Month: June 2023

Case Studies in Hematology: Hemoglobin S Beta Thalassemia Compound Heterozygosity in a 61 Year Old Female

A 61 year old Black woman with a diagnosis of sickle cell beta thalassemia presented to the ER with fatigue, dyspnea, and back and leg pain with some swelling in the hands and feet. Splenomegaly was noted on exam. The patient has a history of moderate to severe symptomatic anemia and is being followed by a hematologist. Her baseline Hgb is 9-10 g/dL. Her treatment plan includes Hydroxyurea, 500 mg daily, and transfusions, as needed. Her last sickle cell crisis was 2 years ago. CBC was ordered. Hgb on admission was 6.1 g/dL. Her RBC morphology showed polychromasia, target cells, sickle cells, anisocytosis, and numerous nucleated RBC forms.

The patient was admitted to the hospital. Type and crossmatch for 2 units of packed red blood cells was ordered. CT imaging was performed and revealed severe osteopenia and vertebrae deformities consistent with her history of sickle cell disease. Chest CT showed hypoinflated lungs and areas of consolidation in the lower lobes consistent with acute chest syndrome due to sickle cell beta thalassemia. She was transfused with 2 units of pRBCs and treated for sickle cell crisis. The patient remained stable and was discharged 3 days later.

Table 1. CBC results on ER admission
Figure 1. Peripheral blood smear on admission. Patient with sickle cell/beta thalassemia shows sickle cells, target cells, nucleated red cells, anisocytosis, poikilocytosis, polychromasia. (Mercy Medical Center, Baltimore, Md)

Sickle cell anemia (HbSS) and thalassemia are the world’s most common single gene disorders. Both are inherited in an autosomal recessive manner, and result in hemolytic anemias. But what happens when you inherit one gene for sickle cell and one gene for beta-Thalassemia (β-thalassemia)?

Sickle cell disease is caused by mutations in the HBB gene that provide instructions for making beta-globin. Sickle cell anemia is a hemoglobinopathy, a qualitative defect in the structure of globin chains, resulting in the production of abnormal hemoglobin. Normal adult Hemoglobin A has 2 α chains and 2 β chains (α2β2). Hb S results from the substitution of valine for glutamic acid at position 6 of the β globin chain. The resultant Hb S has reduced solubility at low oxygen tensions. Patients with sickle cell anemia have a moderate to severe chronic hemolytic anemia with recurrent painful sickle cell crisis.

Sickle cell disease is inherited in an autosomal recessive pattern from parents who have at least one mutated gene. Anyone with a sickle cell gene can pass this gene on to their children. Sickle cell anemia (HbSS) is the homozygous expression of a sickle gene from both parents and is the world’s most common inherited hematological disease. A heterozygote inherits a sickle gene from only one parent. This person is a carrier of sickle cell (HbSs), often referred to as sickle cell trait. HbSs persons do not generally exhibit symptoms or may exhibit only a mild anemia. However, under stressful conditions, such as at high altitudes, they may experience vaso-occlusive sickle crisis.

While hemoglobinopathies are a qualitative defect due to structural changes in the normal amino acid sequence of globin, thalassemias result from an imbalance in the synthesis of the globin chains that make up the hemoglobin molecule. Thalassemias involve the rate of globin chain synthesis leading to a quantitative defect. Thalassemia is divided into α-thalassemias and β-thalassemia. α-thalassemias involve genes for the α chains on chromosome 16. In α-thalassemia, the deletions involve the α1 and/or the α2 globin genes and result in decreased production of α chains. β-thalassemias mainly affect β chain production. They are disorders of reduced globin chain production from the globin chain cluster on chromosome 11.

(Since this case involves a known diagnosis with a compound heterozygous state involving a β-thalassemia gene mutation, the discussion of α-thalassemia has been limited here. Watch for a case involving α-thalassemia in a future blog!)

Beta thalassemia occurs when the beta globin chains are either produced inadequately or not at all. There are many mutations in and around the β globin gene that result in decreased β chain production. Mutations that result in the complete absence of β chain production are designated as β0. In the most severe form of β-thalassemia the patient is homozygous β0/β0 and does not produce any β chains. Without β chains there is no Hb A (α2β2). β+ is used as the designation for any mutations of the β globin gene that cause a partial deficiency of β chains (5-30% decrease) and therefore result in a decrease in production of Hb A. The βsilent designation is used for carrier state gene mutations that result in only a mildly decreased β chain production. The degree of decrease in the β chain production is related to the degree of anemia and the severity of clinical disease.

Thalassemia, like sickle cell anemia, is a hereditary anemia inherited in the autosomal recessive manner. β-thalassemia is divided into categories based on clinical severity of disease. In β-thalassemia major a child inherits a copy of a β-thalassemia gene mutation from both parents. There are various mutations that cause genes with these mutations and different variants may be inherited from each parent. A person with thalassemia major may be homozygous β+/ β+, homozygous β0/ β0, or the compound heterozygous state β+/ β0. Hb A is only produced in patients with the β+ mutation. β-thalassemia major patients have the most severe hemolytic anemia and symptoms. β-thalassemia intermedia is characterized as homozygous βsilent or heterozygous βsilent with β+ or β0 and mild to moderate disease. β-thalassemia minor, also called β-thalassemia trait or carrier state, presents with mild but asymptomatic hemolytic anemia. These patients are heterozygous with normal β globin and have slightly decreased Hb A.

In people with sickle cell disease, at least one of the beta globin is replaced with hemoglobin S. In homozygous sickle cell anemia, both beta globin subunits in hemoglobin are replaced with hemoglobin S. In compound sickle cell diseases, one beta globin is replaced with hemoglobin S and the other beta globin is replaced with a different abnormal variant. Examples of this are Hb SC disease, and Hb SD syndrome. Compound heterozygosity is the inheritance of two different mutated genes that share the same locus. If mutations that produce hemoglobin S and beta thalassemia occur together, individuals have hemoglobin S-beta thalassemia disease. (sickle cell beta-thalassemia, Hb S β thal or sickle-β-thal). Sickle cell beta thalassemia patients have hemoglobin S (α2β26Glu→Val) and either β0 or β+.

When a qualitative hemoglobinopathy is inherited with a quantitative disorder of hemoglobin synthesis, the severity of the compound disorder is dependent on the β gene mutation. Patients with β0 produce no Hb A and have moderate to severe symptoms comparable to that of Hb SS patients. β+ patients will produce some β chains and therefore have some Hb A and milder or no symptoms.

Figure 2. Peripheral Blood smear on day 3. Sickle cell forms, polychromasia, target cells. nucleated RBCs. (Mercy Medical Center, Baltimore, Md)

Newborn screening can diagnose β0-thalassemia at birth by detecting a complete absence of hemoglobin A. However, it is not possible to make a definitive diagnosis of β+-thalassemia in the newborn because newborns have Hb F, and the reduced amount of hemoglobin A overlaps the range for normal babies. In adults with Hb S – β thal the amount of Hb S is variable. There is some Hb A in β+ patients but no Hb A detected in β0. Hb A2 and Hb F are increased. In addition to hemoglobin electrophoresis, molecular testing may also aid in the diagnosis by identifying genetic mutations. Beta globin gene sequencing can identify beta thalassemia alleles that are caused by point mutations in the beta globin gene. As well, structural variants of the beta globin gene such as Hb S can be identified with this technique. This can lead to a better understanding and clinical management of the disease.

Case Study, continued: This patient inherited a Hb S gene from one parent and a β-thal gene from the other, resulting in sickle cell beta thalassemia. This compound heterozygosity affects red blood cells both by the production of structurally abnormal hemoglobin, and by the decreased synthesis of beta globin chains. Clinical manifestations depend on the amount of beta globin chain production. Symptoms may include anemia, vascular occlusion, acute episodes of pain, acute chest syndrome, pulmonary hypertension, sepsis, ischemic brain injury, splenic sequestration crisis and splenomegaly.

Hemoglobin electrophoresis was sent out to a reference lab and results are shown in Table 2. Based on the Hemoglobin electrophoresis, is this patient Hb S- β0-thal or Hb S- β+-thal?

Table 2. Hemoglobin pattern and concentrations of a S/betathalassemia patient

Hemoglobin electrophoresis results for Hb S beta thalassemia patients are expected to show 60-90% Hb S and 10-30% Hg F. This patient’s Hgb S at 74% is within this range. This result reflexed a sickle solubility test, which was positive. As well, the elevated Hb F and Hb A2 are consistent with this diagnosis. It was noted in the discussion above that Hb S- β+-thal mutations cause a decrease of 5-30% in beta chains and therefore a decrease in Hb A. This patient’s Hb S is greater than the Hb A and her Hb A concentration is 14.8%, which is consistent with this diagnosis. Hb S- β0 mutations produce no Hb A. In this case there is some Hb A on electrophoresis but not as much as would be expected in a β+-thal mutation. Also, of note it that this patient was recently transfused with 2 units of pRBCs. Interpretations of hemoglobin electrophoresis assume that the patient has not been transfused in the last 3 months. The Hb A in this patient can be explained by these recent transfusions. Therefore, it can be concluded that the hemoglobin pattern and concentrations are consistent with transfusion of a Hb S beta0 thalassemia patient. The β0 mutation is also consistent with this patient’s moderately severe symptomatic anemia.

References

  • Keohane, Elaine, et al. Rodak’s Hematology, Clinical Principles and Application, 5th ed, Elsevier, 2016
  • McKenzie, Shirlyn. Clinical laboratory Hematology. Pearson Prentice Hall, 2004.
  • McGann PT, Nero AC, Ware RE. Clinical Features of β-Thalassemia and Sickle Cell Disease. Adv Exp Med Biol. 2017;1013:1-26. doi: 10.1007/978-1-4939-7299-9_1. PMID: 29127675.
  • Origa R. Beta-Thalassemia. 2000 Sep 28 [Updated 2021 Feb 4]. In: Adam MP, Mirzaa GM, Pagon RA, et al.,editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2023. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1426/
  • Turgeon, Mary Louis. Clinical Hematology Theory and Procedures, 6th ed, Jones and Bartlett Learning, 2017.
Socha-small

-Becky Socha, MS, MLS(ASCP)CMBBCM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 40 years and has taught as an adjunct faculty member at Merrimack College, UMass Lowell and Stevenson University for over 20 years.  She has worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. She currently works at Mercy Medical Center in Baltimore, Md. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

Drowning: A Diagnosis of Exclusion

With warmer weather approaching (or already arrived, depending on your location), it’s a good opportunity to review investigation of drowning deaths in forensic pathology. Drowning is the leading cause of deaths for children between the ages of 1 and 4 in the United States, but it can affect any age group.

Drowning is a diagnosis of exclusion – there are no pathognomonic signs of drowning, and a complete autopsy is required to rule out competing causes of death. Keeping an open mind during the investigation is the first step – discovering a body in water doesn’t automatically mean the cause of death is drowning. The body of a homicide victim may be disposed of in water as an attempt to destroy evidence, or someone may die of a cardiac arrythmia while they happen to be swimming.

The questions to answer in possible drowning deaths are much like those when faced with a body found after a fire (see “The Basics of Deaths by Fire” from February 23, 2023). Was the person alive when their body entered the water? And if they were, did they die from drowning – or did they die of another cause, and then become submerged?

Autopsies of drowned individuals commonly reveal findings supportive of, but not specific for, drowning. The lungs are typically over-expanded and edematous, and foamy fluid may be in the airways. A “foam cone” may protrude from the nostrils and/or mouth. Pulmonary effusions can be present, and the petrous ridges at the base of the skull may show red-purple discoloration due to vascular congestion and hemorrhage. The stomach and sphenoid sinus may contain large amounts of watery fluid. Wrinkling and pallor of the skin of the hands and feet (formerly called “washerwoman’s hands”) is often identified but doesn’t necessarily indicate drowning as it can occur with pre-mortem or post-mortem submersion.

A posterior neck dissection is necessary in most drownings to rule out high cervical spine injuries, which are often overlooked without this special autopsy technique. This type of trauma can be seen in divers or jumpers who strike head-first in shallow bodies of water and may cause death by itself or contribute to the decedent’s inability to self-extricate from the water. Pathologists also need to be aware that post-mortem injuries can happen as the body is passively carried by currents and bumps into rocks or other debris, known as “travel abrasions”.

The body of water is another consideration. It would be highly unlikely for a neurologically alert teenager or adult to drown in a bathtub, whereas an infant could easily drown if left unsupervised. In contrast, it may take a river or ocean with strong currents to overpower experienced swimmers. Personal medical history is important, as well – an adult with epilepsy could drown even in shallow water if they experience a seizure. The temperature of the water can also play a role. Cold water can trigger cardiac arrhythmias, contribute to fatigue of skeletal muscles, or incite hypothermia leading to a loss of consciousness. Toxicology testing is an important ancillary test in drowning deaths to provide context and may reveal intoxications that help explain someone’s inability to remove themselves from the water. Alcohol can contribute to the impairment of physical coordination and/or increase risk-taking behaviors, and has been associated with up to 70% of water recreation-associated deaths.

If the autopsy doesn’t show any indicators of drowning but reveals potentially lethal natural disease (such as severe coronary artery stenosis), then it’s likely the person died while they happened to be in the water and not because they were in the water. In every situation, though, the autopsy findings must be correlated with the decedent’s history, the results of scene investigation, and toxicology testing before a final diagnosis can be rendered. In this way, autopsies of water-associated deaths highlight the importance of context and investigation in forensic pathology.

Figure 1. A classic example of the “foam cone” seen at autopsy in instances of drowning. This finding results from marked pulmonary edema, and isn’t specific for drowning – it can be seen in many other conditions including opiate overdoses and heart disease.
Figure 2. Foamy fluid in the trachea and mainstem bronchi can be seen in any condition that causes pulmonary edema, and also is not specific for drowning.
Figure3. Wrinkling and paleness of the hands and feet is often seen in bodies recovered from water, whether or not the cause of death was actually drowning.

References

  • Armstrong EJ, Erskine KL. Investigation of drowning deaths: a practical review. Academic Forensic Pathology, Jan 2018.
  • Centers for Disease Control and Prevention, National Center for Injury Prevention and Control. Drowning Prevention. <https://www.cdc.gov/drowning/facts/index.html&gt; Accessed 6/21/2023.

-Alison Krywanczyk, MD, FASCP, is currently a Deputy Medical Examiner at the Cuyahoga County Medical Examiner’s Office.

Microbiology Case Study: Cryptococcal Meningitis In Immunocompromised Patient

Case History

A 47-year-old male originally from Dominican Republic, with a recent diagnosis of acquired immunodeficiency syndrome (AIDS) and diffused large B cell lymphoma (DLBCL), was admitted because of seizures and a rapidly increasing left neck mass. ​MRI of the brain showed a 2.5 x 1.2 cm (about 0.47 in) lesion in the left inferior parietal lobe – (1.4×0.7cm) in the right frontal lobe, plus multiple scattered bilateral lesions. Because of this, he underwent craniotomy/craniectomy for possible resection. A biopsy was taken from the right temple lesion and sent for aerobic, anaerobic, fungal, mycobacterial culture, surgical pathology and Toxoplasma PCR (Polymerase Chain Reaction). ​

Gram stains, KOH prep, acid-fast stains, and Toxoplasma PCR of the tissue were all negative. Aerobic and anaerobic cultures did not show any growth.

Histopathology slides (GMS and H&E stains in Fig A and B) show budding yeasts morphologically consistent with Cryptococcus. Mucicarmine stain was also positive. Lumber puncture was performed the next day and Cryptococcal antigen was positive, with a titer of 1:640. Interestingly, the CSF culture and Gram stain did not reveal any organisms.

Figure A. H&E shows encapsulated variably sized transparent/gray color yeasts with thin walls. Black arrows show organisms.
Figure B. GMS stains highlight very faint staining of capsule (black arrow). Yellow arrow highlights background inflammatory cells.

Discussion

Among several species of Cryptococcus, C. neoformans and Cryptococcus gattii are pathogenic, with C. neoformans causing meningitis in immunocompromised patients worldwide whereas C. gattii has a preference for immunocompetent individuals.1 Cryptococcal disease remains a major opportunistic infection and a leading cause of mortality in patients infected with HIV in much of the developing world. Most HIV-related meningitis cases are caused by Cryptococcus neoformans.2

Cryptococci are found in soil, due to contamination with pigeon droppings. The infection occurs through inhalation, with or without symptoms of pneumonia, with subsequent dissemination to the central nervous system (CNS) via blood. Imaging findings are often unspecific or negative. CT or MRI examination of the central nervous system is performed to rule out alternative diagnoses. The diagnosis of ‘meningitis’ is made with a lumbar puncture, which typically shows lymphocytosis, an increased protein and decreased glucose concentration. Few neutrophil granulocytes are often found in CSF. This is likely because neutrophil migration is inhibited by specific polysaccharides that are part of the cryptococcal capsule.3

Cryptococci can be seen directly in the sediment of centrifuged CSF stained with India ink. The sensitivity and specificity of India ink is poor; therefore, CSF Gram stain and culture, multiplex meningitis/encephalitis PCR, and lateral flow antigen (LFA) tests have replaced the use of India ink. The cryptococcal lateral flow antigen test should be performed in CSF and serum, in addition to Multiplex ME PCR panel, and is a preferred test because of high sensitivity (93-100%) and specificity (93-98%).4

High organism burden at baseline (indicated by quantitative CSF culture or CSF antigen titre) and abnormal mental status are the most important predictors of death, while high opening pressures and a poor inflammatory response in the CSF have also been associated with poor outcome.5

On H&E it has the characteristic appearance ofencapsulated variably sized yeasts (2-20 microns) with thin walls which can be highlighted with the GMS stain. Although the presence of a capsule differentiates Cryptococcus from Histoplasma capsulatum and Blastomyces dermatitidis with the H&E or mucicarmine stain, additional confirmation can be made with Fontana-Masson stainin the absence of capsules.6 Since both C.neoformans and C. gattii produce melanin, the pathology report by FM silver or H&E/GMS stain cannot further distinguish these two closely resembled species.

Occasionally, cryptococcal meningitis cases with sterile CSF culture and/or negative Cryptococcal CSF antigen are observed in HIV individuals, regardless of the CD4 counts.7,8 However, serum Cryptococcal antigen and blood culture may be positive in those individuals.7 In our case, the diagnosis of Cryptococcal meningitis was made by the pathology report and positive CSF Cryptococcal antigen.

-Fnu Sapna is a 2ndyear AP/CP pathology resident in the Department of Pathology at Montefiore Medical Center in Bronx, NY. She completed her Medical education at Chandka Medical College in Pakistan. Her interests are putting efforts to improve screening guidelines for diagnosis of preventable gynecological and breast cancers.

-Phyu Thwe, Ph.D, D(ABMM), MLS(ASCP)CM is Associate Director of Infectious Disease Testing Laboratory at Montefiore Medical Center, Bronx, NY. She completed her medical and public health microbiology fellowship in University of Texas Medical Branch (UTMB), Galveston, TX. Her interests includes appropriate test utilization, diagnostic stewardship, development of molecular infectious disease testing, and extrapulmonary tuberculosis.

One year of Chronic Cough after Past Resolution of Viral and Bacterial Respiratory Infections

Case History

A 74-year-old female from El Salvador with a medical history of hypertension, diabetes, osteoarthritis presented with persistent productive cough. The patient states that the symptoms started on November 2022 which was believed to be associated with ongoing COVID-19 infection. Despite recovery from COVID-19 infection, significant productive cough still remained. A visit to her primary care doctor revealed streptococcal throat infection and despite completing a course of Zithromax antibiotics, a subsequent chest x-ray revealed potential right upper lobe pneumonia and she reported productive cough with occasional blood streaks. Blood work up in the Emergency Department revealed leukocytosis (20.22 x10e3/mcL) with neutrophilia (14.56 x10e3/mcL neutrophils) and monocytosis (3.03 x10e3/mcL monocytes). A chest x-ray showed bilateral reticular and airspace opacities with an air-fluid level containing opacity overlying the left mid lung, likely representing a cavitary lesion. A follow up with a computerized tomography of the chest identified innumerable randomly distributed pulmonary nodules or cavities with upper lobe predominance. The largest cavity measures approximately 6.6 cm with air-fluid levels and debris. Additionally, a small pericardial effusion with thickened pericardium was also noted. A sputum sample was submitted for Acid-fast bacilli (AFB) culture and molecular testing. AFB stain was positive for acid-fast bacilli (Figure 1). The GeneXpert MTB/RIF assay detected M. tuberculosis with no rifampin resistance marker. Growth on the Lowenstein-Jensen agar after two weeks showed buff colored rough and dry colonies and was confirmed as M. tuberculosis on the MALDI-TOF.

Figure 1. Visualization of acid-fast bacilli directly from patient specimen. Using acid-fast stain, cording was observed (Figure 1A, left) and using fluorescent staining, fluorescent rods were observed (Figure 1B, right).

Discussion

Mycobacteria are aerobic, nonmotile, thin rod shape, non-spore forming bacilli that possess mycolic acid in its cell wall giving its acid-fast stain characteristics. Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis (MTB). TB remains to be the leading cause of death from a single infectious agent worldwide. According to the CDC a total of 1.6 million people died from TB in 2021 (including 187, 000 people with HIV) 1,2. The M. tuberculosis complex (MTBC) includes M. tuberculosis, M. bovis, M. bovis, Bacille Calmette-Guérin strain (BCG), M. caprae, M. pinnipedii, M. mungi,M. africanum, M. microti, and M. canettii 3. M. tuberculosis produces cord factor, a glycolipid which is also known as trehalose dimycolate, that causes the bacteria to grow in parallel strands and that appeared like cord, or rope when cultured in liquid media (Figure 1A). The cord factor is present in the outer envelope and protects the bacteria from the host response 4.

The pathogenesis of human tuberculosis involves a complex interaction between host immune system and bacterial factors 4. M. tuberculosis is carried in airborne particles generated by infected individuals. The droplet nuclei traverse the mouth or nasal passages, upper respiratory tract, and bronchi to reach the alveoli of the lungs. The bacteria are then phagocytosed by alveolar macrophages and can inhibit maturation of phagosome and block formation of phagolysosome, allowing its unchecked replication in the macrophage which results in bacteria proliferation in the alveolar macrophage and air spaces. In immunocompetent hosts, the immune response (via TLR2, TLR9, Th-1 and IFN- ꝩ cascades) may contain the infection before significant tissue destruction or systemic illness 1-4. However, in the immunosuppressed hosts, the primary infection results in a broad clinical spectrum such as meningitis, miliary tuberculosis and extrapulmonary granulomas. Post-primary/secondary tuberculosis (reactivation TB) usually begins months to years after the establishment of systemic immunity in primary TB mostly in a period when the host immune response is weakened, following exogenous or a large inoculum of virulent bacilli overwhelming the host immunity system. Extrapulmonary manifestations will develop based on the organ system affected 5.HIV infection is the greatest risk factor for reactivation of TB as the virus causes functional abnormalities in CD4+ T cells and CD8+ T cells which confer protection against active TB. Other risk factors that promote the reactivation TB include aging, malnutrition, diabetes mellitus, renal failure, cancer and immunosuppressive therapy 5. The disease typically affects the lungs (pulmonary TB) but can affect other sites as regional lymph nodes, apex of the lung, larynx, kidneys, brain, bone, joints and pleura 1,2,5.

Diagnostic tests for TB detect either the bacteria or host immune response. Specimens recommended for diagnosis of mycobacterial infection are sputum, bronchial brushing/washings /biopsies, gastric aspirates (children) urine, blood, CSF, BM, body fluids, stool (only in HIV) 5,8. Specimens from sputum and other nonsterile sites should be liquefied with N-acetyl-L-cysteine and decontaminated with NaOH and for gastric aspirate neutralized with buffer 6-8. For diagnostic purposes, all persons suspected of having TB disease at any site should have at least three consecutive sputum specimens collected in 8 to 24 hours with at least one being an early morning one for AFB smear and culture 1,8.

The organisms can be visualized under a microscope using two principal methods: carbolfuchsin staining (e.g., Acid-fast stain), or using a fluorochrome (auramine-rhodamine and auramine-O stains) procedure (Figure 1B). Microscopy is the most rapid diagnostic method for the detection of tubercle bacilli but is less sensitive; it requires a minimum of 10,000 bacilli/mL of sputum to produce a positive result. Culture is the gold standard and more sensitive method for the detection of tubercle bacilli and is necessary for performing antimicrobial drug drug-susceptibility testing and genotyping. 6,9 However culture requires 3–6 weeks for growth which delays the initiation of anti-tuberculosis drug therapy. Two types of solid media are used for mycobacterial culture: egg based (Löwenstein-Jensen) and agar based (Middlebrook 7H10, 7H11, and selective 7H11). Colony morphology of Mycobacterium tuberculosis on solid media are dry, rough, raised, wrinkled, off white to buff colored. M. tuberculosis is commonly positive for niacin, nitrate reduction test, pyrazinamidase test, but negative for 68C catalase, tween 80 hydrolysis, and 5% NaCl tolerance. Molecular techniques such as nucleic acid amplification tests revolutionized tuberculosis diagnosis since M. tuberculosis nucleic acid material can be detected directly from specimen in less than 2 hours. Two commercial NAATs for the detection of M. tuberculosis complex are available in the United States: The Amplified MTD (Mycobacterium Tuberculosis Direct) test (Hologic, Marlborough, MA) and the Xpert MTB/RIF (Cepheid, Sunnyvale, CA).

Common serological approaches for detection of M. tuberculosis are the Tuberculin skin test (TST) or IFN- ꝩ release assays (IGRA’s) 1,9,10. The tuberculin skin test is performed by intracutaneous injection of purified protein derivative of M. tuberculosis, which induces a visible and palpable induration that peaks in 48 to 72 hours. A false-positive tuberculin skin test may result on individuals with prior vaccination with BCG (Bacillus Calmette-Guerin), an attenuated strain of M. bovis. BCG immunization does not affect the test result of IGRA assay. The IGRA are blood tests that measure a person’s immune reactivity to M. tuberculosis. Both T-Spot and QuantiFERON can aid in diagnosis M. tuberculosis but do not differentiate latent infection from tuberculosis disease. IGRAs are in vitro tests that measure the IFN- γ production by T cells responding to stimulation with specific TB antigens ESAT-6, TB7.7, and CFP-10, which are not present in the M. bovis strains. Results can be interpreted both qualitatively (positive, negative, or indeterminate) and quantitatively.

The regimen currently recommended for treatment of TB is isoniazid, rifampin, ethambutol, and pyrazinamide. The initial M. tuberculosis isolate should be tested for resistance to first-line medication. Second-line drug susceptibility testing should be limited to specimens from patients who have prior TB disease treatment, contact with a patient with known TB drug resistance or positive cultures after more than 3 months of treatment 10. Multidrug-resistant TB (MDR TB) disease is defined as resistance to isoniazid and rifampin, and Extensively drug-resistant TB (XDR TB) is characterized with resistance to isoniazid and rifampin, any fluoroquinolone, and at least one of three injectable second-line drugs (i.e., amikacin, kanamycin, or capreomycin). The duration of therapy depends on the drugs used, the drug susceptibility test results, and the patient’s response to therapy. Most patients are started with a 6-month regimen plan. A difficult challenge to M. tuberculosis treatment is patient compliance with lengthened therapy. Without treatment mortality rate for tuberculosis is more than 50% 11.

References

  1. World Health Organization, Global TB Programme. Global tuberculosis report 2022. 2022 November 19. https://www.who.int/publications/i/item/9789240061729
  2. Kamholz, S. L. 1996. Pleural tuberculosis, p. 483-491. In W. N. Rom and S. Garay (ed.), Tuberculosis. Little, Brown and Co., Boston, Mass.
  3. Yanti, B., et al. The role of Mycobacterium tuberculosis complex species on apoptosis and necroptosis state of macrophages derived from active pulmonary tuberculosis patients. BMC Res Notes. 2020; 13: 415. doi: 10.1186/s13104-020-05256-2
  4. Smith, Issar. Mycobacterium tuberculosis Pathogenesis and Molecular Determinants of Virulence Clin Microbiol Rev. 2003 Jul; 16(3): 463–496. doi: 10.1128/CMR.16.3.463-496.2003
  5. Wells, C.D, et al. HIV infection and multidrug-resistant tuberculosis: the perfect storm. J Infect Dis. 2007 Aug 15;196 Suppl 1:S86-107. doi: 10.1086/518665.
  6. Dunn, J.J., Starke, J.R., Revell, P.A. Laboratory Diagnosis of Mycobacterium tuberculosis Infection and Disease in Children. J Clin Microbiol 2016 Jun;54(6):1434-1441. doi: 10.1128/JCM.03043-15.
  7. Parashar D, Kabra S, Lodha R, Singh V, Mukherjee A, Arya T, Grewal H, Singh S. 2013. Does neutralization of gastric aspirates from children with suspected intrathoracic tuberculosis affect mycobacterial yields on MGIT culture? J Clin Microbiol 51:1753–1756.
  8. Clinical and Laboratory Standards Institute. 2008. Laboratory detection and identification of mycobacteria; approved guideline—1st edition. CLSI document M48-A. Clinical and Laboratory Standards Institute, Wayne, PA.
  1. Pai M, Nicol MP, Boehme CC. Tuberculosis Diagnostics: State of the Art and Future Directions. Microbiol Spectr 2016; 4.
  2. Dheda, K. et al. The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis. Lancet Respir Med. 2017 Mar 15;S2213-2600(17)30079-6.
  3. World Health Organization. Tuberculosis Fact Sheet. 21 April 2023. https://www.who.int/news-room/fact-sheets/detail/tuberculosis

-Dr. Carla Ayala-Soriano was born and raised in Bayamon, Puerto Rico. She attended Universidad Autonoma de Guadalajara School of Medicine where she received her doctorate degree. She completed a Bachelor of Science in Biology at the University of Puerto Rico. She spent an additional year completing a Post Bachelor Certificate in Cytotechnology. Her academic interests include Cytopathology and Gynecologic Pathology. In her spare time, Dr. Ayala-Soriano enjoys cooking, traveling, listen to music, and outdoor activities. She is pursuing AP/CP training.

-Rebecca Yee, PhD, D(ABMM), M(ASCP)CM is the Chief of Microbiology, Director of Clinical Microbiology and Molecular Microbiology Laboratory at the George Washington University Hospital. Her interests include bacteriology, antimicrobial resistance, and development of infectious disease diagnostics.

Thyroid Tales, Part 2

Typically, our patients present to the endocrinology clinic after their thyroid nodules are incidentally found on staging or surveillance after being diagnosed with a primary cancer in another part of the body. Based on TI-RADS criteria, the clinician either monitors the nodule or refers the patient to radiology for a thyroid FNA. When we hear “thyroid nodule,” we rarely assume anything other than thyroid tissue. Whether the imaging favors benign or suggests a high risk of malignancy, we prepare ourselves to assess the FNA smears for follicular cells (and all the levels of atypia), colloid, macrophages, Hurthle cells, lymphocytes, etc. While we must keep an open mind, we are always caught off guard when we see anything other than thyroid-related cells. So as promised in the first edition of this post, here are four thyroid FNA cases with unsuspecting findings.

Case 1

A 47-year-old male presented with throat dysphagia and odynophagia. CT scan revealed a destructive mass within the thyroid gland with compression and invasion of the thyroid cartilage and seemed contiguous with a large pharyngeal mass, spanning approximately 8 centimeters. A follow-up PET scan noted multiple hypermetabolic thyroid masses within both lobes, direct invasion of the subglottic trachea and upper esophagus, and mediastinal lymphadenopathy.

FNA passes were obtained from the right lobe of the palpable thyroid mass.

Images 1-2. Thyroid, Right Lobe, FNA 1: DQ-stained smear; 2: Pap-stained smear.

Smears (Images 1 & 2) revealed poorly differentiated neoplastic cells, follicular cells, and colloid (not visualized). No features of papillary thyroid carcinoma, medullary carcinoma, or Hurthle cell neoplasm/carcinoma were identified.

Immunohistochemical stains performed on cell block sections showed the poorly differentiated neoplastic cells to be negative for thyroglobulin, TTF-1, and calcitonin; Follicular cells, which may probably be differentiated neoplasm, were positive for thyroglobulin, TTF-1, and negative for calcitonin. Unfortunately, the scant cellularity in the cell block specimen precluded additional stains. Giant cell and spindle cell features were not identified in this specimen. Morphological features are compatible with poorly differentiated carcinoma of the thyroid gland; however, metastasis from other sites cannot be excluded.

The patient then underwent a total laryngectomy and thyroidectomy (Images 3 & 4) and level IV neck dissection, bilateral modified radical neck dissection, and tracheostomy with reconstruction performed. The patient then underwent adjuvant radiation followed by palliative re-irradiation and chemotherapy after abnormal activity was noted throughout the neck. Treatment was discontinued due to severe disease progression.

Images 3-4. Thyroid, Thyroidectomy: 3: H&E section (200X); 4: H&E section (600X).

Final diagnosis: Poorly differentiated thyroid carcinoma with squamous differentiation arising in association with differentiated follicle derived carcinoma cells.

Case 2

A 68-year-old female presented with a 3.7 centimeter left lobe-filling thyroid nodule and a history of melanoma of the left anterior tibial region that was excised a decade prior. During that time, a sentinel lymph node biopsy identified microscopic metastasis. Seven years after her initial diagnosis, the patient underwent an excision of a right upper quadrant subcutaneous nodule, demonstrating metastatic melanoma. Three months after that excision, the patient had a low anterior resection of a rectosigmoid metastasis. A breast lesion was then identified five months later, and the patient underwent a mastectomy for melanoma involving the breast. Six months after her mastectomy, the patient had a segmental resection and excision of a left posterior thigh nodule, at which point she was enrolled in a clinical trial. The next month, four additional subcutaneous nodules were excised on the left thigh, calf, and arm. After 2 years of relatively stable disease, the patient underwent a partial gastrectomy, partial small bowel resection, and left lower extremity mass for recurrent melanoma. The last PET avid area to biopsy was the left-lobed thyroid nodule. Under ultrasound guidance, multiple FNA passes of the solid and hypervascular thyroid nodule. The smears (Images 5 & 6) and cell block (Image 7) featuring single cells with eccentric nuclei and prominent nuclei are presented below.

Images 5-8. Thyroid, Left Lobe, FNA 5: DQ-stained smear; 6: Pap-stained smear; 7: H&E Cell Block section (600X); 8: A103-positive.

Immunostains were performed on cell block sections, and the neoplastic cells are positive for A103 (Image 8), HMB45 (scattered cells), and SOX-10, while negative for CD45, TTF-1, thyroglobulin, and calcitonin.

The patient began treatment with temozolomide and completed 33 cycles of pembrolizumab. Her most recent metastasis demonstrated extensive tumor cell necrosis, and disease progression has slowed tremendously.

Final diagnosis: Melanoma.

Case 3

After developing sudden shortness of breath and chest tightness, a 40-year-old female patient presented to the emergency department. A large mediastinal mass compressing the heart and central structures in the chest was identified on CT scan. Two thyroid nodules were also noted during that time. The patient underwent a mediastinal biopsy, which demonstrated small cell lung cancer, and the patient underwent thoracic radiation and six cycles of chemotherapy, as well as whole brain radiation. Two years later, the patient established care with endocrinology for her 1.6 centimeter solid left lobe thyroid nodule and a 1.2 cm complex thyroid nodule in the right lobe. While the right nodule was consistent with a hyperplastic nodule, the smears and cell block of the left thyroid nodule are presented below (Images 9-11).

Images 9-11. Thyroid, Left Lobe, FNA 9: DQ-stained smear; 10: Pap-stained smear; 7: H&E Cell Block section (400X).

Immunohistochemical stains performed on cell block sections demonstrate the neoplastic cells were positive for TTF-1, AE1/AE3, synaptophysin, and CD56.

The patient then completed four subsequent cycles of chemotherapy with concurrent chemoradiation and is currently on active surveillance showing no evidence of disease for over 12 months.

Final diagnosis: Metastatic small cell carcinoma.

Case 4

A 59-year-old female presented to her primary care physician for gross hematuria and fatigue. Her thyroid workup demonstrated hypothyroidism on her thyroid function panel and a 2.3 centimeter solid and hypervascular thyroid nodule in the right lobe. Her urology workup revealed a 6.7 centimeter exophytic left kidney mass, and the follow-up CT scan identified a lytic lesion in the right iliac bone. The thyroid biopsy was performed in the endocrinology clinic while she was also establishing care with the urologic oncology team the same day. The smears and cell block specimen from multiple FNA passes are presented below (Images 12-14).

Images 12-15. Thyroid, Right Lobe, FNA 12: DQ-stained smear; 13: Pap-stained smear; 14: H&E Cell Block section (400X); 15: Vimentin-positive.

Immunocytochemical stains were performed on paraffin sections of the cell block. Tumor cells show positive staining for vimentin (Image 15), focal staining for e-cadherin, and negative staining for CK7, TTF-1, thyroglobulin, CD10, and RCC.

The patient was referred to radiology for a CT-guided biopsy of the lytic bone lesion, which demonstrated similar cells. The patient had a radical left nephrectomy, followed by sunitinib. The thyroid nodule was not responding to treatment, so they patient underwent a total thyroidectomy, which showed metastatic high-grade clear cell carcinoma with sarcomatoid progression, consistent with renal primary. In some areas, the thyroid follicles were proliferating and appear atypical, probably reactive to the metastatic carcinoma. A checkpoint inhibitor was added to the patient’s therapy, but the disease continued to progress, and the patient elected for palliative care.

Final diagnosis: Poorly differentiated carcinoma, consistent with metastatic renal cell carcinoma.

That’s a wrap! Stay tuned for the next series of cytology case studies!

-Taryn Waraksa-Deutsch, MS, SCT(ASCP)CM, CT(IAC), has worked as a cytotechnologist at Fox Chase Cancer Center, in Philadelphia, Pennsylvania, since earning her master’s degree from Thomas Jefferson University in 2014. She is an ASCP board-certified Specialist in Cytotechnology with an additional certification by the International Academy of Cytology (IAC). She is also a 2020 ASCP 40 Under Forty Honoree.

Microbiology Case Study: A 21 Year Old Male with Shortness of Breath

Case History

A 21-year-old male with no significant past medical history presented to the emergency department for acute hypoxemic respiratory failure. He also reported a productive cough with green sputum and unintentional 30 lb weight loss over the past 5 months. On presentation, his oxygen saturation was in the 80s on room air and initial labs were significant for lactic acidosis and a reactive HIV Ag/Ab. His HIV viral load was 1,359,029 copies/mL with a CD4 count of 37 cells/mm3 . Imaging included a chest X-ray and chest CT, which revealed left sided pneumothorax and diffuse ground glass opacities with scattered areas of consolidation. Based on his clinical presentation and CD4 counts <200 cells/mm3, the patient was started on trimethoprim/sulfamethoxazole (TMP/SMX) for PCP prophylaxis. A bronchoalveolar lavage was performed but had no growth on bacterial and fungal cultures. On hospital day 5, the left-sided pneumothorax was persistent despite multiple chest tube placements and a right-sided pneumothorax had formed, and the patient underwent video-assisted thoracoscopic surgery and pleurodesis. During the procedure, a ruptured bleb was found at the apex of the upper lobe of the left lung and a limited apical wedge resection was performed. Pathologic examination of the resection was significant for foamy exudate-filled alveoli on H&E staining.  GMS stain revealed cup-shaped organisms confirming the presence of Pneumocystis jirovecii. The patient was continued on TMP/SMX for 21 days total and weaned off supplemental oxygen with follow-up scheduled at an HIV clinic.

Figure 1. Hematoxylin and Eosin lung tissue sections show alveolar spaces filled with
 pink, foamy amorphous material (20x and 100x magnification)
Figure 2. GMS stain show alveolar spaces filled with Fungi 4 – 6 microns, cup / boat shaped cysts (100x magnification)

Discussion

Pneumocystis jirovecii pneumonia (PCP pneumonia) is a life-threatening infection found in immunocompromised patients, with approximately a third of patients affected being HIV-positive.1 Although the incidence of infection in HIV-positive patients is declining with modern therapies, it is still commonly seen in undiagnosed HIV-positive patients who present late in the course of the disease.2

Clinically, PCP pneumonia is characterized by dyspnea, tachypnea, cough, and fever in an immunocompromised patient. Chest imaging features include bilateral interstitial infiltrates and a “ground glass” appearance on CT. Because Pneumocystis is extremely challenging to culture, diagnosis relies on these clinical findings with confirmation by staining or PCR testing of bronchoalveolar lavage fluid or lung biopsy.3 Stains that can be used to identify PCP pneumonia include Gromori-methenamine silver (GMS) stain, calcofluor white (CW) stain, Toluidine Blue O (TBO) stain, with GMS and CW stains having the highest sensitivity.3,4 On GMS stain, the cyst wall of Pneumocystis will appear black with a “crushed ping-pong ball” or crescent shaped appearance.

Treatment with TMP/SMX should be started in patients with suspected PCP pneumonia while work-up is pending. Corticosteroids can also be added to the treatment regimen in patients with more severe respiratory symptoms (5). After completion of 21 days of therapy, a lower-dose of TMP/SMX should be continued in HIV-positive patients with CD4+ counts less than 200 for prophylaxis.5

References

  1. Roux, Antoine, et al. “Pneumocystis Jirovecii Pneumonia in Patients with or without AIDS, France.” Emerging Infectious Diseases, vol. 20, no. 9, 2014, pp. 1490–1497, https://doi.org/10.3201/eid2009.131668.
  2. White, P. Lewis, et al. “Pneumocystis Jirovecii Pneumonia: Epidemiology, Clinical Manifestation and Diagnosis.” Current Fungal Infection Reports, vol. 13, no. 4, 2019, pp. 260–273, https://doi.org/10.1007/s12281-019-00349-3.
  3. Bateman, Marjorie, et al. “Diagnosing Pneumocystis Jirovecii Pneumonia: A Review of Current Methods and Novel Approaches.” Medical Mycology, vol. 58, no. 8, 2020, pp. 1015–1028, https://doi.org/10.1093/mmy/myaa024.
  4. Procop, G. W., et al. “Detection of Pneumocystis Jiroveci in Respiratory Specimens by Four Staining Methods.” Journal of Clinical Microbiology, vol. 42, no. 7, 2004, pp. 3333-3335, https://doi.org/10.1128/jcm.42.7.3333-3335.2004.
  5. Vilar, et al. “The Management of Pneumocystis Carinii Pneumonia.” British Journal of Clinical Pharmacology, vol. 47, no. 6, 1999, pp. 605–609, https://doi.org/10.1046/j.1365-2125.1999.00966.x.

-Alice Ann Lever is a fourth-year medical student at the Medical College of Georgia. She is interested in hematopathology and surgical pathology.

-Hasan Samra, MD, is the Director of Clinical Microbiology at Augusta University and an Assistant Professor at the Medical College of Georgia.

Journey into Mystery: Unknown Source Exposures

In 1962, Marvel Comics introduced a new super-hero in their comic book titled “Journey into Mystery!” That character would become famous both in the book and eventually on the big screen. He was the mighty Thor. Through the years this Norse god of thunder would have many adventures and travel into many strange and unusual places all to protect his home of Asgard and to save the people of his adopted home planet, Earth. While the character of Thor willingly chose to journey into those many unknown places, those who work in the laboratory with bloodborne pathogens should not.

Evan popped the tops off of the serum separator tubes and placed them into the analyzer rack. He used a counter-mounted shield to protect himself from a splash. He picked up the rack containing five specimens and walked over to the chemistry analyzer to run them, but as he neared the analyzer his grip loosened, and he dropped the rack. It fell about an inch onto the analyzer and serum splashed up into Evan’s eyes. He did not know from which tube or tubes was the source of his exposure.

Rose was running late when she started her shift in the histology grossing lab. She did not notice that the small sharps container for scalpel blades was over full at the bench. When it was time to change her blade, Rose reached up without looking to eject the blade into the sharps container. She felt a sharp pain and saw that she had cut herself on several used blades that were sticking up out of the container access hole. Her injury had to be treated as an unknown source exposure.

If a bloodborne pathogen exposure occurs in the lab, there are several regulations that should be in place to help protect the exposed employee. OSHA’s Exposure Control Plan includes hepatitis vaccinations for employees, and follow up source testing instructions to discover the HIV and hepatitis status of the known source patient. Prophylaxis for an HIV exposure in the lab must be administered quickly to be effective, usually within 2 hours of the exposure, so rapid testing is key.

There are, unfortunately, accidents that occur for which the bloodborne pathogen source cannot be determined. The incidents described above could have been prevented, and they should have been, because treatment for an unknown source exposure is a journey no ne should want to make. In some cases, like with the sharps exposure, it is impossible to determine the source. In other cases, as with a rack of tubes, it is too costly and there is no time to test all possible exposure sources.

In some facilities, after an unknown source exposure, the policies call for complete serological testing of the exposed victim for HIV and hepatitis. This does not provide useful information, however, it only provides the serological status before the exposure, it does not alter the necessary treatment.

Treatment for an unknown source exposure usually consists of the immediate administration of prophylactic drugs. While these drugs are designed to help prevent the post-exposure development of HIV or hepatitis, they are known to be toxic to the body and can have many ill effects. Personal consequences can occur as well after such an exposure. As a precaution, the exposed victim may be told to avoid intimate relationships for six months. Clearly, this is not a journey anyone would willingly want to take.

All exposure incidents in the laboratory setting should be prevented, and the majority of them can be prevented easily. Pay attention to the surroundings and look for potential sources of exposure. Consistently use proper PPE including face protection whenever handling open specimens or performing maintenance on an analyzer where tubing or reservoirs are involved. Empty sharps containers when ¾ full, and never allow anyone to open them or dig through them, even for a lost specimen. The risk is too high.

In many ways, the work of a laboratorian should be a journey into mystery. There are test results to produce, diagnoses to be made, and new techniques to discover. With the work in the lab environment, all exposure risks should be assessed, and they should be mitigated using engineering controls, safe work practices, and PPE so that this work can be performed safely. Let the scientific mysteries be those that prevail and not the scary alien consequences of an unknown source exposure.  

–Dan Scungio, MT(ASCP), SLS, CQA (ASQ) has over 25 years experience as a certified medical technologist. Today he is the Laboratory Safety Officer for Sentara Healthcare, a system of seven hospitals and over 20 laboratories and draw sites in the Tidewater area of Virginia. He is also known as Dan the Lab Safety Man, a lab safety consultant, educator, and trainer.