ASCLS Today Volume 34, Number 5

ASCLSToday Masthead 680

Volume 34, Number 5


James Hollowell, MLS(ASCP)CM, 2019-20 ASCLS Developing Professionals Forum Vice Chair

During this past year in my clinical rotations, I was able to experience a wide variety of laboratories and learn from a diverse and talented multitude of laboratory scientists. I interacted with hundreds of hospital employees and worked closely with dozens of laboratory workers at six different clinical sites, ranging from rural hospitals to large urban medical centers and everything in between.

"The employees of the laboratory should reflect the multifaceted patient base we serve in our communities."

Each site was different, and each was equipped with a unique laboratory team willing and ready to teach the next generation, in addition to performing their daily workload. I am incredibly thankful I had the opportunity to work with a spectrum of people who taught me about every aspect of the laboratory in their own unique ways. I feel as though my education in these laboratories left me well-rounded and fortified me with a new perspective on what it takes to meet the gold standard of patient care.

The employees of the laboratory should reflect the multifaceted patient base we serve in our communities. It is our responsibility as healthcare professionals to care for the under-represented and diminished members of society. The further development of healthcare facilities across the globe increases the need for a diverse workforce of laboratory scientists whose varied backgrounds and life experiences equip them with the tools they need to make the world a better place.

Diversity is not limited to our differences in ethnicities, gender, age, or religion. It is also a conscious practice that involves understanding how our cultural differences are interdependent on each other. To say that America is a melting pot is no longer true in the traditional sense because it is no longer standard to try and completely assimilate to one true “American” culture.

The modern laboratory reflects this change, and now we understand the importance of holding on to these special aspects of culture to create a thriving workplace. The laboratory must grow to meet the demands of the expanding world; as a member of the next wave of medical laboratory scientists, I have hope that we will be an integral part of a brilliant future.

James Hollowell is a Medical Laboratory Scientist at Merit Health Natchez in Natchez, Mississippi.


Jie Gao, PhD, MLS(ASCP)CM, and Floyd Josephat, EdD, MT(ASCP)

The recent joint American and European guidelines on the diagnosis of acute myocardial infarction (AMI) rely heavily upon the measurements of cardiac troponins (cTn), especially troponin T (cTnT) and troponin I (cTnI), which are the most useful cardiac biomarker today.1 However, an increasing number of studies have revealed the cTn elevation is not specific to AMI, and it has been observed in a variety of diseases, such as end-stage renal disease, pulmonary embolism, sepsis, cirrhosis, diabetes mellitus, and so on, regardless of whether the symptoms of acute coronary syndrome (ACS) are present or not. Therefore, it is critical to be aware of pathological conditions that may cause elevated cTn levels in conditions other than AMI. This is also essential to prevent misdiagnoses and can provide an opportunity for laboratory professionals to become actively involved in the diagnostic process as well as to provide accurate results. Below are two examples of disease states or conditions where cTn levels were identified to be elevated.

“[I]t is critical to be aware of pathological conditions that may cause elevated cTn levels in conditions other than AMI.”

End-Stage Renal Disease

End-stage renal disease (ESRD), also called kidney failure, is the advanced stage of chronic kidney disease when kidneys are no longer able to work as they should to meet the body’s needs. A great number of clinical studies have reported the blood levels of cTn increase over time as the chronic kidney disease progresses.2, 3 Fortunately, the elevated levels of cTn in ESRD are divergent from the ones in AMI.

The cTn values in AMI are usually as high as 20-50 times of the upper reference limit. However, modest elevations are found in ESRD patients who were asymptomatic or present with nonspecific ACS symptoms such as dyspnea, fatigue, or nausea.4 In addition, the cTn elevation in ESRD carries important prognostic information.

ESRD patients with elevated blood levels of cTn, particularly cTnT, are closely associated with a high risk of death despite being ACS asymptomatic.5 Several theories have been proposed to explain the mechanisms of cTn release into blood in renal diseases, but additional studies are still needed to clarify the patterns of cTn changes over time in patients with chronic kidney disease, especially in the absence of myocardial ischemia.6

Acute Pulmonary Embolism

Acute pulmonary embolism (APE) is the occlusion of the pulmonary artery by thrombus, fat, air, and other materials. The pulmonary artery obstruction can lead to an acute right ventricle dilatation, which significantly increases the pulmonary vascular resistance, reduces coronary artery perfusion, and eventually causes severe myocardial ischemia and elevated cTn levels.7

Patients with pulmonary emboli may clinically mimic myocardial ischaemia, but the management for them is completely different; therefore, it is critical to differentiate AMI and APE in patients with elevated cTn and ACS related symptoms. From the laboratory perspectives, cTn elevation is found in approximately 40-50 percent of patients with APE.8, 9 Despite the low diagnostic accuracy, the elevation process of cTn in APE is significantly different from the one in AMI.

It has been widely known that in AMI, the blood levels of cTn usually start to elevate from four to six hours after the onset of symptoms and remain high for up to four to seven days (cTnI) or 10 to 14 days (cTnT). By contrast, the increase of cTn in APE may occur from 6-12 hours after the symptomatic occurrence and its retention lasts no longer than three days.10 In addition, the blood levels of cTn in APF are moderately increased compared to AMI cTn elevations, which are at least 20 times higher than the upper reference limits.

It has been well known that elevated cTn levels do not always indicate AMI. A variety of diseases should be considered in the differential diagnosis of patients with cTn elevations, despite whether the acute coronary syndrome related symptoms are present or not. The cTn elevations may not be diagnostic for most of these diseases except AMI, however, its strong prognostic implications have been clearly proven with predictive values of increased risk of mortality and cardiovascular incidents. Therefore, it should be recommended that patients with elevated cTn levels receive more intense follow-ups and guidance on the appropriate approach to treatment.

  1. Thygesen, K.; Alpert, J. S.; Jaffe, A. S.; Chaitman, B. R.; Bax, J. J.; Morrow, D. A.; White, H. D.; Executive Group on behalf of the Joint European Society of Cardiology /American College of Cardiology /American Heart Association /World Heart Federation Task Force for the Universal Definition of Myocardial, I., Fourth Universal Definition of Myocardial Infarction (2018). Circulation 2018, 138 (20), e618-e651.
  2. Chesnaye, N. C.; Szummer, K.; Barany, P.; Heimburger, O.; Magin, H.; Almquist, T.; Uhlin, F.; Dekker, F. W.; Wanner, C.; Jager, K. J.; Evans, M.; dagger, E. S. I.; Investigatorsdagger, E. S., Association Between Renal Function and Troponin T Over Time in Stable Chronic Kidney Disease Patients. J Am Heart Assoc 2019, 8 (21), e013091.
  3. Stacy, S. R.; Suarez-Cuervo, C.; Berger, Z.; Wilson, L. M.; Yeh, H. C.; Bass, E. B.; Michos, E. D., Role of troponin in patients with chronic kidney disease and suspected acute coronary syndrome: a systematic review. Ann Intern Med 2014, 161 (7), 502-12.
  4. Skeik, N.; Patel, D. C., A review of troponins in ischemic heart disease and other conditions. Int J Angiol 2007, 16 (2), 53-8.
  5. Khan, N. A.; Hemmelgarn, B. R.; Tonelli, M.; Thompson, C. R.; Levin, A., Prognostic value of troponin T and I among asymptomatic patients with end-stage renal disease: a meta-analysis. Circulation 2005, 112 (20), 3088-96.
  6. Kanderian, A. S.; Francis, G. S., Cardiac troponins and chronic kidney disease. Kidney Int 2006, 69 (7), 1112-4.
  7. Kilinc, G.; Dogan, O. T.; Berk, S.; Epozturk, K.; Ozsahin, S. L.; Akkurt, I., Significance of serum cardiac troponin I levels in pulmonary embolism. J Thorac Dis 2012, 4 (6), 588-93.
  8. Meyer, T.; Binder, L.; Hruska, N.; Luthe, H.; Buchwald, A. B., Cardiac troponin I elevation in acute pulmonary embolism is associated with right ventricular dysfunction. J Am Coll Cardiol 2000, 36 (5), 1632-6.
  9. Konstantinides, S.; Geibel, A.; Olschewski, M.; Kasper, W.; Hruska, N.; Jackle, S.; Binder, L., Importance of cardiac troponins I and T in risk stratification of patients with acute pulmonary embolism. Circulation 2002, 106 (10), 1263-8.
  10. Muller-Bardorff, M.; Weidtmann, B.; Giannitsis, E.; Kurowski, V.; Katus, H. A., Release kinetics of cardiac troponin T in survivors of confirmed severe pulmonary embolism. Clin Chem 2002, 48 (4), 673-5.

Jie Gao is Assistant Professor in the Clinical Laboratory Sciences Program/Department of Clinical and Diagnostic Sciences at the University of Alabama at Birmingham.

Floyd Josephat is Program Director for Clinical Laboratory Science and Clinical Pathologist Assistant Programs at the University of Alabama at Birmingham.


Meera J. Patel, MS; Subhasis B. Biswas, PhD; and Esther E. Biswas-Fiss, PhD, MB(ASCP)CM*

Novel variants of unknown significance are often discovered through genetic testing, which may give rise to the unclear prognosis of pathogenicity. Integration of molecular diagnostic testing with in silico bioinformatic tools and biomedical research can provide enhanced clarification on disease association of these variants. In this report, we describe how integrated approaches were used to predict the pathogenicity of a variant of unknown significance in the ABCA4 gene identified through next-generation sequencing. This case has shown that bioinformatic tools used in conjunction with genetic testing can enable healthcare providers with a holistic assessment for pathogenic variants identified through molecular diagnostic technologies.

Recently, N. Wangtiraumnuay et al. reported the case of a healthy 11-year-old female of asymptomatic parents with a gradual vision decline in both eyes over a four-year period. The patient’s clinical phenotype was determined to be consistent with the features of Stargardt disease through standard ophthalmologic testing. The patient presentation prompted molecular testing for specific genes associated with visual diseases, particularly Stargardt disease. Through next-generation sequencing, a heterozygous paternally inherited ABCA4 variant of unknown significance, c.6184_6187delGTCT, was identified.1

Figure 1. (A) ABCA4 protein is localized in the photoreceptor cells in the retina of the eye, specifically localized to the outer segment discs, and is a key player in the visual transduction cycle. (B) The ABCA4 polypeptide harbors a well-conserved VFVNFA motif in the NBD2 region. (C) Four-nucleotide deletion (c.6184_6187delGTCT) of ABCA4 in the NBD2 region (solid black line) resulted in an altered polypeptide sequence (light green shaded region), deletion of the VFVNFA motif (solid yellow line), and premature truncation of the protein.

ABCA4, a retina-specific membrane transporter localized in the photoreceptor outer segment discs, is essential for the visual cycle (Figure 1A). ABCA4 variants have been associated with a spectrum of autosomal recessive retinal degeneration which culminate in blindness, including Stargardt disease, fundus flavimaculatus, and cone-rod dystrophy.2 The disorders differ from one another with respect to their age of onset and rate of disease progression. Despite the identification of approximately 1,200 disease-associated ABCA4 alleles, the phenotypic-genotypic correlations remain unclear.3 Recently molecular diagnostics has made significant strides in clinical ophthalmological practice for diagnosis and genetic counseling.4

A variety of methods were utilized to understand the genetic and proteomic consequences of the clinically reported c.6184_6187delGTCT variant in ABCA4. Bioinformatic analysis recognized that the mutation is localized in the mid-region of nucleotide binding domain 2 (NBD2) (Figure 1B). Further in silico studies revealed that this four-nucleotide deletion resulted in an alteration of 51 amino acid residues and a premature stop codon, ultimately, truncating the 2273 amino acid protein by 161 amino acids, including the well-conserved VFVNFA motif in the C-terminal domain (Figure 1B). Multiple-sequence alignment of other ABCA transporters show that the VFVNFA motif is a well-conserved sequence which may have more profound functional implications on the overall ABCA4 transporter function. Molecular diagnostic testing and bioinformatic assessments suggested that the ABCA4 sequence variant is pathogenic, however, still lacked the functional validation at the protein level.1

Biomedical research was further used to validate the loss-of-function of the Stargardt-associated ABCA4 variant by functional characterization of recombinant proteins.6 Interestingly, the ABCA4 variant resulted in the loss of less than 7 percent of the protein, which included the VFVNFA motif (Figure 1B). Structurally, the variant harbored a disrupted C-terminal region and led to the deletion of the Walker B motif, the VFVNFA motif, and crucial secondary protein structures (Figure 1C).6 Data obtained from subsequent biochemical studies demonstrated the significant attenuation of functional activity and intra-protein molecular interactions. Thus, it was crucial to assess the importance of this genetic variant on the overall biologic function of the protein.

Overall, a holistic combination of molecular diagnostic testing, clinical assessment, bioinformatics, and biologic functional analysis correlated the ABCA4 variant to the proteomic consequences and its pathogenicity. Genotyping is standard practice for identifying pathogenic variants, however, as technology is becoming more sophisticated, an increasing number of novel variants of unknown significance are discovered. Through a collaborative approach involving medical laboratory scientists, clinicians, bioinformaticians, and biomedical researchers, explanations of novel variant phenotypes identified by molecular diagnostic testing may provide evidence-based genetic counseling and therapeutic decisions in the future. This case further emphasizes the importance of molecular theory and practice in clinical laboratory science curricula.

Financial Disclosure

Work supported, in part, by NIH (EY013113 to EEBF) and funds from the University of Delaware College of Health Sciences (Newark, DE).


We thank Drs. A. Levin and N. Wangtiraumnuay, and Ms. J. Capsso (Wills Eye Hospital, Philadelphia) for helpful discussions and the Graduate College at the University of Delaware (Newark) for the Summer Doctoral Fellowship (to MJP).

  1. N. Wangtiraumnuay, J. Capasso, M. Tsukikawa, A. Levin, E. Biswas-Fiss, Novel ABCA4 mutation leads to loss of a conserved C-terminal motif: implications for predicting pathogenicity based on genetic testing, Eur J Ophthalmol 28 (2018) 123-126. 10.5301/ejo.5001019.
  2. N. Zhang, Y. Tsybovsky, A.V. Kolesnikov, M. Rozanowska, M. Swider, S.B. Schwartz, E.M. Stone, G. Palczewska, A. Maeda, V.J. Kefalov, S.G. Jacobson, A.V. Cideciyan, K. Palczewski, Protein misfolding and the pathogenesis of ABCA4-associated retinal degenerations, Hum Mol Genet 24 (2015) 3220-3237. 10.1093/hmg/ddv073.
  3. F.P.M. Cremers, W. Lee, R.W.J. Collin, R. Allikmets, Clinical spectrum, genetic complexity and therapeutic approaches for retinal disease caused by ABCA4 mutations, Prog Retin Eye Res (2020) 100861. 10.1016/j.preteyeres.2020.100861.
  4. K.A. Sadagopan, J. Capasso, A.V. Levin, Genetics for the ophthalmologist, Oman J Ophthalmol 5 (2012) 144-149. 10.4103/0974-620X.106092.
  5. T. Can, Introduction to bioinformatics, Methods Mol Biol 1107 (2014) 51-71. 10.1007/978-1-62703-748-8_4.
  6. M.J. Patel, S.B. Biswas, E.E. Biswas-Fiss, Functional significance of the conserved C-Terminal VFVNFA motif in the retina-specific ABC transporter, ABCA4, and its role in inherited visual disease, Biochem Biophys Res Commun 519 (2019) 46-52. 10.1016/j.bbrc.2019.08.121.

Meera J. Patel is a PhD candidate in the Department of Medical and Molecular Sciences, University of Delaware, College of Health Sciences, in Newark, Delaware.

Subhasis B. Biswas Professor in the Department of Medical and Molecular Sciences, University of Delaware, College of Health Sciences, in Newark, Delaware.

*Esther E. Biswas-Fiss is Professor and Chair of the Department of Medical and Molecular Sciences, University of Delaware, College of Health Sciences, in Newark, Delaware.

*Corresponding author