hematology – 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.

Hematology Case Study: 75 Year Old Man with Leukopenia

A 75 year old male first presented earlier this year with abnormal CBC results. The patient has a history of Type 2 diabetes, high blood pressure and atrial fibrillation. He was diagnosed with non-small-cell lung cancer (NSCLC) 6 years ago. His stage II NSCLC was completely removed with surgery. Surgery was followed up with adjuvant cisplatin-based chemotherapy to reduce the chance that the cancer would return. In June, he was referred to the hematology oncology department following consecutive CBCs that revealed leukopenia and thrombocytopenia. The CBC results from these specimens are shown below in Table 1.

Table 1. CBC results from a 75 year old male.

The peripheral blood sample from June was sent for flow cytometry. A leukemia/lymphoma phenotype was performed. Result comments noted proportionately decreased granulocytes with a left shift and 4% blasts. The blasts were CD34+, CD117+, HLA-DR+, CD13+ and CD33+ and were identified as myeloblasts. There were proportionately increased atypical monocytes with CD23 expression. Lymphocytes were also proportionately increased and included an increased population of CD57+, CD3+ T cells consistent with T-cell large granular (LGL) expansion. Immunophenotypic findings raised a concern for a myelodysplastic process. The hematologist discussed the findings with the patient and the patient was scheduled for a bone marrow biopsy. The procedure was performed 3 weeks later. CBC results on the day of the procedure are shown below in Table 2.

Table 2. CBC results day of bone marrow procedure. Pre-op diagnosis: Anemia, Leukopenia.

Bone marrow aspirate showed markedly increased myeloblasts (55%), consistent with acute myeloid leukemia (AML), nonacute promyelocytic leukemia (APL) type. The phenotype of the blasts was CD13+, CD33+, CD117+ and HLA-DR+. Blasts were negative for CD34. Several genomic variations were found in the specimen. These included variations in IDH2, SRSF2, STAG2 and ASXL1. Diagnosis: Increase in myeloblasts consistent with AML, nonAPL type.

In July, 20 days after the bone marrow procedure and AML diagnosis, the patient was scheduled to begin his first cycle of Azacitidine (Vidaza). Based on his critical hemoglobin, the patient received 1 unit of packed RBCs followed by his first Vidaza injections. This Cycle 1, Day 1 chemotherapy was well tolerated, and he returned home. The following day he returned for his second treatment. His CBC showed good response to the previous day’s transfusion and his Cycle 1, Day 2 Vidaza was administered without incident. However, that evening the patient presented to the ER with nausea, vomiting and nose bleeds. The patient was admitted to the hospital and received another RBC transfusion. For the next several days the patient continued to do poorly, requiring additional RBC transfusions, and the Vidaza treatments were deferred, then discontinued. The patient had several ER visits and hospital admissions with transfusions over the next 2 weeks. During this time, we saw his blast% on his differential peak at over 60%. The patient was transferred to the palliative care team with care and comfort measures. CBC results from Cycle 1, Day 1 and subsequent CBC results are shown below. Note the sharp increase in blasts over a 2-week period.

Table 3. CBC results after chemotherapy initiated, then discontinued

Image 1. Cells classified as blasts on CellaVision

AML is the most common acute leukemia in adults. In AML with minimal differentiation, evidence of bone marrow failure is characterized by anemia, neutropenia, and thrombocytopenia. The median age for patients with AML in the US is 66-67, and those who are older than 55-65 at diagnosis often have challenges and lower odds for long term survival. These older patients tend to have poor tolerance to traditional aggressive chemotherapy because of other health issues. This patient was likely not a good candidate for strong chemotherapy because of his age and health history. In these more fragile patients, Vidaza may be used. Vidaza is a class of drug called a hypomethylating agent that works by switching off DNA methyltransferase. This switches on genes that stop the cancer cells growing and dividing. The goal is to reduce the number of abnormal blood cells and to control cell growth.

As you can see from the CBC results, the onset of this patient’s AML was very abrupt, and the disease progressed rapidly. He has several risk factors that made him more likely to be diagnosed with AML. Older age is a risk factor for AML, and AML is more common in males than females. He has a history of smoking which is a behavioral risk factor associated with AML. Additionally, patients with cancer who are treated with certain chemotherapy drugs are more likely to develop AML in the years following treatment. This patient was treated with cisplatin following lung cancer surgery. Cisplatin is an alkylating agent which has been linked to an increased risk of AML.

Also interesting is the note on the peripheral blood phenotype interpretation that a T-cell large granular lymphocyte (LGL) expansion was present. These are an increased population of CD57+, CD3+ T cells. LGL clones have been described in AML and a hallmark of this association is cytopenia, as is observed in this patient. The patient’s poor prognosis can partly be attributed to the p.Gly646TrfsTer12 alteration in the ASXL1 gene, identified in the bone marrow interpretation. This alteration is associated with decreased overall survival and poor prognosis which was observed in this patient.

I work in a hospital with a large hematology/oncology practice, and we see a lot of adult leukemia patients. Many of the patients we see regularly have Chronic Lymphocytic Leukemia (CLL). We feel like we get to know these patients, because even though we never see them, we see their CBCs every week, sometimes for many years. This was an interesting case because it reminded me of the sudden onset and rapid progression of AML. It was amazing to see the differentials change so dramatically in a matter of weeks. This patient is currently receiving care and comfort end of life measures.

References

Fattizzo, B, Bellani, V, et al. Large Granular Lymphocyte Expansion in Myeloid Diseases and Bone Marrow Failure Syndromes: Whoever Seeks Finds. Front. Oncol., Sec. Cancer Immunity and Immunotherapy. 01 October 2021.

Pratcorona M, Abbas S, Sanders MA, Koenders JE, et al.Acquired mutations in ASXL1 in acute myeloid leukemia: prevalence and prognostic value. Haematologica. 2012 Mar;97(3):388-92. doi: 10.3324/haematol.2011.051532. Epub 2011 Nov 4. PMID: 22058207; PMCID: PMC3291593.

https://www.cancer.net/cancer-types/lung-cancer-non-small-cell/types-treatment

Thomas XG, Dmoszynska A, Wierzbowska, et al. Results from a randomized phase III trial of decitabine versus supportive care or low-dose cytarabine for the treatment of older patients with newly diagnosed AML. Journal of Clinical Oncology 29:2011

Turgeon, Mary Louis. Clinical Hematology Theory and Procedures, 6^th ed, Jones and Bartlett Learning, 2017.

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: Temporal Arteritis or COVID-19?

What is your least favorite test in hematology? The first things that come to my mind are those tests that are time consuming, tedious, and manual. I’ve worked in a hematology lab that did Kleihaur betke (KB) tests, and whenever I worked, I seemed to get one, or sometimes more, in a given shift. And when I worked in blood bank, we did KBs in blood bank, and I certainly did my share there, too. KBs seem to follow me around! Those, I must admit, are probably my least favorite, but I know that many techs dread parasite smears or % parasitemia, reviewing 150 or more fields or counting thousands of cells on a smear. Manual body fluid counts, manual reticulocyte counts, and manual platelet counts are likely some others on our lists of “not favorites.” Basically, anything that requires a lot of time, manual counting, and math!

One other test that probably doesn’t make many “favorites” list is the Erythrocyte Sedimentation Rate (ESR), or sed rate. Remember old fashioned Westergren Sed Rates that took an hour to do, while the ER doctor kept calling looking for their “STAT” results? There are still labs that set up manual sed rates that take an hour, and modified manual methods that take “only” 15 or 30 minutes. Some semi-automated methods can give us results in a couple minutes, but still require techs to fill a capillary tube and load the instrument. Fortunately, real help may have arrived, in the form of fully automated ESR instruments! There are instruments now that actually make ESRs almost fun. I’ve never seen techs so excited about a new instrument as they were when we got iSeds. This thing is amazing! It’s like a little Ferris wheel for sed rates. You pop the whole tube in, they go for a little ride around the Ferris wheel, then drop out, in less than 30 seconds. And you can keep loading tubes even while it’s running. A truly Stat ESR. Now that’s amazing!

Image 1. Alcor iSED Automated ESR Instrument

While these new instruments make ESR’s easier to run, with more reproducible results, and less hands-on time, they still don’t get much love, because, well…there are newer tests available for inflammation, and we know that the ESR is not a specific test for diagnosis. Across the years, some lab tests have become antiquated and obsolete…bleeding times come to mind, along with CK-MB. In 2010 an article was published that supported discontinuing laboratory tests that no longer have clinical utility in the lab. The ESR was on this list. Yet, many labs still perform ESRs. Should the ESR be phased out, or are there still valid reasons for ordering them?

Even though the test is considered non-specific, the ESR test is considered helpful in diagnosing two specific inflammatory diseases: temporal arteritis (TA) and polymyalgia rheumatica. A high ESR is one of the main test results used to confirm these diagnoses. It is also used to monitor disease activity and response to therapy in both these conditions. Almost all cases present with an elevated ESR, though a normal ESR should not be used to rule out these conditions.

Case 1: A 70 year old White female was admitted to the ER complaining of throbbing headache and blurry vision. She stated that the headache started 2 days ago, had been at her temples at first but in the past few hours was getting worse. She stated that she was prompted to come to the ER because now her whole scalp hurt, and her vision was blurry. A CBC, Basic panel, CRP and ESR were ordered. The CBC results were unremarkable, other than and increased platelet count of 480,000/µL. ESR was 110 mm/hr. Basic panel results were normal. CRP was 2.51 md/dL.

The patient was started on prednisone immediately, and a temporal artery biopsy was scheduled, with a suspicion of temporal arteritis (TA), also known as giant cell arteritis (GCA). TA is an autoimmune disease that causes inflammation of the temporal arteries. Under the microscope, the inflamed cells of these arteries look giant, which is how the disease got its name. The inflammation causes constriction of the arteries, can affect chewing and eating, and may cause blindness if not treated promptly. Treatment of choice are corticosteroids, often prescribed for at least a year. Symptoms are monitored frequently and lab results, including the ESR, can be used to monitor the condition and response to treatment.

If you are still wondering if the ESR should be discontinued as a useful test, we are now seeing patients with COVID infection and elevated ESRs. Over the past 2 years, several articles have been written about elevated ESRs in COVID-19 patients. One study aimed to evaluate the usefulness of ESR in distinguishing severe from non-severe COVID-19 cases. The study suggests that severe COVID-19 cases are associated with higher elevations of ESR, as compared to non-severe cases. A case report of a patient recovering from COVID described an increased ESR. The high ESR persisted for a long time even after the patient recovered from COVID-19, while no other inflammatory processes or other conditions known to raise ESRs were found.

Case 2: My second case is a case of a 58 year old woman who presented with an earache and a pulsing temporal headache. Ear infection was ruled out and the patient was referred to ophthalmology for possible TA. The patient’s CRP was elevated but her ESR and platelet counts were within normal reference range. The patient was COVID tested as part of a pre-op workup before temporal artery biopsy and the COVID-19 test came back positive. There have been cases in literature in the last year of this new set of symptoms in COVID-19 patients. The conclusion from these cases is that if a patient appears with symptoms consistent with TA with an elevated CRP but with a normal ESR and platelet counts, that the patients should be tested for COVID.

The ESR is one of the oldest laboratory tests still in use. The study of the sedimentation of blood was one of the principles on which ancient Greek medicine was based. In the 1700’s, physicians noticed that the rate of red blood cell sedimentation changed during illness. This theory was first introduced as a laboratory test over 100 years ago. Depending on the historic accounts and articles you read, it was first described by a Polish physician, Edmund Biernacki, in 1897, or by a Swedish physician, Robert Fahraeus, in 1915. Biernacki proved the connection between the rate of sedimentation and the amount of fibrinogen in the blood and suggested using the ESR in diagnostics. Alf Vilhelm Albertsson Westergren (a familiar name!) also presented a similar description of the ESR. In the early 1920’s. Dr Westergren went further to develop the blood drawing technique and defined standards for the ESR. To this day, the Westergren Erythrocyte Sedimentation Rate method is recognized as the gold standard reference method for ESR measurement.

The sed rate measures the rate at which erythrocytes sediment by gravity, in mm/hour. RBCs usually repel each other due to zeta potential and aggregation is inhibited. In conditions with increased fibrinogen or immunoglobulins, these proteins coat the RBCs, promoting aggregation. The RBCs form rouleaux which settle faster than individual RBCs. In conditions such as anemia, the ESR will be high because with a lower hematocrit, the velocity of the upward flow of plasma is altered and red blood cell aggregates fall faster. In polycythemia the increased blood viscosity can cause a lower ESR. In sickle cell anemia, and other conditions such as spherocytosis, the RBCs are abnormally shaped and will not form rouleaux easily, thus decreasing the ESR.

The ESR is an easy, inexpensive, non-specific test that has been used for many years to help diagnose conditions associated with acute and chronic inflammation. An elevated ESR is not associated with a specific diagnosis; therefore, it must be used in conjunction with other tests. Conversely, a normal ESR cannot be used to exclude the presence of significant disease. The ESR should also not be used as a screening test in asymptomatic patients. Since fibrinogen is an acute-phase reactant, the ESR is increased in many inflammatory and neoplastic conditions that increase fibrinogen, including diabetes, infection, pelvic inflammatory disease, lupus. rheumatoid arthritis, acute coronary syndrome, and neoplasms. However, noninflammatory factors such as older age, female gender, and pregnancy can also cause elevation of the ESR.

Historically, the ESR was used to indicate inflammatory conditions and monitor disease progression or response to treatment. More specific tests have been developed for many of these conditions, but the ESR still has its advantages. Interestingly enough, for a test that 12 years ago was on the ‘antiquated’ list, in the past 2 years there have been over 50 scientific journal articles written about the ESR. The ESR can eliminate unnecessary testing and help decrease medical costs. It has its advantages in small labs and in rural areas because it can provide quick results without expensive instrumentation. For labs that do not perform more sophisticated tests such as CRP and procalcitonin, the ESR can provide answers without waiting for results from reference laboratories. Even though an ESR may take 1 hour, it is much faster than send out testing. It can therefore expedite a diagnosis, or normal results can give the physician and patient timely reassurance.

What is your favorite or least favorite test in hematology? Let me know and I can highlight it in a future blog!

References

Au, Benjamin Wai Yin MBBS, MMed (Ophth); Ku, Dominic J. BMed, MSurg; Sheth, Shivanand J. MBBS, MS (Ophthal) Thinking Beyond Giant Cell Arteritis in COVID-19 Times, Journal of Neuro-Ophthalmology: March 2022 – Volume 42 – Issue 1 – p e137-e139
Brigden ML. Clinical utility of the erythrocyte sedimentation rate. Am Fam Physician. 1999 Oct 1;60(5):1443-50. PMID: 10524488.
Hale AJ, Ricotta DN, Freed JA. Evaluating the Erythrocyte Sedimentation Rate. JAMA. 2019;321(14):1404–1405. doi:10.1001/jama.2019.1178
Pu, Sheng-Lan et al. “Unexplained elevation of erythrocyte sedimentation rate in a patient recovering from COVID-19: A case report.” World journal of clinical cases vol. 9,6 (2021): 1394-1401. doi:10.12998/wjcc.v9.i6.1394
Tishkowski K, Gupta V. Erythrocyte Sedimentation Rate. [Updated 2021 May 9]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK557485/
Alan H. B. Wu, PhD, Kent Lewandrowski, MD, et al. Antiquated Tests Within the Clinical Pathology Laboratory. The American Journal of Managed Care. September 2010, Volume 16, Issue 9
https://emedicine.medscape.com/article/332483-workup

Hematology Case Study: An Unusual case of Leukemic Reticuloendotheliosis (aka Hairy Cell Leukemia)

Leukemic Reticuloendotheliosis (LRE) is a term that was first used in 1923 but is a name that most of us would not recognize today. In 1958, Bournocle et al. published a paper that characterized LRE as a separate clinical disorder and described the clinical course, pathologic features, treatment options and prognosis. The study also described an unusual morphology of the malignant cells seen in this condition. The malignant cells were noted to be small mononuclear cells with projections around the circumference of the cytoplasm. Another decade went by before these cells were given the nickname “hairy cells”. At the time, though LRE was considered a fatal disease, splenectomy appeared to be a beneficial treatment, thus pointing to a lymphocytic disorder. Later, in 1976, an article was published that suggested that these hairy cells were monocytic rather than lymphocytic in origin. The true lineage of these hairy cells was unknown until the development of newer immunophenotypic methodologies in the mid to late 1970s. Today, hairy cell leukemia (HCL) is considered a rare, chronic B cell leukemia that comprises 2% of lymphoid leukemias and responds well to therapy.

Patients may be entirely asymptomatic at diagnosis, and the finding of hairy cells on the peripheral smear from a routine CBC prompts further investigation. Patients do not usually require treatment at diagnosis, and many patients live a normal lifespan. Originally, diagnosis was based on clinical and laboratory result correlation: CBC results, observation of the characteristic hairy cells, and splenomegaly. One of the first tests used for diagnosis of HCL was tartrate-resistant acid phosphatase activity (TRAP stain). Today, standard practice is immunophenotyping by flow cytometry. HCL is characterized by the expression of B-cell antigens CD19, CD20, and CD22 in addition to bright CD11c expression with CD103, CD25, CD123 and Annexin A1 (ANXA1) co-expression. Annexin A1 is the most specific immunohistochemical marker for HCL. In 2011, the BRAF-V600E mutation was identified as the genetic causal event of HCL, allowing even more advances in the diagnosis and therapy for HCL.

As the disease progresses, most patients experience increasing cytopenia, including monocytopenia, and persistent splenomegaly. Treatment is usually started when a patient meets certain guidelines, which include a severe cytopenia or pancytopenia, malignant lymphocytosis, increased susceptibility to infection or symptomatic splenomegaly. Historically, the only available treatment was splenectomy. In the 1980’s, interferon therapy was introduced and was able to induce partial responses in some patients. In the 1990’s the purine analogs, cladribine or pentostatin, became available as the preferred first line treatment for HCL. Treatment response is good and offers prolonged remission rates. For patients who experience relapse, rituximab may be used in combination with a purine analog. Most recently, anti-CD22 immunotoxins and molecular targeted therapy with BRAF inhibitors have been introduced for cases that do not respond to other therapies.

Additional discoveries into the biology of the disease have identified new subtypes of HCL. It is important to distinguish between classic HCL and Hairy Cell leukemia variant (HCLv) because they are treated differently. HCLv may be more aggressive and does not respond well to purine analogs alone. HCLv is often diagnosed at older age than classic HCL In HCLv the WBC is often elevated, with lymphocytosis, and there is a lack of monocytopenia. The hairy cells seen on a peripheral blood smear may be more abundant than in classic HCL. These HCLv cells also often have a distinct nucleolus not seen in HCL cells. As well, these cells can have a morphology that appears to be somewhere between prolymphocytes and hairy cells. Unlike HCL, HCLv cells are negative for CD25 and BRAF-V600E. HCLv represents only about 10% of HCL cases. Because of its rarity, and the gray areas surrounding differential diagnosis between HCL and HCLv, studying these rare cases can help lead to a better understanding and management of both HCL and HCLv patients.

About 1200 new cases of HCL are diagnosed each year in the US. HCL is 4-5 times more common in males, with a median age at diagnosis of 55-60. This is an unusual case because the patient is female, was older at diagnosis, with no cytopenia or splenomegaly noted. This patient is a 79-year-old female who, one year ago, was referred to a Hematology Oncology practice with a several year history of a mildly elevated WBC with increased lymphocytes, without absolute lymphocytosis. She was referred after a peripheral smear exhibited prolymphocytes and the “hairy’ appearing lymphocytes shown below in Image 1.

Image 1. Hairy Cells seen on peripheral blood smear.

Peripheral blood was sent for myeloid/lymphoid disorders and acute leukemia analysis by flow cytometry. Remarkable in this case were the results of the flow cytometry studies. Flow cytometry performed on the peripheral blood revealed 2 distinct morphological populations of lymphocytes. The majority of lymphs appeared to be small, with scant cytoplasm, round nucleus, clumped chromatin, and inconspicuous nucleoli. These cells were identified as a monoclonal kappa restricted B cell population exhibiting co-expression of CD23 and CD5, consistent with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). A second population of lymphs were larger, with more abundant granular cytoplasm and hairy projections, large nuclei, condensed chromatin, and inconspicuous nucleoli. This second population displayed CD20 expression and was positive for CD11c, CD103 and FMC-7. CD25, CD5 and CD23 were negative.

The immunophenotyping of this second population of cells suggests a diagnosis HCL; or is it suggestive of HCLv? The patient was older at diagnosis, leukocytosis and lymphocytosis are present, and monocytopenia is absent. Hairy cells were over 8% of the differential, though lacking the distinct nucleoli of HCLv. Prolymphocytes were noted. CD25 was negative in this patient and is usually exhibited in HCL.

An immunological scoring system for HCL has been proposed with one point given to each of markers for CD11c, CD103, CD123 and CD25. One point is given if the marker is expressed and no point when it is not expressed. A score of 3 or 4 is observed in 98% of cases of HCL and is usually 0-1 in other HCL-like disorders. This patient’s cells showed expression of CD11c and CD103, was CD25 negative and CD123 was not evaluated so would score at least a 2, which puts her somewhere in an inconclusive score. Additionally, a bone marrow biopsy has not been done and there therefore results for TRAP or annexin A1 immunostaining, or BRAF-V600E mutations are not available.

With a diagnosis of a B-lymphocytosis consistent with CLL/SLL and a simultaneous HCL, or HCLv, this patient is an interesting case. Several articles and reviews in literature of other patients with CLL and HCL give further insight into the biology of HCL. Literature suggests that concurrent HCL and CLL may indicate a common origin. Patients with HCL may subsequently develop CLL, which can mimic a relapse of HCL. Therapy requires treating each case individually and watchful waiting in asymptomatic cases. Rituximab with or without purine analogs have been useful to treat both disorders simultaneously.

Table 1. CBC results from a patient in 2022 and 2022.

This patient at 1 year following diagnoses has developed a mildly increasing lymphocytosis and is being monitored. Both her CLL/SLL and HCL still appear to be in the indolent, “wait and see” stage. The patient has declined further workups at this time.

References

Bain, Barbara J. Blood Cells: A Practical Guide. 5th ed. Wiley Blackwell, 2015. Print.
Chang-Hun Park, Hyun-Young Kim, M.D.et al. Efficacy of Annexin A1 Immunostaining in Bone Marrow for the Diagnosis of Hairy Cell Leukemia. Laboratory Medicine Online 2019; 9(4): 236-241
Falini B, Tiacci E. New treatment options in hairy cell leukemia with focus on BRAF inhibitors. Hematol Oncol. 2019; 37(Suppl. 1): 30– 7..Maitre, E.; Cornet, E.; Troussard, X. Hairy cell leukemia: 2020 update on diagnosis, risk stratification, and treatment. Am. J. Hematol. 2019, 94, 1413–1422.
Obiorah IE, Francischetti IMB, Wang HW, Ahn IE, Wang W, Raffeld M, Kreitman RJ, Wiestner A, Calvo KR. Concurrent chronic lymphocytic leukemia/small lymphocytic lymphoma and hairy cell leukemia: clinical, pathologic and molecular features. Leuk Lymphoma. 2020 Dec;61(13):3177-3187.
Scheinberg M, Brenner AI, Sullivan AL, Cathcart ES, Katayama I. The heterogeneity of leukemic reticuloendotheliosis, “hairy cell leukemia”. Evidence for its monocytic origin. Cancer. 1976 Mar;37(3):1302-7
Shao, Haipeng et al. “Distinguishing hairy cell leukemia variant from hairy cell leukemia: development and validation of diagnostic criteria.” Leukemia research vol. 37,4 (2013)
Verma V, Giri S, Bhatt VR, Amador-Ortiz C, Armitage JO. Synchronous or Metachronous Hairy Cell Leukemia and Chronic Lymphocytic Leukemia: A Case Series and Literature Review. Front Oncol. 2017 Jan 9;6:270.
X. Troussard, M.R. Grever. The revised guidelines for the diagnosis and management of hairy cell leukaemia and the hairy cell leukaemia variant. r J Haematol, 193 (1) (2021), pp. 11-14

Validations/Verifications of Alternative Anticoagulants for Platelet Clumping

Platelet clumping can cause a falsely lowered platelet count on hematology instruments and can be difficult to resolve. With thrombocytopenia, physicians need an accurate count to diagnose, treat, or monitor patients. Clumping can be due to pre-analytic issues with specimen handling, can be caused by medications, or may be an in vitro phenomenon caused by anticoagulants. The clumping makes precise counting impossible and even estimates can be very tricky. If there are clumps, and recollection of the sample still yields platelet clumping, then many labs will have an alternate tube drawn or an alternative method to help resolve clumping.

Many of us have heard of using sodium citrate tubes for patients who have clumped platelets in EDTA. So, if you are having platelet clumping headaches, you can just order some sodium citrate tubes and start using those on your hematology analyzers, right? Not so fast. There are many published references of the use of sodium citrate tubes to resolve EDTA induced thrombocytopenia but we still see samples in which the clumping is not resolved with the sodium citrate tube. Published studies have shown that several other alternate methods have been helpful in resolving platelet clumping issues. These include drawing specimens in CTAD, ACD, or ‘ThromboExact’¹ tubes, or adding amikacin or kanamycin to the EDTA after the specimen is drawn.

So, why can’t we just order one of these other tubes and start reporting results? Hematology analyzers are only FDA approved for EDTA tubes. Before you can use any modified method, and before you can report any patient results, your laboratory must do validation or verification studies to prove that the method produces valid results.

A validation provides objective evidence that a test performs as intended. A validation uses a defined process and is used when setting up and implementing a new test. One example is a laboratory developed test (LDT), which is a test performed by the clinical laboratory in which the test was developed. A LDT can be one that is neither FDA-cleared nor FDA-approved or can be one that is FDA cleared/approved but has been modified by the performing laboratory. The use of sample types or the use of collection devices not listed in manufacturer instructions constitute modifications, by this definition. In a validation, accuracy should be tested with at least 40 samples across the analytical measurement range (AMR). Correlations are then performed. Precision should be tested over approximately 20 days. A verification, on the other hand, uses an abbreviated process and is used when setting up and implementing new tests that are cleared or approved by FDA. Before reporting patient results, the laboratory must demonstrate that a test performs in agreement with prior claims and must demonstrate performance specifications are comparable to the manufacturer’s specifications. Verification therefore is a confirmation that a test method meets specified requirements and would be applied to a method which has already been validated. For a verification, a smaller sample size may be used, and precisions tested over 5 or more days.

So, which would you do if you wanted to use an alternate method for reporting platelet counts? Hematology analyzers are only FDA approved for platelet counts on EDTA, but the by which the sample is analyzed does not change with an alternate tube, so it may be possible to do a limited validation or verification with a smaller sample size. A laboratory needs to prove correlation, accuracy, and precision. Follow your laboratory SOPs for validation/verification and consult with your accrediting agencies, if necessary. A plan needs to be written and signed off by laboratory director. Choose the alternative method you wish to investigate and run correlations for platelet counts on EDTA and the alternate anticoagulant. If your instrument has more than one platelet mode, it is important to run samples in the mode which you would normally use for thrombocytopenia or flagged platelet counts. Apply any dilutional factors and calculate correlations. This data will be Included in your report, which, along with a procedure needs to be signed by the laboratory director.

The most important thing is to write a plan and a follow-up report according to your SOPs and to make sure any requirements of accrediting agencies are included. There can be some differences in interpretation of standards, so it is the laboratory’s responsibility to make sure what you have done meets the standards that apply to your lab.

The use of alternate tubes for platelet counts has been well reviewed in literature. Sodium citrate tubes are the most common, likely because they are the easiest to use and the most cost effective. Remember though that sodium citrate and other methods cannot resolve all case s of pseudothrombocytopenia. There are several special notes to consider. Counts from sodium citrate tubes are known to be stable for approximately 3 hours, after which counts decrease. As well, it has been shown in literature that sodium citrate tubes do show a negative bias. It has been reported that the 10% dilutional factor may be too low. Some studies have been done to determine dilution factors that correlate more closely with EDTA tubes, and researchers have suggested factor of 17%-25%. If your laboratory wishes to determine its own dilutional factor for sodium citrate or other tubes, this will also have to be included in your platelet studies. Lastly, CBCs are calibrated for EDTA, so only the platelet count should be reported from an alternative anticoagulant.

The end of another busy and challenging year is upon us, and at this time of year we can find ourselves rushed to finish ‘end of year’ tasks such as competencies and continuing education requirements. and a response to Sysmex’s recent webinar “Those Sticky, Tricky Platelets – Solving the Puzzle of Platelet Clumping” (Oct.20,2021). After the webinar I had many questions from techs asking, “Do we need to validate our alternative method?” and “How do we go about doing that?” The webinar discusses pseudothrombocytopenia and its causes in more detail than my earlier blog from Oct 2019, “Hematology Case Study: The Story of the Platelet Clump: EDTA-Induced Thrombocytopenia”. The webinar can be found at https://webinars.sysmex.com/webinars/11ae743e-ac99-47e7-acb7-2b24cedc1a1a and is available for CEU, free of charge.

References

Baccini V, Geneviève F, Jacqmin H, et al. Platelet Counting: Ugly Traps and Good Advice. Proposals from the French-Speaking Cellular Hematology Group (GFHC). J Clin Med. 2020;9(3):808. Published 2020 Mar 16. doi:10.3390/jcm9030808
Bizzaro N. (2013): Pseudothrombocytopenia. In: Platelets, Vol. 3, ed Bizzaro N, Elsevier, Amsterdam, pp. 989–997
Chae H, Kim M, Lim J, Oh EJ, Kim Y, Han K: Novel method to dissociate platelet clumps in EDTA-dependent pseudothrombocytopenia based on the pathophysiological mechanism. Clin Chem Lab Med 50, 1387–1391 (2012)
Socha, Becky. Calibration and Calibration Verification: Who, What, Where, When, Why, How & Did I Pass or Fail?. AMT 81^st Educational Program and annual meeting, 2019
Zhou X, Wu X, Deng W, Li J, Luo W: Amikacin can be added to blood to reduce the fall in platelet count. Am J Clin Pathol 136, 646–652 (2011)
https://www.cms.gov/Regulations-and-Guidance/Legislation/CLIA/downloads/6065bk.pdf
https://www.cap.org/laboratory-improvement/proficiency-testing/calibration-verification-linearity
https://www.westgard.com/cal-verification-criteria.htm
https://labmedicineblog.com/2019/10/29/ hematology-case-study-the-story-of-the-platelet- clump-edta-induced-thrombocytopenia/

Hematology Case Study: Too Many Platelets?

Too many platelets? We know that low platelet counts can pose problems for hematology analyzers and that reporting accurate results is vital for good patient care. We learn and read a lot about thrombocytopenia and its various symptoms, causes, and treatments. But, what about thrombocytosis? What happens when there are too many platelets?

In my last blog I compared 2 cases of newly diagnosed CML. Lately I have seen so many new leukemia cases and myeloproliferative diseases that I have become fascinated with them. When I was in college and grad school (many moons ago), nomenclature, diagnoses and knowledge of these disorders were very different, so it’s been fun learning about them all over again!

Today’s case is of a 55 year old woman who was referred for a hematology consult because of a finding of increased RBC and platelet counts. White blood cells appeared normal with few reactive lymphocytes noted. The peripheral smear showed mild anisocytosis and dacrocytes. Platelets were markedly increased with large forms present. No giant platelets were noted. A bone marrow biopsy was ordered. Pre-Op diagnosis: Thrombocytosis.

Bone marrow results reported increased myeloid forms with full spectrum of maturation, erythroid elements normal in number with normoblastic maturation, and markedly increased megakaryocytes with numerous large hyperlobated forms. M:E ratio was increased. No iron was seen on iron stain. A reticulin stain showed mildly increased reticulum fibrosis (1+). Next generation sequencing studies demonstrated a JAK2 V617F mutation. BCR-ABL mutation was not detected. Diagnosis: Myeloproliferative neoplasm most consistent with Essential Thrombocythemia (ET).

Myeloproliferative neoplasms (MPN) are a group of disorders characterized by the over proliferation of WBCs, RBCs, or platelets. These can be separated into the Philadelphia chromosome (Ph) positive Chronic Myelogenous Leukemia (CML) and Ph negative neoplasms. The BCR-ABL oncogene is formed on the Ph and is responsible for the unregulated proliferation of cells seen in CML. At diagnosis over 90% of CML cases are BCR-ABL positive. (See Case Studies in Hematology: Presenting a double feature starring Chronic Myelogenous Leukemia). On the other hand, Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are the three classic Ph negative neoplasms.

Many ET patients have no symptoms at diagnosis but are found to have a high platelet count on a routine CBC. Diagnosis is based on ruling out other disease and testing for genetic mutations, which can be done from a peripheral blood sample or bone marrow. In addition to any blood tests, a bone marrow biopsy is typically recommended for differential diagnosis of MPNs. The most common mutation found in PV, ET or PMF, is in the JAK2 gene. The JAK2 V617F mutation is found in nearly all PV patients, and about 50-60% of ET and PMF patients.Othermutations found in the classic MPN group include CALR, the second most common genetic abnormality after JAK2 mutations,and MPL W515L.Normally, blood cells are only produced when the body has a need for the cells, but these genetic mutations turn a gene ‘on’, causing the unregulated production of the affected blood cell line. Until recently it was believed that a patient with PV, ET or PMF will have a mutation in only one of these genes. However, in 2018, a French group reported that CALR or MPL mutations may co-exist in a small percentage of patients with a low burden of JAK2 V617F mutation. (Accurso) Some patients are triple-negative for the JAK2, MPL and CALR mutations and always have a poor prognosis.

The identification of a genetic marker in MPNs is valuable because a JAK2 mutation distinguishes PV from other disorders that may cause polycythemia. As well, a JAK2 or other mutation can distinguish ET from other causes of reactive thrombocytosis and PMF from secondary causes of myelofibrosis. In addition, most CML cases are diagnosed with a very high WBC, but occasionally patients with CML have a normal or only slightly elevated WBC with a high platelet count. Therefore, patients with suspected ET are also evaluated for CML with a test for the Philadelphia chromosome. Our patient was found to have a JAK2 V617F mutation, BCR-ABL negative and was diagnosed with ET.

ET was first recognized in the 1950’s and was termed a myeloproliferative disorder. At this time, it was not known what was causing the over proliferation of platelets. Theories were broad and ranged from ‘something environmental’ to ‘an internal defect’. Over the decades, it became more apparent that the myeloproliferative disorders were caused by internal defects in stem cells, and they were renamed MPN. In 2005, four separate research groups, using different methods, all identified the JAK2 V617F allele, which led to further understanding of PV, ET and MPN. The MPL mutation was discovered in 2006 and CALR mutations were discovered in 2013.

ET is a type of chronic leukemia and patients with ET generally have a normal life expectancy. Of the 3 BCR-ABL negative MPN, ET has the best prognosis. Treatment is often not needed, other than aspirin for prevention of blood clots. Patients are placed in risk factor groups based on risk of clots or bleeding. A patient <60 years with no JAK 2 mutation and no prior thrombosis is considered very low risk and would be simply observed or prescribed low dose aspirin. Patients <60, JAK2 V617F +, with no prior thrombosis have low risk and would be treated with aspirin, dosage dependent on any cardiac risk factors. Older patients over 60 with JAK2 wild type and no history of thrombosis may be treated with aspirin alone or with cytoreductive therapy. Lastly, the highest risk patients are those over 60, JAK2 V617F + or with prior thrombosis, and would be treated with cytoreductive therapy, such as hydroxyurea. With very high platelet counts, there is a risk of both blood clots and hemorrhage. Blood clots that develop in thrombocythemia can use up the body’s platelets and result in bleeding. For this reason, cytoreductive therapy such as hydroxyurea is recommended to reduce hemorrhage in high-risk patients with very high platelet counts over 1,000 x 10³/ μL. Hydroxyurea can also be used as treatment in patients who have a mixed population of PV and ET. CALR mutated patients with ET tend to be young with a much lower thrombotic risk and do not generally require therapy. Aspirin in this group is considered overtreatment because CALR+ patients suffer more risk of bleeding with aspirin.

While there is some risk of a MPN transforming to another type, ET is the MPN least likely to transform or to progress to acute myeloid leukemia. ET also has a better prognosis than the other MPN. Even so, there is often not one clear cut entity. There can be overlap between the disorders, causing some difficulty in diagnosis and treatment decisions. For instance, a physician may have a patient, as our patient does, with a high RBC and Hgb, with thrombocytosis, and with a JAK2 mutation. Bone marrow biopsy may detect hyperlobated megakaryocytes which would indicate a diagnosis of ET; however, the physician may choose to monitor and possibly treat as PV due to the RBC counts and symptoms.

Many advances in the understanding of ET and molecular techniques for diagnosis have been made in the last 10 years. Unfortunately, many times, diagnosis is not made until after a thrombotic event. In addition, many patients with thrombocytosis are not referred for hematology consults in a timely fashion or until they too experience a thrombotic event. In 2016 WHO published a new diagnostic criterion for PV, ET and PMF. There is an effort amongst research and physician groups to ‘spread the news’ throughout the medical community to promote early detection of ET, minimize the risk of thrombotic events and improve prognosis.

References

Accurso V, Santoro M, Mancuso S, et al. The Essential Thrombocythemia in 2020: What We Know and Where We Still Have to Dig Deep. Clin Med Insights Blood Disord. 2020;13:2634853520978210. Published 2020 Dec 28. doi:10.1177/2634853520978210
Bose P, Verstovsek S. Updates in the management of polycythemia vera and essential thrombocythemia. Ther Adv Hematol. 2019;10:2040620719870052. Published 2019 Aug 30. doi:10.1177/2040620719870052
Kilpivaara, O., Levine, R. JAK2 and MPL mutations in myeloproliferative neoplasms: discovery and science. Leukemia 22, 1813–1817 (2008). https://doi.org/10.1038/leu.2008.229
Panjwani,Laura. Management of ET, PV Requires 2 Distinct Approaches. Special Reports, Hematologic Malignancies: Polycythemia Vera, Volume 3, Issue 3. September 28, 2016
https://rarediseases.org/rare-diseases/essential-thrombocythemia/
https://www.mpnconnect.com/pdf/who-diagnostic-criteria-mf-pv-et.pdf

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

Follow the Indicies. It’s Not Always Cold!

One of my favorite things about working in Hematology is handling those “difficult” samples. You know the ones. The one that some techs put aside to work on “later,” or they might decide it’s time to take a break when they see them coming. I love investigating and working on these interesting but perhaps uncooperative samples. At times this involves running samples in different modes, making new slides or albumin smears, and diluting samples. At other times, we investigate a delta or unusual results by checking patient diagnosis and previous results or by calling the care provider for more information and clues to help us resolve the problem.

I’m sure you’ve all seen the sayings “Without the Lab, you’re only guessing” and “Laboratory Professionals get results.” Physicians rely on the lab every day for information used to help diagnose and treat patients. Therefore, our goal is to deliver to the care provider the best possible results in a timely manner. Which means that we don’t just report results because that’s the answer the instrument gave us. With today’s instruments and middleware, we get very accurate and precise results, and about 85% or more of hematology specimens autovalidate. This is important because it leaves us time to work on those specimens with flags, and discrepancies; the ones that need a little more time and attention.

When faced with unusual or conflicting results, we first need to ask ourselves if we are dealing with a spurious sample, interfering substances or true abnormal results. Many labs today use middleware that will give the operator alerts when a sample needs to be investigated. These alerts give us suggestions as to how to handle the specimen but are usually short phrases triggered by certain values or flags and cannot be all encompassing. Operator alerts cannot tell us all the steps we may need to follow to resolve, for example, deltas, platelet clumps, abnormal scattergrams or a possible cold agglutinin. The alerts are great guidelines but it is often necessary to do more. We may need to refer to procedure manuals for SOPS or check instrument manuals or technical bulletins to decide how to handle these specimens. Sometimes we need to be detectives to report the most accurate results. We must review results with a critical eye, use all that “stuff” we learned in school, and be able to make educated decisions based on this investigation.

In my experience, one of the most common troublesome and perhaps misunderstood specimens I see is the one with a “hemoglobin (Hgb) interference” flag. An instrument flag “suspect, turbidity /Hgb interference?” is generally initiated when the MCHC is above a certain value. In our hematology lab, we see this flag when the MCHC is above 37.5 g/dL. What this is telling us is that turbidity may be present in the diluted and lysed sample. This turbidity can interfere with the Hgb detection light path and falsely increase the Hgb. Because the MCH and MCHC are calculated using the Hgb, these parameters are also affected. BUT, an MCHC >37.5 g/dL is not always something that can be or that needs to be corrected. With any parameter 95% of normal values will fall within 2SD of the mean. This means that 5% of normal healthy individuals have MCHC results <32 g/dL or >36 g/dL, and a few may have an MCHC over 37.5 g/dL. An MCHC >37.5 g/dL therefore can indicate a normal specimen, such as in a healthy young male with a Hgb at the high end of the reference range. High MCHCs can also be seen routinely in specimens from patients with spherocytosis or hemoglobinopathies such as Hgb SS, Hgb SC or Hgb C disease. In these conditions the RBCs are hyperdense due to altered surface volume and this leads to a high MCHC.

On our instrument, an MCHC >37.5 g/dL will cause a Hgb/Turbidity flag. An asterisk (*) will appear next to the Hgb, MCH and MCHC. The middleware triggers an operator alert that says “MCHC >37.5. Incubate at 37C for 30 mins. Evaluate for lipemia, icterus, hemolysis, Plasma replacement if indicated, rerun”. So, what’s the first thing to do?? Incubate? Hold on…not so fast. This is one of those instances where hematology is not just black and white. This operator alert is giving us suggestions of how to handle a specimen, but techs need to evaluate the specimen before jumping on the ‘cold’ wagon. Incubating will usually help resolve a cold agglutinin, but won’t help with a sickle cell specimen, or resolve one that’s icteric or lipemic. A grossly hemolyzed sample can give a spurious high MCHC result and, if so, needs to be recollected, not warmed. Putting a specimen that’s hemolyzed or lipemic or icteric in the heating block for 30 or more minutes would only delay reporting of results. My first case example involves a 45 year old female. The MCHC on initial run was 38.1 and the specimen gave a Hgb turbidity flag. The sample was incubated and rerun several times. After 1 hour of incubation, the MCHC was reported as 37.1 with a comment “repeated after warming for 1 hour at 37C”. In this case the patient was a known sickle cell patient. Previous results show that this patient’s MCHC is typically high and previously reported results ranged from 36.1- 37.8 g/dL. When evaluating a specimen with a high MCHC it is important to check the pattern of results. In this case the MCHC was high but the MCV was low. This does not fit the pattern for a cold agglutinin. As noted above, super dense RBCs in sickle cell patients may cause a high MCHC. This specimen was warmed, and even though the MCHC was a bit lower after warming, it would have been acceptable to report the original run MCHC. Checking patient history and previous results, and reviewing the smear for morphology would have allowed these results to be reported in a timely fashion. The operator alert does say “incubate the specimen” but it also says to evaluate. Be sure to check the MCV and MCHC along with patient history before warming specimens that don’t fit the pattern of a cold agglutinin.

Table 1. Case 1 CBC. The patient is a 45 year old known sickle cell patient.

The second example is from a 75 year old male. The CBC flagged Hgb turbidity with an MCHC of 45.8 g/dL. The MCHC >37.5 operator alert triggered Checking the pattern of results for the indicies, the MCHC was very high and the MCV was low. In a specimen with a low or normal MCV and a high MCHC, lipemia, icterus, abnormal proteins or severe leukocytosis can be affecting the Hgb. On evaluation, this sample’s Hgb and Hct did not meet the ‘rule of 3’. The rules of 3 are now generally recognized to be valid only for samples when the RBCs are normal, but the * here is telling us that there is an interference affecting the Hgb. In these cases it is valuable to know what the interference is so we know how to handle the specimen. By spinning down a small aliquot, (or asking chemistry!) we can investigate for lipemia or icterus. The specimen was found to be grossly lipemic. Flagging guidelines for lipemic specimens suggest diluting the specimen 1:5 and rerunning. Alternately, with severely lipemic or icteric samples, plasma replacement procedure may be necessary to correct the results. In this case, a plasma replacement was performed. After a plasma replacement, the WBC, RBC, Hct, MCV and platelet count are reported from the original run. The Hgb interference is what was causing the problem. Thus, when you correct the Hgb you must always correct any indicies that are calculated with the Hgb. The Hgb from the plasma replacement sample is used and the MCH and MCHC are recalculated. Notice that the new lower Hgb value now matches the Hct.

Table 2. Case 2, a 75 year old male with lipemic specimen. Plasma replacement performed. WBC, RBC, Hct, MCV, and Plt were reported from original run. Hgb was reported from plasma replacement sample. MCH and MCHC were recalculated.

Case 3 is a sample from an 80 year old woman. This was an interesting sample because there were multiple things going on here. This patient had an initial result with a high MCHC and MCH, with decreased RBC and Hct. In this patient the initial WBC was 0.64 and the RBC was 0.31. The Hgb of 9.1 /dL was less than the Hct of 3.1 %. MCV was 116 fl and the MCHC was 293.5 g/dL! In specimens with a high MCV and high MCHC we can suspect a cold agglutinin. When the MCV is very high it is because the RBCs are going through the aperture as one big bunch and this is measured as the size of one RBC. Often the Hct is less than the Hgb. Sometimes the RBC and Hct are so low that it causes the MCV to be appear within normal range. On our instrument, a RBC count of <0.5 x10⁶/μL will give a flag “abnormal RBC scattergram” but no other indicies related flags are generated, so we didn’t even get an operator alert to evaluate the MCHC. But, it’s clear there is something very wrong with these results. Warming the sample is used to loosen clumping of RBCs, which lowers the MCV and allows the RBCs to be counted. Make a smear to examine for RBC clumping and look at the sample tube. Many cold agglutinin samples will appear to be ‘grainy’ or have agglutination along the side of the tube. This is the time when we want to incubate the sample. To resolve a cold agglutinin, warming the sample is necessary. Sometime 30 minutes is enough, sometime they need to be incubated longer. Some cold agglutinins are so strong that after incubation a dilution or plasma replacement still needs to be done. Warming this sample did not lower the MCHC. After incubating, I diluted this sample, and also did a plasma replacement to see how results would compare. The new results matched. This sample took a bit more time than others but the cold agglutinin was resolved and we were able to report valid results.

Table 3. CBC results from 80 year old woman with cold agglutinin.

Image 1. Tube from cold agglutinin specimen. Note agglutination in sample along sides of tube.

There are other factors that can affect the Hct or Hgb and cause a high MCHC. Icteric specimens act much like lipemic ones and the Hgb can be corrected with dilution or a plasma replacement. An electrolyte balance can affect the Hct. Abnormal proteins and severe leukocytosis can affect the Hgb. Grossly hemolyzed samples can have a high MCHC. It is important to evaluate the indicies in these samples and correlate the values with previous results and patient history. What concerns me is that I have seen samples being warmed that do not match the indicies patterns for cold agglutinins. I have seen samples from sickle cell patients signed out with a comment “warmed at 37C. Possible cold agglutinin.” I have seen lipemic or icteric samples that are reported out with high MCHCs, erroneously high Hgb or parameters that are not reported at all. While warming these samples may actually lower the MCHC a bit, it still usually remains on the high side and does not give us the clean results that dilution or plasma replacement will. A little extra time looking at the indicies can give us important clues as to how to handle these samples. Doctors use our results every day to make patient care decisions. We need to make sure that we are making decisions every day to give them the best possible results so that patients can get the best care possible.

Table 4. Evaluating high MCHC specimens.

References

Costa, B. M. B., Vellés, M. C., Viana, M. M. F. B., & Rebelo, C. I. M. (2018). Interference of cold agglutinin autoantibodies in erythrogram interpretation: a case report and literature review. Jornal Brasileiro De Patologia e MedicinaLaboratorial, 54(4). doi: 10.5935/1676-2444.20180043
Sysmex USA. XN-Series Flagging Interpretation Guide. Document Number: 1166-LSS, Rev. 6, March 2021
It’s not Black and White: Unraveling the puzzles of Hematology. Becky Socha MS, BB, MLS(ASCP) Mercy Medical Center, Baltimore, MD