hematopathology – Lablogatory

Bandemia: What is it and Why is it Important?

Recently our lab was asked by physicians to start reporting band counts of ≧10% as critical values. While we have always reported bands when a manual differential is performed, we have also heard for years about other labs that have stopped reporting bands. Mayo Clinic stopped reporting bands in the 1990’s- other nationally and internationally known hospitals and community hospitals alike have followed suit. CAP proficiency surveys for cell ID do not separate segmented neutrophils and band neutrophils for cell IDs. Our hematology analyzer reports Immature granulocytes (IG) and we have learned about the benefits of IG over the band count. Thus, when our pathologists asked us to add a critical value for bandemia, we wondered if we were moving ‘backwards’.

Bandemia is defined as elevated band neutrophils in the peripheral blood. Neutrophils are produced to help fight infection. With infection, there is an increase in WBCs being released by the bone marrow into the peripheral blood. Bands are considered mature neutrophils and can fight infection, and in an effort to keep up with demand, some of these infection fighting cells which are released are bands. However, we are reminded that this is a nonspecific clinical finding. Bands can be elevated in many situations including inflammation, autoimmune disease, metabolic abnormalities, pregnancy, and treatment with granulocyte colony stimulating factors. Band counts of ≧10% have been used clinically as an indicator of serious bacterial illness. A finding of bandemia can help providers decide what steps need to be taken in evaluation of a patient, and in conjunction with clinical findings, can help in making a differential diagnosis.

The trouble with bands is that they are notoriously difficult to measure accurately and precisely. Bands are somewhat controversial and there are conflicting opinions on the utility of bands. Should bands be reported or included with neutrophils? A left shift reflects bone marrow response to bacterial infection, and this has been quantified as band count or immature granulocyte count. Is the IG a better parameter than the band count?

If your Hematology analyzer reports the absolute neutrophil count (ANC) with an automated differential, this includes all mature neutrophils. The theory that supports this is that neutrophilic bands and segmented neutrophils are both mature cells, and both fight infection. A true left shift would therefore be the presence of immature granulocytes (metamyelocytes, myelocytes and promyelocytes). Yet, we also now have analyzers that offer a 6-part differential that includes the immature granulocyte (IG) count. And we know that an automated differential is based on counting thousands of cells, and a manual differential is based on counting 100 cells. So, which is better? We can theorize that the IG is a better indicator of left shift because the automated diff count looks at more cells than a 100-cell manual diff.

Let’s say that we have a patient with a WBC of 20 x 10³/μL. Left shift is defined as an absolute IG count >0.1 x 10³/μL. Automated diffs can count >30,000 cells depending on the WBC count. If the auto diff counts 32,000 cells and finds 1% IG, this means that the analyzer identified 320 IG. Absolute counts are calculated as % x the WBC, in this case 0.01 x 20,000=.2 x 10³/μL. This meets the definition of a left shift. If we do a manual diff and count 100 cells and see 1 meta, the absolute IG would be .2 x 10³/μL, again meeting the criteria for a left shift. However, if we did not see any metas or other IG on the manual diff, the absolute IG would be 0. Thus, performing a manual diff, the difference in seeing 1 cell or 0 would make the difference of reporting a suspected infection versus no suspicion of infection.

Table 1: Left shift? comparison of absolute IG when 1 or no IGs are seen on manual diff

With the advent of the IG count on automated differentials, labs have moved away from reporting bands. I have attended conferences and heard presentations about “banning” bands. A few years ago, I wrote a blog called “Beyond Bands: The Immature Granulocyte Count”, describing the benefits of using the IG count over bands in manual diffs. The above example would support “Ban the bands” arguments. Using the IG count from analyzers can take advantage of analyzing many more cells and give us statistically more precise values.

Results from auto diff can get to patients’ chart faster than a manual diff result, leading to faster treatment. To report bands, a manual differential must be done. We must wait for a slide to be made, dry, and a diff to be analyzed either under the microscope or on CellaVision. Bands are subjective, relying on technologist interpretation.

So, why are we suddenly being asked to make bandemia a critical value?

Physicians asked for this change and have cited cases where patients were seen in the emergency department (ED) and subsequently released, later to return, or experiencing negative outcomes. These patients had bands reported on differentials. Other area hospitals are reporting bands and have critical values for bandemia. Because patients often are seen at more than one area hospital, and doctors may have privileges at more than one of these, for consistency, this makes sense. But is also makes sense clinically.

Recent data has drawn renewed attention to bands as a reliable predictor of severity of patient condition. A number of research papers have been published that indicate that bands may indeed be important for patient care. A 2012 study investigated bandemia in patients with normal white blood cell counts. This cohort study found that patients with normal WBC counts with moderate (11%-18%) or high (>20%) band counts had increased odds of having positive blood cultures and in-hospital mortality. (Drees, 2012)

In 2019 a study showed that there was an “increasing risk for death with increasing bandemia, irrespective of leukocyte count. (Davis, 2019) A 2021 study done at Rhode Island Hospital showed a strong correlation between increasing percentages of bands on an initial emergency room CBC and the likelihood of significant positive blood cultures and in-hospital mortality. This was noted even at band levels below 10%. (Hseuh, 2021) S. Davis, MD, from the Department of Emergency Medicine, George Washington University School of Medicine and Health Sciences, in Washington, DC wrote that “While emergency physicians may find reassurance in a normal leukocyte count, the balance of evidence strongly suggests a more prudent approach would be to wait for the bands.” (S. Davis, 2021) In other words, wait for that manual differential. He stated that emergency room physicians get results from automated CBCs before the manual diff and do not see or are aware of any internal laboratory flags on these specimens. Physicians should be aware of reporting processes to avoid early discharge of otherwise well-appearing patients before band counts are reported. Last year, trends in bandemia and clinical trajectory among patients was reviewed in a retrospective chart review at George Washington University Hospital. They noted that “Bandemia clearance and trending, in conjunction with other existing clinical tools, may be of use as a marker of improvement in sepsis. Conversely, worsening bandemia may be predictive of a deteriorating clinical status and possibly a higher mortality.” They also noted that following trends of band levels in patients with sepsis or septic shock may help to predict a clinical course and overall prognosis. (Prasanna, 2022) Additionally, a band count greater than 10% is one of the American College of Chest Physicians/Society of Critical Care Medicine’s systemic inflammatory response syndrome (SIRS) criteria used to diagnose sepsis. (Chakraborty, 2022) These are just a few of the many articles that support a clinical utility of reporting the band count.

When we first learned that we would be reporting bandemia as a critical value, we realized that we would need to get everyone on the same page. We do most of our diffs on CellaVision, so, in theory, that should be easy, but, and a big but, is that bands are notoriously subjective. Different technologists may have been taught or have used their own definitions of bands. Variation can occur depending on slide quality, tech training, definition of bands used, and number of cells counted. So, how do we make this work? We need to be sure that all our technologists are reporting bands using the same criteria, so we are not reporting differing or confusing information to physicians. The concern lies in a physician making a significant clinical decision based on apparent changes in band counts that are not real but only reflect predictable statistical factors and unpredictable technologist variability.

In our laboratory, we approached the implementation of this new critical value as an educational opportunity. Differentials that had been performed on our CellaVision were reviewed, and it was apparent that bands were not being categorized consistently among techs. (See Figure1) We started with reviewing the definition of bands with all technologists and writing updated detailed procedures that includes these definitions. We use CAP’s definition of bands, which is the definition used in most textbooks and references for over 60 years. (See Table 1) Many examples of both bands and segmented neutrophils were added to our reference library on CellaVision. These included textbook perfect bands and some that may be more subjective. These reference cells can be used by techs to compare cells when making decisions as to in which category they belong. It was also stressed to techs that they need to look at cells carefully, and in a view that is large enough to see both detail and differentiation.

Figure1. Cells reported as segmented neutrophils on Differential. How many bands do you see?

Table 2. CAP Definition of bands vs segmented neutrophils

Figure 2. Are these bands or segmented neutrophils?

Patient samples that had bands reported were located on CellaVision and multiple slides were made from these samples to be used as competency slides. In developing the differential evaluation tool, Rumke’s data showed that for a differential with a reported 12% bands, a second differential would have to have greater than 23% bands or fewer than 3% bands before the difference could be considered statistically significant. But this can be significant to our patients and patient care. In this example, diffs with <10% bands would not indicate bandemia, and diffs over 10% would initiate a critical bandemia call. And this could happen on the same slide depending on who did the diff, or on sequential samples on the same patient over a short period of time. These competency slides were assigned to techs to collect statistics on the mean and SD of the bands reported on each slide. Retraining and coaching will be provided as necessary. Follow up competency slides will be assigned, and statistics will be recalculated. The goal is to decrease variability in our band counts and to show that we have done so. This ongoing quality project has involved writing procedures, offering continuing education, assigning and reviewing competency slides, coaching technologists and reviewing slides with them and calculating statistics. Our goal is consistency and reporting meaningful results to our physicians.

As we saw in the studies cited above, % bands and trends are both important when evaluating clinical correlations. The chart below shows examples of how this might affect patient care. If a patient on presenting at the ED had a 19% band count, this would be called as a critical, and the patient would be further evaluated, and depending on clinical symptoms and medical history would likely have blood cultures drawn and be admitted to the hospital. If a second tech did the diff and reported 5% bands, the patient may be sent home without further evaluation. On subsequent CBCs, we could be giving confusing results to the physician if we are not consistent in our reporting. With multiple techs doing differentials on different shifts, it could look like this patient is getting better,

getting worse, or it could look like the patient is responding to therapy, and then the next day they had a setback. While we understand that bands will probably always be somewhat subjective, we need to narrow this down. By adhering to one definition, our goal is to report consistent and accurate results.

Table 3. Various results on differentials on the same patient over the course of 3 days, showing technologist dependent results.

ED physicians are looking for an early marker that can be used to identify septic patients as early as possible. Bandemia may be used as this marker. We therefore need to be as objective as possible when reporting bands. “Ultimately the band count is only one factor amongst several others which will be used in assessing the patient’s clinical state and in determining any subsequent medical management. Yet, identifying bands is important, and emphasizes the key role that our laboratory professionals play in identifying causes for concern” Dr Edgar Alonsozana, Mercy Medical Center, Baltimore, Md.

I welcome your comments about how your laboratories report bands and if bandemia is a critical value in your facilities!

References

P.Joanne Combleet, Clinical utility of the band count, Clinics in Laboratory Medicine, 2002;22:101-136

Al-Gwaiz, Layla A. and H H Babay. “The Diagnostic Value of Absolute Neutrophil Count, Band Count and Morphologic Changes of Neutrophils in Predicting Bacterial Infections.” Medical Principles and Practice 16 (2007)

S. Davis, R. Shesser, K. Authelet, A. Pourmand. “Bandemia” without leukocytosis: A potential Emergency Department diagnostic pitfall. The American Journal of Emergency Medicine,Volume 37, Issue 10, 2019

Harada T, Harada Y, Morinaga K, Hirosawa T, Shimizu T. Bandemia as an Early Predictive Marker of Bacteremia: A Retrospective Cohort Study. Int J Environ Res Public Health. 2022 Feb 17;19(4):2275.

Christine DeFranco DO and Terrance McGovern DO MPH
St. Joseph’s Regional Medical Center, Paterson, NJ. Isolated Bandemia: What Should We Do with It? Critical Care, Oct. 2016,

Harmening, Denise. Clinical hematology and Fundamentals of Hemostasis, 4^th ed. 1997

Prasanna N, DelPrete B, Ho G, et al. The utility of bandemia in prognostication and prediction of mortality in sepsis. Journal of the Intensive Care Society. 2022;0(0).

Click to access IG-Ban-the-Bands.pdf

https://www.mlo-online.com/home/article/13007276/answering-your-questions

Takayuki Honda, Takeshi Uehara, Go Matsumoto, Shinpei Arai, Mitsutoshi Sugano, Neutrophil left shift and white blood cell count as markers of bacterial infection,Clinica Chimica Acta, Volume 457, 2016

Drees M, Kanapathippillai N, Zubrow MT. Bandemia with normal white blood cell counts associated with infection. Am J Med. 2012 Nov;125(11):1124.e9-1124.e15.

Leon Hsueh, Janine Molino, Leonard Mermel,

Elevated bands as a predictor of bloodstream infection and in-hospital mortality,The American Journal of Emergency Medicine,2021

https://www.ncbi.nlm.nih.gov/books/NBK547669/

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

Lymphocyte Subset Panels (AKA T4T8 Assays)

While writing my last blog, I asked “What is your least favorite test to do in Hematology?” (I’m not ignoring our favorite tests! I will get to those in another blog.) And then, I started thinking about why we may not like certain testing. Is it because they are time consuming, or repetitive? Is it because they hurt our eyes, or necks, or fingers? Or is it because it’s a test that we perform but we may not be sure what the test is for, or we don’t understand the theory behind it? I started thinking about my coworkers and other tests that could be on those lists and I immediately decided that a good candidate in our lab is the T4T8 panel. Probably the primary reason is that the instrument we do these on has given us many problems over the last year. The instrument has spent most of the year with an “instrument out of service” sign on it. Service has been here many times, but the instrument just appears to have exceeded its life expectancy. In normal times, when the instrument was in its prime, setting up and running a T4T8 panel does require a number of steps, and some time. In the last year we have had to add lots of coaxing, even more time, and some luck to get the test to run. This can be frustrating in any lab situation, but is particularly frustrating when we are short staffed, trying to train new staff, and very busy. So, I don’t think it’s the test itself that techs dislike, it’s the time it takes, not being comfortable with setting up the test, juggling our other work while struggling with another instrument, and the fact that even after we get results, a percentage of the samples have results that don’t meet our criteria and still need to be sent out to the reference lab.

Another reason why this test may be a little intimidating is its unfamiliarity. It’s not a test that is done in every lab. I have worked as a Medical Laboratory Scientist for many years. I’ve worked in 6 labs since the mid 1980’s and the introduction of CD4 testing for human immunodeficiency virus (HIV) patients, yet my current place of work is the first place that we have done these in house. Before this job, if you asked me or any of my coworkers what a T4T8 panel was, we probably would have answered “a send out test”. A few weeks ago, we had a call from a doctor asking questions about his patient’s T4T8 assay results. The tech answering the phone got a blank look on their face and quickly handed the phone to me. This told me that techs, and even doctors, may not really understand what this test is testing and what the results mean. This further confirmed to me that the lack of knowledge about these tests may be another reason why these don’t win any popularity contests in our lab.

So, what exactly is a T4T8 panel?

Some other names for the test are a Lymphocyte subset panel, an Immuno T-cell (CD3/4/8) assay, T-Cell subsets Percent and Absolute panel or T-Lymphocyte Helper/Suppressor Panel. As a quick review, we know that lymphocytes are either B-lymphocytes or T-lymphocytes. Immunotyping lymphocytes can provide information for disease diagnosis and monitoring. All T-lymphocytes express CD3 antigens on their surfaces, which can be used to differentiate B-cell disorders from T-cell disorders. T-lymphocyte subsets include T-helper/inducer cells which express both CD3 and CD4, and T-cytotoxic/suppressor cells, which express CD3 and CD8. In a T4T8 panel we are concerned with identifying T-lymphocytes, and the percentage of each subset both individually, and compared to one another.

The test we perform uses monoclonal antibodies, anti CD3, anti CD4 and anti CD8, which recognize specific human lymphocyte subsets. Our reagents come as antibody containing tubes and are run on the Cell-Dyn Sapphire. After performing a CBC on the sample, the instrument is programmed to add an aliquot of the sample to the CD3 +CD4 reagent tube and a second aliquot to the CD3 + CD8 reagent tube. Immunophenotyping is performed by flow cytometry on these 2 aliquot tubes. The CD3 antibody in both tubes separates out all T-lymphocytes, and the addition of the CD4 in the first tube identifies the cells which are also CD4 positive, the T4 or helper cells. The CD3 + CD8 tubes identifies the percentage of T cells that are T8 or suppressor cells. The assay uses the CBC results and the immunophenotyping runs to calculate the helper/suppressor ratio, also known as T4/T8 ratio or CD4/CD8 ratio.

Why is this test performed?

After the discovery of lymphocyte subset abnormalities in human immunodeficiency virus (HIV) patients in the 1980s, lymphocyte immunophenotyping has become widely used in this patient population for the evaluation of their prognosis, immune deficiency status, response to therapy, and diagnosis of AIDS. The test is most often done to assess HIV infection status but may also be useful in the diagnosis and monitoring of other diseases or after organ transplantation. Some examples of conditions in which this assay may be useful include other viral and bacterial infections, severe combined immunodeficiency, Hodgkin disease, certain leukemias, multiple sclerosis, and myasthenia gravis. A newer application of CD4/CD8 ratios are as potential biomarkers of cancer progression. The most interesting new use of T-cell subset testing that I have read about has been with the recent COVID-19 pandemic. Several studies have shown that CD4 and CD8 T- cell counts reflected disease severity and can predict clinical outcomes of COVID-19 infection. These studies have concluded that COVID-19 patients presenting with relatively low CD4 and CD8 T-cell counts are more severely infected and may have a worse prognosis. The Abbott test we use was designed to be used to monitor immune status in (HIV)-infected individuals. It is not intended for screening for leukemic cells or for phenotyping samples in leukemia patients.

What do the results mean?

The absolute CD4 count and CD4/CD8 Ratio can be used as a snapshot of immune system health. Normal absolute CD4 counts are 600 to 1200 /mm³. In immune suppression, values drop below 500/mm³and in advanced infection, values of less than 200/mm³are consistent with a definition of acquired immunodeficiency syndrome (AIDS). In advanced disease, some patients may have a normal CD4 count but experience a weakening immune system. Or the immune system can become exhausted and unable to produce sufficient T-cells. The CD4/CD8 ratio is useful for judging the strength of the immune system. A normal CD4/CD8 ratio is between 1.0 and about 3.0-4.0.

T-helper cells start the defensive immune response by signaling other cells that infectious pathogens are present. At initial infection with HIV, T-suppressor cells increase in an effort to destroy infected cells. We see an increase in CD8 cells as the CD4 cells are destroyed. These events result in a low CD4/CD8 ratio. When HIV antiretroviral therapy (ART) is initiated, the ratio will usually, gradually return to normal. However, if ART is not started or if the immune system is severely affected, the body may not be able to make adequate new CD4 cells and the ratio may never return to normal.

With the availability of very effective therapies available for the treatment of HIV, the CD4/CD8 ratio has become more important in patients with long term HIV infection. Recent studies have suggested that people with a low CD4/CD8 ratio who have been on treatment for years are at an increased risk from non-HIV illnesses such as cardiovascular and renal disease.

CD4 counts are important in HIV management and used to guide treatment including the decision to initiate prophylactic treatment against opportunistic infections. It is recommended that CD4 counts be performed every 3-6 months after initiation of ART. After the first 2 years on ART, CD4 monitoring can be decreased in frequency to every 12 months for people whose CD4 count is between 300 and 500 and may be considered optional for those with CD4 counts over 500. Table 1 and 2 shown below are examples of patient reports for the T4T8 assay.

Table 1. Patient with AIDS, CD4 count 200, T4/T8 ratio 0.16*

Table 2. Patient with absolute CD4 within normal range, but CD4/CD8 below 1.0*

*There are times when the absolute or % CD3T may be less than the sum of the CD4T and CD8T. This is due to averaging of CD3T counts from the 2 monoclonal tubes

In our lab, these tests are performed daily, as they are received, from 7am to 7PM, 7 days a week. There are no commercial quality control materials available for the test, so we must choose negative and positive QC from our patient population. For the QC we choose patients with CBC and WBC differential values within normal ranges, with no flags. There are additional age and diagnosis/treatment related restrictions on samples that can be used as controls. Our in-house patients often have abnormal results, and our patient population also includes our large outpatient hematology/oncology center. Thus, at times, finding appropriate controls can be challenging. I can add this to the list of ‘problems’ with this test and why techs don’t like them. Call me weird, but I actually like doing these! I like the challenge of finding QC, I like that they are ‘different’ from the hundreds of CBCs we perform each day, and I look at them as a little change in routine and a chance to do something unique. Though I wish the instrument would run perfectly every day, I even (sort of) don’t mind troubleshooting when it’s not working. I like solving problems! I enjoy teaching others how to run these, and I enjoy answering questions about the test.

Many thanks to my great coworker Jacky Olive for her assistance always and inspiration for this blog. I know these are not your favorite test!

*There are times when the absolute or % CD3T may be less than the sum of the CD4T and CD8T. This is due to averaging of CD3T counts from the 2 monoclonal tubes

Becky Socha MS, MLS(ASCP)^CMBB

References

Abbott Laboratories, Cell Dyn Immuno T-Cell (Cd3/4/8 )ReagentsPackage Insert. Abbott Park, Il.
Li Raymund; Duffee Doug; Gbadamosi-Akindele Maryam F.CD4 Count. NIH National Library of Medicine. May 8, 2022
Domínguez-Domínguez L, Rava M, Bisbal O, et al. Cohort of the Spanish HIV/AIDS Research Network (CoRIS). Low CD4/CD8 ratio is associated with increased morbidity and mortality in late and non-late presenters: results from a multicentre cohort study, 2004-2018. BMC Infect Dis. 2022 Apr 15;22(1):379.
Liu Z, Long W, Tu M et al. Lymphocyte subset (CD4+, CD8+) counts reflect the severity of infection and predict the clinical outcomes in patients with COVID-19. Journal of Infection. Vol 81, Issue 2. P318-356, AUGUST 01, 2020
Kagan JM, Sanchez AM, Landay A, Denny TN. A Brief Chronicle of CD4 as a Biomarker for HIV/AIDS: A Tribute to the Memory of John L. Fahey. For Immunopathol Dis Therap. 2015;6(1-2):55-64
McBride JA, Striker R (2017) Imbalance in the game of T cells: What can the CD4/CD8 T-cell ratio tell us about HIV and health? PLoS Pathog 13(11)
Sinha A, Mystakelis H, Rivera AS, Manion M, et al. Association of Low CD4/CD8 Ratio With Adverse Cardiac Mechanics in Lymphopenic HIV-Infected Adults. J Acquir Immune Defic Syndr. 2020 Dec 1;85(4)
Wang YY, Zhou N, Liu HS, Gong XL, Zhu R, Li XY, Sun Z, Zong XH, Li NN, Meng CT, Bai CM, Li TS. Circulating activated lymphocyte subsets as potential blood biomarkers of cancer progression. Cancer Med. 2020 Jul;9(14)

Hematology Case Study: Presenting a Double Feature Starring Chronic Myelogenous Leukemia

One of the reasons I love working in Hematology is because when we have unexpected results they are often accompanied by visuals… and a picture is worth a thousand words! Unusual or critical results in Chemistry can be interesting, sometimes there are dilutions to perform, results to compare or puzzles to solve, I have always loved working up a good antibody or complicated multiple antibodies in Blood Bank or calculating how many units I may need to screen to find compatible ones, gram stains of unusual organisms in Microbiology can be exciting, but nothing beats some of the cells we see in Hematology! It’s always fascinating when we find unusual cells and follow up with smear reviews with our pathologists. And, being able to save these visuals in CellaVision or saving the slides for teaching, is a plus. These cases are a gift that keeps on giving! Lately I’ve had my share of “exciting” specimens, usually on a Saturday or Sunday afternoon! It never fails to get the adrenaline going when you are the first one to see a CBC with a WBC of 400,000, a differential that is over 90% blasts, rare lymphoma cells, malarial parasites, or a body fluid smear full of malignant cells. The following 2 cases are a very remarkable looking smear and a not so remarkable one, from 2 different patients with the same diagnosis.

The first patient is a 71 year old male who had a routine CBC done by his primary care physician. The blood was collected as an outpatient on a Saturday morning, and brought to our lab by a routine courier that afternoon (of course, right before change of shift!). We had one previous CBC result on this gentleman, from several years earlier, which was essentially normal. CBC result shown below:

Table 1. Case 1, CBC results. [Editor’s note: a previous version of this table noted a Hct of 231.8. The correct result is 31.8.]

Table 2. Case 1, Manual Differential results.

Image 1. Peripheral smear, Case 1, WBC 363.14.

As soon as I saw the results, I called the provider with the WBC and alerted them that I would be contacting the pathologist on call and calling back with the differential. Our pathologist confirmed blasts on the peripheral smear and requested that the sample be sent out for flow cytometry. The pathology report stated “Marked leukocytosis with left shift and <5% blasts. The presentation is suspicious for a myeloproliferative neoplasm (e.g. chronic myelogenous leukemia (CML)). Immunophenotypic studies have been ordered and will be reported separately. Clinical correlation and Hematology consult recommended.” Flow cytometry results showed left shifted maturation and FISH studies demonstrated t(9;22) BCR-ABL with 98% of positive nuclei in bone marrow. No other mutations were detected. Diagnosis: chronic myelogenous leukemia. Five days later, we had a bone marrow scheduled on a 50 year old male. A CBC done 2 weeks earlier showed a mild leukocytosis and thrombocythemia. (WBC 12.4, Hgb 17.8, Hct 52%, PLT 539). Diagnoses under consideration were possible CML, polycythemia or a myeloproliferative neoplasm (MPN). The patient’s CBC the day of the procedure is shown below.

Table 4. Case 2, Manual Differential results.

Cytogenetic analysis showed an abnormal clone characterized by the Philadelphia chromosome translocation, t(9;22). The BCR/ABL gene rearrangement was detected by FISH, with 78% of positive nuclei in bone marrow. The bone marrow was negative for other mutations. Flow cytometry analysis reported no evidence of abnormal myeloid maturation or increased blast production. There was no evidence for a lymphoproliferative disorder. Diagnosis: chronic myelogenous leukemia.

In 1959, at a time when techniques for preparing chromosomes for visualizing under the microscope were still very unsophisticated, 2 researchers in Philadelphia detected a tiny abnormality in the chromosomes of patients with CML. They noticed that part of chromosome 22 appeared to be missing. It was not until 1970, when techniques for chromosome banding became available, that this discovery was shown to be a translocation between chromosomes 22 and 9. The shortened chromosome 22 was named the Philadelphia (Ph) chromosome after the city where it was discovered.

Image 2. The Philadelphia chromosome. A piece of chromosome 9 and a piece of chromosome 22 break off and trade places (cancer.gov).

At diagnosis, over 90% of CML cases have the t(9;22) translocation which has become a hallmark for a diagnosis of CML. However, the Ph chromosome is also detected in about 30% of adult acute B cell lymphoblastic leukemia (B-ALL), and a very small number of acute myeloid leukemias (AML) and childhood B-ALL so testing must be done for differentiation. t(9;22) is a translocation of the proto-oncogene tyrosine-protein kinase ABL1 gene on chromosome 9 and the breakpoint cluster region BCR gene on chromosome 22. The newly formed chromosome 22 with the attached piece of chromosome 9 is called the Philadelphia chromosome. The BCR-ABL oncogene is formed on the Philadelphia chromosome and the product of the Ph translocation is an abnormal fusion protein, p210, which has increased tyrosine kinase activity. This, in turn, is responsible for the unregulated proliferation of cells seen in CML. Tyrosine kinase inhibitors (TKIs) have been developed as targeted therapy for Ph+ CML.¹

So, how can these 2 patients with very different CBC results both be diagnosed with CML? CML can be classified into phases of CML-chronic phase (CML-CP), accelerated phase (CML-AP), and blast crisis (CML-BP). The WHO Classification of 2017 proposed a system of cutoffs to define each phase. The phases are based mainly on the number of blasts in the blood or bone marrow. Progression from CML-CP to CML-AP is also generally recognized to correlate with an increase in BCR-ABL1 levels. Several studies have been done that discuss another phase, pre-leukemic (pre-clinical) CML. These pre-leukemic patients have the Philadelphia chromosome, the genetic hallmark of CML, without other abnormalities. They have a normal to mildly elevated WBC and are asymptomatic. In these cases, progression to CML-CP can be several months to several years. One interesting factor common in this phase, which can help in diagnosis, is the presence of an absolute basophilia (ABC) >200/mm^3. This basophilia is also seen in CML-CP and often progresses with the disease.²

Results from both patients are compared below. While we may more readily recognize a new CML that presents with very high WBC, left shift, and blasts, FISH, flow and cytogenetics of both these patients indicated a diagnosis of CML. This second patient may be one that could be classified as a pre-CML. The patient is certainly fortunate to have physicians who suggested further workup so he can benefit from his early diagnosis.

Table 5. Comparison of results from 2 cases.

References

Huma Amin*, Suhaib Ahmed. Characteristics of BCR–ABL gene variants in patients of chronic myeloid leukemia. Open Medicine, 2021.16:904-912.
Aye, Le Le; Loghavi, Sanam; Young, Ken H et al. Preleukemic phase of chronic myelogenous leukemia: 2. morphologic and immunohistochemical characterization of 7 cases Annals of Diagnostic Pathology. April 2016 21:53-58 Language: English. DOI: 10.1016/j.anndiagpath.2015.12.004.
Kuan JW, Su AT, Leong CF, Osato M, Sashida G. Systematic review of pre-clinical chronic myeloid leukaemia. Int J Hematol. 2018 Nov;108(5):465-484. doi: 10.1007/s12185-018-2528-x. Epub 2018 Sep 14. Erratum in: Int J Hematol. 2018 Nov 7;: PMID: 30218276.
https://www.cancer.gov/publications/dictionaries/cancer-terms/def/philadelphia-chromosome

Hematology Case Study: Unusual Lymphocytes Seen in an Apparently Healthy Young Adult

A healthy 30 year old woman visited her primary care physician concerned about a rash with questionable infection on her hands. The physician prescribed an antibiotic for infection and ordered a CBC. From the results below, it can be seen that the patient had a pancytopenia and a relative lymphocytosis.

A manual differential was performed on CellaVision and the presence of large, clefted lymphocytes with immature features was noted. A request for pathology review was sent to the pathologist. The pathologist’s review stated “ Atypical lymphocytosis, specimen to be submitted for flow cytometry. Report to follow. Occasional atypical lymphocytes with immature features also noted. Lymphocyte population is predominantly mature”

The peripheral blood sample was sent out for immunophenotyping by flow cytometry and FISH studies. Flow cytology reported “precursor B-cell population expressing CD19, CD10, HLA-DR, and CD34 is identified. Percent of abnormal cells, 30%. These findings are consistent with precursor B-lymphoblastic leukemia.” While we tend to associate a leukemia diagnosis with a high white blood cell count, and the presence of blasts, this patient was unusual in that she did not have a high WBC or blasts seen on the peripheral smear. Pancytopenia in ALL has been noted in literature. A study of new onset pancytopenia in adults showed that the majority of cases were acute myeloid leukemia, but ALL and other lymphomas also caused pancytopenia³. Another study noted that “pancytopenia followed by a period of spontaneous recovery may precede the diagnosis of acute lymphoblastic leukemia.”¹ While the pathologist did not identify blasts on this differential, and cells were predominately mature, WBC was very low, and our analyzer did flag “?blasts/abnormal lymphs” and reflexed the manual differential.

Image 1. Clefted lymphocytes seen on peripheral smear.

Image 2. Clefted lymphocytes on CellaVision.

Leukemia is a broad term that includes a number of different chronic and acute diagnoses. Chronic and acute forms are further broken down into myeloid and lymphoid and then into subtypes. The French-American-British (FAB) classification of acute leukemias was devised in the 1970’s and 1980’s and was based on cytochemical staining and morphology of cells. These tests were performed manually and relied on what the cells look like under the microscope. The series of stains were used to differentiate myeloblasts from lymphoblasts. I’m old enough that I remember learning about these stains when they were being developed and thinking how amazing they were!

We’ve come a long way since the early 1980’s! Although the FAB diagnostic criteria are not entirely forgotten, the World Health Association (WHO) classification, first published in 2001, has largely replaced the FAB classification. The newest guidelines for Acute Lymphoblastic Leukemias (ALL) were published by WHO in 2016. These new guidelines supplement morphology and cytochemical staining with newer testing which can now identify and distinguish B cell and T cell ALL. In making a diagnosis, peripheral blood and/or bone marrow aspirate samples are subject to flow cytometry immunophenotyping and chromosome testing such as cytogenics or fluorescence in situ hybridization(FISH). Molecular tests can also be done to look for specific gene changes in the leukemia cells. The WHO classification has become preferred because these new tests can give more information that is important for treatment. Prognosis for ALL depends on patient age, WBC counts at diagnosis and these specific test results which tell us which subtype of ALL is present. The presence and identification of chromosomal alterations is important for diagnosis and therapy decisions. Identifying chromosomal alterations can also lead to better risk classification which is significant because of the knowledge that, while rearrangements tend to have poorer outcomes, some rearrangements actually offer a better prognosis. With the future era of individualized, targeted therapy for leukemia, combining conventional cytogenics with molecular and FISH methods will greatly enhance the accuracy of information and provide patients with more specific and customized treatment options.

While ALL is the most common childhood leukemia, it is not as commonly seen in adults. B cell ALL is more common than T cell ALL in all ages, and accounts for about 90% of ALL cases in children and about 75% of ALL cases in adults. Cure rates in children exceed 90% but in adults varies with age and depending on chromosomal alterations. Most B cell ALL subtypes with chromosome translocations tend to have a poorer outcome than those without translocations. As well, younger adults, <50 years old, have better prognosis than older adults.

This patient did not have a BCR/ABL rearrangement or MLL gene locus 11q23 translocation, which both carry poorer prognoses, but she also did not have a translocation between chromosome 12 and 21 or more than 50 chromosomes, both of which offer more favorable prognoses. This young woman therefore would be in an average risk category and appears to have been diagnosed very early in the course of her disease. We have not seen any further workup, as the patient is being treated at another facility. We wish her well in her leukemia treatments.

References

Hasle H, Heim S, Schroeder H, et al. Transient pancytopenia preceding acute lymphoblastic leukemia (pre-ALL). Leukemia. 1995 Apr;9(4):605-608.
Iacobucci I, Mullighan CG. Genetic Basis of Acute Lymphoblastic Leukemia. J Clin Oncol. 2017 Mar 20;35(9):975-983. doi: 10.1200/JCO.2016.70.7836. Epub 2017 Feb 13. PMID: 28297628; PMCID: PMC5455679
Bone Marrow evaluation in new onset pancytopenia. Human Pathology. Vol 44, Issue 6. June 2013
Hematology: Basic Principles and Practice, 7th Edition. Ronald Hoffman, Edward J. Benz, et al. 2018 Elsevier

Coagulation Case Study: 14 Year Old Female With a History of Bleeding Episodes

Case Study

A 14 year old female arrived at the emergency room with her mother and grandmother complaining of extremely heavy menstrual bleeding. Patient history reported by her mother included a history of “a bleeding problem” for which she had been treated a few times since age 4. Petechiae were noted on the girl’s abdomen, arms and thighs. There was no history of aspirin or other NSAID use. Blood work was ordered.

Patient results are shown in Table 1 below.

The mother called home to ask her husband for details and reported that her daughter had been diagnosed with Immune Thrombocytopenic Purpura (ITP) 10 years earlier but was not very clear on the treatments. She stated that other than frequent nose bleeds, some petechiae, and occasional bruising that the girl had seemed ok until she started menstruating. They had not seen the specialists in a number of years. Further questioning of the mother revealed that the parents had both immigrated from Iran with their families as infants. The patient was an only child. The grandmother reminisced about the village “in the old country” and mentioned that her daughter and son in law were related, the families being from the same village. When asked about any other family with bleeding disorders, the mother reported that neither she nor her husband had ever met any other relatives in Iran and were unaware of any bleeding tendencies in the family. The grandmother interjected that she did remember that several of her cousins and an uncle experienced frequent epistaxis.

The ER physician noted the normal PT/INR, APTT and slightly decreased platelet count but felt the extensive petechiae and hypermenorrhagia were out of proportion to these results. A manual differential was ordered. Differential results were within normal ranges, RBC morphology reported sight polychromasia and anisocytosis. Platelet estimate was slightly decreased with giant platelets noted. The physician suspected an inherited platelet disorder and the patient was referred to a hematologist for further workup.

Image 1. Giant platelets on peripheral blood smear.

Discussion

I have written a few blogs about different thrombocytopenias. This case interested me because the patient was first diagnosed with ITP. ITP is an autoimmune bleeding disorder in which the immune system makes anti-platelet antibodies which bind to platelets and cause destruction. Even though the exact cause of ITP remains unknown, it is recognized that it can follow a viral infection or live vaccinations. In children this tends to be an acute disease which is self-limiting and self resolves in several weeks. However, in a small number of children, ITP may progress to a chronic ITP, as was thought to be the case in this patient.

A new hematologist saw the patient and reviewed the medical history. In this patient, the diagnosis of ITP had been followed for a short period of time in which the platelet count did not increase. She was treated with immunoglobulin. When her platelet count dropped below 30 x 10³/μL, the patient was transfused several times. Early platelet transfusions increased her counts, but the patient became refractory and was then given HLA matched platelets, with some improvement. After a period of time, the patient did not return to the specialist and the parents described her condition as improved. However, as reported to the ER physician, she still experienced frequent epistaxis and other bleeding symptoms unrelated to accidental injury. The mild thrombocytopenia and giant platelets on the blood smear with normal PT and APTT in a patient with abnormal bruising or bleeding alerted the physician to the possibility of the diagnosis of Bernard Soulier Syndrome (BSS). The family history also suggested BSS.

The hematologist ordered further testing. Noted in the patients chart from 10 years ago was a prolonged bleeding time. This test was not repeated at this time because it has largely been replaced by platelet function analyzers (PFAs.) The PFA test analyzes platelet function by aspirating citrated blood through membranes to induce platelet adhesion and platelet plug formation. The test is first performed with a collogen and epinephrine membrane (Col/Epi). If the closure time is normal, platelet function can be considered normal. If the closure time with Col/Epi is increased, then the test is repeated with a collogen and ADP membrane (Col/ADP). A prolonged closure time with Col/Epi with normal Col/ADP closure time may indicate an aspirin induced platelet disorder, whereas an increased closure time with both membranes may indicate a platelet defect that is not aspirin related.³ The PFA closure times were increased in both the Epinephrine and ADP cartridges.

Platelet aggregation was normal with all agents except ristocetin. BSS can be differentiated from von Willebrand disease(vWD) by the addition of normal plasma to the ristocetin agglutination test. The addition of normal plasma adds vWF to the suspension, and in vWD the ristocetin agglutination is corrected. Agglutination with ristocetin requires vWF and GPIb/IX. Since GPIb/IX is absent or reduced in BSS, he ristocetin agglutination is not corrected in BSS, as seen in this patient.³ Flow cytometric analysis of platelet glycoproteins demonstrated reductions in CD42a (GpIX) and CD42b (Gp1bα).

Bernard Soulier syndrome (BSS), also known as Hemorrhagiparous thrombocytic dystrophy, was first described in 1948 as a bleeding disorder characterized by a prolonged bleeding time and giant platelets seen on a peripheral smear. It is an inherited platelet adhesion disorder caused by platelet glycoprotein (GP) deficiencies. The disorder is rare, affecting only about 1 in 1,000,000, though it is more common in families where parents are related. BSS is typically autosomal recessive, though a small number of cases have been found that are autosomal dominant. Most cases are diagnosed at a young age, with the autosomal dominant type often less severe and diagnosed later in life.¹

Platelets are involved in primary hemostasis, the initial arrest of bleeding that occurs with vascular injury. As we know, platelets’ functions include adhesion and aggregation. Platelets first stick to the blood vessel wall (adhesion), followed by binding to each other (aggregation). In primary hemostasis, platelets first adhere to von Willebrand factor (vWF) which is bound to the subendothelial collogen fibers. This is followed by aggregation, a complex process that results in the formation of the platelet plug and the initial arrest of bleeding.. In BSS, platelet membrane GPs Ib, V and IX are missing, resulting from an inherited mutation in one of the genes that code for proteins in the complex. This affects the binding of the platelets to vWF, which subsequently interferes with primary hemostatic plug formation.⁴ If the platelets don’t adhere, aggregation is also affected.

Patient Results

In order to make a differential diagnosis of platelet function disorders, laboratory testing is necessary:

Tests of secondary hemostasis, PT and APTT, are normal in this patient so a disorder of primary hemostasis would be suspected.

In this patient, the platelet count was slightly decreased. In BSS, the platelet count is variable, from normal is moderately decreased, and can vary from time to time in the same patient.

Platelet adhesion tests (PFA) performed with both Col/Epi and Col/ADP were abnormal.

Light transmission aggregometry revealed platelet aggregation was normal with ADP, collogen and epinephrine. Aggregation with ristocetin was abnormal.

Giant platelets observed on peripheral smear

Flow cytometric analysis of platelet glycoproteins demonstrated reductions in CD42a (GpIX) and CD42b (Gp1bα).

Diagnosis: Bernard Soulier syndrome.

Conclusion

BSS is rare and is commonly mistaken for ITP. Reports have been published that analyze cases of BSS patients long treated as ITP. These misdiagnosed cases have been treated with immunoglobulins, steroids, IV anti-D, and other drugs used to treat refractory ITP. Splenectomies have even been reported in some cases. Platelet aggregation to ristocetin and flow cytometry have provided the correct diagnoses. Molecular studies can also be done to identify the abnormal genotype.² Clues that can lead to a correct diagnosis are childhood ITP that does not spontaneously resolve and does not respond to treatments, other family members with bleeding problems or low platelet counts, platelet counts that are not low enough to explain bleeding or prolonged bleeding times, increased MPV and the presence of giant platelets on the peripheral smear.

This patient was diagnosed with ITP as a child, but treatments did not improve her platelets counts. She continued to have bleeding episodes which increased with the onset of menses. Her grandmother reports a history of bleeding tendencies in other family members. In addition, her parents are related. Her peripheral smears noted giant platelets. Laboratory tests confirmed a diagnosis of BSS.

Bernard Soulier syndrome (BSS) is a rare but important long-term bleeding disorder.

Patients do not require routine prophylactic treatment, so the management of BSS focuses on prophylactic treatment before certain procedures or after injuries. Patients should be advised not to take NSAIDS. The patient should be advised that treatment may be necessary prior to procedures or in response to common bleeding events such as bleeding gums, epistaxis, and menorrhagia. Antifibrinolytic therapy can be used in bleeding episodes. Platelet transfusions are considered for patients before surgery or if anti-fibrinolytics have failed. For severe cases, stem cell transplants have provided a cure. BSS may also be a candidate disorder for gene therapy in the future.¹

References

Grainger JD, Thachil J, Will AM. How we treat the platelet glycoprotein defects; Glanzmann thrombasthenia and Bernard Soulier syndrome in children and adults. Br J Haematol. 2018 Sep;182(5):621-632. doi: 10.1111/bjh.15409. Epub 2018 Aug 17. PMID: 30117143.
Reisi N. Bernard-Soulier syndrome or idiopathic thrombocytopenic purpura: A case series. Caspian J Intern Med. 2020;11(1):105-109. doi:10.22088/cjim.11.1.105
Perumal Thiagarajan, MD; Chief Editor: Srikanth Nagalla, MBBS, MS, FACP. https://www.medscape.com/answers/201722-90211/what-is-the-platelet-function-analyzer-100-pfa-100-and-how-is-it-used-in-the-workup-of-platelet-disorders
Turgeon, Mary Louise. Clinical Hematology, Theory and Procedures. Fifth ed. 2012. Lippincott Williams and Wilkens. Baltimore.

Hematology Case Study: CBC with >80% Blasts

The patient is a 67 year old male who first visited his dentist at the end of December complaining of pain in the jaw that he had been experiencing since early Dec. He had put off making an appointment because he didn’t want to have to go to the doctor with COVID precautions, but the pain was now radiating to his teeth, so he made a dentist appointment. The dentist found no evidence of abscess or other infection but ‘adjusted his bite’. The patient was advised to take over the counter NSAIDs as needed or pain but no prescriptions was needed. Three weeks later the patient visited an urgent care because he had no improvement of the jaw pain. At this time he relayed symptoms of cough, fever, chills, night sweats and chronic fatigue. Patient history included an active lifestyle with vigorous aerobic exercise several times a week, but the he stated that he had been feeling too fatigued to exercise for over a month. On exam the patient was found to be tachycardic with bilateral tonsillar lymphadenopathy and oropharyngeal exudate. The patient was tested for COVID, influenza and Group A Strep. The COVID-19 was negative, as was the influenza A and B, but the Group A Strep was positive. The patient was sent home with a prescription for antibiotics.

One week later, the patient called his PCP because he still had cough, fever and chills and now was experiencing shortness of breath. The office directed the patient to go to the ER but the patient was reluctant to go to the hospital and stated he would rather be seen at the office. On review of the patients chart, the PCP agreed to see him in the office because he had had a negative COVID test in the past week. Two days later the doctor examined the patient in his office and still suspected COVID-19. He ordered a PCR COVID-19 test along with CBC/differential and erythrocyte sedimentation rate (ESR). We received a routine CBC on the patient. Results are shown below.

The patient had no previous hematology or oncology history and no previous CBC received at our lab. The critical WBC was called to the physician. Based on the WBC and flags on the auto differential, a slide was made and sent to our CellaVision (CV). On opening the slide in CV, we immediately called our pathologist for a pathology review. A rare neutrophil was seen on the peripheral smear, with immature appearing monocytes, few lymphocytes and many blasts.

The pathologist reviewed the slide and the sample was sent for flow cytology studies and FISH. The pathologist’s comment ”Numerous blasts (>60%) consistent with Acute Myeloid Leukemia(AML). Specimen to be submitted for flow cytometry. Hematology consult recommended” was added to the report.

Image 2. Image from CellaVision. Predominately blasts with one neutrophil seen in field of unremarkable RBCs.

The myeloid/lymphoid disorders and acute leukemia analysis by flow cytometry reported myeloblasts positive for CD117,CD33, and CD13. Final interpretation was Acute Myeloid leukemia (non-M3 type).

AML is the most common form of leukemia found in adults. AML was traditionally classified into subtypes M0 through M7, based on the cell line and maturity of the cells. This was determined by how the cells looked under the microscope after a series of special staining techniques, but did not take into account prognosis. It is now known that the subtype of AML is important in helping to determine a patient’s prognosis. In 2016 World Health Organization (WHO) updated the classification system to better address prognostic factors. They divided AML into several broad groups, including AML with certain chromosomal translocations, AML related to previous cancer or cancer therapy, AML with involvement of more than one cell type, and other AML that don’t fall into the first three groups.²Once a case has been placed in one of these broad groups, the AML can be further classified as poor risk, intermediate risk and better risk based on other test results. Better risk is associated with better response to treatments and longer survival.³The European LeukemiaNET (ELN) first recommended integrating molecular and cytogenic data into classification to create such a risk classification system for AML in 2010 (ELN-2010). In 2017, this was again revised (ELN-2017) to further improve risk stratification. The ELN-2017 can be used to more accurately predict prognosis in newly diagnosed AML.¹

What this means is that AML is now classified by abnormal cell type as well as by the cytogenetic, or chromosome, changes found in the leukemia cells. Certain chromosomal changes can be matched with the morphology of the abnormal cells. These chromosomal changes can help doctors determine the best treatment options for patients because these changes can predict how well treatment will work.

Examples of risk classification include the knowledge that some chromosome rearrangements actually offer a better prognosis. For example, a translocation between chromosomes 15 and 17 [t(15;17)] is associated with acute promyelocytic leukemia (APL or M3). APL is treated differently than other subtypes and has the best prognosis of all the AML subtypes. Other favorable chromosomal changes include [t(8;21)] and [inversion (16) or translocation t(16;16)]. Examples of intermediate risk prognosis are ones associated with normal chromosomes and [t(9;11)]. Poor prognosis is associated with findings such as deletions or extra copies of certain chromosomes or complex changes in many chromosomes.³

The patient was diagnosed with AML, non M3 type. AML prognosis is based on CBC results, markers on the leukemia cells (flow cytometry), chromosome (cytogenic) abnormalities found and gene mutations (molecular abnormalities). In this patient the FISH studies did not demonstrate any chromosome rearrangements, which alone would place him in an intermediate risk group. In addition, our patient was over age 60 and had a WBC over 100,000/mm³which have both been linked to worse outcomes.

Here’s one more photo for your enjoyment! It’s not often that we see so many blasts in a patient with no previous history. As a side note, I was contemplating titling this blog “Fatigue and Shortness of Breath in the Time of COVID.” I can’t help but wonder if this patient would have been diagnosed 6-8 weeks earlier if this was another year and he had been seen when he first experienced symptoms. This year, emergency rooms and physicians have reported a decrease in numbers of patients being seen for chest pain, ketoacidosis, shortness of breath, strokes and other serious conditions. Many patients are reluctant or afraid of sitting in crowded waiting rooms, fearful they will catch COVID. And many doctors are only offering virtual visits or have reduced the number of patients being seen so it is harder to get appointments. This patient expressed his reluctance to seek medical help because of fears of COVID. He did not want to go out in public and waited almost a month for symptoms to go away on their own before first being seen. After going to the walk in center, he called his PCP a week later and was still averse to going to the ER as suggested by the doctor. Then he waited another 2 days for an office appointment. The doctor still suspected COVID, but fortunately for the patient, ordered a CBC. The flow cytometry and FISH studies were available the following day. The patient was referred for hematology consult but has not been seen again at our hospital.

References

Boddu, P.C., Kadia, T.M., Garcia‐Manero, G., Cortes, J., Alfayez, M., Borthakur, G., Konopleva, M., Jabbour, E.J., Daver, N.G., DiNardo, C.D., Naqvi, K., Yilmaz, M., Short, N.J., Pierce, S., Kantarjian, H.M. and Ravandi, F. (2019), Validation of the 2017 European LeukemiaNet classification for acute myeloid leukemia with NPM1 and FLT3‐internal tandem duplication genotypes. Cancer, 125: 1091-1100. https://doi.org/10.1002/cncr.31885
Mandel, Ananya. Acute Myeloid Leukemia Classification. Medical Life Sciences. https://www.news-medical.net/health/Acute-Myeloid-Leukemia-Classification.aspx
Ari VanderWalde, MD, MPH, MA, FACP; Chief Editor: Karl S Roth, MD. Genetics of Acute Myeloid Leukemia. Medscape. Updated: Dec 17, 2018

Hematology Case Study: A 20 Year Old with Anemia

Case History

A 20 year old Black male with a known history of HbS trait went to the primary care office for a pre-surgical evaluation for elective laparoscopic cholecystectomy for symptomatic cholelithiasis. All physical exam findings were negative. The patient had blood work completed and was found to have mild anemia with microcytosis. On previous imaging, the spleen was noted to be slightly enlarged. Further workup included a peripheral blood smear, finding target cells, microspherocytes, folded cells, and rod-shaped Hb C crystals (see image below). No sickled RBCs were noted.

Image 1. Peripheral blood smear with anemia, increased polychromatphilic RBCs, numerous target cells and rare HbC crystals

Discussion

Hemoglobin C disease is an intrinsic red cell disorder caused by Hemoglobin C (Hb C). Hb C is a variant of normal Hemoglobin A (Hb A) that results from a missense mutation in the β-globin protein, replacing the glutamic acid at position 6 with a lysine molecule. The disease can be either in the homozygous state (Hb CC) or in the heterozygous states (Hb AC or Hb SC). The origin of this mutation was traced back to West Africa and is found to confer protection against severe manifestations of malaria. In the United States, the Hb C allele is prevalent in about 1-2% of the African American population. There is an equal incidence between gender, and the incidence of the homozygous disease (i.e., Hb CC) is only 0.02%. Nevertheless, these statistics may be under-representative, since the disease is generally asymptomatic.

Heterozygous individuals with Hb AC usually show no symptoms, while homozygous individuals with Hb CC can have mild hemolytic anemia, jaundice, and splenomegaly. When Hb C is combined with other hemoglobinopathies, such as Hemoglobin S (Hb S), more serious complications can result. Hb S is similar to HbC in that it arises from a missense mutation; ie, a valine is substituted for the glutamic acid at the 6th position on the β-globin protein. As a result of this mutation, HbS abnormally polymerizes when in the presence of low oxygen tension, leaving the red blood cells (RBCs) rigid and irregularly shaped. Sickle cell disease (SCD) typically is a result of homozygous Hb S mutations (i.e., Hb SS), but the disease can also come from Hb SC.

All clinical features of Hb SS can be seen in Hb SC, including painful vaso-occlusive crises, chronic hemolytic anemia, stroke, acute chest syndrome, etc. Nevertheless, Hb SC is generally a milder disease. The complications from HbSC disease are less severe and less frequent when compared to Hb SS. Fortunately, unlike those with Hb SS disease, patients with Hb SC disease do not experience autosplenectomy, but they can develop splenomegaly. There are two complications that occur in HbSC disease occur more frequently than in HbSS disease, and they include proliferative sickle cell retinopathy and avascular necrosis of the femoral head (the latter case presents especially in peripartum women). Therefore, patients with HbSC disease should follow up with ophthalmology and obstetrics to monitor these complications. Furthermore, patients with Hb SC disease can vary in the severity of symptoms and the resulting complications. For example, some patients may develop a severe anemia and require blood transfusions; whereas, other patients are minimally affected by the disease. Overall, patients with Hb SC disease tend to have a better life expectancy compared to those with Hb SS disease. Patients with Hb SS disease have an average life expectancy of 40 years, while those with Hb SC disease are expected to live into their 60s and 70s. In contrast to Hb SS and Hb SC disease, Hb CC disease does not have an increase in mortality. As mentioned earlier, Hb CC disease results only in mild anemia, asymptomatic splenomegaly, and largely absent clinical symptoms.

Pathologic features of Hb SC and Hb CC diseases can be seen on a peripheral blood smear (PBS). Hb CC disease does not show sickled RBCs, while Hb SC can show sickled RBCs though very rarely. More importantly, Hb C is prone to polymerize into characteristic crystals. Depending on the zygosity of the individual, the crystals take on a defining shape. In heterozygous individuals (Hb SC), the crystals are found as irregular, amorphous, or bent appearing, and the RBCs can take on a “spiked and hooked” appearance. In homozygous individuals (Hb CC), the crystals are elongate, straight, and uniformly dense (as seen in the case above). In addition to crystals, the PBS shows numerous target cells, scattered folded cells, and microspherocytes.

Ancillary studies for diagnosis of these diseases include Hb variant analysis, such as electrophoresis and high-pressure liquid chromatography. Cellulose acetate (alkaline) electrophoresis is a standard method used to separate Hb A, Hb A₂, Hb F, Hb C, Hb S, and other variants according to charge. Some hemoglobin variants comigrate using this described method, so citrate agar (acid) electrophoresis can be used additionally to distinguish between these variants. In Hb CC disease, analysis shows nearly all Hb C with small amounts of Hb F (i.e., fetal hemoglobin) and HbA₂ (i.e., a normal variant of Hb A, in which the hemoglobin molecule is made up of 2 α chains and 2 δ chains). In Hb SC disease, analysis demonstrates almost equal amounts of Hb S and Hb C.

References

Aster JC, Pozdnyakova O, Kutok JL. Hematopathology: A Volume in the High Yield Pathology Series. Philadelphia, PA: Saunders, an imprint of Elsevier Inc.; 2013.
Gao J, Monaghan SA. Hematopathology. Chapter 1: Red Blood Cell/Hemoglobin Disorders. 3rd edition. Philadelphia, PA: Elsevier; 2018.
Karna B, Jha SK, Al Zaabi E. Hemoglobin C Disease. 2020 Jun 9. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan–. PMID: 32644469.
Mitton BA. Hemoglobin C Disease. Medscape, 9 Nov. 2019, emedicine.medscape.com/article/200853-overview.
Saunthararajah Y, Vichinsky EP. Hematology: Basic Principles and Practice. Chapter 42: Sickle Cell Disease: Clinical Features and Management. Philadelphia, PA: Elsevier; 2018.

-Amy Brady is a 4th-year medical student at the Philadelphia College of Osteopathic Medicine. She is currently applying to AP/CP pathology residency programs. Follow her on Twitter @amybrady517.

-Kamran Mirza, MD PhD is an Associate Professor of Pathology and Laboratory Medicine and Medical Education, and the Vice-Chair of Education in the Department of Pathology at Loyola University Chicago Stritch School of Medicine. Follow him on Twitter @KMirza.

Hematology Case Study: Is it Pelger-Huët anomaly or Pseudo Pelger-Huët?

A 73 year old African American female had a CBC ordered as part of routine pre-op testing before knee surgery. The order for a CBC/auto differential and was run on our Sysmex XN-3000. CBC results were unremarkable, with the exception of a decreased platelet count. However, the instrument flagged “Suspect, Left shift?” and a slide was made for review. The CBC results are shown in Table 1 below.

Table 1. CBC results on 73 year old female.

Pelger-Huët anomaly (PHA), is a term familiar to medical laboratory professionals, but mostly from textbook images. PHA is considered to be rare, affecting about 1 in 6000 people. PHA has been found in persons of all ethnic groups and equally in men and women. The characteristic, morphologically abnormal neutrophils were first described by Dutch hematologist Pelger in 1928. He described neutrophils with dumbbell shaped, bi-lobed nuclei. The term ‘pince-nez’ has also been used to describe this spectacle shaped appearance. Pelger also noted that, in addition to hyposegmentation, there is an overly coarse clumping of nuclear chromatin. In 1931, Huët, a Dutch pediatrician, identified this anomaly as an inherited condition.

Pelger-Huët anomaly is an autosomal dominant disorder caused by a mutation in the lamina B receptor (LBR) gene on band 1q42. This defect is responsible for the abnormal routing of the heterochromatin and nuclear lamins, proteins that control the shape of the nuclear membrane.² Because of this mutation, nuclear differentiation is impaired, resulting in white blood cells with fewer lobes or segments. In classic inherited PHA, cells are the size of mature neutrophils and have very clumped nuclear chromatin. About 60-90% of these neutrophils are bi-lobed either with a thin filament between the lobes, or without the filament. About 10-40% of total neutrophils in PHA have a single, non-lobulated nucleus. Occasional normal neutrophils with three-lobed nuclei may be seen.¹ Despite their appearance, Pelger-Huët cells are considered mature cells, function normally and therefore can fight infection. It is considered a benign condition; affected individuals are healthy and no treatment is necessary for PHA.

Automated instruments may flag a left shift when they detect these Pelger-Huët cells. In this patient, the analyzer flagged a left shift and a slide was made and sent to CellaVision. The CellaVision pre-classified the Pelger-Huët cells as neutrophils, bands, and myelocytes. All of the neutrophil images were either bi-lobed or non-lobed forms. None of the neutrophils had more than 2 lobes. Eosinophils also had poorly differentiated nuclei. Cell images from this patient can be seen in Images 1-4.

Image 1. Images from CellaVision of bi-lobed “pince-nez” neutrophils with thin filament

Image 2. Non-terminally differentiated neutrophils pre-classified as bands on CellaVision. Bilobed variant without the thin filament.

Image 3. Non-lobed neutrophils with extremely coarse clumping of nuclear chromatin.

Image 4. Eosinophils in Pelger-Huët Anomaly.

If PHA is considered benign, with no clinical implications, why is it important to note these cells on a differential report? This slide was referred to our pathologist for a review. The patient had several previous CBC orders, but no differentials in our LIS. The pathologist reviewed the slide and, based on 100% of these neutrophils being affected, he reported “Pelger-Huët cells present. The presence of non-familial Pelger-Huët anomaly has been associated with medication effect, chronic infections and clonal myeloid neoplasms.” Thus, the importance of reporting this anomaly if seen on a slide. If the instrument flags a left shift, this is typically associated with infection. If these cells are misclassified as bands and immature granulocytes, with no mention of the morphology, there would be a false increase in bands reported and the patient may be unnecessarily worked up for sepsis.

An additional reason for reporting the presence of Pelger-Huët cells is that pelgeroid cells are also seen in a separate anomaly, called acquired or pseudo-Pelger-Huët anomaly (PPHA). PPHA is not inherited and can develop with acute or chronic myelogenous leukemia and in myelodysplastic syndrome. A type of PPHA may also be associated with infections or medications. Certain chemotherapy drugs, immunosuppressive drugs used after organ transplants, and even ibuprofen have been recognized as triggers for PPHA. PPHA caused by medications is typically transient and resolves after discontinuation of the drug. To add to causes, most recently, there have been studies published that report PPHA in COVID-19 patients.³

With several different causes of PHA/PPHA, a differential diagnosis is important. Is this a benign inherited condition, a drug reaction that will self-resolve after therapy is stopped, or something more serious? If Pelger-Huët cell are reported, it is important for the provider to correlate this finding with patient symptoms, treatments and history. There was no medication history and little other medical history in our case patient’s chart, and no mention of inherited PHA. The patient had also been tested for COVID-19 with her pre-op testing and was COVID negative. On initial identification of Pelger-Huët, a benign diagnosis that needs no treatment or work up would be the best outcome, so an attempt could be made to determine if the patient has inherited PHA. If other family members are known to have this anomaly, this would be the likely diagnosis as PHA is autosomal dominant. Family members can also easily be screened with CBC and manual differential. Molecular techniques are available to confirm PHA but are not routinely used. In the absence of this anomaly in other family members, it would need to be determined if the patient was on any medications that can cause pelgeroid cells. Inherited PHA and drug induced PPHA should be ruled out first because PPHA can also be predicative of possible development of CML or MDS. Considering this cause first could lead to unnecessary testing that might include a bone marrow aspirate and biopsy. Additionally, the entire clinical picture should be reviewed because in PPHA associated with myeloproliferative disorders there is usually accompanying anemia and thrombocytopenia and the % of pelgeroid cells tends to be lower.

Today most clinical laboratories have instruments that do automated differentials, and we encourage physicians to order these because they are very accurate and count thousands of cells compared to the 100 cells counted by a tech on a manual differential. Automated differentials are desirable for consistency and to improve turnaround times. Yet, it is important to know when a slide needs to be reviewed under the scope or with CellaVision. If a patient presents with a normal WBC and a left shift on the auto diff with no apparent reason, pictures can reveal important clinical information. Awareness of different causes of PHA/PPHA can relieve anxiety in patients and prevent extensive, unnecessary testing and invasive procedures.

References

https://emedicine.medscape.com/article/957277-followup updated 8/4/2020
Ayan MS, Abdelrahman AA, Khanal N, Elsallabi OS, Birch NC. Case of acquired or pseudo-Pelger-Huët anomaly. Oxf Med Case Reports. 2015;2015(4):248-250. Published 2015 Apr 1. doi:10.1093/omcr/omv025
Alia Nazarullah, MD; Christine Liang, MD; Andrew Villarreal, MLS; Russell A. Higgins, MD; Daniel D. Mais, MD. Am J Clin Peripheral Blood Examination Findings in SARS-CoV-2 Infection . Pathol. 2020;154(3):319-329.

-Becky Socha, MS, MLS(ASCP)^CMBB^CMgraduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

Case Study Hematology: The Mouse Strikes again! Lymphocytes with Intracytoplasmic Inclusions

If you read my last blog, you heard the about the story “if You Give a Mouse a Cookie” by Laura Numeroff.⁴ The curious little mouse has a mind that never rests. As his mind wanders and hops from one thing to another, he keeps discovering more things to check out along the way. Medical laboratory lcientists are a lot like this. We’re a curious bunch, and, in investigations, one thing often leads to the next. Well folks, the mouse has struck again! We were given another cookie in the form of these beautiful cells.

Image 1. Lymphocytes with intracytoplasmic inclusions.

These cells were found by my coworker Liz Marr, MLS(ASCP), and the adventure began! First, we wanted to know what those were, and then we needed to find out more about them, and then, mostly, I wanted to know why in almost 40 years of working in and teaching hematology that I have never before seen this!

The story begins with our case history. We received a CBC from a 71 year old female with a 4 year history of untreated chronic lymphocytic leukemia/ small lymphocytic lymphoma(CLL/SLL). The patient’s recent history included a myocardial infarction(MI) 5 months prior. The patient was found to have leukocytosis (WBC 25.38 x 10³/μL) and absolute lymphocytosis (18.25 x 10³/μL) with normal hemoglobin and hematocrit (Hgb 13.4 g/dL, Hct 40.8%) and normal platelet count (272 x 10³/μL). The differential had 71.9% lymphocytes with many abnormal forms noted. The slide was sent for a pathology review. The pathologist reported “Atypical lymphocytosis consistent with patient’s known chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) Filament-like inclusions are present in the cytoplasm which has been previously reported in patients with CLL.”

Image 2. Lymphocytes with crystalline-like inclusions.

A curious tech can’t stop at just that description. If you tell me they are filament-like inclusions, I will have all kinds of questions. What are these filaments made of? Are they crystals, or something else? How common are these? Are these diagnostic of CLL? Are these only seen in CLL? What is their significance? And, of course, the most puzzling question, why have I never seen these before??

CLL is a form of non-Hodgkin lymphoma and is the most common leukemia in the Western world. It is generally a leukemia of older age with a median age at diagnosis around 67-72. The disease is widely variable, with some patients asymptomatic and requiring no treatment for many years, while others have a more rapidly progressive course of disease requiring treatment. About 60% of patients are diagnosed before they exhibit any symptoms. CLL and SLL are considered to be different manifestations of the same disease. In CLL, the abnormal B lymphocytes are found mostly in the peripheral blood and bone marrow, but in SLL, there is lymph node involvement, with abnormal cells mostly found in the lymph nodes. CLL is diagnosed based on absolute B lymphocyte counts ≥5 x 10⁹/L. Flow cytometry typically reveals a distinctive cell immunophenotype with expression of CD19, CD5, CD23, and Κ/λ; and weak expression of CD20, CD79b, and surface immunoglobulin.¹

The most recent flow cytometry report on our patient was from one year ago. An 8 color analysis with CD45/SSC gating was performed by LabCorp. The flow revealed an abnormal cell population representing 56% of total cells. Two monoclonal B cell populations were detected with identical phenotypes except for light chain expression. These cells expressed CD45, CD19, CD20, CD22, CD5, and CD23., CD38-. This phenotype was consistent with her previous diagnosis of CLL/SLL.

A literature search revealed only a few articles about intracytoplasmic inclusions in CLL. Cytoplasmic inclusions in lymphomas are uncommon, but have been noted as vacuoles, crystals, and pseudocrystals. These crystalline inclusions represent immunoglobulin(Ig) heavy and light chain that precipitate in the cytoplasm. Using electron microscopy it has been found that theses Ig deposits localize in the rough endoplasmic reticulum (RER).⁵ When surface Ig can be demonstrated on the B lymphocytes, it has been found to be same as Ig in the inclusions.⁶

In two published studies that describe these crystal like inclusions, photographs are very similar to the ones we found on our patient.^3,5 It is interesting to note that, in these two studies, neither of the subjects was a known CLL patient. The inclusions were noted in the patients’ cells and the peripheral blood was subsequently sent for flow. Phenotypes reported confirmed monoclonal B-cells representing a large percentage of cells. Huang reported monoclonal B-cells which expressed CD45, CD19, CD20, CD22, CD79b, CD5, CD23, CD148 and CD200(hi), with partial expression lambda, and negative for FMC7, CD10, CD11c, CD49d, CD103, CD38, CD25, CD160, IgM, CD81, kappa and Ki67.³ In the Ramlal case study, phenotype was CD5, CD19, CD20, CD23, positive, CD10, FMC7 negative.⁵ On the basis of flow, along with the CBC results, the patients were diagnosed with CLL.

Of course, while researching this, the little mouse in me kept asking questions and finding more questions to ask. One question that I still had questions about was if these inclusions have any prognostic value. In three recent studies^3,5,6 it was indicated that these inclusions can be used to help with diagnosis, but are not prognostic for course of disease. Rodriguez followed a patient with asymptomatic Rai stage 0 CLL. This patient consistently had inclusions noted in lymphocytes for 9 years before any progression of disease was noted.⁶ In the medical field even if one study reports no prognostic significance, this opinion could change in the future with more studies. Could these crystalline inclusions be used to forecast time to first treatment (TFT) or overall survival?(OS). So far, because of the rarity of these cytoplasmic inclusions, there is no evidence of prognostic value. As well, the mechanism related to their formation and their role in CLL is yet to be determined.

Our case study patient and the various reports found in literature had common flow cytometry immunophenotypes. Patients were all either previously diagnosed with CLL or lymphocytic lymphoma, or were diagnosed at the time of the findings of these inclusions. While these crystalline inclusions alone are not considered diagnostic for CLL, their recognition can be used to assist in a prompt diagnosis of a lymphoproliferative disease. And they are so pretty! What medical laboratory scientist doesn’t love pretty cells? Be like that mouse. Be curious, keep your eyes open, and be on the lookout for these interesting cells in CLL patients, but, more importantly, in patients with lymphocytosis without a known diagnosis of a lymphoproliferative disorder.

References

AJMC, January 7, 2019
Chronic Lymphocytic Leukemia: An Overview of Diagnosis, Prognosis, and Treatment
Huang, Y., Zhang, L. Intracellular rod-like crystals in chronic lymphocyte leukemia. Int J Hematol 112, 267 (2020). https://doi.org/10.1007/s12185-020-02933-7
Numeroff, Laura If You Give a Mouse a Cookie. 1986
Ramlal, B, DiGiuseppe, JA. Intracytoplasmic crystalline inclusions in chronic lymphocytic leukemia. Clin Case Rep. 2019; 7: 1460– 1461. https://doi.org/10.1002/ccr3.2250
Cecilia M. Rodríguez, Carmen Stanganelli, Claudio Bussi, Daniela Arroyo, Darío Sastre, Viviana Heller, Pablo Iribarren & Irma Slavutsky (2018)Intracytoplasmic filamentous inclusions and IGHV rearrangements in a patient with chronic lymphocytic leukemia, Leukemia & Lymphoma, 59:5,1239-1243, DOI: 10.1080/10428194.2017.1370549

The Utility of Flow Cytometry in Establishing the Correct Diagnosis of a Rare Aggressive Lymphoma

Case history

A 73 year old woman presented with shortness of breath and was found to have bilateral pleural effusions. She had a history of marginal zone B-Cell lymphoma involving the bone marrow, which was diagnosed 3 months before this presentation and was treated with Rituximab.

Thoracentesis revealed an atypical lymphoid population comprised of intermediate and large sized cells with eccentrically placed nuclei, multiple prominent nucleoli and scant to moderate amounts of basophilic cytoplasm (Image 1). Initial evaluation of the cytology material was concerning for large-cell transformation of the patient’s previously diagnosed marginal zone B cell lymphoma. A representative portion of the fine needle aspirate sample was sent for flow cytometric immunophenotyping.

Image 1. Cytology (Diff Quik, 400X). The atypical lymphoid population is comprised of intermediate and large sized cells with eccentrically placed nuclei, multiple prominent nucleoli and scant to moderate amounts of basophilic cytoplasm.

Flow cytometric immunophenotyping showed a distinct population of atypical cells with moderate CD45 expression and increased side scatter in keeping with cytoplasmic complexity (Figure 1, black arrows). On an initial screening B cell lymphoma panel these cells were negative for CD19 and positive for CD30 (partial), and CD44 (Figure 2).

Figure 1. The neoplastic population shows expression of CD30 and CD44.

The population of interest lacked expression of CD10, CD20, CD22 and surface immunoglobulin light chains and CD138 (Figure 2 and 3).

Figure 2. The neoplastic population lacks expression of CD10, CD19, CD20, and CD22.

Figure 3. The neoplastic population of cells are negative for surface immunoglobulin light chains and CD138.

CD30 expression prompted the investigation of additional T-cell markers to rule out a T cell lymphoma (Figure 4). This population showed dim expression of CD7 but was otherwise negative for pan T cell markers (CD2, CD3, CD5) as well as CD4 and CD8 (Figure 4).

Figure 4. The neoplastic population of cells are positive for CD7 (dim) and CD30 and negative for CD3, CD4, CD8, and CD26.

Given the unusual immunophenotype of the neoplasms, a diagnosis of diffuse large B cell lymphoma (transformation of the known marginal zone lymphoma) seemed less likely and other possibilities were considered.

The presence of CD30 expression and the plasmablastic morphologic features together with the clinical presentation with effusions raised the possibility of primary effusion lymphoma. IHC for anti-HHV8 was performed on the cell block sample (Image 2).

Image 2. Cytology (cell block 400X) A. Hematoxylin and eosin stain of the cell block reveals large, atypical lymphoid cells; Small and large atypical lymphoid cells are highlighted by CD30 (B), HHV8 (LANA-1) (C), and CD138 (focally) (D).

Final diagnosis

Primary effusion lymphoma (HHV8 positive).

Discussion

Primary effusion lymphoma (PEL) is a large B-cell neoplasm usually presenting as serous effusions without a detectable tumor mass [1]. It is universally associated with the human herpesvirus 8 (HHV8). It usually occurs in the setting of immunodeficiency [2]. Some patients with PEL secondarily develop solid tumors in adjacent structures such as the pleura [3-5].

Immunophenotype of PEL:

POSITIVE: CD45, HLA-DR, CD30, CD38, VS38c, CD138, EMA, HHV8 (LANA1).

NEGATIVE: pan- B-cell markers (CD19, CD20, and CD79a), surface and cytoplasmic Ig, and BCL6.

PEL is usually negative for T/NK-cell antigens, although aberrant expression of T-cell markers may occur. PEL is usually positive for EBV-encoded small RNA (EBER) by in situ hybridization but negative for EBV latent membrane protein 1 (LMP1) by IHC. This could be explained by EBV virus latency. It is ability of a pathogenic virus to lie dormant (latent) within a cell, denoted as the lysogenic part of the viral life cycle. EBV expresses its genes in one of three patterns, known as latency programs. EBV can exhibit one of three latency programs: Latency I, Latency II, or Latency III. Each latency program leads to the production of a limited, distinct set of viral proteins and viral RNAs. The Epstein-Barr virus encoded RNAs (EBERs): EBER1 and EBER2 are expressed during all latency forms [6], whereas LMP1 is expressed only in latency 2 and 3 rendering it a less sensitive marker for detection of EBV infection. EBV-negative PEL is common in elderly, HIV-negative patients from HHV8-endemic regions (Mediterranean) [7].

Differential Diagnosis

Most common cavities involved by PEL: pleural, pericardial, and peritoneal [8-10].

It was thought that PEL can involve an artificial cavity related to the capsule of a breast implant [11] although it was described only in one case report without appropriate HHV8 staining and before recognition of breast implant-associated anaplastic large cell lymphoma (BIA-ALCL), which this case probably was presenting [12].

Primary effusion lymphoma (PEL) Prognosis

The prognosis is very unfavorable. Median survival is < 6 months. Rare cases have been reported that responded to chemotherapy and/or immune modulation [13].

Flow Cytometry Utility

The importance of utility of flow cytometry in establishing a diagnosis of PEL has been previously shown by others [14]. In the series by Galan et al. the authors described a case of PEL in an 88-year-old HIV-negative female with right-sided pleural effusion without significant lymphadenopathies or other effusions. The cytological study of the pleural fluid revealed a dense proliferation of large plasmablastic cells. A six-color multiparametric flow cytometry immunophenotyping study revealed 45% of large in size and high cellular complexity cells positive for CD45 (dim), CD38, CD138, CD30 and HLA-DR; and negative for CD19, CD20, cytoplasmatic CD79a, surface and cytoplasmic light chains Kappa and Lambda, CD3, CD4, CD5, CD7, CD8, CD28, CD56, CD81, and CD117. In situ hybridization for EBV-encoded small RNA was negative and immunohistochemistry for Kaposi sarcoma herpesvirus (HHV8) confirmed the diagnosis of PEL. These results in addition to the current case highlight the utility of flow cytometry in the diagnosis of lymphomas involving body cavities.

In Summary

PEL is associated with a proliferation of large B-cells which are positive for HHV8, CD45 (dim), CD30, CD38, and CD138 and negative for lineage defining B cell markers (CD19, CD20, and CD79a). Although PEL is a very rare lymphoma, it is important to consider it in patients with pleural, pericardial, and peritoneal effusions by sending a sample for cytological examination and flow cytometric immunophenotyping. Due to the absence of a mass lesion, cytology and flow cytometry are essential for establishing the diagnosis of PEL.

References

Said, J.a.C.E., Primary effusion lymphoma, in WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th edition), C.E. Swerdlow SH, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Editor. 2017: Lyion. p. 323–324.
Song, J.Y. and E.S. Jaffe, HHV-8-positive but EBV-negative primary effusion lymphoma. Blood, 2013. 122(23): p. 3712.
Dotti, G., et al., Primary effusion lymphoma after heart transplantation: a new entity associated with human herpesvirus-8. Leukemia, 1999. 13(5): p. 664-70.
Jones, D., et al., Primary-effusion lymphoma and Kaposi’s sarcoma in a cardiac-transplant recipient. N Engl J Med, 1998. 339(7): p. 444-9.
Luppi, M., et al., Molecular evidence of organ-related transmission of Kaposi sarcoma-associated herpesvirus or human herpesvirus-8 in transplant patients. Blood, 2000. 96(9): p. 3279-81.
Khan, G., et al., Epstein Barr virus (EBV) encoded small RNAs: targets for detection by in situ hybridisation with oligonucleotide probes. J Clin Pathol, 1992. 45(7): p. 616-20.
Dupin, N., et al., Distribution of human herpesvirus-8 latently infected cells in Kaposi’s sarcoma, multicentric Castleman’s disease, and primary effusion lymphoma. Proc Natl Acad Sci U S A, 1999. 96(8): p. 4546-51.
Otsuki, T., et al., Detection of HHV-8/KSHV DNA sequences in AIDS-associated extranodal lymphoid malignancies. Leukemia, 1996. 10(8): p. 1358-62.
DePond, W., et al., Kaposi’s sarcoma-associated herpesvirus and human herpesvirus 8 (KSHV/HHV8)-associated lymphoma of the bowel. Report of two cases in HIV-positive men with secondary effusion lymphomas. Am J Surg Pathol, 1997. 21(6): p. 719-24.
Beaty, M.W., et al., A biophenotypic human herpesvirus 8–associated primary bowel lymphoma. Am J Surg Pathol, 1999. 23(8): p. 992-4.
Said, J.W., et al., Primary effusion lymphoma in women: report of two cases of Kaposi’s sarcoma herpes virus-associated effusion-based lymphoma in human immunodeficiency virus-negative women. Blood, 1996. 88(8): p. 3124-8.
Lyapichev, K.A., et al., Reconsideration of the first recognition of breast implant-associated anaplastic large cell lymphoma: A critical review of the literature. Ann Diagn Pathol, 2020. 45: p. 151474.
Ghosh, S.K., et al., Potentiation of TRAIL-induced apoptosis in primary effusion lymphoma through azidothymidine-mediated inhibition of NF-kappa B. Blood, 2003. 101(6): p. 2321-7.
Galan, J., et al., The utility of multiparametric flow cytometry in the detection of primary effusion lymphoma (PEL). Cytometry B Clin Cytom, 2019. 96(5): p. 375-378.

This case was previously presented by authors as eCSI Case on International Clinical Cytometry Society website. For more information please follow: https://www.cytometry.org/public/newsletters/eICCS-10-1/article7.php

-Dr. Loghavi is an Assistant Professor of hematopathology and molecular pathology MD Anderson Cancer Center in Houston, TX. She received her MD degree from the Azad University in Tehran, Iran. Shen then completed an Anatomic and Clinical Pathology residency training at Cedars Sinai Medical Center in Los Angeles, CA, followed by Surgical pathology, Hematopathology and Molecular pathology fellowship training at the University of Texas, MD Anderson Cancer Center. Dr. Loghavi is passionate about medical education. Her clinical and research interests are focused on hematologic malignancies, with particular focus on myeloid neoplasm and the applications of flow cytometric immunophenotyping and molecular methods in detection of minimal/measurable residual disease. She has authored 100 peer-reviewed articles, 5 book chapters, and numerous abstracts in the fields of hematopathology and molecular pathology.

-Kirill Lyapichev, MD, FASCP, is a board-certified anatomical and clinical pathologist who completed 2 years of hematopathology fellowship at MD Anderson Cancer Center. He is currently a molecular genetic pathology fellow at MD Anderson Cancer Center. Additionally, he is interested and involved in other research projects including neoplastic as well as non-neoplastic entities: MALT lymphoma, Castleman Disease, Kikuchi-Fujimoto Disease, and others. In 2020 he was selected as one of ASCP’s 2020 Top 40 Under Forty. Follow him on Twitter: @KirillLyapichev.