Novel method for autologous peripheral blood stem cell harvest using highly concentrated sodium citrate solution replacing acid citrate dextrose solution A

Abstract

Background

As the processed blood volume increases, a larger amount of anticoagulant (AC) is required, which leads to a serious issue of fluid dilution in large‐volume leukocytapheresis (defined as ≥3‐fold total blood volume). We previously reported a novel method for allogeneic peripheral blood stem cell harvest (PBSCH) using highly concentrated sodium citrate (HSC; 5.32%), which shortened the procedure time and reduced the need for an AC solution without heparin. In this study, we extended this novel method to autologous PBSCH (auto‐PBSCH) and compared it with patients who received auto‐PBSCH using normal concentrated sodium citrate (NSC; 2.2%).

Study Design and Methods

We retrospectively analyzed consecutive auto‐PBSCH data obtained using the Spectra Optia continuous mononuclear cell collection mode between May 2017 and May 2025 at our institution.

Results

Leukocytapheresis was performed using NSC in 36 patients and HSC in 22. In the HSC group, patients tended to be younger, had significantly lower body weight, and had significantly fewer hematopoietic tumors as primary diseases compared to the NSC group. After propensity score‐matched cohort adjusted for patient background, the total amount of AC solution was significantly lower (694 [range, 77–1648] vs. 298 mL [range, 64–797], p = .02), and procedure time was significantly shorter (224 [range, 117–395] vs. 181 min [range, 103–309], p = .048) in the HSC group. Furthermore, the loss rates of magnesium and potassium were lower in the HSC group.

Conclusion

This novel leukocytapheresis method demonstrated the efficacy and safety in auto‐PBSCH, while minimizing the patient burden.

Abbreviations

AC

anticoagulant

ACD‐A

acid citrate dextrose solution A

B‐NHL

B cell non‐Hodgkin lymphoma

BW

body weight

CE2

collection efficacy 2

CIC

circulating immature cell

cMNC

continuous mononuclear cell collection

G‐CSF

granulocyte‐colony stimulating factor

Hct

hematocrit

HL

Hodgkin lymphoma

HSC

highly concentrated sodium citrate

LVL

large‐volume leukocytapheresis

MRTK

malignant rhabdoid tumor of the kidney

NSC

normal concentrated sodium citrate

PBSCH

peripheral blood stem cell harvest

PBV

processing blood volume

PCN

plasma cell neoplasm

Plt

platelet

PS

propensity score

RBC

red blood cell

SEM

standard error of the mean

TBV

total blood volume

T‐NHL

T cell non‐Hodgkin lymphoma

WBC

white blood cell

1. INTRODUCTION

Peripheral blood stem cell (PBSC) transplantation requires the collection of mononuclear cells using a centrifugal leukocytapheresis system and requires an anticoagulant (AC) to prevent clotting of the extracorporeal circuit and the collected blood. ¹ , ² Citrate exerts its AC effect by reversibly chelating divalent cations such as calcium and magnesium, thereby inhibiting their normal physiological functions, whereas citrate toxicity can occur because citrate‐containing blood is reinfused into patients. ² , ³ , ⁴ , ⁵ Furthermore, large‐volume leukocytapheresis (LVL), an alternative to repeated leukocytapheresis to obtain sufficient target CD34⁺ cell yields, may increase the risk of fluid overload by the accumulation of acid citrate dextrose solution A (ACD‐A), generally used as the AC, especially in patients with low total blood volume (TBV), including children or small adults. ⁶

We previously devised a novel method of initiating leukocytapheresis at an inlet blood flow‐to‐AC ratio (hereafter referred to as the AC ratio) of 24:1 with increasing concentrations of sodium citrate solution used for AC up to 5.32% and examined its efficacy and safety in healthy donors for allogeneic PBSC harvest (allo‐PBSCH). Compared with standard concentrations of sodium citrate solution, leukocytapheresis with highly concentrated sodium citrate (HSC) demonstrated a significant reduction in procedure time with an increased maximal inlet flow rate and a significant decrease in the total amount of AC solution. ⁷ In this study, we expanded this novel leukocytapheresis procedure to patients undergoing autologous‐PBSCH (auto‐PBSCH) and evaluated its efficacy and safety by comparing patients who received auto‐PBSCH using the conventional procedure (i.e., using normal concentration of AC solution [2.2%]).

2. STUDY DESIGN AND METHODS

2.1. Patient selection

Patients who underwent leukocytapheresis for the treatment of primary disease at Okayama University Hospital between May 2017 and May 2025 were included in this study. Patient data, including diagnoses and tests for leukocytapheresis, were retrospectively collected from medical records. The study protocol was approved by the Institutional Review Board of Okayama University Hospital, and participation was conducted using an opt‐out approach. This analysis was performed in accordance with the ethical standards of the Institutional and National Research Committee and the 1964 Declaration of Helsinki and its later amendments.

2.2. Peripheral blood stem cell mobilization and transfusion prior to leukocytapheresis

PBSC mobilization was performed using granulocyte‐colony stimulating factor (G‐CSF) (Filgrastim Biosimilar, 400 μg/m²; Mochida Pharmaceutical Co., Ltd., Tokyo, Japan or Lenograstim, 10 μg/kg; Chugai Pharmaceutical Co., Ltd., Tokyo, Japan) in combination with chemotherapy or G‐CSF alone for 5 days. In addition, patients with peripheral blood (PB) CD34⁺ cell counts <20/μL on the day before leukocytapheresis received plerixafor (0.24 mg/kg; Sanofi Co., Ltd., Paris, France). Leukocytapheresis was initiated 2 h after the final G‐CSF injection. If the target CD34⁺ cell yield was not achieved on the first attempt, additional G‐CSF ± plerixafor administration and leukocytapheresis were performed the following day, which were continued until the third day. To ensure safe leukocytapheresis, red blood cell (RBC) and/or platelet (Plt) concentrate transfusions were administered, based on our previous report. ⁸

2.3. Leukocytapheresis procedures

Adequate blood access was established via peripheral venous puncture, inner jugular or femoral central venous access, or radial arterial access. All leukocytapheresis procedures were performed in the continuous mononuclear cell collection (cMNC) mode using Spectra Optia (Terumo BCT, Tokyo, Japan) according to local standard operating procedures. The Spectra Optia cMNC procedures at our institution used a collection flow rate of 1 mL/min or a 0.05‐fold initial inlet flow rate (if the initial inlet flow rate was <20 mL/min), and a packing factor of 4.0, which indicated the centrifugation forces. In pediatric patients requiring sedation during leukocytapheresis, dexmedetomidine (4 μg/mL, Nipro Co., Ltd., Osaka, Japan), fentanyl (0.05 mg/mL, Nichi‐Iko Pharmaceutical Co., Ltd., Toyama, Japan), or secobarbital (10 mg/mL, Nichi‐Iko Pharmaceutical Co., Ltd.) was administered under the management of anesthesiologists or pediatricians. Vital signs were continuously monitored using a mobile bedside monitor.

According to a previous report, ⁷ we used a mixed solution of ACD‐A (Terumo BCT) and 10% sodium citrate hydrate (Fuso Pharmaceutical Industries, Osaka, Japan) as the AC solution in the HSC group, and this procedure has been performed since November 2022. The AC ratio was initially set at 12:1 in the normal concentrated sodium citrate (NSC) group and 24:1 in the HSC group. To avoid abrupt changes, the AC infusion rate was initially started at 0.8 mL/min/L TBV and manually adjusted 30 min after initiation. To prevent hypocalcemia, all patients received a continuous intravenous infusion of 85 mg/mL calcium gluconate hydrate (Nichi‐Iko Pharmaceutical Co., Ltd.) during leukocytapheresis (5–15 mL/h depending on the patient body weight [BW]). Experienced physicians assessed hypocalcemic symptoms, such as dysesthesia, cramps, and nausea, and graded them using a previously described approach. ⁹ , ¹⁰

The TBV was measured by the Spectra Optia device calculator. If the patients BW was less than 25 kg, TBV was calculated by the operator as 80 mL/kg for patients aged <1 year or 70 mL/kg for patients aged ≥1 year. The processing blood volume (PBV) for each leukocytapheresis was determined by the attending physician based on the pre‐leukocytapheresis (pre‐) PB CD34⁺ cell counts, target CD34⁺ cell yields, and estimated CD34⁺ cell collection efficiency 2 (CE2). ¹¹ Based on our experience and previous reports, ¹² , ¹³ , ¹⁴ we estimated the CD34⁺ CE2 at 30%. Following previous studies, ⁸ , ¹⁵ LVL was defined as PBV ≥3‐fold TBV. The CD34⁺ CE2 was calculated as follows: CD34⁺ CE2 (%) = ([product CD34⁺ cell counts × product volume]/[pre‐PB CD34⁺ cell counts × PBV]) × 100. ¹⁶ The target CD34⁺ cell yields were specified by our institution as follows: ≥2 × 10⁶ cells/kg for patients with hematopoietic tumors excluding multiple myeloma (MM) and ≥4 × 10⁶ cells/kg for those with solid tumors and MM. For patients undergoing PBSCH for two or more consecutive days, only data from the first day were analyzed in this study.

2.4. Sample evaluation

Complete blood cell counts were determined using an ADVIA 2120i hematology analyzer (Siemens Healthineers, Erlangen, Germany). Circulating immature cell counts, morphologically identified as myeloblasts, promyelocytes, myelocytes, metamyelocytes, and erythroblasts, were visually assessed by clinical laboratory technicians. Potassium and magnesium levels were measured using a JCA‐BM8040 automatic biochemical analyzer (Japan Electron Optics Laboratory Co., Ltd., Tokyo, Japan), and ionized calcium level was measured using an ABL800 FLEX Radiometer (Copenhagen, Denmark). CD34⁺ cell counts were determined in pre‐leukocytapheresis PB samples collected immediately before procedure and in the final product using Stem‐Kit Reagents (Beckman Coulter, Brea, CA, USA) and were analyzed using a NAVIOS EX flow cytometer (Beckman Coulter). Electrolytes (potassium, ionized calcium, and magnesium) or Plt loss rate was calculated using the following equation: loss rate (%) = ([pre‐leukocytapheresis level − post‐leukocytapheresis level]/pre‐leukocytapheresis level) × 100.

2.5. Statistical analysis

The Mann–Whitney U test was used to compare continuous variables, and Fisher's exact test was used to compare categorical variables. All statistical tests were two‐tailed, and statistical significance was set at p < .05. All statistical analyses were performed using GraphPad Prism v.9 software (GraphPad Software, San Diego, CA, USA).

3. RESULTS

3.1. Patient characteristics

The patient background at leukocytapheresis are summarized in Table 1. A total of 58 patients (NSC, n = 36; HSC, n = 22) were included in this study. The median age tended to be younger in the HSC group than in the NSC group (48 [range, 1–69] vs. 11 years [0–68], p = .07), resulting in a significantly lower median BW in the HSC group (54.7 [range, 9.0–94.1] vs. 33.8 kg [7.9–90.2], p = .02). The primary diseases differed significantly between both groups (p = .009), with approximately 80% in the NSC group consisting of patients with hematopoietic tumors, compared to approximately 45% in the HSC group. Except for a trend toward higher hematocrit (Hct) in the HSC group (32.7 [range, 25.1–43.3] vs. 34.4% [25.4–42.5], p = .09), the median cell counts in the PB at leukocytapheresis showed no significant differences between the two groups. No significant differences were observed between the two groups in terms of sex, PBSC mobilization, or the use of plerixafor.

TABLE 1.

Patient characteristics.

		NSC (36)	HSC (22)	p‐Value
Age (years), median (range)		48 (1–69)	11 (0–68)	.07
Sex, n (%)	Male	20 (55.6)	13 (59.0)	1.00
	Female	16 (44.4)	9 (41.0)
Body weight (kg), median (range)		54.7 (9.0–94.1)	33.8 (7.9–90.2)	.02
Disease, n (%)	Hematopoietic tumors	29 (80.6)	10 (45.5)	.009
	B‐NHL	20 (55.6)	9 (40.9)
	T‐NHL	2 (5.6)	0 (0.0)
	HL	4 (11.1)	1 (4.5)
	PCN	3 (8.3)	0 (0.0)
	Solid tumors	7 (19.4)	12 (54.5)
	Germ cell tumor	1 (2.8)	1 (4.5)
	Glioblastoma	1 (2.8)	0 (0.0)
	Medulloblastoma	2 (5.6)	1 (4.5)
	MRTK	0 (0.0)	1 (4.5)
	Neuroblastoma	3 (8.3)	5 (22.7)
	Rhabdomyosarcoma	0 (0.0)	2 (9.1)
	Retinoblastoma	0 (0.0)	1 (4.5)
	Wilms tumor	0 (0.0)	1 (4.5)
WBC (×103/μL), median (range)		27.74 (5.64–74.19)	35.00 (4.75–85.95)	.72
CIC (×103/μL), median (range)		2.71 (0.00–7.46)	2.96 (0.26–10.31)	.50
Hct (%), median (range)		32.7 (25.1–43.3)	34.4 (25.4–42.5)	.09
Plt (×103/μL), median (range)		169 (56–432)	214 (65–684)	.19
Mobilization, n (%)	G‐CSF	20 (55.6)	11 (50.0)	.79
	G‐CSF + chemotherapy	16 (44.4)	11 (50.0)
Use of plerixafor, n (%)		15 (41.7)	7 (31.8)	.58

Note: Bold values were statistically significant.

Abbreviations: B‐NHL, B cell non‐Hodgkin lymphoma; CIC, circulating immature cell; G‐CSF, granulocyte‐colony stimulating factor; Hct; hematocrit; HL, Hodgkin lymphoma; HSC, highly concentrated sodium citrate; MRTK, malignant rhabdoid tumor of the kidney; NSC, normal concentrated sodium citrate; PCN, plasma cell neoplasm; Plt, platelet; T‐NHL, T cell non‐Hodgkin lymphoma; WBC, white blood cell.

3.2. Peripheral blood stem cell harvest characteristics

All leukocytapheresis variables are summarized in Table 2. Approximately 15% of patients in both groups received leukocytapheresis under anesthesia. Vascular access for leukocytapheresis was performed via peripheral venous, central venous, or arterial puncture, with no significant difference between the two groups. The median pre‐PB CD34⁺ cell counts were significantly higher in the HSC group (33 [range, 25–43] vs. 83 /μL [1–428], p = .04). The number of patients with target CD34⁺ cell yields of 4.0 × 10⁶ per patient BW was significantly higher in the HSC group (25.0 vs. 54.5%, p = .04). Due to the significantly lower BW in the HSC group, the median TBV and PBV were significantly lower in the HSC group (3,594 [range, 630–5,530] vs. 2,509 mL [range, 635–5,401], p = .02 and 11,141 [range, 900–22,501] vs. 7,370 mL [range, 1,388–20,251], p = .02, respectively); however, there was no difference in the median PBV/TBV or the proportion of patients meeting the LVL criteria. In addition, although the median maximum inflow rate was equivalent between both groups, the maximum inlet flow rate per patient BW was significantly higher in the HSC group (1.15 [range, 0.70–1.75] vs. 1.69 mL/min/kg [range, 0.90–2.34], p < .001), and there were no cases of inability to maintain the inlet flow rate for physical reasons related to vascular access. Since the concentration of sodium citrate solution was changed, the AC infusion rate and AC ratio were significantly different between the two groups. In the HSC group, the total amount of the AC solution was significantly lower (925 [range, 77–1,952] vs. 293 mL [range, 64–797], p < .001), and the procedure time was significantly shorter (219 [range, 92–365] vs. 177 min [range, 103–309], p = .01) than in the NSC group. No procedures were interrupted because of extracorporeal circuit obstruction. The CD34⁺ CE2 was significantly lower (48.7 [range, 24.2–73.4] vs. 34.7% [9.4–70.1], p = .001). There was no significant difference between the two groups in CD34⁺ cell yields per patient BW or the proportion of patients requiring leukocytapheresis on subsequent days.

TABLE 2.

Peripheral blood stem cell collection characteristics.

		NSC (36)	HSC (22)	p‐Value
Use of anesthetics		5 (13.9)	3 (13.6)	1.00
Vascular access, n (%)	Peripheral venous	6 (16.7)	9 (40.1)	.14
	Central venous	25 (69.4)	11 (50.0)
	Radial arterial	5 (13.9)	2 (9.1)
CD34+ cell (/μL), median (range)		33 (25–43)	83 (1–428)	.04
Target CD34+ cell yields/patient BW (/kg), n (%)	2.0 × 106	27 (75.0)	10 (45.5)	.03
	4.0 × 106	9 (25.0)	12 (54.5)
TBV (mL), median (range)		3,594 (630–5,530)	2,509 (635–5,401)	.02
PBV (mL), median (range)		11,141 (900–22,501)	7,370 (1,388–20,251)	.02
PBV/TBV, median (range)		3.11 (1.00–6.37)	3.08 (1.52–5.75)	.62
LVL (PBV/TBV ≧3‐fold), n (%)		21 (58.3)	12 (54.5)	.79
Time to interface formation (min), median (range)		20 (13–53)	30 (12–82)	.008
Inlet flow rate (mL/min), median (range)		66.7 (9.5–97.8)	58.0 (10.0–105.0)	.36
Inlet flow rate patient BW (mL/min/kg), median (range)		1.15 (0.70–1.75)	1.69 (0.90–2.34)	<.001
AC infusion rate (mL/min/L TBV), median (range)		1.3 (0.8–1.8)	0.9 (0.5–1.2)	<.001
Blood to anticoagulant ratio, median (range)		12 (12–15)	28 (24–30)	<.001
AC solution volume (mL), median (range)		925 (77–1,952)	293 (64–797)	<.001
Procedure time (min), median (range)		219 (92–395)	177 (103–309)	.01
CD34+ CE2 (%), median (range)		48.7 (24.2–73.4)	34.7 (9.4–70.1)	.001
CD34+ cell yields/patient BW (×106/kg), median (range)		4.50 (0.10–16.64)	6.50 (0.14–12.78)	.24
Number of leukocytapheresis (days), n (%)	1 day	31 (86.1)	19 (86.3)	1.00
	≥2 days	5 (13.9)	3 (13.7)

Note: Bold values were statistically significant.

Abbreviations: AC, anticoagulant; BW, body weight; CE2, collection efficacy 2; HSC, highly concentrated sodium citrate; LVL, large‐volume leukocytapheresis; NSC, normal concentrated sodium citrate; PBV, processing blood volume; TBV, total blood volume.

Next, we evaluated the clinical tolerability of this novel method. In the HSC group, the median potassium and magnesium loss rates tended to be lower or were significantly lower than those in the NSC group (Figure 1A,C; 14.6 [range, −7.7 to 25.6] vs. 8.5% [range, −5.1 to 25.6], p = .08 and 15.8 [range, 0.0–28.6] vs. 8.1% [range, 0–26.3], p = .005, respectively). In contrast, the median ionized calcium and Plt loss rates and the incidence of citrate toxicity were similar in both groups (Figure 1B,D,E).

FIGURE 1.

Analysis of leukocytapheresis‐related adverse events. Comparison of the potassium (A), ionized calcium (B), magnesium (C), and platelet (D) loss rates and the incidence and severity of citrate toxicity during leukocytapheresis (E) between the normal concentrated sodium citrate (NSC) and highly concentrated sodium citrate (HSC) group. Five cases in the NSC group and three cases in the HSC group underwent leukocytapheresis under anesthesia, thus preventing evaluation of citrate toxicity. Data are presented as the mean ± standard error of the mean (SEM) (A–D).

3.3. Effect of using highly concentrated sodium citrate solution on anticoagulant solution volume and procedure time

Since there were significant differences in patient background, such as primary disease (i.e., target CD34+ cell yields/patient BW) or TBV between the NSC and HSC groups (Tables 1 and 2), we performed propensity score (PS) matching based on the 1:1 matching ratio to obtain more robust results regarding the effectiveness of this novel method. The patient characteristics in the PS‐matched cohort are shown in Table 3. The pre‐leukocytapheresis variables were generally well balanced between the two groups, but the HSC group tended to have a higher pre‐CD34⁺ cell counts than the NSC group, resulting in a lower proportion of patients receiving plerixafor. There were no significant differences between the two groups in the median TBV and PBV/TBV. The median maximum inlet flow rate also did not differ in both groups; however, the maximum inlet flow rate per patient BW was significantly higher in the HSC group (1.16 [range, 0.84–1.56] vs. 1.44 mL/min/kg [range, 0.90–2.34], p = .01). Furthermore, the total amount of AC solution significantly decreased (694 [range, 77–1648] vs. 298 mL [range, 64–797], p = .02) and the procedure time was significantly shortened (224 [range, 117–395] vs. 181 min [range, 103–309], p = .048) in the HSC group. The median CD34⁺ CE2 was significantly lower in the HSC group, consistent with our previous results for the allo‐PBSCH cohort. ⁷ Approximately 90% of patients in both groups achieved the target CD34⁺ cell yields in a single procedure. Similar to the cohort before PS matching, the median loss rates of potassium and magnesium tended to be lower or were significantly lower in the HSC group than in the NSC group (Figure 2A,C; 13.2 [range, −7.7 to 25.6] vs. 7.9% [range, −2.9 to 19.5], p = .09, and 22.2 [range, 0.0 to 28.6] vs. 10.0% [range, 0–26.3], p = .049, respectively). Furthermore, the median loss rates of ionized calcium and Plt and the incidence of citrate toxicity did not differ between the two groups (Figure 2B,D,E).

TABLE 3.

Patient variables after propensity score matching in the normal and highly concentrated sodium citrate solution group.

		NSC (n = 17)	HSC (n = 17)	p‐Value
Pre‐leukocytapheresis variables
Age (years), median (range)		35 (1–69)	48 (0–68)	.84
Sex, n (%)	Male	10 (58.8)	10 (58.8)	1.00
	Female	7 (41.2)	7 (41.2)
Body weight (kg), median (range)		51.6 (9.0–85.0)	48.5 (7.9–90.2)	.80
Target CD34+ cell yields/patient BW (/kg)	2.0 × 106	10 (58.8)	9 (52.9)	1.00
	4.0 × 106	7 (41.2)	8 (47.1)
WBC (×103/μL), median (range)		24.53 (5.64–67.39)	37.72 (4.75–50.16)	.57
CIC (×103/μL), median (range)		2.41 (0.20–5.06)	2.99 (0.26–6.24)	.25
CD34+ cell (/μL), median (range)		37 (1–267)	87 (4–350)	.10
Hct (%), median (range)		31.7 (25.1–43.3)	35.7 (25.4–42.5)	.10
Plt (×103/μL), median (range)		131 (57–387)	213 (65–517)	.13
Mobilization, n (%)	G‐CSF	9 (52.9)	9 (52.9)	1.00
	G‐CSF + chemotherapy	8 (47.1)	8 (47.1)
Use of plerixafor, n (%)		8 (47.1)	4 (23.5)	.28
Leukocytapheresis variables
Use of anesthetics		5 (29.4)	3 (17.6)	.69
TBV (mL), median (range)		3303 (630–5480)	3182 (635–5401)	.74
PBV/TBV, median (range)		3.05 (1.43–6.37)	3.04 (1.52–5.75)	.61
Time to interface formation (min), median (range)		22 (13–53)	26 (12–82)	.21
Inlet flow rate (mL/min), median (range)		60.0 (9.5–80.0)	70.0 (10.0–105.0)	.64
Inlet flow rate/patient BW (mL/min/kg), median (range)		1.16 (0.84–1.56)	1.44 (0.90–2.34)	.01
AC solution volume (mL), median (range)		694 (77–1648)	298 (64–797)	.02
Procedure time (min), median (range)		224 (117–395)	181 (103–309)	.048
CD34+ CE2 (%), median (range)		54.6 (30.5–73.4)	34.3 (9.4–66.0)	.001
CD34+ cell/patient BW (×106/kg), median (range)		4.62 (0.10–15.26)	6.20 (0.45–10.50)	.77
Number of leukocytapheresis (days), n (%)	1 day	16 (94.1)	15 (88.2)	1.00
	≥2 days	1 (5.9)	2 (11.8)

Note: Bold values were statistically significant.

Abbreviations: AC, anticoagulant; BW, body weight; CIC, circulating immature cell; CE2, collection efficacy 2; G‐CSF, granulocyte‐colony stimulating factor; Hct, hematocrit; HSC, highly concentrated sodium citrate; NSC, normal concentrated sodium citrate; PBV, processing blood volume; Plt, platelet; TBV, total blood volume; WBC, white blood cell.

FIGURE 2.

Analysis of leukocytapheresis‐related adverse events in the propensity score‐matched cohort. Comparison of the potassium (A), ionized calcium (B), magnesium (C), and platelet (D) loss rates and the incidence and severity of citrate toxicity during leukocytapheresis (E) between the normal concentrated sodium citrate (NSC) and highly concentrated sodium citrate (HSC) group. Five cases in the NSC group and three cases in the HSC group underwent leukocytapheresis under anesthesia, thus preventing evaluation of citrate toxicity. Data are presented as the mean ± SEM (A–D).

4. DISCUSSION

We calculated the PBV from the pre‐CD34⁺ cell counts based on previous reports to obtain the target CD34⁺ cell yields with a single procedure. ¹¹ This PBV adjustment might reduce the emotional and physical burden for the patient and the expenditure required for leukocytapheresis, although more than half of the patients eventually met the LVL criteria in this study. When procedure time is prolonged to process large blood volume, the possibility of undesired citric acid accumulation and fluid retention increases because of the large amounts of ACD‐A volume, especially in patients with low TBV, including children or small adults. ³ , ⁴ , ⁶ , ¹⁷ As the inlet flow rate is proportional to the AC injection rate, AC ratio, and TBV in Spectra Optia, a gradual increase in this flow rate to reduce procedure time requires an elevated AC injection rate, but this leads to an increased risk of citrate toxicity. To address the limitation of the inlet flow rate of the device, the use of heparin as an AC and increasing the AC ratio have been reported; ¹⁸ , ¹⁹ however, this method carries a risk of bleeding and requires frequent activated partial thromboplastin time monitoring. ²⁰ , ²¹ Therefore, we developed a novel method to increase the inlet flow rate by increasing the AC ratio using an HSC solution, for which the efficacy and safety of PBSC collection for healthy donors had been previously reported. ⁷

In this study, we compared 22 patients, including children, who underwent leukocytapheresis using HSC with 36 patients who received conventional auto‐PBSCH using NSC in the past. Our results showed that the HSC group had a lower amount of AC solution and significantly shorter processing time. These results are consistent with our previous report on healthy donors. ⁷ Furthermore, the loss rates of potassium and magnesium were lower in the HSC group. Not only hypocalcemia, but also hypokalemia and hypomagnesemia are electrolyte abnormalities that should be monitored during leukocytapheresis. ²² These electrolyte abnormalities have been reported to be caused by K⁺/H⁺ exchange via Na⁺/H⁺ exchangers and Na⁺/K⁺‐ATPase across RBC membranes associated with metabolic alkalosis induced by citric acid and glucose contained in ACD‐A, and magnesium chelation with citric acid. ²² , ²³ , ²⁴ , ²⁵ We previously monitored potassium dynamics during leukocytapheresis and identified a higher amount of AC solution as a potential risk factor for hypokalemia. ²⁶ Thus, this novel method might reduce electrolyte loss by decreasing the amount of AC solution. In addition, leukocytapheresis, especially in younger pediatric patients, is usually performed under anesthesia and is known to cause more frequent complications than those observed in adult patients. ³ , ²⁷ Therefore, we believe that this novel method is advantageous for pediatric patients because it allows for the collection of target CD34⁺ cells in a shorter time with a lower amount of AC solution. As the next step, it is desirable to examine whether leukocytapheresis using HSC is useful for CD3+ cell collection for the manufacturing of tisagenlecleucel. The CD3⁺ cell collection is performed using the similar cMNC protocol for auto‐PBSCH; however, this procedure requires the same target CD3⁺ cell yields independent of age and BW, which can take several days especially in pediatric patients. We believe that using this novel method could allow for more blood processing by increasing the inlet flow rate and shortening the procedure time, potentially eliminating the necessity for leukocytapheresis over multiple days.

The current study was limited by its retrospective nature and single‐center design with a small group of heterogeneous patients. Nevertheless, our results will be useful for future studies aimed at improving leukocytapheresis procedures.

In conclusion, we extended a novel leukocytapheresis method using HSC, which was previously found to be effective in healthy donors, ⁷ to auto‐PBSCH and demonstrated the efficacy and safety of this procedure. Further studies with larger sample sizes are required to validate our findings.

AUTHOR CONTRIBUTIONS

WK performed conceptualization, data curation, formal analysis, investigation, methodology, validation, visualization, and writing the original draft. KF performed conceptualization, formal analysis, investigation, methodology, supervision, validation, visualization, writing, review, and editing. MA, KI, JS, KW, and FO performed review and editing. YM and NF performed supervision, review, and editing. All authors approved the final manuscript.

CONFLICT OF INTEREST STATEMENT

The authors have disclosed no conflicts of interest.

Kitamura W, Fujii K, Abe M, Ikeuchi K, Shimono J, Washio K, et al. Novel method for autologous peripheral blood stem cell harvest using highly concentrated sodium citrate solution replacing acid citrate dextrose solution A. Transfusion. 2025;65(9):1662–1672. 10.1111/trf.18360