A 60-year-old man with a past medical history of AL amyloidosis presented to the therapeutic apheresis treatment unit for collection of an autologous peripheral blood stem cell transplant after five days of mobilization with G-CSF and plerixafor for a peripheral CD34+ of <10 cells/µL. As part of his pre-transplant evaluation, it was noted that he had an asymptomatic cold agglutinin titer of >512. When collected, his day one product “gelled” and did not separate on centrifugation (Figure 1).

Figure 1
Figure 2

What is the most likely problem, and what further steps could be performed to prevent subsequent processing issues?

  • Potential product contamination. Wash product and filter through 170 µm filter prior to cryopreservation. 
  • Cold agglutination of peripheral blood stem cell product. Warm human albumin replacement of plasma in collection and consideration of therapeutic plasma exchange to reduce CAD titer.
  • Elevated neutrophil fraction in product resulting in increased cell free DNA levels resulting in clumping/gelling. Consider treatment with DNase prior to cryopreservation. 
  • Clotting from processing error without heparin addition to product bag. Add anticoagulant and attempt manual massage to break up clots.

The correct answer is ...

Cold agglutination of peripheral blood stem cell product. Warm human albumin replacement of plasma in collection and consideration of therapeutic plasma exchange to reduce CAD titer.

This is a case of cold agglutination of a peripheral blood stem cell product. Cold agglutinins (CA) are common, but rarely present symptomatically due to the low temperatures required for red cell binding. The thermal amplitude is the temperature range at which cold agglutinins will agglutinate and typically falls within 2-4° C. In the setting of cold agglutinin disease (CAD), the thermal amplitude can reach or exceed body temp (28-30° C) leading to clinical symptoms such as acrocyanosis, Raynaud’s and hemolysis. CAs are often seen with a titer of ≥64 during serial dilutions, but symptomatic disease is more frequently seen at a tier of ≥512.

While uncommon, elevated neutrophil fractions with lysis of these cells can result in significant elevations in cell free DNA. This elevated free DNA can cause clumping of the stem cell product, which may cause infusion issues after thawing. In this circumstance, with fresh cells, the collected cells would not be expected to have lysed to such a degree as to cause this phenomenon. Lack of addition of the anticoagulant is a potential processing error that can lead to clotting within a product bag. Normally, red cell agglutinins will be seen, which may break up with addition of anticoagulant after the fact. Lastly, product contamination is always a concern during collection of a product. No information from the clinical vignette points us to a processing error resulting in lack of anticoagulant or introduction of a contaminant during collection. If such concerns were raised, all of these manipulations are reasonable to successfully cryopreserve the collected stem cell product. Given the clinical vignette, the best answer is cold agglutination resulting from the patient’s known CAs.

CAs are predominately pentameric IgM molecules and exist within the intravascular space. Due to the structure of the autoantibody, complement fixation can occur in vivo with frank intravascular hemolysis occurring (Figure 2). Onset of this disease is often insidious; however, unlike the intravascular hemolysis seen with an incompatible red blood cell transfusion. Instead, chronic hemolysis ensues, resulting in hemosiderinuria and complement consumption/deficiency. The specificity (of the antibody) is variable, but most frequently target the “I” antigen; a highly expressed antigen on adult red blood cells starting at 18 to 24 months of age. Bona fide CAD is now recognized as a unique entity associated with a lymphoproliferative disorder with monoclonal lymphocytes.

The presence of CAs during stem cell collection and processing can lead to complications arising from agglutination at cold temperatures below body temperature. In these settings, special manipulations can be done to improve the donation experience and stem cell product processing. The ambient temperature of the room for collection can be raised to improve potential peripheral symptoms in the donor. Additionally, an inline blood warmer can be placed both on the draw and return line of the apheresis machine to prevent agglutination in the machine and post centrifugation prior to reinfusion to the donor. In the most symptomatic of patients/donors, a preemptive therapeutic plasma exchange can be done to reduce circulating cold agglutinins prior to collection. Lastly, special consideration should be made with infusion of these stem cell products. Due to agglutinations at low temperature, these products should be warmed to a higher temperature prior to infusion to prevent complement fixation when entering the intravascular space after thawing.


  • Linz, Walter J. et al. Cold agglutinin disease. Transfusion. Vol. 43,9 (2003):1185. doi:10.1046/j.1537-2995.2003.00502.x
  • Thompson, T.Z., Larsen, R., Savage, N.M., Shikle, J. 2017 A Case of Mixed-Type Autoimmune Hemolytic Anemia with a Complex Autoantibody Specificity including a Ficin Sensitive Cold Agglutinin and a Warm Autoanti-e Treated with Therapeutic Plasma Exchange. College of American Pathologists. Washington, DC.
  • Sigbjørn Berentsen; How I treat cold agglutinin disease. Blood 2021; 137 (10): 1295–1303. doi: https://doi.org/10.1182/blood.2019003809
  • Berentsen S. Cold agglutinin disease. Hematology Am Soc Hematol Educ Program. 2016;2016(1):226-231. doi:10.1182/asheducation-2016.1.226
Thomas Thompson Profile picture square

Thomas (Zach) Thompson, M.D.

Fellow, Cellular Therapy
Mayo Clinic

Margaret DiGuardo profile photo square

Margaret (Maggie) DiGuardo, M.D.

Senior Associate Consultant, Transfusion Medicine
Mayo Clinic

A 49-year-old retired sailor presented to the Emergency Department with an intermittent rash with left-sided facial numbness and weakness. He was treated with a 7-day course of prednisone for presumed Bell’s palsy. A month later, he returned with worsening vision in his right eye, high-pitched tinnitus, alopecia, arthralgias, and a ~10 lb. weight loss. Initial imaging of the head/neck were unremarkable. Notably, his creatinine was elevated and nephrology was consulted for acute kidney injury. Subsequent kidney biopsy revealed an acute plasma cell-rich tubulointerstitial nephritis (Image 1). Immunohistochemical stains demonstrated spirochetes in the tubular epithelial cells and interstitium confirming active renal infection (Image 2).  

Figure 1: Inflammation with clusters of plasma cells within the interstitium 
Figure 2: Immunohistochemical stains positive for spirochete organisms within the tubules and interstitium

What is the infectious etiology?

  • Leptospira interrogans
  • Treponema pallidum
  • Borrelia burgdorferi
  • Brachyspira pilosicoli

The correct answer is ...

Treponema pallidum

Treponema pallidum is the infectious agent causing syphilis. T. pallidum is a stealthy pathogen due to its ability to evade the host immune system and cause disseminated infection (1). Historians believed that the “sailor’s handshake” served as a way to assess the presence of epitrochlear lymphadenopathy associated with syphilis, but it is nonspecific and frequently observed in non-syphilis infections (2). 

Syphilis has four distinct stages of disease, presenting with different signs and symptoms at each stage (3). Primary syphilis generally presents with painless lesions at the original site of infection, referred to as ‘chancres,’ typically around the genitals, anus or in and around the mouth. Symptoms of primary syphilis can be relatively mild and go unnoticed (1). Symptoms of secondary syphilis include a maculopapular skin rash, swollen lymph nodes, and fever (1,3). When infections with T. pallidum are left untreated, syphilis can progress to a latent stage, during which time patients are entirely asymptomatic, but years later may develop tertiary syphilis (1). Tertiary syphilis can present with disseminated symptoms and can affect the heart, brain, and other organs. Tertiary syphilis can occur 10-30 years after initial infection (1). 

There are two types of serologic tests used to diagnose syphilis: nontreponemal and treponemal-specific tests (1). Nontreponemal tests detect antibodies to cardiolipin and lecithin antigens that are released from infected host cells and are thus considered nonspecific biomarkers of infection (1). Nontreponemal tests are semi-quantitative and are reported with an endpoint titer. Changes in this titer are followed after treatment to monitor for therapeutic response. In contrast, treponemal tests detect antibodies against specific treponemal antigens and are thus considered more specific (1). Once a patient has a positive treponemal test, they can remain positive for life, making serial testing for these antibodies not useful as a monitor for response to therapy or for confirming diagnosis in a patient with previously treated disease. 

There are two algorithms for diagnosis of syphilis infections: the traditional and the reverse syphilis screening algorithms (3). The traditional algorithm begins with a non-treponemal assay, which if positive is followed with a treponemal assay to confirm the result. In contrast, the reverse screening algorithm begins with a treponemal, T. pallidum specific immunoassay as the initial screen, which if positive is followed by the rapid plasma reagin (RPR) as the confirmatory test. If the results are discordant results for these tests, the T. pallidum particle agglutination assay (TTPA) is performed. The advantage of the reverse algorithm over the traditional version is multi-fold, including the ability to screen samples initially using high-throughput automated assays rather than manual nontreponemal methods, and the reverse algorithm has been associated with higher sensitivity during primary disease. Nucleic acid amplification tests (NAATs) have also been developed and evaluated for detection of T. pallidum DNA, but to date none are currently available commercially (3). NAATs are highly specific, but sensitivity of the assay is largely dependent on the specimen source (3). 

For diagnosis of neurosyphilis, additional testing on cerebrospinal fluid needs to be performed (1). A positive cerebrospinal fluid venereal disease research laboratory (CSF-VDRL) is highly specific for neurosyphilis but is associated with lower sensitivity. T. pallidum is a thin, tightly coiled, microaerophilic spirochete that does not grow well on standard culture media. Darkfield microscopy was utilized in the past to identify the organisms directly from the chancre, however, it is infrequently used today (3). T. pallidum can also be visualized by histopathology as was done in this case (3). Although the spirochetes cannot be seen on H&E, they can be highlighted by silver impregnation methods, specific immunohistochemistry (IHC), or immunofluorescence (3). Silver impregnation methods are nonspecific but T. pallidum IHC is recommended for optimal sensitivity (3). 


  1. Henao-Martinez, A F., Johnson, S C. Diagnostic tests for syphilis. Neurol Clin Pract. 2014 Apr; 4(2): 114-122.
  2. O’Glasser, A Y., Kent C. M., J Med. 2016; 129(4): 379-381.
  3. Theel, E. S., Katz, S., Pillay, A. Molecular and direct detection tests for Treponema pallidum subspecies pallidum: A review of the literature, 1964-2017. CID. 2020 June; 71(1): S4-S12.

Anisha Misra, Ph.D.

Fellow, Clinical Microbiology
Mayo Clinic


Photo of Elitza Theel, Ph.D.

Elitza Theel, Ph.D.

Consultant, Clinical Microbiology
Mayo Clinic

Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science


Audrey Schuetz, M.D.

Consultant, Clinical Microbiology
Mayo Clinic

Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science


A 54-year-old man underwent a right lobectomy for a right thyroid nodule. At time of surgery the thyroid was very firm, nodular, and adherent to the trachea. On gross examination, within the lower pole, there was a 2.2 cm, well-circumscribed, nonencapsulated, firm and white mass. 

Figure 1
Figure 2
Figure 3
Figure 4

Which of the following is the most likely diagnosis?  

  • Fibrous variant of chronic lymphocytic thyroiditis
  • Squamous cell carcinoma
  • Sclerosing mucoepidermoid carcinoma with eosinophilia
  • Hodgkin's lymphoma

The correct answer is ...

Sclerosing mucoepidermoid carcinoma with eosinophilia.

Sclerosing mucoepidermoid carcinoma with eosinophilia (SMECE) is a rare thyroid neoplasm and a described entity in the World Health Organization Classification of Tumours of Endocrine Organs. (1) SMECE occurs most often in women (female-to-male ratio of 7:1) and has been documented in a wide age range (32-89 years). (1,2) Patients usually present with a slow growing, painless thyroid mass. Unlike the association between conventional salivary gland mucoepidermoid carcinoma and the CRTC1-MAML2 fusion transcript, no consistent molecular alteration has been identified in SMECE. (3) 

Microscopically SMECE is characterized by a dense hyalinized fibrotic stroma, a mixed inflammatory infiltrate with abundant eosinophils, and a neoplastic cellular infiltrate with squamous and glandular differentiation. The neoplastic cells form thin strands and variable sized nest. By immunhistochemically staining the tumor cells are strongly reactive to antibodies against p63 and show variable reactivity to antibodies against TTF-1. (4,5) Special stain mucicarmine highlights the scattered mucocytes. 

SMECE invariably occurs in a background of chronic lymphocytic thyroiditis, therefore, a main differential is the fibrosing variant of chronic lymphocytic thyroiditis with squamous metaplasia. The squamous nests in lymphocytic thyroiditis should be relatively bland and not have malignant features, such as vascular or perineural invasion. Additional distinguishing features include fibrosis confined to the thyroid and lack of the following mucocytes, mucin pools, and abundant eosinophils. Primary thyroid squamous cell carcinoma (SCC) is a rare occurrence and is more likely to represent metastasis. Differentiating SCC from SMECE may be challenging, especially if the glandular component is not prominent. However, SCC tends to show more pronounced atypia, lacks mucin pools, and is less commonly associated with a prominent fibrohyalinized stroma with abundant eosinophils. Approximately one-third of SMECE cases will metastasize to lymph nodes (6) and can easily be mistaken for nodular sclerosing Hodgkin’s lymphoma. However, cohesive groups of cells and lack of Reed-Sternberg cells will help make the correct diagnosis.


  1. Lloyd, Ricardo V., et al. WHO Classification of Tumours of Endocrine Organs. International Agency for Research on Cancer, 2017.
  2. Chi-Yun, Lai., et al. “Sclerosing mucoepidermoid carcinoma with eosinophilia of thyroid gland in a male patient: a case report and literature review” International Journal of Clinical & Experimental Pathology, vol. 8, no. 5, 2015, pp. 5947-5951
  3. Shah, Akeesha A., et al. “Thyroid sclerosing mucoepidermoid carcinoma with eosinophilia: a clinicopathologic and molecular analysis of a distinct entity.” Modern Pathology, vol 30, no. 3, 2017, pp. 329–339.
  4. Hunt, J., et al. “p63 expression in sclerosing mucoepidermoid carcinomas with eosinophilia arising in the thyroid.” Modern Pathology, vol 7, no. 5, 2004, pp. 526-529.
  5. Albores-Saavedra, J., et al. “Clear cells and thyroid transcription factor I reactivity in sclerosing mucoepidermoid carcinoma of the thyroid gland.” Annals of Diagnostic Pathology, vol 7, no. 6, 2003, pp. 348-353. 
  6. Shehadeh, N., et al. “Sclerosing mucodepidermoid carcinoma with eosinophilia of the thyroid: a case report and review of the literature.” American Journal of Otolaryngology, vol 25, no. 1, 2004, pp. 48-53. 

Lacey Schrader, M.D.

Fellow, Surgical Pathology
Mayo Clinic

Michael Rivera, M.D.

Consultant, Anatomic Pathology
Mayo Clinic

Assistant Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

A 23-year-old man presented with anasarca with serum albumin of 1.5 g/dL, and urine protein to creatinine ratio of 4.8 g/g. Serologic studies for hepatitis B and C were negative. A renal biopsy was performed that showed the following lesion.

Figure 1: Trichrome stain
Figure 2: EM

What is this lesion?

  • Focal segmental glomerulosclerosis, perihilar variant
  • Focal segmental glomerulosclerosis, tip variant
  • Focal segmental glomerulosclerosis, NOS
  • Focal segmental glomerulosclerosis, cellular variant

The correct answer is ...

Focal segmental glomerulosclerosis, tip variant.

Focal segmental glomerulosclerosis (FSGS) is a pattern of glomerular injury that typically presents with moderate to heavy proteinuria. It can be classified based on etiology or morphology. Based on etiology, FSGS can be idiopathic (primary); genetic (non-syndromic or syndromic); infection-related (e.g., HIV, parvovirus B19); drug-induced (e.g., interferon alpha and beta, lithium); immunologic disease-related (e.g., SLE, Guillain-Barre syndrome); lymphoma/leukemia-related (e.g., Mantle cell lymphoma); vascular-related (e.g., hypertension, sickle cell disease); or adaptive (e.g., obesity, nephron loss). Based on glomerular morphology, Columbia classification defines five variants of FSGS, which are: 1. collapsing variant, 2. tip variant, 3. cellular variant, 4. perihilar variant and 5. FSGS not otherwise specified (NOS).

Tip variant of FSGS is characterized by intracapillary foam cells near the “tip” of the glomerular tuft adjacent to the origin of proximal tubule and an adhesion of the capillary loop to Bowman capsule. The glomerular tuft can herniate into the origin of the proximal tubule. On immunofluorescence, the glomeruli do not show immune complex deposits. There can be IgM and C3 deposition in the segmental scar, which is considered nonspecific trapping. On electron microscopy, the non-sclerosed glomeruli show diffuse (>80%) podocyte foot process effacement.

Patients with tip variant of FSGS are more likely to respond to glucocorticoid therapy compared to other FSGS variants. It has the most favorable prognosis among the variants of FSGS.


  1. Stokes MB, Markowitz GS, Lin J, Valeri AM, D'Agati VD. Glomerular tip lesion: a distinct entity within the minimal change disease/focal segmental glomerulosclerosis spectrum. Kidney Int. 2004;65(5):1690-1702.

Sarwat Gilani, M.B.B.S.

Fellow, Renal Pathology
Mayo Clinic

Lynn Cornell, M.D.

Consultant, Anatomic and Clinical Pathology
Mayo Clinic

Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

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