Gram-negative bacteria carrying extended-spectrum β-lactamase (ESBL) enzymes now represent a significant proportion of all bacteria isolated from different countries worldwide. The increased prevalence of isolates carrying carbapenemases in recent years constitutes a greater challenge leading to multidrug-resistant (MDR), extensively-drug resistant (XDR), and pandrug-resistant (PDR) bacteria. The Countries in the Gulf Cooperation Council (GCC) have high prevalence of β-lactamase -producing Gram-negative bacteria possibly due to misuse of antibiotics, suboptimal compliance with infection control practices and hand hygiene, lack of antimicrobial stewardship programs, and the large population of immigrants, especially from the Indian subcontinent where β-lactamases are very common.

Mechanisms of antimicrobial resistance

Antimicrobial resistance may be intrinsically expressed by chromosomal genes or acquired through plasmids. Plasmid-mediated resistance is more worrisome because resistance genes can spread to different bacteria through horizontal gene transfer. Different mechanisms of antimicrobial resistance in Gram-negative bacteria have been identified including target modification, efflux pumps, hydrolyzing enzymes (e.g. β-lactamases). The commonest mechanism of resistance in Gram-negative bacteria is the presence of β-lactamases that hydrolyze the β-lactam ring present in penicillins, β-lactam and β-lactamase inhibitor combinations, cephalosporins, monobactam (e.g. aztreonam) and carbapenems. β-lactamases constitute a heterogeneous group of enzymes and they are classified according to two general schemes: the Ambler molecular classification (classes A through D) based on protein homology and the Bush–Jacoby–Medeiros classification (groups 1 through 4) based on functional properties of enzymes. β-lactamases of class A, C, and D are serine β-lactamases whereas class B enzymes are metallo-β-lactamases (MBL).

The most clinically important β-lactamases include extended spectrum β-lactamases (ESBL), AmpC enzymes and carbapenemases. ESBL enzymes (e.g. TEM, SHV, CTX-M) are plasmid mediated and are mainly present in the Enterobactericeae family (e.g. E. coli and K. pneumonia) (6). Bacteria carrying ESBL enzymes are usually resistant to penicillins, cephalosporins except cephamycins (cefotetan and cefoxitin), and aztreonam . The enzyme is inhibited by β-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam. The predominant ESBL enzyme in United Arab Emirates (UAE) is CTX-M-15, present mainly in E. coli and K. pneumoniae.

AmpC β-lactamases are mainly present on the bacterial chromosome although some species like E. coli and K. pneumoniae can acquire AmpC on plasmids. AmpC enzymes hydrolyze penicillins, cephalosporins including cephamycins and aztreonam. Unlike ESBLs, AmpC enzymes are not inhibited by β-lactamase inhibitors but can be inhibited by cloxacillin. Modifications in regulatory mechanisms in some bacteria can result in AmpC overexpression and therapy failure. This occurs in SPICE organisms (Serratia marcescens, Pseudomonas aeruginosa, Indole-positive Proteeae (Morganella morganii & Providencia), Citrobacter freundii and Enterobacter spp.

Carbapenem resistance can be due to efflux pumps (e.g. in P. aeruginosa), loss of Porins leading to impermeability to carbapenem and more commonly production of carbapenemases. Carbapenemases can be classified into group A (e.g. Klebsiella pneumoniae carbapenemase (KPC)), group B (e.g. New Delhi MBL (NDM)) and group D (e.g. oxacillinase (OXA)). Group A enzymes hydrolyze all β-lactams and are usually plasmid- mediated. KPC is the most common carbapenemase in USA, it is usually expressed at high levels, and plasmids carrying KPC enzymes also carry other resistance genes (e.g. ESBL). NDM is the most common MBL worldwide and it is common in India and Pakistan. The first report of NDM was in 2008 in a Swedish patient who was hospitalized in India. MBLs require zinc for activity and are inhibited in vitro by zinc chelator (e.g. EDTA). OXA enzymes hydrolyze carbapenems and extended-spectrum cephalosporins at low levels. The predominant carbapenemases in UAE are OXA-48 and NDM-1.

Recently, due to the increased spread of multidrug resistant bacteria especially carbapenem-resistant isolates, colistin (polymyxin E) has been increasingly used. Colistin is a lipopeptide that was used in the 1960s and 1970s for Gram-negative bacteria but it was abandoned due to renal and neurologic toxicity and the availability of other agents. Colistin targets the cell membrane through electrostatic interaction with lipopolysaccharides (LPS) leading to displacement of the divalent cations (Mg²⁺ and Ca²⁺), cytoplasmic leakage and cell death. Intrinsic resistance to colistin is present in Morganella morganii, Brucella, Proteus, Serratia, and Burkholderia. Acquired resistance in E. coli, Acinetobacter, K. pneumoniae and P. aeruginosa is mostly due to modification of LPS target. Recently, resistance to colistin due to plasmid mediated mcr-1gene has been described.

Laboratory testing of β-lactamases

There are many laboratory tests available to detect β-lactamases. If the laboratory is using the current Clinical & Laboratory Standards Institute (CLSI) guidelines for cephalosporins and carbapenems, there is no need to use these tests except for infection control purposes when an outbreak is suspected or for epidemiology and research purposes to understand the mechanisms of emerging resistance. However, the microbiologist maybe faced with certain situations or discrepancies where confirmation of resistance is necessary. If the laboratory is using the old CLSI guidelines, these tests are necessary to confirm or exclude resistance. For ESBLs, testing is accomplished by disc diffusion, E-test, and broth microdilution and the comparison of minimum inhibitory concentrations (MICs) or zone of inhibition with and without β-lactamase inhibitor (e.g. clavulanic acid). These tests are standardized for E. coli, Klebsiella spp. and P. mirabilis, yet, ESBLs occur in other species. In addition, ESBLs are not always detected if multiple resistance mechanisms are present producing false negative ESBL test. For AmpC, there is no FDA-approved assays available in the US but some laboratories use AmpC tests for suspected plasmid-mediated AmpC resistance based on inhibition by cloxacillin. For bacteria carrying chromosomal AmpC, accurate identification of species (e.g. Enterobacter cloace, Citrobacter freundii, Serratia marcescens) is adequate to assume that these bacteria are AmpC producers. Detection of carbapenemase activity in a clinical isolate in the laboratory is a difficult but an elevated carbapenem MIC should raise suspicion that a carbapenemase is present. Several methods including modified Hodge test, Carba NP test, and molecular assays can be used.

Antimicrobial susceptibility tests for colistin in the US are not FDA- cleared and all available tests are research use only (RUO). Testing colistin is difficult because it is a large molecule and its diffusion in the agar is slow. Several studies have reported unacceptable categorical agreement for both disc diffusion and E-test (very major error rates up to 32% for disc diffusion and up to 53% for E-test). In addition, colistin adsorbs to plastic microtiter plates yielding falsely high MICs due to loss of colistin to the surface. One study found that only 7.5% of colistin is available in broth and the rest is all adsorbed in the wells of a polystyrene broth microdilution plate. The use of disc diffusion, agar dilution, E-test is unreliable and only broth microdilution is recommended at this time as recommended by the joint CLSI-EUCAST polymyxin breakpoints working group.

Treatment and prevention of MDR infections

The number of antimicrobial agents reliably effective against multidrug resistant bacteria is very limited (5). Carbapenems can be used to treat infections caused by organisms carrying AmpC and ESBL enzymes. Cefepime is a poor inducer of AmpC and may have a role in treating organisms producing AmpC (5). Treatment modalities for multidrug resistant Gram-negative bacteria especially carbapenem-resistant isolates include polymyxins (Colistin, polymyxin B), tigecycline, minocycline, aminoglycosides, fosfomycin, ceftazidime-avibactam (Avycaz) ™, ceftolozane-tazobactam (Zerbaxa) ™, and a combination of antimicrobial agents.

From the laboratory perspective, there are many strategies the laboratory can implement to combat resistance. Susceptibilities should be performed only when it is required. Selective reporting of antibiotics can improve the clinical relevance of test reports and minimize the selection of multidrug resistant strains. Broad-spectrum agents are reported only when the isolate is resistant to narrower-spectrum agents. In addition, each laboratory should develop a protocol to deal with MDR bacteria including testing additional agents in-house or at a reference lab. Screening for multidrug resistant strains especially carbapenem resistant Enterobacteriaceae (CRE) from stool using chromogenic agars or molecular methods will help to isolate colonized patients and prevent spread of resistant bacteria.

Indeed, there has been a call for a beta-cell centric approach to classifying diabetes. At a research symposium “The Differentiation of

Diabetes by Pathophysiology, Natural History and Prognosis” convened by the American Diabetes Association, JDRF, the European Association for the Study of Diabetes, and the American Association of Clinical Endocrinologists in October 2015, it was concluded by strong consensus that the primary defect resulting in hyperglycaemia is insufficient beta-cell number and/or beta-cell function, and that biomarkers and imaging tools are needed to assess beta-cell mass and loss of functional mass and to monitor progression and response to therapeutic interventions.

In research settings, methods for assessing beta-cell secretory function include the hyperglycaemic clamp, and intravenous or oral glucose tolerance tests with application of minimal models. These protocols are labour intensive to perform or technically challenging, and are thus not practical to carry out in routine clinical practice. 

It should also be noted that the assessment of insulin secretion should be differentiated from the assessment of beta cell function, as the latter requires the rate of insulin secretion to be interpreted in relation to the prevailing glucose concentration and the insulin sensitivity of the individual.

Beta-Cell Function Assessment in Clinical Practice

C-peptide measurements

C-peptide is produced in equal amounts to insulin and can be used to assess endogenous insulin secretion, even in patients on exogenous insulin treatment. C-peptide does not undergo hepatic extraction unlike insulin, thus could reflect endogenous insulin secretion more directly. Furthermore, advances in assays have made assessment of insulin secretion using C-peptide cheaper, more reliable and more accessible.

Measuring C-peptide following stimulation may be advantageous in the assessment of beta cell function. The concurrent measurement of glucose would allow the exclusion of hypoglycaemia, while a glucose of >8 mmol/L could be considered a stimulated value. While it has been proposed that C-peptide results are corrected for concurrent glucose measurements, with better correlation with beta cell mass and glucose intolerance after islet transplantation, the interpretation of the ratio may be difficult due to limited published data. To distinguish subjects unlikely to achieve glycaemic control with non-insulin therapies, cut-offs of <0.25 nmol/L for fasting and <0.6 nmol/L for non-fasting (random) C-peptide have been suggested.

HOMA- β

A popular and easy method of assessing beta cell function that takes into account both insulin secretion indices and glucose readings is the HOMA-β, derived from basal measurements of insulin and glucose. The use of C-peptide instead of insulin in HOMA- β further avoids the confounding effect of hepatic insulin extraction.

The computer model for calculating HOMA- β can be downloaded online*, and was updated to account for insulin resistance and to allow for an increase in insulin secretion when glucose is ≥ 10 mmol/L. The computer model can be used to determine insulin sensitivity (%S) and β -cell function (%B) from paired fasting plasma glucose and RIA insulin, specific insulin, or C-peptide concentrations, and can be used for populations and individuals, although in individuals, the average of triplicate readings are recommended to improve the coefficient of variation. In individuals, HOMA can be used to identify whether insulin resistance or beta cell failure is predominant or to track changes in insulin sensitivity and beta-cell function longitudinally.

A consideration is the application of the HOMA- β in individuals on insulin or insulin secretagogues. While theoretically the use of C-peptide HOMA model may be used for people on insulin therapy, the use of the model has not been verified in this situation. For those on insulin secretagogues, results should be interpreted with caution, as the HOMA- β may simply reflect the secretagogue action of the drug.

In addition, HOMA- β should be interpreted together with the HOMA-IR/%S since the model apportions the basal state of insulin and glucose in terms of resistance and β-cell function. Thus, an individual with improved insulin sensitivity over time may have an appropriate reduction in HOMA- %β, and this would not reflect failing beta cell function.

Insulin/glucose or C-peptide/glucose ratios

Even simpler than HOMA-β would be the expression of C-peptide/glucose.

A close correlation with fractional beta-cell area was found with fasting and post-oral glucose insulin/glucose and C-peptide/glucose ratio in a study of 33 patients undergoing pancreatic surgery. In contrast, HOMA- β was not able to predict beta-cell area in this study. The C-peptide /glucose ratio after oral glucose ingestion gave the best estimation of beta-cell area in this study. However, it should be noted that this study was carried out in individuals who were undergoing pancreatic surgery for various underlying pancreatic disorders such as chronic pancreatitis, and were thus different from typical patients with type 2 diabetes.

Among Japanese patients with type 2 diabetes, it was found that the post-prandial (2 hours after breakfast) C-peptide to glucose ratio (C-peptide in ng/ml / Glucose in mg/dL x 100) was well-correlated with fasting values, but performed better than the fasting indices in predicting insulin therapy, with specificity of 80.5% and sensitivity of 61.0% using a cut-off value of 1.53.

In a study of Korean patients with newly diagnosed type 2 diabetes, it was found again that the post-prandial (90-minutes after standardized liquid meal of Ensure) C-peptide/glucose ratio was well correlated with other indices of beta cell function such as HOMA-β, insulinogenic index [(insulin 90min – insulin 0 min)/(glucose 90 min – glucose 0 min)] and index for C-peptide [(C-peptide 90 min – C-peptide 0 min)/(glucose 90 min – glucose 0 min)], and showed a stronger correlation with HOMA- β than post-prandial C-peptide alone. The post-prandial C-peptide/glucose ratio also showed a stronger correlation with indices of glycaemic control (HbA1c and glycated albumin) than HOMA- β.

In the second part of the above study, it was found that the post-prandial C-peptide/Glucose ratio was able to discriminate subjects who would have good response to various treatment modalities, better than fasting C-peptide/glucose ratios, or fasting/post-prandial C-peptide measurements alone. In patients with good glycaemic control (HbA1c<7%), the post-prandial C-peptide/glucose ratio decreased in order according to the following treatment groups: lifestyle modification, insulin sensitizers, insulin secretagogues, and exogenous insulin. Using receiver operating characteristic curve analysis, the cut-off values of post-prandial C-peptide/glucose ratio for insulin vs. insulin secretagogues was 1.457 (specificity 92.6%, sensitivity 60.0%), 2.870 for insulin secretagogues vs. insulin sensitizers (specificity 75.5%, sensitivity 51.4%), and 3.790 for insulin sensitizers vs. lifestyle modification (specificity 69.5%, sensitivity 49.1%).

In Japanese subjects with type 2 diabetes, it was found that both fasting and post-prandial C-peptide/glucose ratios were significantly correlated with various indices of insulin secretion including HOMA-β, first phase and total insulin secretion during hyperglycaemic clamp and serum C-peptide 6 minutes after glucagon challenge. Furthermore, the post-prandial C-peptide/glucose ratio was correlated with both clamp and OGTT derived disposition indices, while fasting indices were not, suggesting that the post-prandial indices takes into better account information related not only to insulin secretion but also insulin sensitivity.

The C-peptide/glucose ratio, particularly in the postprandial state, thus shows potential to assist in the prediction of treatment choices, correlated well with more complex dynamic tests and may be able to reflect beta cell mass.

Conclusion

The assessment of beta-cell function remains challenging both in clinical practice and in the research setting. There continue to be significant knowledge gaps in the clinical application of various indices of beta-cell function, for example, if these can be used to guide treatment choices or prognosticate outcomes.

The current clinical utility of various measurements of beta-cell function may be in assisting classification of diabetes, choosing appropriate therapies and for longitudinal follow-up.

*https://www.dtu.ox.ac.uk/homacalculator/

Preanalytics covers all steps between laboratory test order and sample analysis. Most errors occur during this key step.

In this paper, various variables that may impact sample characteristics or integrity, including patient’s status, sample collection, management and storage will be discussed.

Patient’s status:

One prominent question patients and clinical laboratory may have before drawing blood for a haemostasis workup relates to patient’s fasting.

Scarce data are available on the duration of fasting and there is no well described or standardized definition of fasting on national or international grounds. Furthermore, the nature of clot detection system, i.e. optical versus viscosity-based detection system (also called mechanical detection) may also influence the sensitivity of the assay to patient’s fasting, especially for what regards possible post-prandial lipemia. In a recent study, Lima-Oliveira et al. studied the influence of a standardized light meal (563 kCal) on various haemostasis assays, including APTT, INR, fibrinogen, antithrombin, protein C and protein S. They demonstrated that a light meal had a minimal impact on these assay results.

The preferred time for blood sampling is 7 – 9 am when possible. Patients should refrain from smoking during at least 30 minutes before blood sampling: smoking increases platelet aggregability and induces a procoagulant state due to increased fibrinogen and PAI-1 levels and decreased tPA and plasminogen levels. Consumption of caffeine within two hours before blood draw is discouraged because of its influence on fibrinolytic activity. Physical activity should also be avoided within two hours before blood draw is also to be avoided as it increases leukocyte and platelet counts and results in coagulation activation. Moreover, strenuous physical exercise favors platelet microparticle release and induces a transient procoagulant state. On the other hand, stress should be avoided as it results in an increase of acute phase proteins, especially von Willebrand factor, factor VIII and fibrinogen. Blood should preferably be drawn from patients who have rested for a short period of time (5 minutes).

Devices for specimen collection:

Glass or plastic evacuated tube with a non-activating surface containing the appropriate additive (preferably trisodium citrate 105 – 109 mM, alternatively 129 mM provided labs have standardized to one citrate concentration and established citrate concentration-specific reference ranges), directly connected to the needle should be used. Winged devices may be preferable in certain situation (babies, children, patients with small veins or requiring frequent venipuncture). Syringe is an alternative to evacuated tube systems: in that case, small volume syringes (< 20mL) with blood added slowly to the appropriate volume of anticoagulant within one minute of blood draw and specimen immediately and properly mixed is recommended. The preferred needle gauge is 19 – 22; smaller gauge may induce hemolysis. In case of vascular access device (VAD), components of blood collection system must be checked to avoid air leaks that would result in an incorrect volume and hemolysis. Collection through lines previously flushed with heparin must be avoided (flush the line with 5mL of saline and discard the first 5mL of blood or six dead volumes of VAD). In case of blood obtained from a normal saline lock, two dead spaces volume of catheter + extension must be discarded.

Blood / anticoagulant ratio:

Citrate volume may have to adapt depending on patient’s hematocrit. Otherwise, in patients with an elevated hematocrit, plasma can be artificially diluted; this results in increased clotting times. CLSI recommends that citrate volume should be adapted for hematocrits above 0.55 and provides a formula and an abacus for determining the citrate volume versus hematocrit. On the opposite, there are insufficient date to support citrate volume adjustment for low hematocrits.

Specimen labelling:

As a general rule, specimen management should respect patient privacy. Patient and patient’s specimen should be positively identified at the time of collection labeled in the patient’s presence after the blood is collected The label should contain patient’s full name, unique identification number, date and time of collection, name of the person collecting the specimen, specimen type if a secondary or aliquot tube is used (anticoagulant type versus serum) and assay(s) performed (optional). An information request form may be transmitted to the lab along with the specimen.

Discard tube and order of blood draw:

PT (INR) and APTT results not adversely affected if tested on the first tube drawn, without discard tube. Current published data to support the asumption that a discard tube is necessary or unnecessary when drawing samples using a standard evacuated tube system for other tests are not available. When using a winged blood collection set and coagulation tube is the first tube drawn, draw a discard tube first (fill the blood collection tubing dead space, ensure maintenance of the proper blood / anticoagulant ratio, need not to be completely filled, non-additive or coagulation tube).

CLSI recommends that tubes are drawn in the following order, so that contamination by another anticoagulant agent than citrate or by a clot activator that may impact test result is avoided: (1) blood culture tube, (2) coagulation tube (light blue closure), (3) serum with or without clot activator, with or without gel (red closure), (4) heparin tube with or without gel separator (green closure), (5) EDTA tube with or without gel separator (lavender closure) and (5) glycolytic inhibitor (gray closure).

Influence of underfilling tubes:

The appropriate filling of coagulation tubes up to the nominal volume (as typically indicated on the tube) is essential, to produce the most appropriate blood-to-additive ratio, which is typically established at 1:10 (i.e., one part of anticoagulant plus nine parts of blood). Lippi et al. studied the influence of underfilling tubes on various coagulation assays and have identified a clinically significant bias in test results when tubes are drawn at less than 89% of total fill for activated partial thromboplastin time, less than 78% for fibrinogen, and less than 67% for coagulation factor VIII, whereas prothrombin time and activated protein C resistance remain relatively reliable even in tubes drawn at 67% of the nominal volume.

Effect of EDTA or heparin sodium versus citrate as the anticoagulant:

More and more plasma samples are referred to remote technical platfo resulrms. The nature of the anticoagulant of the anticoagulant present in the primary blood collection tube is not available in most of the cases. One frequent error is that EDTA plasma is referred instead of citrate plasma. This may result in erroneous results that are not necessarily easy to identify as results may mimic real clinical situations. PT and APTT are prolonged in EDTA versus citrate plasma samples whereas factor V and factor VIII levels are decreased; moreover, low factor V and factor VIII levels may make the laboratory testing for factor inhibitor, the result of which test will be falsely positive. Thrombophilia screening can also be affected in EDTA versus citrate plasma samples with decreased protein C and protein S activity, possible false positive lupus anticoagulant and possible no clot for activated protein C test.

Another possible error is testing on sodium heparin plasma aliquots instead of citrate plasma aliquots. Heparin samples are characterized by no clot PT, APTT and thrombin time, low factor VIII and factor IX levels, low von Willebrand ristocetin cofactor activity, low protein C and protein S activity (clotting assays) and high anti-Xa activity.

Wrong anticoagulant can easily be detected using routine chemistry tests: typical values for citrate plasma samples are 160mM for sodium, 3.2mM for potassium, 2.0mM for calcium and 0.79 for magnesium. EDTA plasma samples are characterized by a high potassium level (around 20mM), and very low or undetectable calcium and magnesium levels. Heparinized samples can be detected by high anti-Xa activity (roughly 3 IU/mL).

Whole blood sample transport:

Whole blood samples should be transported at room temperature. Transport on ice is not recommended as it may cause cold activation of factor VII, loss of von Willebrand factor, and platelet disruption. Extreme high and low temperatures should be avoided.

Ideally, whole blood samples should be transported to the lab within one hour of collection. However, PT has been demonstrated to be stable up to 24 hours, whereas the use of CTAD tubes can prevent platelet heparin neutralization up to four hours.

Special handling requirements should be provided to carriers.

Date, time shipped / received by the lab, and approximate temperature of the blood sample should be recorded upon arrival at the laboratory.

When using pneumatic tubes, specimens should be protected from vibrations and shock to avoid protein denaturation and platelet activation. Thalen et al. have studied the impact of pneumatic transport on platelet function and shown that the use of pneumatic tubes can affect platelet functions.

Precentrifugation sample examination:

Upon arrival at the laboratory, whole blood samples should be checked for gross clot formation by gentle inversion and observation by removing the cap and inserting and then removing two wooden sticks. However, (Micro)clots may not be detected by these methods. In a prospective study conducted on 1,334 samples, 0.6% (8) samples were found clotted. Differences observed between paired clotted and not clotted samples were not clinically relevant in routine tests (PT, APTT, fibrinogen) as well as for factor II and factor VII+X. In contrast, factor V levels were consistently higher in clotted versus not clotted samples.

Specimens that are clotted or collected in the wrong anticoagulant as well as collection containers that have other than the appropriate blood/anticoagulant ratio (under- / overfilled tubes) and mislabeled or unlabeled specimen should be rejected.

Centrifugation:

Centrifugation should be performed on capped specimen tubes for a time and at a speed that allow to produce platelet poor plasma (platelet count < 10 x 10/L or 10,000/µL). These conditions have to be established locally; this can be typically achieved by centrifuging whole blood samples at 1,500g for no less than 15 minutes. Higher speed and shorter duration (“Stat-fuge”) may be used in some instances (e. g. emergency situation).

Samples should be centrifuged at room temperature in order to avoid coagulation protein cold activation, with use of a swing-out bucket rotor to minimize contamination of plasma with platelets and other blood cells.

When critical, double centrifugation to ensure that the sample is platelet-poor (e.g. screening for lupus anticoagulant or before freezing plasma samples) is recommended.

The influence of centrifuge brake has been studied by Daves et al. who showed no statistical difference for APTT, a significant difference but with no clinical relevance (mean bias: 0.2 seconds) for PT and a statistical and clinically relevant although limited bias (mean bias: 0.29g/L) for fibrinogen.

Hemolysis, icterus and lipemia Interferences:

Hemolysis should be noted when visible. Lysis of red cells and resultant release of intracellular or membrane components may cause clotting factor activation that can have an impact on clotting time results whether using an optical or mechanical end-point detection system.

Icteric or lipemic samples or samples that contain substances that interfere with light transmission may generate erroneous results when using an optical end-point detection without accurate multi-wave length detection.

In a recent study, Wolley et al. studied the influence of hemolysis on PT run with two different reagents when using a viscosity-based dectection system (mechanical detection) in paired hemolyzed versus non-hemolyzed plasma samples. No significant difference was observed between hemolyzed versus non-hemolyzed samples.

In the same study, the impact of hemolysis for APTT when using three different APTT reagents was studied. No statistical difference was observed with one reagent, whereas a statistical difference was observed for the two other reagents, although the difference had no clinical impact for one these two reagents.

No statistical difference was observed for fibrinogen between hemolyzed and non-hemolyzed plasma samples.

Plasma sample storage:

Specimens should be stored capped unless sitting for brief periods (<30 minutes). Storage at refregirated temperature is not recommended: it may induce cold activation of factor VII, gradual loss of von Willebrand factor and factor VIII Plasma samples are usually stable for four hours at room temperature, two weeks frozen at -20°C and several months at -80°C. Stability should be checked locally for specific analytes.

For frozen sample storage, the use of frost-free freezers (automatic freeze/thaw cycles) is not recommended. Frozen samples should be rapidly thawed at 37°C, then gently mixed and tested immediately.

Importance of anticoagulant treatment information:

All anticoagulants may impact coagulation testing: vitamin K antagonists (e.g. warfarin, acenocoumarol, fluindione, phenprocoumon…), unfractionated heparin (UFH), low molecular weight heparins (e.g. enoxaparin, dalteparin, nadroparin, bemiparin, tinzaparin…), as well as the new direct oral anticoagulants, direct thrombin inhibitor dabigatran etexilate (PRADAXA®) and direct factor Xa inhibitors (rivaroxaban – XARELTO®, apixaban – ELIQUIS®, edoxaban – LIXIANA®, SAVAYSA®, betrixaban) can impact routine (PT, APTT, Thrombin Time – dabigatran) and specialized (factor assays, thrombophilia assays) clotting assays. Furthermore, even chromogenic assays if they use factor Xa or factor IIa (thrombin) in the assay principle may be impacted in presence of an anti-Xa or anti-IIa drug respectively.

Laboratories must know the assay principle of the test they use and the potential impact of anticoagulant treatment on these assays.

Information on patient’s anticoagulant treatment is crucial to avoid misinterpretation of test results.

Quality indicators in the preanalytic phase

Quality indicators are key for lab accreditation / certification. Quality indicators have been proposed as well as quality specifications in order to help clinical laboratories better manage their quality for preanalytics.

In conclusion, preanalytics is a multifaceted topic. Laboratories must be aware of the possible detrimental impact of preanalytical conditions on test results. Guidelines or similar documents are available from various organizations including the Clinical Laboratory Standards Institute (CLSI) that is quoted extensively in this paper, the International Council for Standardization in Haematology (ICSH), the College of American Pathologist (CAP), the Deutsches Institut für Normung (DIN), the Groupe Français d’Etudes sur l’Hémostase et la Thrombose (GFHT) or others. Clinical laboratories are highly encouraged to refer to them. They should, in the light of the relevant guideline(s), check their own procedures in order to minimize the risk of erroneous results caused by improper preanalytical conditions and monitor their performance using quality indicators.

Germline mutations in BRCA1 and BRCA2 are highly penetrant genetic susceptibility factors that predispose carriers to develop breast and ovarian cancer. Across different populations, an estimated 5 – 10% of breast cancer arises in individuals who inherit mutations in BRCA1 and BRCA2 genes. The protein products of these two genes play important functions in DNA homologous recombination repair (HRR). In normal cells, the HRR pathway is activated in response to DNA double-stranded breaks. However in BRCA-deficient cells, HRR is abrogated and therefore the more error-prone DNA repair mechanisms, such as single-strand annealing and nonhomologous end joining (NHEJ) are activated. These latter pathways are particularly sensitive to the DNA damaging effects of chemotherapy. HRR defect due to BRCA-deficiency can be exploited as a therapeutic strategy by the use of poly (ADP-ribose) polymerase (PARP) inhibitors which inhibit the PARP proteins commonly PARP1 and 2 that take part in base excision repair (BER). While HRR or BER pathway disruption on its own is not lethal, the combined defect in both pathways lead to cell death through synthetic lethality. In normal cells of carriers heterozygous for BRCA gene mutation, the remaining wild-type allele is active and the residual protein product produced can repair double-stranded breaks through HRR. As a result, the treatment of BRCA gene mutation carriers with PARP inhibitors is highly specific for cancer cells and should spare normal cells.

Clinical significance of genetic testing

Genetic testing for BRCA1 and BRCA2 mutations is indicated for patients who fulfill inclusion criteria for hereditary and high-risk breast and ovarian cancer, to be followed by family study for carrier detection in positive index patients. Any genetic testing must be performed with consent before testing. Of equal importance is the provision of genetic counseling before and after genetic testing. To mitigate the risk of breast and ovarian cancer, enhanced surveillance, prophylactic surgery and chemoprevention may be offered to mutation carriers. Also for the patient the documentation of BRCA1 and BRCA2 mutation status may be of prognostic significance. A recent large scale analysis shows that, among patients with invasive epithelial ovarian cancer, having a germline mutation in BRCA1 or BRCA2 is associated with improved 5-year overall survival, and that BRCA2 carriers shows the best prognosis. Furthermore, in ovarian cancer a mutated BRCA1 or BRCA2 status predicts for platinum sensitivity and response to PARP-1 inhibitor therapy.

Genetic testing by conventional methods

Based on the lower incidence of breast cancer in Asia cohorts, high-risk female patients satisfying the clinical criteria for genetic testing are generally defined as those who: 1) had at least one first- or second-degree relative with breast and / or ovarian cancer, regardless of age; 2) were less than 45 years of age at diagnosis; 3) had bilateral breast cancer; 4) had triple negative (TN) or medullary type pathology; 5) had at least one relative with cancers other than breast and ovarian cancer such as stomach and prostate that are known to be related to BRCA mutations; 6) male breast cancer; or 7) they were ovarian cancer patients with a family history of breast cancer. Since 2015, ovarian cancer patients without family history are also accepted.

The conventional mutation detection methodology is a combination of BRCA1 and BRCA2 full gene DNA sequencing and multiplex ligation dependent probe amplification (MLPA). Mutation detection is performed on genomic DNA extracted from peripheral blood samples and mutation analysis is performed by direct DNA sequencing of all coding exons of BRCA1 and BRCA2 and partial flanking intronic sequences. Sequencing results are compared with the reference DNA sequences using variant reporter software such as Mutation Surveyor and then reviewed manually. Computational analysis for potential cryptic splice site mutation is performed using splice site prediction programs when sequence changes were identified. All mutation and sequence variants are named according to the recommendations for the description of sequence variants of Human Genome Variation Society (HGVS). DNA sequencing is supplemented by MLPA to detect large deletions or rearrangements of the BRCA1 and BRCA2 genes.

An important observation is the detection of recurrent and founder BRCA mutations in the Chinese population, many of which are hitherto unreported. The most common is the founder mutation c.3109C>T, p.Q1037X of the BRCA2 gene. Significantly, genetic screening methods such as high-resolution melting study or mini-sequencing that were simple, rapid and economical may be employed upfront to focus the detection of these recurrent and founder mutations before proceeding to detailed genetic analysis.

The era of next-generation sequencing (NGS)

The advent of NGS or massively parallel sequencing allows an increase in both the capacity (more patient samples) and capability (more genes to the exome or even genome level) of DNA sequencing at a lower cost than conventional Sanger sequencing. Since November 2011, our laboratory has started to utilize microfluidic access array for PCR amplification of a gene panel (BRCA1, BRCA2, TP53 and PTEN) for 454 pyrosequencing on the Roche GS Junior. A total of 91 PCR primer pairs are used to target all exons of the 4 genes and 10-bp intron-exon boundaries (except downstream of BRCA1 exon 2 where 8 bp was covered) for amplification of genomic DNA samples. Access Array System (Fluidigm) was used to generate separate pools of 91 amplicons from 48 DNA samples per run. A 10-bp barcode nucleotide sequence is incorporated in both ends of each amplicon for sample identification. Resulting amplicon pools of on average 12 patients are further pooled for sequencing library preparation. Each library is subjected to multiple sequencing runs of 454 GS Junior system so that each nucleotide position in target regions is sequenced at a minimum depth of 30-fold. It is also equivalent to the average depth of at least 150-fold. Since 2014, the NGS workflow has migrated from Roche GS Junior to Illumina MiSeq benchtop genome sequencer, for which the minimum depth is 50-fold and the average depth is 300-fold.

A robust and properly validated bioinformatics pipeline is the key to success in NGS diagnostics. In Roche GS Junior, sequencing reads are assigned to corresponding samples according to sequence barcodes using Amplicon Variant Analyzer (AVA). Reads are then aligned to reference sequence by AVA. Each patient-specific BAM sequence alignment was processed by three variant callers: 1) AVA using default settings, and 2) SAMtools and 3) an in-house algorithm for homopolymer variant detection. Multiplicity is essential and therefore the adoption of more than one variant caller (including in-house scripts) is useful. Variant effect on corresponding protein coding genes is annotated using Ensembl Variant Effect Predictor. Annotated variants are automatically prioritized for manual review according to standardized laboratory criteria Variants are designated according to the recommendations from HGVS. Putative mutations are validated by bi-directional Sanger sequencing. All patient samples subjected to massively parallel sequencing are simultaneously examined for large genomic rearrangement of BRCA1 and BRCA2 by MLPA. For MiSeq, BWA-MEM and variant callers SAMtools and GATK HaplotypeCaller are adopted.

Another important aspect of NGS is appropriate variant interpretation. Searching through the literature and various database can be time consuming. To characterize missense variants of unknown significance (VUS) identified in our patient cohort, the allele is checked against 107 healthy local individuals and 1000 Genomes project samples, and analyzed by in-silico prediction methods including PolyPhen and SIFT. Segregation of VUS with cancer phenotype is documented by family study as far as possible.

Among 948 high-risk breast and/or ovarian patients who are subject to genetic testing by NGS in Hong Kong, the prevalence of BRCA1 and BRCA2 germline mutations was 9.4% in our Chinese cohort, of which 48.8% of the mutations arose from hotspot mutations. The frequencies of PTEN and TP53 were 0.21% and 0.53% respectively. High-throughput NGS approach allows the incorporation of control cohort that provides an ethnicity-specific data for polymorphic variants. Our data suggest that hotspot mutations screening such as SNaPshot or mini-sequencing can be an effective preliminary screening alternative adopted in a standard clinical laboratory without NGS setup.

Future perspective

Recent studies from large consortium have resulted in the identification of additional breast cancer susceptibility loci through candidate gene or whole genome approaches. While familial breast cancer comprises approximately 20 – 30% of all breast cancers, the two major high penetrance genes BRCA1 and BRCA2 associated with hereditary breast and ovarian syndromes explain less than 10% of all breast cancer cases. Mutations in CHEK2 contribute to a substantial fraction of familial breast cancer. Carriers of TP53 mutations develop Li-Fraumeni syndrome and are at high risk of developing early-onset breast cancer, but these mutations are very rare. Susceptibility alleles in other genes such as PTEN, ATM, STK11/LKB1, MSH2/MLH1, BRIP1 and PALB2 are also rare causes of inherited breast cancer. Nevertheless, around half of the familial clustering of breast cancer remains unexplained. The susceptibility to breast cancer in this group is presumed to be due to additional but hitherto unidentified high-penetrance genes or variants at many moderate to low-penetrance loci each conferring a moderate risk of disease. The NGS approach can be harnessed to provide comprehensive gene panel testing that extends beyond BRCA mutations, especially for those high risk but BRCA negative breast cancer patients. It should be borne in mind that may only provide the information on genetic risk but not all the answers to the patient since the standard of care for many of these rare genetic susceptibility genes awaits more evidence for clinical practice to be generated.

PARP inhibitors induce synthetic lethality in tumours with HRR defect due to loss of function BRCA gene mutations. Irrespective of whether the origin of the BRCA mutation is germline or somatic, tumours in patients with a BRCAmutation should be sensitive to PRAP inhibition because of the loss of function of the gene within the tumour cells. The NGS approach can be applied to germline or somatic testing or both. Olaparib is a potent oral PARP inhibitor that has demonstrable anti-tumour activity in clinical trials of patients with BRCA-mutated or sporadic high-grade serous ovarian cancer. Also PARP inhibitors may have a wider application in the treatment of cancers defective in DNA damage repair pathway such as breast, prostate, endometrial and pancreatic cancer. Due to the therapeutic implication, testing for BRCAmutation status in breast and ovarian cancer is in high demand and the NGS is going to be the method of choice in this situation.