Diagnosis and clinical management of red cell membrane disorders

Abstract

Heterogeneous red blood cell (RBC) membrane disorders and hydration defects often present with the common clinical findings of hemolytic anemia, but they may require substantially different management, based on their pathophysiology. An accurate and timely diagnosis is essential to avoid inappropriate interventions and prevent complications. Advances in genetic testing availability within the last decade, combined with extensive foundational knowledge on RBC membrane structure and function, now facilitate the correct diagnosis in patients with a variety of hereditary hemolytic anemias (HHAs). Studies in patient cohorts with well-defined genetic diagnoses have revealed complications such as iron overload in hereditary xerocytosis, which is amenable to monitoring, prevention, and treatment, and demonstrated that splenectomy is not always an effective or safe treatment for any patient with HHA. However, a multitude of variants of unknown clinical significance have been discovered by genetic evaluation, requiring interpretation by thorough phenotypic assessment in clinical and/or research laboratories. Here we discuss genotype-phenotype correlations and corresponding clinical management in patients with RBC membranopathies and propose an algorithm for the laboratory workup of patients presenting with symptoms and signs of hemolytic anemia, with a clinical case that exemplifies such a workup.

Graphical abstract

Figure 1.

HS. (A)Ektacytometry of blood samples from the patient before and after splenectomy and from his parents. The patient had AR HS due to compound heterozygosity of SPTA1 c.4295del (p.L1432*), shared with the mom, and SPTA1 c.4339-99C>T, shared with the dad. The dad and the proband were also found to carry the PIEZO1 VUCS c.6205G>A (p.Val2069Met). The parents had a normal ektacytometry with no evidence of xerocytosis for the father, offering reassurance that the PIEZO1 VUCS is benign, while the patient after splenectomy had a typical HS curve. Before splenectomy, the patient was chronically transfused. Ektacytometry at that time was performed at a nadir before the next transfusion and 2 months after a previous one. Transfused RBCs have obviously modified the curve, giving a falsely normal Omin (ie, the hypotonic osmolality where the elongation index is minimal). The value of Omin provides information on the initial surface-to-volume ratio of the cell sample. A shift to the right reflects a decrease in the surface area/volume ratio and corresponds to increased osmotic fragility. (B) Blood smear of the patient about 18 months post partial splenectomy before starting to require transfusions again, demonstrating significant anisopoikilocytosis and polychromasia as well as many spherocytes, typical for AR SPTA1-associated HS. (C) Blood smear of the patient 5 years after total splenectomy, stable and without need of transfusions since the time of that surgery. Moderate anisopoikilocytosis and several spherocytes are still noted. Scale bar = 14 mm. Details on the ektacytometry assay and parameters can be found at https://www.cincinnatichildrens.org/service/c/cancer-blood/hcp/clinical-laboratories/erythrocyte-diagnostic-lab/ektacytometry.

Figure 2.

CDA-II is in the differential diagnosis for HS. A 4-year-old African American girl presented with DAT-negative, mild hemolytic anemia (Hgb, 10 g/dL) with a reticulocyte count of 2.4%, a normal ARC of 86 × 10³/µL, and mild jaundice. She had a history of prolonged neonatal jaundice treated with phototherapy and no transfusion requirement. Ektacytometry showed a curve that resembled HS, and a blood smear showed spherocytes, marked poikilocytosis, and no polychromasia. Inadequate reticulocytosis and a ferritin of 80 ng/mL, at a generous level for her age with no concurrent inflammation, triggered further evaluation with sequencing of an HHA gene panel that revealed 2 SEC23B mutations, c.40C>T (p.R14W) and c.367-3A>G. Follow-up targeted sequencing of her parents confirmed that these 2 variants were in trans, causing CDA-II. Scale bar = 14 mm.

Figure 3.

Proposed algorithm for laboratory workup of a patient presenting with hemolysis with or without anemia. In many cases of mild HS and most cases of PIEZO1-associated HX, anemia may be well compensated by reticulocytosis. Although our focus here is on RBC membrane disorders, the differential includes other causes of hemolytic disorders that are mentioned in this algorithm. Evaluation for autoimmune or, especially in an infant, alloimmune hemolytic anemia with DAT and IAT is the first testing recommended since such a diagnosis is frequently acute and evolving, requiring immediate action. Of note, warm autoimmune hemolytic anemia in children and occasionally adults with underlying immune dysregulation may be atypical and conventionally DAT-negative. Consideration should also be given to the possibility of MAHA, PNH, and Wilson disease. The cases described in this review, especially that presented in Figure 6, demonstrate utilization of this algorithm. A blood smear review of the patient and parents and attention to the RBC indices including MCV, MCHC, and RDW, along with hemolytic markers (unconjugated bilirubin, lactate dehydrogenase, haptoglobin—of note, haptoglobin is reliable after 6 months of life since earlier it may be low due to decreased production by the infant's liver rather than increased consumption) and ferritin and transferrin saturation to consider iron-loading inefficient erythropoiesis, can provide hints as to the differential diagnosis. In a non-chronically transfused patient, we suggest phenotypic evaluation considering the differential of globin disorders, followed by RBC membranopathies and enzymopathies. Rare causes of HHA such as unstable Hgb disorders and CDAs also need to be considered. When suboptimal reticulocytosis or iron overload or skeletal abnormalities are noted in a patient with hemolytic anemia, the possibility of CDA should be considered and pursued. The combination of blood smear review and osmotic gradient ektacytometry frequently helps to narrow the differential while alerting clinicians of rare possibilities. Osmotic gradient ektacytometry evaluates the deformability of RBCs as they are subjected to constant shear stress in a medium of increasing osmolality in a laser diffraction viscometer and is the reference technique for differential diagnosis of erythrocyte membrane and hydration disorders when a recent transfusion does not interfere with phenotypic evaluation of the patient. Flow cytometry with eosin-5′-maleimide binding of band 3 and Rh-related proteins is a rapid screening test for RBC membrane disorders characterized by membrane loss. Osmotic fragility is increased in HS and expected to be decreased in HX (however, it is reported as normal in patients with KCNN4 Arg352His mutation). When the patient is recently or chronically transfused, as is typically the case for infants with HHA, genetic evaluation with clinically available NGS panels or research-based whole-exome sequencing or whole-genome sequencing may provide an accurate diagnosis necessary for appropriate management decisions. Laboratories that offer sequencing on genes or panels associated with HHAs and red cell membrane disorders can be found by searching the Genetic Testing Registry: https://www.ncbi.nlm.nih.gov/gtr/. e.g. https://www.ncbi.nlm.nih.gov/gtr/all/tests/?term=RBC%20membrane%20disorders. aHUS, atypical hemolytic uremic syndrome; HUS, hemolytic uremic syndrome; MAHA, microangiopathic hemolytic anemia; PNH, paroxysmal nocturnal hemoglobinuria; RDW, RBC distribution width; TTP, thrombotic thrombocytopenic purpura.

Figure 4.

Transient infantile HPP. An African American girl presented within the first day of life after delivery at term with nonimmune hemolytic anemia and neonatal jaundice. She was treated with triple phototherapy but did not require RBC transfusion. (A) The blood smear of the patient with HPP showing marked anisocytosis and poikilocytosis with bizarre microcytes and fragmented cells along with elliptocytes (scale bar = 14 µm). (B) The blood smear of the mother, who had a normal Hgb and reticulocyte count, was notable for elliptocytes indicating HE. (C) Ektacytometry showed the trapezoid curve characteristic for HE and HPP with decreased deformability for both mother and patient. The patient's sample was sequenced by NGS on an RBC membrane gene panel, which revealed the SPTA1 HE-causing variant c.460_463insTTG (p.L155dup) as well as the SPTA1 c.6531-12C>T polymorphism known as α^LELY. Targeted sequencing for these variants revealed that the mother was heterozygous for SPTA1 p.L155dup. The mother did not carry α^LELY, which was presumably inherited by the father. The compound heterozygosity of an HE-causing SPTA1 mutation in trans to α^LELY confirmed the diagnosis of infantile HPP. The child continued to have a mild anemia with reticulocytosis (Hgb, 10.2 g/dL with 3.8% reticulocytes) and poikilocytosis, with RDW up to 18.7% at her 2-year-old follow-up, but a CBC with reticulocyte count and blood smear at 5 years of age indicated transition to a nonhemolytic HE phenotype. RDW, RBC distribution width.

Figure 5.

Persistent (true) HPP. A boy of Iranian descent presented with severe anemia since infancy. He remained transfusion dependent; therefore, his erythrocyte phenotype was not evaluable. (A) A blood smear of the father (practically identical with the blood smear of the mother) and (B) ektacytometry showing a trapezoid shape typical for HE for both parental samples indicated that they both had elliptocytosis. NGS on a panel of RBC membrane disorder genes revealed that the patient was homozygous for a novel missense mutation in the SPTB gene (c.6040T>G, p.F2014V), affecting the spectrin self-association site. Both parents were heterozygous for the same mutation.

Figure 6.

HX due to heterozygousPIEZO1 mutation. A European American girl, born at term, developed neonatal hyperbilirubinemia that prevented discharge from the nursery for 4 days while receiving treatment with phototherapy. Scleral icterus was again noted at 10 months of age, and a CBC indicated reticulocytosis but a normal Hgb. She continued to have chronic hemolysis and jaundice and had an extensive workup including negative DAT, normal Hgb electrophoresis, RBC enzyme activity testing showing normal or increased activity, osmotic fragility testing that was (probably mistakenly) reported as normal rather than as decreased, alpha and beta globin genetic testing that revealed no mutation or copy number variation, a negative PNH screen by flow cytometry, and bone marrow studies showing erythroid hyperplasia, no dyserythropoiesis, and markedly increased iron stores. (A) At 10 years of age, she had osmotic gradient ektacytometry performed that demonstrated a typical HX curve, with left shift due to decreased Omin and Ohyp (Figure 2A). The CBC at the time indicated an Hgb of 14.2 g/dL, an MCV of 96 fl, an MCH of 36.5 pg, an MCHC of 37.9 g/dL, a reticulocyte count 16.9%, and an ARC of 643 × 10³/µL. (B) A blood smear was significant for polychromasia, macrocytosis, and occasional stomatocytes, target cells, and dense fragmented cells. Of note, sometimes the blood smear may be deceptively normal, with only a few target cells and no stomatocytes. A determination of the RBC intracellular cation confirmed a reduced K⁺ content without a corresponding increase in Na⁺ content. NGS for an RBC membrane disorder panel revealed 1 of the most common PIEZO1 variants (c.7367G>A, p.R2456H) causing HX. PNH, paroxysmal nocturnal hemoglobinuria.