Leading in a VUCA World

Leading people can be a challenging task regardless of the industry or size of an organization. Adding volatile, uncertain, complex, and ambiguous (VUCA) environment into the mix and the leadership challenge increases. Today’s organizations are increasingly complex, ambiguous, uncertain, and volatile because change is accelerating and intensifying. How can leaders equip themselves to manage a VUCA workplace? The first step is understanding what each terms means.

Volatile Situations describe circumstances that change constantly and unexpectedly, and a certain level of instability of a task or challenge is present. However, the best leadership approach is to use available information, be proactive, and have multiple plans and strategies in place. An example of a volatile circumstance is a natural disaster. In such a circumstance not only is the natural disaster a volatile situation, but also the constantly changing nature of the aftermath; which emergency agencies are coming and when, where are people stuck, etc. There are a lot of changes occurring in a volatile situation.   Being proactive and prepared in volatile circumstances can be expensive, but that preparation is necessary to handle these situations.

Uncertain Situations are situations known for a lack of information, so on some level they are the opposite of volatile situations. In uncertain circumstances there is no reliable information about cause and effect and it is not known if change will happen, can happen, or have a positive effect if it does happen. The best approach in these circumstances is to find more information, more data, and more analytics. Once leaders have access to more data, they need to make sure the data is analyzed and implemented into new strategies and change processes. An example of an uncertain situation is when a competitor suddenly emerges that takes direct aim at your company by undercutting prices. In this case, it is important to collect as much data and information as possible to respond to the situation appropriately through new strategies.

Complex Situations have several interconnected and interdependent aspects which have a clear relationship. In these situations, there is partial information available but because everything is interlinked, it is a challenge to process the information in a way that reliably predicts the future. The approach is to reduce the number of linkages, or at least to make them clearer, so the complexity of the situation or task is easily understood and managed. An example of a complex situation is when implementing a process change affects all departments in an organization. In such a circumstance, everything is interconnected and it can be hard to predict how this change will impact everyone and to prepare for it. The key here is to make the change as simple as possible and to assess the impact it makes on every aspect of the organization before implementing the change.

Ambiguous Situations are situations which have relationships that are completely unknown and ambiguous; there appears to be no rhyme or reason. The phrase that comes to mind in these situations is “you don’t know what you don’t know.” In such ambiguity, leaders need to learn from mistakes, hypotheses, and test rounds so it is important to experiment in order to gain information. An example of an ambiguous situation is when you are launching a new product or starting a new business. There are a lot of unknowns in these circumstances so making hypotheses and learning from mistakes is essential for leaders’ success.

In order to lead in a VUCA world, leaders need to analyze these four situation types to confirm which one they are currently leading in. Next is to find the right approach in order to lead people, a department, or an organization through the volatile, uncertain, complex, or ambiguous situation. Knowing is half the answer, so the next time you find yourself in a VUCA situation, start by not only analyzing the situation and possible solutions, but also by analyzing your own reaction to each of the four situations. Being able to understand and control your own reaction will increase your leadership skills in all VUCA and non-VUCA worlds.



-Lotte Mulder earned her Master’s of Education from the Harvard Graduate School of Education in 2013, where she focused on Leadership and Group Development. She’s currently working toward a PhD in Organizational Leadership. At ASCP, Lotte designs and facilitates the ASCP Leadership Institute, an online leadership certificate program. She has also built ASCP’s first patient ambassador program, called Patient Champions, which leverages patient stories as they relate to the value of the lab.

Beyond the CBC and Reticulocyte Count: Early Detection of Iron Deficiency Anemia

In my May 2018 post (Not your Grandmother’s Hematology), I discussed the history of hematology and chronicled how far we have come in the last 60 years. We have progressed from manual counting of cells to the first Coulter Counter in 1956, which revolutionized hematology by being able to automate the counting of red blood cells, to modern instruments that can report up to 30 parameters and perform up to 400 CBCs an hour. Among these parameters are what are termed advanced clinical parameters, new parameters which provide physicians with additional information about the state of blood cells. In this blog I will explore how one of these advanced clinical parameters, the Reticulocyte Hemoglobin content, can provide physicians with information that can assist them with earlier detection, differential diagnosis and better management of iron deficiency and iron deficiency anemia. 

Case Study 

A 29 year old female was seen by her gynecologist reporting a history of heavy menstrual bleeding with current bleeding lasting 15 days. The doctor discussed various treatment options with the patient and a CBC was performed. CBC results are shown below.

Test Result Flags Reference
WBC 7.23   4.5-10.5 K/CMM
RBC 4.38   3.70-5.30 M/CMM
HGB 12.0   12.0-15.5 GM/DL
HCT 36.2   36.0-46.0 %
MCV 82.6   80-100 FL
MCH 27.4   27.0-34.0 PG
MCHC 33.1   32.0-36.0 %
PLT 243   150-450 K/CMM
MPV 11.0   9.6-12.0 FL
RDW 12.5   0-15.1 %

This CBC shows no abnormal flags. Based on patient history and presentation, the physician questioned iron deficiency despite normal hemoglobin and hematocrit, MCV and MCHC. He ordered a reticulocyte profile on the same specimen with the following results:

Test Result Flags Reference Range
Retic 1.55   0.5-2.0 %
Abs Retic 0.0679 H 0.0391-0.057 M/CMM
Imm Retic Frac 14.9   2.3-15.9 %
Ret-Hgb 24.6 L 30-35 PG

Reticulocyte counts are the quantity of the youngest red blood cells released from the bone marrow into the peripheral blood. Reticulocytes are reported as a % and the absolute reticulocyte count is calculated by multiplying the Retic% by the RBC. The immature reticulocyte fraction (IRF) is the rate of production of reticulocytes and depends largely on the ability of the bone marrow to respond to erythropoietin. The reticulocyte hemoglobin (Ret-He) content is the amount of hemoglobin in newly formed red blood cells. (There are two different hematology systems that report reticulocyte hemoglobin content. The two nomenclatures used for reticulocyte hemoglobin are Ret-He and CHr and studies have been done that demonstrate their equivalence)

Note that the Ret-He reflects the quality of the newly formed reticulocytes. Ret-He is a direct measurement of the amount of hemoglobin in each reticulocyte, which indicates the amount of iron available for incorporation into the precursors of mature red cells. This patient’s retic% and IRF are within normal ranges, but her absolute reticulocyte count is high. A Ret-He less than 29 pg in an adult is indicative of iron deficiency. With a normal CBC and low Ret-He, this is an early indication that iron deficiency is indeed present. With the absence of sufficient iron, this patient would eventually develop a microcytic, hypochromic anemia. Therefore, Ret-He can measure and indicate inadequate hemoglobin production before the hemoglobin and hematocrit decrease.

In this case the importance of clinical awareness is illustrated. This physician remembered a recent laboratory technical bulletin announcing implementation of a new hematology analyzer system with the availability of new parameters for reticulocyte counts. When the CBC results came back from the laboratory, the patient had already gone home, and no serum had been drawn to perform a ferritin level. Rather than calling the patient back to have another sample drawn, the Ret-He could be done from the same blood sample already in the lab. Ret-He is a faster, easier and less expensive test than additional iron studies and bone marrow iron stains. Ret-He can easily be used at a very low cost to get that first piece of information to decide whether or not iron deficiency is a concern. A high or normal Ret-He would have ruled out an iron deficiency with a fairly high confidence level. In this case, the low Ret-He could be used to guide further workups. A subsequent blood drawn revealed a low ferritin and iron deficiency was confirmed. The patient was advised to take an iron supplement along with ongoing treatment for the bleeding.

This case is just one example of the clinical utility of the Ret-He. Using the Ret-He, physicians can determine iron deficiency before iron deficiency IDA develops. A low Ret-He can alert a physician to iron deficiency without the presence of anemia, microcytosis or hypochromia. Ret-He can also be used to monitor and show early response to iron therapy before any other parameters change. A case example is that of a 5 month old who was brought to the emergency room with a Hgb of 7 g/dl and a Ret-He of 11.9 pg. In pediatric patients, a Ret-He less than 27.5 is an indicator of IDA. In this child, treatment with oral iron showed that the Ret-He had risen to 24.6 pg seven days after the onset of iron therapy, while the CBC remained virtually the same. This provided a very early indication that the iron therapy was effective.1 The Ret-He can also been used to minimize transfusions. The AABB Choosing Wisely Campaign lists 5 things that physicians and patients should questions before transfusion. One of the guidelines states “Don’t transfuse red blood cells for iron deficiency without hemodynamic instability.“2 Historically, physicians have used a ‘wait and see’ approach and watched Hgb levels drop before they start looking at iron. Using a Ret-He, iron deficiency could be determined, for example, in a patient with a Hgb of 11 g/dl. Oral or intravenous iron could be started before the Hgb drops below 7 g/dl and transfusion becomes necessary. The AABB Choosing Wisely Campaign emphasizes this by stating that patients with chronic iron deficiency or pre-operative patients with iron deficiency should be given iron therapy before transfusion is considered.2 Ret-He can give the earliest indication of iron deficiency and can be used to monitor the response to iron therapy. Another clinical utility of Ret-He has been to help diagnose or rule out iron deficiency in oncology patients. Additionally, Ret-He has been included in guidelines for anemia management in end stage renal disease patients on dialysis and who get erythropoietin.

The Ret-He parameter has proved clinically useful in early determination of functional iron deficiency. Traditionally ordered chemistry iron studies are indirect measures that have certain inherent inaccuracies due to the presence of inflammation and infection, or in patients on iron therapy. Ret-He is a direct and very effective screening tool and physicians can use Ret-He with other RBC indicies to improve anemia diagnosis and management in many patient populations. Ret-He can be used as a screening measure, and used to reflex for iron studies. Therefore, laboratories who have instruments that can report Ret-He and CHr should develop an education program to help clinicians effectively use Ret-He. Together physicians and laboratorians can develop their own guidelines for reflex testing and improvement for patient care.


  1. Case Studies Demonstrating the Clinical Application of the Advanced Clinical Parameters (1/20/2016) Chantale Pambrun, MD, FRCPC, Head of Division of Hematopathology and Assistant Professor of Pathology and Laboratory Medicine, IWK Children’s & Women’s Health Centre and Dalhousie University
  2. https://www.aabb.org/pbm/Documents/Choosing-Wisely-Five-Things-Physicians-and-Patients-Should-Question.PDF
  3. Advanced parameters offer faster, surer guidance to cancer care. Anne Paxton. CAP Today. Sept 2017
  4. The Value-driven Laboratory. Reticulocyte Hemoglobin Content (Ret-He): A Parameter Well-Established Clinical Value. Sysmex America White Paper.
  5. Sysmex Clinical Support Team. Utility of RET-He, August 10. 2015
  6. Brugnara C, Schiller B, Moran J. Reticulocyte hemoglobin equivalent (Ret-He) and assessment of iron-deficient states. Clinical Laboratory Hematology 2006;28:303 – 308.



-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 22 Year Old Female with Wound Infection


Case History

22 year old female with a past medical history of scoliosis presents for routine follow-up after hospital discharge for post-op wound infection following a spinal fusion surgery. Patient had an anterior and posterior spinal fusion with allograft and hardware on 1/18/18. She had a laminectomy and irrigation for post-op epidural hematoma on 1/19/18. Subsequently, she developed a lumbar spine abscess and underwent irrigation and debridement of the abscess on 3/1/18. Two operative cultures of the left paraspinal musculature grew only tiny clear colonies on the anaerobic blood plates. Gram stain of these colonies did not show any organism. MALDI-ToF MS identified these colonies as Mycoplasma hominis which was confirmed at a reference laboratory by PCR. The patient was given daptomycin plus levofloxacin. Since discharge from the hospital, she had wound healing with intermittent discharge.

Lab Identification

Mycoplasma hominis requires a specific rich and complex agar medium for growth and grows tiny colonies on standard media such as Columbia agar. In a patient with urogenital disease, Mycoplasma hominis is diagnosed with a urogenital specimen culture and confirmed by PCR. In a patient with spinal hardware infection, Mycoplasma hominis is diagnosed by a culture of infected tissue with PCR confirmation.


Mycoplasma is a bacteria that lacks a cell wall and contains the smallest bacterial genome totally sequenced. Due to its lack of cell wall, Mycoplasma cannot be visualized with a Gram stain, and it is innately resistant to b-lactams.1 Due to its small bacterial genome, 580 kpb, it cannot be detected by light microscopy and requires complex nutrients for growth1.

Mycoplasmas are frequently part of the oropharyngeal and genital tract flora among healthy subjects.1 There are more than 200 Mycoplasma species, of which 13 have been isolated from humans. Only 6 species, among which 5 are pathogens, live in the urogenital tract.2 As one of the Mycoplasma species detected in the genitourinary tract, M. hominis can be either a pathogen or part of the normal flora.1 Colonization with M. hominis is associated with younger age, lower socioeconomic status, multiple sexual partners, African American ethnicity, and hormonal status.1 Infection with M. hominis is more common among pregnant women.1

Mycoplasma hominis is associated with genital infections in females but not in males. Examples of infections include pelvic inflammatory disease and bacterial vaginosis.1 In addition, it is responsible for pregnancy-related infections such as chorioamnionitis and post-partum fever secondary to endometritis.1 Moreover, M. hominis is associated with infections of the newborns, meningitis among premature babies, and low birth weight among neonates.1 Lastly, M. hominis can lead to extragenital infections including spinal hardware infections, septic arthritis, retroperitoneal abscess, hematoma infection, and osteitis.1

Infections by Mycoplasma hominis are infrequent and difficult to confirm prior to the start of empiric therapy.2 Urogenital and systemic infections due to Mycoplasma hominis are treated with oral tetracycline.1 For organisms resistant to tetracycline, fluoroquinolones are recommended.1 For wound infections or abscesses, doxycycline, clindamycin, or fluoroquinolones are recommended for at least 2 weeks.1 Drainage and debridement may be necessary.1


  1. Pereyre S. et Mycoplasma hominis, M. genitalium and Ureaplasma spp.  Antimicrobe http://www.antimicrobe.org/m06.asp
  1. Baum S. Mycoplasma hominis and ureaplasma urealyticum infections. (2017, Dec. 7th).  Last retrieved on March 27, 2018 from https://www.uptodate.com/contents/mycoplasma-hominis-and-ureaplasma-urealyticum-infections


-Ting Chen, MD 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.

Managing Up For Safety

Several employee injuries over a six-month period did nothing to get the attention of the laboratory leadership. The Occupational Health nurse was nearing retirement, and she didn’t pay attention to the fact that these injuries came from the same area- the autopsy suite- and that many had a common cause. The pathologist knew that the employees were getting hurt because of bad conditions in the morgue area. The autopsy table was old and had rusted sharp edges that frequently caused cuts on the hands of those handling it. The body storage refrigerator was small, and staff members from the security department and nursing suffered back injuries from the awkward positions needed to load and unload bodies on the shelves. However, the pathologist’s complaints to the lab manager were unheeded, mainly because he complained about something different every day.

The new lab safety officer noted the lab injury reports and very quickly noticed a pattern. She interviewed the affected staff and took a look around the autopsy suite. She used her camera and took pictures of the old rusty table and the high shelves in the tiny body storage refrigerator. She tallied the cost to the facility of the accumulated injuries and placed the information in a presentation that included the photographs. She made an appointment with the hospital administrator and gave her brief presentation. Before the week was out, the lab had approved funding for updated autopsy furniture and a mechanical lift for moving bodies.

In life, each person has a specific “sphere of influence,” those things you are able to touch and on which you have an effect. It is typically a waste of time to expend energy on those things you cannot change- like a traffic jam, for instance. Stewing about that truly is a waste and accomplishes little. If your role deals with lab safety, then you do have influence on every safety issue in the department, even though it may not always seem that way.

As a lab safety professional, it can be frustrating to see safety issues go unnoticed or unattended, especially after they have been reported. The apparent roadblocks to solutions may be a lack of funds, busy or disinterested leadership, and even an overall poor culture of safety. There are steps you can take, however, which can help you move around the roadblocks and bring those unattended safety issues toward a solution.

Finances is a common hindrance to making changes in the laboratory such as remodeling a space or even getting new or improved safety equipment. Safety is always value-added, but it is important to be able to prove it to those holding the financial reins. First, tally the cost of any injuries that may have occurred due to the safety issue. That total should include any medical treatment, time off of work, the cost of replacement employees or overtime incurred, and time to make any temporary fixes and to communicate to staff. If there is a possibility of penalties or fines should the issue be noted by an outside regulatory agency, those should be considered as well. Many times, the total of the costs for the safety issue are greater than the cost of the fix. In the healthcare setting where finances are getting more attention each year, this can be a powerful tool to get things done.

If lab leadership is uninterested or too busy to help you with safety issues, there are some long-term solutions. First, make sure you act as the safety role model and build trust with peers and leadership. If your discussions with them are reasonable, and if your focus is on sensible, realistic solutions, you will have a better response than if you get angry or try to control everything. That relationship-building can be critical to your ability to influence changes when needed. If the overall safety culture in the lab is poor, you can still have a positive effect on it even without the full support of leadership. That leadership support always helps, but making positive changes can occur without it, and that also comes through being a role model and working well with the lab staff.

A successful lab safety professional develops and increases their sphere of influence over time, but it can be an uphill battle depending on the location and the other people involved. Knowing what the important issues are and when to tackle them is key, and learning that while navigating through a particular culture and organizational structure can take time. Have patience, and you will eventually be able to leverage your safety knowledge to be able to manage upward in order to create a safer laboratory.


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.

Hematopathology Case Study: A 67 Year Old Female with a Sore Throat


A 67 year old female presents with a two-month history of sore throat. She endorses dysphagia and left-sided otalgia but denies voice changes, shortness of breath, hemoptysis, weight loss, fever or night sweats. She has smoked 1 pack/day for 41 years and occasionally drinks alcohol. Her past medical history is notable for systemic lupus erythematosus for which she takes Plaquenil.

Physical examination slightly elevated systolic blood pressure. She is afebrile. Pertinent neck exam findings include mild tonsillar asymmetry (left slightly larger than right), and a firm mass at left base of tongue, and a 3 cm lymph node in the neck (left level III). A biopsy sample was taken from the tongue mass. 



H&E stained sections reveal sheets of large lymphocytes. The lymphoid cells are medium to large in size with irregular nuclear contours and prominent nuclei. Areas of necrosis are prominent. No specific areas of epithelial ulceration are noted. Immunophenotypic characterization of the larger cells reveals positivity for CD20, CD30, CD79a, PAX5, MUM1, Epstein Barr virus encoded RNA (EBER) and a variable Ki-67 proliferation index, which is up to 60-70% in the larger cells, but around 20-30% overall. Only rare cells are positive for BCL-2 and BCL-6. The lymphoma cells are negative for keratin AE1/AE3, CD10, CD4, CD8, CD21, CD23, CD7, CD5, Cyclin D1, CD68, CD56, and CD43. The background T cells express CD5 and CD7 and are a mixture of CD4 and CD8 with CD4 predominance.

We considered the diagnosis of EBV-positive mucocutaneous ulcer (a more indolent entity); however, the lack of history of an ulcer/ulceration and the presence of a mass-lesion (with additional adenopathy) does not support this diagnosis.

The findings are most consistent with EBV-positive DLBCL, NOS (WHO 2017), previously known as EBV positive DLBCL of the elderly (WHO 2008). 


Epstein Barr Virus, a member of the Herpesviridae family is mostly known for causing Infectious Mononucleosis. However, the ubiquitous virus which is present in about 90% of adults but often asymptomatic1, has a predilection for epithelial cells including B-cells.2 Incorporation of the viral genome and viral takeover of the cells proliferative machinery underlies the pathogenesis of any EBV-related disease/malignancy. It has been associated with a gastric carcinoma, fulminant hepatitis, undifferentiated nasopharyngeal carcinoma, and B cell, T cell and NK cell lymphomas3, including EBV+ diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NOS).

EBV-positive diffuse large B-cell lymphoma, not otherwise specified (EBV+ DLBCL-NOS) was formerly known as EBV-positive diffuse large B-cell lymphoma (DLBCL) of the elderly. The WHO classification substituted “not otherwise specified” in place of “for the elderly” to reflect two things: 1) EBV is associated with other specific neoplastic Large B-Cell diseases such as lymphomatoid granulomatosis, and 2) EBV+DLBCL can affect younger individuals as well as the elderly. 2

EBV+DLBCL-NOS patients may occur in nodal or extranodal sites, with up to 40% presenting with extranodal sites at least in the early stages. Patients may be asymptomatic with or without B symptoms but usually, patients present with rapidly enlarging tumors at single or multinodal sites, as well as at extranodal sites. 4

The patient’s presentation with sore throat and the finding of neck mass with EBV-positive large B-cells associated with ulcer-like necrosis raises a differential diagnosis that ranges from reactive to malignant. Table 1 shows a comparison between three differential diagnoses: EBV+DLBCL-NOS; EBV-positive mucocutaneous ulcer; and infectious mononucleosis.

Table 1. Comparison of 3 EBV-positive differentials in the head and neck

Unfortunately, there is currently no uniformly agreed standard of treatment for EBV+DLBCL which has a worse prognosis than EBV negative DLBCL.2 The standard treatment for DLBCL (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone- R-CHOP) is used but it responds poorly to treatment, with a median survival of 2 years.

Therefore, early detection by clinical suspicion and testing all DLBCL patients for EBV is very important.2 


  1. Tsuchiya S. Diagnosis of Epstein–Barr virus-associated diseases. Critical Reviews in Oncology and Hematology. 2002;44(3):227-238. https://www.sciencedirect.com/science/article/pii/S1040842802001142. doi: 10.1016/S1040-8428(02)00114-2.
  2. Murthy SL, Hitchcock MA, Endicott-Yazdani T, Watson JT, Krause JR. Epstein-barr virus–positive diffuse large B-cell lymphoma. Proceedings (Baylor University.Medical Center). 2017;30(4):443-444. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595389/.
  3. Okano, Motohiko, MD, PhD|Gross, Thomas G., MD, PhD. Acute or chronic life-threatening diseases associated with epstein-barr virus infection. American Journal of the Medical Sciences, The. 2012;343(6):483-489. https://www.clinicalkey.es/playcontent/1-s2.0-S0002962915309435. doi: 10.1097/MAJ.0b013e318236e02d.
  4. Swerdlow S, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Arber D, Hasserjian R, Le Beau M. WHO classification of tumours of haematopoietic and lymphoid tissues. 2017.
  5. Dunmire SK, Hogquist KA, Balfour HH. Infectious Mononucleosis. Current topics in microbiology and immunology. 2015;390:211-240. doi:10.1007/978-3-319-22822-8_9.


Adesola picture-small

-Adesola Akinyemi, M.D., MPH, recently earned his MPH-Health Policy and Management from New York Medical College. He plans on pursuing residency training in pathology. His interests include cytopathology, neuropathology, and health outcomes improvement through systems thinking and design.


-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.

Microbiology Case Study: A 70 Year Old Female with Bronchiectasis

Case History 

A 70 year old female presents with bronchiectasis with acute exacerbation. She is a non-smoker, although claims to have been exposed to secondhand smoke, and she has chronic sinusitis. The patient recently traveled to Savannah, Georgia where she developed a productive cough. She was prescribed doxycycline and was then sent home. She returned to the pulmonary clinic for a follow up consultation after her cough worsened.

Laboratory Identification

Image 1. Intracellular gram negative coccobacilli with polymorphonuclear cells found in the sputum smear (100x oil immersion).
Image 2. The predominant organism found in this patient’s sputum culture is growing 4+ on chocolate agar, but not growing on blood and MacConkey agars.
Image 3. Close up of chocolate agar showing 4+ growth of wet, translucent colonies.

The Gram stain and smear showed 4+ neutrophils, 4+ gram negative coccobacilli and little to no mixed respiratory flora. The following day, the culture grew 1+ respiratory flora on the blood plate, no growth on the MacConkey plate, and 4+ translucent colonies on the chocolate plate. 


The predominant organism was identified by the MALDI-TOF as Haemophilus influenzae. The Gram stain and culture findings are consistent with the MALDI-TOF identification. H. influenzae is an oxidase positive, gram negative coccobacilli known for its requirement of X (hemin) and V (NAD) factors found in chocolate agar. Because of its growth requirements, H. influenzae will not grow on MacConkey agar despite being a gram negative organism. It may be cultured on blood agar if the agar is inoculated with an organism such as Staphylococcus aureus, which can provide the V factor, while the X factor is provided by the agar itself. This phenomenon is known as satelliting. Identification of H. influenzae may also be done using a Haemophilus ID Quad plate. Each section of the plate contains varying factors and allows for Haemophilus identification to the species level based on the growth and hemolysis pattern.

H. influenzae is normal flora of mucous membranes and frequently colonizes the human oral cavity and upper respiratory tract. Commonly, H. influenzae causes pneumonia, as with our patient, bronchitis, and ear infections. However, it is also a known cause for bacterial meningitis, endocarditis, and osteomyelitis. Transmission of H. influenzae occurs through respiratory droplets so proper PPE precautions must be taken by clinicians when working with infected patients. It is important for laboratory professionals to work with the organism using proper PPE and BSL-2 practices and plating of respiratory specimens should occur in a biosafety cabinet.

Susceptibility testing is not routinely performed on isolates of H. influenzae. β-lactamase production can be determined by using nitrocefin, a chromogenic cephalosporin spot test. 


  1. Haemophilus influenzae Disease (Including Hib). (2018, February 13). Retrieved June 28, 2018, from https://www.cdc.gov/hi-disease/index.html
  2. (2012, March 15). Retrieved June 28, 2018, from https://www.cdc.gov/meningitis/lab-manual/chpt09-id-characterization-hi.html. Identification and Characterization of Haemophilus influenzae
  3. Manual of Clinical Microbiology, 11th edition



-Madaine Saguinsin, MLS (ASCP), graduated from Purdue University with a BS in Medical Laboratory Sciences and is a medical technologist at NorthShore University Health System. Her interests are microbiology and parasitology.

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois.

Next Generation Sequencing – Ion Torrent Semiconductor Sequencing

We’ve finally made it to the sequencing step of the NGS workflow. This post we will discuss the technology and process behind the Ion Torrent sequencing step. Next time, we will review the Illumina sequencing process.

When we left off, the final product of the clonal amplification had been prepared – Ion Sphere Particles (ISPs) covered in single stranded amplicons (hopefully all of the same amplicon). Next, control Ion Sphere Particles are added to the mix, along with sequencing primer, which is complimentary to one of the adapter sequences added back in library preparation. The primer is annealed to each of the amplicons on every ISP. This mixture of control ISPs and specimen ISPs is then loaded onto the chip. The size of the chip is determined by the number of bases needing to be sequenced. There are three different types of chips for the Personal Genome Machine (PGM) – 314, 316, 318 – and five different types for their GeneStudio S5 system (510, 520, 530, 540, 550), offering enough coverage for a single sample of a hotspot panel, all the way up to enough coverage for a specimen of exome sequencing. Each of the chips contains a top layer covered in tiny wells. Each well is just large enough to fit a single ISP. The ISP solution is loaded onto the chip, then flowed over it by centrifuging it in different directions, in order to attempt to get as many ISPs into wells as possible. The chip is then ready for sequencing.

Each well of the chip can be thought as of the smallest pH meter in the world. So before sequencing can be started, the instrument must be prepped (initialized) so that all of the reagents added to the chip are in the correct pH range. On the PGM, this takes approximately an hour and requires some hands-on steps and high quality 18MΩ water. On the GeneStudio S5, the reagents are added and the initialization is begun and, as long as everything works correctly, doesn’t require any other hands on time.

After the initialization is complete, the chip is loaded onto the instrument. The sequencing run is started and runs according to the plan prepared before the run. Thermo Fisher’s Ion Torrent uses semiconductor sequencing technology. Nucleotides are flowed over the chip one at a time. If the nucleotide is incorporated, a hydrogen ion is released. This release of hydrogen decreases the pH of the liquid surrounding the ISP. This pH change is then detected by the sensing layer beneath the well, where it is converted to a voltage change and is picked up by the software and recorded as that nucleotide. Let’s say two nucleotides in a row are incorporated (two G’s complementary to two C’s) – double the hydrogen is released, which results in double the signal, so the software will record two G’s in a row. The benefit of this type of technology is that it is fast – it only takes 15 seconds for each nucleotide flow, so a 200bp fragment can be sequenced in less than 3 hours.

Image courtesy of http://www.genomics.cn/en/




-Sharleen Rapp, BS, MB (ASCP)CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine.