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. 2023 Oct 4;163(3-4):121–130. doi: 10.1159/000534433

Assessment of Micronuclei Frequency in the Peripheral Blood of Adult and Pediatric Patients Receiving Fractionated Total Body Irradiation

Karthik Kanagaraj a,✉, Michelle A Phillippi a, Pratyush Narayan a, Barbara Szolc a, Jay R Perrier a, Amanda McLane b, Suzanne L Wolden b, Christopher A Barker b, Qi Wang a, Sally A Amundson a, David J Brenner a, Helen C Turner a,✉
PMCID: PMC10946645  NIHMSID: NIHMS1935742  PMID: 37793357

Abstract

The cytokinesis-block micronucleus (CBMN) assay is an established method for assessing chromosome damage in human peripheral blood lymphocytes resulting from exposure to genotoxic agents such as ionizing radiation. The objective of this study was to measure cytogenetic DNA damage and hematology parameters in vivo based on MN frequency in peripheral blood lymphocytes (PBLs) from adult and pediatric leukemia patients undergoing hematopoietic stem cell transplantation preceded by total body irradiation (TBI) as part of the conditioning regimen. CBMN assay cultures were prepared from fresh blood samples collected before and at 4 and 24 h after the start of TBI, corresponding to doses of 1.25 Gy and 3.75 Gy, respectively. For both age groups, there was a significant increase in MN yields with increasing dose (p < 0.05) and dose-dependent decrease in the nuclear division index (NDI; p < 0.0001). In the pre-radiotherapy samples, there was a significantly higher NDI measured in the pediatric cohort compared to the adult due to an increase in the percentage of tri- and quadri-nucleated cells scored. Complete blood counts with differential recorded before and after TBI at the 24-h time point showed a rapid increase in neutrophil (p = 0.0001) and decrease in lymphocyte (p = 0.0006) counts, resulting in a highly elevated neutrophil-to-lymphocyte ratio (NLR) of 14.45 ± 1.85 after 3.75 Gy TBI (pre-exposure = 4.62 ± 0.49), indicating a strong systemic inflammatory response. Correlation of the hematological cell subset counts with cytogenetic damage, indicated that only the lymphocyte subset survival fraction (after TBI compared with before TBI) showed a negative correlation with increasing MN frequency from 0 to 1.25 Gy (r = −0.931; p = 0.007). Further, the data presented here indicate that the combination of CBMN assay endpoints (MN frequency and NDI values) and hematology parameters could be used to assess cytogenetic damage and early hematopoietic injury in the peripheral blood of leukemia patients, 24 h after TBI exposure.

Keywords: Total body irradiation, Micronucleus, Neutrophil-to-lymphocyte ratio, Leukemia

Introduction

The cytokinesis-block micronucleus (CBMN) technique was originally developed by Fenech and Morley in 1985 and has been used to assess DNA damage induced by ionizing radiation (IR) in peripheral blood lymphocytes (PBLs) for more than 3 decades. Micronuclei can arise from an acentric chromosomal fragment or a centric whole chromosome that are excluded from the daughter nuclei at the end of mitosis [Fenech and Morley, 1985]. They appear as small nuclei located in the cytoplasm adjacent to daughter nuclei. MN can arise from exposure to various clastogenic agents such as benzene, ethylene oxide, arsenic, phosphine, chemotherapy drugs, and IR, which is one of the strongest clastogenic agents and results in non- or mis-repaired DNA double-strand breaks. For the CBMN assay, MN are visualized and numerically scored within binucleated (BN) cells, or once-divided cells that have been halted before cytokinesis during cell division using a cytochalasin block.

The Organization for Economic Co-operation and Development (OECD) has approved guidelines for both in vivo mammalian erythrocyte MN [OECD 2016a], and the in vitro mammalian micronucleus test in epithelial cells and human peripheral blood cells [OECD 2016b]. The CBMN assay is well-regulated to ISO standards which provide guidelines on how to perform the assay for dose assessment using documented and validated procedures for biodosimetry [ISO 17099, 2014]. The CBMN assay is also a known biomarker for the toxicity of mutagenic agents that induce DNA double-stranded breaks and has been widely used as an indicator of genomic instability and in vivo exposures by genotoxins in biomonitoring studies [Speit et al., 2011]. The CBMN assay is currently applied for the assessment of radiation exposure in patients in vivo as well as environmental and occupational exposures. In a series of studies conducted by Thierens and colleagues, they used the CBMN assay for the biomonitoring of in vivo radiation in patients treated with radiotherapy for cervical cancer and, on a larger scale, applied the assay for routine testing of the PBLs from nuclear power plant workers and potentially occupationally exposed hospital workers [Thierens and Vral, 2009].

The CBMN assay has been successfully used to estimate absorbed dose in cancer patient PBLs after partial and total body fractionated radiation [Venkatachalam et al., 1999; Dossou et al., 2000; Vorobtsova et al., 2001; Lee et al., 2002; Wojcik et al., 2004]. More recently, the prognosis of patients with rectal cancer after radiation therapy was evaluated using the MN assay as a biomarker for individual radiosensitivity after in vitro irradiation and in vivo radiochemotherapy [Dröge et al., 2021]. Further, a strong correlation has also been shown between MN frequency in peripheral blood samples irradiated in vitro and the appearance of acute side effects of radiotherapy for cervical cancer patients who have undergone chemotherapy [Borges da Silva et al., 2021].

In the present study, we used this established CBMN biomarker to assess cytogenetic DNA damage in blood PBLs of adult and pediatric cancer patients, exposed to three 1.25 Gy fractions of total body irradiation (TBI), as part of a conditioning regimen prior to a hematopoietic stem cell transplant (HSCT). Fresh blood samples (pre-irradiation and after 1.25 Gy and 3.75 Gy TBI exposures) were collected from patients diagnosed with acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), multiple myeloma (MM), myelodysplastic syndrome (MDS), and non-Hodgkin lymphoma. Hematology parameters based on differential blood counts recorded before and after three fractions of TBI at 24 h after TBI exposure were correlated with the formation of MN in peripheral blood T lymphocytes.

Materials and Methods

Patient Recruitment and Blood Collection

Patients undergoing HSCT preceded by TBI were recruited to this study following informed consent at Memorial Sloan-Kettering Cancer Center (MSKCC), using protocols approved by the Institutional Review Boards of MSKCC (IRB#: 07-158) and the Columbia University Irving Medical Center (CUIMC IRB#: AAAB5846). The TBI patients received three fractions of 1.25 Gy treatments within 1 day (dose rate = 0.1 Gy/min), about 4 h apart for a total accumulated dose of 3.75 Gy. Complete blood count with differential (CBC w/diff) including white blood cells (WBCs), neutrophils, lymphocytes, monocytes, eosinophils, and basophils and red blood cells (RBCs), hemoglobin (HGB), hematocrit test (HCT), and platelets (PLTs) were recorded before and after 3 TBI fractions at approximately 20–24 h after start of the first fraction (online suppl. Table 1; for all online suppl. material, see https://doi.org/10.1159/000534433). Fresh peripheral blood samples were collected at the following time/dose points for the CBMN assay: (1) before the start of the first TBI treatment, (2) at approximately 4 h after the first 1.25 Gy TBI dose (but before the second), and (3) at 20–24 h after the first TBI treatment (total dose = 3.75 Gy). Apparently healthy adult volunteer donors (2 male and 2 females; aged 35–45 years) were recruited at CUIMC after obtaining informed consent (IRB Protocol#: AAAB5846). The CBMN assay was performed using these samples to assess baseline levels of MN as a comparison with the cancer patients.

CBMN Assay Protocol

The CBMN assay cultures for each patient were prepared on the same day following the collection of the 3rd TBI fraction. Whole blood samples (2 mL) were transferred into T25 flasks containing 20 mL of pre-warmed (37°C) RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal calf serum, 0.4 mL phytohemagglutinin (PHA; final concentration, 25 μg/mL), and 1% antibiotics (5,000 IU penicillin and 5,000 μg/mL streptomycin). All reagents were purchased from Invitrogen (Carlsbad, CA, USA) unless separately indicated. To block cytokinesis, 200 µL of cytochalasin-B (600 μg/mL stock prepared in DMSO; Sigma-Aldrich, St. Louis, MO, USA) was added after 44 h to all the flasks at a final concentration of 6 μg/mL. After a 72-h total culture time (37°C, 5% CO2, 98% humidity), the blood/media mix was transferred into two 15-mL centrifuge tubes and centrifuged at 1,000 RPM for 10 min. To swell the cells, 0.075 M KCl was added to the sample at room temperature for 10 min and then fixed with a 4:1 ratio of methanol/acetic acid. Approximately 30–50 µL of fixed cell suspension was dropped onto a clean glass slide, air-dried, and stained with VECTASHIELD® mounting medium containing 1.5 μg/mL DAPI (Vector Laboratories, Burlingame, CA, USA), covered by a coverslip placed on top, and sealed with clear nail varnish. The cells were observed under UV light using an Olympus epifluorescence microscope (Olympus BH2-RFCA; Center Valley, PA, USA). Binucleated cells (BNCs) were manually scored for the presence and absence of MN formation. In samples with very few BNCs [Bertucci et al., 2023], we counted as many BNCs per side as possible or stopped scoring after a minimum of 250 BNCs (online supplementary Table 2). The nuclear division index (NDI) was calculated based on the formula NDI = (M1 + 2*M2 + 3*M3 + 4*M4)/N, where M1–M4 represents the proportion of mononucleated, BN, tri-nucleated, and quadri-nucleated cells, respectively, and N is the total number of cells scored [Fenech 2000; Fenech et al., 2003; Eastmond and Tucker, 1989].

Statistical Analyses

Statistical analyses were performed using GraphPad Prism version 9.4.0 for Windows (GraphPad Software Inc., La Jolla, CA, USA). All means are presented as the mean ± standard error of the mean. Statistical difference for each of the blood cell subtypes before and after TBI exposure was determined using a multiple paired t test. Linear regressions were used to determine the relationship between age and MN frequency before and after TBI. To compare MN frequency (ratio of micronuclei/binucleate cells) and NDI values between pre-irradiated and irradiated samples, a one-way ANOVA (Tukey’s multiple comparisons test) was employed. Differences between means were considered statistically significant when p value <0.05. A two-tailed Pearson correlation coefficient test was used to statistically compare the association between hematology and cytogenetic damage, considered statistically significant when p value <0.05.

Results

Patients recruited for HSCT were diagnosed with either AML, ALL, MM, MDS, or non-Hodgkin lymphoma.

Hematology

CBC w/diff was collected at MSKCC from all patients (n = 28) prior to irradiation at ∼24 h after the start of radiotherapy treatment. Differential cell counts, demographic, cancer diagnosis, and previous chemotherapy treatments are provided in online supplementary Table 1. Figure 1 shows the mean relative percentage of leukocyte subtypes before and after TBI. All patients received three fractions of radiation except patient 85 (diagnosis = MDS), who only received two. After TBI, the percentage of neutrophils was significantly elevated from a mean of 63.96–80.09%, and the percentage of lymphocytes was significantly reduced from a mean of 18.02–7.52%. The change in the neutrophil-to-lymphocyte ratio (NLR) measured as the ratio of relative % neutrophil to % lymphocyte showed an increase from a mean of 4.62 ± 0.49 to 14.45 ± 1.85 after a total TBI dose of 3.75 Gy and therefore was indicative of high physiological stress and a systemic inflammatory response within 24 h after radiation exposure. A recent study in a healthy adult (non-geriatric; n = 413) population by Forget and colleagues [Forget et al., 2017] reported that normal NLR values were between 0.78 and 3.53. Compared to neutrophils and lymphocytes, the levels of monocytes, eosinophils, and basophils in the peripheral blood were relatively low. We also recorded a significant decrease in eosinophils with no significant change in monocytes and basophils after radiation exposure. Also, there was a significant decrease in the absolute counts of RBCs, HGB, and HCT with no significant change in PLT cell levels measured at 24 h after fractionated TBI. Statistical difference for each of the blood cell subtypes before and after TBI exposure is provided in Table 1.

Fig. 1.

Fig. 1.

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Differential white blood cell counts in patients receiving TBI, recorded before and at 24 h after radiation exposure. Error bars represent the standard error of the mean. Statistical significance before and after TBI across the WBC subtypes was determined by a multiple paired t test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Table 1.

Mean blood cell levels in patients (n = 28) before and after three fractions of 1.25 Gy TBI (3.75 Gy total)

Cell type Mean cell levels before TBI Mean cell levels after TBI p value
Neutrophils, % 63.13 79.08 0.0001
Lymphocytes, % 18.30 7.73 0.0006
Monocytes, % 9.97 8.54 0.0880
Eosinophils, % 5.52 2.32 0.0072
Basophils, % 0.48 0.33 0.0880
WBC, K/μL 4.83 4.93 0.7534
RBC, M/μL 3.52 3.37 0.0006
HGB, g/dL 10.95 10.44 0.0002
HCT, % 32.45 31.04 0.0008
PLTs, K/μL 206.00 193.80 0.1034
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Statistical differences between the cell levels before and after TBI exposure were determined using a multiple paired t test.

In Figure 2, we grouped the blood leukocyte data according to cancer diagnosis and age (13 ALL and 11 AML patients; 18 adult and 10 pediatric patients). AML patients showed a significant increase in the relative % of neutrophils (p = 0.003) and decrease in the relative % of lymphocytes (p = 0.003), eosinophils (p = 0.019), and basophils (p = 0.014), highlighting the radiosensitivity of the leukocyte cell population. For the ALL patients, there was a higher degree of individual variability across the leukocyte subtypes, leading to reduced significance before and after TBI. Both adult and pediatric groups showed a significant increase in neutrophils (p = 0.006 and 0.020, respectively) and decrease in lymphocytes (p = 0.006 and 0.030, respectively). Additionally, adult patients showed a significant decrease in eosinophils (p = 0.010) and basophils (p = 0.011), while pediatric patients did not.

Fig. 2.

Fig. 2.

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Differential white blood cell counts in four subgroups of the 28 total patients (patients diagnosed with ALL, AML, adult and pediatric patient groups) receiving TBI, recorded before and at 24 h after radiation exposure. Error bars represent the standard error of the mean. Statistical significance before and after TBI across the WBC subtypes was determined by a multiple paired t test (*p < 0.05; **p < 0.01).

Micronucleus Frequency

CBMN assay cultures were prepared from a subset of 12 TBI patients that included 6 adult and 6 pediatric patients. MN frequency was measured in peripheral blood samples collected before and at 4 and 24 h after the start of TBI, corresponding to TBI doses of 1.25 Gy and 3.75 Gy after irradiation, respectively. Table 2 summarizes patient demographics and MN frequency in the PBL cells. A minimum of 250 BNCs to a maximum of 1,000 were scored before and after TBI exposure. Overall, micronuclei yield in the adult patients showed a dose response starting at 0.07 ± 0.04 (pre-irradiation), 0.24 ± 0.08 (after the 1st fraction), and 0.63 ± 0.13 (after the 3rd fraction of TBI). MN yields measured in the pediatric blood samples showed a dose response of 0.05 ± 0.04 (pre-irradiation), 0.20 ± 0.07 (after the 1st fraction), and 0.44 ± 0.11 (after the 3rd fraction). For both age groups, there was a significant increase in MN yields with increasing dose (p < 0.05). There was no significant difference in MN yields between the adult and pediatric blood samples in either the pre-irradiated baseline (p = 0.542) or after TBI exposure (p = 0.140). Figure 3 shows the pooled data and dose response for MN frequency for both age groups. Online supplementary Table 2 shows the MN distribution per BN cell in each sample across the patient cohorts; MN frequency and distribution in the unirradiated samples collected from 4 healthy adult patients are also included. The data show significantly higher baseline levels of MN frequency in the TBI patients compared to the healthy adults (p = 0.027). In addition, linear regression analysis showed that there was no significant relationship between age and MN frequency in the lymphocytes of patients with leukemia before or after TBI exposure (Fig. 4).

Table 2.

Overview of the TBI patient demographic and cancer diagnosis recruited to this study

Patient No. Demographics MN frequency (MN/BNs) NDI
age, years gender diagnosis time between last chemo + TBI pre-exposure (0 Gy) 1st fraction (1.25 Gy) 3rd fraction (3.75 Gy) pre-exposure (0 Gy) 1st fraction (1.25 Gy) 3rd fraction (3.75 Gy)
1 30 M AML 3 days 0.09 0.23 0.71 1.86 2.23 1.68
2 23 M AML 3 months 0.09 0.25 0.69 1.71 1.49 1.49
5 16 F ALL 26 days 0.07 0.20 0.64 2.06 1.37 1.21
16 36 M ALL 13 days NA 0.25 0.73 NA 1.39 1.30
20 50 M MM 1.5 months 0.08 0.25 0.31 1.51 1.37 1.21
91 53 M AML 2 months 0.06 0.28 0.69 1.91 1.74 1.47
27 8 F ALL 8 days 0.08 0.27 0.69 2.44 1.71 1.36
85* 7 M MDS 7 days 0.02 0.15 NA 2.35 2.24 NA
93 6 F ALL 5 days 0.09 0.25 0.27 2.44 2.09 1.22
96 6 M ALL 19 days 0.04 0.07 0.34 2.34 1.71 1.29
120 6 F ALL 7 days 0.04 0.24 0.50 2.33 1.71 1.36
152 9 M ALL 21 days 0.03 0.23 0.51 1.62 1.59 1.28
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MN and NDI frequency before TBI and after the 1st and 3rd fractions are shown.

For patient 85*, only two fractions of TBI were given.

Fig. 3.

Fig. 3.

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Micronucleus frequency in leukemia patients undergoing TBI. Solid dots represent the adult population, and unfilled ones represent pediatric patients. Statistical significance was determined by a multiple paired t test (**p < 0.01; ****p < 0.0001).

Fig. 4.

Fig. 4.

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Effect of age on MN frequency in stimulated cytokinesis-blocked lymphocytes before and after TBI exposure. Red dots represent pre-TBI MN frequencies. Blue dots represent post-TBI (3 fractions, 3.75 Gy total) MN frequencies. The red and blue lines represent linear regressions of their respective deviation.

When the hematological cell subset counts were tested for correlation with cytogenetic damage, only the lymphocyte subset survival fraction (after TBI compared with before TBI) showed a negative correlation with increasing MN frequency from 0 to 1.25 Gy (r = −0.931; p = 0.007; n = 6 with 1 adult and 5 pediatric). Although we observed a negative relationship between lymphocyte cell survival and MN after an accumulated TBI dose of 3.75 Gy (n = 5; with 1 adult and 4 pediatric), the correlation coefficient was not significant. We speculate that this could be due to the larger variability in MN frequency across the pediatric cohort with increasing fractions of TBI. Despite the limited number of patients, the data suggest that measurement of increased MN formation in the peripheral blood T lymphocytes could potentially highlight individual overall sensitivity to lymphocyte cell loss/death.

Calculation of the NDI across all the patient samples showed that the NDI decreased with increasing dose, indicating a reduction in cell proliferation capacity (Table 2). NDI values typically range from 1 to 2, where a value of 1.0 signifies that all viable cells have failed to divide and are visualized as mononucleated cells, whereas an NDI value of 2.0 signifies that all viable cells have completed one division and are scored as a BNC [Eastmond and Tucker, 1989]. We observed a significant reduction in the NDI after the 1st and 3rd TBI fractions (p < 0.0001) across the RTx patients with no significant difference in NDI values between adult and pediatric patient groups, indicating a similar radiosensitivity in the peripheral blood T-cell lymphocyte population. There was a significantly (p = 0.015) higher NDI in the pediatric pre-irradiation samples (range 1.62–2.44; mean = 2.25) compared to the adult (range 1.51–2.06; mean = 1.66). The reason for the higher NDI (>2) in the pediatric cohort is because a substantial proportion of the viable cells completed more than one nuclear division during the cytokinesis-block phase and contain more than two (bi-) nuclei (tri- and quadri-nucleated cells), suggesting an increased rate of cell division. The percentage of mono-, bi-, tri-, and quadri-nucleated cells in the pediatric and adult population were 27.52%, 37.44%, 19.04%, 16%, and 30.45%, 47.6%, 12.6%, 9.35%, respectively.

Discussion

CBMN assay is an established and standardized method used to quantify DNA damage caused by IR. The relative ease of scoring MN in cytokinesis-blocked blood T lymphocytes has led to the development of automated, high-throughput platforms, using commercial biotech robotic systems and fully automated microscopy and image analysis, providing practical use to rapidly evaluate in vivo radiation exposure across occupational, medical, and victims of a mass-casualty radiological incident or accident. In the present work, we used a human radiotherapy (RTx) model for the early (at day 1 after TBI exposure) assessment of cytogenetic damage, and hematology based on the measurement of MN frequency, reduced cell proliferation capacity (nuclear division index; NDI) and differential blood cell counts.

The hematopoietic system is one of the most vulnerable organ systems to radiation exposure since the limited lifespan of blood cells requires continuous cell divisions of hematopoietic stem cells in the bone marrow. Acute exposure to moderate doses (2–6 Gy) of radiation leads to hematopoietic acute radiation syndrome, in which bone marrow is severely compromised and severe hemorrhage and infection are common [Singh et al., 2016]. Impairment of hematopoiesis will result in pancytopenia of various degrees. In cancer patients, immunodeficiency from the suppression of hematopoiesis in the bone marrow is a severe side effect of RTx [Heylmann et al., 2014]. It is also likely that the combination of myelosuppressive chemotherapy and TBI exposure will have contributed to increased cell death of the blood lymphocytes and other specific blood cell subtypes prior to blood sample collection. In preparation for a HSCT, adult and pediatric patients received myeloablative TBI (three fractions of 1.25 Gy TBI). Hematology parameters (CBC w/diff) recorded in both age groups before and after RTx showed a significant increase in circulating neutrophils and decrease in lymphocyte, eosinophil, and basophil cell levels as well as a significant decrease in RBCs (also HGB and HCT) after three fractions (total accumulated dose = 3.75 Gy) of TBI (Fig. 2; online suppl. Table 1). A large change in the mean NLR from 4.62 ± 0.49 to 14.45 ± 1.85 reflected a hyperinflammatory immune response after 3.75 Gy TBI of radiotherapy treatment. Although there was no significant change in the absolute platelet cell counts after TBI, there was an increase in the platelet-to-lymphocyte ratio from 13.55 ± 1.59 to 33.58 ± 5.03 which is also an emerging and informative marker for acute inflammatory and prothrombotic states [D’Emic et al., 2016; Gasparyan et al., 2019].

Previous studies have shown that peripheral blood cell counts are important biomarkers of radiation exposure that can provide useful diagnostic information of the severity of hematopoietic acute radiation syndrome [Macia`i Garau et al., 2011; Till and McCulloch, 2012; Thrall et al., 2013]. It is well documented that across the peripheral blood cell population, mature lymphocytes (T and B cells) are considered among the most radiosensitive compared to erythrocytes and PLTs which have been shown to be more radioresistant [Heylmann et al., 2014, 2021; Swanson et al., 2022]. Apoptosis is proposed to play a major role in dose-dependent radiation-induced cell death of the highly sensitive PBLs after exposure to doses in the low dose range, from as low as 0.125 Gy up to 2 Gy [Nakamura et al., 1990; Heylmann et al., 2021]. Based on data from historical accidents, Goans and colleagues [Goans et al., 1997; Goans et al., 2001] reported that the onset of lymphopenia in vivo is rapid and follows an approximate exponential decline within the first 24–48 h following gamma ray exposure. A rapid rise in neutrophil cell counts and decrease in lymphocyte levels in vivo has also been reported in the non-human primate model within 24 h after TBI [Farese et al., 2021; Ghandhi et al., 2023]. Hematology kinetics showed that with increasing time post-exposure, WBCs, neutrophils, lymphocytes, PLTs, and RBCs decreased, highlighting radiation-induced myelosuppression with pancytopenia.

Measurements of the mean MN frequency in adult and pediatric blood T-lymphocyte cells showed a significant dose-dependent increase with increasing TBI dose up to 3.75 Gy (Table 2; Fig. 3). Although there was no significant difference in MN levels in the pre- and post-TBI samples between the adult and pediatric patients, individual variability in MN frequency across the patients is apparent. Measurement of MN levels in the pre-irradiation samples collected from the adult RTx patients was significantly greater than in the healthy adult control group (online suppl. Table 2), suggesting that the PBLs in the cancer patients were sensitive to the previous chemotherapy treatment, leading to an increased level of chromosomal damage. Previous studies have also reported significantly higher basal levels of MN in lymphocytes of untreated patients with leukemia [Hamurcu et al., 2008; Wang et al., 2013] as well as multiple other cancers [Baciuchka-Palmaro et al., 2002; Jagetia et al., 2001] compared to age-matched control subjects. These authors suggest that this may reflect genomic instability, alterations in the immune system, or deficiency of DNA repair capacity because of the disease process.

Age and sex are important demographic variables affecting MN frequency in cytokinesis-blocked lymphocytes. In healthy adult human blood lymphocytes, studies have shown that MN frequency increases with age [Fenech and Morley, 1985; Ganguly, 1993; Nefic and Handzic, 2013]. Fenech and colleagues [Fenech et al., 1994] identified a significantly elevated spontaneous baseline MN frequency in women relative to men, with a greater dispersion of MN frequency in elderly females (>40 years), relative to males of a similar age. Mechanistic evidence using specific centromeric X- and Y-specific DNA probes showed that the X chromosome was present in 72.2% of the micronuclei in BN cells scored and increased with age [Hando et al., 1994], and in situ hybridization studies on human interphase nuclei demonstrated a significant correlation of X chromosome loss versus age in women >51 years [Guttenbach et al., 1995]. Presently, we found no significant relationship between age and MN frequency in patients with leukemia before and after TBI exposure (Fig. 4), which may be due to interindividual variability, cancer type, and/or the limited number of RTx patients recruited to the study.

In this study, we show that the NDI in stimulated peripheral blood T-cells from both adult and pediatric RTx cohorts showed a significant dose-dependent decrease with increasing TBI dose up to 3.75 Gy (Table 2). This is not surprising as it is well known that increasing doses of radiation can cause delays in cell cycle, reduced cell proliferation capacity, and MN/BN cell yields in PBLs. The NDI in the adult and pediatric groups after the TBI fractions were not significantly different, suggesting a similar radiosensitivity between adult and pediatric patients [Lee et al., 2002; Borgmann et al., 2008; Fenech et al., 2011; Rajaraman et al., 2018]. The significantly higher NDI measured in the pediatric pre-irradiated blood samples and increased number of tri- and quadri-nucleated cells scored in the blood cultures compared to the adults suggests that the frequency of cell division may be higher in the pediatric T lymphocytes than that of adults. Presently, we are unaware of other studies that directly support these findings, although we could speculate that a larger proportion of the lymphocyte cells in the pediatric cohort may reside in early G1-phase and could be more prone to external proliferation [Obermann et al., 2007].

In summary, it is widely accepted that there is a large variation in individual DNA repair capacity and responses to IR [Marchetti et al., 2006; Slyskova et al., 2011; Turner et al., 2014; Sharma et al., 2015], which could potentially affect estimation of absorbed dose and clinical radiotherapy outcomes. Radiation sensitivity is likely multifactorial in its origin; therefore, understanding the effects of confounding factors such as interindividual variation, demographics, inflammation stress, and chemo/radiotherapy treatments on radiation-responsive biomarkers is important in practical human exposure scenarios [Budworth et al., 2012; Cruz-Garcia et al., 2018]. RTx patients represent a valuable model toward the development of biomarker signatures for the prediction of organ injury and pathology after radiation exposure. This highlights the important need for future prospective population studies to comprehensively assess individual radiosensitivity using validated biomarkers of radiation exposure. In future work, our goal is to build on these patient datasets for dose and time after TBI (and PBI) exposures and use novel machine learning methods developed in our group to combine MN/BN, NDI, and NLR endpoints to more accurately determine the magnitude of cytogenetic DNA damage in peripheral blood T lymphocytes and likelihood of developing hematopoietic injury following radiation exposure [Shuryak et al., 2022, 2023].

Statement of Ethics

This consent protocol was reviewed, and the need for written and informed consent was approved by Memorial Sloan-Kettering Cancer Center (MSKCC; IRB Protocol #07-158) and the Columbia University Irving Medical Center (CUIMC; IRB Protocol #AAAB5846) in New York City. Written consent was obtained from the participants (or their parent/legal guardian where appropriate) for both the TBI patients and the healthy volunteers.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This work was supported by the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, National Institute of Allergy and Infectious Diseases (NIAID) grant No. U19 AI067773 (P.I. David Brenner), and the National Cancer Institute at NIH Cancer Center Support Grant P30 CA008748 (P.I. Craig Thompson). Investigator support was also provided by NIAID funding U01-AI148309 (P.I. Helen Turner) and a further development grant from the Opportunity Funds Management Core of the Centers for Medical Countermeasures against Radiation, No. #U19 AI067773 (P.I. Helen Turner).

Author Contributions

Sally A. Amundson, David J. Brenner, Christopher A. Barker, and Helen C. Turner designed the study of radiation response after TBI. Karthik Kanagaraj and Helen C. Turner wrote the manuscript, and Karthik Kanagaraj analyzed the CBMN data. Michelle A. Phillippi and Qi Wang analyzed the blood count data and provided statistical analyses, figures using GraphPad and text as well as editing the manuscript. Sally A. Amundson and Pratyush Narayan contributed to overall editing of the manuscript. Helen C. Turner and Barbara Szolc performed the CBMN assay and sample processing; MN scoring was performed by Pratyush Narayan, Barbara Szolc, and Jay R. Perrier. Suzanne L. Wolden, Christopher A. Barker, and Amanda McLane were responsible for the informed patient consent and recruitment at MSKCC, coordination of sample collection, and recording complete blood counts. All authors read and approved the manuscript.

Funding Statement

This work was supported by the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, National Institute of Allergy and Infectious Diseases (NIAID) grant No. U19 AI067773 (P.I. David Brenner), and the National Cancer Institute at NIH Cancer Center Support Grant P30 CA008748 (P.I. Craig Thompson). Investigator support was also provided by NIAID funding U01-AI148309 (P.I. Helen Turner) and a further development grant from the Opportunity Funds Management Core of the Centers for Medical Countermeasures against Radiation, No. #U19 AI067773 (P.I. Helen Turner).

Data Availability Statement

Data are available from the corresponding author and submitted as supplementary material.

Supplementary Material

Supplementary Material

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Data Availability Statement

Data are available from the corresponding author and submitted as supplementary material.

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