5

/1.8

)8.9

28.7

6(9

7.1

/5.8

)8.8

68.9

7(1

00.5

/4.6

)8.0

27.6

7(9

4.6

/4.5

)

RB

Ci,

·10

12/l

3.6

93.6

9(9

9.9

/0.6

)3.7

33.7

5(1

00.5

/1.5

)3.8

23.9

1(9

9.6

/0.6

)3.9

63.9

3(9

8.3

/4.0

)

RB

Co,

·10

12/l

3.7

83.7

8(1

00.1

/0.5

)3.8

33.8

3(9

9.9

/1.7

)3.9

33.9

1(9

9.6

/0.7

)4.1

04.0

6(9

8.1

/3.9

)

Hem

oglo

bin

,g/l

114

114

(99.4

/0.5

)116

118

(100.9

/1.8

)118

118

(100.3

/0.6

)124

123

(98.9

/3.1

)

Hem

ato

crit

,l/

l0.3

40.3

4(9

9.7

/0.7

)0.3

50.3

5(1

01.4

/1.9

)0.3

60.3

6(1

01.6

/1.0

)0.3

70.3

7(9

9.9

/3.8

)

MC

V,

fl93.4

93.3

(99.8

/0.3

)93.7

94.5

(100.9

/0.7

)93.4

94.7

(101.3

/0.3

)93.5

95.1

(101.7

/0.9

)

MC

H,

pg

31.1

30.9

(99.5

/0.7

)31.2

31.3

(100.5

/1.2

)31.1

31.1

(99.7

/1.1

)31.2

31.4

(100.4

/1.4

)

MC

HC

,g/l

332

331

(99.6

/0.9

)333

331

(99.5

/1.2

)333

328

(98.6

/1.3

)*334

330

(98.8

/1.7

)*

RD

W,

%C

V13.4

13.4

(99.9

/0.5

)13.4

13.3

(99.1

/1.7

)13.1

13.1

(99.5

/0.9

)12.9

12.8

(99.2

/1.3

)

PLTo,

·10

9/l

262

268

(102.2

/2.5

)286

268

(91.5

/15.9

)354

338

(94.8

/5.0

)294

298

(98.8

/3.0

)

PLTi,

·10

9/l

275

277

(100.7

/2.9

)301

286

(92.8

/14.5

)375

373

(98.4

/6.7

)308

317

(100.3

/3.5

)

MPV

,fl

8.3

8.5

(101.9

/2.1

)8.1

8.6

(106.4

/3.4

)*7.7

8.7

(110.2

/3.5

)*8.0

9.0

(111.8

/4.2

)*

RE

TC

,·1

09/l

73.5

74.5

(100.4

/5.9

)97.2

96.3

(95.5

/12.5

)76.0

75.4

(98.8

/5.7

)86.1

83.3

(95.5

/6.4

)

R%

2.2

2.2

(100.7

/5.7

)2.9

2.8

(92.0

/18.7

)2.1

2.1

(98.8

/5.7

)2.5

2.4

(94.3

/6.1

)

IRF

0.4

20.4

3(1

03.7

/5.5

)0.4

30.4

4(9

9.4

/13.2

)0.3

80.3

8(1

02.1

/6.8

)0.4

40.4

5(1

04.0

/5.3

)

WV

F0.9

92

0.9

80

(98.3

/2.2

)0.9

88

0.9

5(9

5.4

/4.6

)*0.9

90

0.9

10

(90.5

/5.4

)*0.9

82

0.8

00

(74.9

/16.1

)*

Neu

troph

ils,

·10

9/l

7.5

77.6

3(1

01.2

/2.5

)6.6

66.5

3(9

4.4

/12.4

)6.1

26.1

8(1

00.7

/3.9

)5.1

84.8

5(8

9.5

/13.2

)

Lym

ph

ocy

tes,

·10

9/l

1.2

71.2

1(9

4.5

/6.9

)1.4

01.4

2(9

5.5

/13.9

)1.6

21.5

8(9

2.7

/14.8

)1.9

01.8

8(9

3.9

/18.1

)

Mon

ocy

tes,

·10

9/l

0.9

30.8

4(1

00.8

/8.0

)0.6

30.6

7(1

01.2

/16.1

)0.7

90.9

0(1

08.4

/13.0

)0.6

90.7

2(1

01.6

/12.2

)

Eosi

noph

ils,

·10

9/l

0.1

40.1

6(1

09.4

/19.7

)0.2

50.1

8(7

5.6

/56.2

)0.3

10.3

1(9

8.4

/17.7

)0.2

40.2

3(9

4.7

/14.9

)

Baso

ph

ils,

·10

9/l

0.0

30.0

4(6

4.4

/88.5

)0.0

30.0

4(8

1.3

/81.3

)0.0

40.0

3(4

4.0

/125.6

)0.0

40.0

4(9

2.6

/74.0

)

WB

C,

wh

ite

blo

od

cell

;R

BC

i,im

pedan

cem

easu

rem

en

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during storage at +4 �C. However, a statistically significant

change was seen for WVF at 24-, 48- and 72-h storage.

A slight decrease in the absolute counts of lymphocytes

and absolute counts of neutrophils was present with

the storage at +4 �C, which was not seen at +23 ± 2 �C.

One sample analyzed at baseline and at 6 h had a

positive NRBC value in both measurements (0.88 and

1.00 · 109/l, respectively). None of the samples ana-

lyzed at baseline and 24 h had a positive NRBC value.

Five of the samples in the group analyzed at baseline

and 48 h had positive NRBC values in the latter mea-

surements (0.30, 0.30, 0.30, 0.25 and 0.18 · 109/l).

Finally, four of the samples in the group analyzed at

baseline and 72 h had positive NRBC values at 72 h

(0.47, 0.44, 0.24 and 0.57 · 109/l).

Aging stability at refrigeration and +23 ± 2 �C (room

temperature) cycles

To further evaluate the effect of storage at +4 �C and

rewarming to +23 ± 2 �C, 10 normal specimens were

assayed at baseline, 6, 24 and 48 h after collection

(Table 3). The samples were same for all the time-

point measurements. The only changes from the origi-

nal values at 6 h were seen with absolute basophil

counts ()24.6%). They progressively decreased 150%

from the original value at 72 h. The statistically signif-

icant change (P < 0.05) was observed at 48 h. A pro-

gressive loss of lymphocytes was also seen during

cycles up to 48 h. All the other parameters were sta-

ble under these conditions, reaching a less than 10%

change from the original values. However, a statisti-

cally significant change was observed for WVF at 24

and 48 h. Positive NRBC values were detected with

one sample at each measuring time point (0.85, 0.88,

0.95 and 1.08 · 109/l, respectively) and with two

samples 48 h (0.28 and 0.26 · 109/l, respectively).

CELL-DYN Sapphire flagging stability

Additional analysis of CELL-DYN Sapphire flagging

stability (Table 4) revealed better storage conditions

5060708090

100110120130

WBC

RBCi

RBCoHGB

HCTMCV

MCH

MCHCRDW

PLToPLTi

MPV

WBC

RBCi

RBCoHGB

HCTMCV

MCH

MCHCRDW

PLToPLTi

MPVW

BCRBCi

RBCoHGB

HCTMCV

MCH

MCHCRDW

PLToPLTi

MPV

WBC

RBCi

RBCoHGB

HCTMCV

MCH

MCHCRDW

PLToPLTi

MPV

% c

han

ge

fro

m in

itia

l val

ues

6 h

5060708090

100110120130

% c

han

ge

fro

m in

itia

l val

ues

24 h

5060708090

100110120130

% c

han

ge

fro

m in

itia

l val

ues

48 h

5060708090

100110120130

% c

han

ge

fro

m in

itia

l val

ues

72 h

Figure 1. Changes of CBC parameter values induced by storage of blood at +4 �C. Data are presented as mean

changes (% from the original value, which is 100%) at different time points (error bars represent the ±SDs). WBC,

white blood cell; RBCi, impedance measurement of red blood cells; RBCo, optical measurement of red blood cell;

HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCHC, mean cell hemoglobin concentration;

RDW, red cell distribution width; PLTo, optical measurement of platelets; PLTi, impedance measurement of plate-

lets; MPV, mean platelet volume.




for samples at +4 �C. IG flag appeared at 24 h at both

temperatures. BAND and IG flags also appeared at

24 h and the number of samples with IG flag progres-

sively increased over time at both temperatures.

BLAST cell flagging did not appear to be affected by

storage time or temperature.

DISCUSSION

Manufacturers of automated analyzers and published

literature often quote that blood specimens, kept at

either at room temperature or at +4 �C for up to 24 h,

generally yield reliable results for complete blood

count and automated differential count.

Recently, Muller et al. (2006), in a multicenter

evaluation of Abbott CELL-DYN Sapphire, studied the

aging stability with 10 normal EDTA-anticoagulated

samples at +4 �C and room temperature. They found

that proportions of nonviable leukocytes (predomi-

nantly neutrophils) progressively increased between

12- and 72-h storage at room temperature compared

with +4 �C, where viability remained above 90% for

up to 36 h. At both temperatures, the absolute WBC

count remained stable. Relatively little variation was

seen in the analysis of leukocyte population stability

stored at +4 �C up to 72 h, whereas at room tempera-

ture all parts of the leukocyte differential were stable

for up to 48 h, with the exception of a significant

decline (statistical processing was performed using

ANALYSE-IT Software, Leeds, UK) in the eosinophil frac-

tion after 12-h storage. Storage changes at +4 �C did

not exceed 10% during the 72 h period for RBC, reti-

culocytes and platelet count. However, at room tem-

perature, all parameters were stable for up to 24 h

with subsequent progressive changes exceeding 10%

being seen with the MPV (increase), immature reticu-

locyte fraction (decrease), percentage reticulocytes

(decrease) and optical platelet count (decrease). A less

significant trend for increasing MCV values after

hours of storage was noted. They also followed the

flagging stability of the CELL-DYN Sapphire and they

noted that, with the exception of IG flag (room

WVF

NEU#

LYMF#

MONO#

EOS#

BASO#

WVF

NEU#

LYMF#

MONO#

EOS#

BASO#

WVF

NEU#

LYMF#

MONO#

EOS#

BASO#

0

50

100

150

200

% c

han

ge

fro

m in

itia

l val

ues

6 h 24 h

020406080

100120140160180

% c

han

ge

fro

m in

itia

l val

ues

0

20

40

60

80

100

120

140

160

% c

han

ge

fro

m in

itia

l val

ues

48 h 72 h

0

50

100

150

200

WVF

NEU#

LYMF#

MONO#EOS#

BASO#

% c

han

ge

fro

m in

itia

l val

ues

Figure 2. Changes of WBC differential parameter values induced by storage of blood at +4 �C. Data are presented as

mean changes (% from the original value, which is 100%) at different time points (error bars represent the ±SDs).

WVF, white blood cell viability fraction; NEU, neutrophil; LYMP, lymphocyte; MONO, monocytes; EOS, eosino-

phils; BASO, basophils.




temperature), reliable flagging could be obtained for

up to 24 h, independent of storage temperature.

Flagging efficiencies of hematology analyzers have

also been found to be associated with total WBC

counts, with the flagging sensitivities being rather

poor in leukocytopenic samples and highest in sam-

ples with more than 10 · 109 WBC/l (Ruzicka et al.,

2001). In contrast, the flagging specificities were

found to be poorer with rising WBC counts. Leuko-

cytopenic and thrombocytopenic specimens have also

been found to have the greatest day-to-day mean per-

centage changes at room temperature and the widest

day-to-day standard deviations compared with the

changes observed for specimens with normal or high

initial respective counts (Gulati et al., 2002). Further-

more, the platelet counts of thrombocytopenic speci-

mens tend to increase over time, and samples with

higher initial platelet count tend to decrease.

Although the precision of new automated hemato-

logy analyzers have been improved, the precision per-

formance of each parameter has to be considered

when finalizing the results. In particular, the precision

of WBC count and differential count have been found

to be different during storage at room temperature

and at +4 �C (Wood et al., 1999). Also, the precision

might be different in different concentration levels. In

our analytical evaluation of CELL-DYN Sapphire (data

not shown), we found that between-day precisions

were higher for monocytes (17.8% at 0.2 · 109/l vs.

7% at 1.7 · 109/l), eosinophils (19.5% at 0.1 · 109/l

vs. 6.2% at 0.5 · 109/l) and basophils (42% at

0.1 · 109/l vs. 10% at 0.7 · 109/l) in the low concen-

trations compared with high concentration levels. Also

the imprecision of PLTo and RETC were �4.6% in

our study, thus may have had some effect on the

results of these stability studies.

Table 3. Changes of CBC parameter and WBC differential values induced by storage of blood at +4 �C and rewarming

before analyzing. Data are presented as means of parameter values, changes (% from original value, which is 100%) at

different time points and SDs of change percentages in parenthesis. The statistically significant changes (P < 0.05;Student’s t-test) between the means are marked with asterisks (*)

<2 h 6 h 24 h 48 h

Mean Mean (CV%/SD) Mean (CV%/SD) Mean (CV%/SD)

n = 20

WBC, ·109/l 9.30 9.31 (100.3/1.2) 9.26 (99.2/1.0) 9.10 (98.0/2.8)

RBCi, ·1012/l 3.72 3.73 (100.1/0.6) 3.72 (99.9/0.9) 3.73 (100.2/0.8)

RBCo, ·1012/l 3.80 3.80 (100.1/0.5) 3.78 (99.5/0.8) 3.78 (99.5/0.8)

Hemoglobin, g/l 116 115 (99.4/0.4) 173.2 (105.5/9.9) 116 (100.1/0.4)

Hematocrit, l/l 0.35 0.35 (99.7/0.6) 0.35 (101.0/0.9) 0.35 (101.2/0.9)

MCV, fl 94.1 93.7 (99.6/0.3) 95.1 (101.1/0.2) 95.1 (101.0/0.2)

MCH, pg 31.3 31.0 (99.3/0.7) 31.3 (100.2/0.9) 31.2 (100.0/0.9)

MCHC, g/l 332 331 (99.7/0.8) 329 (99.1/1.1) 328 (98.9/1.0)

RDW, %CV 13.4 13.4 (99.7/0.7) 13.5 (100.1/0.6) 13.4 (99.4/1.2)

PLTo, ·109/l 198 202 (102.0/2.6) 194 (98.1/2.9) 194 (98.0/4.5)

PLTi, ·109/l 207 211 (101.9/3.3) 205 (98.2/3.9) 204 (96.6/8.7)

MPV, fl 8.82 9.0 (102.1/2.5) 9.35 (105.7/2.5) 9.63 (108.5/2.4)

WVF 0.990 0.970 (97.2/4.6) 0.975 (98.6/0.9)* 0.950 (96.1/1.5)*

Neutrophils, ·109/l 6.95 7.04 (101.6/1.8) 7.06 (101.6/1.3) 6.99 (100.9/2.7)

Lymphocytes, ·109/l 1.30 1.22 (92.5/6.2) 1.15 (84.9/8.3) 1.10 (79.8/11.6)

Monocytes, ·109/l 0.87 0.85 (98.5/7.5) 0.88 (100.2/8.5) 0.86 (98.4/8.7)

Eosinophils, ·109/l 0.15 0.17 (108.4/18.5) 0.15 (100.3/15.7) 0.14 (92.3/19.6)

Basophils, ·109/l 0.03 0.04 (75.8/64.1) 0.03 (20.3/129.9) 0.02 ()48.3/191.6)*

WBC, white blood cell; RBCi, impedance measurement of red blood cells; RBCo, optical measurement of red blood

cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCHC, mean cell hemoglobin concentra-

tion; RDW, red cell distribution width; PLTo, optical measurement of platelets; PLTi, impedance measurement of plate-

lets; MPV, mean platelet volume; WVF, white blood cell viability fraction.




In general, if we only contemplate the stability of

the parameter changes to be acceptable within ±10%

of the original values, not from the clinically signifi-

cant point of view (MCV increasing over time etc.),

the CBC parameters were stable for up to 24–48 h at

room temperature, except PLTo.

In the CELL-DYN Sapphire system, thrombocytes

are measured by both PLTi and PLTo and the analyzer

creates an alert if the difference between PLTo and

PLTi exceeds the present limit. For specimens aged

over 24 h, the PLTi could be used instead of the rou-

tine PLTo to yield more reliable results. WBC differen-

tial parameters, except eosinophils, were stable to

48 h at +23 ± 2 �C, confirming the results of Muller

et al. (2006). CBC parameters were stable at 72 h,

except MPV, which slightly increased between 48 and

72 h, at +4 �C. WBC differentials were stable at

48–72 h, a slight decrease was seen in absolute neu-

trophils, lymphocytes and lymphocyte percents. The

imprecision of basophils using the CELL-DYN Sap-

phire is high, reflecting that the cell types that occur

in low numbers can yield possible unreliable results in

the stability studies. Differences in the sample size and

sample type between our study and that of Muller

et al. (2006) may at least partly account for the differ-

ent findings. Additionally, the exact calibration of the

WBC differential channels may affect the results,

especially the flagging efficiencies. For example, in

our results, the BAND flag appeared as early as 24 h,

but Muller et al. (2006) reported that this flag

appeared at 36 h. However, the accuracy of the flags

should be checked by microscopic review by experi-

enced observers, which was not undertaken in our

study. Also, a fault of our study was that the changes

were not observed in the low and high levels, for

example, using leukopenic and thrombocytopenic

specimens.

From the review of the literature and according to

our results, we conclude that CBC and differential

parameters should be analyzed as soon as possible

after collection. If analysis is delayed for a longer per-

iod of time specimens should preferably be main-

tained at +4�C. The date and time collection should

be clearly indicated and, in cases where a delay in

analysis does not enable accurate results, the corre-

sponding parameter should be omitted and substituted

by a comment.

ACKNOWLEDGEMENTS

We would like to thank Oulu University Hospital Lab-

oratory staff and Dr Veli Isomaa for the valuable help.

Table 4. Stability of CELL-DYN Sapphire flagging

alerts. Data indicate the number of samples with flags

at each time point at +23 ± 2 �C and at +4 �C

Flag <2 h 6 h 24 h 48 h 72 h

+23 ± 2 �C, n = 25

VARLYM 0 1 0 0 13

IG 3 6 23 25 22

FP? or NRBC 0 1 4 16 23

BAND 0 3 12 17 14

BLAST 0 0 0 0 0

+4 �C,

n = 40

Batch 1

(n = 40)

Batch 2

(n = 40)

Batch 3

(n = 39)

Batch 4

(n = 30)

<2 h 6 h <2 h 24 h <2 h 48 h <2 h 72 h

VARLYM 1 0 0 6 5 6 0 19

IG 3 6 5 23 1 23 4 21

FP? or NRBC 1 1 0 13 1 24 1 26

BAND 0 6 4 21 1 24 3 18

BLAST 1 3 0 3 0 0 0 1

VARLYM, variant lymphocyte; IG, immature granulo-

cytes; FP?, fluorescent population; NRBC, nucleated red

blood cell absolute count.

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