Processing

Placental blood analysis and processing

The blood sample that reached the laboratory will successively pass through several stages:

Step 1. Integrity control of the placental blood kit used for harvesting, of the labelling with the unique bar code and the accompanying files of each sample.

Step 2. The introduction of personal identification data of the mother and information on birth, in the database.

Step 3 is the phase of qualitative assessment of the harvested blood unit:
- Verification of the sterility of the harvested samples, by taking an aerobic, anaerobic and mycological bacteriological analysis (in 2-3% of the cases, during the harvesting, there is the possibility of blood contamination with germs, most commonly commensal-ordinary, in the genitor-anal area)
- Screening test to check the viability of cells

Step 4 This is the processing stage
4.1 Quantitative assessment of the placental blood unit:
- assessment of the harvested blood volume
- assessment of the total concentration of nucleated cells in the sample (is done automatically by a haematology analyzer)
- calculation of the weight of the receiver, according to the quantitative parameters of the sample (volume and cellularity)

4.2 Preparation of the placental blood unit in view of its cryopreservation:
The placental blood bag, sealed immediately after harvesting, will be subjected to a centrifugal split, with the purpose of separating erythrocytes (red blood cells), nucleated cells and plasma.

A cryopreservation agent will be introduced in the blood unit obtained, with the purpose of preventing formation of water crystals during cryopreservation and namely, of preventing the occurrence of cell ballooning on the occasion of de-cryopreservation.

This preparatory step is necessary because the water crystal formation, or ballooning, that is the excessive accumulation of water inside cells can cause alteration of cell membranes and affect the cells.

Currently, there are 2 main possibilities of processing placental blood. The first procedure would be the preservation of all cellular elements and of a part of the placental blood plasma component and the second, the separation and cryopreservation of nucleated cells, strictly, after the prior removal of plasma and of erythrocytes from the blood.

Within our laboratories, the procedure for processing and cryopreservation of the whole blood is applied. Blood processing is done in a closed system which ensures a sterile working environment (blood manipulation is achieved in a sterile box).

Also, inside the laboratory we maintain a strictly controlled environment with an air filtration system and through periodic sterilization of the air, all these measures being taken for keeping the sterility of the harvested blood during the entire duration of processing and cryopreservation.

We opted for the procedure of processing and cryopreservation of whole blood because it is one that ensures the preservation of the maximum cell concentration in each sample of processed blood, secondary processing cell loss being insignificant.

The technique of cell separation, or reduction in volume, with the elimination of the plasma and the red blood cell component of the blood sample, there is the risk of cell loss assessed, by the manufacturers of automatic processing systems, at 10 -15%, but according to the findings from practical activity, cell loss would be even higher.

This should be evaluated in the context of the clinical activity, that is the administration of the placental blood graft in a stem cell transplant. In such a situation, the cellularity of the transplanted graft is extremely important in terms of transplant outcome and further development of the patient. Any team of transplant doctors desires to have a graft with a larger cellularity, available for transplantation.

Moreover, the good results of the first placental blood transplants have been obtained by administration of non-manipulated grafts, of grafts of whole blood, confirming the safety and efficiency of their administration. Therefore, it is not necessary to separate the cellular component of the placental blood sample tin order to save the life of a patient.

Maximum cell concentration preservation is important not only in terms of haematological indications for transplantation. It is also important in terms of the development of organic and tissue regenerative medicine.

The cell population of placental blood is heterogeneous, in addition to hematopoietic stem cells and haematopoietic progenitors there are other types of cells (e.g. mesenchymal stem cells, endothelial progenitor cells). These cells could form the basis of new therapeutic applications in the future.

Another important issue arises from medical studies published in recent years on the cryopreservation agent (DMSO).

Initially, it was considered that "washing" the de-cryogenic placental blood unit before its administration as a transplant graft, was necessary, in order to remove the DMSO and, if needed, the haemoglobin resulted from eritrocitary haemolysis. In recent years, however, studies have been published to evidence that in low concentrations of the DMSO, it is no longer necessary to "wash" the placental blood unit prior to its administration, because the risk of adverse effects associated with the DMSO or the risk of haemolysis associated to cryopreservation / de-cryopreservation and DMSO, become insignificant. Instead, by giving up "washing" before the graft administration the risk of cell loss associated with this procedure is eliminated.

The decision on whether or not this procedure of post de-cryopreservation should be applied and, also, on how the graft should be issued, is taken by the transplant centre where the transplant is to be made.

Therefore, we opt for the whole blood processing procedure by which you are ensured the preservation of the maximum cell concentration in the harvested blood sample, both during processing and in the graft preparation stage in view of the transplant performance.

Step 5 – The proper cryopreservation step

After the preparatory phase, the unit will be progressively frozen, on heat levels, a process assisted and monitored digitally, up to the achievement of the final temperature of -196 g C.

The cryopreservation (freezing) technique and the strict control of the computer freezing curve decisively influence cell survival after thawing.

Step 6 – Storage stage

The cryogenic blood sample will be kept in liquid nitrogen, in stable thermal and pressure conditions at a temperature of -196g C. Placental blood stem cell grafts have been used for over 20 years, the first placental blood transplant being performed in 1988, in Paris, in a case of severe Fanconi anaemia.

According to the guidelines developed by the Department of Health of New York, on the activity of collecting, processing and storing the placental stem cells until now, there is no evidence of loss of cell viability and of cell biological activity, in vitro testing of the blood samples stored cryogenically at -196 Celsius degrees, under constant thermal and pressure parameters.

Consequently, the placental blood grafts constantly stored in liquid nitrogen, would have no validity term.

The results of tests carried out will be brought to attention, at an interval of about 2 to 3 months, being sent by express courier service, together with invoices. If the cells are not viable or the harvested blood is contaminated bacterially or mycologically, you will pay nothing for the provided services.