• Normally, centrifugation for 15 minutes at approximately 1500 g yields PPP (platelet poor plasma) and

centrifugation at approximately 2000 g for 15 minutes

yields PFP (platelet free plasma). The ‘g’ is a function of

length of rotor head and RPM. It is for this reason each

laboratory must calibrate its own equipment to achieve

satisfactory samples depending on test performed and

kind of plasma sample required.

280 Concise Book of Medical Laboratory Technology: Methods and Interpretations BUFFERED 3.2% CITRATE SOLUTION (PROFACT)

(Courtesy: Tulip Group of Companies)

Ready to use 3.2% buffered citrate solution for coagulation

assays and ESR by Westergren method.

Summary

Accurate coagulation testing is dependent on numerous

preanalytical variables, which may affect the results of

routine coagulation assays. To improve the precision and

accuracy of laboratory testing, it is important to identify

these variables and control their potential effect on results.

Preanalytical variables pertinent to routine coagulation

testing can be classified into three major categories:

specimen collection, specimen processing and specimen

storage and transport. 3.2% citrate is also the anticoagulant

of choice for performing ESR by Westergren method.

Reagent

Laboratory reagent: Ready-to-use solution.

Profact is a unique ready-to-use 3.2% buffered trisodium

citrate solution formulated for collection of blood for

routine coagulation assays. Profact can be used for sample

preparation in the following clot based assays such as PT,

APTT, TT, quantitative estimation of fibrinogen, test for

factor deficiency, test for lupus anticoagulants, protein C

and protein S tests. Profact can also be used for collection

of blood to perform ESR by Westergren method.

Principle

About 3.2% trisodium citrate is the anticoagulant of choice

for coagulation studies. When anticoagulated blood is

centrifuged for preparing PPP for routine coagulation

assays, the centrifugation process leads to release of

carbon dioxide (CO2). The end result being shift in pH,

which has an adverse impact on the results of clot-based

assays. Profact incorporates 3.2% trisodium citrate in a

unique protective solution, which arrests shift in pH due

to the release of carbon dioxide during centrifugation. Also

labile factor V and VIII are well preserved and the results of

clotbased assays are more accurate.

Also Profact incorporating 3.2% citrate is the anticoagulant of choice for ESR by Westergren method.

Storage and Stability

¾ Store the reagent at 2 to 8°C

¾ Stability of unopened vial: 12 months from the date of

manufacturing.

¾ Stability of opened vial: 90 days from the date of

opening, provided it is not contaminated.

Material Required But Not Provided

Sterile and clean 0.5/1 mL micropipettes, micropipette

tips or glass blow out pipettes, ESR tube.

Sample Collection and Preparation

For Coagulation Assays

Though no special preparation of the patient is required

prior to sample collection by approved techniques, it is

preferable that patients are not heavily exercised before

blood collection. Fasting or only light non-fatty meals

prior to blood collection provides sample with a desirable

low opacity.

Withdraw blood without undue venous or frothing into

a plastic syringe fitted with a short needle of 19 to 20 SWG.

The venipuncture must be a ‘clean’ one and if there is any

difficulty, take a new syringe and try another vein. Transfer

the blood into tubes containing Profact, after detaching the

needle from the syringe. Do not delay mixing blood with

Profact. Avoid foam formation during mixing. Mix exactly

nine parts of freshly collected blood with one part of

Profact. For occasional patients with hematocrit less than

20% or greater than 50%, this ratio must be readjusted

to ensure valid results. Centrifuge immediately for 15

minutes at 1500 to 3000 rpm (approximately 1500 g) on a

laboratory centrifuge and transfer the plasma into a clean

test tube. It should be ensured that the plasma is free from

platelets (PPP). Cap the test tubes to prevent deterioration

of samples. Plasma must be tested preferably immediately.

However, if the specimen is held at 22 to 24°C then they

may be tested within 2 hours and if the specimen is held

at 2 to 4°C then they may be tested within 3 hours. Also

plasma samples obtained after collection with Profact

may be stored at –20°C for 2 to 3 weeks before testing.

For ESR by Westergren Method

For performing the test, venous blood is mixed accurately

in the proportion of 1 part of Profact and 4 parts of whole

blood.

The sedimentation rate is reduced in stored blood,

hence, the test should be carried out within 4 hours

of collecting the blood, and a delay up to 6 hours is

permissible provided that the blood is kept at 4°C.

Precautions

1. Take every possible aseptic precaution to minimize

contamination while drawing the reagent.

Clinical Hematology: Bleeding Disorders 281

2. Avoid dipping contaminated pipettes/micropipette

tips in the reagent vial. Ideally pour the required

quantity for the day's work into another sterile clean

vial.

3. Recap and replace the reagent vial immediately back

at 2 to 8°C.

Remarks

1. Since most of the routine coagulation assays use PPP,