Optia – Efficiency Considerations With MNC Collection PBCS harvesting in children Some of the...

Optia – Efficiency Considerations With MNC Collection

Dr. Volker Witt

OPTIA™ system (CaridianBCT, Lakewood, Dnv, CO, USA)

• Introduced as the replacement for the COBE spectra system

• TPE, RCE available all over the world

• MNZ, Granulo, Leukodepletion are available in Europe

• BM processing is pending • Universal apheresis system

for therapies and collection of blood and tissue products

Harvesting autologous PBMNC in children

• Routine procedure in many pediatric oncology therapy protocols – Neuroblastoma

– Ewing sarcoma

– Nephroblastoma

– Hepatoblastoma

– Medulloblastoma

– CNS tumours

– Lymphoma

Autologous PBCS harvesting in children

Some of the problems • Extracorporeal blood volume

of the apheresis systems • Difficult venous access • Different physiological aspects

in children • Apheresis procedures in

children are accounting for less than 5% of the total number of procedures performed

• Only a few centres are specialized in pediatric apheresis.

Systems in use

• Fenwal CS 3000

– expired

• COBE spectra

– will expire 2014 in Europe

• AMICUS

– still in use

• Fresenius COMTTEC

– rarely used

• Optia

– No published data in pediatrics

Apheresis procedures in the St. Anna Kinderspital from 1996 to QI-2013

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AUTO DC FD DLI ERY ECP RCE TPE IA WBC XX

The use of the apheresis systems in children at St. Anna Kinderspital in 2011+2012

OPTIASpectra

AMICUSothers

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Spectra

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collection efficiency : What is efficient?

• Which cell type do I want to collect (HPC, MNC, monocytes, CD3+ T-cells,…)?

• Collecting enough cells for the poposed therapy?

• Collecting enough cells per kg bodyweight?

• Collecting all cells which are running into the apheresis system?

• Collecting all cells per liter whole blood running into the apheresis system?

Factors influencing the calculation of the collection efficiency

• Cell number of the target cell population

• Total cell number

– Cells who can interfere with the target cell population

• Relation between the target cell population and the cells of no interest

• A sufficient and continous blood flow for optimal separation and harvesting

• Cell loss in the set

• A constant and valid method to describe and enumerate the target cell population

Model of apheresis

BM PB AP

Recruitment phenomenon

• The number of cells mobilized into the peripheral blood is even higher than the number of collected cells.

[Mayer J. Bone Marrow Transplantation, 2003, 749-757]

Yield of a cell population

cells in the product [/ml] X harvest volume [ml]

[ cells x 10E06]

[ cells x 10E06/kg bw]

[Strasser E.F. Transfusion 2005, 445-452]

Cell loss

(1-(postdonation cell count/predonation cell count))X(-100)[%]

pre- or postdonation cell count =

absolute number of cells in blood pre- and postdonation =

blood cell count [x10E09/L] x total blood volume [L]


Cell yield per liter of processed blood volume (/L PBV)

Y ÷ PBV [/L]

Y = Yield

PBV = processed blood volume [L]


Recruitment factor (RF)

(postdonation cell count + cell yield) ÷ predonation cell count

pre- or postdonation cell count =

absolute number of cells in blood pre- and postdonation =

blood cell count [x10E09/L] x total blood volume [L]


Collection Efficiency (CE)

Efficiency of the procedure [%]

= cells in the harvest ÷ cells processed

Witt V. Journal of Clinical Apheresis; 2001; 161-168

Collection Efficiency (CE)

a = cells/mL

b = harvest volume in mL

c = cells/mL before harvest

d = cells/mL after harvest

e = processed blood volume [mL]

absolute cell count in the harvest = a * b

processed blood volume (e) = whole blood volume processed – ACD-A volume used

Witt V. Journal of Clinical Apheresis; 2001; 161-168

Manzone et.al. • JOURNAL OF NUCLEAR MEDICINE TECHNOLOGY • 2007 • 55-63

Blood volume is the volume of blood (both red blood cells and plasma) in a person's circulatory system. A typical adult has a blood volume of approximately between 4.7 and 5 liters, with females generally having less blood volume than males. Blood volume is regulated by the kidneys. Blood volume (BV) can be calculated given the hematocrit (HCT; the fraction of blood that is red blood cells) and plasma volume (PV):

Total Body Volume (TBV)

Weight in kg x BV in mL/kg = TBV

Patient Fat Thin Normal Muscular

Male 60 65 70 75

Female 55 60 65 70

Infant/Child - - 80/70 -

G. Herold Innere Medizin 2007

Tronier W., Goodfellow E., Larson K. Apheresis Math and Useful Physical constants in Principles of Apheresis Technology, 4th edition Linz W. (editor) 2010

Normal BV = 70 mL/kg bw or 1/14 part of the bodyweight

Collection efficiency


• Harvested cells (HPC harvested) are calculated by the formula:

Cellsharvested = cell countbuffy [cells/ml] x volumebuffy [ml]


• The cells processed (HPC processed) are calculated by the forumla:

cellsstart – cellsend

2

• The processed blood volume is calculated by the formula:

Inlet volumeproc.– ACD-A used = blood volumeproc.

X processed blood volume

Collection efficiency st

art

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• The cells processed are calculated by the formula:

cellsstart – cellsend

2

• This formula describes the mean of cells present during the procedure.

• The cell yield is represented by the area under the curve.

• This approach reflects only an approximation to the real conditions during an apheresis procedure.

X processed blood volume


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•A > B

•A < C

Collection ratio

• CR = ((CD34+ /µl product x PV) /kg donor BW) / CD34+ pre

• This formular gives an idea about the collection efficiency of one cell populationen independently from the donor or patients weight.


• Ideal times points for target cell count to calculate the „true“ collection efficiency:

Immediately before start of the apheresis

End of priming (saline and blood)

Before reinfusion

Immediately after end of the apheresis


• For practical reasons target cell counts are performed only before and after apheresis procedure

• For economic reasons target cell count are performed before apheresis procedure only


• How good could the target cell yield predicted by measuring the target cell population before the apheresis procedure only?

N = 7 Median Min - Max

Bodyweight 35 kg 10 – 55

Bodyweight < 20 kg 3/7 10.25, 12.7 and 19 kg

Body lenght 1.45 m 0.75 – 1.72

Body surface 1.19 m² 0.46 – 1.61

Age 11.8 years 1.03 - 20

Sex 2 female 5 male

Underlyingdiseases neuroblastoma, hepatoblastoma, ATRT, PNET, AML and 2 Ewing’s sarcoma

Patients_AUTO


Bodyweight 30,50 8 – 89

Bodyweight < 20 kg 7/16 12.70, 10.25, 19, 17, 10.7, 12.7 and 8 kg

Body lenght (cm) 134 72 – 187

Body surface (m²) 1,07 0.4 – 2.17

Age 9,88 1,03 - 20,03

Sex 4 female 12 male

Underlying diseases 2 ATRT, AML recurrency, Hepatoblastoma, 3 Neuroblastoma, PNET, 5 Ewing's sarcoma, Retinoblastoma recurrency, germ cell tumor and Rhabdoid tumor


Bodyweight (kg) 36 27.4 – 45

Bodyweight < 20 kg 0/9 -

Body lenght (cm) 130 125 - 158

Body surface (m²) 1,12 0.99 - 1.41

Age (years) 8,65 7.12 - 20.13

Sex 3 female 6 male

Underlying diseases 8 GVHD and ALL/GVHD

Patients_ECP

Parameter settings_AUTO

N=16 Median Min – Max

Total whole blood drawn (ml) 5267 1687 - 18882

Whole blood (ml/kg bw) 211 123 - 322

Time used [hh:mm] 4:15 1.44 - 6:34

Product volume (ml) 90 31 - 332

N cycles 4 2 - 20

ACD-A ratio 13 12 - 15

Collection preferency 30 20 - 40

Parameter settings_ECP

N=9 Median Min – Max

Total whole blood drawn 3617 2328 - 5100

Whole blood / kg bw 98 58 - 158

Time used 3:04 2:00 - 4:23

Product volume 54 18 - 72

N cycles 3 1 - 4

ACD-A ratio 12 12

Collection preferency 30 20 - 40

N=16 Median Min - Max

Hemoglobin (g/dl) 10,5 8 - 12

Hematocrit (%) 30,80 21 - 34

Platelets (G/l) 173 41 - 255

Leukocyte (G/l) 15,5 6 - 74

Lymphocytes (%) 4,35 1 - 15.4

Monocytes (%) 11,75 5.5 - 41

MNC (%) 18,55 7 - 59.8

Granulocytes (%) 81,45 40.2 - 93

CD34+ cells (%) 0,33035 0.05 - 1.39

cells/µl 46,5 18 - 270

Pre apheresis values_AUTO

Pre apheresis values_ECP


Hemoglobin (g/dl) 11,4 10 - 12.1

Hematocrit (%) 33,00 30 - 36

Platelets (G/l) 158 31 - 362

Leukocyte (G/l) 4,72 1.3 - 7.07

Lymphocytes (%) 31,1 10 - 52.2

Monocytes (%) 6,2 3.7 - 10.3

MNC (%) 38,3 15.7 - 67.8

Granulocytes (%) 61,6 32.5 - 84.5


Hemoglobin (g/dl) 9,5 8.2 - 12.8

Hematocrit (%) 27 23 - 35.2

Platelets (G/l) 105 30 - 167

Leukocyte (G/l) 18,8 4.9 - 69

Lymphocytes (%) 4,15 1 - 16.8

Monocytes (%) 10 2.5 - 39.6

MNC (%) 14 4 - 44.4

Granulocytes (%) 86 51 - 95.5

CD34+ cells (%) 0,21 0.01 - 1.34

cells/µl 32 0.2 - 194

Post apheresis values_AUTO

Post apheresis values_ECP


Hemoglobin (g/dl) 10,2 8.6 - 11.6

Hematocrit (%) 29,00 25 - 34

Platelets (G/l) 114 9 - 320

Leukocyte (G/l) 4,85 1.5 - 7.6

Lymphocytes (%) 36,1 8.7 - 59.4

Monocytes (%) 5,3 1.6 - 11.3

MNC (%) 45,7 14.7 - 74.1

Granulocytes (%) 54,4 25.8 - 85.3


RBC (T/l) 0,25 0.1 - 0.5

Hematocrit (%) 2,65 1 - 7

Platelets (G/l) 1044,5 171 - 2330

Leukocyte (G/l) 154,5 106.7 - 275

Lymphocytes (%) 25,00 4.5 - 48.5

Monocytes (%) 35 5.5 - 58

MNC (%) 68,50 21 - 96.5

Granulocytes (%) 31,50 3.5 - 79

CD34+ cells (%) 0,86 0.35 - 4.18

cells/kg bw 6,1 2.2 - 17

Apheresis product values_AUTO

Apheresis product values_ECP


RBC (T/l) 0,2 0.1 - 0.5

Hematocrit (%) 2 1 - 6

Platelets (G/l) 563 70 - 2310

Leukocyte (G/l) 50,9 0.9 - 73

Lymphocytes (%) 71,50 6 - 81.5

Monocytes (%) 14 7 - 42.5

MNC (%) 97,50 13.5 - 99.5

Granulocytes (%) 2,50 0.5 - 86.5

patient Eff_CD34 Eff_lympho Eff_Ery Eff_G Eff_M Eff_Th Eff_MNC Eff_L

AUTO20110001 68,48 57,56 0,1 0,87 8,02 18,9 23,56 7,49

AUTO20110006 67,95 69,64 0,2 7,25 49,46 22,41 59,56 34,75

AUTO20110007 59,56 58,97 0,13 0,63 51,26 23 58,46 13,96

AUTO20110008 26,79 56,34 0,32 5,16 57,86 16,79 55,66 14,76

AUTO20110010 72,38 20,5 0,09 13,75 71,26 18,41 73,35 31,01

AUTO20110011 66,23 66,29 0,19 1,34 55,23 14,65 57,92 6,4

AUTO20110013 83,86 80,31 0,24 11,97 75,64 38,42 79,04 16,06

AUTO20120005 277,38 86,73 0,12 5,13 52,58 11,15 67,41 19,04

AUTO20120008 55,12 50,24 0,12 50,14 54,03 9,56 52,41 51,21

AUTO20120009 74,46 76,54 0,04 6 61,43 1,97 66,63 18,63

AUTO20120008 37,39 60,51 0,1 6,46 25,6 8,23 39 10,14

AUTO20120012 166,94 177,49 0,19 7,91 33,11 17,04 84,15 16,52

AUTO20130001 53,32 132,56 0,24 2,74 22,96 19,79 49,37 10,05

AUTO20130002 117,68 112,41 0,23 3,04 60,86 24,58 87,26 8,61

AUTO20130003 113,92 116,68 0,16 16,03 26,99 19,68 112,16 25,92

AUTO20130004 65,24 59,23 0,07 27,3 2,78 19,6 45,56 29,81

median 68,215 67,965 0,145 6,23 51,92 18,655 59,01 16,29

Collection efficiencies_AUTO

Collection efficiencies_ECP

patient Eff_lympho Eff_Ery Eff_G Eff_M Eff_Th Eff_MNC Eff_L

ECP20110009 55,81 0,09 8,93 39,55 16,81 52,45 31,46

ECP20110023 32,38 0,14 0,09 16,99 0 26,97 6,62

ECP20110026 21,63 0,05 36,23 64,23 7,92 30,28 35,29

ECP20110031 15,39 0,03 0,15 28,33 0 18,84 4,69

ECP20110056 46,4 0,13 1,15 68,96 18,57 48,45 24,39

ECP20110060 16,62 0,03 0,19 29,39 1,83 19,24 5,41

ECP20110085 66,65 0,19 0,8 56,94 22,46 64,47 46,19

ECP20110088 56,84 0,12 0,2 56,15 29,16 56,48 23,56

ECP20120005 1,93 0,15 0,31 1,5 8,72 0,79 0,61

median 32,38 0,12 0,31 39,55 8,72 30,28 23,56

HPC IN DETAIL

Correlation between CD34 positive cell yield and CD34 positive cell number in the peripheral blood_AUTO

y = 15,09x - 20,97 R² = 0,609

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Correlation between Eff_CD34 and Leukocyte count pre apheresis in peripheral blood_AUTO

y = 0.0019x + 29.404 R² = 0.3182

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Eff

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Leuko_PB_vor

Correlation between CE CD34 positive cells and whole blood volume processed_AUTO

y = 0.3507x + 11.216 R² = 0.1306

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WBV_kg BW

Correlation between CE CD34 positive cells and number of cycles performed_AUTO

y = 2.9646x + 65.498 R² = 0.0814

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Correlation between Eff_CD34 and collection preference in the peripheral blood_AUTO

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Collection preferency

± Standarddeviation

IN MONONUCLEAR CELLS =>

Correlation between pre apheresis MNC and MNC_kg_buffy_AUTO

y = 5,3006x + 2338,6 R2 = 0,215

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Whole blood processed / kg bw 1 = HPC and 2 = ECP

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± Standardabweichung

Number of cycles processed 1 = HPC and 2 = ECP

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MNC count in blood pre 1 = HPC and 2 = ECP

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U – Test: p = 0.012

MNC % in the product 1 = HPC and 2 = ECP

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U – Test: p = 0.0035

MNC content in the product 1 = HPC and 2 = ECP

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U – Test: p = 0.001

MNC content in the product 1 = HPC and 2 = ECP

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U – Test: p = 0.00049

MNC collection ratio 1 = HPC and 2 = ECP

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U – Test: p = 0.95

CAN WE PREDICT ANYTHING?

Formel A CD34+ 106/kg = CD34+/µl PB / 10

• Simple formular

• Is only valid in aphereses procedures standardized for blood volume processed, stable collection efficiency (=> parameter settings!)


y = 1.509x - 2.0975 R² = 0.6096

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CD 34_10E06_kg


y = 7.014ln(x) - 31.585 R² = 0.6052

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Eff_CD34

Formel B C = [(AxD):Bx0.5]:1000

• A = CD34+ cells in peripheral blood [1003/ml]

• B = bodyweight [kg]

• C = CD34+ cells/kg bw in the product

• D = processed volume

• Estimation of an efficiency of 50%


y = 1.0115x + 0.1793 R² = 0.5085

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y = 5.6977ln(x) - 24.851 R² = 0.6935

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WE CAN CALCULATE THE BLOOD VOLUME NEEDED TO BE PROCESSED

D = [(C:0.5)xB:A]x1000

• A = CD34+ cells in peripheral blood [1003/ml]

• B = bodyweight [kg]

• C = CD34+ cells/kg bw in the product

• D = processed volume

• Estimation of an efficiency of 50%

COLLECTION EFFICIENCY IN PLATELETS =>

Platelet count in blood pre / CE 1 = HPC and 2 = ECP

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Platelet count pre

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y = -0.0594x + 27.137 R² = 0.1603

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Correlation between platelets collection efficiency and number of cycles_AUTO

y = 0.8442x + 11.377 R² = 0.3657

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Correlation between platelets collection efficiency and number of cycles_ECP

y = 4.9335x - 2.5341 R² = 0.1888

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Platelet collection ratio versus number of cycle performed

y = 11.336ln(x) + 4.8427 R² = 0.3762

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Side effects

• Hypocalcemia in a 10 kg child (Ca++ 0.86 mmol/l) complete restitutio after Ca 10% i.v.

• Leackage in the set => change of set, procedure was finished with 30 min delay

Engraftment data

patient disease bw transplanted engrafted

AUTO20110001 ATRT 40,00 y y

AUTO20110006 AML recurrency 54,50 y y

AUTO20110007

Hepatoblastom

/hepatozelluläres

Karzinom 12,70

n n.a.

AUTO20110008 NB 10,25 n n.a.

AUTO20110010 PNET zentral 19,00 n n.a.

AUTO20110011 Ewing Sarkom 35,00 n n.a.

AUTO20110013 Ewing Sarkom 51,30 n n.a.

So far 2 / 7 patients are treated with Mega therapy and autologous rescue, both engrafted regulary.

Triggering first chamber flush

• „Less operator input was required, although 26% of Spectra Optia apheresis procedures required triggering of the first chamber flush.” [1]

1 Brauninger Transfusion 2011

• Flow pauses were 40% less frequent with the Spectra Optia (Table 2), likely by virtue of electronic interface management and an algorithm that automatically restarts the flow if pressure recovers quickly.


Allogeneic stem cell apheresis outcomes with the COBE Spectra MNC

and the Spectra Optia v.5.0. (A) Collection efficiency for CD34+ cells is

negatively correlated with preapheresis CD34+ cell counts.

Preapheresis circulating CD34+ cells counts (X-axis) are plotted over

the corresponding collection efficiency CE1 (Y-axis) for this donor. (

,—) Results from the Spectra Optia; (, - - -) data fromthe COBE Spectra

MNC.

Spectra Optia apheresis procedures are associated with better

collection efficiency in less well-mobilized donors. Preapheresis

circulating CD34+ cell counts were stratified as indicated. CE1

(mean + SEM) for each group is drawn to the Y-axis. Significant

differences between the groups. ( grey) Spectra Optia apheresis

procedures; (black ) COBE Spectra MNC collections.

• PLT attrition was calculated as (1—postapheresis PLT count/preapheresis PLT count) x 100.

Spectra Optia PLT attrition correlates with preapheresis PLT counts.

Preapheresis PLT counts (X-axis) are plotted over the corresponding

value for PLT attrition (Y-axis). ( ,—) Results from the Spectra Optia; (, -

- -) data fromthe COBE Spectra MNC.Markedly lower PLT attrition with

the Spectra Optia was observed particularly in donors with fewer PLTs.

Conclusion

• Safe and efficient for HPC cell harvest in young children and adolescents

• Good correlation of PB HPC cell count and yield and easily predictable

• The same is found for MNC

• The number of cycles processed might be an independent factor for collection efficiency, not only for white blood cells

• Loss of platelets seems to be dependent on the number of cycles necessary

Optia – Efficiency Considerations With MNC Collection PBCS harvesting in children Some of the...

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