July 2015 – Lablogatory

Five Things to Know … Granulocyte Transfusions

This is the first in series of “5 Things to Know” updates on various Transfusion Medicine topics, posted on Lablogatory and the Blood Bank Guy website. Today, we will cover a topic that is a mystery to many: Granulocyte transfusions!

1. We aren’t totally sure that granulocytes actually work! In the nearly half-century that granulocyte concentrates have been transfused, numerous retrospective articles, case reports, and small series about granulocytes have been published. If you ask Hematology-Oncology docs, you will likely find a general consensus that granulocytes can be effective (including a really cool, almost miraculous-sounding story!). Sadly, stories are not evidence. A study begun in 2008 called The “RING study” (Resolving Infection in people with Neutropenia with Granulocytes) was designed to gather evidence by randomizing recipients into either a group that received granulocytes or one that did not. Unfortunately, the study failed to gather enough participants before closing in 2013 to be truly meaningful (though the data that was gathered did not show a substantial beneficial effect, according to data presented at the 2014 American Society of Hematology Annual Meeting).

So, does this mean that granulocyte transfusions are stupid? Not from my perspective! Honestly, if the standard for producing a product is proof positive that the product is effective, we blood bankers would have NOTHING on our shelves! Some of my colleagues disagree with me, but from my perspective, granulocytes have value in some limited settings (outlined below).

2. Modern granulocyte collections are a multiple day process. Historically in the United States, granulocytes were collected by apheresis either from “walk-in” unstimulated donors (more common) or from donors given a single dose of oral steroids to increase their white blood cell count. The unstimulated collections resulted in a product with a total granulocyte count of about 1.0 x 10¹⁰ (that number is the minimum requirement in at least 75% of collections, according to AABB Standards, and is a GREAT number to remember if you are studying for an exam!). However, if we have learned anything about granulocytes, we know that 1.0 x 10¹⁰ just isn’t enough! A “modern” U.S. granulocyte collection is from an apheresis donor stimulated the day before collection with an injection of Granulocyte Colony Stimulating Factor (“G-CSF”), in many cases supplemented by an oral dose of steroids (typically dexamethasone); this regimen typically results in a yield of 4.0 x 10¹⁰ granulocytes or more (please note that G-CSF is not FDA-approved for use in stimulating donors, so donors should have a formal informed consent prior to undergoing stimulation). As an aside, granulocytes may be produced in different ways outside of the US. The United Kingdom, in particular, does not permit G-CSF or steroid stimulation of granulocyte donors that are not family or friends of the patient. As a result, non-family member UK granulocyte products are produced from pooled buffy coats from ten random donors, with a yield of approximately 1.0 x 10¹⁰ granulocytes. These collections require lots of extra effort by numerous people (including our donors!), which leads us to number three…

3. Granulocytes should only be ordered in specific clinical settings Granulocyte collections are not easy, and they should not be ordered carelessly. While there is no universally agreed-upon set of conditions, granulocytes are most commonly utilized in one of the following clinical situations:

Patients with hematologic malignancies and low neutrophil counts due to chemotherapy
Stem cell transplant recipients during pancytopenic phase
Neonates with sepsis
Patients with chronic granulomatous disease

This is not to say that everyone in one of those situations will or should receive granulocytes. Instead, we are looking for some specific clinical and laboratory findings before agreeing to start the granulocyte collection process, especially in the first three groups of patients on the list above. Here’s what we like to see:

Proven or highly probable bacterial or fungal infection (NOTE: Available data suggests granulocytes work better with bacterial infections)
No response to appropriate antimicrobial therapy
Absolute neutropenia (<500 granulocytes/microliter)
A reasonable expectation that the patient will begin producing granulocytes soon

Traditionally, blood bankers have strongly resisted “prophylactic” granulocyte transfusions (for immunosuppressed patients without current evidence of infection but at high risk of acquiring one) or use in fever of unknown origin. There has been some debate about this recently, but most still believe (as do I) that granulocytes should only be given for patients who have an infection.

4. Granulocytes look funny, and have unusual storage, matching, and modification requirements. If you have never seen a granulocyte product, you might guess that granulocyte and platelet concentrates look alike, since both cells reside in the “buffy coat” and both are collected in the US primarily by apheresis technology. Your guess would be wrong! Apheresis granulocyte units typically contain between 30 and 50 mL of RBCs, because it is essentially impossible to get a good granulocyte yield without harvesting some RBCs as well (they are immediately adjacent to each other by density separation in the apheresis machine). 30-50 mL sounds like a small amount, but that quantity makes the granulocyte product look almost as “red” as a unit of RBCs! (see images below)

Notice how the same 30 mL that makes the bottom of the bag look red during collection (on left) makes the whole bag look REALLY red after mixing!

Granulocytes have pretty much the shortest shelf life of any product that we collect directly from donors. They should be transfused “as soon as possible” after collection, but definitely within 24 hours of collection. They are stored at room temperature, like platelets, but they should not be continuously agitated, unlike platelets. This out-of-the-ordinary storage is really another reason that granulocytes are only collected on an “as-needed” basis. Granulocytes are also unusual in that they are almost always issued prior to the availability of the infectious disease screening results! For that reason, essentially all unrelated apheresis granulocyte donors will be recent apheresis platelet donors who have had negative results on a donation within the previous 30 days. We still perform the testing, of course, but the results just aren’t available before the product has to be transfused. Because there are so many RBCs in each granulocyte product, the donor must be ABO compatible with the recipient, and the unit must be crossmatch-compatible with the recipient. By AABB Standards, if more than 2 mL of RBCs are present in any product, those RBCs must be compatible with the recipient’s plasma antibodies. In addition, if the recipient has an unexpected RBC antibody (like anti-D, anti-K, or any others), the granulocyte unit must come from someone who is negative for that antigen, as well. To make matters even more challenging, if the patient has developed anti-HLA antibodies (most commonly in previously pregnant females), then the donor should be HLA matched or at least HLA compatible with the patient’s antibodies. Finally, to complete the weirdness, it is essential to remember which modifications CAN and CAN’T be done to granulocytes. First, granulocytes CAN (and must) be irradiated to prevent Transfusion-associated Graft vs Host Disease (TA-GVHD). This is an extremely fresh product, full of highly active T-lymphocytes in addition to the granulocytes, and the recipient is immunocompromised, by definition. As a result, irradiation is essential to deactivate those donor T-lymphocytes and protect the patient (see image below). Second, granulocytes CAN’T be leukocyte-reduced. Every now and then, people will ask me about leukoreducing granulocytes for prevention of Cytomegalovirus (CMV) transmission. Rather than make fun of them, I usually just sit quietly while they work it out themselves (“OK, let’s see: Granulocytes are WBCs, which some people call ‘leukocytes,’ so if I leukocyte-reduce a unit of a product that is primarily composed of leukocytes, I would be left with…nothing! OHHHH!”). As a result of our inability to make a granulocyte product “CMV-safe” by leukoreduction, CMV-seronegative donors will be recruited to provide products for CMV-seronegative patients whenever possible. Note that it is totally fine to run granulocyte concentrate through a “standard” transfusion filter, just not a leukoreduction or microaggregate filter.

Irradiation is not only OK for granulocyte concentrate, it is essential! Leukocyte reduction, on the other hand, makes no sense for granulocytes, and shouldn’t be done.

5. Granulocytes very commonly cause reactions in recipients. Granulocytes are famous for causing transfusion reactions much more often than other components. The two issues that are seen most often are fever and chills (without hemolysis) and pulmonary reactions. I won’t discuss the self-explanatory fever and chills issue, but the pulmonary reactions are worth mentioning here. Considering that current thought suggests that neutrophils are the primary cell involved in the pathogenesis of Transfusion-related Acute Lung Injury (TRALI), it is not surprising that granulocyte transfusions are thought to cause pulmonary compromise quite often (5% in one study). Granulocytes LOVE the lungs, and they localize there very quickly (especially when the infection being treated is pneumonia). So, even if full-blown TRALI does not occur (which certainly CAN happen!), granulocyte transfusions are notorious for at least transient pulmonary compromise. Clinicians should be prepared to manage dypsnea and hypoxia during granulocyte transfusion. Note that the previously suggested “link” between more pulmonary reactions when granulocytes were given with the antifungal Amphotericin B seems to be disproven (though many still advise avoiding transfusing granulocytes within a few hours of administration of that medication). As mentioned in item 4, TA-GVHD is a risk for a fresh product, so irradiation is required. Further, HLA alloummunization can certainly occur as a result of granulocyte transfusion. Final thoughts: There is no question to me that granulocytes CAN, in some circumstances, appear to make a difference for patients. Unfortunately, there’s not a lot of objective proof that they are a predictably helpful treatment. However, given our current ability to collect granulocyte products with substantially higher yields than in years past, I expect that interest in this product will continue despite the lack of “proof” of its effectiveness.

I hope that this quick “5 Things to Know” has been useful for you!

-Joe Chaffin, MD, is the new Vice President and Chief Medical Officer for LifeStream, a Southern California blood center headquartered in San Bernardino, CA. He has a long history of innovative educational efforts and is most widely known as the founder and chief author of “The Blood Bank Guy” website (www.bbguy.org).

The Law of Unintended Consequences

Has the guidelines that encourage less Pap testing over the course of a woman’s life contributed to less women being screened for STDs such as Chlamydia? According to a study published this week, yes. The cohort is rather small–3000 teenagers and young women–but even so, the results are striking. Before 2009, 30 percent of patients were screened for Chlamydia; after 2009, only 1 percent were.

While this is discouraging, the CDC found that teenagers are having less sex than they were twenty-five years ago, which some attribute to the HPV vaccine and its accompanying education.

Further reading on the relationship between HPV vaccines, Pap screening, and STD testing:

Chicago Tribune

NPR

Healthday

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

Mass Transfusion Protocol: A Poll

The Aftermath of Ebola

The Ebola outbreak may no longer make front page news in the United States and Liberia may have been declared Ebola free in May, but the consequences of the outbreak are still ever present, and will be for years to come.

The first reported Ebola case was in December 2013 in Guinea. By June of 2014 the outbreak had rapidly spread and the rest of the world was taking note. In May 2015 Liberia was declared Ebola free. Currently, the situation in Guinea and Sierra Leone is improving and each country is now able (from a health care standpoint) to isolate and treat current patients. However, new cases are still being reported in both countries so continued vigilance is paramount.

As the disease itself abates and it is possible to see the light at the end of the tunnel, it is clear that Ebola may be (nearly) gone, but its effects will linger for decades and it has left devastation in its wake.

The CDC estimates that, in the hardest hit countries of Guinea, Liberia, and Sierra Leone, the death toll from Ebola cases is: Guinea: 2,509, Liberia: 4,806, Sierra Leone: 3,947. The total number of cases is hard to track but estimates of total cases, including those that are suspected, probable, and confirmed are as high as 3,784 in Guinea, 10,666 in Liberia, and 13,241 in Sierra Leone (numbers are as of July 17, 2015).

The socio economic consequences of the outbreak are numerous. Much of the day-to-day workings of the economy ground to a halt as people stayed home or fled jobs in factories, mines, and fields. Panicked investors fled as the disease began to spread. Disruption during the agricultural season caused diminished agricultural yields which had both economic consequences and resulted in severe food insecurity across the region. Internal and regional trade were dramatically affected due to boarder closures and movement restrictions to help staunch the transmission of the disease. Those who survived the outbreak are now left to pick of the pieces of their daily lives and learn to live without loved ones. Parents were left without children, children were left without parents. The courage it would take to find jobs, housing, a support system after watching loved ones die (and possibly being sick oneself) is hard to fathom.

A portion of the damage left in Ebola’s wake includes health care systems and infrastructure. Already weak to begin with, Ebola wreaked havoc on the human and infrastructural health care resources in all three countries. The Economist reports that an estimated 509 health care workers died in Guinea, Liberia, and Sierra Leone. This is an enormous loss of human resources in countries where, before the outbreak, the average doctor to patient ratio was less than 10 doctors per 100,000 people. Looking to the future, one of the biggest health care concerns is the number of children who went unvaccinated during the height of the outbreak. Because of this, vaccine-preventable diseases, such as measles and polio, will contribute, indirectly, to Ebola’s death toll.

To end on a positive note, however, the local and international communities are now left with an opportunity: to remake the systems better than they were before. In many places physical infrastructure, human resources, and other necessary systems will be starting anew. With the right expertise and resources, using best practices and with cooperation among international agencies, private donors, and local governments, among others, health care can be built stronger than before with an eye toward preventing such devastating outbreaks in the future and caring for the day-to-day health needs of the local populations.

For further reading:

-Marie Levy spent over five years working at American Society for Clinical Pathology in the Global Outreach department.

Markers of Inflammation

I thought today I’d do a little discussion related to two of the more non-specific, questionably useful tests that we have in the laboratory test arsenal, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), and their use as markers of inflammation. I’ve left out procalcitonin on purpose since I’ve posted about that inflammatory marker previously. And I won’t discuss hs-CRP and its use in cardiovascular disease risk assessment.

CRP and ESR are referred to as inflammatory markers because both rise when inflammation is present. However neither marker provides much more information other than the presence of inflammation. That leads to the questions: how are these tests useful, and why do we need both?

Good questions! First though, what exactly are these markers? Both of these markers will increase in inflammation, infection and tissue destruction, but at different speeds and to different degrees. CRP is a protein and an acute phase reactant. It is produced by the liver and released in response to inflammatory cytokines, usually within hours of a tissue injury, an infection, or any other cause of inflammation. ESR on the other hand isn’t any kind of analyte at all, but rather a measure of the ability of the red cells to settle out in a blood sample. This settling is affected by the fibrinogen and globulin concentration in the blood as well as by the red cell concentration and how normal the red cells are. Thus besides inflammation, things like anemia, polycythemia and sickle cell disease also will affect an ESR. ESR also increases in malignancies, especially paraproteinemias and other states with abnormal serum proteins, and in autoimmune diseases. ESR elevations are used to support the diagnosis of specific inflammatory diseases, like systemic vasculitis and polymyalgia rheumatic. CRP is useful for monitoring patients after surgery and since it rises rapidly in response to bacterial sepsis, it is often used to monitor response to antimicrobial therapy. Considering the differences in these two “markers” it’s perhaps not surprising that they do not correlate well when compared against each other. Nor is it surprising that the lab has been unable to retire either test.

The pattern of usage for these tests in my lab has shifted in the last several years. In 2007 we ran almost equal numbers of both tests, about 500 per month of each. Eighty percent of the time, both tests were ordered simultaneously. Of those, 20 percent had one normal result and one abnormal result, 50 percent were both abnormal and 30 percent were both normal. Surprisingly, 50 percent of the time, only one single CRP and ESR was ordered, even though these tests are probably more useful when used to trend response and the majority of the time, one or both results were abnormal. This year in 2015 we are running about 1400 CRP per month and 900 ESR per month, and still 70 percent of those ESRs that we do run, are run simultaneously with CRP samples. The same services tend to order both tests, with many of the orders coming from GI, the ED, Orthopedics, Rheumatology, Oncology or Infectious Diseases. CRP tends to be ordered more frequently by general hospitalists, intensivists and Cardiology. ESR orders are STAT 30 percent of the time, while CRP orders are STAT about 22 percent of the time.

Both of these analytes are markers for the presence of an inflammatory process. CRP seems to reflect bacterial or septic processes and response to therapy to a better degree than ESR does, probably because CRP is one of the liver’s acute phase proteins and reflects liver response to injury. CRP also tends to respond more quickly than ESR, rising faster and then falling more rapidly. ESR on the other hand tends to reflect a more systemic response. With either analyte, a one-time order is a snap-shot in time. Thus often one of these markers is normal while the other is abnormal, which may explain why physicians tend to order both. Ordering either analyte as a one-time order will only tell you that inflammation is present, and the results of the tests must always be used in conjunction with other tests and clinical signs and symptoms in order to have any diagnostic efficacy. Sequential CRP or ESR samples allow for trending and helping to determine response to therapy, thus providing more useful information.

-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.

CLSI Publishes New and Revised Standards on POC Testing

From the press release:

“CLSI recently released new and revised standards on point-of-care testing in relation to glucose measuring and monitoring. Effects of Different Sample Types on Glucose Measurements, 1st Edition (POCT06-Ed1), provides information to assist the clinical and point-of-care staff in result and measurement procedure comparisons of glucose tests. Glucose Monitoring in Settings Without Laboratory Support, 3rd Edition (POCT13-Ed3), focuses on performance of point-of-care glucose monitoring systems, with an emphasis on safety practices, quality control, training, and administrative responsibility.“

These documents, including their sample pages, can be found on the CLSI Shop.

Microbiology Case Study: 22 Year Old Female with Joint Pain

A 22 year old female with no significant past medical history presented with a fever, joint pain and a petechial rash. She endorsed having cold/flu symptoms for two weeks prior. The patient was admitted to the hospital where blood cultures were drawn and antibiotics were initiated. One set of blood cultures from the patient flagged positive at 14 hours of incubation with the following gram stain and colony morphology.

Gram stain showing Gram negative diplococci.

Chocolate agar plate with small gray, slightly mucoid colonies. Photo courtesy of pixgood.com — Chocolate agar plate with small gray, slightly mucoid colonies.
*Photo courtesy of pixgood.com*

Laboratory identification:

The patient’s blood was cultured on aerobic blood agar and chocolate agar plates. The gram stain revealed gram negative diplococci. Medium sized, round, gray to white, slightly mucoid colonies grew on blood and chocolate agars. The organism was definitively identified as Neisseria meningitidis by VITEK-MS. Prior to adoption of mass spectrometry, biochemical tests were performed for further characterization of the organism. Neisseria meningitidis is catalase positive, ferments glucose and maltose but not lactose, is oxidase positive, and does not reduce nitrate.

Discussion:

Neisseria meningitidis asymptomatically colonizes the oropharynx and nasopharynx of humans. It is transmitted by person-to-person spread of contaminated respiratory droplets. Infection causes a spectrum of disease including life-threatening meningitis. Bacteremia causes the characteristic petechial rash, thrombocytopenia, DIC, and shock. The organism may also cause conjunctivitis, pneumonia, and sinusitis. Its virulence factors include surface structures to facilitate attachment to and invasion of epithelial cells. Once the organism gains access to the vascular system, its survival is mediated by the polysaccharide capsule. Endotoxin release mediates many of the systemic manifestations of infection such as shock.

The differential diagnosis for this organism based on the gram stain and colony morphology includes Neisseria gonorrhoeae and Moraxella species. Different Neisseria species can be identified by the sugars they are able to ferment. For example, N. gonorrhoeae ferments only glucose, but N. meningitidis ferments both glucose and maltose.

There is a vaccine that is available for N. meningitidis that includes serogroups A, C, W-135, and Y. There are 12 different serogroups that can be distinguished based on the polysaccharide capsule. Our patient had been fully vaccinated. The isolate was sent to the state public health lab and it was reported back as non-typable and was sent to the CDC.

Treatment of N. meningitidis consists of supportive therapy for shock plus antimicrobial therapy with penicillin, ceftriaxone, or cefotaxime.

***Rare, fatal cases of meningococcal disease have been reported in laboratory staff. Any potential N. meningitidis should be worked with under a class II biological safety cabinet.

-Lauren Pearson, D.O. is a 2^nd 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.

New Technology for Transfusion Medicine

Late last year, the FDA approved Cerus’s INTERCEPT blood system for platelets and plasma. This system reduces the risk of transmitting blood-borne pathogens through platelets and plasma. INTERCEPT accomplishes this by inserting molecules into the DNA of pathogens that makes them incapable of replicating. While the pathogen still exists, it can’t replicate, and therefore can’t cause disease in the recipient. This is useful for well-known agents such as HIV or hepatitis as well as emerging diseases such as Chikungunya. Since it targets DNA, it would also neutralize undiscovered pathogens.

One potential downside of using this system is potentially increasing the cost of blood products for hospitals. Also, there is a bit of risk involved with being the so-called “first kid on the block” when using any new technology. While the FDA approval process is rigorous, unforeseen complications can arise with widespread use. Since the FDA approval, several blood centers—Delmarva, SunCoast, and Bonfils—have signed agreements to use this this system to ensure the functional sterility of their blood products. It will be interesting to see how widely this new technology is adopted and if blood products are made safer than with current methodologies.

If you’d like to read papers about this technology, you can find them here.

Reconsidering Mass Transfusion Protocols

In a new article exclusive to the Lab Medicine website, Gregory et al discuss mass transfusion protocols and argue against the 1:1:1 (1 unit each of platelets, plasma, and packed red blood cells) dogma. You can follow this link to read the paper.

What do you think? Is it time to reevaluate mass transfusion protocols?

What’s That Interference?

I’ve heard it said that there is no such thing as a lab test with no interferences, and I have to admit, I believe that to be true. For every method devised to measure a specific analyte, something else can interfere with that measurement. For example, photometric measurements using absorbance assume that only the analyte of interest absorbs light at the wavelength being used. Quite often, many other compounds absorb light at that wavelength as well. In chromatography methods, we assume only the compound of interest elutes from the column at a specific time point, and again, many other compounds often do. Various types of mass spectrometry are touted as specific for the compounds being measured, however, even using mass spectrometry, compounds may fragment in similar patterns when looking at mass spectra, or fragment into the same size precursor and/or product fragments using tandem MS.

Thus, we routinely report test results knowing that most often what we are reporting is accurate. However, we must always be aware that the result we’re reporting may not be accurate due to interferences.

I recently had an occurrence related to test interference. Like all such cases, the tech responding to the clinician’s call used our standard response. He located the original sample and repeated the test. The assay gave the same results on the repeat and the result was reported back to the clinician as real and accurate, even when questions were raised by the healthcare staff about the result not fitting the clinical picture. And in fact, although the result was reproducible and in the realm of possibility, in this case the result was wrong.

The analyte in this case was plasma free hemoglobin which is performed in our lab by an assay which measures absorbance at one of the wavelengths at which hemoglobin absorbs light and subtracts a background wavelength reading. The test was persistently giving very high plasma free hemoglobin results even though the patient had no other evidence of hemolysis. When the healthcare staff became adamant about the discrepancy, the sample was sent to an outside lab which performs the assay using a full spectrophotometer, and the sample was found to have no hemoglobin present. An interferent in this patient’s sample was being measured as hemoglobin by our method.

Of course, once it’s been determined that a test is experiencing interference the next question from the healthcare provider is always, what is interfering? That’s a much more difficult question to answer, although occasionally it can be answered with some investigation. Looking into the patient’s drug regimen can help, as well as checking other health parameters to see what else is occurring. In the case of the elevated plasma free hemoglobin, the patient did have an elevated myoglobin which may have interfered.

The take home message here is that no matter how reproducible the results are, interferences are possible. As laboratory professionals, we should always be ready to look for ways to prove our results other than by repeating them, especially when the result does not fit the clinical picture and is being questioned by our healthcare colleagues. Sending the test to be run by a different method is one good way of determining interference. Another way is to check the patient’s chart for drugs or other substances that are known to interfere and are listed in the package insert. Finally, understanding the realities of assay interferences, and being willing to continue looking for answers is also important in the laboratory.