A 25 year old female presented to the ED with a mild fever and a blister on her hand, which arose from a rat bite. She stated that she worked with different kinds of animals including rodents. The patient was discharged with clindamycin after two sets of blood cultures were drawn. Anaerobic bottles became positive 48 hours after incubation. Gram stain showed unusual gram negative bacilli (Figure 1). Rapid Multiplex PCR Blood culture identification (FilmArray BCID-2) panel was negative. Poor growth on Blood agar was observed after 48 hours of sub-culture. The organism was identified as Streptobacillus monoliformis by Bruker Biotyper MALDI-ToF.

S. monoliformis appears as pleomorphic gram negative rods, with its classic characteristics of “bulges” or swollen-rods in tangled chains and filaments (Image 1). Sodium polyanethol sulfonate (SPS) inhibits S. monoliformis growth. As aerobic blood culture bottles made by certain manufacturers contain SPS, the growth is mostly seen in anaerobic bottles devoid of SPS. In liquid broth, S. monoliformis grows as “puff balls.”

Since S. moniliformis is a fastidious organism, the Gram stain from direct smears, such as positive blood culture bottles, in the absence of growth on solid agar media provides an adequate preliminary diagnosis. In such circumstances, 16srRNA sequencing provides a definitive identification. When it grows on agar media, it takes from 2-3 days to as long as 7 days and may appear as “fried-egg” colonies.

Wild rodents as well as laboratory rats carry S. moniliformis in their upper respiratory tract. Cats or dogs preying on rodents also carry the organism. Hence, rat bite fever (RBF) typically occurs upon animal bites.

RBF is a systemic disease wherein patients present with fever, vomiting, headache, and muscle pain or joint swelling. While S. monoliformis is the only known cause of RBF reported in the United States, RBF in Asia is caused by Spirillum minus. Signs and symptoms of the RBF caused by these two organisms slightly differ in addition to the fact that S. minus cannot be cultured in vitro. Several cases of endocarditis and septic arthritis due to S. moniliformis have been reported in the United States.

The notion of rat bite fever as a rare infection is likely due to the fact that 1) S. moniliformis is not a reportable disease and 2) the challenges associated with recovery and identification of this organism from culture media. Thus its occurrence maybe underestimated in the past decades. However, the advancement in technology, such as 16srRNA sequencing and MALDI-ToF, has allowed better diagnosis of RBF in recent years. S. moniliformis is generally responsive to penicillin, cephalosporins, carbapenems, aztreonam, clindamycin, clarithromycin, and tetracycline while it may have intermediate susceptibility to aminoglycoside and fluroquinolones. Since there are no definitive minimal inhibitory concentration (MIC) breakpoint guidelines for this organism, clinical laboratories do not generally perform antimicrobial susceptibility testing.

Most laboratories are designed with eyewash stations and at least one safety shower depending on the size of the department. The use of these safety showers is not common, but it does happen, and the staff needs to be prepared for such an event. That preparation not only involves testing and training on equipment use, but also in making sure the physical space is ready for a potential deluge of water that can pour down into the department for potentially up to fifteen minutes. Other flooding incidents may occur as well. A floor drain can back up, a water line connected to an analyzer might break, or water might even come through the ceiling from a pipe above the department. Being prepared and responding efficiently to these types of flooding events should be part of the overall lab safety program.

One reason safety specialists and some regulatory agencies require that items in the lab not be stored directly on the floor is so they will not be damaged in the event of a departmental flood. It is generally acceptable to store plastic items (waste bins, etc.) on the floor since they cannot be damaged by water. Cardboard, computer hard drives, and other like items should be stored on palettes or shelves. Securing electrical wires and raising multi-plug adaptors off the floor is also a best practice.

When designing or remodeling a laboratory, consider the possibility of floods when choosing the type of flooring to be installed. The best laboratory flooring is monolithic, like a sheet vinyl that has few seams. It should bend up to the walls to create a coved base that is integral with the floor. This design (recommended by the CDC and CLSI) keeps liquids from going under tiles or through walls which will create more problems (like mold) down the road.

Floor drains where safety showers exist are not required, and many labs have showers where there is no drain at all. Remember that in a typical situation where a shower would be used, hazardous chemicals are involved. Any hazardous waste that might go into the sanitary sewer should be routed through a neutralization station or into a hazardous waste collection tank. The ANSI requirements for a safety shower include the ability to deliver 20 gallons of water per minute for 15-20 minutes. That’s a total of 400 gallons. The requirements also state that the water pattern must be at least 20” in diameter and 60” above the floor. Therefore, a majority of the water will not even travel to the drain. It will go to the lowest point of the floor in the department. The bottom line is, if the safety shower must be used, a flood should be expected.

In order for the lab to be prepared for a flood emergency, materials should be on hand that will help contain large amounts of water. Those materials may include large volume spill kits with booms or dikes that are capable of holding water back. Staff should be trained how to use these materials as spill training is provided, and drills should be conducted so they can use the supplies comfortably. Make sure these spill materials are easily accessible and that signage clearly indicates where they are stored.

What does the physical anatomy of your lab look like? Is it designed for safety in the event of a hazardous material spill or exposure? Is the department set up to handle a sudden flood situation, and can staff identify the steps to take to respond efficiently and safely? Take a look around your lab today, and make any necessary corrections so that all will be ready should a laboratory flood occur for any reason.

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

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

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

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

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

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

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

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

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

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

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

References

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