Summary:
A key therapeutic strategy in haematological malignancies such as Hodgkin's
lymphoma, non-Hodgkin lymphomas, multiple myeloma and amyloidosis is
megatherapy followed by haematopoietic blood stem cell transplantation, where the
readministration of a sufficient number of functional AAKs is necessary for the
restoration of haematopoiesis and patient survival. Today, the main source of AAK for
autologous transplantation is peripheral blood, in which AAKs can be mobilized by
administration of granulocyte growth factor (G-CSF) with or without chemotherapy with
or without plerixafor.
The collection of AAK from peripheral blood is performed by leukapheresis in a
central venous catheter cytoseparator. The day of AAK collection depends on the
mobilization protocol and the absolute number of CD34+ in peripheral blood. The most
commonly used cell separator today is the Spectra Optia Apheresis System
(TerumoBCT), a portable system for automatic separation of blood components, which
uses centrifugation and optical detection and operates under the control of an
experienced operator. During collection, the operator is responsible for the correct
selection of the collection preference and the achievement of the collection target as
well as for ensuring the uninterrupted operation of the cell separator and good
condition of the donor.
In this thesis, the parameters that affect the AAK CE2 collection performance
were studied. The efficiency of the collection reflects the percentage of the number of
cells collected in relation to the total number of cells contained in the volume of blood
processed during collection. For this purpose, the data of AAK collections from
autologous donors conducted at the Collections Department of the Cell Therapy
Program of the Hematology Clinic of NKUA at the Popular Hospital of Athens between
2/3/2021 and 17/09/2024 were studied with the continuous flow cell separator Spectra
Optia Apheresis System (Terumo-BCT) version 11, with the execution of a continuous
mononuclear cell collection (CMNC) program in semiautomatic mode. For the
parametric and non-parametric analysis of the data, the statistical program IBM SPSS
Statistics 25 was used. The effect on collection performance was considered
statistically significant when p<0.05.
87 collections of patients (43 females with a median age of 53 years and 44
males with a median age of 54.5 years) with Multiple Myeloma (31%) or Hodgkin
Lymphoma (18.4%) or B-non Hodgkin Lymphoma (14.9%) or T-non Hodgkin
Lymphoma (1.1%) were studied, while 1 patient had scleroderma. For the mobilisation
of AAK in peripheral blood, patients received growth factor alone (6.9% of patients) or
growth factor with chemotherapy such as cyclophosphamide (52.9%), ESHAP
(11.5%), R-ESHAP (5.7%), IGEV (9.2%), NICE (9.2%), R-ICE (1.1%), MT-ARAC
(1.1%). 31 patients needed concomitant administration of Plerixafor, mainly those
mobilised with growth factor and NICE alone. The majority of patients were collected
on the scheduled day (75.8%), 4.5% of patients were collected one day earlier, 17.2%
one day later and 2.2% two days later. The collection of blood samples before
collection and the start of collections were 1.5±1.4 hours apart and 87.4% of
collections started in ≤3 hours. The concentration of WBC in the peripheral blood of
patients was 32±18.4K/μl with a mononuclear cell rate of 17.2±5.4%, the haematocrit
was 30.7±4.6%, the haemoglobin 10.2±1.5 g/dL and the PLT concentration was
111±72.3 K/μl. Seventeen patients required platelet transfusion before collection while
2 required transfusion and concentrated red blood. The concentration of CD34+ cells
in peripheral blood was 96.05±113.83/μl, of CD45+ cells was 29.030±17.300K/μl, the
percentage of CD34+ cells in the CD45+ cell population was 0.30±0.44% and the
percentage of viable CD34+ cells was 99.04±1.47%.
The majority of collections were performed with the Spectra Optia 1 cell
separator (N=81) and the remainder with the Spectra Optia 2 cytoseparator (N=6).
Operator A was responsible for 80.5% of the collections and operator B for 19.5%.
During the collections, a blood volume of 13.2±4.0L was processed, corresponding to
3±0.77 cycles of whole blood. The duration of collections was 225±55.8min, the
median blood flow rate was 70±7.6ml/min and the anticoagulant addition ratio was
18:1 in 42.5% of collections, 20:1 in 42.5% and 16:1 in 14.9%. The collection layer
preference ranged between 10-59. "Sawtooth" flow in the collection tube was
observed in 39.1% of the collections, while layer disturbances and/or interruptions of
blood supply were observed in 3.4% of the collections.
Evaluation of the collection product by taking an intermediate sample from the
product was carried out in all collections after collecting at least 50ml of product. The
concentration of WBC in the intermediate samples was 408.7±150.6K/μl, where the
majority (N=47.54%) was 10 times the concentration of WBC in peripheral blood
before collection. In 20 collections (23%) the WBC concentration of the intermediate
sample was about 5 times their concentration in peripheral blood and in 20 (23%) it
was about 20 times. A statistically important parameter for the tenfold increase in the
concentration of WBC in the collection product was found to be the "sawtooth" flow in
the collection tube. In collections that did not have "sawtooth" flow, the concentration
of WBC in the product was ≥10 times higher in 84.6% of cases, while in collections
that had sawtooth flow the concentration of WBC was ≥10 times higher only in 64.7%
of cases (p = 0.038). Apart from the sawtooth flow, the concentration of mononuclei
(p<0.0005) and the percentage of CD34+ cells in the CD45+ population (p<0.0005)
were found to have a statistically significant positive correlation with the degree of
WBC proliferation in the collection product, while the concentration of WBC (p<0.0005)
and the concentration of platelets (p0.019) were found to have a statistically significant
negative correlation with the degree of proliferation of WBC.
In the final collection samples, the concentration of WBC was 288.14±
58.31K/μl, the percentage of mononuclei was 39.5±13.2%, the haematocrit 3.1±1.8%
and the concentration of PLT 671±462K/μl. The concentration of CD34+ cells was
2364.04±2260.9/μl with viability rates of 95.71-100% and the concentration of CD45+
cells was 270.94±55.17K/μl with a percentage of CD34+ cells in the CD45+ cell
population of 0.18-4.22%. In all products the final result was the collection
7.78x106±3.26x106 CD34+/Kg (range 1.20x10 6-20.19x106/Kg) corresponding to
5.71x108±3.23x108 CD34+ cells (range 1.68x108-21.41x108). Collections yields ranged
between 22.3-90.5% with a median of 47.8±12.0%. 21 collections (24.1%) returned >
40%, while 66 (75.9%) returned ≥40%.
In this study, 36 parameters were tested for their impact on the performance of
the collections. Parameters related to patient characteristics (gender, age, disease,
weight, TBV), mobilisation of AAKs in peripheral blood (mobilisation schedule,
Plerixafor administration and timing of administration, day of collection),
characteristics of peripheral blood before collection (time interval between blood
collection and start of collection, white blood cell and platelet concentration,
percentage of mononuclei, haematocrit, concentrations of CD34+ and CD45+ cells,
percentage of CD34+ cells in CD45+ and percentage of their viability, administration
of red and/or platelets before collection), the parameters of the collection (cell
separator, operator, number of processing cycles, duration of collection, requirement
to correct the haematocrit in the initial phase of layer formation, time for formation of
the initial layer, anticoagulant:blood ratio, flow rate, collection preference, sawtooth
flow in the collection tube, frequent interruptions of flow flow and frequent disturbances
of the collection layer) and finally, the characteristics of the intermediate sample at
collection (degree of multiplication of WBC concentration in the intermediate sample
relative to peripheral pre-collection, haematocrit, percentage of mononuclei, platelet
concentration). In the literature, several of the above parameters are mentioned as
statistically significant or not for the performance of the collection. This paper is the
first in which so many parameters are examined simultaneously and several are
examined for the first time.
Of the above parameters, 13 were found to have a statistically significant effect
on the performance of the collection. Negative effects on collection performance were:
1) the increase in the age of the donor (p=0.038), but no reduction in yield was
foreseen <44.6% even in donors 70 years old, 2) the collection was performed one
day earlier than planned (yield 29.0±5.3% vs. 47.9±11.4%, p<0.0005), 3) the delayed
start of collection >3 hours after blood collection from the peripheral (collections started
within ≤3 hours of collection of blood yielded ≥40% in 80.3% of cases versus 45.6%
of collections initiated within >3 hours of blood collection, p=0.011); 4) increased WBC
and CD45+ concentrations in peripheral blood before collection (among collections
with yields less or greater than 40% WBC concentrations were 50.4±17.3K/μl versus
27.7±17.0K/μl respectively; p<0.0005 and CD45+ concentrations were
114.4±168.6K/μl versus 90.5±83.9K/μl, p<0.0005), 5) increased CD34+ concentra
