Hematology Case Study: Thrombocytopenia in a 50 Year Old Male

A 50 year old male patient receiving chemotherapy for treatment of gastric cancer presented to ER. Labs reported: 

WBC = 5.4 x 103/μL

Hgb = 8.9 g/dL

PLT (impedance) = 26 x 103/μL

PLT-F (fluorescent) = 9 x 103/μL

IPF = 21%

The hemoglobin was consistent with the patient history. Flags on the original impedance platelet count included thrombocytopenia, platelet clumps and platelet abnormal distribution. The sample was checked for clots, with no clots found. A fluorescent platelet count (PLT-F) was reflexed and the critical platelet count was called to the ER physician. The high immature platelet fraction (IPF%) indicates increased platelet production. Despite the increased production, the patient still had a severe thrombocytopenia. This would suggest thrombocytopenia caused by platelet destruction or consumption. Examination of the blood smear showed the presence of moderate numbers of schistocytes.

Image 1. Schistocytes seen on peripheral blood smear

Additional labs were ordered. BUN and Creatinine were slightly elevated. PTINR and APTT were within normal range. LDH was markedly increased. The physician was able to use this information, along with the clinical presentation and history, to diagnose Thrombotic Thrombocytopenic Purpura (TTP). Plasma exchanges were initiated. The patient expired 3 days later.

The difference between the impedance platelet count and the fluorescent platelet count in this patient is actually related to the presence of schistocytes. With thrombocytopenia, platelet counts can be less reliable than with normal counts. Automated platelet counts were originally performed by impedance methods, then better accuracy and precision was obtained with optical platelet counts. Physicians rely on precision with very low platelet counts to make informed decisions about treatment. The problem with the impedance counts at the low end is that RBC fragments, schistocytes and microcytic RBCs can be counted as platelets, giving a falsely high count, as we see in this case. On the other hand, measuring platelets by size (optical) can miss large platelets leading to a falsely low count. The PLT-F is more reliable because it uses a platelet specific dye which eliminates these interferences. The fluorescent dye labels the RNA. Forward scatter is used to determine size while fluorescence is used to measure RNA content. With gating set based on cell volume and RNA content, the PLT-F can be measured. When there is an abnormal scattergram or a low platelet count, the PLT-F is reflexed and the IPF% is also reported.

The Immature platelet fraction (IPF) can also be used to help understand the etiology and aid in diagnosis. Historically, the MPV has been used as an indirect marker for platelet production. However, an inherent problem with the MPV is that, similarly to the impedance platelet count, this count can be unreliable because any RBC fragments or particles may interfere with the measurement. Reticulated or immature platelets are the youngest platelets, within 24 hours of being released from the bone marrow. Measurement of these is a concept that first emerged in the late 1960s, before automated hematology analyzers performed platelet counts. Thus, the original method was staining with new methylene blue and manually counting, much like a manual reticulocyte count. These manual methods tend to be tedious and imprecise. In the last 20 yeas we have developed flow cytometry methods for performing a reticulated platelet count. Reticulocytes are stained with Thiazole Orange and passed through a flow cytometer. Unfortunately, there is no standardization for the procedure as there are variations in dye concertation, timing and gate settings. As well, this method is also time consuming, labor intensive, costly, and requires highly trained technologists to perform.

Newer flow cytometry methods to count these youngest platelets are available on Sysmex and Abbott CELL-DYN analyzers. The IPF (Sysmex) or RetPLT(Abbott) can be performed along with the routine CBC with no additional sample or time required. Knowing the reticulated or immature platelet fraction can help physicians to differentiate pathogenesis. A decreased percent of newly formed platelets may indicate that thrombocytopenia is caused by deficient platelet production, as seen in bone marrow failure. Increased circulating immature platelets with a low platelet count may suggest that the bone marrow is making adequate platelets and the thrombocytopenia is caused by platelet destruction or consumption. Treatment for these scenarios is different, and the physician must determine the etiology in order to determine treatment

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathic hemolytic anemia with thrombocytopenia and organ failure caused by microvascular thrombosis. Platelets clump in the small blood vessels and cause the low platelet count. The hemolytic anemia causes schistocytes which can be seen on the peripheral blood smear. In this case, the low platelet count and high IPF, schistocytes on the smear and the patient presentation were all important factors that led to a speedy diagnosis and start of therapy.

Plasma exchange is the treatment of choice for TTP. With the advent of therapeutic plasma exchange, mortality from TTP has decreased from about 90% to 10-20%. In patients who have relapses or become refractory, vincristine has been used successfully as an adjunct to plasma exchange.4 The exact etiology of TTP is unknown. It can be secondary TTP, often triggered by chemotherapy drugs, or can be sporadic. Sporadic, or idiopathic, TTP is now thought to be associated with an acquired autoimmune deficiency of a plasma metalloprotease named ADAMTS13. The ADAMTS13 gene controls this enzyme, which is involved in blood clotting. In acquired TTP, the ADAMTS13 gene isn’t faulty. Instead, the body makes antibodies that block the activity of the ADAMTS13 enzyme. In these cases, a lack of activity in the ADAMTS13 leads to TTP. Almost all cases of recurrent TTP have severe ADAMTS13 deficiency. These patients benefit from immunosuppressive therapy with vincristine along with plasma exchange.

However, despite the decreased mortality seen with plasma exchange, patients with cancer, infections, transplant patients, or those receiving certain drug therapy have a much worse prognosis.4 In this case study, this was this patient’s first episode of TTP and he was undergoing chemotherapy for gastric cancer. The patient’s unfortunate outcome is most likely linked to this finding.

References

  1. Arshi Naz et al. Importance of Immature platelet Fraction as a predictor of immune thrombocytopenic purpura. Pak J Med Sci 2016 Vol 32 No 3:575-579
  2. Johannes J. M. L. Hoffmann, Nicole M. A. van den Broek, and Joyce Curvers (2013) Reference Intervals of Reticulated Platelets and Other Platelet Parameters and Their Associations. Archives of Pathology & Laboratory Medicine: November 2013, Vol. 137, No. 11, pp. 1635-1640.
  3. M Meintker, Lisa & Haimerl, Maria & Ringwald, Juergen & Krause, Stefan. (2013). Measurement of immature platelets with Abbott CD-Sapphire and Sysmex XE-5000 in haematology and oncology patients.
  4. J. Evan Sadler, Joel L. Moake, Toshiyuki Miyata, James N. George Clinical chemistry and laboratory medicine : CCLM / FESCC. 51. 1-7. 10.1515/cclm-2013-0252.; Recent Advances in Thrombotic Thrombocytopenic Purpura. Hematology Am Soc Hematol Educ Program 2004; 2004 (1): 407–423. doi: https://doi.org/10.1182/asheducation-2004.1.407
  5. Sysmex White Paper. The role of the Immature Platelet Fraction(IPF) in the differential diagnosis of thrombocytopenia. www.sysmex.com/us

-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 60 Year Old with Non-Healing Wound

Clinical history

A 60 year old patient with a past medical history of type II diabetes mellitus, right Charcot foot, and cirrhosis presented to the emergency department with altered mental status and several days of subjective fevers, as well as a 2 month history of right lateral malleolar non-healing ulcer which had subacutely increased in size and volume of drainage.

The patient’s spouse reported the wound had been showing purulent discharge for 3 weeks. Nine days before presentation, the patient had seen a foot and ankle specialist for evaluation of his Charcot foot and the ulcer. Radiographs were taken at this time, but no treatment was initiated. Worsening of the wound was associated with an episode of long travel, after which the patient reported being able to see bone.

Infectious disease noted that the patient had a pet corgi.

On exam, the patient was febrile with dry mucous membranes and oriented only to person. MRI showed evidence of possible osteomyelitis. The patient subsequently underwent a right below the knee amputation.

Laboratory findings

Gram smear of a sample taken from the patient’s ankle wound in the emergency department showed many neutrophils, moderate gram positive cocci and moderate gram negative bacilli, with intraleukocytic organisms seen. Growth was observed on blood and chocolate agar plates, but there was no growth on the MacConkey plate. The organisms were identified as few Pasteurella multocida, few vancomycin resistant Enterococcus faecalis, and few usual skin flora.

Image 1. Gram stain of the sample taken from the patient’s ankle wound.

Blood cultures drawn in the emergency department were positive at 10 hours in both bottles, and again on planting showed growth on blood and chocolate agar, but no growth on MacConkey. The organism was identified as P. multocida, consistent with that which grew from the ankle wound culture.

The patient underwent a right below the knee amputation, and anaerobic cultures taken from the right foot again grew P. multocida.

Discussion

Pasteurella multocida is a nonmotile gram negative bacillus which is part of the normal oropharyngeal flora in domestic dogs and cats. It is a facultative anaerobe, positive for oxidase, catalase, and indole. It grows on chocolate and blood agar, forming small, gray, non-hemolytic colonies. It does not typically grow on MacConkey agar.

P. multocida is classically associated with a zoonotic soft tissue infection in humans who suffer bite wounds from a pet, as well as licking of any broken skin by a pet. These infections have a characteristic rapid onset and intense inflammatory response, and can progress to necrotizing fasciitis. Cases of Pasteurella osteomyelitis can be associated with significant wound infections. Conditions such as diabetes, liver dysfunction, and organ transplantation can predispose patients to Pasteurella bacteremia.

Pasteurella spp. are susceptible to beta-lactam antibiotics in most cases, and since Pasteurella wound infections are usually polymicrobial, recommended treatment is broad-spectrum such as amoxicillin-clavulanate. In isolated Pasteurella infections, first line treatment is penicillin, although there are some that favor testing isolates from sterile sites for the presence of beta-lactamase production, and treating those infections with ampicillin-sulbactam, pipercillin-tazobactam, or ceftriaxone. (Weber)

References

  1. Weber, David J., and Sheldon L. Kaplan. “Pasteurella infections.” UpToDate, Wolters Kluwer, 15 June 2018, http://www.uptodate.com/contents/pasteurella-infections?search=pasteurella%20treatment&source=search_result&selectedTitle=1~25&usage_type=default&display_rank=1#H14. Accessed 4 Feb. 2020.

-Tom Koster, 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.

Respiratory Protection in the Days of the Novel Coronavirus

In the peak of the flu season we might see many people wearing masks in physician offices and hospitals. In the news today, as the 2019 Novel Coronavirus (SARS-CoV-2) continues to spread, we see many images of people wearing different types of respirators, some are N95 respirators and others are surgical masks. Not all experts agree on the efficacy of these pieces of personal protective equipment in the face of viruses, but if you’re going to use them, it is important to know how, when and why.

OSHA’s Respiratory Protection standard (1910.134) provides information about requirements for staff who may potentially be exposed to airborne pathogens. These requirements include specific instructions for choosing the proper respirator, for providing fit-testing, and for user training. The College of American Pathologists (CAP) also expects labs to determine the risk of airborne pathogen exposure for each employee, and they require labs to have a plan which outlines engineering and work practice controls that reduce exposure potential.

The purpose of a respirator is to protect the employee from contaminated or oxygen-deficient air. Therefore, two classes of respirators are common; air-purifying respirators which use filters to remove contaminants from the air you breathe, and atmosphere-supplying respirators which provide clean air from an uncontaminated source. These types of respirators can also be classified further as tight-fitting or loose-fitting.  Tight-fitting respirators need a tight seal between the respirator and the face and/or neck of the user in order to work properly. For now, let’s focus on the air-purifying respirators which are in high demand these days as a potential 2019-nCoV pandemic looms.

In the laboratory, N95 respirators are probably the most commonly-used respirators, often used for protection against tuberculosis and other airborne pathogens. These respirators filter out 95% of airborne pathogens that are 0.3 microns or larger. While the exact size of the 2019-nCoV is not yet known, most coronaviruses are slightly larger than 0.1 microns. Does that mean a N95 respirator (recommended by the CDC) will not offer protection from the coronavirus? Not necessarily.

According to biosafety expert Sean Kaufman (www.saferbehaviors.com), the filter in the N95 respirator works three ways- through interception, impaction, and diffusion. Interception collects larger particles which are blocked by mask fibers, and impaction collects larger particles which have too much inertia to be moved around the filter fibers. Diffusion occurs as smaller particles are bombarded with larger air molecules and are pushed against filter fibers. Most of the bacteria or virus particles are removed from the airstream making the respirator quite useful and protective (HEPA filters on a Biological Safety Cabinet work in much the same way).

Employees who may need to wear a tight-fitting respirator as part of their job are required to have fit-testing every year. This is required by OSHA, and contracted employees (such as pathologists) should be fit-tested as well. Employees who may need such respirators would be those who work in microbiology labs, cytology techs who participate in patient procedures, and others. Labs should perform a risk assessment for each job category to determine the type and level of potential harmful airborne exposure.

Procedure masks, such as those handed out when people suspect they have the flu, are not technically considered respirators. Often, the person who is sick will wear these masks in order to prevent the spread of droplets when coughing or sneezing. They can protect others in the area, but they do not protect the user from harmful airborne pathogens or vapors.

Can these surgical masks be useful for the healthy public when a coronavirus is present? Sean Kaufman says “yes. If you wear a surgical mask in a potentially contaminated environment (on a commuter bus, for example),” Kaufman says, “it can keep you from touching your nose or mouth- two major routes of entry for viruses. Behaviorally speaking, these masks do offer some protection.”

Knowing when and why you use a respirator is vital, but knowing how to use it is important as well. Tight-fitting respirators should never be used without fit-testing to make sure the correct size is being used. Otherwise, the protection offered will be limited. Make sure your staff is properly trained and protected to work in environments where the air is not safe to breathe, and teach others about the usefulness of respirators when the flu and other viruses are lurking!

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.

Forget DNA Sequencing, Protein Sequencing is Here!

Every so often I have the distinct pleasure of hearing of a new technology that I’d never imagined before. This last week, a study in Nature Biotechnology described using nanopore to perform protein sequencing. Thinking of the difficulty and complexity of sequencing just 4 nucleotides, it’s hard to conceive of the exponential difficulty to sequence 20 amino acids.

The technology is still in a nascent period working on the ability to differentiate 20 amino acids- some with very similar sizes and charges. However, as has been the case with many technologies, the speed of advancement can increase rapidly. The possibilities of this technology are very exciting.

Figure 1. Aerolysin structure from CyroEM. This pore comes from Aeromonas hydrophila, a Gram-negative bacterium associated with diarrheal diseases and deep wound infections.

Nanopores have been used mostly for sequencing DNA. Pacific Biosciences have created a platform that reads long sequences of DNA by synthesizing DNA from a single strand moving through a pore. Another company, Oxford Nanopore has made several devices that use nanopore technology to perform sequencing in portable devices. The key to most of these technologies involve functionalizing the pores to have properties desirable for specific purposes.

For protein sequencing, amino acids need to move through the pores slowly enough to have their charges accurately measured. To accomplish this goal, the French start-up, DreamPore, used a bacterial derived cytolytic pore called aerolysin. Aerolysin was found to slow down the passage of amino acids as it has a single molecule trap, which binds to single amino acids with carrier cations. This method detects 13 of the20 amino acids. Additional chemical modifications, instrumentation advances, and nanopore imaging allowed two additional amino acids to be detected. The last few were too similar in charge to be differentiated. However, this study provides a path forward where it is conceivable to sequence each of the 20 amino acids.

In the future, detecting post-translational modifications such as glycosylation and phosphorylation will be an important advance. It will be fun to follow the progress of this technology to see how it might be applied clinically.

References

  1. Iacovache I, De Carlo S, Diaz Nuria et al. Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process. Nature Communications 2016. 7:12062.
  2. Ouldali H, Sarthak K, Ensslen T et al. Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore. Nature Biotechnology 2020; 38: 176-181

-Jeff SoRelle, MD is a Chief Resident of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and improving genetic variant interpretation.

Microbiology Case study: A 42 Year Old Male with Bacteremia

Case History

A 42 year old male with past medical history of diabetes mellitus and essential hypertension presented to the emergency department with high fever and chills which developed two days prior. His examination revealed a painful ulcer on the planter aspect of his right toe with surrounding erythema. According to patient, the exact duration of the ulcer is unclear as it was on planter aspect of his foot and he does not inspect his feet regularly. However, the ulcer grew in size and symptoms over the past week. He denies any cough, diarrhea or abdominal pain. He is on oral anti-diabetics with well-controlled blood sugar. Complete blood count revealed leukocytosis. Blood was collected and sent to microbiology laboratory for gram stain and culture.

Laboratory identification

Cultures signaled positive after 32 hours of incubation and gram negative rods were identified on Gram stain (Image 1). The organism grew after 24 hours of incubation on 5% sheep blood, chocolate, and MacConkey agars (Images 2 & 3). MALDI-TOF mass spectrometry identified the isolate as Salmonella spp. The isolate was sent to the public health department for additional testing by molecular typing where it was identified as Salmonella enterica subsp. enterica serovar Brandenburg (Salmonella Brandenburg). Later, MRI revealed osteomyelitis of right third toe which was considered as the likely source of patient’s bacteremia.

Image 1. Gram stain of positive blood culture broth showing gram negative rods
Image 2. Non-lactose fermenting colonies growing on MacConkey agar
Image 3. Colonies producing hydrogen sulfide making them appear black on Hektoen enteric agar

Discussion

Salmonella is a genus of the family Enterobacteriaceae in the order Enterobacterales. They are non-spore forming gram negative facultative anaerobes. Salmonella spp. are lactose non-fermenters and usually produce H2S on triple sugar iron and Hektoen enteric agar.

The genus has only two species: Salmonella enterica, divided into 6 subspecies and containing over 2500 serovars, and Salmonella bongori. Subspecies and serotype determination is necessary for epidemiological investigations. Serotyping is used to classify Salmonella based on bacterial surface antigens; the thermostable polysaccharide cell wall or somatic (“O”) antigens and the thermo-labile flagella proteins or “H” antigens. It is also possible to identify Salmonella serotypes on the basis of phage typing, plasmid profiling, ribotyping and pulsed field gel electrophoresis (PFGE) of DNA fragments generated from restriction enzyme digestion.

Salmonella are zoonotic bacteria that can cause abortion, metritis, and systemic illness in ewes and does. Natural reservoirs of Salmonella are domestic and wild animals, including poultry, swine, cattle, birds, dogs, rodents, tortoises, turtles and cats. Humans also serve as a natural host. The most common source of transmission of Salmonella is the consumption of contaminated poultry and meat products. Person-to-person, fecal–oral transmission does occur and has been a problem in health care facilities traced to inadequate hand washing.

Salmonella brandenburg ranked 16th among the serovars responsible for human infections. It causes acute diarrhea and severe illness in a variety of animals and was first isolated in New Zealand in 1986. Since 1996 Salmonella Brandenburg has been associated with an emerging epidemic of abortions and deaths in sheep in the southern regions of the South Island. Subsequently, the same strain was reported to cause disease in horses, goats, deer, pigs and humans. The disease is known to have high morbidity and mortality within a flock or herd, rapid local spread and an occupational, health and safety risk to farm workers and their families.

There are three clinically distinguishable forms of salmonellosis in humans. These include gastroenteritis, enteric fever and septicemia. Established Salmonella bacteremia requires aggressive antimicrobial treatment with ciprofloxacin, ceftriaxone, or less frequently trimethoprim-sulfamethoxazole. A careful search for focal metastatic disease should be undertaken, especially when relapse follows cessation of treatment. Surgical drainage of metastatic abscesses may be required, with surgical intervention. Resistance to any of the drugs used to treat invasive infection may occur, so treatment should be supported by susceptibility testing.

In the case of our patient, he was treated with ceftriaxone and underwent toe amputation. The patient had an uncomplicated hospital course and made a complete recovery.

References[EMT1] 

  1. Alvseike O., Skjerve E. (2000). Probability of detection of Salmonella using different analytical procedures, with emphasis on subspecies diarizonae serovar 61:k:1,5,(7) [S. IIIb 61:k:1,5,(7)]. International Journal of Food Microbiology, 58, 49-58.
  2. Clark G, Swanney S, Nicol C, and and Fenwick S. Salmonella Brandenburg – the 1999 Season. Proceedings of the Sheep and Beef Cattle Society of the New Zealand Veterinary Association, 151-156, 2000
  3. Bailey K.M. (1997). Sheep abortion outbreak associated with Salmonella Brandenburg. Surveillance, 24(4), 10-11
  4. Baumler A.J., Tsolis R.M., Heffron F. (2000). Virulence Mechanisms of Salmonella and their Genetic Basis. In “Salmonella in Domestic Animals” (Ed. C. Wray and A. Wray). CAB International 2000, pp. 57-72

-Ansa Mehreen, MD. 1st year AP/CP resident at University of Chicago hospital program based at Evanston Hospital. Her academic interests include gastrointestinal pathology.

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois. Follow Dr. McElvania on twitter @E-McElvania. 

EUA for 2019-nCoV Test

On February 4th, the FDA announced an Emergency Use Authorization for the CDC’s 2019 Novel Coronavirus real-time RT-PCR Diagnostic Panel. Here’s the press release:

Audience: Clinical Laboratory Professionals

Subject: Laboratory Update: Information about Emergency Use Authorization for  2019 Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel

Level: Laboratory Update

This message is to ensure that clinical laboratories are aware that CDC has developed a new laboratory test kit called the CDC’s 2019 Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, for use in testing patient respiratory specimens for 2019-nCoV. On February 4, 2020, the Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) to enable emergency use of the test kit in the United States. All EUA documents are available on the FDA website.

The test kit will be available for ordering today from the International Reagent Resource (IRR). Formerly, U.S. diagnostic testing for 2019-nCoV was only being conducted at CDC; however, the FDA EUA and distribution of the tests will allow 2019-nCoV testing to take place at laboratories designated by CDC.  This includes U.S. state and local public health laboratories and Department of Defense (DoD) laboratories.

Clinical laboratories should NOT attempt viral isolation from specimens collected from 2019-nCoV persons under investigation (PUIs). For interim guidelines for collecting, handling, and testing clinical specimens from PUIs for 2019-nCoV, please see the CDC 2019 Novel Coronavirus website

The FDA website lists current EUA assays, and also includes a link to terminated EUA assays. Each pathogen-specific EUA includes the device-specific Letter of Authorization, fact sheets, and manufacturer instructions/package inserts. These documents are updated when amendments are made (e.g., additional specimen types, extraction methods, procedural clarifications), so check the website routinely to ensure your laboratory staff members have the most up-to-date information.

Additional Resources

If you have any questions, please contact LOCS@cdc.gov.