The Human Research Ethics Committee of the South Egypt Cancer Institute, Assiut University, approved this study (SECI-IRB-IORG0006563, 37:10/1/2018). Written informed consent was obtained from patients and healthy volunteers in accordance with the Declaration of Helsinki. Patients and controls provided written informed consent and participated in the study after study clarification. Patients got the maximum care, and the study procedures were in accordance with relevant guidelines and the Declaration of Helsinki. Investigators and researchers collected data using paper data collection forms. Patients were identified by serial study codes, which were linked with patient names and hospital file numbers on a separate sheet and stored in a locked safe cabinet. Data analysis was performed on the de-identified data.

A prospective case-control study was conducted at the Clinical Haematology Unit, Internal Medicine Department, Assiut University Hospital, and Medical Oncology Department, South Egypt Cancer Institute, Assiut University, in Assiut, Egypt, from October 2018 to August 2019. Twenty newly diagnosed untreated MM patients diagnosed according to the World Health Organization criteria for MM, and 20 matched (age and sex) healthy volunteers were included in this study. Patients who were under treatment or refused to participate were not included.

Multiple myeloma patients were classified using the Durie-Salmon staging system¹⁸ that assesses tumour burden based on three laboratory parameters and evaluates bone involvement based on a radiographic skeletal survey. Patients who had haemoglobin levels over 10 g/dL, normal serum calcium or less than 10.5 mg/dL, normal bone X-ray or solitary lytic lesion, monoclonal immunoglobulin G under 5 g/dL, immunoglobulin A under 3 g/dL, and Bence Jones protein under 4 g/24 h were classified as stage I. Stage II patients were patients with a moderate number of myeloma cells that did not meet either stage I or stage III criteria. Stage III patients were those who met one or more of the following criteria: haemoglobin under 8.5 g/dL, serum calcium value over 12 mg/dL, advanced lytic bone lesions, monoclonal immunoglobulin G over 7 g/dL, immunoglobulin A over 5 g/dL, and Bence Jones protein over 12 g/24 h.

As the Durie-Salmon scoring may not provide good prognostic discrimination, the International Staging System was used in this study to group MM patients according to the serum β₂-microglobulin (β₂M) and serum albumin into 3 stages: stage I characterised by serum β₂M less than 3.5 mg/L and serum albumin 3.5 g/dL or higher, stage II in which serum β₂M was less than 3.5 mg/L and serum albumin was less than 3.5 g/dL or β₂M was between 3.5 mg/L and 5.5 mg/L, and stage III in which serum β₂M was 5.5 mg/L or higher.¹⁹

Patient data were also collected for the study; these included age, sex, and clinical history of bone pain, pathological fracture, weakness and fatigue, recurrent infection, manifestation of hypercalcemia such as thirst, polyuria, manifestation of hyperviscosity (blurring of vision, mental confusion), bleeding tendency, and neuropathy. Clinical signs of anaemia, oedema, and renal impairment were carefully assessed in all patients. A radiographic skeletal survey of the skull and long bones was also done to detect lytic bone lesions, pathological fractures, and abdominal ultrasound.

Complete blood count with differential was done using the Cell-Dyn Ruby fully automated blood counter (CELL-DYN Ruby System, Abbott Diagnostics, Chicago, Illinois, United States; serial number: 36026BG). Examination of peripheral blood smear for rouleaux formation was done as part of routine care. Blood urea, serum creatinine, total protein, albumin, and lactate dehydrogenase (LDH) tests were done using the Cobas Integra 400 automated chemical analyser (Roche Diagnostics GmbH, Mannheim, Germany; serial number: 500558). Serum protein electrophoresis with immunofixation was performed to identify monoclonal protein (M-protein) types among patients at diagnosis using the Pretty Interlab device (Interlab, Rome, Italy; serial number: 38405301). Bone marrow aspiration and biopsy to evaluate and quantify bone marrow plasma cell infiltration were done on all recruited patients. All these investigations were done as routine services for newly diagnosed MM cases.

Fifty microlitres of remnant routine complete blood count samples were added to 5 µL of CD14 fluoroisothiocyanate-conjugated and CD16 phycoerythrinconjugated (Becton Dickinson [BD] Biosciences, San Jose, California, United States). After 15 min of incubation at room temperature in the dark, lysis and washing of erythrocytes was done. The cells were then re-suspended in phosphate-buffered saline, and cells were acquired using the BD FACSCalibur flow cytometry system (BD Biosciences, San Jose, California, United States), with data analysis done using the CellQuest software (BD Biosciences, San Jose, California, United States). An anti-human immunoglobulin G isotype-matched negative control was used for each flow cytometry experiment. Forward and side scatter histograms were used to define monocytes. Afterwards, the expression of CD14 and CD16 were assessed in the monocyte population. The classical subset was CD14⁺⁺CD16^-, intermediate subset was CD14⁺⁺CD16⁺, and non-classical subset was CD14⁺CD16⁺⁺ (Figure 1). Monocyte subset data are expressed as percentages of the total monocytes.

We used Statistical Package for the Social Sciences version 21 (IBM Corporation, Armonk, New York, United States) for data analysis. Quantitative data are presented as mean ± standard error while qualitative data are presented as frequencies and percentages. Mann-Whitney test analysis was performed to determine the statistical significance and differences between the study groups. Spearman’s correlation was performed to determine the association between monocytes subtypes and laboratory parameters. Results were considered significant if p was 0.05 or less.

The mean age of the MM patients was 6.35 ± 4.07 years, and the number of men (13) was double that of women (7) (Table 1). The proportion of plasma cells in bone marrow was 39.7 ± 3.7% and that of the monoclonal band (M-protein) was 4.44 ± 2.8 g/dL.

Laboratory investigations showed that platelet counts, total leukocytic counts, serum total protein, and urea levels were comparable between patients and healthy controls (p > 0.05).

Multiple myeloma patients had significantly reduced haemoglobin (9.3 ± 2.4 g/dL in MM vs 12.8 ± 1.97 g/dL in healthy controls; p = 0.045) and serum albumin (2.46 ± 0.9 g/dL in MM patients vs 4.63 ± 1.1 g/dL in healthy controls; p < 0.0001) when compared to healthy controls.

On the other hand, after comparing the mean levels of serum calcium, LDH, and serum creatinine between MM patients and healthy controls, we found statistically significant increased levels in MM patients (calcium: 11.75 ± 0.4 mg/dL in MM patients vs 8.8 ± 0.1 mg/dL in healthy controls, p < 0.0001; LDH: 886.86 ± 92.6 units per litre in MM vs 155.8 ± 8.23 units per litre in controls, p < 0.0001; serum creatinine: 1.9 ± 0.2 mg/dL in MM patients vs 0.97 ± 0.1 mg/dL in healthy controls, p < 0.0001).

Based on the Durie-Salmon staging system, 12 (60%) of the 20 newly diagnosed untreated MM patients had stage I disease, 6/20 patients (30%) had stage II disease, and 2/20 patients (10%) had stage III disease. Whereas according to the International Staging System, 70% (n = 14) of MM patients had stage I disease, 20% (n = 4) had stage II, and 10% (n = 2) had stage III.

Monocytes present in the peripheral blood displayed a range of intensities of CD16 and CD14 expression. They were divided into classical (CD16⁻CD14⁺⁺), intermediate (CD16⁺CD14⁺⁺), and non-classical (CD16⁺⁺CD14⁺) based on these ranges.

In healthy controls, classical monocytes represented the major component among total monocytes (83.8%), followed by non-classical monocytes (8.9%) and intermediate monocytes (7.4%).

Multiple myeloma patients had statistically significant lower proportions of classical monocytes (77.24% ± 0.66 in MM patients vs 83.75% ± 0.5 in controls), and higher proportions of non-classical (12.44% ± 0.5 in MM patients vs 8.9% ± 0.34 in controls) and intermediate (10.3% ± 0.24 in MM patients vs 7.4% ± 0.29 in controls) monocytes compared to healthy controls (all with p < 0.0001) (Figure 1).

In newly diagnosed untreated MM patients, the proportion (%) of classical monocytes correlated positively with the serum albumin level (R² = 0.731; p < 0.0001), and correlated negatively with serum levels of serum creatinine (R² = 0.389, p < 0.0001), LDH (R² = 0.545, p < 0.0010), calcium (R² = 0.582, p < 0.0001), M-protein (R² = 0.222, p = 0.005), and with the percentage of bone marrow plasma cells (R² = 0.347, p < 0.0001), while urea and total protein did not show significant correlation (Figure 2).

In contrast, the proportion (%) of non-classical monocytes in newly diagnosed untreated MM patients correlated negatively with the serum albumin level (R² = 0.624, p < 0.0001), and correlated positively with serum levels of creatinine (R² = 0.413, p < 0.0001), LDH (R² = 0.533, p < 0.0001), calcium (R² = 0.575, p < 0.0001), M-protein (R² = 0.28, p = 0.024), and with the percentage of bone marrow plasma cells (R² = 0.27, p = 0.018), while urea and total protein did not show significant correlation (Figure 3).

Similarly, the proportion (%) of intermediate monocytes in newly diagnosed untreated MM patients correlated negatively with serum albumin level (R² = 0.649, p < 0.0001) and correlated positively with the serum levels of creatinine (R² = 0.222, p = 0.002), LDH (R² = 0.338, p < 0.0001), calcium (R² = 0.398, p < 0.0001), and with the percentage of bone marrow plasma cells (R² = 0.301, p = 0.016), while urea, total protein, and M-protein did not show significant correlation (Figure 4).

One limitation of this study was the small sample size, but these initial results provide new perspectives on the diagnosis and monitoring of MM.

The higher frequencies of non-classical and intermediate monocytes in newly diagnosed untreated MM patients suggest a possible role of these cells rather than classical monocytes in the disease process. The participation of these monocyte subsets in MM pathogenesis should thus be part of future investigations as they may provide important information for the diagnosis and follow-up of MM patients. More studies are needed to address the possibility of using flow cytometry results as a primary staging criterion.