Hirudin as an anticoagulant for both haematology and chemistry tests

Hirudin, an extract from the leech, has powerful antithrombin activity affecting 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 (EDTA) in the complete blood count (CBC), including white blood cell (WBC) differential 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. Thus, 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.

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.

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 …nˆ5 † were assayed ® ve times, and the coe cients of variation (CV) for CBC and WBC di erential counts were calculated.

Statistics
Statistical analysis was carried out using the Stat View-4.5J 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.

Haematology tests
No signi® cant di erences were noted for any item between EDTA-treated and hirudin-treated blood (table 1) . Any possible e ects of hirudin on blood cell morphology were meticulously sought. No e ects were seen on white cells, red cells or platelet counts.

Stability of haematology specimens
For 2 h after blood collection, no signi® cant di erence was seen in CBC or di erential WBC counts between EDTA-treated blood and hirudin-treated blood. However, when the measurement was performed 3 h 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.

Reproducibility of haematology parameters
Good reproducibility was demonstrated for each item and no signi® cant di erences were noted between EDTA-treated and hirudin-treated blood (table 2) . The results indicated that the reliability of determinations performed by the haematology analyser was not a ected by hirudin.

Discussion
Most physicians order multiple laboratory tests to manage patients, thereby requiring substantial volumes of blood. count results using EDTA has worked against development of new reagents. Nevertheless, our results showed that hirudin-treated and EDTA-treated blood gave similar values for CBC and WBC di erential counts, and no morphologic changes due to hirudin could be observed within 2 h of blood collection. While hirudin inhibits thrombin selectively, clotting can eventually get underway because of the incredible rapidity with which thrombin is generated, faster than the association rate of hirudin unless very high concentrations are used [11].
EDTA-treated plasma is used for chemistry tests in some emergency laboratories. Thus, as it usually takes 40± 50 min to obtain a serum specimen for chemistry tests, importance was attached to the experiment with plasma. However, measurements of calcium, iron, alkaline phosphatase, leucine aminopeptidase, and sodium and potassium are distorted by chelation and by sodium or potassium ions associated with EDTA. Correspondence between results using serum and hirudin-treated plasma was found to be high. Signi® cant di erences between hirudin-treated plasma and serum were seen for potassium …P < 0:01 † and TP …P < 0:01 † concentrations. The di erence for potassium may have resulted from release of platelet-derived potassium in serum during blood coagulation which does not occur in hirudin-treated plasma. The TP value was higher in hirudin-treated plasma than in serum because hirudin-treated plasma contains ® brinogen. Because di erences between such plasma and serum were limited and predictable, hirudin may be useful for emergency laboratory use as well as for maximizing diagnostic yield from small blood samples.
The number of samples evaluated in the present small pilot study was not large, and the ranges of values compared were limited, but our data indicate suitability of hirudin anticoagulation for haematology and chemistry test specimens. While these data suggest that hirudin is an attractive candidate as an anticoagulant for haematology and chemistry tests, further laboratory trials for coagulation tests are necessary.