Lablogatory

Are We Creating Wisdom After Y2K?

We all remember the panic and drama over the centennial turn of the century? Well, here we are in the middle of the second month of the fifteenth year of this “new millennium.” Who could imagine we’d be this far into the future this fast?

As the world turns, we balance on the precipice of our “seasoned and experienced” laboratory professionals reinventing, repurposing, redefining their careers, or just moving on to enjoy life after laboratory service—and leaving the bench at a rate that looks like a diabetic insulin spike. We are also experiencing a surge of new laboratory interest, (thanks, CSI!) and it’s refreshing to see students eager to learn and practice in our labs and specialty departments. But there is a “gap” in the middle, a desert of years when schools closed, students went into nursing or pharmacy or radiology instead of laboratory science, and we didn’t “feel” it because we were in the prime years of our careers. This “gap” is very soon going to appear on the horizon and it’s looking a bit like the Olduvai Gorge…deep and wide with not too many ways to cross unscathed.

It is with urgency that those of us still active in the field begin to engage, mentor, sponsor and grow new laboratory professionals around the world. Seek out that student you don’t know yet, take that youngest tech to lunch, make friends with a student/young tech from another country, take a turn at teaching and training (yep, without the stipend or salary bonus—just do it because someone did it for you a long time ago, and I’m guessing they weren’t compensated either…) I’d like to challenge everyone to leave some knowledge behind as you take the next step on life’s path. There are so many ways to do it, find a way to give a little bit back, help complete the circle.

Just remember…the next generation of medical technologists and laboratory scientists are going to be performing tests and releasing results for us one of these days. Wouldn’t it be great if they were as good as I want them to be when they’re doing my CBC and chemistries, and cross matching that unit I hope I don’t need during surgery? I’ll be crossing my fingers, and counting on the fact that YOU were one of their mentors!

Here’s to the next 15 years in our Y2K world! Cheers!

–Beverly Sumwalt, MA, DLM, CLS, MT(ASCP) is an ASCP Global Outreach Volunteer Consultant.

The ABCs of Vitamin D

Vitamin D is produced from 7-dehydrocholesterol in the skin when the skin is UV-irradiated by sunlight. In humans, vitamin D3 or cholecalciferol is specifically produced. Plants produce predominately vitamin D2 or ergocalciferol. While human bodies can utilize vitamin D2, they preferentially use D3 and the rest of this post will be talking about D3.

Vitamin D is actually more of a steroid hormone than a vitamin. Unlike vitamins, Vitamin D is produced in the body, and like hormones, it is produced in skin cells and acts on cells at sites distant from its site of production. The primary functions of vitamin D include actions to increase blood calcium levels. Low blood calcium levels cause a release of parathyroid hormone (PTH), which in turn activates vitamin D. Vitamin D then actively increases calcium absorption from the intestine and helps mobilize calcium from the bone. Vitamin D has been studied and associated with health benefits ranging from decreasing the risks of getting various types of cancer to lowering the risks of heart attacks and type 1 diabetes, causing it’s measurement to become an almost routine part of most physicals and resulting in a large testing volume in the lab.

In the body, Vitamin D exists in multiple forms. The vitamin D produced in the skin is hydroxylated in the liver to give 25-hydroxy-vitamin D (25-OH-D), the main circulating form of Vitamin D. This form is not biologically active. When the correct physiological signals are received however (low calcium and high PTH), another hydroxyl group is added to 25-OH-D in the kidneys, to form the biologically active form, 1,25-dihydroxy-vitamin D (1,25-diOH-D). 1,25-diOH-D is present in very low concentrations.

25-OH-D is the form measured when assessing a person’s overall vitamin D status. It is in the greatest concentration in the body and has a half-life of 2 to 3 weeks. It can be measured by a variety of immunoassays as well as by tandem mass spectrometry. Unfortunately not all assays measure the same forms of 25-OH-D, and thus values can differ significantly depending on the assay used to measure them. This is a major problem because vitamin D has health-based reference intervals, not population based. This means that studies have determined that 25-OH-D concentrations below 30 µg/dL suggest vitamin D deficiency. So all assays use this 30 µg/dL cut-off, even though all assays don’t measure the same amount of vitamin D in samples. Cholesterol is another example of an analyte with health-based reference intervals. We say a person’s cholesterol should not exceed 200 mg/dL, rather than establishing the population-based reference intervals for cholesterol for our population.

1,25-diOH-D is much more difficult to measure because it occurs in much lower concentrations, with a half-life in the body of 4 to 6 hours. It is generally only measured when renal function is impaired, or to check for diseases involving vitamin D metabolism. It is often ordered in error when the healthcare provider actually wants to know the patients overall vitamin D status, the 25-OH-D concentration. Assays for measuring 1,25-diOH-D include radioimmunoassays and extraction followed by liquid chromatography-tandem mass spectrometry. This testing is usually performed in reference labs.

-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.

Microbiology Case Study–Back and Flank Pain in a Transplant Patient

Clinical Summary:

59 year old male with a history of acute myeloid leukemia, status post allogeneic bone marrow transplant complicated by graft-versus-host disease and relapse presented to the ED complaining of flank and back pain. His work-up identified multiple pulmonary lesions, thought to be infarcts and a left upper extremity deep vein thrombosis. He was started on cefepime for neutropenic fever and lovenox for the emboli. His symptoms worsened and imaging revealed progressive lesions in the lung that were concerning for invasive aspergillosis. A biopsy of one of the lesions was non-diagnostic but an aspergillus serum antigen test was positive. He was started on voriconazole. He remained hospitalized and began showing improvement, but then again developed a febrile neutropenia and became hypotensive with a decreased hemoglobin level. A CT of the abdomen showed typhlitis in the cecum and possible liver phlegmons, and a CT of the chest showed bilateral pulmonary effusions. He went into respiratory failure and passed away. An autopsy was performed and they sent lung tissue for fungal cultures.

Microbiology:

Plates:

Potato flake agar shows a brown, rapid growing mold that was raising the lid of the plate. No growth on the Mycosel plate.

Scotch-tape prep shows ribbon-like hyphae with few septations.

Temperature Studies:

The mold grew at both 37° and 42°C.

Discussion:

The above findings lead to the classification of a Zygomycete which are hyaline, pauciseptate molds that include Rhizopus, Mucor, Absidia, Rhizomucor, Synecephalastrum, Cunninghamella, and others.

The colonies are fluffy, white to gray or brown. They are rapid-growers and diffusely cover the agar within 24-96 hours. The hyphae appear to be coarse and fill the entire culture dish with loose, grayish hyphae dotted with brown or black sporangia. It is not possible to differentiate the organisms based on colony morphology. Temperature studies can differentiate between some species: Rhizopus grows best between 40-50°C; Rhizomucor grows best around 38-58°C; Mucor grows best at less than 37°C; Absidia grows between 45-50°. Since our specimen grew at both 37° and 42°C, that would lead us to have Rhizomucor high on our differential.

Zygomycetes produce large, ribbon-like hyphae that are irregular in diameter and contain occasional septae. Classification of specific organisms relies on identifying the characteristic saclike fruiting structures called sporangia. The sporangia produce sporangiospores, which are within the sporangia and are spherical and yellow or brown. Each sporangium is formed at the tip of a sporangiophore which is a supporting structure. The sporangiophores are connected by hyphae with occasional septations called stolons. These are contact points where rootlike structures called rhizoids attach to the hyphae. The presence and location of the rhizoids helps to identify the organism. Rhizopus has unbranched sporangiophores with rhizoids at their base where the stolon arises. Mucor has singularly produced or branched sporangiophores that have round sporangium filled with sporangiospores at their tips. It does not have rhizoids or stolons. Absidia has rhizoids that are between sporangiophores, and the sporangia are pyriform and have a funnel-shaped area called apophysis at the junction of the sporangium and the sporangiophore. Usually a septum is formed in the sporangiophore just below the sporangium. Our microscopic exam did not show any rhizoids at first, but a second exam after a longer growth period showed potential rhizoids at the base of the sporangiophores, which would lead us to have Rhizopus on our differential. Our case did not clearly define itself at the species level, so it was signed out as a Zygomycete and there were no treatment implications.

Zygomycetes are not a common cause of infection, but are an important cause of morbidity and mortality in patients who are immunocompromised. They have a worldwide distribution and are commonly found on decaying vegetable matter, soil, or old bread. Infection occurs by inhalation of spores, and once established, it is rapidly progressive, particularly in patients with diabetes mellitus who have infections that involve the sinuses. The organisms have a propensity for vascular invasion and rapidly produce thrombosis and necrosis of tissue. A common presentation is invasion within the nasal mucosa, palate, sinuses, orbit, face, and brain showing massive necrosis with vascular invasion and infarction. Perineural invasion can also occur which can spread retro-orbitally into the brain. They can also infect the lungs and GI tract as well as have disseminated infection. They can cause skin infections in patients who have severe burns and infections of subcutaneous tissue of patients who have undergone surgery.

Follow up:

Lung tissue, area of necrosis; H&E stain, 10x

Lung tissue, vasculature; H&E stain, 40x

Lung tissue, vasculature; silver stain, 10x

Lung tissue vasculature; silver stain, 40x

Lung tissue, areas of necrosis; silver stain, 10x

The histology on H&E stain shows areas of necrosis with faint septate hyphae as well as broad, ribbon-like hyphae within the vasculature. The silver stain nicely highlights the broad hyphae which we can identify as a zygomycete. The silver stain also accentuated the massive amounts of thinner hyphae with parallel walls and 45 degree branching which is consistent with aspergillus. This patient was found to have both an aspergillus infection which caused the positive serum antigen test, but then also developed a zygomycete infection which led to his death. We did not identify aspergillus on our fungal culture which may be explained by several possibilities. Our patient had been treated with voriconazole for a potential aspergillus infection which may make it more difficult for the aspergillus to grow on fungal culture. Zygomycetes are rapid growers which could have inhibited the growth of another organism or could have inhibited our ability to identify a second organism growing on the plate.

Kirsten Threlkeld, MD is a 4^th 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 Assistant Professor at the University of Vermont.

CAP Inspections and the Resident

Hello, fellow blog readers! It’s about 4 weeks since we communicated last. Since my first half of the year was loaded with lighter CP rotations to allow me to complete interviews for two successive fellowships, this half of the year is surgpath heavy and so that’s why I took a short hiatus from posting.

Well, I have a 4 week reprieve before I have another surgpath rotation and I am on what we refer to as our “comprehensive CP” rotation. Basically, it’s a combination chemistry-microbiology rotation. Since both of those rotations don’t always have enough work to require a resident to remain at the hospital for the usual 8-5 schedule, we cover both rotations simultaneously. We also have 11 comp CP rotations throughout the 4 years at my new program which is quite a lot but after the initial 2 months of “wet lab” rotating through all the stations in the chemistry and microbiology labs, we have the flexibility to tailor our comp CP rotation. And so, right now, I write as I sit in a hotel in Baltimore about to meet for our third preparation meeting before my attending and I go inspect a new molecular genetic pathology laboratory for the College of American Pathologists tomorrow. Since this is this lab’s first inspection, unlike the usual CAP inspection, this one is announced – they know we are coming and can prepare for our visit. The two of us will complete the entire inspection; my program counts this as rotation duties even though I am off-campus.

This is the second CAP inspection that I’ve been asked to assist with since I transferred to my program as a PGY-3. I think it’s great that my program gives our residents this opportunity since as attendings (whether we are AP or CP), we will also have to either assist in or enforce adherence to CAP or other accreditation standards and supervise preparations for lab inspections every other year and self-inspections on the alternate off-years. At my program, residents assist in both the preparations for CAP and off-year inspections. I’ve said it before, but residency is the transition from passive learning to active learning where we should participate in the daily responsibilities that our attendings oversee and that we will have in the future.

So, this inspection will be much more work than when I inspected the chemistry and special chemistry sections with my last team. Since there are only two of us, we are responsible for splitting the duties for the lab director, general, common, and molecular pathology accreditation checklists. CAP suggests a “ROAD” approach: read (through their binders of policies, SOP’s, etc), observe (a sample from receipt in the lab and though processing and interpretation of results), ask (open ended questions), and discover.

Well, I guess it’s time for me to go inspect but before I leave, I’d like to encourage all trainees (residents and fellows) to apply to serve as a junior member on one of the CAP’s committees or councils. You need to be a junior member but membership is free as a resident. Each committee or council (that oversees multiple committees in a topic area) usually has one junior member on it, very rarely, two. I’m currently serving my second year as the junior member on the Council on Education and I can say it has been a very rewarding experience where I have met many role models who definitely take an interest in what I have to say about the trainee opinion and who also think of me when opportunities arise that they think might be good for me. You can access both the instructions to apply (which includes a list of the committees/councils with junior member positions opening up in 2016) and the junior member application here – you will need a letter of recommendation from your program director and email in your app before the deadline of March 31^st. Good luck guys! If you have any questions, feel free to email me.

-Betty Chung, DO, MPH, MA is a third year resident physician at Rutgers – Robert Wood Johnson University Hospital in New Brunswick, NJ.

Microfluidics + Consumer Electronics = The Future of Point of Care Testing?

In a paper published yesterday in Science Translational Medicine, researchers tested a low-cost smartphone attachment (a “dongle”) that detects the presence of HIV and syphilis antibodies using ELIZA technology. While the research took place in Rwanda and highlights the usefulness of such technology in low-resource settings, the implications are potentially far-reaching in terms of point-of-care and direct-to-consumer testing.

What do you think? Do you think using smartphone attachments could replace full laboratories in the not-so-distant future?

–Kelly Swails, MT(ASCP), is a laboratory professional, recovering microbiologist, and web editor for Lab Medicine.

Pancytopenia in a 67-Year-Old Female

A 67-year-old female presents with pancytopenia, a markedly enlarged spleen, and extramedullary hematopoiesis. Her blood smear is shown here. She is found to have a JAK-2 mutation. What is the diagnosis?

Chronic myelofibrosis
Chronic myeloid leukemia
Hairy cell leukemia
Metastatic breast carcinoma
Renal cell carcinoma

The diagnosis in this case is chronic myelofibrosis. Chronic myelofibrosis is one of the four main chronic myeloproliferative disorders (the others are chronic myeloid leukemia, essential thrombocythemia, and polycythemia vera). In this disorder, the bone marrow is initially hypercellular, with proliferation of all of the myeloid cell lines (neutrophils, red cells, and megakaryoblasts). Over time, however, the marrow becomes progressively fibrotic. Eventually, there is not enough room for normal hematopoiesis, and the body starts making hematopoietic cells elsewhere (most notably in the spleen, which becomes markedly enlarged).

In these later stages of chronic myelofibrosis, the blood is characterized by pancytopenia (a decrease in white cells, red cells and platelets). Teardrop-shaped red cells (dacryocytes) may also be seen as a result of the red cells wending their way through a fibrotic marrow. Red cell precursors, such as the normoblast present in this image, are also commonly present, as there is less and less room for red cells to mature fully before leaving the marrow.

These blood smear findings are not specific for chronic myelofibrosis. Teardrop-shaped red cells may be seen when the marrow is fibrotic for other reasons, such as metastatic cancer, and pancytopenia and normoblasts may be seen in many other conditions. The JAK-2 mutation, however, is seen most frequently in three of the four chronic myeloproliferative disorders: polycythemia vera, essential thrombocythemia, and chronic myelofibrosis.

-Kristine Krafts, MD, is an Assistant Professor of Pathology at the University of Minnesota School of Medicine and School of Dentistry and the founder of the educational website Pathology Student.

New Study Suggests No Difference in Mortality Rate in Two Different Transfusion Ratios

From the study published in the Journal of the American Medicine Association: “Among patients with severe trauma and major bleeding, early administration of plasma, platelets, and red blood cells in a 1:1:1 ratio compared with a 1:1:2 ratio did not result in significant differences in mortality at 24 hours or at 30 days. However, more patients in the 1:1:1 group achieved hemostasis and fewer experienced death due to exsanguination by 24 hours.”

You can read the NIH press release here.

You can read the abstract for the study here.

Doctor, There’s a Snake in My Poop

Case history:
A mother brought her 5 year old son into his pediatrician with a “snake” that she found in his stool. Otherwise, the patient was completely asymptomatic.

Specimen sent for ID:

Ascaris is the most common Helminth affecting humans. It is also the largest of the roundworms (nematodes), growing up to 35 cm in length. There are several species of Ascaris however only A. lumbricoides affects humans.

Epidemiology
Ascaris occurs in areas with poor sanitation, hygiene and solid waste practices. Worldwide it is most common in tropical and subtropical areas.

Transmission & Life Cycle
Ascaris is transmitted through the fecal-oral route, and is therefore most prevalent in areas with poor sanitation and waste practices. Transmission occurs when an uninfected person swallows a fertilized egg which was originally passed through the stool of an infected person.

The adult male and female forms live in the small intestine. The female passes up to 200,000 eggs per day. If there is also a male worm living in the small intestine, these eggs may then be fertilized. Both fertilized and unfertilized eggs are eventually passed in the stool.

Unfertilized eggs are not infective and do not cause disease if ingested. Fertilized eggs are only infective after approximately three weeks of maturation. The exact amount of time required before the fertilized egg becomes infective will depend on environmental conditions, such as temperature and humidity.

Once ingested, the fertilized egg travels to the small intestine, where it hatches and becomes a larva. The larval form invades the small intestinal mucosal wall and enters the bloodstream. Upon reaching the lung, the larva invades the capillary and alveolar walls and continues to grow within the alveoli. After about two weeks of maturation, the larva then migrates up through the airspaces and into the trachea, where it is eventually swallowed and transported back down into the small intestine. The fully mature larval forms are now adult worms and will continue to live in the small intestine for the rest of their lifespan (up to 1 to 2 years).

Laboratory Diagnosis
In most cases, Ascaris is diagnosed in the egg form on ova and parasite exam. The fertilized egg is round, 45-70 microns in diameter and has a thick, mammillated outer wall which stains brown with bile. The unfertilized egg is larger (90 microns), are has a more oval shape with a less regular mammillated contour. Patients may also pass adult worms in the stool or less commonly they may cough them up through the mouth. The adult worms have tapered ends with a three-lipped mouth (“tricuspid” mouth). The female is larger than the male (female: 20-35 cm, male: 15-30 cm).

Clinical Symptoms
Clinically, most people affected with Ascaris are asymptomatic. With a very high worm load however patients may begin to develop complications related to obstruction, including abdominal pain, constipation, appendicitis and obstructive cholangitis. In younger children infection with Ascaris may result in stunted growth. Of note, immune reaction to larva in the lung may result in an eosinophilic immunologic response known as Löffler’s pneumonitis.

Treatment
Ascaris is treated with anthelminthic medication (albendazole, mebendazole or ivermectin). Therapy for Ascaris extremely effective and rids the patient of all adult, larval and egg forms.

-Javier De Luca-Johnson, MD is a 2^nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

–Christi Wojewoda, MD, is certified by the American Board of Pathology in AP/CP and Medical Microbiology. She is currently the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.

Something for Nothing

I’m probably going to be a little bit on a soap box with this post, but this is something that is bothering me. It’s about the society we live in and how it’s becoming more and more of a “something for nothing” society. We expect to get things without having to pay for them. In fact we’re so used to it that we even get angry when someone asks for payment. Let me give you some examples:

How many times have you gone to Wikipedia to look for an answer to a question? Or any other site on the Internet for that matter? And how many times have you donated any funds at all to the upkeep and maintenance of that site? Guess what. It costs money to maintain a website.

How many times have you downloaded a song off the Internet without paying for it in any way? People spend money to record songs and money to make movies. Where does the money come from to allow them to continue doing those things if nobody pays for the ones already made?

The reason this is bothering me is in relation to our professional associations. Even here, people want to receive benefits without paying for a membership – something for nothing. Our professional associations are worth supporting. They offer us educational opportunities, networking opportunities and a host of other benefits. All of these things cost our associations money to produce and provide. And even big associations cannot afford to continue eating the costs without eventually being financially unable to continue. You might be surprised to know how much of any given board meeting for your association is spent discussing staying financially viable.

Nobody, including your professional associations, can stay in business if they cannot make enough of a profit margin to survive, basically if they give away too much for free. I think it’s time for us to stop expecting everything to be handed to us without needing to give anything in return. So go for it. Donate to Wikipedia, buy your CDs, blue-rays and downloads, and join your professional societies. You will not regret it.

CLSI Publishes New Guide for Laboratory-Developed Tests

From the press release:

“The Clinical and Laboratory Standards Institute (CLSI) has published Quality System Regulation for Laboratory-Developed Tests: A Practical Guide for the Laboratory.This guide converts the requirement complexities of US Food and Drug Administration (FDA) regulations into plain language, offering intuitive assistance on how to conform to the Quality System Regulation (QSReg), 21 CFR 820, when creating laboratory-developed tests (LDTs). This CLSI practical guide can help laboratorians learn how to address the new demands, beyond the Clinical Laboratory Improvement Amendments (CLIA) regulations, within their unique laboratory settings.

“LDTs are in vitro diagnostic devices that are intended for clinical use and are designed, manufactured, and used within a single laboratory. This practical guide is intended to clarify how to implement the QSReg that may be required for some classifications of LDTs. On October 3, 2014, the FDA issued draft guidance for regulating LDTs that includes notification or registration of LDTs with the FDA, reporting adverse events, and other requirements. This document only addresses the QSReg that is currently applicable to manufacturers and is expected to become applicable for some classifications of LDTs when the final guidance is published.”

To purchase this guide for your laboratory, visit the CLSI website.