Cytopenia associated with copper deficiency

Abstract Introduction Due to an increased incidence of copper deficiency, we investigated adult patients who had low serum levels of copper with cytopenia at our hospital from March 2014 to March 2021. Methods We retrospectively reviewed the clinical data of patients who had been diagnosed with cytopenia due to copper deficiency at the Aichi Medical University Hospital from March 2014 to March 2021. Results In the 15 patients with cytopenia secondary to low serum copper level, 11 had cytopenia of two to three lineages; three (27%) had pancytopenia, and eight (73%) had bicytopenia. Of the 15 patients, nine (60%) underwent bone marrow examinations; three (30%) showed typical morphologic features associated with copper deficiency, such as multiple clear cytoplasmic vacuoles in erythroblasts and myeloid cells, and three (30%) showed dysplastic features as observed in myelodysplastic syndrome. Among the 14 (93%) patients who were treated with copper supplements, had cessation of zinc supplements, or both, 11 (73%) and eight (53%) showed normal copper levels and hematological improvement, respectively. Conclusion Copper deficiency is more common than expected and should be considered in patients with unexplained cytopenia.

Copper deficiency is less common in healthy individuals because copper is a ubiquitous element and is present in almost all foods [4].
In the past, copper deficiency has been observed in preterm infants and patients with hereditary conditions, such as Menkes disease [5].
Increased use of enteral nutrition (EN) and total parental nutrition (TPN), advances in medical techniques, and an increase in the aging population has led to increased copper deficiency [6]. Moreover, copper deficiency induced by excessive zinc has also increased because zinc supplements have been common.
Copper deficiency causes hematological abnormalities such as anemia, leukopenia, neutropenia, and thrombocytopenia. However, diagnosis of cytopenia due to copper deficiency is difficult because the bone marrow morphology in copper deficiency cannot be distinguished from myelodysplastic syndrome (MDS) [2,[7][8][9]. This difficulty prompted us to retrospectively investigate and analyze the clinical and hematologic features of patients with copper deficiency.
We retrospectively collected data on the clinical history, hematologic examination findings, and treatments of copper deficiency. Complete blood count and bone marrow morphology were reviewed, and their relationship with the copper concentration was investigated.
Moreover, we divided the patients into two groups according to the median value of serum copper level (<16 and ≥16 µg/dL) and compared the hematological features and therapeutic effect between the two groups.

Data management and statistical analyses
The student's t-test was applied to compare the means of continuous variables and normal distribution data and was carried out using the EZR software package [10].

Patients' characteristics
A total of 15 patients (ten men and five women) with cytopenia secondary to low serum copper level were identified. The median (range)

Hematological and laboratory features
Cytopenia in two to three lineages was observed in 11 (73%) patients, with pancytopenia in three (20%), anemia and leukopenia in three (27%), anemia and thrombopenia in four (27%), and leukopenia and thrombopenia in one (6.7%) patient (Table 3); four patients (27%) had isolated anemia. Neutropenia was seen in four patients (27%). Seven patients (47%) with anemia developed normochromic anemia and eight (53%) developed macrocytic anemia. Vitamin B12 and folic acid levels were measured in 11 patients (73%) and were within the normal range in nine patients. Two patients with low folic acid level did not improve their cytopenia after increasing their levels of folic acid.
Among the patients with copper deficiency, three patients (20%) had higher serum zinc levels than 130 µg/dL, which is the upper limit of the normal range in our laboratory.

Reference ranges Median Range
Hemoglobin (  We divided the patients into two groups according to the median value of serum copper level (< 16 and ≥16 µg/dL) and compared the hematological features and therapeutic effect between the two groups.  The patient with malabsorption showed persistent anemia, although his serum copper level increased to normal. Only one patient died before the initiation of copper supplements.

DISCUSSION
The present study showed the results of the analysis of 15 patients with cytopenia secondary to copper deficiency. Zinc supplementation and bicytopenia were most commonly associated with copper deficiency.
Low serum copper level (<16 µg/dL) was also associated with a lower

Number of cases (%)
Types of therapy

Median duration (range)
Time from diagnosis to normalization of serum copper level (days) 84  neutrophil count. Not all patients had typical morphologic bone marrow features associated with copper deficiency, and in some patients dysplastic features seen in MDS were observed.
The mechanisms of copper deficiency-induced cytopenia have not yet been fully investigated. Anemia and neutropenia are common, whereas thrombocytopenia is reported to be uncommon in copper deficiency [6]. Copper is an important component of ceruloplasmin and hephaestin, which are necessary for the export and absorption of iron and synthesis of heme [11,12]. Moreover, copper deficiency has been shown to reduce the lifespan of red cells [13]. We attempted to verify the relationship between copper and iron metabolism in our study. Although some patients showed low total iron-binding capacity, unsaturated iron-binding capacity, and high ferritin levels, it was difficult to determine whether copper deficiency induced iron unavailability because these patients had chronic inflammation and underwent repeated red blood cell transfusion, which increases ferritin level.
Some studies have also suggested that copper deficiency arrests neutrophil mutation and leads to the development of anti-antibodies resulting in neutropenia [14,15]. Our finding that some patients with copper deficiency developed leukopenia and neutropenia supports these previous reports. Moreover, we found that severe low copper deficiency frequently led to much lower neutropenia than mild low copper deficiency. To our knowledge, this is the first report on the relationship between neutrophil count and the extent of decrease in serum copper levels.
Although isolated anemia, bicytopenia, and pancytopenia have been reported, there is no report about isolated thrombocytopenia because of copper deficiency and its mechanisms. In our study, five patients (33%) with thrombocytopenia coexisted with anemia and leukopenia and improved their platelet count after treatment. Therefore, we suspected that thrombocytopenia caused by copper deficiency might have occurred in the case of severe and long-term copper deficiency.
However, there was no significant difference between the platelet count and the extent of decrease in serum copper level. Moreover, it makes the analysis difficult because the details regarding the duration of copper deficiency were not within the scope of the current retrospective study, and thrombocytopenia is caused by copper deficiency and various conditions. Therefore, further studies with a larger sample size are necessary to investigate our hypothesis and establish the mechanisms of thrombocytopenia caused by copper deficiency.
The results of our study suggest that the most common cause of copper deficiency was zinc supplementation in this study. Reports of copper deficiency due to zinc supplements have increased since the 2000s, and the mechanism that zinc induces copper deficiency suggested [16][17][18][19][20][21][22][23][24][25]. Excessive zinc stimulates enterocytes to produce metallothionein, and copper has a higher affinity for metallothionein than zinc. Therefore, copper binds to metallothionein and then sloughs into the stool, resulting in copper deficiency [26]. Zinc deficiency is developed frequently in patients on hemodialysis or who have liver cirrhosis [27][28][29][30]. In the present study, we also experienced that the patients who were on maintenance hemodialysis or had liver cirrhosis developed copper deficiency, possibly due to zinc supplements.
Although our patient developed anemia 8 months after she started PPZ, there was a report that cytopenia due to copper deficiency with PPZ developed earlier than in our patient [31]. Moreover, patients prescribed ZAH, which contains more zinc than PPZ, were reported to develop copper deficiency more frequently and earlier than patients prescribed PPZ [32]. This report is compatible with our result that more patients prescribed with ZAH experienced cytopenia due to copper deficiency than patients prescribed with PPZ. Therefore, in patients with cytopenia, it is important to elicit history zinc supplements and consider the risk of copper deficiency induced by excessive zinc, especially in patients undergoing hemodialysis and liver cirrhosis.
Our study also suggests that the second most common cause of copper deficiency was EN or TPN in this study. Total copper intake from the 900-1500 kcal/day tube feeding formula, which contains 5-12 µg of copper per 100 kcal, is approximately 50-180 µg/day [33]. and is easy to administer to tube-feeding patients because it is in powder form. We recommended other copper-rich foods such as nuts, shellfish, or liver to patients with diabetes mellitus because almost all patients add sugar to suppress the bitterness of cocoa. We also used other copper-rich foods for patients who could not continue oral cocoa intake due to diarrhea because cocoa contains fibers [35]. We suggest that copper supplements should be chosen depending on the patient's underlying conditions. This study has some limitations. One major limitation is the small number of patients included in this study. In addition, detailed information on the clinical presentation induced by copper deficiency other than cytopenia, such as neurologic symptoms, duration of copper deficiency, laboratory parameters, and bone marrow examination, was not available for all patients because this study was retrospective.
In conclusion, most cytopenia secondary to copper deficiency occurred in patients receiving zinc supplements, EN, or TPN. The number of patients with impaired hematopoiesis due to copper deficiency is expected to increase because the number of patients undergoing dialysis, EN, or TPN is also expected to increase with the advancement of medicine. Diagnosis of cytopenia due to copper deficiency is difficult because these patients have many problems that can cause impaired hematopoiesis. Additionally, the bone marrow morphologies in copper deficiency, which showed dysplastic features similar in MDS, complicate the diagnosis. Therefore, in cytopenia patients with zinc supplements or prolonged EN or TPN, copper deficiency should be considered in the differential diagnosis.

ACKNOWLEDGMENTS
We would like to acknowledge all clinicians and their administrative staff who assisted in the study.

FUNDING STATEMENT
The funders played no role in the study design, data collection, and analysis, the decision to publish or the preparation of the manuscript.