Does the Inclusion of CD34+ B-cell Progenitors (Hematogones) in Stem Cell Enumeration of Apheresis Product Using ISHAGE Protocol Affect the Final Harvest dose Adequacy and the Outcome of Transplantation? A Single Institution Experience from Southern India

Abstract

Purpose

Hematogones have similar antigenic and light scatter properties when compared to CD34+ hematopoietic stem cells (HSC) but they form a separate cluster with dimmer CD45 expression. These should be excluded while enumerating HSC, as their inclusion can overestimate and hence affect the final dose of HSC. However, their exact impact on the outcome of HSC transplant (HSCT) is not entirely known and hence this study was undertaken to address these issues, if any.

Methods

This was a retrospective study in which patients undergoing HSCT were included, and flow cytometric enumeration was done on the apheresis product using single platform ISHAGE protocol. The gating of all plots was reviewed and carefully studied for hematogone population which would have otherwise been included in the original gating.

Results

Totally 78 patients underwent HSCT during the study period. On re-analysis, it was found that 10/78 (12.8%) cases had a separate hematogone population which was included in the HSC in the original analysis. Out of these 10 cases, 7/51 and 3/27 were in autologous and allogenic subgroup respectively. However, all the ten cases ultimately had adequate final stem cell dose and had successful engraftment.

Conclusion

The inclusion of hematogones in CD34+ HSC enumeration of apheresis products did not yield any impact on neither the final dose nor the outcome of transplant in this study. However, it is recommended to exclude them from the final count when they are > 10% of the final HSC lest it overestimate the final harvest dose and outcome of HSCT.

Introduction

Enumeration of CD34+ stem cells from pre-apheresis sample after mobilisation helps in predicting the final dose adequacy in hematopoietic stem cell transplants (HSCT). Most often, if the pre-apheresis hematopoietic stem cell (HSC) count is ≥ 10 cells/µl, it is the time to collect HSC which will maximise the yield of stem cell harvest during apheresis [1, 2]. The International Society of Hematotherapy and Graft Engineering (ISHAGE) protocol lays down guidelines for this enumeration using flow cytometry and the following parameters such as bright expression of CD34, dim expression of CD45, low side scatter and low to moderate forward scatter with respect to lymphocytes as defining criteria for the identification of HSC [3]. It has been reported that the normal B-cell progenitors (hematogones) are also CD34+ and also have similar light scattering properties comparable to the CD34+ HSC and small lymphocytes [4]. However, these hematogones have lower forward scatter when compared to the CD34+ stem cells and are CD19+ and CD10+ [4]. These cells have the risk of being included in the CD34+ stem cell population while using the standard ISHAGE protocol (as only CD34 and CD45 antibodies are used) and by overestimating the total CD34+ cell count, this could potentially affect the final dose of HSC and have an effect on the final engraftment. However, their exact impact on the outcome of HSC transplant (HSCT) is not entirely known and hence this study was undertaken to address these issues, if any.

Methods

This was a retrospective study conducted over a period of 3 years from January 2019 till December 2021, from the Department of Pathology, Transfusion Medicine and Medical Oncology, tertiary care centre from Southern India. Patients undergoing both autologous and allogenic HSCT was included in this study and flow cytometric enumeration of CD34+ HSC was done on the apheresis stem cell harvested product using single platform ISHAGE protocol in 3-laser 10-color Navios flow cytometer (Beckman Coulter). The gating of all flow cytometric enumeration was reviewed and carefully studied for identifying a separate hematogone population which would have otherwise been included in the original gating and report. In this study, hematogones were identified based on lower forward scatter when compared to CD34+ stem cells (similar light scatter property) but they are seen to form a separate cluster and have dimmer CD45 expression [4, 5]. In order not to compromise the single platform ISHAGE protocol, no additional B-cell markers were utilised for this purpose [4]. In all these cases, we specifically looked for the percentage of hematogones (> 01%), type of transplant, absolute CD34 count (per µl) initially reported and corrected for the hematogones (revised absolute CD34 count), final dose of CD34 in the harvest and the engraftment status on follow-up.

Results

A total of 78 patients underwent HSCT in our hospital during the study period of three years including 51 autologous and 27 allogenic HSCT. On re-analysis of all the initially gated CD34 plots, it was found that 10/78 (12.8%) cases had a separate hematogone population (> 01%) which was also included in the CD34+ count in the original analysis and report. Hematogone population was identified as events forming a separate cluster with lower forward scatter than the stem cells (Fig. 1). Out of these 10 cases, 7 were in autologous subgroup and 3 were in allogenic subgroup. However, all the ten cases ultimately had adequate final stem cell dose even after correcting for the hematogones and had successful engraftment on follow-up till date (Table 1). There was no evidence of graft failure or delayed engraftment in all these 10 cases.

Fig. 1.

Identification of hematogones population (blue) and stem cells (black) in single platform ISHAGE gating strategy for CD34 + stem cell enumeration: (A) Data acquisition vs. time with beads (B) SSC vs. 7-AAD viability plot (C) SSC vs. CD45 plot [R1 gate] (D) SSC vs. CD34 plot [R2 gate] (E) SSC vs. CD45 plot [R3 gate] (F) SSC vs. FSC plot [R4 gate] (G) SSC vs. FSC plot [R5 gate showing lymphocytes] (H) CD34 vs. CD45 plot

Table 1.

Analysis of hematogones% and original and revised CD34 cell count with final engraftment

Sl. No.	Age/ gender	Type of transplant and primary disease	Hematogones (%)	Original CD34 count (per µL)	Revised CD34 count (per µL)	Final stem cell dose harvested	Final outcome of HSCT
1	14/Male	Autologous (relapsed Hodgkin’s lymphoma)	13.6	2202.5	1902	9.0	Engrafted
2	11/Male	Autologous (relapsed Hodgkin’s lymphoma)	6.5	1767.5	1653	12.1	Engrafted
3	52/Male	Autologous (Multiple myeloma)	7.7	1355	1250	3.2	Engrafted
4	26/Male	Autologous (relapsed Hodgkin’s lymphoma)	8.1	1678	1542	2.9	Engrafted
5	51/Male	Autologous (Multiple myeloma)	1.8	1102	1082.5	3.6	Engrafted
6	36/Male	Autologous (Multiple myeloma)	5.3	892	845	2.1	Engrafted
7	58/Male	Autologous (Multiple myeloma)	6.4	1090	1020	4.9	Engrafted
8	36/Male	Allogenic donor	1.1	3279	3243	12.4	Engrafted
9	44/Male	Allogenic donor	25	1400	1050	4.0	Engrafted
10	33/Female	Allogenic donor	22.7	2752.5	2127.5	10.3	Engrafted

Discussion

Hematogones have similar property when compared to CD34+ stem cells but they are seen to form a separate cluster and have dimmer CD45 expression [5]. These should be excluded when gating for stem cells, as their inclusion can overestimate the stem cell count and hence the final dose of stem cells. However, their exact impact on the final harvest dose adequacy is not entirely known.

Thiago and Sutherland [5] have reported two cases where the hematogone population was greater than 10% of all CD34+ cells counted i.e. 30% and 65% of the total CD34 + cells. In the former case, they found that the final stem cell dose was adequate and the patient went on to achieve timely and successful engraftment. However, in the latter case, since almost two-thirds of the total CD34+ cell counts were hematogones, they advised remobilisation and re-apheresis for the patient. They recommended that whenever the hematogones account for more than 10% of the total CD34+ cells counted, the hematogone population should be separately quantified and excluded from the final CD34+ stem cell count.

In another study conducted by Ondrejka et al. [6], they reviewed 274 apheresis sample in 12 months period and found that 61% included hematogones, with a median percentage of 7.1% of total CD34+ cells (ranging from 1.3 to 78.0%). The found that the patients with sample positive for hematogones ultimately had a better likelihood for neutrophil engraftment, however there was no impact on platelet engraftment. Recently Matos et al. [7] used anti-CD19 APC antibody to identify the hematogones, however they stated that it was not strictly required and currently do not know whether it would be beneficial or not. Also, they commented that additional validation should be required for the appropriate clone and fluorochrome combination.

In our study, we found a total of 10/78 (12.8%) cases had a separate hematogone population which was also included in the CD34+ count in the original analysis. We found that contrary to Thiago and Sutherland, all the ten cases ultimately had adequate final stem cell dose and had successful engraftment with no change seen after re-analysis [5]. This was in concordance with the results of Ondrejka et al. [6]. It could be attributed to a smaller sample size where hematogones were identified. It has been reported [5] that in cases with hematogones population > 10%, they should be excluded from the final CD34+ cell count and a corrected CD34+ cell count should be provided before issuing the report as it could otherwise adversely affect the outcome of the transplants.

In our study, we found that three of our cases had > 10% hematogones population, with all others having < 10% hematogones. We found that the re-analysed CD34+ cell count did not affect the final outcome of HSCT in any of the ten cases even after correcting for the presence of hematogones. This could be attributed to 2 out of the 3 cases with > 10% hematogones being healthy allogenic donors with adequate CD34+ cell count and 1 case of an 11 year old male child who is expected to have higher hematogones in the marrow [4]. We also found that younger age patient/ donor had higher percentage of hematogones and also adequate HSC final dose in the harvest product.

Conclusion

The inclusion of hematogone population in CD34+ stem cell enumeration of apheresis products did not yield any impact on the final dose adequacy and the outcome of HSCT in this study. However, it is recommended to have much larger studies prospectively evaluating whether to exclude them or not from the final CD34 count, lest it overestimate the final harvest dose, and their effect on HSCT to be carefully validated.

Author contribution

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Naadia Fatima Nadeem, Prabhu Manivannan, Abhishekh Gowda and Smita Kayal. The first draft of the manuscript was written by Naadia Fatima Nadeem and all authors commented and corrected the previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

Source(s) of support/ funding: Intramural grant received from the Institute (Rs.1,98,500/-).

Declarations

Conflicting Interest

Nil.

Ethics Approval

Ethical approval was obtained from the local Ethics Committee of the institute and all the procedures performed met the ethical guidelines and waiver of consent obtained from the Ethics committee.