Hematopathology Case Study: A 7 Year Old Transplant Patient with Neck Swelling

A 7 year old male with a history of restrictive cardiomyopathy status-post orthotopic heart transplant in June, 2010 that was on maintenance doses of tacrolimus and mycophenolate mofetil presented to his primary pediatrician left neck swelling. Starting in January 2017, the patient began with neck pain and swelling in the context of a recent gastrointestinal illness. Per CT report of the neck, a rim enhancing well-defined suppurative level III lymph node measuring 1.4 x 1.2 x 2.1 cm with adjacent soft tissue inflammatory changes extending into the left parapharyngeal space was identified. The patient was subsequently started on antibiotics and was discharged home with some improvement of swelling and pain.

The patient then presented again with continued neck swelling, although painless this time, and the patient’s cardiologist was contacted, who recommended a decrease in tacrolimus dosing. An otolaryngology evaluation was requested and given the concerning findings, the patient was admitted for further work-up, including a biopsy with a lymphoma protocol.

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BCL6
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BCL2
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EBER

 

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Flow Cytometry

 

Results

Flow cytometry revealed a kappa restricted CD10 positive mature B-cell population.

On biopsy examination, a population of monotonous lymphoid cells that are large in size with round to mildly irregular nuclear contours, open chromatin, and multiple inconspicuous nucleoli are present in a diffuse pattern. Abundant apoptotic bodies and mitotic figures are noted and occasional “starry sky” features are present. By immunohistochemistry, BCL6 highlights the neoplastic lymphocytes while BCL2 highlights background T-cells. EBER is negative.

Overall, despite a negative t(8;14) IGH/MYC translocation, the findings are best considered to be of an EBV-negative post-transplant lymphoproliferative disorder with morphologic features consistent with Burkitt lymphoma.

Discussion

Post-transplant lymphoproliferative disorders (PTLD) are a relatively rare complication in a variety of transplants that occurs in 2-10% of post-transplant patients. Overall, following a solid organ transplant (SOT), PTLD development is 1-5% of recipients with the highest incidence in intestinal and multivisceral transplantations (5-20%). Another factor is EBV status of the recipient, for which those that are EBV-naïve and lack cellular immunity to EBV are susceptible to graft-mediated EBV infection and ultimately developing an increased incidence in early PTLD. This population is overrepresented by pediatric transplant recipients1.

The presentation is highly variable and ranges from benign proliferations to overt lymphoproliferative disorders. Classifications for PTLD include early lesions, which are oligo- or polyclonal proliferations of EBV positive B cells have either a predominant infectious mononucleosis-like proliferation or a plasmacytic hyperplasia form. Polymorphic PTLD is a similar concept to the early proliferative lesions but the host architecture of the native structure is disrupted. Lastly, monomorphic PTLD is an entity that fulfills criteria for a non-Hodgkin lymphoma and is diagnosed according to the criteria of non-transplant associated lymphomas. Within pediatric registry studies, monomorphic PTLD accounts for 35-83% of all PTLD cases. B-cell lymphomas, particularly DLBCL, comprise the vast majority of monomorphic PTLD with plasmacytoma and T-cell lymphoproliferative disorders much less common2.

In this particular case, with the patient having been 7 years post-transplant and negative studies for EBV present, it is not surprising that germinal center phenotypic markers are highly expressed, such as CD10 and BCL6, which has been well elucidated by Jagadeesh, et al. Although not many genetic studies have been performed on post-transplant B-cell lymphomas, regardless of EBV status, there is some data demonstrating trisomies of 9 and/or 11 with translocations 8q24.1 (C-MYC), 3q24 (BCL6), and 14q32 (IGH). Rinaldi et al. noticed a lack of genetic lesions characteristic of postgerminal center derivation, such as gain of chromosome 3 (FOXP1, BCL6, and NFKBIZ) and 18q (BCL2 and NFATC1) together with losses of 6q (PRDM1 and TNFAIP3) in post-transplant DLBCL.  A number of DNA mutations have also been described including genes associated with somatic hypermutation (SHM) such as PIM-1, PAX5, C-MYC, and RhoH/TTF. These particular mutations are also found to be independent of EBV status1.

Overall, post-transplant lymphoproliferative disorders occur in a variety of transplant settings across many age groups and can be dependent on EBV and CMV status as well as the type and degree of immunosuppression. Although many variations take place in PTLD, patients with the monomorphic type are diagnosed according to their non-transplant counterparts. Current perspective includes further analysis of molecular and cellular mechanisms incorporated into research projects, which could better aid in prognostic implications and future therapeutics.

  1. Morscio, et al. “Molecular pathogenesis of B-cell posttransplant lymphoproliferative disorder: What do we know so far?” Clinical and Developmental Immunology 2013.
  2. Mynarek, et al. “Posttransplant lymphoproliferative disease after pediatric solid organ transplantation,” Clinical and Developmental Immunology 2013.

 

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-Phillip Michaels, MD is a board certified anatomic and clinical pathologist who is a current hematopathology fellow at Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. His research interests include molecular profiling of diffuse large B-cell lymphoma as well as pathology resident education, especially in hematopathology and molecular genetic pathology.

Sickle Cell Anemia

Sickle cell disease is a blood cell disorder that affects a lot of Americans, especially those of African descent. The disease is a result of a genetic defect that changes the structure of hemoglobin. This alteration in hemoglobin causes normally round red cells to become sickle in shape as well as sticky and deformed. As a result, these deformed red cells block perfusion of red blood cells into circulation and vital organs and affects the vasculature resulting in severe pain, organ damage and sometimes stroke.

Management of sickle cell disease includes blood transfusion, hydorxyurea and pain management. The long-term administration of hydroxyurea to stimulate the production of fetal hemoglobin is a form of therapy that provides relief in that fetal hemoglobin essentially protects the red cells from sickling. The transfusion of red cells lowers the amount of sickle hemoglobin levels.

Transplantation of hematopoietic stem cells from HLA-identical siblings can be curative in several nonmalignant hematologic disorders, including aplastic anemia, β-thalassemia major, congenital immunodeficiency disorders, and certain inborn errors of metabolism. Pilot studies of bone marrow transplantation for the treatment of young patients with symptomatic sickle cell disease have demonstrated eradication of the underlying disease with low transplantation-related mortality (Bhatia and Walters, 2007).

The process for successful bone marrow transplant to cure sickle cell involves administration of chemotherapy or immunosuppressive drugs to eradicate all the cells.  Now, doctors have developed a more successful regimen where patients take immunosuppressive drugs with a low dose body irradiation, a treatment much less harsh than chemotherapy. Next, donor cells from a healthy and tissue-matched sibling are transfused into the patient. Stem cells from the donor produce healthy new blood cells in the patient, eventually in sufficient quantity to eliminate symptoms. In many cases, sickle cells can no longer be detected. Patients must continue to take immunosuppressant drugs for at least a year.

In the reported trial, published online in the journal Biology of Blood & Marrow Transplantation, physicians from the University in Illinois Chicago transplanted 13 patients, 17 to 40 years of age, with a stem cell preparation from the blood of a tissue-matched sibling. In a further advance of the NIH procedure, the physicians successfully transplanted two patients with cells from siblings who matched but had a different blood type (Parmet, 2015)

Stem cell transplantation has proven itself to provide cures for a lot of hematologic malignancies, now it is successfully finding its way to cure other hematologic abnormalities, including sickle cell anemia.  This continued advancement in medicine will provide relief to a lot of patients who are suffering from sickle cell disease.

References

  1. Bhatia, M., & Walters, M. C. (2007). Hematopoietic cell transplantation for thalassemia and sickle cell disease: past, present and future. Bone Marrow Transplantation, 41(2), 109-117. doi:10.1038/sj.bmt.1705943
  1. Parmet, S. (2015). Adults with sickle cell disease cured with stem cell transplants. Retrieved March 25,2017, from https://news.uic.edu/cure-for-sickle-cell

 

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-Carlo Ledesma, MS, SH(ASCP)CM MT(ASCPi) MT(AMT) is the program director for the Medical Laboratory Technology and Phlebotomy at Rose State College in Midwest City, Oklahoma as well as a technical consultant for Royal Laboratory Services. Carlo has worked in several areas of the laboratory including microbiology and hematology before becoming a laboratory manager and program director.

Interference in Lab Assays

A 69 year old patient with cirrhosis presented to the ER with fever. Her bilirubin was markedly elevated at 7.4 g/dl and her hemoglobin and hematocrit were measured at 13.4 g/dl and 35.6% respectively with a MCV of 103.2 fl and MCH of 38.5 pg. The next day her H/H were 11.9 g/dl and 31.3 % respectively. While her hemoglobin one day later was 11.9 g/dl, the reported hematocrit was 39.3%. Patient had a bilirubin level of 8.7 g/dl at this time.

The fluctuating numbers together with the discrepancy between hemoglobin and hematocrit over a very short period of time was concerning. We realized that presence of markedly icteric plasma was responsible for these discordant values. Saline replacement and spun crit were performed in order to correct interference by bilirubin. Subsequent measurements of H/H revealed hemoglobin in the range of 12.9 g/dl with a hematocrit of 38% and a MCV of 113 fl. As the bilirubin levels started dropping (in the range of 6.5 g/dl) the hemoglobin level measured by the analyzer fell in the range of 10.3 to 11 g/dl. The instrument (XN-200) gave no error codes and therefore we were able to report out the analyzer results without correction. It was however very important to convey to the clinical team that the H/H values did not truly represent a fall from the previous values. As the two methodologies were different (spun crit and plasma replacement was being no longer performed) the numbers should be interpreted accordingly. Patient was not bleeding actively and did not require any blood transfusion.

Interference occurs when a substance or process falsely alters an assay result. Interferences are classified as endogenous or exogenous. Endogenous interference originates from substances present in the patient’s own specimen. Exogenous interferences are substances introduced into the patient’s specimen. Interference from hemolysis, icterus and lipemia are most frequently studied. Protein interferences are most often associated with paraproteins and predominantly with IgM or IgG and rarely with IgA. Drug interference may be due to the parent drug, metabolite(s) or additives in the drug preparation. Determining if interference is significant requires deviation limits from the original result. Once interferences are identified there is a need to establish procedures for handling affected results as part of the quality system.

Hemoglobin is quantified based on its absorption characteristics. Conditions such as hyperlipidemias, hyperbilirubinemia, a very high white blood cell count, and high serum protein can interfere with this measurement and result in falsely elevated hemoglobin values. When the values of hemoglobin, red cell count, and MCV are affected, MCH and MCHC also become abnormal, since these indices are calculated and are not directly measured. Sometimes a set of spurious values may be the first clue to an otherwise unsuspected clinical condition (e.g., the combination of low hematocrit, normal hemoglobin, and high MCV and MCHC is characteristic of cold agglutinins).

Although one must pay attention to very high amounts of bilirubin within the plasma, most hematology analyzers do not presently demonstrate any interference with bilirubin, at least for concentrations up to 250 mg/l. Above these values attention is however needed.

High serum or plasma bilirubin concentrations can cause spectral interference with assays near the bilirubin absorbance peak of ~ 456 nm. Chemical interference e.g. with peroxidase-catalysed reactions may also occur.

 

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-Neerja Vajpayee, MD, is the director of Clinical Pathology at Oneida Health Center in Oneida, New York and is actively involved in signing out surgical pathology and cytology cases in a community setting. Previously, she was on the faculty at SUNY Upstate for several years ( 2002-2016) where she was involved in diagnostic work and medical student/resident teaching.

Hematopathology Case Study: A 54 Year Old Male with Acute Onset of Progressive Neck Swelling

Case History

A 54 year old male with a diagnosis of HIV (last CD4 count was 301 on 11/2016) currently on HAART presented to the Beth Israel Deaconess Medical Center (BIDMC) ED on 2/28/2017 with an acute onset of progressive neck swelling over the course of 4-5 days. Laboratory values on presentation was significant for a LDH of 1061 IU/L. Other laboratory values were stable. Upon CT imaging with contrast of the neck, an extensively necrotic right cervical lymphadenopathy was present and was extending into the supra- and infraclavicular chain. No mediastinal or hilar lymphadenopathy was noted.

On 3/1/2017, the patient underwent an ultrasound guided core needle biopsy of the right cervical mass (see images). By immunohistochemistry, the neoplastic cells are positive for CD138 and MUM1. PAX5 shows dim and heterogeneous staining in a subset of cells while CD79a highlights a minor component of the lymphoid population. CD3 and CD5 are positive in T-cells occupying a small subset of the lymph node. CD20, BCL2, BCL6, BCL1, CD30, CD56 and HHV8 are negative. By Ki-67 immunostaining, the proliferation index approaches 100%. In-situ hybridization for Epstein-Barr virus encoded RNA (EBER ISH) is positive in a major subset of cells.

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CD20 (left) and CD3
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MUM1 (left) and CD138
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EBER ISH (left) and Ki-67

By cytogenetic analysis, only two cells were available for metaphase interpretation and it showed a translocation between the long arms of chromosomes 8 and 14 and by FISH, a t(8;14)(q24.1;q32) was noted, indicating an IGH/MYC rearrangement.

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Overall, the morphologic, immunophenotypic, and cytogenetic findings in conjunction with the clinical features of a HIV positive male and EBV association, the diagnosis is in keeping with a plasmablastic lymphoma.

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Discussion

Plasmablastic lymphoma is a diffuse proliferation in which the cells resemble immunoblasts but share an immunophenotype similar to that of plasma cells. First described in the oral cavity, especially among HIV infected patients, it can present in a variety of extranodal sites, such as skin, soft tissue, and gastrointestinal tract. Although uncommon, plasmablastic lymphoma has its highest incidence among HIV infected individuals. Most patients are at stage III or IV at presentation with an intermediate to high risk IPI score. The tumor cells of plasmablastic lymphoma are invariably infected by Epstein-Barr virus (EBV) and are consistently negative for HHV8. According to Balague et al.2, up to 39% of plasmablastic lymphomas demonstrate a MYC translocation, all of which involved the IGH gene. Generally, plasmablastic lymphoma displays a complex karyotype, although some cases display an isolated MYC rearrangement without a complex karyotype. Taddesse-Heath et al.3 has shown a small cohort that is positive for gains in odd-numbered chromosomes 3, 5, 7, 9, 11, and/or 15, similar to that seen in plasma cell myeloma. The clinical course of plasmablastic lymphoma is quite aggressive with most patients dying within one year after diagnosis. Current first line treatment for plasmablastic lymphoma is dose-adjusted EPOCH with or without bortezomib, intrathecal prophylaxis, and possible autologous stem cell transplantation in first remission candidates. Future directions of therapy include chimeric antigen receptor (CAR) T-cells and small molecular inhibitors against the MYC bromodomain4.

References

  1. Swerdlow, S., et al., WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th. ed., IARC press: 2008
  2. Balague, O., et al., “Plasmablastic lymphomas are genetically characterized by frequent MYC translocations [abstract],” Mod Pathol 2009; 22:255A.
  3. Taddesse-Heath, L., et al., “Plasmablastic lymphoma with MYC translocation: evidence for a common pathway in the generation of plasmablastic features,” Mod Pathol 2010; 23:991-999.
  4. Castillo, J., et al., “The biology and treatment of plasmablastic lymphoma,” Blood 2015; 125:2323-2330.

 

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-Phillip Michaels, MD is a board certified anatomic and clinical pathologist who is a current hematopathology fellow at Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. His research interests include molecular profiling of diffuse large B-cell lymphoma as well as pathology resident education, especially in hematopathology and molecular genetic pathology.

Automated Body Fluid Cell Counts

Body fluid cell count has been part of the hematology laboratory and remains a time-consuming manual task for technologists. The cell count test provides valuable information to clinicians in the diagnosis and treatment of a various medical conditions. Albeit the diagnostic prowess of cell counts, there has also been an intrapersonnel variation in counts that proves the lack of precision among testing personnel. As laboratory professionals, we are trained that precision is important in the performance of cell counting procedures; therefore the implementation of automated body fluid counts will improve these quality parameters.

Automated methods for body fluid cell counts have been rapidly replacing manual hemacytometer methods. Advances in medical technology, especially in hematology instrumentation, have decreased the turnaround times and improved precision counts for body fluids. Technological advances in hardware and software engineering have developed instruments with expanded analytical capabilities that enable processing multiple specimen types including urine, CSF, peritoneal fluid, pleural fluid, synovial fluid, and lavages on a single analyzer.1 Most body fluid instruments like the Sysmex XE-5000 have analyzed body fluids easily and quickly. In a study published in Lab Medicine, the Sysmex XE5000 technology showed significant improvement in the ability of automated hematology analyzers regarding body fluid analysis.2 This technology provides counting nucleated cells in an acellular fluid(i.e. Cerebrospinal fluid). This technology also offers differential capabilities between mononuclear and polymorphonuclear cells, providing laboratory technologists and clinicians rapid, cellular differential analysis.

Laboratory technologists should not fear that their jobs will be replaced by these instruments. In fact, laboratory professionals should be enthused that it provides ease in their work, improves quality, decreases work load, and increases efficiency in their processes. The limitations that need to be considered in automated cell counts analyzers are the use of purulent specimens where the main concern is clogging the instrument’s flow cell apertures. Crystals in synovial fluids may cause a false increase in counts; in these cases, manual intervention in cell count may be warranted. Extremely clear fluids with low cell counts also limit the application of automated methods and may warrant manual analysis. Of important consideration as well is the microscopic review of cellular distribution when malignancy is of diagnostic consideration.

Modernization of laboratory equipment and analysis provide ease in operation from a management stand point but also efficiency, accuracy and precision in reporting of results. Automation of body fluid counts provides help to technologists and a rapid diagnosis tool for clinicians.

 

Reference

 

  1. Scott, G. (2014, June 9). An automated approach to body fluid analysis. Medical Laboratory Observer.

Williams, J., MD. (2011). Gaining Efficiency in the Laboratory – Automated Body Fluid Cell Counts: Evaluation of the Body Fluid Application on the Sysmex XE-5000 Hematology Analyzer . Lab Medicine, 42(7).

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Carlo Ledesma, MS, SH(ASCP)CM MT(ASCPi) MT(AMT) is the program director for the Medical Laboratory Technology and Phlebotomy at Rose State College in Midwest City, Oklahoma as well as a technical consultant for Royal Laboratory Services. Carlo has worked in several areas of the laboratory including microbiology and hematology before becoming a laboratory manager and program director.

Hematology Quiz: A Patient with Multiple Myeloma

What is the red cell phenomenon in this blood smear from a patient with multiple myeloma?

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  • Agglutination
  • Erythrocytosis
  • Polychromasia
  • Rouleaux
  • Anisocytosis

 

 

The red cell phenomenon in this blood smear is rouleaux. The massive amount of serum immunoglobulin in patients with multiple myeloma interferes with the normal repellent force between red cells, allowing the cells to pile up on top of each other in formations that resemble stacks of coins. Rouleaux formation can also occur in hyperproteinemia due to other causes, such as chronic inflammation or hyperfibrinogenemia of pregnancy, and it usually occurs artifactually in the thick end of any blood smear.

 

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-Kristine Krafts, MD, is an Assistant Professor of Pathology at the University of Minnesota School of Medicine and School of Dentistry and the founder of the educational website Pathology Student.

 

 

 

Stomatocytes: Inherited or Acquired?

A 40 year old patient presented with macrocytic anemia.

CBC Results:

  • WBC: 8.6 K/uL                     Normal
  • RBC: 3.60 M/uL                   Decreased
  • Hb : 13.1 g/dl                       Normal
  • MCV: 111.3 fl                        Increased
  • MCH: 36.4 pg                      Increased
  • MCHC:  32.7                        Normal
  • RDW:     15.5%                     Increased
  • Platelet: 360 K/uL              Normal

Review of peripheral smear showed several stomatocytes (Figures 1-3).

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Figure 1

 

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Figure 2
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Figure 3

Serum vitamin B12 and folate levels were normal and serum iron studies were consistent with anemia of chronic disease. Typically anemia of chronic disease is normocytic normochromic. Based on presence of numerous stomatocytes and macrocytosis (which can be seen in patients with hereditary stomatocytosis) it was recommended that patient be investigated for hereditary stomatocytosis and acquired causes of stomatocytosis.

STOMATOCYTES

“Stomatocyte” describes the appearance of red blood cells (RBCs) on the peripheral blood smear. Stomatocytes (also called hydrocytes) contain a mouthlike or slitlike pattern that replaces the normal central zone of pallor.

Mechanism of stomatocyte formation — When the normal biconcave disc becomes a uniconcave cup red blood cell (RBC) will appear as a stomatocyte on the peripheral blood smear. There are several mechanisms by which this change can occur:

  • In hereditary stomatocytosis (HSt), the mechanism of stomatocyte formation often involves changes in cell volume caused by reduced intracellular ion content.
  • In most cases of acquired stomatocytosis and rare inherited conditions that affect lipid metabolism formation often involves either a decrease in RBC membrane surface area or qualitative changes in the composition of the membrane lipid bilayer.

 

DISORDERS WITH STOMATOCYTES ON PERIPHERAL BLOOD

  1. Hereditary stomatocytosis (HSt) is an inherited autosomal dominant condition characterized by an excess of stomatocytes Patients have variable degrees of hemolysis and anemia. Hereditary stomatocytosis (HSt) is a rare disorder that presents with various degrees of hemolytic anemia and abnormal red blood cell (RBC) morphology. The genetic abnormalities responsible for these conditions remain incompletely characterized. Some of the defects associated with this condition involve membrane transporters such as Piezo1, Gardos, Rhesus antigen-associated glycoprotein, and the anion exchanger band 3.
  2. Several rare inherited defects affecting membrane lipid composition have been reported to have stomatocytosis on the peripheral blood smear.
  • Tangier’s disease
  • Rh null disease
  • Phytosterolemia
  1. Liver disease/medications. Stomatocytes can be seen with some acquired conditions such as chronic liver disease (most often due to alcoholism) or acute alcohol intoxication. The stomatocytosis with acute alcohol intoxication appears to be transient, and it may affect a significant proportion of RBCs. The mechanism is thought to be due to a reduction in RBC membrane surface area rather than an increase in RBC volume. Also, dministration of some medications can cause transient stomatocytosis. This was demonstrated in a study that demonstrated formation of stomatocytes upon exposure of RBCs to drugs like vinblastine and chlorpromazine. Intercalation of the drug into the inner half of the lipid bilayer may be responsible for creating the abnormal morphology.
  1. In some healthy individuals, stomatocytes occasionally can be found on the peripheral blood smear. This is thought to be due to a drying artifact; hence, it is important to evaluate several different areas of the peripheral smear before determining that a patient has circulating stomatocytes.

Hereditary stomatocytosis (HSt)

HSt can be completely asymptomatic or can present with chronic hemolytic anemia of varying severity. The age of presentation depends on the specific gene mutation, presence of other inherited conditions, and other environmental factors. The increasingly routine use of the complete blood count (CBC) in asymptomatic individuals has resulted in earlier diagnosis in some individuals who otherwise might never have come to medical attention. There does not appear to be a relationship between the degree of peripheral stomatocytosis on the blood smear and the severity of hemolytic anemia

The diagnosis of HSt is made by demonstrating the presence of anemia associated with the characteristic changes in RBC morphology (stomatocytosis) in conjunction with altered RBC indices and osmotic fragility. Genetic testing for PIEZO1 or Gardos channel mutations is confirmatory but not required.

The evaluation for HSt includes review of the complete blood count (CBC) and peripheral blood smear, which may show stomatocytes.The blood smear should be reviewed closely to ensure there are no abnormalities of white blood cells (WBCs) or platelets. The RBC indices typically show an increased mean corpuscular volume (MCV) in the range of up to 140 femtoliters (fL) and abnormally low or high mean corpuscular hemoglobin concentration.

 

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-Neerja Vajpayee, MD, is an Associate Professor of Pathology at the SUNY Upstate Medical University, Syracuse, NY. She enjoys teaching hematology to residents, fellows and laboratory technologists.