One versus two: How much does it matter? A single‐center retrospective study evaluating 1‐day extracorporeal photopheresis schedule for treating patients with chronic lung allograft rejection

Abstract

Background

Several studies show that extracorporeal photopheresis (ECP) might benefit chronic lung allograft dysfunction (CLAD). A standard ECP cycle consists of two consecutive procedures regardless of the technique employed.

Study Design and Methods

Evaluation of ECP cycle (from two to one procedure) modification due to pandemic restrictions in 25 patients with CLAD under chronic treatment by off‐line ECP in the 6 months preceding cycle modification (one procedure processing 1.5 patients blood volumes [1.5 ECP]). Assessment of any significant change in lung function decline and the relationship with product characteristics compared to pre‐ECP cycle modification.

Results

ECP patients (23 obstructive and two mixed) were enrolled in 2020 during the COVID pandemic. Two hundred and thirty five ECP procedures followed the standard protocol and 121 the 1.5 ECP. There was little or no variation in lung function during the study period. The mean number of mononuclear cells (MNC) per kg administered over time was higher in the 1.5 ECP than in the standard ECP protocol (p = .014). No association was found between respiratory function and MNC infused. Persistent Forced Expiratory Volume in 1 s decline >10% was observed in two patients over the 6 months preceding 1.5 ECP (due to CLAD progression) and in three patients after 1.5 ECP initiation (one for CLAD progression, two for bronchial colonization).

Conclusion

Our study shows that respiratory function is maintained over time and is comparable between both ECP strategies in responders. The shift from two to one procedure per cycle may be reasonable in CLAD patients treated by off‐line ECP.

Abbreviations

8‐MOP

8‐methoxypsoralen

ACD‐A

acid citrate dextrose formula A

ACR

acute cellular rejection

ASFA

American Society for Apheresis

BMI

body mass index

BOS

bronchiolitis obliterans syndrome

CBC

cell blood count

CLAD

chronic lung allograft dysfunction

COVID

coronavirus disease

ECP

extracorporeal photopheresis

ERS/ATS

European Respiratory Society/American Thoracic Society

FEF 25–75

forced expiratory flow at 25%–75% of the pulmonary volume

FEV1

forced expiratory volume in 1 second

FVC

forced vital capacity

GVHD

graft vs host disease

ISHLT

International Society for Heart and Lung Transplantation

miRNAs

small non‐coding RiboNucleic acid

MNC

mononuclear cells

PLT

platelets

RAS

restrictive allograft syndrome

SD

standard deviation

UV‐A

UltraViolet‐A

WBCs

white blood cells

1. INTRODUCTION

Lung transplantation is the last option for end‐stage lung diseases refractory to conventional treatments. Despite the advances in medical treatments, the median survival after lung transplantation remains at 6.2 years, ¹ primarily due to the onset of chronic lung allograft dysfunction (CLAD). According to the latest consensus statement from the Pulmonary Council of the International Society for Heart and Lung Transplantation (ISHLT), CLAD is defined as a permanent reduction (≥20%) in Forced Expiratory Volume in 1 s (FEV1) from baseline, calculated using the mean of the two best values. CLAD has four phenotypes based on functional and radiological patterns: bronchiolitis obliterans syndrome (BOS), restrictive allograft syndrome (RAS), mixed, and undefined. CLAD leads to progressive dyspnea and deterioration of graft function and quality of life, and it is the leading cause of death in lung transplants. ²

Despite CLAD's significant impact on survival, there are few randomized clinical trials ³ , ⁴ , ⁵ and there is no consensus on optimal treatment strategies. Current approaches include the change of immunosuppressive regimens and the addition of immunomodulating agents. Among these interventions, extracorporeal photopheresis (ECP) proved to be a promising option, with an excellent safety profile to limit lung function decline over time. ⁵ , ⁶ , ⁷ , ⁸

ECP is an immunomodulatory therapy based on the autologous collection of mononuclear cells (MNC) which are irradiated with UV‐A (UltraViolet‐A) rays in the presence of 8‐methoxypsoralen (8‐MOP) before being reinfused to the patient. Notwithstanding its mechanism of action remaining incompletely understood, ECP seems to exert an immunomodulatory effect mainly via induction of T regulatory lymphocytes, reducing inflammatory cytokines, ⁹ , ¹⁰ , ¹¹ and exhibiting antifibrotic activity through modulation of alveolar macrophage decorin expression. ⁹ Of note, in the original studies of ECP, patients with cutaneous T‐cell lymphoma were treated with two consecutive daily procedures. ¹¹ Although the treatment schedule was selected to meet the specific logistics of the study, the practice of performing two sequential ECP procedures (cycle) has continued despite improvements in available technologies, regardless of treated cells and expanding indications. To date, ECP is the first‐line therapy for erythrodermic cutaneous T‐cell lymphoma. ¹²

Although the ECP frequency and treatment length are highly variable among centers, it is usually recommended to start patients on a weekly cycle (two procedures/week) and progressively extend the interval between cycles based on a specific protocol schedule or patient response. ¹² , ¹³ , ¹⁴

At present, ECP is a valid option in both Graft versus Host Disease (GVHD) and solid organ transplant rejection. ¹⁵ However, despite its wide use, the optimal treatment protocol has not yet been defined. Key questions persist regarding procedure frequency, blood volume to process during each procedure, and the target MNC collection number for optimal outcomes. As a result, each center has developed its protocol and published data report heterogeneous treatment schedules.

The COVID (COronaVirus Disease) pandemic presented unprecedented challenges to healthcare delivery necessitating modifications in the follow‐up and treatment of chronic illnesses, including CLAD. Our hospital, located in Lombardy, Italy, was among the first to face the initial COVID wave in March 2020.

Given previous research ¹⁶ demonstrating negative outcomes following cessation in CLAD and our positive experience with long‐term ECP in reducing organ decline, ⁵ our Transfusion Service team and the lung transplant physicians sought to maintain patients under chronic ECP treatment.

To minimize COVID transmission risk, while complying with safety measures in place, we modified the standard ECP protocol of our institution (providing for two ECP procedures each cycle), optimizing cell treatment to a single procedure per cycle. This adaptation was supported by positive outcomes reported by other groups in GVHD treatment ¹⁷ and our sporadic preliminary experience indicating a favorable safety profile with this modified schedule.

In this paper, we share our positive experience with the modified off‐line ECP protocol in a cohort of 25 CLAD patients during the COVID pandemic, demonstrating the feasibility and effectiveness of this adapted approach.

2. STUDY DESIGN AND METHODS

This single‐center, national retrospective observational study evaluated the efficacy of ECP in CLAD. The study compared a modified protocol processing 1.5 patient's blood volume in a single procedure (1.5 ECP) against the standard two‐procedure protocol, processing one patient's blood volume during each procedure.

All patients provided written informed consent for the use of pseudo‐anonymized personal and clinical data before treatment; the study was approved by the local Ethical Committee (N.5/D.G./1359 on October 26, 2023).

2.1. Study design

2.1.1. Primary endpoint

The primary endpoint of this study assessed changes in FEV1 measured over 12 months, spanning 6 months before and 6 months after switching to the modified 1.5 ECP protocol. This evaluation aimed to determine the impact of protocol modification on CLAD progression in patients undergoing chronic treatment (>6 months). The treatment schedule in place at our center, using the off‐line method, was borrowed from the protocol adopted for GVHD patients and modified as follows: one cycle per week for 3 weeks followed by one cycle fortnightly for three times and then one cycle per month if patients improved/stabilized. Patients are maintained chronically under ECP, progressively lengthening the treatment intervals from 1 to 2 months, according to the clinical status. The treatment is discontinued only in non‐responders. The ECP protocol adopted until the COVID pandemic included two close procedures for each cycle (within 1 week), processing one patient's blood volume during each procedure (standard ECP) regardless of the cycle frequency. During the pandemic, the protocol was changed for patients under chronic treatment and a cycle included a single procedure processing 1.5 blood volumes. In a sensitivity analysis of the primary endpoint changes, Forced Expiratory Flow at 25%–75% of the pulmonary volume (FEF 25–75) and FEV1/Forced Vital Capacity (FVC) were also evaluated and compared between the two periods.

Since FEV1 is the most important data to assess CLAD evolution and its stage, we measured FEV1 at different time points before and after switching the ECP procedure over a time window of 6 months (6 months before the switch, at the time of the switch, and 3 and 6 months after it). We compared the changes over time of respiratory function by FEV1 failure (>10% drop) after the 1.5 ECP procedure. Due to national COVID preventive strategies, we were not allowed to perform spirometry during the first pandemic wave; therefore, we could not collect functional data 3 months before the switch for most of the patients. The data from the two protocols were used to compare the FEV1 trend before and after the beginning of the single ECP procedure/cycle and to evaluate its possible efficacy in preventing FEV1 decline.

2.1.2. Secondary endpoints

The study evaluated changes over time in administered MNC per kg and platelets (PLT) concentration between groups; it analyzed the association of respiratory function parameters (FEV1, Tiffeneau index, FVC, and FEF 25–75) with cell collection content at the ECP procedure.

2.2. Patient population

From March to September 2020, patients with established CLAD (regardless of phenotype and stage) under chronic ECP treatment were selected at the Transplant Center of our hospital to undergo a single procedure (1.5 ECP), instead of two per cycle (standard ECP).

Additional inclusion criteria were: a minimum of 6 months of prior ECP treatment post‐CLAD diagnosis (completion of the induction cycles) and active chronic treatment (1–2 months frequency). Patients were submitted to usual follow‐up and immunosuppressive treatment irrespective of ECP schedule, as previously described. ⁵

Exclusion criteria were: early CLAD occurrence (<12 months post‐transplantation), lack of functional data, non‐CLAD ECP indication; recent ECP initiation (<6 months before the study period), active infection at enrolment in ECP schedule change, absolute MNC count of less than 200 × 10⁹/L, acute heart and/or acute renal failure, and hemorrhagic diathesis; active cancer or cancer history within 5 years (excluding non‐melanoma skin cancer).

The screening process to meet inclusion/exclusion criteria was performed by looking at the personal history and demographics of the patients and pulmonary function tests performed during the usual follow‐up.

2.3. Extracorporeal photopheresis

ECP was performed using the off‐line technique. The procedure in use at the Immunohaematology and Transfusion Service provided for MNC collection from the patient's peripheral blood employing the Spectra Optia cell separator device (Terumo BCT Inc., USA) using the MNC collection protocol. Subsequently, collected MNC were diluted with a saline solution to reach a final volume of 300 mL, and 8‐MOP was added to the bag (at a 200 ng/mL concentration). Afterward, the product was transferred in a UV‐A permeable bag (Macogenic, Macopharma, France) and irradiated with UV‐A rays (at 2 J/cm). The whole process was performed under sterile conditions. Finally, the photo‐activated MNC were immediately reinfused into the patient. The 1.5 ECP protocol consisted of one procedure (corresponding to one cycle) during which 1.5 patients' blood volumes were processed. All patients were under chronic treatment and ECP treatment frequency was maintained at 1–2 months even after the introduction of 1.5 ECPs. Any procedure‐related side effect was recorded, as per routine.

2.4. Laboratory tests

A complete cell blood count (CBC) from the patient's peripheral blood immediately before the ECP procedures and from the leukapheresis collection bag before manipulation was assessed, using the Sysmex XN‐1000 automated cell counter.

2.5. Pulmonary function tests

Pulmonary function tests were performed every 3–6 months due to the restrictions of the COVID pandemic.

Spirometry (Vinctus One, Vyaire Medical) was conducted to determine the FEV1, the FVC, the Tiffeneau index, and the FEF 25–75, following the latest European Respiratory Society/American Thoracic Society (ERS/ATS) criteria for spirometry standardization. ¹⁸

FEV1 values were compared to the best‐FEV1, calculated as the mean of the two best values measured more than 3 weeks apart as required by the 2019 Pulmonary Council of the ISHLT consensus statement ² to follow the course of CLAD and stage it.

2.6. Statistical analysis

2.6.1. Sample size

In this retrospective study, we were able to retrieve information from 25 patients. With this sample size, we can elicit an effect size of about 0.6 standard deviations between changes in FEV1 with the standard procedure versus the 1.5 ECP procedure if the power is 80%, the type I error is 5%, and the correlation between paired measures is 0.5.

2.6.2. Data analysis

The data were summarized with the mean and standard deviation, the median and quartiles (25th–75th) if continuous, and as counts and percent if categorical. All patients were included in the analysis according to the intention to treat principle.

For the analyses of changes over time, we used a mixed model for repeated measures, with a random effect for subject. The model included an interaction term of period (pre–post) and time of assessment to test the hypothesis of no difference in time profile between periods. The association of respiratory function with each ECP procedure data was assessed with generalized linear regression models with calculation Huber‐White to account for intra‐subject correlation, while adjusting for time of assessment and period as well as for the number of MNC/kg. The partial correlation of ECP variables and respiratory function was calculated. A 95% confidence interval (95% CI) was computed for all estimates.

All analyses were performed using the Stata software, release 18, StataCorp, College Station, TX, USA. A two‐sided p‐value <.05 was retained for statistical significance.

3. RESULTS

3.1. Population

Twenty‐five CLAD patients were enrolled, including CLAD Grade 0 and 0p, following institutional protocol for maintenance therapy in ECP responders. Patients' demographics and clinical characteristics at the beginning of the 1.5 ECP procedures are shown in Table 1.

TABLE 1.

Patient demographics and clinical characteristics at baseline. All patients were under chronic maintenance during the study period (from 6 months preceding 1.5 extracorporeal photopheresis [ECP] shift).

Patients characteristics
Sex, no. of patients (%)
Male	16 (64)
Female	9 (36)
Weight (kg), median (25th–75th)	70 (59–74)
BMI (kg/m2), mean (SD)	24 (5)
Age (years), median (25th–75th)	49 (42–65)
Comorbidities, no of patients (%)
Chronic renal failure	15 (60)
Chronic heart failure	2 (8)
GERD	7 (28)
Cancer	5 (20)
Frequent infections	3 (12)
Indication for lung transplant, no. of patients (%)
Cystic fibrosis	8 (32)
IPF	6 (24)
COPD	4 (16)
Progressive fibrosis	3 (12)
Pulmonary arterial hypertension	1 (4)
BOS	1 (4)
Ebstein disease	1 (4)
RA‐ILD	1 (4)
Time from transplant (months), median (25th–75th)	129 (83–172)
Time from first ECP and 1.5 ECP (months), median (25th–75th)	53 (16–85)
Type of lung transplant, no. of patients (%)
Bilateral	20 (80)
Monolateral	4 (16)
Heart–lung	1 (4)
Type of CLAD, no. of patients (%)
BOS	23 (92)
RAS	2 (8)
Grade of CLAD, no. of patients (%)
0	2 (8)
0p	4 (16)
1	6 (24)
2	8 (32)
3	5 (20)
Therapy, no. of patients (%)
Tacrolimus	22 (88)
Cyclosporine	3 (12)
Mycophenolate mofetil	8 (32)
Everolimus	12 (48)
Azathioprine	1 (4)
Prednisone	25 (100)
Azythromicin	15 (60)
Montelukast	2 (8)

Abbreviations: BMI, body mass index; BOS, bronchiolitis obliterans syndrome; CLAD, chronic lung allograft dysfunction; COPD, chronic obstructive pulmonary disease; ECP, extracorporeal photopheresis; GERD, gastroesophageal reflux disease; IPF, idiopathic pulmonary fibrosis; RA‐ILD, rheumatoid arthritis‐interstitial lung disease; RAS, restrictive allograft syndrome.

3.2. Extracorporeal photopheresis

There were no significant differences between the standard and 1.5 ECP protocols (Table 2), except for the number of procedures performed, the total number of white blood cells (total WBCs) collected, the number of MNC administered per kg, the procedure time, the total blood volume processed, and the collection bag volume. Two hundred and thirty‐five ECP procedures were performed following the standard protocol, whereas 121 were performed with the 1.5 ECP. The difference between the mean absolute number of WBCs collected during the 1.5 ECP and standard ECP was at the limit of statistical significance (4.5 × 10⁹; standard deviation [SD] 2.2 vs. 3.7; SD 1.6, p .049). In parallel, the mean number of MNC per kg was higher in the 1.5 ECP than in the standard protocol (54 × 10⁶, SD 21.6; vs. 62 × 10⁶, SD 28, p .155). The mean collection time during 1.5 ECP was 109 (SD 19) versus 84 min (SD 8.6) for each standard ECP procedure, with a median MNC collection volume of 56 mL (SD 24.6) versus 44 mL (SD 15.5), respectively. The mean percentage of lymphocyte and monocyte content (purity) was very high and did not change between the two protocols, as expected. Details on the pre‐apheresis peripheral blood count and collection characteristics for each standard and 1.5 ECP are shown in Table 2.

TABLE 2.

Patient's peripheral blood and apheresis collection characteristics for standard extracorporeal photopheresis (ECP) (one patient's blood volume processed) versus 1.5 ECP (1.5 patient's blood volumes processed) procedures.

		1 volume ECP	1.5 ECP	p‐Value
Peripheral blood	WBC (×109/L), mean (SD)	6.9 (1.97)	7.3 (1.97)	.256
	Lympho (%), mean (SD)	17 (9)	15 (6)	.293
	Mono (%), mean (SD)	10 (3)	9 (3)	.736
	HCT (%), mean (SD)	37.7 (3.8)	37.7 (4)	.989
	PLT (×109/L), mean (SD)	210 (68.3)	213 (177–259)	.215
Collection	Total number of procedures	235	121	p < .001
	Collection time (min), mean (SD)	84 (8.6)	109 (19)	p < .001
	TBV processed (mL), mean (SD)	4110 (830)	5536 (1498)	p < .001
	Bag volume (mL), mean (SD)	44 (15.5)	56 (24.6)	.002
	WBC (×109/L), mean (SD)	91.2 (35.6)	87.9 (38.7)	.711
	MNC (×106/kg), mean (SD)	54 (21.6)	62 (28)	.155
	Total WBC (×109), mean SD)	3.7 (1.6)	4.5 (2.2)	.049
	MNC purity (%), mean (SD)	93 (12)	93 (12)	.942
	Lympho (%), mean (SD)	56 (15)	57 (18)	.683
	Mono (%), mean (SD)	37 (16)	36 (19)	.712
	PLT (×109/L), mean (SD)	2006 (919)	1857 (450)	.344
	Bag HCT (%), mean (SD)	3.6 (1.9)	3.4 (1.2)	.552

Abbreviations: Bag HCT%, contaminating red blood cells in the collection bag; Bag volume, leukapheresis bag volume; HCT%, hematocrit; Lympho, lymphocytes %; MNC, mononuclear cells; MNC purity, percentage of MNC in the collection bag; Mono, monocytes%; PLT, platelets; SD, standard deviation; TBV, total blood volume; Total WBC, absolute number of WBC collected during apheresis; WBC, white blood cells.

No adverse events were reported during the study period.

3.3. Primary endpoint

As shown in Figures 1, 2, 3 and described in detail in Table 3, there was no difference between the time profile of the 1 and 1.5 ECP procedures, with little or no change over the 6 months of follow‐up, neither before nor after the standard to 1.5 ECP procedure switch in the patients participating in the study. The change in FEV1 was −0.09 L, 95% CI −0.19 to 0.02 and −0.04 L, 95% CI −0.04 to 0.05, in the 1 ECP and 1.5, respectively; p between groups over time .148. Similar small changes are observed for FEF 25–75 and FEV1/FVC (p between groups over time .327 and .537, respectively).

FIGURE 1.

(A) Changes in Forced Expiratory Volume in 1 s (FEV1) slopes over time (months) before and after the switch from one extracorporeal photopheresis (ECP) to 1.5 ECP. Red lines show the patients with a FEV1 decline >10% and blue lines show the patients who improved or stabilized lung function over the analysis time. (B) The figure shows the five patients who experienced a decline of FEV1 >10% during the study period (before or after 1.5 ECP). Each patient is distinguished by a different color to highlight the FEV1 trend before and after the 1.5 ECP switch. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2.

Changes in respiratory function over time (6 months) before and after the switch from one extracorporeal photopheresis (ECP) to 1.5 ECP. Starting from the top left: the blue and red lines show the correlation with the trend of Forced Expiratory Volume in 1 s (FEV1), Forced Expiratory Flow at 25%–75% of the pulmonary volume (FEF25–75) (top right), and FEV1/Forced Vital Capacity (FEV1/FVC) (bottom left) before and after the 1.5 ECP switch. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3.

Association of respiratory function and mononuclear cells (MNC) ×10⁶/kg administered to the patients after manipulation, adjusted for time, according to the off‐line technique protocol. Starting from the top left: correlation with the trend of Forced Expiratory Volume in 1 s (FEV1), Forced Expiratory Flow at 25%–75% of the pulmonary volume (FEF25–75) (top right), and FEV1/Forced Vital Capacity (FEV1/FVC) (bottom left). [Color figure can be viewed at wileyonlinelibrary.com]

TABLE 3.

Changes over time. Comparison before (pre) and after (post) the switch to 1.5 extracorporeal photopheresis (ECP).

Parameters change over time
Primary endpoint	Pre 1.5 ECP			Post 1.5 ECP			p between groups over time
	Mean (SD)	∆0 (95%)	p within groups	Mean (SD)	∆0 (95%)	p within groups
FEV1 (L)
T0	1.99 (0.83)	0	.113	1.91 (0.86)	0	.652	.148
T3	NA	NA		1.93 (0.87)	−0.02 (−0.06 to 0.03)
T6	1.90 (0.85)	−0.09 (−0.19 to 0.02)		1.91 (0.88)	0 (−0.04 to 0.05)
FEF 25–75 (L)
T0	1.31 (1.30)	0	.911	1.31 (1.29)	0	.320	.327
T3	NA	NA		1.38 (1.26)	0.07 (−0.03 to 0.16)
T6	1.35 (1.35)	0 (−0.09 to 0.08)		1.37 (1.24)	0.06 (−0.03 to 0.16)
FEV1/FVC (%)
T0	64 (14)	0	.358	62 (14)	0	.896	.537
T3	NA	NA		62 (14)	−0.41 (−2.10 to 1.29)
T6	63 (14)	−1.16 (−3.59 to 1.27)		62 (14)	−0.20 (−1.84 to 1.44)
Secondary endpoints
MNC ×106/kg
T0	54 (22)	0	.138	62 (28)	0	< .001	.014
T3	62 (32)	7.2 (−5.4 to 19.9)		79 (29)	16.7 (5.9 to 27.4)
T6	48 (28)	−5.6 (−17.3 to 6.2)		82 (32)	19.5 (8.8 to 30.3)
PLT ×109/L
T0	2007 (919)	0	.343	1857 (450)	0	.409	.797
T3	1722 (530)	−256.7 (−601.1 to 87.6)		1733 (450)	−135.5 (−334.5 to 63.4)
T6	1887 (568)	−120.1 (−440.7 to 200.5)		1797 (751)	−59.9 (−256.1 to 136.2)

Abbreviations: FEF25–75, Forced Expiratory Flow at 25%–75% of the pulmonary volume; FEV1, Forced Expiratory Volume in 1 s; FEV1/FVC, Forced Expiratory Volume in 1 s/Forced Vital Capacity; MNC/kg, mononuclear cells per kg bw reinfused to the patient; NA, data were not retrievable from the repository; PLT, contaminating platelets in the collected cells; SD standard deviation; T0, baseline of the study; T3, 3 months, T6, 6 months.

Among the 25 analyzed patients, two experienced a drop in FEV1 >10% before the protocol switch due to COVID‐related interruption of ECP for 3 and 5 months; they recovered their respiratory function after the switch to 1.5 ECP. All other patients had continuity in ECP treatment. After the beginning of the 1.5 ECP three patients (12%) had a drop in FEV1 >10%; this was due to an episode of severe acute cellular rejection (ACR) in one patient. A second patient declined right after an acute bronchitis sustained by Escherichia coli, and finally, the third patient suffered from an episode of acute renal failure. Their individual time course is shown in Figure 1B.

3.4. Secondary endpoints

The amount of MNC per kg infused into the patient changed significantly over time after the 1.5 ECP switch (p within groups .014), whereas PLT did not (Table 3).

Moreover, as shown in Figure 3, there is no association between respiratory function and the infused MNC per kg concentration (while adjusting for time). Also, a large variability of the cell concentration is evident. Pearson correlation coefficients were 0.20 or below.

4. DISCUSSION

Our study shows sustained respiratory function across both ECP strategies (single vs. double procedures per cycle), with comparable outcomes between the two approaches.

Among all participants, respiratory function decline was observed in only two patients before protocol modification and three patients following the transition to 1.5 ECP. In the former case, we can speculate that the functional decline of these patients could be explained by the prolonged time the treatment was withheld due to COVID. This interpretation is supported by two key observations: the subsequent FEV1 stabilization upon resumption of ECP under the new protocol and consistency with findings reported by Robinson et al. ¹⁶ The intercurrent events observed after the switch to 1.5 ECP are common causes of functional decline other than CLAD, ² and ACR is also known to be one of the risk factors for CLAD. ¹⁹ Therefore, the observed FEV1 reductions likely stem from these underlying pathologies rather than the inefficacy of the 1.5 ECP protocol in CLAD management.

Our findings indicate no correlation between the concentration of MNC infused into the patient and respiratory function. Our results show that the total WBC collected is slightly higher when processing 1.5 blood volumes compared to 1 blood volume/procedure, while the MNC purity is comparable using the off‐line method with the MNC collection program, as expected.

Peripheral blood cell analyses revealed comparable lymphocyte and MNC counts between standard and the 1.5 ECP cycles, without ECP‐induced lymphopenia.

Consistent with previous studies, ²⁰ , ²¹ we found a positive correlation between peripheral blood MNC count and content in the collection bag. Notably, both standard and 1.5 ECP protocols achieved higher MNC yields per body weight compared to previous studies using the MNC system, ²⁰ validating our earlier findings. ²² In addition, the cellular product administered following the 1.5 ECP exceeded the therapeutic threshold reported by Worel et al. for GVHD response. ²³ Moreover, both standard and 1.5 ECP protocols demonstrated exceptional quality of the collected cells, characterized by high MNC purity and minimal red blood cell and granulocyte contamination.

These results offer potential benefits: reduced long‐term anemia risk and minimized patient discomfort associated with granulocyte degranulation during reinfusion. ²⁴

While the 1.5 ECP protocol required approximately 30% more anticoagulant (ACD‐A, Acid Citrate Dextrose Formula A) administration than standard ECP, we observed no differences in symptomatic hypocalcemia incidence, bleeding episodes, or transfusion requirements between protocols. Although the 1.5 ECP procedure duration was significantly longer, it remained clinically feasible with average completion times under 2 h.

The optimal number of cells to be infused, treatment duration, and cycle frequency for ECP response remain the subject of ongoing debate. Current understanding suggests that ECP efficacy in responding patients involves multiple mechanisms: the activation of immature dendritic cells triggered by the inflammatory cascade, plasmacytoid dendritic cell expansion, and the induction of a tolerogenic response. However, comprehensive mechanistic data remain limited. ²⁵ , ²⁶

Recent research has identified epigenetic factors, particularly miRNAs (small non‐coding RNA) aberrant expression, as influential in CLAD progression. In this context, ECP's therapeutic mechanism may exert molecular regulation through modulation of specific miRNAs that influence adaptive immunity and antibody‐related T‐cell response. ²⁷ Furthermore, recent experimental investigations highlighted that not only leukocyte‐mediated effects but also platelet activation induced by 8‐MOP and UV‐A rays treatment, along with subsequent immune cell interactions, may contribute to the ECP's immunomodulatory effect. ²⁸ , ²⁹

These findings suggest that the therapeutic response to ECP likely depends on factors beyond the number of MNC treated alone. This broader mechanistic understanding could help explain why varying approaches give comparable results, despite different treatment cycles (e.g., one procedure per cycle) when using the off‐line method. This is potentially due to the increased blood volume processed and subsequent higher cell collection rates.

The decision to perform one single procedure per cycle (instead of two, as per routine) processing a higher patient's blood volume (1.5 instead of 1) was based on positive outcomes reported in GVHD treatment using the off‐line technique. Notably, Cid et al., ¹⁷ demonstrated that processing one patient's blood volume using the off‐line technique, achieves more than double the blood volume processed by the on‐line system (1500 mL), with corresponding higher MNC collection. Additionally, as reported by Bueno et al., ³⁰ the amount of MNC collected during a single off‐line procedure is approximately 95% of peripheral blood circulating cells.

These findings support the rationale for a single procedure/cycle with the off‐line technique potentially avoiding redundant processing of the cells collected the day before and their uncertain immunologic effect. This approach is also addressed in the American Society for Apheresis (ASFA) 2023 guidelines for GVHD management. ¹² Our preliminary results suggest that processing one patient blood volume/cycle may be sufficient, specifically with the off‐line ECP technique.

Reducing the number of procedures per cycle would bring several benefits: reduced patient burden, preservation of peripheral venous access, cost savings for both patient and healthcare system, decreased workload for healthcare professionals, and increased treatment accessibility for a larger number of patients (including those at an early stage of CLAD to mitigate FEV1 decline) with the same resources.

This benefits are particularly significant given the approximately 60% response rate reported across multiple studies on the subject. ² , ⁶ , ⁸ , ³¹ , ³² , ³³ , ³⁴ , ³⁵

The inherent limitations of retrospective studies leave several questions unresolved, including the relationship between graft functional evaluation and CLAD subtypes. Evidence suggests that patients with a rapid deterioration of graft function often show limited ECP benefit, potentially due to the delayed intervention. Similarly, a restrictive phenotype (RAS) shows a poorer response, ⁶ , ³⁵ , ³⁶ although notably, none of our declining patients had RAS.

Limitations of our study are the relatively small sample size (25 patients) and a lack of a concurrent control group, though we used a before/after design with each patient serving as his/her own control. Furthermore, the majority of patients had obstructive CLAD, limiting generalizability to other CLAD subtypes. A randomized controlled trial might provide stronger evidence for 1.5 ECP non‐inferiority. However, the limited lung transplant population and patients' heterogeneous characteristics and type of transplant make such a study impractical.

To the best of our knowledge, only one prospective randomized trial is investigating ECP‘s efficacy in this setting. ³⁷ Nevertheless, based on the available evidence, ECP is classified as a valid second‐line treatment for CLAD in the ninth ASFA guidelines. ¹²

Although our preliminary findings warrant validation through a prospective comparative study, they support transitioning from two to one procedure per cycle, processing one patient's blood volume, also in CLAD management, similar to what other groups have proposed in GVHD management.

To the best of our knowledge, this represents the first study examining a modified treatment cycle for CLAD using the off‐line technique.

CONFLICT OF INTEREST STATEMENT

FM is in Therakos advisory board for lung transplantation; the other authors do not have disclosed no conflicts of interest.

Del Fante C, Vertui V, Klersy C, Mortellaro C, Briganti DF, Morlacchi LC, et al. One versus two: How much does it matter? A single‐center retrospective study evaluating 1‐day extracorporeal photopheresis schedule for treating patients with chronic lung allograft rejection. Transfusion. 2025;65(8):1490–1501. 10.1111/trf.18318