Hello Fangfang Hu

Abnormal hemolysis may be caused due to several factors which includes inappropriate handling during processing of blood, inappropriate storage conditions, bacterial hemolysins, antibodies that cause complement lysis, defects in the RBC membrane, or an abnormality in the blood donor. Hemolysis could also result from phlebotomy may be by incorrect needle size, improper tube mixing, incorrect filling of tubes, excessive suction and prolonged tourniquet.

If you are in doubt about storing blood samples in ice boxes for too long, then the answer is yes. The red blood cells become haemolyzed at temperature below 2 degree C. So the blood samples must never be allowed to freeze. During the freezing process at near-zero temperatures, mechanical damage to the red cell membrane is one of the most important causes of hemolysis of erythrocytes.

So, whole blood must always be stored at a temperature between +2 to +6 degree C. At mobile collection centers, blood is directly placed into a well insulated cold chain shipper with sufficient cryophase change materials that is capable of cooling blood below +10 degree C but not less than 2 degree C in under 6 hrs. and thereafter maintaining the temperature below +10 degree C for a maximum transportation time of 24 hrs.

Hence it is very important to make sure that the quality of the cold chain shipper or icebox and the appropriate refrigerant ice pack being used in storing and transporting blood samples is appropriate.

Best Wishes.

Good day, Fangfang Hu! That's a thoughtful question.

In Vitro Hemolysis is a common pre-analytic error in samples that leads to the rejection in order to avoid interference. It is caused by several causes such as inappropriate collection of the samples, careless transportation of the sample, and prolonged storage. (Wan Azman et. al, 2019).

According to Hasan Kara et. al (2014), during the collection period, the phlebotomist or the health professional tasked to obtain sample may cause hemolysis due to selecting the wrong needle size, incorrect inversion amd filling of tubes, forcing the equipment to draw blood which produces too much suction, leaving the torniquet attached for more than a minute, and difficulty in collection.

During the transportation of sample, the use of pneumatic tube system may cause hemolysis. The pressure on the red blood cells during the transport in the system may possibly increase due to the length of the system and speed of the transport. Manual transport can also cause hemolysis if carelessly handled. (Kara et. al, 2014).

As per the storage of blood samples in ice boxes for too long, hemolysis is possible. If not careful, direct contact of the ice and the sample may cause freezing which will result to lysis of blood cells. (Nei, 1967).

References:

Eclinpath. (n.d.). Sample Collection. https://eclinpath.com/hematology/sample-collection-heme/

Kara, H., Bayir, A., Ak, A., Degirmenci, S., Akinci, M., Agacayak, A., Marcil, E., & Azap, M. (2014). Hemolysis associated with pneumatic tube system transport for blood samples. Pakistan journal of medical sciences, 30(1), 50–58. https://doi.org/10.12669/pjms.301.4228

Nei, T. (1967). Mechanism of hemolysis of erythrocytes by freezing at near-zero temperatures: II. Investigations of factors affecting hemolysis by freezing. https://www.sciencedirect.com/science/article/abs/pii/S0011224068801282

Wan Azman, W. N., Omar, J., Koon, T. S., & Tuan Ismail, T. S. (2019). Hemolyzed Specimens: Major Challenge for Identifying and Rejecting Specimens in Clinical Laboratories. Oman medical journal, 34(2), 94–98. https://doi.org/10.5001/omj.2019.19

Thank you very much for your answers. I have to say that it truly helped me a lot. I have since then found the final reason. It could be the blood sample was kept in ice for too long, causing the cells to hemolyze and rupture. To solve this, I tried to replace the ice with ice packs so that the blood sample does not come into direct contact with the ice yet at the same time maintains a cool environment and finally the hemolysis stops.

Anyway, thank you very much , without your sincere help and hints,I wouldn't have found the solution so smoothly, and I wish you all the best of luck from the bottom of my heart!