Hematology Case Study: Monocytosis in An Elderly Patient

An 81 year old presented with fatigue and not feeling too well. CBC revealed marked leukocytosis and monocytosis.

  • White cell count: 85.1 K/uL (elevated)
  • Hemoglobin: 8.6 g/dl (decreased)
  • Platelet count: 79 K/uL ( decreased)

Review of peripheral smear revealed leukoerythroblastosis with monocytosis (19.57 K/uL) along with presence of numerous immature monocytoid cells, dysplastic myeloid precursors and 7% blasts, consistent with myelodysplastic/myeloproliferative neoplasm such as chronic myelomonocytic leukemia-1.

 

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For further evaluation of disease progression and/or transformation to acute leukemia, bone marrow evaluation was recommended.

Diagnostic criteria for chronic myelomonocytic leukemia

  1. Persistent peripheral blood monocytosis >1 K/uL
  2. No Philadelphia chromosome or bcr-abl 1 fusion gene
  3. No rearrangement of PDGFRA or PDGFRB
  4. Fewer than 20% blasts in the blood or bone marrow**
  5. Dysplasia in one or more myeloid lineages.

 

**Blasts include myeloblasts, monoblasts and promonocytes

Prognosis and predictive factors

Survival of patients with CMML is reported to vary from one to more than 100 months, but the median survival time in most series is 2- to 40 months. Progression to AML occurs in approximately 15-30% of cases. A number of clinical and hematological parameters, including splenomegaly, severity of anemia and degree of leukocytosis, have been reported to be important factors in predicting the course of the disease.

 

Vajpayee,Neerja2014_small-Neerja Vajpayee, MD, is the director of Clinical Pathology at Oneida Health Center in Oneida, New York and is actively involved in signing out surgical pathology and cytology cases in a community setting. Previously, she was on the faculty at SUNY Upstate for several years ( 2002-2016) where she was involved in diagnostic work and medical student/resident teaching.

Microbiology Case Study: A Routine Sputum Culture on a 20 Year Old Cystic Fibrosis Patient

Case History

A 20 year old woman with cystic fibrosis was routinely screened with bacterial sputum cultures.  The patient reported feeling well and was compliant with her treatment regimen.  She had no history of Nocardia colonization in the past and her last hospitalization was four years prior due to a pulmonary exacerbation in which cultures grew Stenotrophomonas and Pseudomonas.

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Image 1: Modified acid fast stain showing two unique traits of Nocardia: long, delicate rods and weak acid fastness.

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Image 2: Nocardia colonies on buffered charcoal yeast extract (BCYE) agar.

Discussion

Nocardia are slow growing, aerobic gram negative rods. Nocardia are unique for being weakly acid fast and displaying aerial hyphae.  They are ubiquitous organisms that can cause a variety of infections in immunocompromised individuals. Most often in the United States, nocardiosis causes a lung infection. If left untreated, it can spread to the brain or spinal cord, where up to 44% die [1].

The above patient showed no signs of an infection, making the possibility of colonization by Nocardia more likely.  Cystic fibrosis patients can be colonized by Nocardia and the clinical approach is to treat regardless of the patient’s overall health. However, there is a lack of reporting on whether Nocardia is the cause of an infection when it happens. Host and pathogen interactions are also not well known. Thorn et al. showed that treating cystic fibrosis patients that are colonized with Nocardia with oral antibiotics did not affect their clinical outcome [2]. More studies are needed to be done to see if antibiotics are warranted in circumstances like the above patient.

References:

  1. Nocardiosis. https://www.cdc.gov/nocardiosis/transmission/index.html
  1. Pulmonary nocardiosis in cystic fibrosis. Thorn, Shannon T. et al.. Journal of Cystic Fibrosis, Volume 8 , Issue 5 , 316 – 320

 

-Angela Theiss is a pathology resident at the University of Vermont Medical Center.

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-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.

Planning Lab Testing for Medical Missions, Part 2

Last month I blogged about key points to consider when preparing to do lab testing in the field. Here I will expand on using point of care testing in medical missions. Point of care testing is easy to use and relatively easy to access, making it very attractive for use in the field or on medical missions. In fact, it is tempting to take these tests and go rogue – it’s not uncommon for point of care diagnostics to be obtained by non-laboratory professionals and tossed in luggage to be used by short-term medical teams. However, this is not in the best interest of the patients or the community. Helping establish point of care testing for medical missions is one very important way that a laboratory professional can get involved in this kind of outreach.

Proper utilization and quality assurance practices are just as critical in the outreach situation as at home in a large lab. Perhaps even more so; for example, in areas with high disease prevalence, false positives and negatives can significantly affect patient care and population health. Under-diagnosis due to false negatives means that those who need treatment might not get it, just as over-diagnosis due to false positives may cause patients to get unnecessary treatment. Unnecessary treatment, especially for infectious diseases, harms the community by contributing to drug resistance.

Most point of care tests, especially lateral flow tests, have built-in controls which lessens the need to run QCs with patient testing. However, it is important to know the limitations of the testing. Sometimes point of care testing systems that are not available in the United States are selected for use in outreach in foreign countries. It’s more likely that an American medical team would be unfamiliar with the tests. A laboratory professional can help establish or at least verify the validity of the tests, including limits of detection and accuracy, before they are deployed. Also, it is often helpful to have the results interpreted for the end user. Little interpretation is needed for the more straightforward qualitative tests that simply give a positive or negative result. Even with these tests, the limit of detection should be available to the provider, especially if this is significantly different from that which the provider is accustomed. Tests that involve titration, such as some of the rapid typhoid and syphilis testing, benefit from having an explanation of what the titers mean clinically available to the end user.

Other tests with results that are prone to confusion are point of care versions of assays more commonly performed in clinical laboratories. Difference in reference intervals for the POCT compared to a conventional test can be particularly confusing. For example, the results of a lateral flow point of care C-reactive protein assay have a different reference interval than results from high-sensitivity C-reactive protein assays used in clinical labs. Using the incorrect reference interval to determine whether a result is normal can lead to over- or under-treatment, which is contrary to the purpose of diagnostic testing. Yet, when using point of care tests in the field, there is not a neat little interpretive comment accompanying the result.

So, how can this be remedied? If the laboratory professional is also on the team, they can be available to provide information as needed. However, if the team is not so fortunate as to have their own laboratory professional, another way to provide the information is to provide a short guide to cheat sheet that briefly explains how to use test results.

Proper utility is also important, especially in areas with high burden of disease or in areas where there is no confirmatory testing. Consider rapid tests for H. pylori. These typically detect antibody to H. pylori, which can be found in up to 70% of asymptomatic populations. The rapid test is of little utility since positive results only indicate the presence of antibody and not necessarily an active infection. Consider using rapid screening tests, such as for HIV, when confirmatory testing is not available. Sometimes a second screening test that employs a different method than the first can be used as a confirmatory test if nothing else is available.

Consider environmental limitations of the testing when selecting tests for use in the field. Many tests are unreliable at extremes of temperature and humidity. This might not always be obvious even when quality controls are used properly. For example, Tang et al (1) showed that the effect of temperatures and humidity similar to what was experienced in Louisiana after Hurricane Katrina on quality control material for a POCT glucose meter system caused significantly depressed results. Also keep in mind that exposure to environmental extremes can reduce the shelf life of POCT and related reagents. If using POCT long term, it is good practice to routinely test a known standard – even on tests with built in quality controls such as the test line on lateral flow tests – to ensure there has not been degradation in quality due to the environment.

Preparing POCT for medical missions is a great way for a laboratory professional to get involved in global health and outreach. From helping to select appropriate tests, to verifying test validity, to teaching proper utilization of testing and providing interpretive guideline, the laboratory professional is a vital part of a medical mission – even if they never leave their lab!

  1. Tang CS, Ferguson WJ, Louie RF, Vy JH, Sumner SL, Kost GJ. Ensuring quality control of point-of-care technologies: effects of dynamic temperature and humidity stresses on glucose quality control solutions. Point of Care 2012;11:147-51.

 

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Sarah Riley, PhD, DABCC, is an Assistant Professor of Pediatrics and Pathology and Immunology at Washington University in St. Louis School of Medicine. She is passionate about bringing the lab out of the basement and into the forefront of global health.  

Owning Safety in the Autopsy Suite

The hospital security guard placed the deceased patient into the morgue refrigerator while chatting with his co-worker. They walked away without realizing the door did not close completely. Within the hour the automated temperature recording system sent an alert to the lab on the third floor.

The body had been unclaimed, and it stayed on the bottom shelf in the morgue. No one in the hospital wanted to take ownership of it. After a couple of months, fluids began to fill the shelf where the body was. The environmental services staff refused to clean up the mess since some staff were afraid.

The pathologist wanted to finish the autopsy quickly, so he started before the complete patient chart arrived. When the phone rang in the morgue, the physician on the other end of the phone said he believed the patient may have Creutzfeldt-Jakob Disease (CJD).

Managing safety in the autopsy suite can be difficult, but as these case studies show, it is important. One reason for the struggle is that clear ownership of the area is often not defined. Multiple internal departments and even external agencies may work in the morgue and autopsy suite. Pathologists, medical examiners, research physicians, security personnel, nurses, and organ procurement staff are just some of the various people that may perform tasks in the autopsy suite. This can create some unique and unwanted problems. The laboratory should take the lead in making sure all safety regulations are followed and that other users of the suite comply to avoid any unfortunate mishaps.

The morgue should be treated as a laboratory space, and it should be designed similarly to a BSL-3 laboratory space which includes an anteroom. Warning signs indicating the presence of biological and chemical materials should be placed on entry doors. Whenever work is performed in the area, proper personal protective equipment should be utilized. This PPE may include lab coats, gowns, gloves, respirators, and face protection. Make sure PPE is available in the area at all times. The autopsy space should be adequate, such that procedures may be performed effectively and that items such as knives and saws can be stored and used safely. Ventilation should be adequate (with a recommended minimum 12 air exchanges per hour), and the ambient temperature should be monitored as well.

While other personnel may access the morgue body storage refrigerator, it is often the lab or security departments who monitor the temperature. Since CAP inspectors set specific morgue refrigerator temperature ranges (1.1 to 4.4° Celsius), it can be important to communicate with the people who utilize the unit often. If placing or removing a body takes longer than expected, make sure there is adequate communication so that proper documentation of the temperature outages can be made. If a department other than the lab is responsible for temperature monitoring, make sure it is done correctly so there are no citations during an inspection.

Proper decontamination in the morgue is crucial. Instruments, tables, and counters must be disinfected to remove contamination of bloodborne pathogens. Use a chemical germicide for instrument and surface decontamination such as a 10-percent solution of sodium hypochlorite (or bleach). This intermediate-level disinfection will eliminate most bacteria (including Mycobacterium tuberculosis), and all fungi, and it inactivates viruses such as the hepatitis B virus. Rinsing with water or ethanol after disinfecting will help prevent the pitting of any stainless-steel surfaces.

Dealing with Creutzfeldt-Jakob Disease (CJD) in the autopsy suite requires special safety measures. Procedures should be posted in the area directing staff how to handle tissue and clean up in cases where patients are infected with CJD. The intact brain should be fixed in formaldehyde for one to two weeks before handling or cutting in order to reduce the prion activity. Non-disposable implements used with such patients should be immersed in 1N sodium hypochlorite (NaOH) for one hour before reuse. Surfaces on which autopsies occurred should also be immersed in NaOH for one hour for disinfection purposes.

Chemicals are stored and used in the autopsy suite, and standard safe lab practices should be used. Make sure staff is trained in proper the handling, labeling, and storage of chemicals as well as prepared to handle spills. Spill kits should be available and suitable to the chemicals used in the area. If formaldehyde is used, be sure an appropriate neutralizer is available for spill incidents.

As the most involved and best educated about its dangers, laboratory personnel should take the lead in making sure safety is a priority in the morgue, and educate all who may enter the area. Make sure communication is clear about who will use the suite and when- it’s never good to have someone walk in during an autopsy or organ removal. Use signage when necessary, and be willing to help in any unusual situations, because with a morgue, they definitely will arise. Work together as a team with all who utilize the area, and that ownership of safety will translate into safety for all.

 

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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.

Chemistry Case Study: Falsely Elevated Methotrexate

High dose methotrexate infusion is widely used in the treatment of malignancies such as leukemia, high risk lymphoma, and osteosarcoma. It can be associated with multiple adverse effects, especially renal toxicity, which could leads to acute kidney injury (AKI), delaying drug elimination and worsening its toxicity. Leucovorin, a reduce folic acid, is commonly used with methotrexate treatment to lessen its toxicity. After administration of methotrexate, serum creatinine and methotrexate concentration should be closely monitored. The levels of serum methotrexate to be associated with a high risk for nephrotoxicity are: 24 h, > 10 μmol/L; 48 h, > 1 μmol/L; 72 h, > 0.1 μmol/L.

In this case, the patient is a 33-yo old male with T-lymphoblastic leukemia in complete remission. He was given consolidation therapy with high dose methotrexate. Leucovorin rescue was given 24 hours after methotrexate administration. Patient’s methotrexate level was at 4.7 μmol/L 3 days postinfusion due to AKI and poor methotrexate clearance. An alternative rescue, glucarpidase (Garboxypeptidase G2), was then given to patient to rapidly lower serum methotrexate level. Glucarpidase cleaves methotrexate molecule to inactive metabolite, DAMPA (2,4-diamino-N-methylpteroic acid). After glucarpidase rescue, patient’s methotrexate level were still remained above the toxic level on the following two days (1.02 μmol/L and 0.68 μmol/L).

In most clinical laboratories, serum methotrexate is measured by immunoassays, and the inactive metabolite of methotrexate after glucarpidase rescue, DAMPA, cross-reacts with immunoassays and interferes the measurement of methotrexate. After glucarpidase treatment, patient’s methotrexate level can be falsely high for 5-7 days, before accurate measurement can be obtained using immunoassays. In this case, the concentrations of methotrexate after glucarpidase rescue were falsely high results due to DAMPA interference. There are laboratory-developed LC-MS methods to detect methotrexate. LC-MS methods are more specific and have no interference from the metabolite, can be used for accurate methotrexate measurement in the case of glucarpidase rescue.

 

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-Xin Yi, PhD, DABCC, FACB, is a board-certified clinical chemist, currently serving as the Co-director of Clinical Chemistry at Houston Methodist Hospital in Houston, TX and an Assistant Professor of Clinical Pathology and Laboratory Medicine at Weill Cornell Medical College.

Microbiology Case Study: An Elderly Man with Abdominal Pain

Case History

The lab received two sets of admission blood cultures from an elderly man with a history of atrial fibrillation, aortic valve replacement six years ago, and idiopathic pancytopenia who presented with abdominal pain and symptoms of decompensated heart failure.

Lab Identification

At 59 hours, one aerobic bottle showed growth of a gram negative bacillus. The organism grew pure yellow-pigmented colonies on the blood and chocolate plates with no growth on the MacConkey plate.

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Gram stain from blood agar plate.

The Verigene microarray was not able to identify the organism against 12 gram negative targets. The organism was identified by MALDI-TOF mass spectrometer as Brevundimonas vesicularis. The organism could not be grown for susceptibility testing.

Discussion

B. vesicularis is an aerobic, glucose non-fermenting, spore non-forming, gram negative bacillus closely related to Pseudomonas species. It will demonstrate slow growth of yellow-pigmented colonies on chocolate and blood agar plates with variable growth on MacConkey agar plates. It has in the past been classified as Corynebacterium and Pseudomonas vesicularis. It has been identified globally in environmental sources such as soil, spring water, and healthcare related water containers. It has been identified as a community-acquired, and increasing a hospital-acquired, pathogen in all age groups, and particularly in immunocompromised patients [1, 2]. It has been most commonly reported as a cause of bacteremia, but has also been implicated as the causative agent in cases of arthritis, meningitis, endocarditis, peritonitis, and urinary tract infection [1]. Interesting in vitro studies have demonstrated that the presence of B. vescularis may facilitate the growth of Legionella [3].

Even in cases of the septicemia in immunocompromised patients the mortality rate from B. vesicularis is relatively low. Strains have demonstrated resistance to most forms of antibiotics though is most consistently susceptible to amikacin and piperacillin-tazobactam [1, 2].

 

References

  1. Shang ST, Chiu SK, Chan MC, Wang NC, Yang YS, Lin JC, Chang FY. Invasive Brevundimonas vesicularis bacteremia: Two case reports and review of the literature. J Microbiol Immun Inf. (2012) 45: 468-472.
  2. Zhang CC, Hsu HJ, Li CM. Brevundimonas vesicularis bacteremia resistant to trimethoprim-sulfamethoxazole and ceftazidime in a tertiary hospital in southern Taiwan. J Microbiol Immunol Infect. (2012) 45(6): 448-452.
  3. Koide M, Higa F, Tateyama M, Cash HL, Hokama A, Fujita J. Role of Brevundimonas vesicularis in supporting the growth of Legionella in nutrient-poor environments. New Microbiol. (2014) 1:33-39.

 

-Taylor Goller is a Pathology Student Fellow at University of Vermont Medical Center.

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-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.

Jaffe vs. Enzymatic Method for Serum Creatinine Measurement

The Jaffe and enzymatic methods are the two most common methods for measuring serum creatinine. The Jaffe method is less expensive than the enzymatic assay ($0.30 vs $2.00 per test based on 2014 list prices) but is more susceptible to interferences. Although these tests are not expensive, they are high-volume tests and the savings could be substantial. We were using the enzymatic assay at the University of Utah and estimated that we could save about $50,000 per year by switching to the Jaffe assay; however, we were uncertain whether the Jaffe assay was safe to use due to the potential for interferences. For that reason, we decided to conduct a risk assessment to evaluate the suitability of the Jaffe assay.

Risk is defined as the expected cost of an action. The expected cost has two components: 1) the probability that an event will occur and 2) the consequences or cost of an event:

Risk = prob(event) x cost(event)

The event of interest was misclassification of a patient due to an error in serum creatinine measurement. Nephrologists classify kidney disease based on the estimated glomerular filtration rate which is based on the creatinine value. The distribution of eGFR for patients at our hospital is shown in Figure 1. The dashed lines show decision limits that nephrologists use to classify kidney disease. An eGFR is considered normal or healthy.

We spoke with the nephrologists and learned that they were relatively unconcerned about errors in eGFR in healthy patients (eGFR above 60 ml/min) because there was no potential for harm. Similarly, they felt there was relatively little risk of harm to patients with low eGFRs because these patients are routinely monitored and no major decision would be based solely on a single eGFR measurement. An error in creatinine measurement in a low eGFR patient would be detected by repeat measurements or be inconsistent with other measurements. From the nephrologists’ point of view, the only area of concern was in the region around 60 ml/min.  Patients about 60 ml/min are considered healthy whereas those below 60 ml/min are diagnosed with stage 3a chronic kidney disease. In this zone, an error in serum creatinine could result in a false negative (i.e. observed eGFR greater than 60 ml/min when the true eGFR was less than 60 ml/min). In such cases, a patient may go without care and their disease could progress.  The nephrologists believed that the potential for harm was relatively minor, but potential for harm did exist.

We compared the eGFRs provided by the enzymatic and Jaffee methods to estimate how often patients might be misclassified (Figure 2).1 Focusing on the 60 ml/min decision limit, we found that 17 of 500 (3.4%) of measurements were discordant. Some of these discordant results would be due to imprecision. Discordance due to imprecision would have small differences (bottom of Figure 2) and are unavoidable – they would occur using any method. Discordance due to interference would be expected to have larger differences (top of Figure 2) and could be avoided by using the enzymatic method. We used statistical techniques to estimate the proportion of discordances that were due to interference vs imprecision and found that about 60% of the discordance at the 60 ml/min limit was due to interference. In summary, our risk analysis showed that using the Jaffe method would pose about a 2% rate of avoidable misclassification which presented some potential risk to patients. The nephrologists felt the risk was low but, in theory, disease could unnecessarily progress in a patient with a false negative diagnosis.

Our risk analysis was based on analytical error. We compared magnitude of analytical error to the biological variation in eGFR and found that the analytical error was relatively small in comparison to biological variation (data not shown).  Biological variation was likely to be a more significant cause of misclassification than analytical error.

So, what to do? Was the potential savings of the Jaffe method worth the risk? Some experts recommend against using the Jaffe method. 2-4 On the other hand, most US laboratories use the Jaffe assay. A recent College of American Pathologists proficiency challenge found that 70% of the submitted results were based on the creatinine assay.5

We decided to get the best of both worlds by using BOTH methods. We defined a zone of risk surrounding the 60 ml/min eGFR decision limit (Figure 3). Results in this zone would have some risk of misclassification whereas results outside of the zone would be unlikely to be misclassified using the Jaffee method. All creatinine measurements are initially performed using the Jaffe method. If the result is outside the risk zone, the result is reported. If results fell within the risk zone, they were repeated with the enzymatic method and the results of the enzymatic method are reported. This reflex procedure saves money while avoiding risk. The reflex rate is approximately 15%.

There are circumstances in which one would want to order the best possible test. To that end, we created a special orderable test, based on the enzymatic method, that the nephrologists could use to insure the most accurate results when required. For example, the enzymatic test may be indicated when making decisions regarding biopsies for renal transplant patients. The order volume for the special test has been less than 100 orders per year. 

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Figure 1. Distribution of Estimated Glomerular Filtration rates (eGFR). The distribution is for outpatients at University of Utah for calendar year 2014. The dashed lines indicate decision limits used for classification of chronic kidney disease (15, 30, 45 and 60 ml/min). eGFRs greater than 60 ml/min are considered disease free.
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Figure 2. Discordances in estimated glomerular filtration rate (eGFR) at the 60 ml/min decision limit. The length of each arrow, represents the difference between estimates based on the Jaffe (head) and enzymatic (tail) methods. The dashed line represents two standard deviations of expected imprecision of the difference. Differences greater than 2 standard deviations would most likely be due to analytical interference (loss of specificity).
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Figure 3. Reflex test strategy. The figure shows the distribution of eGFR values for outpatients at the University of Utah.  The dashed lines represent clinical decision limits. The yellow zone represents the range of eGFR values where misclassification could pose a risk to patients. Creatinine is first measured by the Jaffe method. The Jaffe result is reported if the estimated eGFR is outside the yellow zone. If the eGFR is within the yellow zone, the measurement is repeated using the enzymatic method and the result based on the enzymatic method is reported.

References

  1. Schmidt RL, Straseski JA, Raphael KL, Adams AH, Lehman CM. A Risk Assessment of the Jaffe vs Enzymatic Method for Creatinine Measurement in an Outpatient Population. PloS one. 2015;10(11):e0143205.
  2. Cobbaert CM, Baadenhuijsen H, Weykamp CW. Prime time for enzymatic creatinine methods in pediatrics. Clinical Chemistry. 2009;55(3):549-558.
  3. Drion I, Cobbaert C, Groenier KH, et al. Clinical evaluation of analytical variations in serum creatinine measurements: Why laboratories should abandon Jaffe techniques. BMC Nephrology. 2012;13(1).
  4. Panteghini M. Enzymatic assays for creatinine: time for action. Scand J Clin Lab Invest Suppl. 2008;241:84-88.
  5. College of American Pathologists. Chemistry/Therapeutic Monitoring, Participant Survey. 2014.

 

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-Robert Schmidt, MD, PhD, MBA, MS is currently an Associate Professor at the University of Utah where he is Medical Director of the clinical laboratory at the Huntsman Cancer Institute and Director of the Center for Effective Medical Testing at ARUP Laboratories.