Tips for Performing Internal Lab Audits

In previous blog posts we discussed some hints and tips for how to survive when your lab is being inspected. Today we get to flip things around and let you be the inspector. Whether it’s an internal audit of your own laboratory, or an external inspection of a peer laboratory, we’ll discuss some ways to help keep you on track to cover the most important aspects of the overall testing process in a limited amount of time.

For external audit preparation, the CAP has a wonderful training program that all volunteer inspectors are required to take prior to participating in an inspection. For labs that are not CAP accredited, they still have helpful information on their website that is free and open to all: https://www.cap.org/laboratory-improvement/accreditation/inspector-training. CLSI document QMS15-A (Assessments: Laboratory Internal Audit Program; Approved Guideline) is another great resource to use when planning your audit.

The primary role of an auditor is to review policies, processes, and procedures to identify any inconsistencies (does your SOP match the manufacturer recommendations, and is staff following the SOP as written). Audits should focus on collecting objective evidence and facts, rather than subjective opinions. For example, staff failing to document required weekly maintenance tasks, as opposed to an auditor simply not liking the particular form the tasks are being documented on.

Define the Objective of the Audit

Laboratory leadership should be involved in the planning process to help define the scope and expected goal of performing the audit. This can range from an overall assessment of general laboratory quality and safety, to a more directed and focused audit on either a single department, instrument/test, or test process (specimen collection, physician notification of critical values, etc). The format for the audit findings should also be discussed – will the site require a formal, written report outlining all observations detected, or will a simple informal summation discussion be sufficient?

Draft a Schedule for the Audit

Once the scope of the audit is defined, a tentative schedule should be created so all staff involved in the audit process are aware and available to participate. If the audit will encompass multiple departments and all phases of testing (pre-analytic, analytic, post-analytic), it may be necessary to split the audit up over multiple days, or to recruit multiple auditors. The frequency of audits will depend on the perceived risk to quality based on previous findings or complaints received, but at a minimum should be completed annually.

Prepare for the Audit

Reach out to the local management team of the site being audited for help in gathering the information you’ll need to prepare. This can include things such as a testing activity menu, list of new instrumentation or new test validation studies, employee roster if personnel and competency records will be reviewed, and copies of previous audit/inspection results to check for corrective action implementation and sustainability. Review the information provided, and use it as a guide for where you feel your efforts should be focused on based on highest risk.

Utilize a Patient Tracer

Ask the site to pull all related records and reports for a particular patient sample by choosing a date, and specifying any particular characteristics for the specimen that you want to follow (such as age of the patient, sex, or focusing on abnormal/critical results). By asking the sites to prepare a patient tracer ahead of time, this will reduce the amount of time spent waiting and digging for specific files or log sheets as they are already organized and ready when you walk in for the audit. Tracers should adhere to the defined scope/objective of the audit, and will help you follow the path of a specimen through the entire process from pre-analytical, analytical, and finally post-analytical phases.

Pre-analytical: Include any specimen collection instructions or a printout/photocopy from the test directory for each test requested. This information should be compared to the information within the applicable SOPs to ensure they match and are both current and accurate. Physician orders can be included to confirm that the correct test was ordered and performed based on what was requested by the clinician.

Analytical: Copies of the related SOPs for the test being reviewed should be included. Ensure the SOPs have all required elements, including a current, valid signature of approval from the medical director. Instrument QC and maintenance logs for the day of testing, calibration records, and patient correlation studies should also be reviewed, along with the reagent lot-lot validation performed. When available, copies of the actual instrument printouts should be included to check for accuracy in result transcription. Training and competency records for the staff who performed any handling or testing of the specimens in question may also be reviewed.

Post-analytical: Check for supervisory review of patient log sheets and QC records, along with appropriate corrective actions documented as applicable. Review the patient results in the same format that is seen by the physician: confirm reference ranges and units of measure are accurate, interpretive notes are valid and appropriate, test methodology is stated when applicable, abnormal values are flagged, and confirm result transcription accuracy from the original instrument printout. Proficiency testing results should be reviewed for any unsuccessful events to confirm sustainability of corrective actions.

Conduct the Audit

Perform an objective review of the documents provided, along with any affiliated records and logs based on the scope of the audit (temperature logs, reagent inventory records, decontamination records, etc). As with an official inspection, be transparent with the staff as issues are identified so they can have an opportunity to clarify any confusion, or locate additional records that may be missing or incomplete. Document any discrepancies or possible issues noted, as well as any good lab practices observed that should be celebrated. When logging your findings, be specific and provide as much details as possible so the staff can quickly identify what was found and make the needed corrections (SOP numbers, dates, instrument serial numbers, etc).

In addition to reviewing documentation, perform a direct observation of the staff doing specific tasks. Are they following the steps outlined in their procedures, or are deviations noted? Rather than a formal interview, ask the staff to explain what they are doing, or why they are performing certain steps in a particular order. Again, the audit is not meant to be punitive or to ‘catch someone in the act’, but rather to help identify areas for improvement or clarification so that testing processes can be improved and standardized among all staff members. Asking open ended questions will provide more information than directed ones. For example, “Show me how you would access testing instructions if your computer network was down” as opposed to “Where are the paper versions of your SOPs?”

Prepare an Audit Report

The audit findings should be summarized for the site based on the format agreed upon during the initial planning stage (written report, verbal discussion). Whenever possible, similar findings should be grouped together so the location can identify systemic problems that need to be addressed on a more global level (expired reagents found in multiple departments, staff failing to utilize appropriate PPE in multiple departments, etc). Depending on the number and severity of the issues identified, sites may prefer to have the observations grouped by department as well for easy assignment of follow-up action items to the department leaders. Issues should also be ranked by risk severity so that the site knows where to focus their improvement efforts first: 1) Patient care and employee safety issues; 2) Regulatory compliance gaps; 3) Recommendations for improved overall good laboratory practice.

Implement Corrective Actions

Any issues identified during the audit should be assigned to a specific person for follow-up, along with an anticipated date of completion. Perform a proper root cause analysis to identify why the issue happened, and then decide how to correct it and prevent it from happening again. Depending on the scope of the audit, the audit team members may be involved with these tasks, or this may fall to the sole responsibility of the management team being inspected.

Evaluate the Effectiveness of the Audit

The utility of the audits will depend greatly on the commitment of laboratory leadership to both implement, and sustain, effective corrective actions based on the quality gaps identified. This can be assessed by the overall level of compliance with the regulations being checked, and comparing the results of this audit to previous and subsequent ones to hopefully show a downward trend in potential citations detected. The audit team should obtain feedback on the audit process to assess the inspected lab’s overall satisfaction with the program, the amount of support offered to the inspected laboratory, effectiveness of communication between the teams, and any potential areas for improvement in the process.

Performing internal audits is a great way to meet regulatory, accreditation, and customer requirements. It allows you an opportunity to identify non-conformances and risks that can affect both quality, and patient/employee safety. By performing regularly scheduled internal audits, not only will staff members become more experienced and better prepared for the official external inspections from regulatory and accrediting agencies, but the laboratory will move from a culture of reactive, corrective actions to that of a proactive model of continual improvements.


-Kyle Nevins, MS, MLS(ASCP)CM is one of ASCP’s 2018 Top 5 in the 40 Under Forty recognition program. She has worked in the medical laboratory profession for over 18 years. In her current position, she transitions between performing laboratory audits across the entire Northwell Health System on Long Island, NY, consulting for at-risk laboratories outside of Northwell Health, bringing laboratories up to regulatory standards, and acting as supervisor and mentor in labs with management gaps.

What’s in a Differential?

When a complete blood count (CBC) and differential is ordered by a physician, most labs today have instrumentation capable of performing an automated differential. Depending on the instrument results and flags, we may need to perform a scan, review of the slide, or a manual differential. However, the definition of a manual differential today may be a bit different than the historical definition. A typical manual differential, when I first started working as a technologist, consisted of counting and differentiating 100 white blood cells under a microscope, and performing a red blood cell morphology along with a platelet estimate. Today, the 3 components of the manual differential have not changed, but more and more  labs are using an automated digital counting device, such as CellaVision. Whether counting cells under the microscope or scanning and verifying or reclassifying cells in CellaVision, it is important to always address all 3 parts of the manual review.

When an automated CBC has flagged that abnormal RBC morphology may be present, a peripheral blood smear should be reviewed. Reporting the red blood cell (RBC) morphology is an important component of a differential. Evaluation and interpretation of RBC morphology may provide the physician with important diagnostic information regarding the underlying cause of a variety of disorders, including anemia and systemic disease. Therefore, it is important to be able to accurately recognize and identify RBC morphologic abnormalities.

Red blood cell morphology can be subjective, and therefore inconsistent. Therefore, Laboratories must have training and competency programs as well as  procedures which dictate how they will report RBC morphology. Some labs use a numbering system, 1+, 2+, 3+, and others report, ‘rare’, ‘few’, moderate’ or ‘many’. Some morphological, such as rouleaux, can just be reported as present, with no quantified. Any method is acceptable, as long as there is consistency in reporting.

When performing RBC morphology,  these semi-quantitative report formats for should be based on clinical significance. Some RBC morphologies and inclusions are clinically significant,even when they are present in very low numbers. Sickle cells are one of these abnormalities that are significant even if only seen in very small numbers. Malaria or other parasites are clinically significant in any number. Fragmented cells such as schistocytes and helmet cells should also be noted if seen in any number. Other abnormalities which can be clinically significant in very low numbers are polychromasia, spherocytes and teardrop cells.

There are many other abnormal RBC morphologies which are only clinically significant if seen in larger numbers. Laboratories may choose to only report the presence of ovalocytes, target cells, burr cells, macrocytes, microcytes or hypochromia when greater than a defined percentage of cells exhibit these morphologies. Other laboratories choose to not report macrocytes, microcytes and hypochromia at all, instead relying on the physician to use the RBC indicies for their indication. The 2 most important things to remember, whatever your procedures are, is to be consistent, and not to ignore the:RBC morphology.

In addition to performing RBC morphology, a manual differential also requires platelet examination. A smear should be examined for a platelet estimate and abnormalities. This is particularly important when platelet clumps or an abnormal platelet scattergram are flagged on the CBC.  If an instrument uses optical platelet counts, large platelets can be missed. A fluorescent platelet count (PLT-F) , performed on Sysmex analyzers, will stain only platelets and give an accurate platelet count. The fluorescent count eliminates interferences seen with other methods. However, even when reporting a PLT-F, it may still required to review the smear for a platelet estimate, particularly with a very low count, or with clumped platelet flags. Clumped platelets are not an uncommon phenomenon, and an accurate platelet count can not be reported if significant clumping is present. The presence of giant platelets or hypogranular platelets, seen on the slide,  can also aid the physician in diagnosis or patient management.

CellaVision users have the added benefit of automation which simplifies the process of performing manual differentials. The system automatically locates and takes digital images of cells, including white blood cells, red blood cells and platelets.This simplifies the process of performing a manual differential. White  blood cells are pre-classified, RBC images are provided, and platelet images allow platelet estimates to be performed easily. The new advanced RBC application software can pre-classify RBCs.  This makes it even easier than before to perform reliable, standardized RBC morphology. (Watch for my next Hematology blog about the new RBC software!)

Particular disorders or abnormalities often involve characteristic changes to RBC morphology “Assessment of RBC morphology can be the best tool for laboratory hematology professionals to recommend clinical and laboratory follow‐up in a patient with anemia and to select the right tests for definitive diagnosis.”1 Too often, I have seen technologists perform a manual differential and either superficially skim over the RBC and platelet components, or totally forget them. Don’t forget your RBC morphology and platelet estimate and morphology! With today’s automated differential and autovalidation, 75-85% of CBCs are autovalidated. This allows us to spend quality time on those manual reviews that need to be done. Be sure to spend your time thoroughly reviewing the slides. A scan, slide review or manual differential, whether done under the scope of with CellaVision,tells the physician that we have looked at the slide or cells, which must include all 3 parts of manual review… WBCs , RBCs and platelets. Don’t sign it out until it’s complete!

References

  1. J. Ford, Red Blood Cell Morphology. International Journal of Laboratory Hematology. 2013
  2. https://www.labce.com/red-cell-morphology.aspx

-Becky Socha, MS, MLS(ASCP)CM BB CM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

Regulating Your Lab Medical Waste

In general, there are two reasons employees in the laboratory should care about proper waste disposal. Improper disposal is expensive. Laboratorians like raises, bonuses, and updated equipment, but there is less money for those things when paper items are tossed into sharps containers or when used gloves go into red bag trash containers. Labs in many states also risk large fines if items with biohazard symbols are disposed of into regular trash containers. The other reason to care about trash disposal involves the environment. Regulated Medical Waste (red bag trash and sharps) has to be treated, and some of it is incinerated while some ends up in special biohazard landfills. Both of those are things we want less of in our environment.

As a lab safety professional, you may know of several other reasons to implement and maintain proper lab waste segregation, but in my years of safety training, money and the environment are the two that tend to hit home with staff. There are multiple waste streams generated in the lab setting, and while management in some departments may choose to offer only biohazard waste receptacles for everything, the safety savvy professional knows this is wasteful and perhaps a bit lazy. With proper education and training, laboratorians are capable of goo trash segregation that meets the regulations and meets best practice standards.

Appropriate trash segregation in the lab requires knowledge about what waste goes into what type of container, and it requires availability and proper placement of those containers. If a processing department only uses red bag trash cans, for example, then much of the non-hazardous waste will end up there. Assess the laboratory areas for proper placement of all necessary types of waste receptacles.   

In one lab, it was discovered that staff was throwing out urine containers with embedded needles into red bag trash containers. Why? There simply were no sharps containers in the area. It was a simple fix to move containers nearby, but no one was paying attention, and there could have been an unnecessary needle stick exposure. In another lab staff emptied urine sample cups into the sink and tossed them into regular trash bins. From a waste standpoint, that was fine, but because there was patient information on the container labels, HIPAA violations occurred.

Many venipuncture sample tubes used today are plastic, and they cannot be broken to create sharp edges. Given that, those items could be disposed of into biohazard trash bags. That can save a lab some money by reducing the volume of sharps containers used (they are more expensive to handle). However, glass specimen tubes are still available for purchase. Be sure to check for these in your racks before throwing out all lab tubes into a plastic bag. A broken tube can cause a very unfortunate exposure event.

Place patient information and extra labels into bins for shredding if available. Teach staff that in most cases it is acceptable to place used disposable lab coats and gloves into regular trash receptacles provided they are not visibly bloody. Other items can go into the regular waste stream such as plastic transfer pipettes, gauze pads, and paper towels (again, provided there is no blood visible on them).

If items can be broken to create a sharp edge, they should be disposed of into a sharps container. That includes specimen cups made of hard plastic, sharp pipette tips, and any glass item. Agar plates and wooden applicator sticks should also go into a sharps container. Remember, if the item breaks while a trash bag is handled, an employee may become exposed, and the incident would need to be treated as an unknown source exposure, something that should always be avoided.

Make sure staff know the proper disposal of chemical waste as well. Never pour chemical waste down the drain unless your facility has a permit to do so. Place chemical waste containers in appropriate locations and label them according to EPA regulations. Provide proper training for employees who sign waste manifests when hazardous waste is hauled away from the lab. If you take the easy route and combine all of your laboratory waste, you would be responsible for both increased departmental expenses and for unnecessarily adding bio-waste to the environment. Talk regularly to your group of trained lab scientists about proper waste segregation, use signage as reminders, and assess their lab waste knowledge regularly. Proper waste management takes work. Mistakes can be made easily, and some of them can cause injury and invoke heavy fines. Invest in a robust laboratory waste management program to avoid those issues and to create a safety savvy example for others.

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.

Hormone Blockers = Blood Letting for Female Athlete with high T?

Caster Semenya celebrates as she wins gold in the women’s 800 meters in the Commonwealth Games on April 13, 2018, on Australia’s Gold Coast (1). Jason O’Brien/Getty Images

I will continue this month along the thread of last month’s post, which addressed the controversy surrounding South African female mid-distance runner Caster Semenya. Caster has won many international mid-distance races (400-800m), but she has been suspected of naturally producing higher levels of testosterone.

Since last month, I’ve learned the reason for the higher testosterone is uncertain: it could be due to natural production (hyperandrogenism) or rumors of her being intersex1. Regardless, what I will discuss here is how the proposed actions of the International Olympic Committee would be expected to affect Semenya’s performance. Specifically, how would lowering testosterone levels affect her athletic performance?

Last month, we saw that muscle mass might be expected to decrease, but this may not affect athletic performance significantly.

Another important effect of testosterone is on red blood cell levels including hemoglobin, which by carrying oxygen to muscle is a central part of calculating VO2max. VO2max is maximal oxygen consumption. This is strongly linked to performance in cardiovascular athletic events.

Mid-distance running requires a large cardiovascular capacity. Maybe not the same level of Tour-de-France long distance bikers in the Alps, but still substantial. As a runner that feels pretty proud at having run a sub-3 minute 800m, I can say Caster’s feat of running it in less than 2 minutes is incomprehensible. From the burning feeling in my lungs and thudding, maximum heart rate at the end of the half-mile, I can attest that this event requires substantial cardiovascular efficiency.

Maximal oxygen consumption (VO2max) by exercising skeletal muscle is principally limited most by cardiac output and oxygen-carrying hemoglobin levels. This has been shown quite convincingly in a series of experiments in the 1950’s-70’s2,3 that probably wouldn’t be approved by the IRBs of today charged to protect research subject rights.

First, transfusing blood increased hemoglobin concentration and similarly the VO2max and exercise endurance of participants.  (This practice was exploited most notably later on in the Tour de France).  In other studies3, blood was removed from participants before assessing their exercise tolerance (10% loss of hemoglobin à 13% reduction in VO2max). Another study removed 400mL, 800mL and 1,200mL over several days, which decreased hemoglobin by 10%, 15%, and 18% respectively. There was a concomitant decrease in endurance time (-13%, -21%, -30%) and VO2max as well (-6%, -10%, -16%)3.  A summary of blood transfusion and hemodilution studies is shown in Figure 1 from Otto JM et al4.

Figure 1. Reproduced from Otto JM et al (4)

In transgender women (gender incongruent with sex assigned male at birth), hormone therapy to increase estrogen levels (oral estradiol) and block testosterone (anti-androgen: spironolactone) reduces hemoglobin by 9% on average (from 15.2 g/dL to 13.9 g/dL)5. I would expect a smaller decrease for Semenya as she will likely not get a full dose hormone regimen used for transgender transition and because her testosterone levels wouldn’t be as high as biologic males’.  However, she would still be expected to have lower hemoglobin- similar to donating a half or whole unit of blood. If hemoglobin decreased even just 5%, that could affect her performance substantially when the difference between competitors boils down to seconds in mid-distance races.

Arguably, forced blood donation could produce the same effects as testosterone-lowering therapy. But it would be far too dramatic to suggest something like bloodletting by the International Olympic Committee.

In the end, I don’t feel qualified to say what should be done in this case. All I can say is that I don’t think lowering Caster Semanya’s testosterone levels will have the intended effect of decreasing muscle mass. On the other hand, it would decrease hemoglobin levels tempering her performance. But who should determine the point where her hormone levels should be? There is such a strong biologic connection between hormone levels and physiology that manipulating them for athletic fairness could be akin to playing puppeteer.

References

  1. North, Anna. ““I am a woman and I am fast”: what Caster Semenya’s story says about gender and race in sports” Vox. May 3, 2019
  2. BALKE B, GRILLO GP, KONECCI EB, LUFT UC. Work capacity after blood donation. J Appl Physiol. 1954 Nov; 7(3):231-8.
  3. Ekblom B, Goldbarg AN, Gullbring B. Response to exercise after blood loss and reinfusion. J Appl Physiol. 1972 Aug; 33(2):175-80.
  4. Otto JM, Montgomery HE, Richards T. Haemoglobin concentration and mass as determinants of exercise performance and of surgical outcome. Extrem Physiol Med. 2013; 2: 33.
  5. SoRelle JA, Jiao R, Gao E et al. Impact of Hormone Therapy on Laboratory Values in Transgender Patients. Clin Chem. 2019; 65(1): 170-179.

-Jeff SoRelle, MD is a Molecular Genetic Pathology fellow at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and advancing quality in molecular diagnostics.

New at the Multiplex: The Syphilis Shuffle

Hello again everyone and welcome back!

If you caught my previous post it was a rather long twofer for my March rotation at the Mayo Clinic’s Department of Pathology and Laboratory Medicine as well as a great case study of a patient’s therapy-related AML in the setting of Li-Fraumeni Syndrome. Now, as I inch closer and closer to my last months of medical school, I’m doing another pathology clerkship at Danbury Hospital in southwestern Connecticut. It’s an excellent community-based pathology program with a great staff. As you’ve read in my posts before, community hospitals are no stranger to the leading edge of laboratory innovation. A fellow medical scientist in a recent ASCP membership video on social media said, “Laboratory medicine is at the precipice of change.” As a beacon of translational medicine, labs turn routine medical unknowns into answers. Often, they act as leaders in the lab medicine community because of certain population-specific testing and reporting that goes on at their institutions. You might recall my discussion of Bronx-Care Hospital leading the charge in New York City with the newest, 5th generation troponin testing or my experiences at Swedish Covenant Hospital in Chicago with lab automation, software innovation, and CQI.

But back to medical school: the coordinators of this rotation asked be about my interests in pathology and we discussed my past as a medical laboratory scientist. As such, they offered some special projects for me to be a part of in their lab! Specifically, and in addition to my subspecialty objectives and observership, I’ve been helping them with three small projects. First, I assisted in calibrating a freshly validated second Bio-Rad Bioplex2200 analyzer to correlate to a second instrument for some very interesting testing. Second, I’m helping gather ongoing inter-instrument data for a Sebia serum protein electrophoresis instrument. And finally, I’ve been assisting the histopathology section with cross-instrument validation of immunohistochemical stains as well as gathering data for the validation of a great new IHC that would replace PSA.  All of these have both used skills from my MLS foundational experience but taken that one step further into the scope of a pathologist by going over the clinical implications and testing outcomes provided by these analyses.

The Syphilis Shuffle

I already mentioned that Danbury’s lab uses the Bioplex2200, so let me tell you a bit about the analyzer and a bit more about an interesting way to do syphilis testing. It’s an interesting immunoassay instrument that uses something called “multiplex” technology. I won’t go into the details about footprint, throughput, and timing because, well, I’m not exactly expecting any Bio-Rad checks over here. But what I do want to talk to you about is the testing methodology. So, what exactly is “multiplex” testing. Basically, by using magnetic beads with fluorescent dyes associated with various tests you can create bead-set assays. Those beads are coated with detection proteins for each assay and exist in a single reagent pack. That allows numerous analytes to be detected at the same time from a single aspirated specimen. A laser in the instrument detects these immunologic events and reports the values. Now, in my experience with immunoassays for tests like syphilis, hepatitis, EBV, or HIV, there are usually honorable mentions of Abbott’s ARCHITECT and Siemens ADVIA Centaur alongside the Bioplex2200. I’m much more familiar with the former two, so getting a chance to work with the latter was great. But what did I learn?

Figure 1. Immunoassay guests-of-honor from left-to-right: Siemens ADVIA Centaur, Abbott ARCHITECT, and Bio-Rad BioPlex2200. I guess clinical immuno is all about the swivel workstation screen placement. But all three instruments are excellent at what they do; an aspect of laboratory medicine is analyzing your analyzers. Depending on the needs of the institution, your lab might have different demands of what limits of detection, turn-around, and sensitivity/specificity statistics are required for populations of patients.
Figure 2. Final reaction mixture with coated, tagged beads passing through the detector simultaneously. (Source: Bio-Rad)

First of all, other analyzers like the ADVIA Centaur use chemiluminescent immunoassay technology and the Abbott ARCHITECH use photometric, potentiometric, or turbidimetric detection for immuno and chemistry tests. There are published demonstrations of how the three instruments compare regarding various metrics and detection statistics. One of those papers from 2017 demonstrated the sensitivity and specificity for HIV testing across the analyzers. Overall, it said the ARCHITECT was the best performing instrument (spoilers: this study was funded by Abbott), though each had their strengths—read the paper here. The multiplex method, although similar in principle, is unique to the BioPlex. And one of the tests I find interesting is their syphilis assay. On the BioPlex, its tested as a total immunoassay with RPR that improves accuracy and precision in real-time. It’s a dual treponemal/non-treponemal test. Those bead packs contain two types of beads that qualitatively detect IgG and IgM antibodies to Treponema pallidum while also qualitatively detecting RPR antibodies in serum or plasma. It comes with its own internal control and verification beads for internal QC. Fast, simple, easy, unique and just as accurate as most syphilis testing—that’s what I’m talking about.

Figure 3. Multiplex dual treponemal/non-treponemal immunoassay acts as a one-step test with less steps and less requirments for confirmatory testing. Just set it and forget it! Unless your test comes back positive, then call the local health department and get that penicillin injection, STAT! (Source: Bio-Rad)

(Immuno) Fixing Everyone’s Problems

I remember learning a lot about gel electrophoresis, serum/urine protein electrophoresis, and immunofixation in MLS school but never really spent too much time with gels. I didn’t get to do much work with electrophoresis because I spent my time in labs either on a microscope doing diffs, a blood bank shaking tubes, or monitoring chemistry outputs and validating reports. Despite spending years with an ID badge that often said “chemistry” on it, gels were a less common test and not just for me. The primary care clinician usually only orders SPEP and subsequent gels in the investigation of multiple myeloma or other paraproteinemia from a vast array of disease process. As such, the results are often more challenging to interpret and require reporting and education from the pathology department. There are, however, a myriad of interpretable patterns and information within the gels of an SPEP. The principle is standard—proteins separate in media based on charge and mass—and particular patterns tell us more information than one might realize. For example, chronic inflammatory processes might increase production of acute phase reactants like alpha-1-antitrypsin or haptoglobin. Where do the peptide building blocks for those new proteins come from? Wherever the most protein is: Albumin, obviously for those playing at home. So your friendly normal neighborhood SPEP in a chronic inflammatory process might morph into something with a fainter albumin band and some extra attenuation in the alphas 1 and 2. If you see that, you might correlate with an ESR or CRP (or hs-CRP, you fancy laboratorian) …

Figure 4. We’ve all seen one of these before, but there are so many patterns to interpret which translate to real pathology and correlate well with concomitant serum values implicated in anything from myeloma, to infection, inflammation, or hepato-renal disorders. (Source: Univ. of Washington)
Figure 4. We’ve all seen one of these before, but there are so many patterns to interpret which translate to real pathology and correlate well with concomitant serum values implicated in anything from myeloma, to infection, inflammation, or hepato-renal disorders. (Source: Univ. of Washington)

NKX3.1, the Lexus of Prostate IHCs

Now for something different. Let’s talk about prostates. Fun! More specifically, let’s talk about prostate-related immunohistochemical stains. The first one you’ll think off right away is probably …drumroll… PSA, and you’d be absolutely correct. That’s a standard IHC for detecting prostatic adenocarcinoma and is especially useful in finding metastases from a prostate primary. Though not brand new, there is another stain for prostatic IHC detection that, in some recent studies, has been shown to be more sensitive for prostate malignancy than PSA by about 5%. It’s called NKX3.1 and it has PSA’s sensitivity of 94.2% beat at around 98.6%–that could translate to plenty of earlier diagnoses and better outcomes for patients.

Image 1. Immunohistochemical staining of prostatic tissue. In IHC stains, brown is positive for expression. The immunologic technique of marking a specific antigen with a detectable antibody can be translated to a long list of tissue typing and can identify nuclear, cytoplasmic, or membranous patterns. For something like prostate tissue (seen here) sometimes PSA and/or NKX3.1 can identify prostatic malignancy or distant, suspicious metastases. (Source: BioCare medical)

What’s that got to do with my current pathology rotation? Well, I’ve gotten a lot of anatomical pathology exposure in my time here and I even helped correlate IHC stain quality across two instruments. With that done, I’m currently collecting specimens of saved tissue blocks that were both positive and negative for the lab’s current prostate IHC, PSA, and retesting them all with NKX3.1 in order to switch protocols to the new, more sensitive test. At the very least, the addition of a secondary validated prostatic stain would be useful. What’s important in gathering specimens for this kind of correlation is understanding when and where this new stain would be positive and negative and making sure it behaves in your patient population the way you would expect. NKX3.1 is supposed to be positive in nearly 99% of prostatic adenocarcinomas whether they’re primary or metastatic. It is a Chromosome 8 protein which is expressed in the prostate and testis and can even be found in the salivary glands, bronchial submucosal glands, and regions of the ureters. It can be positive in 27% of invasive lobular carcinoma, 25% of metastatic lobular carcinoma, 2-9% of invasive ductal carcinoma, and 5% of metastatic ductal carcinoma. (Source: Pathology Outlines) While I’m looking over specimens with historical orders for PSA IHCs, not all are positive, and not all are prostatic tissue. Conducting validation studies like these in pathology really require a good understanding of how to clinically correlate data with useful decision-making and tailor it to your patient population.

I wrote about a lot of topics this month, I know, but I think there’s a common theme. As a medical laboratory scientist, like many of you, I’ve worked out countless QC problems and instrument validations per protocol. Now that I’m making the transition to medicine in pathology, there’s a lot of forethought and planning that goes into validating or calibrating any test. In chemistry you need to get your limits of detection just right and match your throughput with the test volume your population needs. In hematology, you better know exactly how cells get detected by your analyzers and have a solid algorithm for working up and understanding aberrant flags. When it comes to anatomic pathology, speaking a common language of morphology and pattern-recognition is vital to reporting reliable and critically important data. Laboratory medicine always exists at the forefront of medical testing and methodology, and what that translates to on a day-to-day basis is being able to know how to find, make, or confirm a good, reliable test. As for me, medical school is full of unique experiences and rewarding opportunities to learn. This month, I couldn’t be happier to use my skills in the lab to connect my time at the bench to my work learning, calibrating, and validating for the next step.

And, after all, aren’t we all looking for a little validation now and then?

Thanks to Danbury Hospital’s Department of Pathology and Laboratory Medicine for having me this month and thank you all for reading.

See you next time!

–Constantine E. Kanakis MSc, MLS (ASCP)CM graduated from Loyola University Chicago with a BS in Molecular Biology and Bioethics and then Rush University with an MS in Medical Laboratory Science. He is currently a medical student actively involved in public health and laboratory medicine, conducting clinicals at Bronx-Care Hospital Center in New York City.

Proficiency Testing (PT) Part 3: Quality Indicators

Last month we discussed the rules associated with evaluating your PT results, and how to investigate any unsuccessful surveys. In the last of this 3-part series we’ll review ways to utilize your PT reports to check for trending in your patient values – shifts, trends and bias. Your PT results can help show you developing problems and allow you to correct them, before they become failures or begin to affect patient care. Before declaring a failure as a ‘random error’, be sure that it truly is.

Accuracy & Systematic Errors

Accuracy describes how close your measured value is to the reference value – did you obtain the correct result? This will be affected by systematic errors, such as using expired or degraded reagents, changes in lot numbers or calibration values, or instruments with analytical lamps or lasers near the end of their use life. Systematic errors are reproducible inaccuracies that occur in the same direction; all results will be falsely low or all results will be falsely high. If systematic errors are present, all results will show similar deviations from the true value. Bias is a measure of how far off your results are from their true intended value.

Precision and Random Errors

Precision on the other hand refers to the overall agreement of results upon replicate testing – will you get the same value if you repeat the test? Precision is affected by random errors, such as incomplete aspiration of a sample or reagent due to fibrin clots or air bubbles, operator variability in pipetting technique, or temperature fluctuations. Random errors are statistical fluctuations in the measured data due to the limitations of the assay in use. These errors will occur in either direction from the mean, unlike systematic errors that will be on the same side. Imprecision can be measured and monitored by evaluating the standard deviation (SD) and coefficient of variance (CV) for an assay.

https://blog.forecast.it/the-difference-between-accuracy-and-precision

Let’s look at some example PT results from CAP, and see what hints these reports reveal to us.

  • Albumin: Although all results passed and were graded as ‘acceptable’, there are still issues that should be looked into. For the last 3 surveys in a row, the plot shows that nearly all samples have been on the same right side of the mean. When comparing the value of the % relative distance from the first survey to the most recent one, you can see that the values are trending worse and getting closer to being unacceptable if the pattern continues. Additionally, be mindful of the standard deviation index (SDI) value reported. This is a measure of your bias, and how far off your values are from the mean. It should be defined within your Quality System Manual (QSM) the values which should trigger an investigation, but as a general rule, anything >±2.0 indicates a potential issue. (https://unityweb.qcnet.com/Documentation/Help/UnityWeb/399.htm)
  • Alkaline Phosphatase: Again all results passed, but 3/5 samples have SDI values >±2.0. The first survey had all values to the right of the mean, the second survey was a nice tight even mix of +/- bias, and now with the most recent survey all values are appearing to the left of the mean. If this shift coincides with a change in lot number, a calibration may be necessary to get results back on target to help lower the SDI values.
  • GGT: Although only 1 sample was graded as unacceptable, all of the results for this recent survey were at risk of being failures due to how close they were to the upper limit of acceptability. Results like this should be very carefully evaluated to ensure that there is no impact on patient care. Provided the sample stability has not been exceeded, all 5 samples should be repeated. If the repeat values are closer to the target mean, you will need to identify what went wrong on the day the samples were originally tested. If the repeat values are still grossly far from their intended target, a full patient lookback would need to be performed from the time the samples were originally tested until the day they were repeated, as there is a systemic problem that has now continued for weeks or longer.  
  • Vancomycin: Similar to the albumin example above, these results show a trend occurring between the first survey and the most recent; however unlike albumin these are moving in the correct direction. Values are getting closer to the target mean, and SDI values are decreasing, suggesting that any corrective actions implemented after the last survey were successful.
  • Lithium: This shows a good example of what you hope all of your quantitative proficiency results will look like. There is a nice distribution of results on both sides of the mean, and SDI values are all relatively low. Values such as these allow you to have complete confidence in the accuracy of your patient results.
  • MCH: Focus on sample #2, with an SDI of -1.9. The other samples within this survey all appear fine, but it looks as though there was truly a random error with sample #2. When we look at the affiliated analytes we see a similar issue with the RBC count of sample #2, which coincides with our decreased MCH (a reminder for our non-hematology readers, MCH = (Hgb x 10)/RBC). For any calculated values, be sure to evaluate the all parameters together as well as individually to serve as a common sense check that your results are appropriate and truly make sense.

It is important to have a robust quality assurance program that outlines what to monitor, key decision points for when to take action, and guidance on what those actions should include. Your proficiency testing results can provide you with a ton of useful information to evaluate the overall quality of laboratory, and help provide confidence in the patient values being reported out as well.

-Kyle Nevins, MS, MLS(ASCP)CM is one of ASCP’s 2018 Top 5 in the 40 Under Forty recognition program. She has worked in the medical laboratory profession for over 18 years. In her current position, she transitions between performing laboratory audits across the entire Northwell Health System on Long Island, NY, consulting for at-risk laboratories outside of Northwell Health, bringing laboratories up to regulatory standards, and acting as supervisor and mentor in labs with management gaps.

Sex Hormones in Competitive Athletics

Image 1. Photo from NBC News.

Given my previous work in lab value changes in transgender individuals on hormone therapy, I was recommended to consider discussing the case of Olympic mid-distance runner, Caster Semenya. Although she is not transgender, this professional runner from South Africa has won her last 30 races and been scrutinized for her muscular build as having potentially higher levels of testosterone, a condition called hyperandrogenism. The International Olympic Committee’s (IOC) regulations require testosterone levels to be below a certain threshold for female athletes. 

While no competitor can achieve great victories without hard work and practice, there are certainly examples of outliers whose genetics give them an advantage. However, I don’t think we would endorse shortening Michael Phelps’ arms or lobotomizing chess master Bobby Fisher to decrease their inborn advantages for a level playing field.

But this gets into an area of ethics that I’m not an expert on, so instead I will stick to my area of science and examine what evidence may exist to support the IOC’s policy. Then I will extrapolate the results from our study of transgender individuals to see if hormone regulation may impact contributions to athleticism. The most strongly shifted lab values in hormone therapy for transgender individuals are red blood cells (including oxygen-carrying hemoglobin) and creatinine (byproduct of muscle used to monitor kidney function, but also reflects total muscle mass).

Once looking more closely at this topic, I realized there is a lot to say about the contributions of 1) muscle mass and 2) red blood cells to athleticism. So, I will discuss muscle mass this month and wait until next month to discuss hemoglobin levels (including athletic performance by blood removal/ doping).

Mid-distance running, which is Caster Semenya’s sport, is a mix of anaerobic and aerobic activity. This means having more muscle would be advantageous. This is supported by a study that was commissioned by the IAAF (International Association of Athletics Federation), which shows a 1.8-2.6% increased competitive advantage in short distance track events (400m, 800m and, 400m hurdles)1. However, this study had several limitations. First, the sample size was quite low with only 22 female athletes. Next, they use a p-value of 0.05 for significance without correction for multiple hypothesis testing (21 hypotheses tested representing each event), which increases the likelihood of a false positive result by chance.

What makes me curious is whether following the International Olympic Committee’s recommendations of lowering testosterone levels would even have a meaningful impact and improve competitiveness?

From my research, I know that adding testosterone to individuals assigned female at birth to transition to transgender males (TM ) does substantially increase creatinine (p<0.005, Figure 1)2 to male levels (baseline TW). This is likely not due to changes in kidney function (although this has not yet been proven), but rather due to increased muscle mass.

Figure 1.

However, the inverse is not quite true for transgender women who take combinations of estrogen for feminization and spironolactone to block the effects of testosterone. In these patients, we see a slight decrease in the creatinine (TW). While this decrease is statistically significant, the range is not clinically different from male creatinine levels. This concurs with the observations that musculature in transgender women does not change substantially upon taking hormone altering medication.

A more rigorous examination of muscle mass, performed by MRI measurement, determined that after 1 year of hormone therapy testosterone increased muscle mass in transgender men to biological male levels3, similar to our observations of creatinine. Further, they saw a significant reduction in muscle mass from baseline of transgender women on hormone therapy for 12 months, but it was still much higher than the muscle mass of biologic females4.

Therefore, were Casten Semenya to take testosterone blocking medication, I suspect there would be little impact on her overall muscle mass. Which is one of, if not the explicit purpose of taking testosterone lowering medicine. The strength of my conclusions is limited by the fact that we don’t know Casten Semenya’s testosterone levels, and furthermore a hyperadrogenic female is not the same as a male-to-female transgender woman.

As mentioned above, I will continue this discussion next month with an exploration of how testosterone lowering therapy could affect red blood cell levels, which would affect athletic performance differently.

References

  1. Bermon S and Garnier P. Serum androgen levels and their relation to performance in track and field: mass spectrometry results from 2127 observations in male and female elite athletes. British Journal of Sports Medicine. 2017; 51(17): 1309-1314.
  2. SoRelle JA, Jiao R, Gao E et al. Impact of Hormone Therapy on Laboratory Values in Transgender Patients. Clin Chem. 2019; 65(1): 170-179.
  3. Gooren LJ, Bunck MC. Transsexuals and competitive sports. Eur J Endocrinol. 2004; 151(4): 425-9.
  4. Jones BA, Arcelus J, Bouman WP, Haycraft E. Sport and Transgender People: A Systematic Review of the Literature Relating to Sport Participation and Competitive Sport Policies. Sports Med. 2017;47(4):701-716.

-Jeff SoRelle, MD is a Molecular Genetic Pathology fellow at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and advancing quality in molecular diagnostics.