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As Ege University, Center for Drug R&D and Pharmacokinetic
Applications, we
have established a 24-bed clinic and approved by Turkish Ministry of
Health
(MOH) to perform bioavailability and bioequivalence studies.
Recently, we have performed a study that is approved by ethical
committee on
6 healthy volunteers in order to test our quality documents and clinical
facilities. In our study, the only problem that we could not overcome
was
the hemolysis of our blood samples. We have collected the blood by a
venous
catheter and draw the blood by a syringe and then transfered it to a
test
tube. And then we have let the blood samples stay for 15 minutes in 4
degrees C and then centrifuged them at 4000 rpm for 10 minutes. We have
thought that factors like blood collection velocity, pouring pressure,
patient characteristics, precentrifuge waiting time all affect
hemolysis. We
have tried to change one factor and the other in order to prevent
hemolysis
but at the end, hemolysis was nearly 15 % when all our blood samples are
considered. It will really be appreciated if you can help me find a way
to
prevent hemolysis. And I also would like to know upto what percent,
hemolysis may be acceptable? Thanks a lot for your help beforehand.
Sebnem APAYDIN, M.D.
Associate Prof. Pharmacology
Ege University, Center for Drug R&D and
Pharmacokinetic Applications
35100 Bornova IZMIR TURKEY
Tel: +90-232-3392754
Fax: +90-232-3425088
e-mail: sapaydin.-at-.bornova.ege.edu.tr
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Frequently, the smaller the needle size, the more hemolysis. Have you
varied the size of the venous catheter or the needle?
Dale
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Dear Sebnem,
I would like to know whether u r collecting plasma or serum. In case of
plasma, u might be using EDTA coated tubes. As u said pouring pressure
is one key factor and at the same time, the mixing of blood with EDTA is
very important. The very gentle mixing is required in order to avoid
hemolysis. We found this is the critical step towards avoid hemolysis.
As Dale said, needle size of the catheter also may affect to some
extent.
U have already changed all other possible factors and only this mixing
may help your query.
Regards,
KANTHI KIRAN,
Glenmark Pharmaceuticals LTD.
INDIA.
Ph.: 91-22-55902491/92 ext 315
Fax: 91-22-55902318
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We are using lithium heparin tubes and after we transfer the blood from
the
syringe to the tube we gently make only an upside down motion. Should
we mix
the tube more then one time???
Sebnem
The following message was posted to: PharmPK
We have used the same needle size for all the volunteers.
Sebnem
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plastic - made catheters are sometime reported to perform worser than
equal-gauge metal needles in regard to Hemolisis
Best regards
Cesare Terzi , MD, PhD
Paediatrics Dep.t
University of Parma - ITALY
e-mail : cesare.terzi.-a-.unipr.it
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Sebnem,
Hemolysis may be due to keeping blood samples at 4 degrees C for 15 min
before centrifugation. I have notices that with rat blood -- some
hemolysis whenever samples are kept on ice before centrifuging compared
to no to little hemolysis when kept at room temperature. This is of
course in addition to the technique of blood collection which has been
mentioned by others (e.g., size of the needle [23 and higher gauge
needle is fine], collection into tube, mixing of blood with
anticoagulating agent [slow mixing by inverting vs. shaking etc.]).
Little hemolysis in some of the samples kept at room temperature is
always due to poor collection technique, like using smaller gauge
needle or squeezing the collection site etc. All anticoagulating agents
work same and should not have an impact on the degree of hemolysis.
Shakil A. Saghir, M.S.P.H., Ph.D., D.A.B.T.
Toxicology Specialist
The Dow Chemical Co.
1803 Bldg., Midland, MI 48674
Phone: (989) 636-8708, Fax: (989) 638-9863
Email: ssaghir.at.dow.com
The following message was posted to: PharmPK
Sebnem,
You can mix in more than one time. We keep our tubes on a mechanical
tube rocker until we are ready to centrifuge (after collecting samples
from 6-10 dogs; around 30 min). We collect 3 ml of blood from either
jugular or cephalic vein of the dogs and rarely have mild hemolysis
which are always due to problems during blood collection. We can almost
predict which samples will have hemolysis before centrifuging them.
Hope this will help.
Shakil A. Saghir, M.S.P.H., Ph.D., D.A.B.T.
Toxicology Specialist
The Dow Chemical Co.
1803 Bldg., Midland, MI 48674
Phone: (989) 636-8708, Fax: (989) 638-9863
Email: ssaghir.at.dow.com
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We have experimented with the rate at which blood is withdrawn from a
catheter. This definitely influences hemolysis. We use a very small
diameter catheter placed in a vein or artery of rodents. The rate at
which
blood is withdrawn is controlled by a syringe pump. There is a big
increase in hemolysis at high flow rates (>300 uL/min) and virtually
none
at lower flow rates (<100 uL/min). The catheter is the same, and it's
plastic, so there is nothing inherent in the catheter itself which
creates
the problem. We've collected whole blood into untreated vials for
serum,
and we've collected into vials pretreated with sodium heparin, liquid
EDTA
or liquid sodium citrate/citric acid. To us, the flow rate looks like
the
biggest culprit. We can solve this because we work with rodents and we
can
use a syringe pump in an automated blood sampler so the rate is very
reproducible and tightly controlled. In humans, you're collecting
manually, using another human to withdraw the sample. You might try to
slow down the collection rate into the hand-held syringe and see if that
helps. Practice using a solution of 50 g sucrose in 200 mL of DI water
which has the same viscosity as human blood.
Candice Kissinger
In Vivo Sampling Laboratory
Bioanalytical Systems Inc.
West Lafayette, Indiana USA
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