Hirudin as an anticoagulant for both haematology and chemistry tests
J ournal of Automated Methods & Management in Chemistry, Vol. 22, No. 4 (July–August 2000) pp. 109–112
Hirudin as an anticoagulant for both
haematology and chemistry tests
Takeo Kumura,1 Masayuki Hino,2 Takahisa
Yamane2 and Noriyuki Tatsumi1,2
1
Departments of Laboratory M edicine and 2 Clinical Hematology, Osaka City
University M edical School, Osaka, J apan
Hirudin, an extract from the leech, has powerful antithrombin
activity aå ecting the blood coagulation pathway. We evaluated the
usefulness of hirudin in anticoagulating specimens for routine
laboratory tests. Results using blood anticoagulated with hirudin
corresponded well with results with blood treated with ethylenediamine tetraacetic acid …EDT A † in the complete blood count
…CBC†, including white blood cell …WBC† diå erential count and
morphology of blood cells, when CBC was performed within 2 h of
blood collection. Clinical chemistry results from hirudin-treated
samples were similar to results obtained with serum specimens.
T hus, hirudin may be a useful anticoagulant for emergency
laboratory medicine.
Introduction
Blood tests are used widely for clinical monitoring and
diagnosis. Relatively high volumes of blood and multiple
sampling tubes are usually required: 2 ml of blood in an
EDTA-containing tube for haematology tests, 5 ml in an
anticoagulant-fre e tube for most chemistry tests, and
more blood in citrated tubes for others such as coagulation tests. These tubes of blood are loaded into di erent
automated analysers and the entire sample is usually
needed. Patients object to the frequency of phlebotomy
for clinical tests. To decrease specimen waste, universal
anticoagulant s have been sought for clinical laboratory
use.
EDTA is currently used for haematology tests requiring
whole blood. A chelator of divalent cations, EDTA
interferes with blood coagulation by removing calcium
ions from the sample. While plasma obtained from whole
blood containing EDTA is used for chemistry tests in
some emergency laboratories, results of some chemistry
tests are distorted both by chelation and by the sodium or
potassium ions associated with EDTA. An alternative
anticoagulant to EDTA is hirudin (® gure 1) , known to
strongly inhibit blood coagulation by blocking the action
of thrombin [1± 4]. However, no trials have assessed its
use as a laboratory anticoagulant . We studied the use of
hirudin in specimens for haematology and chemistry
tests.
Materials and methods
Reagents
After recombinant hirudin (American Diagnostica,
Greenwich, CT, USA) was dissolved in distilled water
(20 mg/0.5 ml), quantities of this solution were placed in
blood specimen collection tubes to result in a ® nal
concentration of 4 mg hirudin/2.0 ml blood. Tubes containing 2.4 mg of dipotassium ethylenediamine tetraacetic acid (EDTA-2K) per 2 ml of blood (Terumo, Tokyo,
Japan) were used as controls for haematology tests.
Venous blood
Blood samples were collected from 30 healthy normal
subjects (15 males and 15 females) and divided into
hirudin-containing tubes for haematology and chemistry
tests, EDTA-containing tubes for haematology control
samples, and anticoagulant-fre e tubes as serum controls
for chemistry tests. Tubes for haematology tests were
allowed to stand for at least 15 min at room temperature
to stabilize samples before testing. The serum was
separated by centrifugation at 400 g for 5 min after
leaving for 60 min. Hirudin-treate d plasma was separated by centrifugation at 400 g for 5 min. All tests were
completed within 2 h of blood collection.
Haematology studies
CBC and WBC di erential counts were performed with a
haematology analyser (NE-8000, Toa Medical Electronics, Kobe, Japan). Parameters evaluated were white
blood cell count (WBC), red blood cell count (RBC),
haemoglobin (Hb), haematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular haemoglobin
(MCH), mean corpuscular haemoglobin concentration
(MCHC), platelet count (Plt), and percentages of the
WBC representing neutrophils, lymphocytes, monocytes,
eosinophils and basophils.
M orphology of blood cells
Blood samples taken from tubes within 2 h after collection were smeared on slides, ® xed and stained by the
Giemsa method for microscopic observation.
Stability of haematolog y specimens
CBC and WBC di erential counts were compared over
time immediately and at 1, 2 and 3 h after blood
collection using the NE-8000 analyser at 25 8C for
samples anticoagulate d with either EDTA or hirudin.
Reproducibility of haematology parameters
Samples anticoagulated with either EDTA or hirudin
from healthy adults …n ˆ 5† were assayed ® ve times, and
the coe cients of variation (CV) for CBC and WBC
di erential counts were calculated.
J ournal of Automated M ethods & M anag ement in Chemistry ISSN 1463± 9246 print/ISSN 1464± 5068 online # 2000 Taylor & Francis Ltd
http ://www.tandf.co.uk/journals
109
T. Kumura et al. Hirudin as an anticoagulant for both haematology and chemistry tests
T able 1. CBC parameters and WBC diff erential count with
EDT A and hirudin anticoagulation at 2 h after blood collection.
Item
2=
WBC …£10 ml†
RBC …£104 =ml†
Hb …g =dl†
Hct (%)
MCV (¯ )
MCH ( pg)
MCHC (g/dl)
Plt …£104 =ml†
Neutrophils (%)
Lymphocytes (%)
Basophils (%)
Eosinophils (%)
Monocytes (%)
EDT A
Hirudin
P*
52.3 § 7.5
415.2 § 49.3
13.3 § 1.6
36.5 § 4.2
90.2 § 6.4
30.9 § 0.8
34.3 § 1.1
29.7 § 4.2
51.6 § 7.4
418.1 § 41.5
13.1 § 1.5
35.9 § 3.9
89.4 § 6.7
30.8 § 0.9
34.5 § 1.3
29.1 § 4.1
NS
NS
NS
NS
NS
NS
NS
NS
62.2 § 3.6
29.5 § 10.8
0.6 § 0.3
2.9 § 1.8
4.8 § 1.8
61.5 § 3.5
29.8 § 10.5
0.6 § 0.4
3.2 § 1.7
4.9 § 1.8
NS
NS
NS
NS
NS
Values are mean § SD. * By paired t-test. NS, not signi® cant.
n ˆ 30.
Figure 1. Structural formula of recombinant hirudin.
Chemistry tests
Chemistry tests were performed using a fully automated
analyser (7170 Automatic Analyzer, Hitachi, Tokyo,
Japan) for determinations of aspartate aminotransferase
(AST, IU/l), alanine aminotransferase (ALT, IU/l),
total bilirubin (T-Bil, mg/dl), lactate dehydrogenase
(LDH, IU/l), blood urea nitrogen (BUN, mg/dl),
creatinine (Cre, mg/dl), sodium (Na, mEq/l), potassium
(K, mEq/l) , chloride (Cl, mEq/l), total protein (TP,
g/dl), albumin (Alb, g/dl), total cholesterol (T-Cho,
mg/dl), triglyceride (TG, mg/dl), alkaline phosphatase
(ALP, IU/l), leucine aminopeptida se (LAP, IU/l), cholinesterase (Ch-E, IU/l), gamma glutamyl transpeptidase
(® -GTP, IU/l) and uric acid (UA, mg/dl).
after collection, WBC, RBC and platelet counts
showed decreases due to coagulation in hirudin-treated
blood (® gure 2) . Four hours later, the specimen had
completely coagulated and measurements could not be
performed.
Statistics
Statistical analysis was carried out using the Stat View4.5J software package (Abacus Concepts, Berkeley, CA,
USA). A paired t-test was used for comparison analyses;
P values less than 0.01 were considered signi® cant.
Results
Haematology tests
No signi® cant di erences were noted for any item
between EDTA-treated and (...truncated)
