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.

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

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 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.

Hematopathology Case Study: A 72 Year Old Female with History of Lung Adenocarcinoma

Case history 

A 72 year-old female with a history of stage IA lung adenocarcinoma diagnosed in 2009 s/p resection underwent a surveillance CT scan of the chest which revealed an enlarged right upper lobe paramediastinal lung nodule. A subsequent MRI of the abdomen and PET scan revealed mediastinal lymphadenopathy with numerous boney lesions. Due to the prior history of lung cancer, a right iliac bone biopsy was performed.

Diagnosis

myesar-he-10
H&E, 10x
myesar-he-20
H&E, 20x
myesar-he-50
H&E, 50x
myesar-cd-45
CD45
myesar-cd-117
CD117
myesar-cd-34
CD34
myesar-cd-68
CD68
myesar-cd-56
CD56
myesar-mpo
MPO
myesar-cd-43
CD43

Sections of bone show an extensive intramedullary infiltration by large cells with moderate amounts of cytoplasm, irregular nuclear contours, moderately condensed chromatin and some cells with inconspicuous nucleoli.

By immunohistochemistry, the neoplastic cells are immunoreactive for CD45, MPO, CD68, CD56, and CD43. The cells are negative for cytokeratins, TTF-1, CD20, CD10, PAX5, BCL6, MUM1 and CD79a. CD3 and CD5 highlight rare scattered T-cells.

Overall, in the context of multiple osseous lesions, these findings are representative for a myeloid sarcoma.

Discussion 

Myeloid sarcoma is a tumor mass consisting of myeloid blasts with or without maturation occurring at any site other than the bone marrow. Infiltration of blasts at any site are not classified as a myeloid sarcoma unless there is effacement of tissue architecture. Frequent sites for involvement by a myeloid sarcoma include skin, lymph node, gastrointestinal tract, bone, soft tissue, and testis.

Detection of a myeloid sarcoma is considered as an equivalent diagnosis of acute myeloid leukemia. It may precede or coincide with AML as well as be a presenting finding in those that relapse from AML.

Morphologically, the blasts may or may not show features of maturation and efface the architecture of the involved site. Immunophenotypically, CD68 is considered the most commonly expressed marker followed by MPO, CD117, lysozyme, CD34, TdT, CD56, CD30, glycophorin and CD4. Interestingly enough, CD123 may be expressed in those cases that also have inv(16). It must be emphasized that those cases that meet criteria for a mixed phenotypic acute leukemia (MPAL) cannot be classified as a myeloid sarcoma.

By cytogenetics, 55% of myeloid sarcomas have aberrant cytogenetic findings including monosomy 7, MLL rearrangements, inv(16), and other chromosomal changes. In the pediatric population, t(8;21) may be observed and is less frequent in adults. NPM1 is mutated in 16% of cases.

Lastly, the differential diagnosis should be kept broad in cases that appear lymphoid in nature yet do not mark appropriately. It is often expressed that the primary morphologic differential is a lymphoma, including lymphoblastic lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, blastic plasmacytoid dendritic cell neoplasm, and other small round blue cell tumors of childhood.

Reference

  1. Swerdlow SH, Campo E, Harris NL, et al.  WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2008

 

PhillipBlogPic-small

-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.

Pathologist on Call: There Is No Perfect Lab Test for Smoking Assessment

Cigarette smoking can affect both innate and adaptive immunity, and introduces concerns when evaluating a patient’s eligibility for surgery. It has been shown to hinder time required for healing and long-term survival of patients. It can promote vascular complications, increase the rates of hepatocellular carcinoma and reduce lung function.1 For lung transplantation, one of the common requirements of eligibility is smoking abstinence for at least 6 months. Smoking post-surgery is associated with worse outcomes for the patients including complications and higher rates of mortality.2 Relapse to smoking post lung transplantation has been reported to range from 11% to 23% in various patient populations.3 As a result, clinical testing for cigarette smoking abstinence is an important part of initial workup and follow-up of transplant patients.

In some situations, the burden of lung allocation weighs heavily on a single clinical laboratory result that is perceived to definitively confirm or exclude active cigarette smoking. This subsequently factors into the decision by the physicians to deem the patient eligible to receive a lung transplant. The perception of nicotine testing as definitive proof of smoking is misleading and does not reflect the complexity of situations that can lead to a positive test result.

How can we assess smoking?

Ideally, many factors should weigh into the final smoking status determination including self-reporting (used historically), witnesses to behavior, odor, and past history including cessation attempts. Clinical laboratory testing is important and thought to be more reliable means for smoking assessment. It can involve testing for nicotine (originating from tobacco or nicotine replacement therapy, NRT) and its metabolites: cotinine, 3-hydroxycotinine (3-OH-cotinine), and nornicotine. Moreover, nicotine contains a number of alkaloids that are not usually present in nicotine-replacement therapies (NRTs) including anatabine and anabasine.4 Nicotine testing can involve a combination of metabolites such as cotinine as well as alkaloids like anabasine. Various sample types have been used including saliva, blood and urine.5 In addition, measurements of the exhaled carbon monoxide (CO) have been used to assess recent smoking status (within the last 8 hours).6

Clinical case: patient with detectable nicotine metabolites

A case involving a patient being considered for lung transplantation was received by our department. The patient had been tested for anabasine, nicotine, and its metabolites in urine. Testing of random urine specimens was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS) at different time points from samples collected during hospital visits (days 0, 38, and 62). The urine contained variable concentrations of nicotine and its metabolites, with anabasine concentrations below the detection limit in 2 out of the 3 testing instances. Testing at day 0 showed an interfering substance that prevented the determination of accurate anabasine concentration. The nicotine and its metabolite concentrations in the random urine specimens were lower from day 0 to day 38, but a noticeable increase of 3-OH-cotinine and cotinine concentrations was observed in the specimen collected on day 62. The physician was seeking information about the current smoking status of the patient and was planning to use this information to determine the patient’s lung transplant eligibility.

smoking-1

 

Days 0 38 62
Analyte concentration (ng/mL)
3-OH-cotinine 4074 89 603
Anabasine interf. subst. < 3 < 3
Cotinine 1404 47 425
Nicotine 241 < 2 72
Nornicotine 58 < 2 6

 

Figure and table 1. Nicotine, metabolite and anabasine concentrations (ng/mL) at different time points for a patient evaluated for lung transplantation eligibility. Anabasine was not detected on days 38 and 62, with an interfering substance preventing quantitation on day 0.

How definitive are these results?

No information was available regarding self-reported smoking or NRT use history for this patient. The physician had high suspicion that the patient was an active smoker and was attempting to use higher concentrations of nicotine and metabolites observed on day 62 as evidence of recent tobacco use.

For cotinine, values can range from 20-550 ng/mL for daily tobacco use.5 Nicotine concentrations in urine can approach over 5000 ng/mL with daily use. Together, high nicotine and cotinine can support tobacco or high-dose nicotine patch use. Furthermore, presence of nornicotine above 30 ng/mL along with anabasine greater than 10 ng/mL would be consistent with current tobacco use rather than NRT.7

Given that these were random urine specimen and the urinary creatinine values are not routinely measured, it’s important to consider the possible contributions of the variable urine concentration to the analyte concentrations. It has previously been reported that individuals abstaining from smoking for at least two weeks should present with nicotine of <30 ng/mL, cotinine of < 23 ng/mL, 3-OH-cotinine of <120 ng/mL, nornicotine < 3 ng/mL, and anabasine of < 2 ng/mL in urine.7 Based on these cut-offs, all analytes except anabasine would suggest new nicotine intake within the last two weeks.

In general, a positive anabasine result, in combination with the presence of nicotine metabolites, is consistent with active use of a tobacco product, whereas anabasine values of < 2ng/mL may suggest that NRT is the likely source.8 This can imply that the patient is abstinent from smoked or chewed tobacco if anabasine is not detected. However, anabasine is not a sensitive marker of smoked tobacco. It has been reported that the compound may not be detectable in 60% of self-reported smokers (N=51; 3 ng/mL cut-off in urine)9  and its urinary concentrations do not correlate well with self-reported tobacco use.8

As a result, anabasine has low sensitivity for determining eligibility for UNOS (United network for organ sharing) listing. There are some recommendations that this marker should not be used alone. Given that other alkaloids can originate from tobacco plant, it has been proposed that anatabine should be added to analysis due to higher expected concentration.9 However, this alkaloid is not completely specific to tobacco as it has been proposed to also arise from other plant sources 10,11  leading to possible implications for the patient that may be misclassified. In addition, anatabine sensitivity in detecting smoked tobacco use varies depending on the tobacco source and the clinical cut-off used. Clinical tests that include anatabine are not routinely available.

Can we improve this process?

Unfortunately, there is no definitive marker distinguishing smoking from NRT.

The determination of smoking status has advanced from reliance on self-reporting to quantitative and specific measurements of metabolites of nicotine and minor components of tobacco. Additional analyte incorporation into a test panel leads to additional complexities and considerations in interpretation of the results. Therefore, it is important to educate the physicians about various nicotine sources causing a positive nicotine and/or metabolite test result including NRT or e-cigarettes. It is also important to convey the limitations of tobacco alkaloid testing in such scenarios. Both the lab and the physician need to be cautious about implying active smoking in the absence of indirect supporting evidence and/or positive clinical test results.

At the same time, there is a need to improve the utility and availability of other tobacco alkaloid testing in distinguishing cigarette smoking from NRT in specific transplant populations and consider the value of testing alternative specimens. This may lead to a more effective implementation of secondary markers of tobacco use.

References

  1. Qiu, F.; Fan, P.; Nie, G. D.; Liu, H.; Liang, C.-L.; Yu, W.; Dai, Z., Effects of Cigarette Smoking on Transplant Survival: Extending or Shortening It? Frontiers in Immunology 2017, 8, 127.
  2. Zmeskal, M.; Kralikova, E.; Kurcova, I.; Pafko, P.; Lischke, R.; Fila, L.; Valentova Bartakova, L.; Fraser, K., Continued Smoking in Lung Transplant Patients: A Cross Sectional Survey. Zdravstveno varstvo 2016, 55 (1), 29-35.
  3. Vos, R.; De Vusser, K.; Schaevers, V.; Schoonis, A.; Lemaigre, V.; Dobbels, F.; Desmet, K.; Vanaudenaerde, B. M.; Van Raemdonck, D. E.; Dupont, L. J.; Verleden, G. M., Smoking resumption after lung transplantation: a sobering truth. The European respiratory journal 2010, 35 (6), 1411-3.
  4. Hukkanen, J.; Jacob, P., 3rd; Benowitz, N. L., Metabolism and disposition kinetics of nicotine. Pharmacological reviews 2005, 57 (1), 79-115.
  5. Raja, M.; Garg, A.; Yadav, P.; Jha, K.; Handa, S., Diagnostic Methods for Detection of Cotinine Level in Tobacco Users: A Review. Journal of clinical and diagnostic research : JCDR 2016, 10 (3), Ze04-6.
  6. Sandberg, A.; Skold, C. M.; Grunewald, J.; Eklund, A.; Wheelock, A. M., Assessing recent smoking status by measuring exhaled carbon monoxide levels. PloS one 2011, 6 (12), e28864.
  7. Moyer, T. P.; Charlson, J. R.; Enger, R. J.; Dale, L. C.; Ebbert, J. O.; Schroeder, D. R.; Hurt, R. D., Simultaneous analysis of nicotine, nicotine metabolites, and tobacco alkaloids in serum or urine by tandem mass spectrometry, with clinically relevant metabolic profiles. Clinical chemistry 2002, 48 (9), 1460-71.
  8. Jacob, P., 3rd; Hatsukami, D.; Severson, H.; Hall, S.; Yu, L.; Benowitz, N. L., Anabasine and anatabine as biomarkers for tobacco use during nicotine replacement therapy. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2002, 11 (12), 1668-73.
  9. Feldhammer, M.; Ritchie, J. C., Anabasine Is a Poor Marker for Determining Smoking Status of Transplant Patients. Clinical chemistry 2017, 63 (2), 604-606.
  10. Lanier, R. K.; Gibson, K. D.; Cohen, A. E.; Varga, M., Effects of dietary supplementation with the solanaceae plant alkaloid anatabine on joint pain and stiffness: results from an internet-based survey study. Clinical medicine insights. Arthritis and musculoskeletal disorders 2013, 6, 73-84.
  11. von Weymarn, L. B.; Thomson, N. M.; Donny, E. C.; Hatsukami, D. K.; Murphy, S. E., Quantitation of the minor tobacco alkaloids nornicotine, anatabine, and anabasine in smokers’ urine by high throughput liquid chromatography mass spectrometry. Chemical research in toxicology 2016, 29 (3), 390-397.

 

VG

-Dr. Valentinas Gruzdys developed interest in clinical chemistry early in his academic training which led him to pursue and obtain a PhD in Clinical and Bioanalytical Chemistry at Cleveland State University. Valentinas is enthusiastic about teaching and helping improve the understanding of limitations and utility of clinical laboratory testing. He is currently enrolled in a clinical chemistry fellowship program at the University of Utah. He enjoys learning more about various aspects of clinical chemistry and cannot wait to make his own contributions to the field after his training.