When Rapid Blood Culture Identification Results Don’t Correlate, Part 1: Clinical Correlation Needed

More and more laboratories perform rapid (i.e., multiplex PCR) blood culture identification. For the most part, it has been a wonderful addition to the laboratory workflow, not to mention the added benefits of provider satisfaction and improved patient care. Because the PCR only provides the organism identification (sometimes only to the family-level, i.e.; Enterobacteriaceae), laboratories must continue to culture the positive blood for definitive identification and/or antimicrobial susceptibility results. So what do you do when the results don’t correlate?

The Issue

From time to time, the PCR result is not going to correlate with the direct Gram stain or with the culture results. Although this is an issue one would fully anticipate, what do you do when this happens? Do you take some sort of action to arbitrate? Do you report the results as is?

First of all, the PCR assays do not detect all organisms. They only detect the most common bloodstream pathogens. Therefore, one should fully expect to observe cases in which the Gram stain would be positive, but the PCR results would be negative (scenario 1).  This is not a surprise.

Additionally, one should also assume that the PCR will occasionally detect organisms that were present at the lower limit of detection of the Gram stain. An example of this would be that the Gram stain is positive for one morphology (i.e.; Gram-positive cocci), but the PCR is positive for two organisms (i.e.; Staphylococcus and a Proteus species). Most of these cases tend to correlate with culture. In other words, although the second organism was not originally observed in the Gram stain, it was detected via PCR and then it also subsequently grew in culture (scenario 2).

Another type of discordant result laboratories sometimes experience is when the organism detected via PCR does not grow in culture for whatever reason. Similar to scenario 2 stated above, except that the culture is also negative for the second organism (scenario 3). Perhaps the patient was treated with antibiotics and the organism is no longer viable for culture? Perhaps a sampling or processing error was to blame?

The Solution

Depending on the scenario and how much work you want to do, you can either repeat testing or try an alternative method. Take scenario 2 for example. If the PCR detects two organisms and the Gram stain is only positive for one, then review of the original Gram stain is warranted. It is possible that the Gram-negative was somehow missed. Our eyes tend to go to the darker, more obvious structures. Perhaps the Gram-negative organism was faintly stained and it was overlooked? It is also possible that the Gram-positive is present in much lower numbers and only Gram-negative organism was originally observed. If the Gram stain result remains the same after review (only one organism observed), then there is nothing much left to do except to wait for the culture. That being said, an alternative method, such as acridine orange can be utilized in this type of scenario (two different cell morphologies). Acridine orange is a fluorescent stain that improves organism detection, as it is more sensitive than the Gram stain (1, 2).

If only the Proteus is growing (and the Staphylococcus isn’t from scenario 2) and we normally subculture positive blood to blood, chocolate, and MacConkey agars, then perhaps including an additional media that inhibits Gram-negative growth would be beneficial.

Scenario 3 can be a little more difficult to solve because you can’t make a non-viable organism grow. It just is what it is. [Spoiler alert: in next month’s blog I plan to write about when you should change your thinking from true-positive to false-positive.]

Regardless of why the result is discrepant, our laboratory appends a comment to the discordant result which says, “Clinical correlation needed.” This lets the clinician know that the results are abnormal and that they must use other relevant information to make a definitive diagnosis. In addition to the comment, we also make sure the discrepancy is notified to laboratory technical leadership (i.e.; Doctoral Director, Technical Lead/Specialist). This allows us to keep track of discrepancies as they may become important to know about in the future (see next month’s blog).

The Conclusion

In terms of organism detection, nucleic assays (i.e., NAATs) can provide superior sensitivity over antigen and culture-based methods of organism detection (i.e., sensitivity = PCR > culture > Gram). From the laboratory perspective, other potential benefits of utilizing nucleic acid detection methodologies include decreased TAT, simplified workflows, and reduced hands-on time. In terms of patient care, many have noted improved outcomes due to increased sensitivity and decreased time to result.

Although advances in technology can significantly improve analytical performance, they can also add complexity to the post-analytical process. Making sense of the results can sometimes lead to confusion. It is important to know the product’s limitations and what your risk(s) is. This should already be known and included in your Individualized Quality Control Plan (IQCP). Lastly, guiding the clinician to proper result interpretation is also important to maintain valuable patient care.
References

  1. Mirrett, S., Lauer, B.A., Miller, G.A., Reller, L.B. 1981. Comparison of Acridine Orange, Methylene Blue, and Gram Stains for Blood Cultures. J. Clin. Microbiol. 15(4): 562-566.
  2. Lauer, B.A., Reller, L.B., and Mirrett, S. 1981. Comparison of Acridine Orange and Gram Stains for Detection of Microorganisms in Cerebrospinal Fluid and Other Clinical Specimens. J. Clin. Microbiol. 14(2): 201-205.

 

Martinez Headshot-small 2017

-Raquel Martinez, PhD, D(ABMM), was named an ASCP 40 Under Forty TOP FIVE honoree for 2017. She is one of two System Directors of Clinical and Molecular Microbiology at Geisinger Health System in Danville, Pennsylvania. Her research interests focus on infectious disease diagnostics, specifically rapid molecular technologies for the detection of bloodstream and respiratory virus infections, and antimicrobial resistance, with the overall goal to improve patient outcomes.

Hematopathology Case Study: A 64 Year Old Man with Widespread Lymphadenopathy

Case history

A 64-year-old, previously healthy man presented with a history of cervical and axillary lymphadenopathy of unknown duration. He did not endorse night sweats, weight loss, or fever. Radiologic examination (CT chest and MRI abdomen) revealed numerous enlarged mediastinal, peritracheal, periaortic, periportal and retroperitoneal lymph nodes. He underwent excisional biopsy of a 3.5 cm axillary lymph node.

foll-lymph

Microscopic Description

Histologic examination of the node revealed distortion of nodal architecture by a proliferation of neoplastic-appearing follicles. Follicles were distinct from one another, and closely packed. In areas the follicles were present back-to-back. Follicular centers were comprised of mostly small, cleaved centrocytes and showed no obvious zonation. There was loss of tingible body macrophages.

Immunophenotyping

Immunohistochemical analysis revealed CD20-positive B cells in a follicular pattern. The germinal centers revealed an underlying follicular dendritic meshwork highlighted by staining for CD21. Interestingly, while the germinal centers demonstrated immunopositivity for BCL-6, there was minimal to absent CD10 staining on follicular B cells. Analysis of BCL-2 staining revealed only few cells to be positive within the follicular centers, consistent with resident follicular helper T cells (Th cells). Equivalent numbers of CD3 and CD5 positive T cells were noted in the interfollicular zones. The Ki-67 proliferation index was estimated at 15-20% within follicular centers. Flow cytometric phenotyping demonstrated a lambda light chain restricted clonal B-cell population expressing CD20, CD19 and, FMC7. These neoplastic B-cells were negative for CD5 and CD10 expression.

Diagnosis

The morphologic features were consistent with Follicular Lymphoma; however the phenotype (BCL-2 negativity in follicular centers) was unusual for this diagnosis. Fluorescence in situ hybridization (FISH) was negative for an IgH/BCL-2 fusion; however, a BCL-6 rearrangement at the 3q27 locus was detected in 70% of the cells.  Taken together, a diagnosis of Follicular Lymphoma with a BCL-6 rearrangement was given.

Discussion

Follicular lymphoma (FL) is a germinal center derived B-cell neoplasm. The majority of cases exhibit the pathognomonic translocation t(1418)(q32; q21). This translocation leads to overexpression of the anti-apoptotic BCL-2 protein, which can be detected by immunohistochemistry on germinal center B cells. Lymphoma cells are usually positive for germinal center origin markers BCL-6 and CD10 and do not co-express CD5. As exhibited in this case, FL can exhibit biologic heterogeneity and may not express these typical markers. The follicular proliferation with absence of germinal center zonation and tingible body macrophages as seen in this case represents classic morphology of follicular lymphoma but aberrant phenotypic markers [and absence of t(14;18)] may be a pitfall in this diagnosis.

FL with lacking of CD10 expression, BCL-2 expression, and t(14;18) translocation and harboring only BCL-6 positivity with 3q27 rearrangement is rare. Only few such cases have been reported in the literature. Published data reveals that the hallmark t(14;18) translocation is absent in about 10-15% of FL. The majority of these cases are negative for BCL-2 expression, and 9-14% of them demonstrate BCL-6 rearrangement (3q27 locus). While BCL-6 rearrangement can be present in both the usual t(14;18) harboring FL, and also in cases without t(14;18), the latter is rare. Interestingly, studies have shown BCL-6 rearrangements to be more frequent in in BCL-2 rearrangement negative FL – which is evidence of the anti-apoptotic role of non-rearranged BCL-6 in certain microenvironments.

One third of t(14;18) negative FL are also reported to have rare or negative expression of CD10. Morphologically, this subtype has been shown to have significantly larger follicles than  their t(14;18)-positive counterparts, but the distinction may not be obvious in all cases. Some of these cases are shown to have a component of monocytoid B cells. This findings can be problematic in differentiating these FL cases from marginal zone lymphoma (MZL) that can also harbor BCL-6 rearrangements and lack t(14;18), CD10 and BCL-2 positivity. Absence of prominent marginal zone proliferation, BCL-6 protein expression and characteristic genetic alterations present in MZL, such as trisomies 3, 7, and 18 can help differentiating MZL from t(14;18)-negative FL.

This case highlights the importance of morphologic evaluation of a excisional biopsy tissue, and FISH studies to help identify the rare t(14;18) negative FL. While the reported cases are few, there is no published difference in prognosis or survival when compared to t(14;18)-positive FL. As such, it is not clear whether the follicular lymphoma grading scheme applies to t(14;18)-negative FL; however, no significant grading difficulties or differences have been reported.

References

  1. Jardin F, Gaulard P, Buchonnet G, et al. Follicular lymphoma without t(14;18) and with BCL-6 rearrangement: a lymphoma subtype with distinct pathological, molecular and clinical characteristics. Leukemia. 2002;16:2309–2317
    2. Leich E, Salaverria I, Bea S, et al. Follicular lymphomas with and without translocation t(14;18) differ in gene expression profiles and genetic alterations. Blood. 2009;114(4):826-834.

 

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Aadil Ahmed, MD is a 3rd-year anatomic and clinical pathology resident at Loyola University Medical Center. Follow Dr. Ahmed on Twitter @prion87.

Mirza-small

-Kamran M. Mirza, MD PhD is an Assistant Professor of Pathology and Medical Director of Molecular Pathology at Loyola University Medical Center. He was a top 5 honoree in ASCP’s Forty Under 40 2017. Follow Dr. Mirza on twitter @kmirza.

Hematopathology Case Study: A 57 Year Old Male with History of Malignant Melanoma

Case History

A 57 year old male with a history of stage IA malignant melanoma presented with a new pink nodule on the right shoulder (see image provided) that has persisted for one month following a tetanus shot. Resultant specimen is a punch biopsy of the lesion.

calcl1.jpg

 

Diagnosis

calclhe10
H&E, 10x
calclhe20.jpg
H&E, 20x
calclhe50
H&E, 50x

 

calccd20
CD20
calccd3
CD3
calccd4
CD4
calccd8
CD8
calccd30low
CD30, low power
calccd30high
CD30, high power
calclgranzyme
Granzyme
calclperforin
Perforin

Sections show a punch biopsy of skin with a superficial as well as deep dermal infiltration of small and large lymphocytes. No epidermotropism is noted. An admixed background of inflammatory cells including eosinophils, neutrophils, and histiocytes is present.

By immunohistochemistry, CD2, CD4, and CD5 highlight the abundance of lymphocytes indicating a dominant T-cell population. CD30 highlights a major subset of larger lymphocytes that co-express perforin. Granzyme is positive only in a small subset of cells. CD3 is present in a subset of CD30 positive cells indicating downregulation of CD3 in neoplastic cells. By Ki-67, the proliferation index is focally high (70%). CD20 highlights rare B-cells. CD8 is positive in a small fraction of T-cells. EMA is negative.

Overall, the diagnosis is that of a primary cutaneous CD30 positive T-cell lymphoproliferative disorder. The differential diagnosis includes lymphomatoid papulosis, type C and primary cutaneous anaplastic large cell lymphoma.

Discussion 

Primary cutaneous CD30 positive T-cell lymphoproliferative disorders are the second most common cutaneous T-cell lymphoma (30% of cases). The primary groups within this entity include lymphomatoid papulosis (LyP) and cutaneous anaplastic large cell lymphoma.

Primary cutaneous anaplastic large cell lymphoma (C-ALCL) is composed of larger cells that are anaplastic, pleomorphic, or immunoblastic morphology that express CD30 in over 75% of the tumor cells. C-ALCL most commonly affects the trunk, face, extremities, and buttocks and often present as a solitary or localized nodules or tumors with ulceration. Clinically, the lesions may show partial or complete remission similar to LyP but often relapse in the skin. Interestingly enough, approximately 10% of cases may disseminate to local lymph nodes.

The histologic pattern of C-ALCL demonstrates a non-epidermotropic pattern with cohesive sheets of large CD30 positive T-cells. Ulcerating lesions may show a morphologic pattern similar to LyP with abundant inflammatory cells such as histiocytes, eosinophils, neutrophils with few CD30 positive tumor cells. By immunophenotyping, the tumor cells are CD4 positive with variable loss of CD2, CD5 or CD3 and express cytotoxic markers such as granzyme B, TIA1, and perforin. Unlike systemic anaplastic large cell lymphoma, C-ALCL does not express EMA or ALK.

The 10 year disease related survival of C-ALCL is 90%. Lymph node status or multifocal lesions does not alter prognosis significantly.

The differential diagnosis also include LyP, type C. These lesions often present on the trunk and extremities and are characterized by popular, papulonecrotic and/or nodular skin lesions. After 3-12 weeks, the skin findings may disappear. Up to 20% of LyP may be preceded by, have concurrent, or followed by another type of lymphoma such as mycosis fungoides (MF), C-ALCL, or Hodgkin lymphoma.1

Briefly, there are up to 5 types of LyP (types A-E).2,3 The more recently described LyP type D and E are determined by either simulating an epidermotropic aggressive CD8 positive CTCL and angiocentric and angioinvasive CD8 positive CTCL, respectively.

LyP has an excellent prognosis but since these patients may have other lymphomas, long term follow up is advised.

Overall, C-ALCL and LyP type C show considerable overlap both morphologically and clinically so close clinical follow up is recommended, however both demonstrate an excellent prognosis.

References

  1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2008.
  2. Cardosa J, Duhra P, Thway Y, and Calonie E, “Lymphomatoid papulosis type D: a newly described variant easily confused with cutaneous aggressive CD8-positive cytotoxic T-cell lymphoma.” Am J Dermatopathol 2012 Oct; 34 (7): 762-765.
  3. Kempf W, Kazakov DV, Scharer L, et al. “Angioinvasive lymphomatoid papulosis: a new variant simulating aggressive lymphomas.” Am J Surg Pathol 2013 Jan; 37(1): 1-13.

 

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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.

Microbiology Case Study: 6 Year Old Male with Meningitis

Case History

A 6 year old male presented to the emergency department with a concern for ventriculo-peritoneal shunt (VP) malfunction. His past medical history is significant for myelomeningocele and hydrocephalus since birth. On arrival, symptoms included high fever (102.7°F), headaches and swelling at the VP shunt catheter site in the neck. Over the past week, his mother also noted nausea, vomiting and diarrhea. CT scan of the head revealed increased size of the 3rd and lateral ventricles which was concerning for either a VP shunt malfunction or infection. Lab work showed a white count of 13.5 TH/cm2 and elevated CRP values suggestive of an infection/inflammatory process. He was taken to surgery for VP shunt removal and placement of an external ventricular drain (EVD). Intra-operatively, purulent yellow material was noted at both the proximal and distal ends of the catheter. Cerebrospinal fluid (CSF) was sent for Gram stain and bacterial culture. He was started on vancomycin and ceftriaxone.

 Laboratory Identification

listmon1
Image 1. CSF Gram stain prepared from the cytospin showed many white blood cells and Gram positive bacilli (100x oil immersion).
listmon2
Image 2. Gram stain from the liquid media culture showing gram positive bacilli (100x oil immersion).
listmon3.png
Image 3. Small, grayish colonies with a narrow zone of beta hemolysis grew on blood agar after 48 hours incubation in a 35°C incubator with 5% CO2.

Bacterial cultures collected from a shunt tap and intra-operatively both showed short gram positive bacilli on Gram stain (Image 1&2). The organism grew on blood and chocolate agars as small, gray colonies with a narrow zone of beta-hemolysis when observed closely (Image 3) after incubation at 35°C in CO2. The isolate was positive for catalase and showed a “tumbling motility.” MALDI-TOF MS identified the isolate as Listeria monocytogenes.

 Discussion

Listeria species are gram positive bacilli that grow as facultative anaerobes and do not produce endospores. The major human pathogen in the Listeria genus is L. monocytogenes and it is found in soil, stream water, sewage & vegetable matter and may colonize the gastrointestinal tract of humans and animals.

The most common mode of transmission is ingestion of contaminated foods, in particular, raw milk, soft cheeses, deli meats and ice cream. L. monocytogenes’ ability to grow at cold temperatures (4°C) permits multiplication in refrigerated foods. In a healthy adult, it causes an influenza like illness and gastroenteritis. Pregnant women are especially susceptible to disease and neonates infected in utero can develop granulomatosis infantiseptica which can lead to miscarriage, stillbirth or premature delivery. Elderly or immunocompromised can present with a febrile illness, bacteremia and meningitis (20-50% mortality).

In the microbiology laboratory, L. monocytogenes is usually identified via blood, CSF or placental bacterial cultures. It grows well on standard agars and after overnight incubation, the small, gray colonies show a narrow zone of beta hemolysis on blood agar. L. monocytogenes is positive for catalase & esculin and the CAMP test demonstrates block like accentuated hemolysis. It has characteristic tumbling motility at room temperature and an umbrella shaped motility pattern in semi-solid agar.  Automated methods of identification provide reliable species level differentiation on the majority of current platforms.

Susceptibility testing should be performed on isolates from normally sterile sites. Ampicillin, penicillin, or amoxicillin are given for L. monocytogenes, and gentamicin is often added for its synergistic effect in invasive infections. Trimethoprim-sulfamethoxazole and vancomycin can be used in cases of allergy to penicillin. Cephalosporins are not effective for treatment of listeriosis.

In the case of our patient, after L. moncytogenes was identified, his antibiotic therapy was changed to ampicillin and gentamicin. Antibiotics were administered for 3 weeks before the placement of a new VP shunt. On further questioning, his mother revealed his diet consisted heavily of hot dogs and soft cheeses. She was educated on how to prevent subsequent infections prior to discharge.

 

JKO

-Jaspreet Kaur Oberoi, MD, is a Pathology resident at the University of Mississippi Medical Center. 

 

Stempak

-Lisa Stempak, MD, is an Assistant Professor of Pathology at the University of Mississippi Medical Center in Jackson, MS. She is certified by the American Board of Pathology in Anatomic and Clinical Pathology as well as Medical Microbiology. She is the director of the Microbiology and Serology Laboratories. Her interests include infectious disease histology, process and quality improvement and resident education.

New Year. New Skills.

I do not recall if it was an email or if I saw it on the ASCP website, but the byline caught my attention: New Year. New Skills. My mind quickly started racing. January marks a fresh beginning, the time to make new resolutions, the time to feel the excitement of new possibilities. 

The Issue

We are more than halfway through the month and I have yet to identify the skill I would next like to acquire. So many questions! So much to learn, so little time! How do you choose what to focus on? Where do you start? What can you manage? Is there anyone who can help or teach you? And if you are like me, you might also ask yourself, “Why do I always pile more on my plate?” Maybe this is the year you choose to learn to say no? Nah. So what’s it going to be?

The Solution

Since our lives are all different and there are millions of possible distinct scenarios, I will share what I decided to do. First, I evaluated my work-life balance and determined if I wanted to acquire a skill that would benefit my work (career and ambition) or lifestyle (health, pleasure, leisure, family) (1). I also took into consideration how much more I could fit onto my already overflowing plate.

I decided to work on something that would help me with both work and lifestyle (because who doesn’t like to maximize their return on investment?). I chose something I do not like to do, something that scares me, something I have difficulty with, something I avoid like the plague, but most importantly it’s something that I wish I could do better; a skill that I envy: having difficult conversations.

Communication is a vital component of our lives. We all communicate, but how many of us have mastered the skill of communicating? Also, there are many aspects of communication (2). Poor communication can make or break a situation or relationship. Being able to communicate well is a great skill to possess (3). Reference two provides a long list of skills that I highly recommend you also take a look at (https://www.thebalance.com/communication-skills-list-2063737). I went down the list and individually assessed which skills I feel that I do well with and which ones I do not (2). This little exercise served as a reality check as to where I stand in regard with my aptitude to communicate. I invite you to do the same. You may be surprised at what you find!

The Importance of Good Communication

As a laboratory director, many facets of my job depend on my ability to communicate well. I must communicate with clinicians, technologists, administrators, other coworkers, vendors, students, etc. Not only do I communicate with a variety of groups of people, in a multitude of different platforms (individually, small groups and meetings, or large groups; such as national conferences), but it is also important that my written, verbal, and non-verbal communication skills are clear and easily understood.

As laboratory professionals, one very important aspect of our job is to communicate critical results. It is essential that we not only relay the data, but it is equally important for us to communicate it well so that the clinician completely understands the information so that they can properly care for the patient. Moreover, we must not forget the golden rule: garbage in, garbage out. What I mean by this is that good communication should begin in the pre-analytical phase. We want the clinician to provide the laboratory with the best possible specimen so that in turn, we can provide them with the most accurate result. So how do we ensure that we obtain the best possible specimen? We communicate.

The laboratory communicates our needs to the provider in order to properly do our job. For example, we provide detailed information on how to properly collect specimens, which container type to use, how to handle the specimen, how much (volume) specimen to submit, which temperature to submit the specimen, etc. Properly communicating these details is essential.

The Difficult Conversation

As laboratory professionals, we are just one part of a larger healthcare team. If you stop to think about it, we all have to participate in difficult conversations as part of our jobs. Doctors have to tell patients that they are going to die, laboratory professionals have to tell clinicians we lost their specimen, executive administrators have to tell downstream leadership that the budget has been cut again, managers and supervisors have to tell employees they are being written up or worse. Being able to successfully have a difficult conversation would serve us all well. As such, most institutions provide classes or webinars to help employees develop this skill.

The definition of difficult is: not easily or readily done; requiring much labor, skill, or planning to be performed successfully; hard (4). Carrying out a difficult conversation with grace is an extraordinary skill that encompasses a variety of communication attributes. Regardless of the scenario, the communicator must be clear, articulate, and courteous. However, depending on the scenario, being concise, confident, strategic, diplomatic, convincing, empathetic, motivating, open-minded, and/or quick thinking may also be useful skills to possess during a difficult conversation. Other valuable skills are conflict management, being able to explain, and/or listening. 

The Conclusion

For many, the New Year marks the time to set new goals, to accept new challenges, and welcome new beginnings. Why not use this opportunity to learn a new skill? The good news is that no matter what your new skill will be, it will also benefit your health. In order to acquire a new ability, you must work to actively learn to become proficient in that ability; therefore learning a new skill will also benefit your brain function. There are many studies that demonstrate that active learning keeps the mind sharp (5). Challenging your mind improves brain function and active learning slows cognitive decline (6). If you want to be brave, then don’t only choose a skill that will be fun or helpful, but choose to learn something that also challenges you to face one of your fears. For me, I hope to learn how to master the art of having difficult conversations….successfully. In the words of Marie Curie, “Nothing in life is to be feared, it is only to be understood. Now is the time to understand more, so that we may fear less.”

Happy learning! Happy New Year!

 

The References

  1. Work-life Balance. https://en.wikipedia.org/wiki/Work–life_balance. Accessed January 16, 2018.
  2. The balance. List of Communication Skills for Resumes. https://www.thebalance.com/communication-skills-list-2063737. Accessed January 16, 2018.
  3. The balance. Communication Skills for Workplace Success. https://www.thebalance.com/communication-skills-list-2063779. Accessed January, 16, 2018.
  4. com. Difficult. http://www.dictionary.com/browse/difficult. Accessed January 16, 2018.
  5. Stenger, M. 2013. New Study Shows How Active Learning Improve Cognitive Function. https://www.opencolleges.edu.au/informed/other/new-study-highlights-activities-to-improve-cognitive-function-6008/. Accessed January 17, 2018.
  6. Park, D.C., Bischof, G.N. 2013. The aging mind: neuroplasticity in response to cognitive training. Dialogues Clin Neurosci. 15(1): 109-119. PMC23576894. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3622463/. Accessed January 17, 2018.

 

Martinez Headshot-small 2017

-Raquel Martinez, PhD, D(ABMM), was named an ASCP 40 Under Forty TOP FIVE honoree for 2017. She is one of two System Directors of Clinical and Molecular Microbiology at Geisinger Health System in Danville, Pennsylvania. Her research interests focus on infectious disease diagnostics, specifically rapid molecular technologies for the detection of bloodstream and respiratory virus infections, and antimicrobial resistance, with the overall goal to improve patient outcomes.

Hematopathology Case Study: An 18-year-old Man with Acanthocytosis

Case History

We were asked to review the peripheral blood smear of an 18-year-old male who had presented to the emergency department with shortness of breath and abdominal distension. His past medical history was significant for numerous hospitalizations for recurrent fungal and bacterial pneumonia, pulmonary abscesses, osteomyelitis, necrotizing granulomas, and cervical lymphadenopathy requiring multiple lymphadenectomies. This history dates back to when he was 3 months old.

Blood Smear findings

The CBC demonstrated severe anemia and mild leukopenia. The peripheral blood smear showed numerous acanthocytes and poikilocytosis shown below.

McLeodAcantyocytes3

McLeodAcantyocytes4

Additional Clinical Findings

Abdominal ultrasonography demonstrated hepatosplenomegaly with enlarged porta-hepatis lymph nodes. Additionally, chest CT scanning demonstrated bilateral mass-like consolidations, prominent hilar lymphadenopathy, and osteolytic lesions of the vertebral bodies. A comprehensive investigation for opportunistic infections was negative. Lung and vertebral body biopsies (not pictured here) revealed poorly formed granulomas. A blood transfusion was considered; however, the patient had previously been demonstrated to express anti-Kx antibodies, which would require transfusion with exceedingly rare blood products.

Diagnosis

The preceding case history describes a patient with a contiguous gene deletion syndrome that includes chronic granulomatous disease (CGD) and the McLeod phenotype, demonstrating a fascinating disorder with important implications in hematopathology and several other disciplines of pathology.

Discussion

McLeod syndrome is a rare, X-linked disorder characterized by the deletion of the XK gene which encodes for the Xk protein. Overall, the lack of synthesis of the Xk protein leads to the lack of expression of the Kx antigen which in turn leads to a marked decrease in the quantities of Kell antigens. In this case, due to the presence of an anti-Kx antibody, the patient would require transfusion with either Kell-null or McLeod phenotype blood products. Unfortunately, only one unit of compatible blood was identified when the rare blood donor database was queried. The clinical team therefore elected for treatment with erythropoietin and iron supplementation which eventually lead to a modest increase in the patient’s hemoglobin concentration.

Acanthocytes, or spur cells, are spiculated red cells with a few projections of varying size and surface distribution that can be seen in a variety of clinical conditions including CGD with McLeod red cell phenotype. Other conditions include (but are not limited to) neuroacanthocytosis, malnutrition states, infantile pyknocytosis, (Lu) null Lutheran phenotype, hypothyroidism, myxedema, and Zieve syndrome. Acanthocytes should be distinguished from echinocytes, or burr cells, that also demonstrate multiple small projections but these are uniformly distributed on the red cell surface.

The prominent acanthocytosis seen in McLeod syndrome is thought to be due to an imbalance of the number of lipids in the inner layer relative to the outer layer. Related to this phenomenon is McLeod neuroacanthocytosis syndrome, a disorder with neurologic manifestations including movement disorders, cognitive alterations, and psychiatric symptoms. Although our patient did not exhibit these symptoms, McLeod neuroacanthocytosis syndrome is known to start in early to middle adulthood and the patient will need to be monitored for the onset of neurologic sequelae.

The McLeod phenotype is frequently associated with CGD due to the proximity of the XK gene to the CYBB gene on the X chromosome. The CYBB gene encodes for a subunit of the NADPH oxidase enzyme complex. A deficiency in NADPH oxidase activity leads to the characteristic increased susceptibility to severe bacterial and fungal infections seen in CGD. The nitroblue-tetrazolium test can be used to evaluate NADPH oxidase activity in the white blood cells and can help make a diagnosis of CGD. Histologically, CGD can show prominent necrotizing and non-necrotizing granulomas in various locations throughout the body.

Overall, treatment of CGD with McLeod red cell phenotype is supportive. There is no known cure or definitive treatment. The patient will likely continue to have infections with opportunistic organisms which will be treated on a case by case basis.

References

  1. Heyworth PG, Cross AR, Curnutte JT. Chronic granulomatous disease. Current opinion in immunology. 2003 Oct 31;15(5):578-84.
  1. Jung HH, Danek A, Walker RH, Frey BM, Gassner C. McLeod neuroacanthocytosis syndrome.
  1. Khodadad JK, Weinstein RS, Marsh LW, Steck TL. Shape determinants of McLeod acanthocytes. Journal of Membrane Biology. 1989 Mar 1;107(3):213-8.
  1. Watkins CE, Litchfield J, Song E, Jaishankar GB, Misra N, Holla N, Duffourc M, Krishnaswamy G. Chronic granulomatous disease, the McLeod phenotype and the contiguous gene deletion syndrome-a review. Clinical and Molecular Allergy. 2011 Nov 23;9(1):13.

 

MM

-Michael Moravek, MD is a 2nd year anatomic and clinical pathology resident at Loyola University Medical Center. Follow Dr. Moravek on twitter @MoravekMD

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-Kamran M. Mirza, MD PhD is an Assistant Professor of Pathology and Medical Director of Molecular Pathology at Loyola University Medical Center. He was a top 5 honoree in ASCP’s Forty Under 40 2017. Follow Dr. Mirza on twitter @kmirza.

Hematopathology Case Study: A 45 Year Old Man with Cytopenias and High Ferritin

Case history

A 45 year-old man presented with vomiting and diarrhea for 5 days. Laboratory studies demonstrated anemia and thrombocytopenia, an elevated ferritin level (23,772 ug/L) and methemoglobinemia. Chest roentgenography revealed cardiomegaly. A follow-up ECHO showed a desreased ejection fraction of 15%. Work-up confirmed viral myocarditis and G6PD deficiency as the cause of the cardiac findings and methemoglobinemia respectively. His clinical condition deteriorated despite therapy: he developed acute kidney and liver failure and had worsening cytopenias. A bone marrow biopsy was performed.

Histomorphological findings

image-121
Bone marrow aspirate smear (100x)
BM17-228 HLH 100x-2
Bone marrow aspirate smear (100x)
CPC Core biopsy 20x
Bone marrow core biopsy (H&E, 20x)
CPC Core biopsy 40x
Bone marrow core biopsy (H&E, 40x)
CPC Core biopsy 100x
Bone marrow core biopsy (H&E, 100x)

Evaluation of the peripheral blood (not pictured) confirmed a macrocytic anemia with marked anisopoikilocytosis including schistocytes, polychromasia, nucleated red blood cells, absolute neutrophilia, monocytosis and thrombocytopenia. The marrow aspirate smear demonstrated appropriate maturation in all cell lines. Scattered hemophagocytic histiocytes (pictured above) were noted. The bone core biopsy was high-normocellular for age with progressive trilineage hematopoeisis. Scattered histiocytes with internalized erythroid cells and debris were visualized. There was no increase in blasts. Flow cytometry analysis performed on the bone marrow aspirate did not show a significant increase in blast population. Gating on the lymphocytes did not show a B-cell monoclonal population or T-cell abnormality by markers assayed.

Diagnosis

High-normocellular marrow with progressive trilineage hematopoeisis and prominent hemophagocytic histiocytes.

Overall the patient met the clinical criteria (see below) for Hemophagocytic Lymphohistiocytosis (HLH); with fever (≥38.5 C), splenomegaly, bicytopenia, presence of hemophagocytic histiocytes in bone marrow and high ferritin level (>500ng/mL).

A clinical diagnosis of HLH was rendered.

Discussion

HLH is an uncommon hematologic disorder that is often fatal.  The underlying pathogenesis involves an exaggerated but ineffective inflammatory response of excessive macrophage and T-cell activation, and impairment of natural killer (NK) and cytotoxic T-cell function. HLH has familial and acquired forms. Secondary, or acquired HLH can be associated with infections (especially viral etiologies), underlying malignancy (particularly lymphomas and leukemias), and medications used for systemic lupus erythematosus. Clinically, autoimmune disease-associated HLH overlaps significantly with macrophage activation syndrome (MAS).

HLH is a clinical diagnosis that can be established with molecular testing or by meeting five of eight clinical and laboratory diagnostic criteria according to the HLH-2004 guidelines.

HLH-2004: Revised diagnostic guidelines for HLH10

The diagnosis HLH can be established if one of the two criteria below is met:

  1. A molecular diagnosis consistent with HLH (i.e., reported mutations found in either PRF1 or MUNC13-4); or
  2. Diagnostic criteria for HLH are fulfilled (i.e., at least five of the eight criteria listed below are present:
    • Persistent fever
    • Splenomegaly
    • Cytopenias (affecting ≥2 of 3 lineages in the peripheral blood):
      • Hemoglobin <90g/L (in infants <4 weeks: <100g/L)
      • Platelets <100 x 109/L
      • Neutrophils <1.0 x 109/L
    • Hypertriglyceremia and/or hypofibrinogenemia:
      • Fasting triglycerides ≥3.0 mmol/L (i.e., ≥ 265mg/dl)
      • Fibrinogen ≤1.5 g/L
    • Hemophagocytosis in bone marrow* or spleen or lymph nodes, no evidence of malignancy
    • Serum ferritin ≥ 500µg/L (i.e., 500 ng/ml)
    • Low or absent NK cell activity (according to local laboratory reference)
    • Increased serum sIL2Rα (according to local laboratory reference)

*In hematopathology, the finding of relevance is the presence of hemophagocytic histiocytes in the marrow or other biopsies organs. While debris-laden histiocytes are commonly noted in marrow aspirates, the findings of engulfed erythroid cells is warranted to call a ‘hemophagocytic’ histiocyte.

Often a bone marrow biopsy will be performed in cases where there is clinical suspicion for HLH. This serves to try and visualize the hemophagocytic activity, and to rule out other diseases with similar clinical presentations as HLH. The pathologic evaluation of HLH is tricky, since there is no established criteria for quantitation of hemophagocytic histiocytes in a bone marrow aspirate. Furthermore, hemophagocytosis is not specific to HLH and can be seen in other conditions such as: post-blood transfusion, chemotherapy, sepsis and major operations. Published data shows that the presence of hemophagocytosis has a sensitivity of 83% and a specificity of only 60% in diagnosing HLH.

What about immunohistochemical staining for the histiocytes? While IHC can help outline histicoytic cells, unfortunately, quantitation of hemophagocytic histiocytes in the core biopsy or clot sections with the aid of CD68 immunostains does not correlate well with disease probability either.

Overall, the nonspecificity of hemophagocytosis in the marrow, even when present in high amounts, should remind both pathologists and clinicians that an isolated finding of hemophagocytosis lacks specificity and does not necessarily suggest HLH when the clinical presentation and laboratory findings are not compatible with the diagnosis. However, there still remains value in bone marrow biopsy examination in cases where clinical suspicion for HLH is high; in order to exclude other marrow processes; and in the rare case where there may not have been clinical suspicion of HLH but the presence of hemophagocytic histiocytes can raise that differential.

For additional images of hemophagocytic histiocytes check out these amazing picture tweets by Dr. Kate Dannheim (@KDannheimMD) https://twitter.com/KDannheimMD/status/933128799818002432) and Dr. Bharat Ramlal (@BeRaad87): https://twitter.com/HHPathology/status/926087105381531648

 

References

  1. Ho C, Yao X, Tian L, et al. Marrow Assessment for Hemophagocytic Lymphohistiocytosis Demonstrates Poor Correlation with Disease Probability. Am J Clin Pathol. 2014 Jan;141(1):62-71
  2. Hunter JI et al. HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer. 2007 Feb;48(2):124-31.

 

AIK

-Ayse Irem Kilic is a 1st year anatomic and clinical pathology resident at Loyola University Medical Center. Follow Dr. Kilic on twitter @iremessa.

Mirza-small

-Kamran M. Mirza, MD PhD is an Assistant Professor of Pathology and Medical Director of Molecular Pathology at Loyola University Medical Center. He was a top 5 honoree in ASCP’s Forty Under 40 2017. Follow Dr. Mirza on twitter @kmirza.