Beyond Bands: The Immature Granulocyte Count

In today’s clinical laboratories there are an increasing number of tests available for physicians to order. In a constant effort to provide the best tools for patient care, laboratories typically issue a Laboratory Technical Bulletin when new platforms or new testing is available. These help introduce physicians to or update them on the ever changing array of test availability. In Hematology, Advanced Clinical Parameters are an extension of the traditional CBC and Automated Differential. Used appropriately, these new tests can provide a wealth of knowledge that can be available to physicians in making diagnoses. In my July post, (Beyond the CBC and Reticulocyte Count: Early Detection of Iron Deficiency Anemia), I discussed the Reticulocyte Hemoglobin (RetHe) and it’s uses in early detection and better management of iron deficiency anemia. In this blog I will explore the Immature Granulocyte Count (IG%), another of the Advanced Clinical Parameters.

Historically, the 100 cell manual differential has been used to enumerate percentages of each cell type present and to detect the presence of abnormal cells, including immature granulocytes. An increase in band count >10 %, was, and is still often used with other screening tests as an indicator of infection or sepsis. The first microscopic image analyzer, the Perkin-Elmer Cellscan in 1966, could recognize neutrophils, lymphocytes and monocytes with 90% accuracy, and a 100 cell differential could be completed overnight. With improved, faster computers this instrument was marketed in the mid 1970’s and then purchased by Coulter Electronics in 1977 to become the Coulter Diff 3. These first hematology analyzers to provide an automated differential yielded a 3 part differential, and then more sophisticated instrumentation gave us the 5 part diff. In the beginning, an automated differential only counted 100-200 cells and manual diffs performed by a trained technologist were still considered superior. Today’s hematology analyzers count over 30,000 cells using impedance and flow cytometry to give us a statistically superior automated differential.

An automated 5 part differential will flag a ‘Left Shift’ when bands are seen. Many laboratories then perform a scan or manual diff. However, bands are very subjective and therefore it has been questioned if the band count is truly useful as a clinical indicator of sepsis. If a band count >10% is an indicator of sepsis, what happens when 2 or 3 technologists perform diffs on the same slide and get a band % range between 5% and 19%? 5% would not indicate sepsis, whereas 19% exceeds the 10% cutoff almost twofold. Two experienced technologists can perform a manual diff on the same slide and get band counts in a fairly wide range depending on their training and which definition of bands they use and which 100 cells they see in their count. On the other hand, a 6 part automated differential counts and differentiates over 30,000 neutrophils, lymphocytes, monocytes, eosinophils, basophils and immature granulocytes. The auto diff separates bands and immature granulocytes. Band cells are considered mature and included in the neutrophil count. The Advanced Clinical Parameter, Immature granulocytes (IG%) include metamyelocytes, myelocytes and promyelocytes. In the peripheral blood these immature granulocytes are an indicator of leukopoiesis, cells just coming out of the bone marrow and represent the earliest information possible and an earlier indication of inflammation and infection than a band count. The example below illustrates this concept.
Case Study 1

A patient admitted to the ER with high fever and chills has a CBC and auto diff ordered. His WBC is elevated and percent neutrophils are high. The physician suspects sepsis despite the normal WBC and orders a CRP and blood cultures. CRP results are within normal limits. The immature granulocyte counts are shown below on admission and in 4 subsequent phlebotomies. Antibiotics were started 3 hours after initial triage. The patient was admitted to the hospital and Streptococcus pneumoniae was isolated from the blood cultures at 48 hours.

left shift 1

The IG% and absolute counts are increased in this patient when other markers of infection are not. An IG% of >1% is indicative of a true left shit and >3% may predict positive blood cultures. The ED can also use the IG to determine that the infection is community acquired and not nosocomial if the IG% is high on admission. Note the band counts here that could be high or low depending on who performed the diff. This demonstrates the inherent subjectivity and imprecision of band counts and manual differential counts because they are done on only 100 cells. The immature granulocyte count is automated, not subjective. In checking the slide for correlation, we see the presence of these immature granulocytes. The IG% is thought to be better as a predictor of sepsis than the WBC and band count. For this reason, with the availability of a 6 part diff, labs have encouraged physicians to order and use the automated differential.
Another application of the utility of the IG% is seen in comparing IG% and ITR, immature to total neutrophil ratio. ITR is used by neonatologists to determine infection. The ITR is a calculation based on a 100 cell manual differential. In this formula, the immature neutrophils are bands, myelos, metas and pros. An ITR <0.2% is a negative predictive value for sepsis.
Immature neutrophils/Total neutrophils=bands+myelos+metas+pros/total neutrophils= ITR

Case Study 2

left shift 2

In 2 babies less than 30 days old, we see again here the phenomenon of a wide variety of bands counted by 2 techs. In the ITR, bands are counted as immature cells. Despite the imprecision of the band count, Baby A has >10% bands counted by both techs and the ITR range is >0.2% in both calculations. However, in Baby B, we see that the band count from one differential was 5% and the second tech counted 17% bands. Since the bands are used to calculate the ITR, one calculation gives an ITR of 0.06%, a negative predictor of infection, and the second count gives an ITR of 0.22%, above the 0.2% threshold. At the same time, if physicians are looking at an absolute neutrophil count(ANC), the number of neutrophils available to fight off infection, this number is calculated using bands and neutrophils. In one formula, bands are lumped with immature cells, and in the other, bands are included with mature cells. This can be confusing information. If we were instead to rely on the automated differential, the IG% gives us a more clear and precise measure of sepsis. Using the previously stated criteria for infection, the IG% of Baby A is clearly above 3% and in Baby B is well below 1%.

How do band counts compare to the IG%? Should bands be eliminated as an indicator of infection? Studies have been done that suggest that the WBC and band counts are not as reliable in predicting infection as the IG% and ANC. Suggestions have been made that the left shift should be redefined with IG% rather than bands. Bands may be too subjective to be the best indicator of infection and they lead to an imprecision in the ITR. The IG count can highlight potential acute infection or inflammation at its earliest stages, even when other parameters are still within normal ranges. IG counts (% and #), reported with the automated differential, reduce turnaround time and are valuable for clinicians to use in conjunction with other current indicators for the diagnosis of infection and inflammation.


  1. Ansari-Lari, M.Ali et al. Immature Granulocyte Measurement Using the Sysmex XE-2100 Relationship to Infection and Sepsis Am. J Clin Pathol. 2003,Nov;120(5):795-9.
  2. Balamurugan Senthilnayagam, et al. Automated Measurement of Immature Granulocytes: Performance Characteristics and Utility in Routine Clinical Practice. Patholog Res Int. 2012: 483670. Published online 2012 Feb 15. doi: 10.1155/2012/483670
  3. Cavallazzi, R et al. Is the band count useful in the diagnosis of infection? An accuracy study in critically ill patients. J Intensive Care Med. 2010, Nov-Dec;25(6):353-7.
    Dutcher, Thomas F. Automated Leukocyte Differentials: A Review and Prospectus. Laboratory Medicine, Volume 14, Issue 8, 1 August 1983, Pages 483–487,
  4. McDaniel, Holly. Ban the Bands. Sysmex News, 2013.
  5. Sysmex America White Paper. Getting Beyond the Flags: Quantitative assessment of immature granulocyte (IG) populations may improve the assessment of sepsis and inflammation.



-Becky Socha, MS, MLS(ASCP)CM BB 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 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

Microbiology Case Study: a 77 Year Old Woman with a Splenic Abscess

Case History

The patient is a 77 year old woman with a past medical history significant for hypertension, hyperlipidemia, diverticulitis, and advanced vascular disease with mesenteric ischemia and post-prandial pain who presented to an outside hospital with severe diffuse abdominal pain. Of note she was schedule to undergo endovascular repair of a known occluded celiac artery. Imaging at the outside hospital showed intraperitoneal free air and a fluid collection by the tail of the pancreas. The inferior mesenteric artery was patent but the celiac and superior mesenteric artery were occluded. The lesion at the tail of the pancreas was previously known to the patient, work up showed it was benign. The patient was transferred to our facility and was taken urgently to the OR for exploratory laparotomy for diffuse peritonitis. During surgery the patient was found to have an infarcted spleen with a splenic abscess; no ischemic bowel was seen. A surgical mesh was in place from a previous hernia surgery and was removed. A drain was placed in the abscess and the abdomen was closed. The patient was placed on Zosyn intraoperatively and remained on Zosyn following transfer to the ICU. On post-operative day 6, Zosyn was switched to Ertepenam due to IV and dosing problems.

Laboratory Identification

Image 1. Box shaped gram positive rod identified on Gram stain of splenic abscess culture (100x oil immersion).
Image 2. Mixed culture anaerobic growth. Pathogen of interest is clear and spreading causing a double zone of beta-hemolysis (Schaedler blood agar plate).

An aspirate from the abscess was sent to the lab for aerobic and anaerobic culture. Cultures from the abscess showed mixed gram positive and gram negative aerobic and anaerobic organisms. Of note, there was a gram positive rod which showed a double zone of beta hemolysis on the Schaedler blood plate, which grew only in anaerobic conditions. The colonies are clear/gray. The organism is catalase negative and indole positive, and was identified by the MALDI as Clostridium perfringens.


C. perfringens is an anaerobic gram positive spore forming rod. This organism is known to cause myonecrosis, gas gangrene, gangrenous cholecystitis, bacteremia, food poisoning, and is a worldwide cause of necrotizing enterocolitis (1). The main virulence factor is the alpha toxin, which is a hemolytic toxin with both phospholipase C and sphingomyelinase activities and is essential in disease. The toxin can rapidly breakdown phospholipid membranes, which is particularly dangerous because it can cause massive intravascular hemolysis in bacteremic patients (1, 2). It is important to recognize the signs of infection early as bacteremia can have a mortality rate of up to 75% (1). Splenic abscess with C. perfringens is rare with only a few case reports in the literature. Typically, splenic abscess are caused by aerobes such as Escherichia coli, Staphylococcus ssp., Streptococcus ssp., and Salmonella ssp. Infection with anaerobes account for only 10% of splenic infections (3). Those with clostridial infections typically had a predisposing condition such as colitis, diabetes, trauma, or malignancy (3).



  1. Hashiba M, Tomino A, Takenaka N, et al. Clostridium Perfringens Infection in a Febrile Patient with Severe Hemolytic Anemia. The American Journal of Case Reports. 2016;17:219-223. doi:10.12659/AJCR.895721.
  2. Awad MM, Bryant AE, Stevens DL, Rood JI.  Virulence studies on chromosomal alpha-toxin and theta-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of alpha-toxin in Clostridium perfringens-mediated gas gangrene. Molecular Microbiology. 1995;15(2):191.
  3. Chalasani, Rajendra MD; Siripurapu, Shantipriya MD; Hasan, Saqib MD. Splenic Abscess due to Clostridium perfringens: A Rare Entity. Infectious Diseases in Clinical Practice. 2007;15(2);137-138.


-Casey Rankins, DO, is a 1st year Anatomic and Clinical Pathology resident at the University of Vermont Medical Center.


-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.

A Response to “Offline: Why has global health forgotten cancer?”

I read with great interest Richard Horton’s comment, “Offline: Why has global health forgotten cancer?” ASCP applauds his bringing light to this issue and his strong call to action for both the global health community and governments to take up the challenge of dealing with cancer. There is no doubt that the world needs a “Global Fund for Cancer” or the “President’s Emergency Plan for Cancer.” There is no question on what those funds could be spent—

prevention, screening, diagnosis, and treatment of cancer has been well worked out in high-income countries (HIC). There is definitely a question of how best to spend those funds, what is the most effective approach in a given population, and what special circumstances exist in a population that must be considered. We thank him for shouting about this and being so direct and for using the Lancet as a platform for this important message.

We would like to clarify, however, that Richard is certainly not the first person to shout this call (and hopefully he will not be the last!). Please review the 17 references below; one of the earliest was authored by pathologists and appeared in Lancet in 2012.  In addition, the three most recent were from a Lancet Series. When I was in Malawi working in a diagnostic laboratory in 2000, more than 75% of what I saw was cancer. Although, at the time, a lot of cases found their etiology in untreated HIV. My senior colleagues told me I was wasting my time because there was “no way to treat cancer in Africa.” As I continued to visit Malawi over the next 15 years, the percentage of cases that were cancers increased. The HIV-related cancers decreased. Lung cancer never crossed the scope because there were no resources to biopsy or resect patients; yet, lung cancer was a leading cause of death in cancer registries. Today, the limited oncologists in Blantyre are overwhelmed by breast cancer cases. A similar story is found in Butaro, Rwanda and Mirebalais, Haiti.

But in all three places, patients can access a diagnosis because pathology services have been installed, bolstered, or maintained through commitments of NGOs, academic institutions, and governments. More importantly, they have access to treatment because oncologists and oncology nurses have joined the fight against cancer in global health in these units. There are many organizations in the United States and around the world that focus on cancer in low- and middle-income (LMIC) countries including (but not limited to) ASCP, PIH, UICC, ACS, CHAI, BVGH, ICCP, ICCR, NIH, APECSA, ASLM, ASCO, and, yes, the WHO. Do all of these organizations need more resources to make their missions more effective? Absolutely! Do more organizations need to join the fight? Absolutely! But, even with limited resources, huge progress can be made for individuals and populations.

In his comment, Richard points out two arguments used to explain why global health has forgotten cancer. The first is that cancer is not a statistical priority in LMICs. This is actually untrue. Advances in treatment for communicable diseases, especially HIV, have “unmasked” cancer in every one of these nations with clear evidence that many are preventable, many are curable, and many require palliative care. Mortality in Africa from cancer reaches 80% compared with only 35% for all cancers in the US. We clearly have a goal to focus on in mortality reduction with measurable targets. The WHO has announced a cancer resolution at the World Health Assembly. National Cancer Control Plans have been written for most LMICs. The stage is set for any one or all LMICs to develop, build, and expand cancer centers of excellence with people in and out of those countries eager to help. What is missing is not desire or resolve. What is missing is funding. And in this challenge, we find an actual barrier for advancing cancer care. Many organizations are drunk with funding for infectious diseases. They have no experience with cancer and no capacity to tackle it. If funding were suddenly diverted from these communicable disease organizations (CDO) to NCD organizations that could deal with cancer, many CDOs would have to close their doors. And millions would suffer at the loss of infrastructure and capacity that these organizations have created. But THAT is the ultimate barrier—the assumption that we have to divert funding. We don’t need to move funding from one program to another. We must find creative ways to finance cancer for every patient everywhere around the world.

Richard second points out that global health people tout “building systems” rather than focusing on specific cancer types (e.g., breast or cervix) as an excuse to not start cancer care. However, this is not accurate because a) health systems ARE needed to treat cancer and b) it is impossible to treat a single entity cancer and maintain an ethical program. For example, focusing on breast cancer or cervical cancer “first” or “only” is highly unethical because all the tools for those cancers also allow one to partially move a non-breast/non-cervical cancer patient through the system (the main difference being the chemotherapy types used). I do not disagree with the concept of “you have to start somewhere” but, if we think back to HIV and malaria, there is a precedent for why this is a flawed approach. HIV was a single test that diagnosed a single disease but the pre-test probability was high (since very few things looked like HIV at the height of the epidemic). RDTs for malaria were a single test that diagnosed a single disease but the pre-test probability was medium (many things look like malaria that are not). But we focused on HIV diagnosis and treatment and we focused on malaria diagnosis and treatment. Now we have HIV patients who are doing great—and getting cancer. We have malaria patients that are doing great with RDTs and ACTs—but any child with a fever of another cause probably dies. If you ask anyone with an understanding of biology or epidemiology to look at the history of the HIV epidemic or malaria in the modern age, they would all predict these findings. It’s not an epiphany…it was deliberate ignorance. Building systems is hard but it IS the answer. So, I 100% disagree with Richard that treating a single cancer will have an impact beyond those few patients that benefit from that disease. Do those patients with a specific cancer deserve treatment? Of course! But so do patients with all cancers. So, the answer IS still systems.

In order to treat cancer, clinicians must have a pathological diagnosis. For example, if clinicians decided that they would by assumption treat all women with Stage 4 breast cancer in Peru (with positive lymph nodes on palpation), 20% of patients would actually have tuberculosis. But a % of the patients will also have metastasis from other tumor types (such as lymphoma, benign lesions, and soft tissue tumors). If we provide chemotherapy for invasive ductal carcinoma and a pathology service to biopsy the patients to prove the diagnosis, what do we do with those that don’t actually have invasive ductal cancer? How is that ethical? Once we expand our breast tumor regiment to cover all tumors that MAY occur in the breast, now we must treat patients that have those tumors in other locations, otherwise we are in an ethical nightmare.

At the heart of this issue is the pathological diagnosis. There is no treatment without a pathological diagnosis and, once you have the ability to make a pathological diagnosis, there is not justifiable excuse for not treating patients who present with any cancer. The curse of a tissue biopsy processed for histology is that it is one test with, literally, thousands of possible results. Remember HIV and Malaria? They are each one test with one actionable result. A histology slide can present thousands of actionable results! So, no, it is not possible within an ethical construct of healthcare or within a paradigm of equity to focus on one cancer. We can deploy thousands of oncologists and nurses across LMICs with truckloads of every chemotherapy known to humankind and there would be NO IMPACT—absolutely none—unless every patient was pathologically diagnosed before treatment was begun. Surgeons could enter a country and remove every breast with a lump in it—the number of women with inappropriate surgical treatment would result in criminal charges. Pathology is the central tool for diagnosing cancer and creating an appropriate treatment plan, but it is also a single tool that can diagnose EVERY cancer so we must be able to fulfill every appropriate treatment plan.

It is for this reason that PIH with assistance from Dana-Farber Cancer Institute and Brigham and Women’s Hospital began diagnosing and treating patients in Haiti, Lesotho, and Rwanda in 2005 with cancer. By 2011, the trickle of patients that would find their way to PIH clinics had become a flood. It was now necessary to not only build pathology laboratories in countries that could handle the volume and range of diagnoses but also import nurses and oncologists to formulate and run programs. Before the pathology laboratory was built in Butaro, Rwanda, patients may have waited for up to 6 months (if ever) to receive a result which may have been incomplete or inaccurate due to the limitation of staffing. In Butaro today, after the construction of a laboratory, training of staff, addition of immunohistochemistry, installation of telepathology, and residence of a permanent Rwandan pathologist, the turnaround time is < 72 hours. There are other success stories like this but these systems need to be replicated within country and in other countries at a rate of at least one cancer treatment center per 5 million people or less. And, as Richard rightly points out, these centers need to have resources to treat every patient.

ASCP has been in the global health arena working with PEPFAR since its inception. In 2015, ASCP launched Partners for Cancer Diagnosis and Treatment in Africa (including Haiti) which was built on the premise that telepathology would be a key tool to diagnose patients more rapidly and accurate in LMICs. Butaro, Rwanda was the first site to receive telepathology with ASCP but there were many examples of other labs with telepathology in place prior to that; however, the bulk of them were focused on single-entity or research-based programs. The ASCP program starts with the premise that the site where telepathology is placed plans to treat all cancers that are diagnosed. Thus, ASCP requires that a system for cancer care is at least planned or in process. So, the old adage, “you have to start somewhere” is great but, for cancer, that first start must be the provision of pathology services. The ethical framework that follows will require that all cancer move into the realm of treatment.

Again, ASCP thanks Richard Horton for bringing this issue up with the Lancet audience and ASCP hopes that we, all shouting together, can move the needle much further along towards funding for cancer across the systems spectrum.


  1. Horton S, Sullivan R, Flanigan J, Fleming KA, Kuti MA, Looi LM, Pai SA, Lawler M. Delivering modern, high-quality, affordable pathology and laboratory medicine to low-income and middle-income countries: a call to action. Lancet. 2018 May 12;391(10133):1953-1964. doi: 10.1016/S0140-6736(18)30460-4. Epub 2018 Mar 15. Review. PubMed PMID: 29550030.
  2. Sayed S, Cherniak W, Lawler M, Tan SY, El Sadr W, Wolf N, Silkensen S, Brand N, Looi LM, Pai SA, Wilson ML, Milner D, Flanigan J, Fleming KA. Improving pathology and laboratory medicine in low-income and middle-income countries: roadmap to solutions. Lancet. 2018 May 12;391(10133):1939-1952. doi: 10.1016/S0140-6736(18)30459-8. Epub 2018 Mar 15. Review. PubMed PMID: 29550027.
  3. Wilson ML, Fleming KA, Kuti MA, Looi LM, Lago N, Ru K. Access to pathology and laboratory medicine services: a crucial gap. Lancet. 2018 May 12;391(10133):1927-1938. doi: 10.1016/S0140-6736(18)30458-6. Epub 2018 Mar 15. Review. PubMed PMID: 29550029.
  4. Sayed S, Cherniak W, Lawler M, Tan SY, El Sadr W, Wolf N, Silkensen S, Brand N, Looi LM, Pai SA, Wilson ML, Milner D, Flanigan J, Fleming KA. Improving pathology and laboratory medicine in low-income and middle-income countries: roadmap to solutions. Lancet. 2018 May 12;391(10133):1939-1952. doi: 10.1016/S0140-6736(18)30459-8. Epub 2018 Mar 15. Review. PubMed PMID: 29550027.
  5. Milner DA Jr. Pathology: Central and Essential. Clin Lab Med. 2018 Mar;38(1):xv-xvi. doi: 10.1016/j.cll.2017.11.001. Epub 2017 Dec 12. PubMed PMID: 29412893.
  6. Milner DA Jr. Global Health and Pathology. Clin Lab Med. 2018 Mar;38(1):i. doi: 10.1016/S0272-2712(17)30139-7. Epub 2018 Feb 3. PubMed PMID: 29412888.
  7. Orozco JD, Greenberg LA, Desai IK, Anglade F, Ruhangaza D, Johnson M, Ivers LC, Milner DA Jr, Farmer PE. Building Laboratory Capacity to Strengthen Health Systems: The Partners In Health Experience. Clin Lab Med. 2018 Mar;38(1):101-117. doi: 10.1016/j.cll.2017.10.008. Epub 2017 Dec 28. Review. PubMed PMID: 29412874.
  8. Milner DA Jr, Holladay EB. Laboratories as the Core for Health Systems Building. Clin Lab Med. 2018 Mar;38(1):1-9. doi: 10.1016/j.cll.2017.10.001. Epub 2017 Dec 1. Review. PubMed PMID: 29412873.
  9. Dayton V, Nguyen CK, Van TT, Thanh NV, To TV, Hung NP, Dung NN, Milner DA Jr. Evaluation of Opportunities to Improve Hematopathology Diagnosis for Vietnam Pathologists. Am J Clin Pathol. 2017 Nov 20;148(6):529-537. doi: 10.1093/ajcp/aqx108. PubMed PMID: 29140404.
  10. Mpunga T, Hedt-Gauthier BL, Tapela N, Nshimiyimana I, Muvugabigwi G, Pritchett N, Greenberg L, Benewe O, Shulman DS, Pepoon JR, Shulman LN, Milner DA Jr. Implementation and Validation of Telepathology Triage at Cancer Referral Center in Rural Rwanda. J Glob Oncol. 2016 Jan 20;2(2):76-82. doi: 10.1200/JGO.2015.002162. eCollection 2016 Apr. PubMed PMID: 28717686; PubMed Central PMCID: PMC5495446.
  11. Sayed S, Lukande R, Fleming KA. Providing Pathology Support in Low-Income Countries. J Glob Oncol. 2015 Sep 23;1(1):3-6. doi: 10.1200/JGO.2015.000943. eCollection 2015 Oct. PubMed PMID: 28804765; PubMed Central PMCID: PMC5551652.
  12. Nelson AM, Milner DA, Rebbeck TR, Iliyasu Y. Oncologic Care and Pathology Resources in Africa: Survey and Recommendations. J Clin Oncol. 2016 Jan 1;34(1):20-6. doi: 10.1200/JCO.2015.61.9767. Epub 2015 Nov 17. Review. PubMed PMID: 26578619.
  13. Mpunga T, Tapela N, Hedt-Gauthier BL, Milner D, Nshimiyimana I, Muvugabigwi G, Moore M, Shulman DS, Pepoon JR, Shulman LN. Diagnosis of cancer in rural Rwanda: early outcomes of a phased approach to implement anatomic pathology services in resource-limited settings. Am J Clin Pathol. 2014 Oct;142(4):541-5. doi: 10.1309/AJCPYPDES6Z8ELEY. PubMed PMID: 25239422.
  14. Mtonga P, Masamba L, Milner D, Shulman LN, Nyirenda R, Mwafulirwa K. Biopsy case mix and diagnostic yield at a Malawian central hospital. Malawi Med J. 2013 Sep;25(3):62-4. PubMed PMID: 24358421; PubMed Central PMCID: PMC3859990.
  15. Berezowska S, Tomoka T, Kamiza S, Milner DA Jr, Langer R. Surgical pathology in sub-Saharan Africa–volunteering in Malawi. Virchows Arch. 2012 Apr;460(4):363-70. doi: 10.1007/s00428-012-1217-z. Epub 2012 Mar 10. PubMed PMID: 22407448.
  16. Roberts DJ, Wilson ML, Nelson AM, Adesina AM, Fleming KA, Milner D, Guarner J, Rebbeck TR, Castle P, Lucas S. The good news about cancer in developing countries–pathology answers the call. Lancet. 2012 Feb 25;379(9817):712. doi: 10.1016/S0140-6736(12)60306-7. PubMed PMID: 22364759.
  17. Carlson JW, Lyon E, Walton D, Foo WC, Sievers AC, Shulman LN, Farmer P, Nosé V, Milner DA Jr. Partners in pathology: a collaborative model to bring pathology to resource poor settings. Am J Surg Pathol. 2010 Jan;34(1):118-23. doi: 10.1097/PAS.0b013e3181c17fe6. PubMed PMID: 19898229.



-Dan Milner, MD, MSc, spent 10 years at Harvard where he taught pathology, microbiology, and infectious disease. He began working in Africa in 1997 as a medical student and has built an international reputation as an expert in cerebral malaria. In his current role as Chief Medical officer of ASCP, he leads all PEPFAR activities as well as the Partners for Cancer Diagnosis and Treatment in Africa Initiative.

Hematopathology Case Study: A 48 Year Old Woman with Left Upper Quadrant Pain

Case History

A 48-year-old female presents with a one-month history of left upper quadrant pain. Laboratory investigation reveals pancytopenia. Radiology work-up demonstrates splenomegaly. CT scan confirms splenomegaly at 22 cm. There is no lymphadenopathy appreciated in the abdomen. A bone marrow biopsy is performed.

Figure HSTCL
Image 1. H&E and CD3 stains at varying magnification.

Bone Marrow Findings

The bone marrow core biopsy reveals a normocellular marrow space (approximately 50% cellular marrow) with progressive trilineage hematopoiesis. Clusters of small, slightly irregular, mature-appearing lymphocytes are seen within the sinusoids. The marrow aspirate smears reveal mild erythroid hyperplasia without morphologic evidence of dysplasia. There is no increase in blasts. Lymphocytes comprise 18% of a 500-cell differential count on the marrow aspirate smears.

The sinusoidally distributed lymphocytes demonstrate immunopositivity (flow and/or IHC) for CD2, CD3, CD7, CD16, CD56, and γδ. These neoplastic lymphocytes are negative for granzyme B, CD4, CD5, CD8, CD57, and αβ.  PCR for T-cell receptor clonality was positive. Cytogenetics revealed a normal female karyotype. FISH for 5p/5q and 7p11/7q31 was normal.


Taken together, the patient’s clinical presentation along with the presence of an abnormal gamma-delta population of T cells in a sinusoidal distribution with PCR evidence of T-cell clonality is diagnostic of a T-cell lymphoma. The pattern of distribution, granzyme B negativity, lack of concurrent adenopathy favor a diagnosis of Hepatosplenic T-cell lymphoma.


Hepatosplenic T-cell lymphoma (HSTCL) is an uncommon entity that represents <1% of all non-Hodgkin lymphomas and 1%-2% of all T/natural killer cell lymphomas. It most commonly affects young adult men, with a median age of 35 years. This high-grade malignancy is most often characterized by γδ T-cells. The most consistent symptoms among patients are fever, splenomegaly, hepatomegaly, bone marrow involvement, peripheral blood cytopenia, and less commonly, adenopathy. Hepatosplenic T-cell lymphoma has a poor prognosis with median survival rates varying from a few months to 16 months in different studies.

Immune suppression (such as solid- organ transplant, or immune dysregulation secondary to malignancy or infection) is thought to play a role in the lymphomagenesis in around 20% of cases. Inflammatory bowel disease and the use of immunosuppressive agents (e.g., antitumor necrosis- α agents) and antimetabolite therapy (e.g., 6TG, 6MP) had also been associated with development of HSTCL.

HSTCL initially infiltrates the cords and sinusoids of the splenic red pulp. The white pulp is often atrophic or absent. Eventually, the neoplastic T cells diffusely replace the spleen. The lymphoma cells often involve the liver and bone marrow sinusoids. At the time of diagnosis, the bone marrow is almost always involved and commonly hypercellular. The neoplastic cells are mostly intermediate in size, with pale agranular cytoplasm and round nuclei with condensed chromatin and inconspicuous nucleoli.

Cytogenetic studies in HSTCL most commonly show isochromosome 7q and trisomy 8. Molecular analysis of HSTCL characteristically shows expression of a γδ T-cell type and flow cytometric analysis typically reveals a CD2+, CD3+, CD7+/−, CD4−, CD5−, and CD8− phenotype with positivity for natural killer cell-associated markers CD11b, CD16, and CD56.

Activating mutations in PI3KCD and STAT signaling genes have also been described in HSTCL, providing potential molecular target therapies for this aggressive lymphoma.

The differential diagnosis of HSTCL includes other types of T-cell lymphoma and leukemia, and non-neoplastic such as immune thrombocytopenia or acute hepatitis. In most instances, the distinctive presentation of spleen, liver and bone marrow involvement, the immunophenotype and T-cell monoclonality distinguishes HSTCL from other entities.

The outcomes of the patients using standard chemotherapy regimens are dismal, and allogeneic SCT appears to be a reasonable approach to achieve the best possible patient outcome.


  1. Yabe M, Miranda RN, Medeiros LJ. Hepatosplenic T-cell Lymphoma: a review of clinicopathologic features, pathogenesis, and prognostic factors. Hum Pathol. 2018 Apr; 74:5-16.
  2. McThenia SS, Rawwas J, Oliveira JL, Khan SP, Rodriguez V. Hepatosplenic γδ T-cell lymphoma of two adolescents: Case report and retrospective literature review in children, adolescents, and young adults. Pediatr Transplant. 2018 Aug;22(5): e13213.


levent photo

-Levent Trabzonlu, MD is a postdoctoral researcher in the department of pathology at Johns Hopkins University in Baltimore, MD. Follow Dr. Trabzonlu on twitter @aflevent


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

The Pyramid and the Power

In 1950 the National Safety Council began describing a safety system known as the “hierarchy of controls.”  This new model was created to show that that design, elimination and engineering controls are more effective in reducing risk to workers than ‘lower level controls’ such as warnings, training, procedures and personal protective equipment (PPE). The National Institute for Occupational Safety and Health (NIOSH) began to use the hierarchy of controls, and it has been an effective safety teaching tool for that organization and others over the years. The philosophy of the hierarchy- or the pyramid- is simple: “Controlling exposures to occupational hazards is the fundamental method of protecting workers.” It is simple, and although it may not be rocket science, it’s a powerful idea.

While this hierarchy is represented differently by multiple organizations, the basic protection levels of the pyramid remain the same; Elimination, Substitution, Engineering Controls, Administrative Controls, and PPE. The most effective part of the pyramid (Elimination) is at the sharp end, or the top, and the least effective (PPE) lies at the bottom.

Unfortunately, the top two most-effective layers of the safety pyramid do not work well in the laboratory setting. We can’t eliminate or substitute the biohazards we work with- that would mean not being able to perform our work. Laboratorians handle and analyze patient samples and chemicals, and they are a necessary hazardous part of the job. There is some substitution possible in the lab when considering chemicals (the use of a non-hazardous xylene substitute, for example), but for the most part, this level of the hierarchy of controls is not very helpful to the lab.

Engineering Controls involve the use of engineered machinery or equipment which reduces or eliminates exposure to a chemical or physical hazard. Engineering Controls are definitely favored over other levels on the pyramid for controlling existing worker exposures in the workplace because they are designed to remove the hazard at the source, before it comes in contact with the worker. Well-designed engineering controls can be very effective in protecting lab employees, and they are typically independent of worker interactions so they can provide that high level of protection. Sometimes the initial price of certain engineering controls can be high, but over the longer term, operating costs are frequently lower, and the controls can ultimately provide a cost savings. Good examples of engineering controls include Biological Safety Cabinets, Chemical Fume Hoods, centrifuges, and glove boxes.

The next level of the hierarchy is represented as Administrative Controls.  These controls seek to improve workplace safety by creating safer policies and procedures in the workplace. Administrative Controls can range from the placement of warning signs throughout a lab, the provision of safety training programs, and the implementation of proper ergonomics. The part of the pyramid may be the most difficult to manage. The onus of workplace safety here begins to shift from management over to staff, and sometimes the results can be… unpredictable.

An off-shoot of Administrative Controls that is discussed often in safety models is known as Work Practice Controls. These controls are not truly part of hierarchy, but they can be important safety practices in the lab setting. OSHA describes Work Practice Controls as “procedures for safe and proper work that are used to reduce the duration, frequency or intensity of exposure to a hazard.” These are the not the actual written procedures, but the actions that put those written policies into action. Following proper hand hygiene and preventing eating or drinking in the laboratory are good examples of those actions.

PPE is at the bottom of the hierarchy of controls- by definition that means that it is the least effective method to keep employees from hazard exposure. It is the last resort for safety in the lab. That’s a powerful point, and it should be discussed when providing lab safety training. All too often lab staff carelessly perform tasks without wearing PPE, and the danger is immediate and potentially disastrous. Even though this level of protection is considered the least effective, this last barrier between the employee and the hazardous material is crucial. Lab staff are required to have PPE education, and they should be able to provide a return demonstration for the proper donning and doffing of that PPE.

The Hierarchy of Controls is typically represented as a pyramid. It’s a simple symbol, but it’s really a powerful and complex model for safety. When you look at each separate level, you can see that there is a great deal of information that can provide a lab safety professional with helpful resources. As a lab leader, you can use the model to provide education, train staff, and help to enforce good safety behaviors which will improve the lab safety culture and keep employees from harm.


Scungio 1

Dan Scungio, MT(ASCP), SLS, CQA (ASQ) has over 25 years experience as a certified medical technologist. Today he is the Laboratory Safety Officer for Sentara Healthcare, a system of seven hospitals and over 20 laboratories and draw sites in the Tidewater area of Virginia. He is also known as Dan the Lab Safety Man, a lab safety consultant, educator, and trainer.

Error Codes in Blood Gas Analysis

We recently received a venous blood sample for blood gas analysis from the operation room. We analyzed the specimen according to manufacturer’s instructions on the ABL800 FLEX blood gas instrument (Radiometer, Copenhagen, Denmark). Multiple error codes were present for the results of ctHb, sO2FO2Hb, FCOHb, FHHb, and FMetHb. Text messages accompanying the report read, “Detection of SHb” and “OXI spectrum mismatch.” The sample was re-tested on the ABL800 but the same error codes were flagged.

A closer look at the patient’s chart revealed that patient is heterozygous for hemoglobin M-Saskatoon variant,  which causes the replacement of histidine by tyrosine in position 63 on the beta chain of hemoglobin (beta codon 63, CAT>TAT/His63Tyr). This renders the NADH methemoglobin reductase system incapable of reducing oxidized iron. A group of mutations in the globin chain gene can result in such dysfunction of ferric iron reduction and are referred to as methemoglobin forming hemoglobin variants (Hgb M).

HbM variants usually have a different absorbance spectrum from the physiologic methemoglobin. Modern day CO-oximeters use more than 100 wavelengths and can detect most unknown substances. We speculated that Hgb M in the patient is the reason the ABL800 reported error codes. The clinical team collected another venous blood sample and  it was tested on the GEM5000 blood gas instrument (Instrumentation Laboratory, Bedford, MA, USA). This specimen also reported with error codes.

Non-invasive pulse-oximetry devices use two wavelengths (660 nm and 940 nm) to calculate hemoglobin oxygen saturation based on oxyhemoglobin and deoxygenated hemoglobin, and thus are unable to report interferences from dyshemoglobins. In a nut shell, Hgb M variants can possibly interfere with CO-oximetry measurements. Caution is needed to interpret the results. Pulse oximetry usage should be avoided for these patients.


  1. Schiemsky T, Penders J, Kieffer D. Failing blood gas measurement due to methemoglobin forming hemoglobin variants: acase report and review of the literature. Acta Clin Belg. 2016 Jun;71(3):167-70.
  2. Stucke AG, Riess ML, Connolly LA. Hemoglobin M (Milwaukee) Affects Arterial Oxygen Saturation and Makes Pulse Oximetry Unreliable. Anesthesiology 4 2006, Vol.104, 887-888.



-Jayson Pagaduan, PhD, is a senior year clinical chemistry fellow Texas Children’s Hospital in Houston, TX.


-Jing Cao, PhD, DABCC, FACB, is a board-certified clinical chemist, serving as the Associate director of Clinical Chemistry at Texas Children’s Hospital in Houston, TX and an Assistant Professor of Pathology and Immunology at Baylor College of Medicine.

Microbiology Case Study: A 14 Year Old Immunocompromised Male with Pneumonia

Case History

A 14 year old male with a history of selective IgG deficiency, asthma, and GE reflux s/p Nissen Fundoplication, presented to the pediatric pulmonary clinic with 2 weeks of cold like symptoms that progressed to a wet sounding cough productive of sputum and chest pain. 5 days prior he had seen his primary care physician who diagnosed him with pneumonia and started him on amoxicillin and 5 days of prednisone (60mg daily). He is using his albuterol nebulizer every 3 hours, and feels as though his asthma may be contributing to his symptoms but is not the main cause as he has not had a wet cough and chest heaviness and pain with previous asthma attacks. He has had low grade fevers for the past several days but denies chills, sweats, or hemoptysis.  Of note his immune deficiency is treated with IVIG and long term Bactrim which was recently stopped. Pediatric pulmonology elected not to restart the Bactrim but changed his antibiotic to Augmentin. The patient continued to have chest pain and coughing, so the decision was made to proceed with bronchoscopy

Laboratory Identification

Image 1. Scotch tape prep showing broad hyphae with rare septation and round sporangia.
Image 2. Colonies with growth at 30° and 37° C showing “fluffy” growth with darkening in the center of the colony.

Cultures from the BAL revealed a rapidly growing fungi with broad hyphae (5 to 15 micron diameter) that are irregularly branched, and have rare septations. These features, paired with the morphology of the sporangia are diagnostic for a Zycomycete; further identification was attempted however, rhizoids characteristic of Rhizopus, and branching characteristic of Mucor were not identified in this culture.


Zygomycetes (Mucormycetes) are widely distributed in the environment in soil and vegetation and infection is through inhalation of spores. Typically, the spores are transported by the muco-cilliary escalader to the pharynx, are swallowed, and then are broken down by the GI tract. In immunocompromised patients, the spores can settle in the nasal turbinates and alveoli causing disease (1). The most common sites of infection are rhino-orbital-cerebral and pulmonary and typically occur in immunocompromised hosts and diabetics (2). The Zygomycetes are also known to invade blood vessels making tissue infarction and necrosis one of the hallmarks of the disease (3). These fungi grow rapidly and are often referred to as “lid lifters” when cultured. Because of this rapid growth, infection with Zygomycetes typically progress quickly and cause periorbital edema, proptosis, and blindness. Facial numbness can occur if there is infarction of sensory branches of the fifth cranial nerve. Infection can spread from the ethmoid sinus to the frontal lobe and result in obtundation. If infection spreads from the sphenoid sinuses to the adjacent cavernous sinus, it can result in cranial nerve palsies, thrombosis of the sinus, and involvement of the carotid artery (4). When the spores are inhaled into the lung, pneumonia with infarction and necrosis results. The infection can spread to contiguous structures, such as the mediastinum and heart, or disseminate hematogenously to other organs such as the GI tract, kidney, and brain (5). Infections are treated with a combination of antifungal drugs such as Amphotericin B, and aggressive surgical debridement. In some situations removal of the palate, nasal cartilage, and orbit are necessary for cure. In patients with pneumonia, often lobectomy is needed for cure (5).

The genera most commonly found in human infections are Rhizopus, Mucor, and Rhizomucor. Rhizopus organisms have an enzyme called ketone reductase, which allows them to thrive in high glucose, acidic conditions, such as in individuals with diabetic ketoacidosis. Rhizopus will have root like rhizoids and are typically located near the sporangiophores. The post-mature sporangiophores can undergo “umbrella like” collapse which is characteristic of Rhizopus. Mucor is more likely to have branching and is often identified when all other species are ruled out; they do not produce a rhizoid.

  1. Ferguson BJ. Mucormycosis of the nose and paranasal sinuses. Otolaryngology Clinics of North America. 2000;33(2):349.
  2. Kauffman CA, Malani AN. Zygomycosis: an emerging fungal infection with new options for management. Current Infectious Disease Reports. 2007;9(6):435.
  3. Greenberg RN, Scott LJ, Vaughn HH, Ribes JA. Zygomycosis (mucormycosis): emerging clinical importance and new treatments. Current Opinions in Infectious Diseases. 2004;17(6):517.
  4. Yohai RA, Bullock JD, Aziz AA, Markert RJ. Survival factors in rhino-orbital-cerebral mucormycosis. Survey of Ophthalmology. 1994;39(1):3.
  5. Tedder M, Spratt JA, Anstadt MP, Hegde SS, Tedder SD, Lowe JE. Pulmonary mucormycosis: results of medical and surgical therapy. Annals of Thoracic Surgery. 1994;57(4):1044.


-Casey Rankins, DO, is a 1st year Anatomic and Clinical Pathology resident at the University of Vermont Medical Center.


-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.