|Year : 2017 | Volume
| Issue : 2 | Page : 97-101
Total leukocyte count-based predictor tool for calculating hematopoietic progenitor cell dose in bone-marrow harvest
Dinesh Arora1, Aseem K Tiwari1, Ruchira Misra2, Ravi C Dara3, Geet Aggarwal1, Nitin Sood2, Gunjan Bhardwaj1
1 Department of Transfusion Medicine, Medanta-The Medicity, Gurgaon, Haryana, India
2 Department of Medical and Hemato-oncology, Medanta-The Medicity, Gurgaon, Haryana, India
3 Department of Transfusion Medicine, Manipal Hospitals, Jaipur, Rajasthan, India
|Date of Web Publication||11-Sep-2017|
Aseem K Tiwari
Department of Transfusion Medicine, Medanta-The Medicity, Gurgaon, Haryana
Source of Support: None, Conflict of Interest: None
Background: Hematopoietic progenitor cell transplantation is increasingly being used as the curative therapy in India for various clinical conditions. There are three main sources of hematopoietic progenitor cells; hematopoietic progenitor cell-apheresis (HPC-A), hematopoietic progenitor cell-bone marrow (HPC-M) and hematopoietic progenitor cell-Umbilical Cord (HPC-C). The number of CD34+ cells in HPC-C is fixed. In HPC-A, the number of CD34+ cells collected is based on the baseline peripheral blood counts and a second harvest can be easily performed. The trickiest calculation of CD34+ cells adequacy is in case of HPC-M harvest, where the end point of harvest cannot be predetermined. Aim: The aim was to study the accuracy of a total leukocyte count (TLC)-based predictor tool in predicting CD34+ dose in comparison to the actual CD34+ cell counts in the harvest. Materials and Methods: This was a prospective study to validate the tool. The data captured included patient and donor demographic data, disease condition, mobilization regimen of the donor, progenitor cell harvest data, dose collected, cryopreservation if any, progenitor cell infusion data, engraftment, and follow-up of the patient including day thirty and day hundred chimerism in a tertiary care hospital. Results: Five patients were included in the study. For each patient, the target dose and volume were collected in a single HPC-M harvest attempt, and no repeat harvests were required. The average volume of HPC-M harvest collected was 195 ml. The average CD-34+ cell dose in HPC-M harvest achieved was 4.3 × 106/kg (Range = 3.39–6.42). This was 85.7% of the targeted dose calculated on the basis of TLC-based predictor tool. Conclusion: This study reiterates the importance of a simple TLC-based predictor tool (formula) for estimation of HPC-M volume to be harvested.
Keywords: Hematopoietic progenitor cell-bone marrow, hematopoietic progenitor cell-umbilical cord total leukocyte count, hematopoietic progenitor cell-apheresis, mononuclear cell
|How to cite this article:|
Arora D, Tiwari AK, Misra R, Dara RC, Aggarwal G, Sood N, Bhardwaj G. Total leukocyte count-based predictor tool for calculating hematopoietic progenitor cell dose in bone-marrow harvest. Glob J Transfus Med 2017;2:97-101
|How to cite this URL:|
Arora D, Tiwari AK, Misra R, Dara RC, Aggarwal G, Sood N, Bhardwaj G. Total leukocyte count-based predictor tool for calculating hematopoietic progenitor cell dose in bone-marrow harvest. Glob J Transfus Med [serial online] 2017 [cited 2017 Sep 23];2:97-101. Available from: http://www.gjtmonline.com/text.asp?2017/2/2/97/214278
| Introduction|| |
Hematopoietic progenitor cell transplantation is increasingly being used as the curative therapy for various clinical conditions in India., There are various sources of hematopoietic stem cells commonly used clinically; hematopoietic progenitor cell-apheresis (HPC-A), hematopoietic progenitor cell-bone marrow (HPC-M), and hematopoietic progenitor cell-Umbilical Cord (HPC-C). The number of CD34+ cells in HPC-C is fixed depending on the volume collected before clamping the cord. In HPC-A, the number of CD34+ cells collected is decided by the microprocessor of the apheresis equipment based on the baseline peripheral blood counts including total leukocyte count (TLC), mononuclear cell (MNC) count, and CD34+ cell count. These peripheral blood counts can be performed immediately before or during the harvest procedure. If a single HPC-A harvest does not provide adequate dose of CD34+ cells, a second harvest can always be performed on a subsequent day. The trickiest calculation of CD34+ cells adequacy is in case of HPC-M harvest, where the end point of harvest cannot be predetermined as the enumeration of CD34+ cells is difficult during the harvest procedure with the donor still in an operation theater under general anesthesia (GA). Further, since the HPC-M transplantation is generally done in pediatric population for non-malignant conditions,,, the recipient and donor are generally young children making the possibility of second harvest difficult.
Thus, hematopoietic cell dose harvested or/and HPC-M volume collected should be based on some predictor tool. We studied the performance of one such TLC-based predictor tool (formula) in predicting CD34+ cells in comparison to the actual CD34+ cell counts in the harvest.
| Materials and Methods|| |
This study was carried out prospectively at a tertiary care center in Delhi-NCR region, India from January to August 2014.
The data for HPC-M harvest and transplantation were prospectively collected. This data included patient and donor epidemiological data, disease condition, mobilization regimen of the donor, progenitor cell harvest data, dose collected, cryopreservation planned (if any), progenitor cell infusion data, engraftment and follow-up of the patient including day 30 and day 100 chimerism.
Patients with HLA-matched (6/6) family donors, suffering from various nonmalignant pediatric clinical conditions who underwent HPC-M transplantation during this period were included in the study. All the patients were screened and cleared for transplant after normal liver function tests (LFTs), renal function tests (RFTs), echocardiography, pulmonary function tests, ear nose throat, dental, and psychiatric evaluation.
The donors of marrow are evaluated using the same selection and exclusion criteria, which is applicable to blood donors as laid down in drugs and cosmetics act. In addition to the basic criteria, donor selection was based on HLA typing with 6/6 (HLA-A, B, and DRB1)-matched siblings. All donors were tested for various infectious disease markers including anti-HIV, anti-HCV, anti-HB core (total), anti-cytomegalovirus (CMV), HBsAg, tests for Syphilis and Malaria, and CMV polymerase chain reaction. ABO and Rh blood group was done for both donor(s) and recipient(s).
Donors were mobilized using granulocyte colony-stimulating factor (G-CSF) at a dose of 10 μg/kg body weight in two divided doses given subcutaneously for 4 days and in a single dose on the 5th day before harvest.,,,
Total leukocyte count-based predictor tool
On the basis of mid-harvest TLC, the target dose was 5–6 × 108 cell/kg body weight of the patient. A dose of 5–6 × 106 CD34+ cells/kg body weight of patient  was targeted and a dose >2 × 106 CD34+ cells/kg body weight was considered as “adequate dose.”
The formula  used to calculate “target” volume of the dose, based on TLC of harvest product, was as follows:
The second formula used to calculate dose of progenitor cells harvested based on TLC counts was as follows:
Harvest and application of formula
All the HPC-M harvests were done on the fifth day of mobilization from bilateral posterior superior iliac spines under GA in an operation theater. Peripheral blood TLC (Sysmex XN 9000, Japan) was done before the harvest to assess mobilization. Mid harvest TLC was performed on the HPC-M product collected. This TLC value was used in the TLC-based predictor tool (formula I) to decide the target volume to be harvested. The maximum volume collected was limited to 20 ml/kg body weight of donor. The second formula (formula II) was used to predict the cell dose.
The CD34+ cells enumeration was done on final harvested product by flow cytometry using FACS verse (BD Biosciences, US). The enumeration was done as per standard International Society of Haematology and Graft Engineering protocol. This actual cell dose was compared with predicted cell dose in all patients.
Stem cell infusion
The infusion was carried out on the same day as HPC-M harvest in a positive pressure room with high-efficiency particulate air (HEPA) filters. The infusion was done through a Hickman line, and all the patients were prophylactically hydrated and given anti-histaminic before infusion. All patients were monitored for blood pressure, pulse, respiratory rate, and oxygen saturation during the infusion. Reverse barrier nursing was practiced according to the institutional protocol. Patient monitoring was done for critical biochemical and hematological parameters at defined frequency (complete blood counts and electrolytes once a day; LFT, RFT and blood glucose twice a week; blood culture as and when deemed necessary). Antimicrobial prophylaxis included Fluconazole and Acyclovir.
Neutrophil engraftment was defined as absolute neutrophil count of 500 cells/mm 3 (0.5 × 109/L) or greater for three consecutive days. Platelet engraftment was defined by the achievement of a continued platelet count of 20,000/mm 3 (20 × 109/L) or greater for two consecutive days, at least 7 days from the last platelet infusion. Those who did not engraft up till 28th day were considered as “Engraftment Failure.” Standard protocols were followed in deciding blood component transfusion for all ABO compatible transplants and major, minor and bi-directional ABO incompatible transplants. The threshold for initiating RBC transfusion was hemoglobin of 7 g/dl and 10,000/μl for afebrile patients in the case of platelet transfusions. After successful engraftment, patients were shifted to non-HEPA rooms and discharged, once they were clinically stable.
After discharge, patients were followed up every alternate day for 1 week, twice a week for the next 2 weeks, then weekly till posttransplant day 100. The follow-up also included engraftment failure, complications, transplant-related death, and all-causes-death.
| Results|| |
Five patients with diseases including pure red cell aplasia (n = 1), Fanconi anemia (n = 2), osteopetrosis (n = 1), and Diamond–Blackfan anemia (n = 1) were included in the study. For each patient, the target volume and predicted dose, calculated from the above-mentioned formula (formula I and II, respectively) on the basis of mid-harvest TLC, was collected in a single HPC-M harvest attempt and no repeat harvests were required.
The average volume of the HPC-M harvest collected was 195 ml. The average CD34+ cell dose in HPC-M harvest achieved was 4.3 × 106/kg (range = 3.39–6.42). This was 85.7% of the predicted dose calculated on the basis of TLC predictor tool [Table 1].
|Table 1: Comparison of hematopoietic cell dose calculated by total leukocyte count method and actual CD34+ cell counts in the harvest|
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Four recipients' engrafted neutrophils on an average in 14.5 days (range: 13–17 days) and platelets on an average in 14 days (range: 13–17 days). The fifth recipient (a case of Fanconi anemia) succumbed to death on 9th posttransplant day as a result of intracranial hemorrhage. One patient was diagnosed with acute graft-versus-host disease (GVHD) on the 65th posttransplant day and was successfully treated for the same. All four patients had full donor chimerism on Day 100.
| Discussion|| |
This single center study included all the bone marrow harvests done for allogeneic transplants for nonmalignant conditions in pediatric patients during the study. Bone marrow harvest is generally preferred for pediatric nonmalignant conditions as this has a lesser incidence of GVHD. In this study, we used G-CSF for 4 days with a harvest done on the 5th day. This is the standard practice followed by most centers.,,, Most centers would plan to infuse a minimum of 2 × 106/kg CD34+ cells and not exceed 8 × 106/kg. We too targeted 5-6 × 106/kg CD34+ cells for harvest.
Since all the donors and recipients were of pediatric age, estimation of HPC-M volume to be harvested was crucial as the HPC-M harvest procedure is done under GA and in an operation theater with relatively low chances of repeat harvest. If the weight of the donor is more than that of the recipient, unnecessary excess amount of HPC-M can be harvested from donor that can result in hemodynamic instability and cellular deficiency. Further, infusion of larger HPC-M volume can lead to fluid overload in the recipient. Similarly, if the weight of the donor is less than that of the recipient, harvested HPC-M volume can be inadequate for successful treatment. Therefore, it makes sense to have some predictor tool to estimate the appropriate volume of bone marrow to be harvested providing adequate CD34+ cells for successful engraftment [Figure 1].
Typically, checking the TLC of the HPC-M product midway through the procedure helps to estimate the volume to be collected. Alternatively, flow cytometry-based CD34+ cell enumeration can be done. However, turn-around-time (TAT) for CD34+ cell enumeration is longer than the HPC-M harvest procedure TAT. Therefore, even in a setting where flow-cytometry is available, as in our institute, it is the mid-procedure TLC that guides the HPC-M volume collection estimate. Postharvest flow cytometry enumeration can be used to check the adequacy of the CD34+ cell in the harvested product. The mean of maximum permissible volume was 376 ml (range: 240-500 ml), however, with the help of predictor tool, the predicted and actual volume harvested (mean– 195 ml; range: 80–300 ml) were much lower [Table 2].
|Table 2: Demographics of donor and recipients and maximum permissible volume and actual volume of bone marrow harvested|
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The target dose of HPC-M was achieved in all the five cases. The postprocedure flow cytometry-based CD34+ cell enumeration yielded a mean dose of 4.29 × 106 CD34+ cells/kg body weight of recipient. This CD34+ cell was 85.7% of the target dose of 5 × 106 CD34+ cells/kg body weight of recipient. Although this actual collected dose was slightly less than the target cell dose, it was adequate as proven by the timely engraftment of neutrophils and platelets in all four patients [Table 1].
Although the sample size is small, based on the observations of the study, it can be said that estimation of volume and dose is crucial at the time of HPC-M harvest and this predictor tool (formula I and II) for dose calculation can very well be used in hematopoietic stem cell transplantation set-ups, especially where CD34+ analysis is not readily available.
| Conclusion|| |
This study reiterates the importance a simple TLC-based predictor tool (formula) for estimation of HPC-M volume to be harvested.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]