Diego Velasco-Rodríguez

δβ-Thalassemia trait: how can we discriminate it from β-thalassemia trait and iron deficiency anemia?

OBJECTIVES To analyze the differences not only in classic hematologic parameters but also in RBC subpopulations among δβ-thalassemia trait (δβ-TT), β-thalassemia trait (β-TT), and iron deficiency anemia (IDA) and to evaluate the role of fetal hemoglobin (HbF) in elevated RBC distribution width (RDW). METHODS Samples from 553 patients with microcytosis (74 δβ-TT, 272 β-TT, and 207 IDA) were run on an Advia 2120i analyzer (Siemens Medical Solutions Diagnostics, Tarrytown, NY). Classic hematologic parameters and RBC subpopulations were assessed. The correlation between HbF and RDW in patients with thalassemia (both β and δβ) was evaluated. An independent sample t test was used to compare classic hematologic parameters and RBC subpopulations among β-TT, IDA, and δβ-TT and receiver operating characteristic curves performed in the significant comparisons. RESULTS RDW was significantly higher in δβ-TT compared with β-TT (18.79% vs 16.04%, P < .001), as was mean corpuscular volume (66.39 vs 64.82 fL, P < .001), mean corpuscular hemoglobin (20.73 vs 20.04 pg, P < .001), and mean corpuscular hemoglobin concentration (31.16 vs 30.66 g/dL, P = .03). Pearson coefficient showed a good correlation between HbF and RDW. The values obtained for all the parameters were significantly different (P < .001) between patients with thalassemia (β and δβ) and IDA. CONCLUSIONS RDW is the best parameter to discriminate δβ-TT from β-TT. The degree of anisocytosis in patients with β-TT and δβ-TT is strongly correlated with HbF.

Metastatic malignant melanoma detected on bone marrow aspiration

A 75-year-old man presented with asthenia and epistaxis of 3 weeks duration. He had been diagnosed with malignant melanoma 4 months previously, and had received intensive chemotherapy. Physical examination showed pallor, petechiae of both legs and splenomegaly. A blood count showed a haemoglobin concentration of 98 g/l, white cell count of 1 7 9 10/l and platelet count of 18 9 10/l. A blood film showed a leucoerythroblastic reaction; metastasis was suspected and a bone marrow aspiration and trephine biopsy were therefore performed. A May-Gr€ unwaldGiemsa stain revealed numerous clusters of non-haemopoietic cells containing black pigment (top left and top right), some with giant granules (bottom). Immunohistochemical staining of the biopsy specimen showed positivity for S-100 protein, thus confirming metastatic melanoma. Liver and spleen metastases were also identified on a computed tomography scan. The patient was treated palliatively and died 2 months later. Although metastasis of malignant melanoma to the bone marrow is infrequent, this possibility should be considered in the presence of pancytopenia or leucoerythroblastosis.

Prostatic-Like Syndrome in a Woman with Chronic Lymphocytic Leukemia: Sequential Kinase Inhibitor Therapy

Chronic lymphocytic leukemia (CLL) is an incurable lymphoproliferative disorder with a heterogeneous genetic and clinical course. Two kinase inhibitors, ibrutinib and idelalisib, have demonstrated achievement of complete and durable remissions in relapse/refractory genetically unselected CLL patients. We present a case of relapsed CLL with extensive disease and hourglass deformity of urinary bladder as a result of the compression of two extraperitoneal paravesical soft tissue bulky masses, with excellent response to sequential kinase inhibitor therapy.

Laboratory parameters provided by Advia 2120 analyser identify structural haemoglobinopathy carriers and discriminate between Hb S trait and Hb C trait

Background Haemoglobinopathies have spread owing to human migration, and the number of people needing diagnosis and management of these conditions is increasing. Clinicians need to accurately identify carriers and provide adequate genetic counselling in order to prevent the occurrence of homozygous or compound heterozygous offspring. Objectives To identify red blood cell (RBC) laboratory parameters that discriminate between structural haemoglobinopathy carriers and healthy subjects, and to compare RBC laboratory indices between HbAS and HbAC individuals. Methods Samples of 500 variant Hb carriers (355 HbAS, 104 HbAC, 19 HbAD, 7 HbAE, 7 HbAO-Arab, 4 α-chain variants and 4 Hb Lepore) and 251 normal controls were run on an Advia 2120 analyser (Siemens). Classic haematological parameters and RBC populations were assessed in all subjects. A multivariable binary logistic regression model was created to predict the probability of a subject carrying any structural haemoglobinopathy. HbAS (n=355, 71%) and HbAC (n=104, 20.8%) subjects were compared. Results A clinical prediction rule was developed by assigning one point to each of the most efficient variables: mean corpuscular volume (MCV) <88.4 fL, RBC distribution width >13.4%, percentage of microcytic RBCs (%MICRO) >0.7% and the ratio of microcytic RBCs to hypochromic RBCs >0.8. A score of 0, 1, 2, 3 or 4, resulted in a probability of 9.6%, 36.3%, 66.7%, 85.2% or 98.3%, respectively. Among the most frequent variant Hb, HbAC subjects had lower values of parameters related to cell size (MCV, %MICRO) and higher values of parameters related to haemoglobin concentration (MCHC, %HYPER) than HbAS subjects. Coexistence of α-thalassaemia in both HbAS and HbAC individuals resulted in decreased Hb, MCV, MCH and MCHC. Conclusions Structural haemoglobinopathy should be investigated in subjects belonging to ethnic groups with high prevalence of variant Hb and with a score of 3 or 4. Erythrocytes of HbAC subjects are smaller and denser than those of HbAS subjects.

Reticulocyte parameters of delta beta thalassaemia trait, beta thalassaemia trait and iron deficiency anaemia

Aims To analyse the differences in reticulocyte indices between delta beta thalassaemia trait (δβ-TT), beta thalassaemia trait (β-TT) and iron deficiency anaemia (IDA), and to correlate those differences with the physiopathological features of these three types of microcytoses. Methods We performed a descriptive study of 428 samples (43 δβ-TT, 179 β-TT and 206 IDA) that were run on Advia 2120 analyser (Siemens). The following reticulocyte indices were assessed: absolute reticulocyte count (ARC), percentage of reticulocytes, mean corpuscular volume of reticulocytes (MCVr), haemoglobin content of reticulocytes (CHr), mean corpuscular haemoglobin concentration of reticulocytes, red blood cell distribution width of reticulocytes (RDWr), haemoglobin distribution width of reticulocytes (HDWr) and reticulocyte subpopulations based on their fluorescence according to mRNA (low (L-R), medium (M-R) and high (H-R)), MCV ratio and MCHC ratio. Correlation between fetal haemoglobin (HbF) and RDWr in patients with thalassaemia was evaluated. Results RDWr was significantly higher in δβ-TT compared with β-TT (15.03% vs 13.82%, p<0.001), and so were HDWr (3.65% vs 3.27%, p<0.001), CHr (23.68 vs 22.66 pg, p<0.001) and MCVr (88.3 vs 85.5 fL, p<0.001). A good correlation was observed between HbF and RDWr (r=0.551, p<0.001). IDA subjects have more immature reticulocytes, but less ARC than β-TT, suggesting a certain degree of inefficient erythropoiesis in IDA in comparison with β-TT. Conclusions Previously described differences between δβ-TT, β-TT and IDA in the corpuscular indices of mature red blood cell can also be observed in reticulocytes. The degree of anisocytosis in reticulocytes from patients with thalassaemia is correlated with HbF.

Type B Niemann–Pick disease

A 39-year-old man was referred to our centre with hepatosplenomegaly that had been present since childhood. He was asymptomatic and his full blood count showed only mild thrombocytopenia (platelet count 123 9 10/l). A blood film was normal. A bone marrow aspirate revealed hyperplasia of foamy and, especially, sea-blue histiocytes (images), with haemophagocytosis. The activity of acid sphingomyelinase from dried blood was below the normal range (54 pmol/ spot*20 h, normal range 200–3500) in leucocytes, leading to the diagnosis of type B Niemann–Pick disease (NPD). Acid lipase activity was within the normal range (0 56 nmol/ spot*3 h, normal range 0 10–2). Molecular analysis showed compound heterozygosity for two mutations in SMPD1 (p.Tyr469Ser/p.Arg610del). No mutation was detected in NPC1 or NPC2. NPD is a sphingolipidosis that includes at least three different disease types (A, B, C), all inherited by autosomal recessive transmission. Complete and partial deficiency of the lysosomal enzyme acid sphingomyelinase (ASM) is responsible for NPD types A and B, respectively. Type A is the most common form in which symptoms begin shortly after birth, with hepatosplenomegaly, failure to thrive, neurological impairment and cherry red retinal spots. Type B, the nonneuropathic form, is a relatively benign disorder with splenomegaly, thrombocytopenia, sea-blue histiocytes in the bone marrow and, rarely, hepatomegaly and pulmonary infiltrates; survival to adulthood is frequent and its incidence is believed to be underestimated. Type C is due to mutations in NPC1, which cause a massive lysosomal accumulation of cholesterol leading to progressive neurological impairment in early childhood. As NPD type C produces a secondary reduction of ASM activity, all three types are considered forms of the same disease. Morphological features of NPD are undistinguishable from those of acid lipase deficiency. Although demonstration of decreased specific enzymatic activity is mandatory, the diagnosis of lysosomal storage disorders can be guided by the morphological findings in bone marrow films.

Cryofibrinogenaemia in a patient with multiple myeloma and necrotizing vasculitis.

A 38-year-old man presented with arthritis affecting both knees and feet, palpable purpura with necrotizing lesions, livedo reticularis, asymmetric multiple axonal mononeuritis involving superficial peroneal nerves, exudative rhinitis, crystalline keratopathy, a serum monoclonal immunoglobulin (Ig) G kappa (24 g/l) and Bence-Jones proteinuria. A bone marrow aspirate showed 12% plasma cells. Due to the absence of lytic lesions, anaemia, hypercalcaemia and renal insufficiency, a diagnosis of IgG kappa Bence-Jones smouldering multiple myeloma was made. A peripheral blood film revealed bluish amorphous “cotton-like” particles between red cells and basophilic cytoplasmic inclusions with compression of the nucleus in neutrophils (top) that disappeared when the blood was warmed to 37°C, suggesting the presence of cryoglobulins. A skin biopsy showed small vessel leucocytoclastic vasculitis, obstruction of small and medium vessels and numerous thrombi suggesting cryoglobulin deposits. Direct immunofluorescence did not reveal immune deposits. Cryoglobulin determinations were repeatedly negative. Serology for hepatitis B, hepatitis C and human immunodeficiency virus was negative. Tests for autoantibodies including rheumatoid factor were also negative. Finally, a cryoprecipitate was detected in the patients’ plasma but not in the serum, so a diagnosis of cryofibrinogenaemia associated with multiple myeloma was made. No response of the skin lesions was observed with methotrexate, prednisone and cyclophosphamide (bottom left). However, after five sessions of plasmapheresis, remarkable clinical improvement of the skin ulcers (bottom right) and normalization of axonal mononeuritis were observed. Bortezomib, thalidomide and dexamethasone (VTD) chemotherapy was started, achieving complete remission after six courses. Autologous stem cell transplantation was then performed. Cryofibrinogenaemia can be associated with infection, malignancy, connective tissue disorders and autoimmune conditions. Skin lesions are usually the first clinical manifestation. Several cases of cryofibrinogenaemia and multiple myeloma have been described.

Gaucher disease and myelodysplastic syndrome with isolated del(5q)

An 80-year-old woman presented with asthenia of several weeks duration. She had been diagnosed with Gaucher disease 34 years earlier (N370S/N370S genotype, acid beta-glucosidase activity 18%, serum chitotriosidase levels: 1557 nmol/ml/h). She had been asymptomatic since then, with only mild splenomegaly, and no treatment had been required. A full blood count showed macrocytic anaemia (haemoglobin concentration (Hb) 109 g/l, MCV 122 fl) and a platelet count of 122 9 10/l. Serum vitamin B12 was reduced (112 pg/ml) and vitamin B12 therapy was therefore initiated. In spite of the normalization of platelet count and vitamin B12 levels, her Hb decreased and several red blood cell transfusions were needed. A bone marrow aspiration and trephine biopsy were therefore performed. A May-Gr€ unwald-Giemsa stain revealed numerous Gaucher cells and megakaryocytes with hypolobated nuclei (images). No other dysplastic features were found. Karyotype showed deletion of 5q in one out of 21 metaphases, and fluorescent in situ hybridization (FISH) for 5q deletion was positive in 51% and 66% of analysed nuclei in bone marrow and peripheral blood respectively. FISH for monosomy 7, trisomy 8, 20q deletion and 17p deletion yielded negative results, confirming a myelodysplastic syndrome with isolated del(5q) Treatment with lenalidomide (5 mg/day, days 1–21 every 28 days) was initiated. The patient is asymptomatic after three months of treatment, with Hb of 125 g/l, MCV 98 fl, leucocyte count 2 56 9 10/l and platelet count 88 9 10/l. It is well known that the incidence of cancer is increased in patients with Gaucher disease, especially multiple myeloma. Association with myelodysplastic syndromes is rare.

Azurophilic inclusions in lymphocytes and plasma cells: a case of Sanfilippo disease.

An 11-year-old boy was referred to our centre because of dementia, hypertrichosis and coarse facies. His facial features had begun to change at the age of 18 months and he had started to loose previously acquired skills with progressive loss of speech and walking ability. A peripheral blood film showed azurophilic inclusions in lymphocytes (left). Alpha-Liduronidase and iduronate-2sulfatase activity were tested on dried blood spots, but normal activity of both enzymes was found. A bone marrow aspirate revealed azurophilic inclusions in all of the plasma cells (right). Absence of heparan N-sulfatase was demonstrated in leucocytes, leading to the diagnosis of mucopolysaccharidosis (MPS) IIIA, also known as Sanfilippo syndrome. Sanfilippo syndrome is an autosomal recessive metabolic disease caused by an absence or malfunction of certain enzymes needed to break down glycosaminoglycans. Deficiency of heparin N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoAlpha-glucosaminide acetyltransferase and N-acetylglucosamine 6-sulfatase lead to Sanfilippo A, B, C and D respectively. Consequently, heparan sulphate accumulates inside the lysosomes of lymphocytes and plasma cells, interfering with their function. Azurophilic inclusions in the shape of dots or commas in lymphocytes, in this clinical setting and with no other morphological features, are characteristic of MPS I, II and III (Hurler, Hunter and Sanfilippo syndromes, respectively). However, if bone marrow plasma cells show the same inclusions the clinical suspicion can only be of MPS III. Cytology of peripheral blood and bone marrow can play a key role in the diagnosis of lysosomal storage diseases, as it can indicate which enzymes should be investigated.

Dehydrated red blood cells in a peripheral blood film: a case of hereditary xerocytosis

A 20-year-old Moroccan woman was referred to a haematologist because of chronic haemolytic anaemia with mild splenomegaly and no transfusion requirement. A full blood count, performed on an Advia 2120i analyser (Siemens Medical Solutions Diagnostics, Tarrytown, NY, USA), showed RBC 2 91 9 10/l, haemoglobin concentration 98 g/l, haematocrit 0 266 l/l, MCV 91 4 fl, MCH 33 5 pg, MCHC 394 g/l, red cell distribution width 20 6%, reticulocyte count 934 3 9 10/l and reticulocyte haemoglobin content 35 2 pg, with normal leucocyte and platelet counts. The volume/haemoglobin concentration erythrogram showed 43 1% hyperchromic red cells (top left). Serum unconjugated bilirubin was 27 7 lmol/l, haptoglobin <75 6 mg/l and lactate dehydrogenase 539 u/l. Direct and indirect Coombs tests were negative. A peripheral blood film revealed polychromatophilia, echinocytes, eccentrocytes and frequent irregularly contracted cells, consistent with dehydrated cells (top right and bottom). Pyruvate kinase and glucose 6-phosphate dehydrogenase concentrations were within normal limits. Neither variant haemoglobins nor abnormal globin chains were identified by ion exchange chromatography or capillary electrophoresis. An isopropanol test was negative. Expression of CD16, FLAER and CD24 by neutrophils and of CD14 and FLAER by monocytes was normal on flow cytometry. Osmotic resistance was abnormally increased, as was autohaemolysis, which was corrected by glucose and ATP. Intracellular potassium was abnormally low at 80 mmol/l (control 100 8 mmol/l) whereas intracellular sodium was increased at 23 5 mmol/l (control 10 3 mmol/l) and intracellular H2O content was 64% (control 77%). A diagnosis of hereditary xerocytosis was therefore made. Hereditary xerocytosis is an autosomal dominant red cell membrane defect characterized by dehydration of erythrocytes and well compensated haemolytic anaemia due to an abnormally increased potassium efflux. Mutations in the PIEZO1 gene, which encodes a mechanosensitive ion channel component, have been recently described. Morphology of peripheral blood and automated detection of hyperchromic erythrocytes can play a key role in the diagnosis of this condition.