Cytological control

All  photographs are  provided by the scientific team of Biohellenika.

Τhree types of measurements are performed before and after the procedure of separation

1. White and red blood cells count with the certified for clinical use Beckman Coulter (CE-IVD)
2. Estimation of the viability and the number of the haematopoietic stem cells (HPC) CD45dimCD34+ according to the approved clinical protocol of ISHAGE in the flow cytometer Epics XM Beckman Coulter and the stem counttm – (CE-IVD).
3. Culture of a small number of haematopoietic cells in a specific medium for colony forming estimations BFU-E CFU-G and CFU-GM.

Cell Types

Image 1. The total blood as first coming in the lab, before processing. We can see three nucleated cells and the rest red blood cells. Red blood cells are not stem cells. Stem cells are included in the nucleated population

The population of leukocytes in the umbilical cord blood contains 2 types of cells: the haemopoietic population of CD45dim/ CD34+ cells and the population of multipotent CD45-/CD34 cells. Both populations show great differences in their morphology and their development. The haemopoietic population comprises of more mature cells which constitute the population that will be transformed to haemopoietic cells after transplantation (Images 2 & 3).

The population of multipotent cells consists of spindle type cells with extensions resembling fibroblasts, and is characterized by the ability to divide 40 times without further differentiation, reaching a population of 1,015 cells without caryotype changes (Gogler et al, 2004, 200: 123-135). This population is a subject of intensive research all over the world and today the in vivo differentiation of the cell population has been accomplished, in neuron, bone, chondrocytes, fat, cardiac cells, muscular and hepatic cells (See Image 4).



Image 4
Appearance of multipotent stem cells of  fibroblast type
in umbilical cord blood after culture for 15 days



Image 5. CFU of red blood cells. Stem cells maintain all the biological abilities.

Εικόνα 6 a	Εικόνα 6 b

CFU of neutrofil-granulocytes 

Therefore, the cellular proliferation for the increase of the number of stem cells for haemopeisis and use for the therapy of leukaemia has no indication, as the haemopoietic population is close to the stage of apoptosis. The number of stem cells required for the treatment of leukaemias depends on the body weight of the recipient. The number of nucleated cells from umbilical cord blood reaching 1.73x107 /Kg body weight and the number of CD34+ cells reaching 2.7x105 /Kg body weight, have been administered successfully to patients with leukaemias (Ren et al 2001, Zhonghua, 22: 621, Cilley et al 2004, Bone Marrow Transplantation, 33: 161-4, Magro et al 2006, Hematologica, 91: 640-8). The number of stem cells is proportional to the total volume of collected blood. However, due to the presence of multipotent cells that allow proliferation, even small quantities of blood  can nowadays be cryopreserved.

Virological control
Virological control for hepatitis B,C,HIV and CMV in the umbilical cord blood with the method RT.PCR.
RT.PCR can detect immediately the virological load the child’s blood and relieves mothers from continuous examination during and after pregnancy. Recently Biohellenika was equiped with Compass instrument by Roche for the direct virus detection in the babys  umbilical cord blood. Today this system is provided only by the capital blood centers for virus detection, and  it is recomended by the European Union for safe  results.

Bacteriological control
Bacterial aerobious and anaerobious control in three certified for clinical use (CE-IVD)  Bactec systems  (Becton Dickinson)  of total capacity of 290 samples. These systems are characterized by their great sensitivity and reliability of the results.
3-12% of umbilical cord blood units worldwide are considered unsuitable due to contamination.  According to our experience in private obstetrics clinics is nearly (0.5-1%) due to special care and skill of obstetricians.
In case of positive bacteriological control, the sample might not be suitable for use but only after the bacterial characterization and the antibiotics and therefore is cryo-preserved for free.

Results of the quality check
The parents receive the medical results of the quality control signed by name, by the doctor in charge (university professor). It is also given a certificate with an identity card, with the unique number of identification of the customer, which escorts the sample at all the phases of the procedures.

All the above mentioned quality methods are accredited by the Hellenic Accreditation System according to 410, 8/2/2008 published document.