The perils of not digging deep enough—uncovering a rare cause of acquired anemia
Authors: Neeraj Saini, Joseph O. Jacobson, Smita Jha, Vivek Saini, Ronald Weinger
Journal: American Journal of Hematology (2011)

In 2004, a 74-year-old Caucasian female was referred to Hematology for normocytic normochromic anemia of about 2-year duration. She related fatigue and dyspnea on exertion. Past medical history was notable for obesity (Body mass index > 45), diabetes mellitus type 2, mild aortic stenosis (valve area 1.6 cm2), hypertension, hyperlipidemia, and a total right hip and knee replacement two years ago for degenerative joint disease. Her hip replacement was complicated by chronic Methicillin resistant Staphylococcus Aureus (MRSA) infection of the prosthesis requiring multiple incision and drainage (I&D) and Girdle stone arthroplasty. Her home medications included losartan, rosiglitazone, atenolol, atorvastatin, gabapentin, multivitamins, and linezolid (LZD). She had been on 600 mg twice daily of LZD for ∼30 months to suppress the right hip infection. Her baseline complete blood count (CBC) before her hip surgery revealed white blood cell (WBC) of 8.39 K/μL, hemoglobin (Hgb) 11.5 g/dL, hematocrit 36%, MCV 90, and platelets 277. Over the last 2 years since the surgery, her Hgb and hematocrit had slowly declined to 8.5 g/dL and 25%, respectively. This patient has a baseline mild anemia with Hgb > 11 g%. Her recent drop in Hgb is concerning. The differential diagnosis for her worsening anemia would be very broad. Iron deficiency anemia (IDA) is the most common form of anemia and prevalent widely in older age group. It could also be anemia of chronic disease (ACD) given chronic MRSA infection, diabetes, and morbid obesity. Drug-induced anemia should be considered as LZD that has been associated with reversible myelosuppression. Aortic stenosis is known to be associated with bleeding from colonic angiodysplasia. She denied recent use of any over the counter drugs or nonsteroidal anti-inflammatory drugs. She denied any family history of hematological cancers or chronic inflammatory conditions. There was no history of tobacco or alcohol abuse. Physical examination was pertinent for obesity, pale conjunctiva and grade 2/6 systolic ejection murmur radiating to neck, and scar marks from her hip procedures. There was no adenopathy, no hepatosplenomegaly and fecal occult blood test was negative. Laboratory tests on presentation revealed: WBC 7,800/μL, Hgb 8.5 g/dL, Hct 25%, platelets 224,000/μL, Mean corpuscular volume (MCV) 90.4, and RDW 18.8. Differentials showed neutrophils 55.7%, lymphocytes 33.8%, monocytes 7.9%, eosinophils 2.3%, and basophils 0.3%. Basic metabolic panel was normal with blood urea nitrogen 29 and serum creatinine 1.1. Absolute reticulocyte count (ARC) was 64,100/μL. Urinalysis was normal. Our patient has isolated anemia without any abnormality in other cell lines. With an ARC well below 100,000/μL and calculated reticulocyte production index (after correction for low hematocrit and maturation time for reticulocyte) of ∼0.65, the patient has hypoproliferative red cell lineage in bone marrow. The differential diagnosis at this stage, although relatively expansive, would primarily include acquired conditions like IDA, ACD, vitamin deficiency, bone marrow disorders (myelodysplastic syndrome), and sideroblastic anemia (SA). Although there could be coexistence of mild grade chronic blood loss or hemolytic anemia but with a markedly depressed bone marrow, it is prudent to first find the etiology for the decreased red cell precursors. Iron panel revealed serum ferritin 233 ng/mL (normal 11–307), iron 43 μg/dL (normal 60–160), total iron binding capacity 315 μg/dL (normal 275–425), and %iron saturation 14%. Serum vitamin B12 level of 712 pg/mL and folic acid >20 ng/mL were normal. Erythrocyte sedimentation rate, serum lactate dehydrogenase (LDH), haptoglobin, and liver function tests were within normal range. Coombs test was negative. Total serum protein was 7.1 g/dL, and serum/urine protein electrophoresis with immunofixation showed normal pattern with no abnormal monoclonal bands. Hgb electrophoresis showed A1 – 97.3%, A2 – 2.7%, F – 0.0%. Serum lead level was 1.0 mcg/dL (normal 1–9). Serum thyroid stimulating hormone was normal. In this case, megaloblastic anemia or thalassemia or hemoglobinopathy does not appear probable due to normal MCV along with normal serum vitamin B12, folic acid, and normal hemoglobin electrophoresis panel. Hemolytic anemia seems less likely with a slow indolent course of anemia and normal serum LDH, haptoglobin, and negative coombs test. Anemia of chronic inflammation is plausible given her chronic hip MRSA infection and other comorbidities. In both ACD and IDA, the serum iron and transferrin saturation are reduced, reflecting absolute iron deficiency in IDA and sequestration of iron by the reticuloendothelial system in ACD. In IDA, transferrin saturation may be lower compared with ACD, because serum concentrations of the iron transporter transferrin are increased, whereas transferrin levels remain normal or are decreased in anemia of chronic disease [1]. With a normal high ferritin value of 233 and iron panel suggestive of iron sequestration, ACD is more likely compared to IDA. Also, there have been case reports of isolated anemia associated with LZD toxicity leading to red cell precursor hypoplasia with a decrease in reticulocytes count (Table I). Multiple myeloma is ruled out with normal SPEP and UPEP, however, other myelopthisic disorders are quite possible. Myelodysplasia (MDS) is another disorder that is very frequent in this age group. Bone marrow examination would help us to differentiate these etiologies. A trial of erythropoietin (EPO) 40,000 units weekly was started with a presumed diagnosis of ACD. Her Hgb increased to 12.1 g% within 3 months with improvement in her symptoms. However, after about a year patient had increasing requirements of EPO and needed transfusions to maintain her counts despite adequate iron stores. She needed 2 units of packed red blood cells in 2005, 4 units in 2007, and 5 units in 2009 along with increasing high doses of EPO agents. Patient had been on LZD 600 mg bid for ∼7 years at this time. Testing for absence of CD55 and CD59 ruled out paroxysmal nocturnal hemoglobinuria. Bone marrow biopsy in 2009 showed hypercellular bone marrow with significant dyserythropoiesis (megaloblastoid changes) with numerous erythroblasts and vacuolated cytoplasm. There were no excess blasts, and other cell lineages were unremarkable. Iron stains showed markedly increased bone marrow storage iron with greater than 50% ring sideroblasts. Cytogenetic and fluorescence in situ hybridization (FISH) were unremarkable. No immunophenotypic abnormalities could be detected through flow cytometry. Bone marrow aspiration shows a hypercellular bone marrow with features suggestive of MDS. According to the WHO 2008 MDS classification, it falls under refractory anemia with ringed sideroblasts (RARS) category. Interestingly, LZD has also been implicated to cause reversible SA [3, 9-11]. In this case, no clonal abnormalities are detected through cytogenetic and FISH. Flow cytometry is normal as well. Except for dyserythropoietic changes, which were primarily megaloblastoid changes, all other cell lineages appear unremarkable. These features make the diagnosis of LZD-induced SA equally, if not more, feasible as MDS. Also, the bone marrow findings are similar to what has been published in the literature regarding LZD-induced myelosuppression (Table I). To differentiate between these two pathogenetic processes, bone marrow biopsy should be repeated after few months of cessation of LZD. Contrary to MDS, LZD-induced dysplasia and bone marrow changes will reverse after drug's cessation. Given concerns for LZD-induced SA, LZD was stopped, and patient was started on Bactrim for lifetime suppression of chronic MRSA infection of hip. Her Hgb and Hct increased from 8.8 g% and 26.4% to 11.8 g% and 35.1%, respectively, within 3 months and has remained stable without any erythropoietin or blood transfusion. Repeat bone marrow biopsy after 5 months of cessation of LZD showed slightly hypercellular bone marrow (∼40–50%) for patient's age, with mild dyserythropoiesis, 11% ring sideroblasts, and no increase in blasts. The degree of dysplastic changes in the red cell lineage was much reduced and involved ∼10% of erythroid precursors. Cytogenetic, FISH, and flow cytometry studies were unremarkable again. The repeat bone marrow examination shows marked improvement from the previous marrow examination. Mild dyserythropoietic changes (primarily megaloblastoid features) are much reduced from previous biopsy and involve only 10% of erythroid precursors. Also, ring sideroblasts are markedly decreased from >50% to 11%. No increase in blasts is noted again, and cytogenetic studies are normal. In conclusion, significant marrow changes since cessation of LZD, along with the lack of identifiable clonal or chromosomal abnormalities, suggest a possible ongoing resolution of her nonclonal dysplasia. We render the diagnosis of LZD-induced SA. The fact that patient's anemia and clinical symptoms worsened after initiation and got better with cessation of LZD lends further strength to our diagnosis. In hindsight, ACD could have contributed to anemia as patient initially responded briskly to erythropoietin and later in the course when LZD-induced bone marrow changes started to worsen, her response progressively waned and required maximal doses of erythropoietin with transfusions. The residual persistent dysplastic changes in bone marrow after 5 months of cessation of LZD could be due to premature timing of bone marrow examination, not allowing the marrow enough time to recuperate. LZD has not been shown to cause irreversible myelotoxicity and incline us to favor that bone marrow would continue to recover with reversal of SA. Contrary to this case, other case reports of LZD-induced SA have shown marrow changes returning to baseline within a month of cessation of drug, however, no patient in literature has been subjected to LZD for a prolonged span of ∼8 years as happened in this case. Another possibility is that there is an underlying MDS in this patient along with drug-induced SA. SA are a heterogeneous group of disorder characterized by abnormal ringed sideroblasts (amorphous iron deposits in mitochondria of erythroblasts that are housed within a distinct mitochondrial ferritin) in bone marrow. These disorders can be hereditary or acquired. Acquired SA is more common and etiologies include alcohol, drugs, toxins, hypothermia, MDS, and deficiency of copper. Drugs commonly associated with SA include isoniazid, chloramphenicol, cycloserine, pyrazinamide, and others. LZD has been implicated in reversible myelosuppression with thrombocytopenia being the major adverse hematological side effects noted so far [12]. Pancytopenia has also been observed with LZD [6, 7]. Case reports of anemia due to LZD toxicity have been mostly due to reversible myelosuppression leading to red cell precursor hypoplasia with a decrease in reticulocytes count (Table I). The bone marrow findings in these cases show intracytoplasmic vacuolation in erythroblasts with decrease in erythropoiesis, highly increased myeloid/erythroid ratio along with corroborative findings of decreased reticulocytes and increased serum iron/ferritin that suggests nonhemolytic nonregenerative red cell hypoplastic anemia. A case report of pure red cell aplasia has also been described [5]. The effect of LZD on iron kinetics has not yet been investigated in clinical trials. On review of literature, using PubMed with keywords "sideroblastic anemia" and "linezolid" four case reports [3, 9-11] were identified. Also, a study in dogs treated with PNU-100592, an Oxazolidinone analog, induced SA in dogs treated with high dose [13]. SA is characterized by imbalances in mitochondrial iron import and its utilization, which could be due to either insufficient generation of heme or deficient incorporation of iron into protoporphyrin to form heme. The mechanisms for mitochondrial iron loading in developing erythroblasts in SA are diverse; however, the mechanism of LZD toxicity has been compared to chloramphenicol (CP), because both inhibit mitochondrial protein synthesis, and appearance of morphologically abnormal vacuolated erythroblasts in bone marrow has been shown in both drugs' toxicity [2]. Similar to CP, LZD toxicity is probably a result of inhibition of key mitochondrial proteins, including 5-aminolevulinic acid and ferrochelatase. Serum protoporphyrin was increased in a pediatric case that further suggests that LZD does inhibit ferrochelatase [5]. LZD binds to the 50S subunit of the prokaryotic ribosome, which are similar to human mitochondrial ribosome, preventing fusion with the 30S subunit resulting in blockage of assembly of functional initiation complex, ultimately preventing translation of mRNA [14, 15]. This mode of action differs from that of CP that allows mRNA translation to begin but then inhibits peptide elongation by inhibiting peptidyl transferases [15]. This mode of action may also account for LZD effectiveness against gram-positive bacteria like Staphylococcus aureus, as it prevents synthesis of virulence factors (e.g., coagulase, hemolysins, and protein A) as compared to other prokaryotic protein inhibitors antibiotics available in the market [16]. LZD also inhibits mitochondrial respiratory chain complex IV activity (partially synthesized by mitochondria) in peripheral-blood mononuclear cells that further proves that LZD-induced SA is due to mitochondrial insults [17]. The mechanism may be similar; however, unlike CP, LZD has not been shown to be genotoxic or cause aplastic anemia. Also, LZD-induced hematologic toxicity has been shown to be reversible with patients returning back to their baseline values within 2–3 months after cessation of therapy. A higher rate of toxicity, thrombocytopenia, and anemia has been noted in patients with end stage renal disease (ESRD) [18]. Two major metabolites of LZD (PNU-142856 and PNU-142300) were found to accumulate in patients with renal impairment, and these metabolites might have a role to play in induction of sideroblasts formation with mitochondrial insults. Ringed sideroblasts are seen in acquired and congenital anemia as well as MDS. The formation of ringed sideroblasts in inherited SA has been linked to various genetic mutations including loss of function mutation in the genes ALAS2, ABCB7, and SLC25A38; however, the clinical significance and mechanisms of formation of ringed sideroblasts in MDS is still unclear. Recently, mutations in multiple components of RNA spliceosomes, including U2AF35, ZRSR2, SRSF2, and SF3B1 have been identified in MDS [19]. In one particular subset of MDS associated with ringed sideroblasts, RARS including RARS-T (RARS with thrombocytosis) a high frequency (60–80%) of mutations in SF3B1 gene has been reported [19, 20]. MDS patients with SF3B1 mutation were found to have higher neutrophil and platelet counts, fewer bone marrow blasts, longer event free survival, and relatively benign course [20]. We contemplate that testing for SF3B1 gene mutation, which could be easily done on peripheral blood, would not only help in identifying MDS patients with benign prognosis but also help in differentiating drug-induced SA from MDS. LZD is the only FDA approved oral agent for MRSA and will potentially replace vancomycin for long-term and outpatient therapy. In conclusion, we want to increase the awareness among clinicians about the potential for myelosuppression and SA induced by LZD. This side effect should be considered in elderly patients where drug-induced SA could be confused easily with MDS. We suggest monitoring of hematologic parameters in patients receiving long-term LZD therapy.