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David Plaut: Off the Cuff

A Change in QC Rules?
September 17, 2014 10:22 AM by David Plaut

In 1974, the College of American Pathologists (CAP) proposed an algorithm for accepting/rejecting analytical runs using 2 controls. The algorithm used 3 rules to reject a run – 1 3SD,* 2 2SDw and 2 2SDa. In 1981, Westgard et al. (JOW) proposed an expanded set of rules. Both groups were clear on the idea that a single value beyond the 2 SD limits was not a reject signal.

In the past 40 years, improvements in instruments, reagents and calibrators have resulted in a significant improvement of precision. This improvement suggests that QC monitoring systems should review rules based on the SDs from current data, not based on data from 30-plus years ago. Do we use 30 year old instruments? Or computers? Or phones?

At the AACC meeting in July, 2014, I presented a poster looking at the changes in precision since 1988. Surveys from 1988 and 2012 were studied. We used %CV to correct for the variation in means from one survey to another. Our survey of 35 analytes from chemistry, hematology and hemostasis indicated a decrease of 40 percent (average; range 24-77) in CVs.

The table shows representative changes in SDs measured as %CV and the change over time.

1988 2012   %Change

Analyte Method Mean %CV Mean %CV

Hemoglobin Coulter 2.9 1.7 41

WBC Coulter 3.8 2.7 24

Cholesterol Abbott 2.9 1.0 66

AST Roche 8.4 5.2 33

Ca Baker 4.9 1.3 77

Prothrombin time    * 6.4 2.8 56

APTT Stago 10.4 2.9 78

*In 1988 most laboratories user a manual method.


•             The changes in analytical precision strongly suggest that QC rules be reevaluated. We "translated" the SD of the year 2012 into a new set of rules to detect both systematic and increased random errors. Our translations indicate that based on a 40 percent decrease the following rules would work very effectively: 1 4 SD, 2 3 SD, and R 5 SD. If you are squeamish about the 1 4 SD rule, use either 1 3 SD or at least 1 2.5 SD

•             The changes in the rules will yield essentially 0% false rejects and an error detection of nearly 100%

•             As before each analyte should be assessed to determine the proper rule(s).

Hops and Sleeping
September 10, 2014 12:36 PM by David Plaut

I ran across an article recently that I thought you, like me, would find it interesting. The title of the article is “Effect of non-alcoholic beer on Subjective Sleep  in  university stressed population.” Of course, you easily relate to the word “stressed.” Who working in a laboratory isn’t stressed? The other part of the title was the idea of “non-alcoholic beer.” You may, as did I, say how can that be? What is beer if not alcoholic? But is does exist – if only to get the hops into your system. It turns out that beer is the only beverage that contains hops, and hops is known to have a sedative effect.

Here is the result of this study and another one carried out on nurses. There is a sleep quality index from Pittsburgh that measures the quality of your sleep and uses the 30 students as their own control for the first week of three. During the last two weeks, the students were asked to drink the NAB at dinner and then fill out the survey. The overall rating improved significantly (“p<0.05”), as was the sleep latency also a level of significance of “<0.05.” As I mentioned, another study measured other aspects of sleep and found the same high level of significance. 

It turns out that there are quite a number of non-alcoholic beers, although they can contain as much as 1.0% (but usually 0.5%) alcohol.


1. Acta Physiol Hung. 2014 Sep;101(3):353-61..Effect of non-alcoholic beer on Subjective Sleep Quality in a university stressed population. Franco L, Bravo R, Galán C, et al.

2. PLoS One. 2012;7(7):e37290. The sedative effect of non-alcoholic beer in healthy female nurses. Franco L  Sánchez C, Bravo R, et al.

Troponin: An update
September 4, 2014 1:18 PM by David Plaut

I have been working with troponin since it came to this country several years ago. Most of my experience has been with troponin I (cTnI). When cTnI first was released to the laboratories the cut-off was set at 0.4. Then it dropped to 0.2 and now it has been lowered by some laboratories even further. These newer variations have pros and cons as you have seen. For example, there are reports of measurable cTn following a marathon.

"The increase in early diagnostic sensitivity of hs-cTn assays for ACS comes at the cost of a reduced ACS specificity, because more patients with other causes of acute or chronic myocardial injury without overt myocardial ischemia are detected than with previous cTn assays." 

These newer assays detect low levels of cTn in apparently healthy people. "In addition, the sensitive assays detect more cTn positive patients who do not have a final diagnosis of ACS. It is unknown if such mild elevations in cTn detected by sensitive assays are of clinical concern. What is certain is that AMI remains a clinical not a biochemical diagnosis and interpretation of cTn concentrations should be made according to the clinical context." It has been demonstrated that the newer assays are better able (using the area under the ROC curve) to identify AMI in patients with existing CAD compared to the ‘standard' cTn.

A diagnostic accuracy study of patients presenting to the emergency department (ED) with symptoms of ACS was performed. Troponin was measured at 0, 2 and 6h post-presentation. AMI was made by 2 cardiologists and incorporated the 0 and 6h troponin values measured by a sensitive troponin assay. There was no significant difference in the diagnostic accuracy of early versus late biomarker strategies when used with the current risk stratification processes. Incorporation of a significant delta did not improve the stratification at 2h post-presentation."


1. Mair J., World J Cardiol. 2014 Apr 26;6(4):175-82.

2. Gaze DC., Curr Med Chem. 2011;18(23):3442-5. Curr Med Chem. 2011;18(23):3442-5.

3. Reiter M, Twerenbold R, Reichlin T, et al. Eur Heart J. 2012 Apr;33(8):988-97.

4. Cullen L, Greenslade J, Than M et al., Heart Lung Circ. 2014 May;23(5):428-34.

Laboratory Errors
August 22, 2014 12:54 PM by David Plaut

Laboratories have a major impact on patient safety: 80-90% of all the diagnoses are made on the basis of laboratory tests. Laboratory errors have a reported frequency of 0.012-0.6% of all test results.

Patient safety is a managerial issue which can be enhanced by implementing active system to identify and monitor quality failures. This can be facilitated by reactive method which includes incident reporting followed by root cause analysis. This leads to identification and correction of weaknesses in policies and procedures in the system. Another way is a proactive method like Failure Mode and Effect Analysis.

Here are synopses of two studies aimed at quantifying preanalytical errors which can be reduced by continuous education and FMEA approaches. In a study of data from 105 laboratories and 4,715,132 tubes during the data collection period, according to determinations by clinicians in the request form, 32,977 (0.7%) were found to be rejected. Whole blood-EDTA samples and serum samples accounted for 76% of all samples collected among laboratories, although they corresponded to only 56% of all rejections. In total, 81% of rejections arose as a result of the following reasons:

  • "specimen not received" (38%),
  • "hemolysis" (29%), and
  • "clotted sample" (14%).

Moreover, plasma-citrate-erythrocyte sedimentation rate exhibited the highest percentage of rejection (1.5%), whereas the lowest rate corresponded to whole blood-EDTA (0.38%).

During a 1-year period, a total of 168,728 samples and 88,655 requests forms were received in a Stat laboratory. The total number of preanalytical errors was 1457, accounting for 0.8% of the total number of samples received in a year. Of the total preanalytical errors, 46% were hemolysed samples (biochemistry), 43% were clotted samples (hematology), 6% were samples lost-not received in the laboratory, 2.9% samples showed an inadequate sample-anticoagulant ratio, 0.7% were requests with errors in patient identification, 0.3% were samples collected in blood collection tubes with inappropriate anticoagulant and 0.1% were requests with errors -- missing test requests.


  1. Alsina MJ, Alvarez V, Barba N, et al. Clin Chem Lab Med. 2008;46(6):849-54.
  2. Grecu DS, Vlad DC, Dumitrascu V. Lab Med. 2014 Winter;45(1):74-81.
  3. Agarwal R. Indian J Clin Biochem. 2013 Jul;28(3):227-34.

For more on Failure Mode and Effect Analysis (FMEA) see http://asq.org/learn-about-quality/process-analysis-tools/overview/fmea.html and http://www.ihi.org/resources/Pages/Tools/FailureModesandEffectsAnalysisTool.aspx

Humans, Guinea Pigs and Bats
August 20, 2014 2:58 PM by David Plaut

Why can humans (and guinea pigs and dry-nosed primates and bats) not make vitamin C and are thus open to scurvy without replacement?

Many years ago, I worked on a study of guinea pigs that had been fed a diet without vitamin C and thus developed scurvy.  I knew that people, like the guinea pigs, could develop scurvy without adding the vitamin to our diet.  This has been known for more than a century and in the last few years we have found out why it happens to these few animals.

The inability of humans to synthesize L-ascorbic acid is known to be due to a lack of an enzyme that is required for the biosynthesis of this vitamin. The enzyme is known as L-gulono-gamma-lactone oxidase (GULO). Isolation of a cDNA for the rat enzyme resulted in a study of the basic defect underlying this deficiency at the gene level and led to isolation of a human genomic clone related to L-gulono-gamma-lactone oxidase as well as three overlapping clones covering the entire coding region of the rat L-gulono-gamma-lactone oxidase cDNA. Sequence analysis study indicated that the human L-gulono-gamma-lactone oxidase gene has accumulated a large number of mutations since it stopped being active and that it now exists as a pseudogene in the human genome.

This genetic defect has not been selected against in natural selection as we are able to consume more than enough vitamin C from our diet. It is also suggested that organisms without a functional GULO gene have a method of "recycling" the vitamin C that they obtain from their diets using red blood cells.


  1. Cell. 2008 Mar 21;132(6):1039-48
  2. Hum Gene Ther. 2008 Dec;19(12):1349-58
  3. Am J Clin Nutr. 1991 Dec;54(6 Suppl):1203S-1208S.
PSA Kinetics
August 6, 2014 2:56 PM by David Plaut

For the past few decades, many clinicians have used the change in PSA over a 1 or 2 year period to determine whether a biopsy was needed (for example when my PSA increased by more than the limit at that time, I was encouraged to have a biopsy. I did.) 

There has historically been considerable uncertainty about PSA kinetics for decisions about biopsy and initial treatment. It turns out that calculation of PSA velocity and doubling time is far from straightforward. More than 20 different methods have been proposed, and many of these give quite divergent results. There is clear evidence that PSA kinetics are critical for understanding prognosis in advanced or relapsed prostate cancer. However, PSA kinetics have no value for men with an untreated prostate: neither PSA velocity nor doubling time have any role in diagnosing prostate cancer or providing a prognosis for men before treatment.

Given current data on PSA velocity and doubling time, Vickers et al. proposed somewhat middle of the road these recommendations:

  1. High PSA velocity is not an indication for biopsy;
  2. for men with a low total PSA but a high PSA velocity, consideration should be given to measuring PSA at a shorter interval;
  3. men with an indication for biopsy should be biopsied irrespective of PSA velocity;
  4. changes in PSA after negative biopsy findings do not determine the need for repeat biopsy;
  5. monitoring PSA over time can aid judgment in decisions about biopsy, as informed by the clinical context;
  6. PSA velocity is uninformative of risk at diagnosis;
  7. high PSA velocity is not an indication for treatment in men on active surveillance;
  8. PSA velocity at the time of recurrence should be entered into prediction models (or "nomograms") to aid patient counseling.


  1. Br J Med Surg Urol. 2012 Jul 1;5(4):162-168.
  2. Urology. 2014 Mar;83(3):592-6
Intriguing Troponin Series
July 28, 2014 11:53 AM by David Plaut

I recently was asked to comment on a series of troponin values from a general hospital.

Since this is a series of extremely high troponin, an MI was suspected. Although the EKG/ECG was not normal, there was no evidence of an AMI.

The apparent "change" in the TnI is probably due to the random error rather than a physiological change.

Table: Troponin Series

Day Time Troponin 1 
 1 1325 2.6
 1 1724 2.4
 1 2035 2.3
 2 0248 2.2
 2 06462.1 

I am sorry to say that, by the time I was asked to comment on this, the sample was gone and the patient had been discharged. My response had the sample still been available was to suggest that one or more of the samples be mixed 1+1 and 1+3 with a sample with a very low TnI level (approx. 0).  Then assay the unmixed sample(s) and the dilutions. If the sample contained a heterophilic antibody (my thought) the values would not show linearity.  If the troponin were truly TnI the sample would show linearity.  Another/additional test would be to measure a not constituent measured by immunoassay such as TSH or hCG.  A high value in these would also indicate a heterophilic antibody.

An interesting article on salivary cTnI appeared recently.  In a group of 30 confirmed AMI and 28 non-MI

the cTnI were measured in both serum and saliva.  The interquartile range for the saliva was 0.08-0.23 and for the AMI patients at 12 hrs. post-admission the range was 2.7-11.6 and at 24 hrs. the range was 2.1-9.0

In a similar study by the same authors higher CK levels in saliva were also recorded.

In both cases there was a positive correlation between serum levels of both CK and cTnI and salivary levels.


  1. Indian J Med Res. Dec 2013; 138(6): 861-865
Iron Deficiency and Hepcidin
July 21, 2014 11:00 AM by David Plaut

Iron deficiency (ID) is relatively common among the elderly population, contributing substantially to the high prevalence of anemia observed in the last decades of life, which in turn has important implications both on quality of life and on survival. In elderly subjects, ID is often multifactorial (i.e., due to multiple concurring causes, including inadequate dietary intake or absorption, occult bleeding, medications).

Moreover, because of the typical multi-morbidity of aged people, other conditions leading to anemia frequently coexist and make diagnosis of ID particularly challenging. Treatment of ID is also problematic in elderly, since response to oral iron is often slow, with a substantial fraction of patients showing refractoriness and requiring cumbersome intravenous administration. In the last decade, the discovery of the iron regulatory hormone hepcidin (an acute-phase reacting protein) has revolutionized our understanding of iron pathophysiology.

In serum samples, age- and gender-dependent reference values were determined using serum samples from healthy volunteers (n = 231). Hepcidin is stable for 1 day at room temperature, 6 days at +4°C and at least 42 days at -20°C. Breakfast and the type of sampling device do not affect hepcidin concentration. Reference values for females aged 18-50 years were 0.4-9.2 nmol/L, for those >50 years 0.7-16.8 nmol/L and for males ≥18 years 1.1-15.6 nmol/L.


  1. Front Pharmacol.2014 Apr 23;5:83.
  2. Bioanalysis.2014 Apr;6(8):1081-91
  3. Arthritis Rheum.2011 Dec;63(12):3672-80.
The Human Gut
July 16, 2014 12:49 PM by David Plaut

The human gut is home to trillions of microbes (the intestinal microbiota) that form a symbiotic relationship with the human host. During health, this intestinal microbiota provides many benefits to the host and is generally resistant to colonization by new species; however, disruption of this complex community can lead to pathogen invasion, inflammation, and disease.

Restoration and maintenance of a healthy gut microbiota composition requires effective therapies to reduce and prevent colonization of harmful bacteria (pathogens) while simultaneously promoting growth of beneficial bacteria (probiotics). Accumulating evidence indicates that the gut microbiota plays a significant role in the development of obesity, obesity-associated inflammation and insulin resistance. 

Important to this subject is the concept of "crosstalk" (i.e., the biochemical exchange between host and microbiota that maintains the metabolic health of the superorganism and whose dysregulation is a hallmark of the obese state). Differences in community composition, functional genes and metabolic activities of the gut microbiota appear to distinguish lean vs obese individuals, suggesting that gut "dysbiosis" contributes to the development of obesity and/or its complications. The current challenge is to determine the relative importance of obesity-associated compositional and functional changes in the microbiota and to identify the relevant taxa and functional gene modules that promote leanness and metabolic health.

As diet appears to play a predominant role in shaping the microbiota and promoting obesity-associated dysbiosis, parallel initiatives are required to elucidate dietary patterns and diet components (e.g., prebiotics, probiotics) that promote healthy gut microbiota.


  1. Mol Aspects Med. 2013 Feb;34(1):39-58.
  2. J Mol Biol. 2014 Jun 6. pii: S0022-2836(14)00279-4.
  3. Gastroenterol Clin North Am. 2012
  4. Curr Opin Clin Nutr Metab Care. 2011
  5. Pharmacol Ther. 2011
Curing HIV
July 14, 2014 11:54 AM by David Plaut

What have we learned about early treatment of HIV-1 from the "Mississippi Baby"?

Due to the increased risk of human immunodeficiency virus (HIV) infection during the child bearing years, voluntary screening during the prenatal period has been suggested. HIV testing was offered as a component of a prenatal laboratory panel, using informed refusal. During the first screening period, there were 20 seropositive women among the 14,143 patients (1.4/1000).  Free treatment with zidovudine (AZT) for both mother and baby began in January 1994. The perinatal transmission rate was 33% before AZT treatment, during the period of assessment, and was reduced to 10% during the next 30 months.

A child, nicknamed the Mississippi Baby, did not receive any prenatal HIV care. Because of a greater risk of infection, she was started on a powerful HIV treatment just hours after labor. She continued to receive treatment until 18 months old, when doctors could not locate her. When she returned 10 months later, no sign of infection was evident though her mother had not given her HIV medication in the interim. Repeated tests showed no detectable HIV virus until last week. She had appeared free of HIV as recently as March, without receiving treatment for nearly 2 years. Doctors do not yet know why the virus re-emerged.

A second child with HIV was given early treatment just hours after birth in Los Angeles in April 2013. Subsequent tests indicate she completely cleared the virus, but that child also received ongoing treatment.

Only one adult is currently believed to have been cured of HIV. In 2007, Timothy Ray Brown received a bone marrow transplant from a donor with a rare genetic mutation that resists HIV. He has shown no signs of infection for more than 5 years.


  1. BBC News Medicine. July 10, 2014 
  2. BBC News Medicine. March 4, 2013
  3. Pediatr AIDS HIV Infect. 1997 Feb;8(1):12-4
Quorum Sensing
July 14, 2014 11:47 AM by David Plaut

Quorum sensing (QS) is a bacterial communication process that depends on the bacterial population density. It involves small diffusible signaling molecules which activate the expression of myriad genes that control a diverse array of functions including virulence. Quorum sensing is a process of cell-cell communication that allows bacteria to share information about cell density and adjust gene expression accordingly.

This process enables bacteria to express energetically expensive processes as a collective only when the impact of those processes on the environment or on a host will be maximized. Among the many traits controlled by quorum sensing is the expression of virulence factors by pathogenic bacteria. 

As QS is responsible for virulence in the clinically relevant bacteria, inhibition of QS appears to be a promising strategy to control these pathogenic bacteria. QS antagonists should be viewed as blockers of pathogenicity rather than as anti-microbials and because QS is not involved in bacterial growth, inhibition of QS should not yield a strong selective pressure for development of resistance. QS inhibitors hold great expectations and we may look forward to their application in fighting bacterial infections. 


  1. EMBO Mol Med. 2009 Jul;1(4):201-10
  2. Recent Pat Antiinfect Drug Discov. 2013 Apr;8(1):68-83.
Biomarkers for Acute Kidney Injury
July 1, 2014 11:28 AM by David Plaut

The kidney has a remarkable capacity to withstand insults for an extended period of time. The sensitivities of individual renal cells to injury vary depending on their type, position in the nephron, local vascularization, and the nature of injury. The resulting kidney injury is a product of the interplay between cell dysfunction, cell death, proliferation, inflammation, and recovery.

Acute kidney injury (AKI) is a common and serious condition in both the inpatient and outpatient settings, and its diagnosis depends on serum creatinine or cystatin C measurements.

Unfortunately, creatinine is a delayed and unreliable indicator of AKI. The lack of early biomarkers has limited our ability to translate promising experimental therapies to human AKI. Fortunately, understanding the early stress response of the kidney to acute injuries has provided a number of potential biomarkers. For example, neutrophil gelatinase-associated lipocalin (NGAL) is emerging as an excellent stand alone biomarker in the plasma and urine for predicting and monitoring clinical trials and in the prognosis of AKI. In recent years, a number of new biomarkers of AKI with more favorable test characteristics.

The two tables indicate the utility of the newer markers, some of which are commercially available indicate the utility of these markers compared to cystatin C.  Because the pattern of appearance in the urine or serum, varies with the markers it may be necessary to use more than one marker to detect and monitor AKI.

Efforts to detect AKI in the earlier stage has resulted in some promising biomarkers such as KIM-1, NGAL, IL-18, and Clusterin. Cystatin C is a biomarker for glomerular filtration function, while 2-microglobulin, 1-microglobulin, NAG, RBP, IL-18, NGAL, Netrin-1, KIM-1, Clusterin, Sodium Hydrogen Exchanger Isoform and Fetuin A are biomarkers for tubular reabsorption function.

For Additional Reading

Geriatric patients and anemia
June 19, 2014 11:28 AM by David Plaut

Anemia is a common condition in the elderly (for example, in a study of .6880 individuals, 2905 men and 3975 women, aged 65-95 [mean age 72.5], mild anemia [hemoglobin levels <10 g/dL] was found in 6.1% of women and 8.1% of men.) The reason that LDH shows up is that there is 20% more LDH in red cells than serum. More than 10% of the population over 70 years have iron and/or vitamin B-12 deficiency.  Here is a flow chart that may help evaluate chronic anemia: http://asheducationbook.hematologylibrary.org/content/2012/1/183/F3.expansion.html

For Additional Reading

Body on a Chip
June 17, 2014 10:55 AM by David Plaut
In a recent blog, I touched on some ideas and tools that are being used to build "labs on chips."  The word nano operates significantly in this area.  If you were as excited and intrigued with that blog, read on for now we are going to talk on "organs-on-a-chip" and even a "body-on-a-chip."  Just how does one model, test, and learn about the communication and control of biological systems with individual organs-on-chips that are one-thousandth or one-millionth of the size of adult organs, or even smaller, i.e., organs for a milliHuman (mHu) or microHuman (μHu)?

With serious work being done to realize functioning artificial livers, kidneys, hearts, and lungs on chips, the next step is not only to interconnect these organs but also to consider the integration of stem cell technology to create interconnected patient-specific organs. Such a patient-specific body-on-a-chip requires a sophisticated set of tools for micropattering cell cultures in 3D to create interconnected tissue-like organ structures. It seems that anticipation that such a technology would have a wide area of application, primarily benefiting drug development, chemical safety testing, and disease modeling.

We are not there, yet.  But it is certain that a large amount of work is going into these projects not just for the ‘fun' of creating, say, a kidney, on a chip but for the results will aid in drug testing and replacement medicine and individual (unique) drug treatment.

For Additional Reading

  1. Wikswo JP. Lab Chip. 2013 Sep 21;13(18):3496-511. Scaling and systems biology for integrating multiple organs-on-a-chip.
  2. Moraes C. Integr Biol (Camb). 2013 Sep;5(9):1149-61. On being the right size: scaling effects in designing a human-on-a-chip.
  3. Williamson A. Lab Chip. 2013 Sep 21;13(18):3471-80. The future of the patient-specific Body-on-a-chip.
Middle East Repiratory Syndrome
June 11, 2014 9:56 AM by David Plaut

Middle East Repiratory Syndrome (MERS) recently was said to be a "public health emergency of international concern." The MERS virus, which appeared in the Middle East in 2012, has spread through that area; cases have been found in Asia, Europe and the United States. The mortality rate is near 30%.

Two health workers at a hospital in Orlando, Fla., who were exposed to a patient with MERS exhibited flu-like symptoms, and one was hospitalized. 

MERS, which causes coughing, fever and occasionally fatal pneumonia, is a virus from the same family as SARS, which has killed about 800 people worldwide since it first appeared in China in 2002. 

MERS, a second coronavirus, SARS being the first, is transmissible from person to person, and its close relationship with several bat coronaviruses suggests that these animals may be the ultimate source of the infection. However, many key issues need to be addressed, including identification of the proximate, presumably zoonotic, source of the infection, the prevalence of the infection in human populations, details regarding clinical and pathological features of the human infection, the establishment of a small rodent model for the infection, and the virological and immune basis for the severe disease observed in most patients. Margaret Chan, Director-General of the World Health Organization, has called MERS-CoV "a threat to the entire world." There is no vaccine for MERS.

For Additional Reading



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