Q&A: Chemical pathology

Questions by:
Jane Diggle
Practice Nurse, West Yorkshire

Q: What are the relative merits of using HbA_1c as opposed to fasting plasma glucose or the oral glucose tolerance test for diagnosis of diabetes?
HbA_1c can be considered an alternative diagnostic tool to fasting plasma glucose (FPG) or the oral glucose tolerance test (OGTT), but it is not generally considered superior to these tests and has advantages and disadvantages comparatively. HbA_1c tends to be used as a first-line screen for diagnosis due to convenience as it does not require fasting and is not a dynamic function test. HbA_1c also is able to capture chronic hyperglycaemia more effectively than a standard paired assessment of FPG or an OGTT as it is describing a longer period of time. In addition, both microvascular and macrovascular disease are better associated with HbA_1c than FPG (DECODE Study Group, 2001; International Expert Committee, 2009). It is not necessary to fast prior to testing HbA_1c, and the analyte itself has greater pre-analytical stability than glucose. It has also been argued that using the same analyte for both diagnosis and then subsequent monitoring of diabetes may be beneficial (Bonora and Tuomilehto, 2011).

Two-hour post-glucose concentration as seen during OGTT better reflects the abnormal physiology seen in diabetes as it describes the post-prandial state with insights into beta-cell function, information that HbA_1c does not provide. It has also been shown that HbA_1c has poorer sensitivity than OGTT (Tuomilehto et al, 2001) and FPG (Zhou at al, 2010) for detecting asymptomatic cases in the early stages of disease. Issues with assay standardisation for HbA_1c unfortunately remain, although the situation has markedly improved in recent years. This is an important issue given the implications for an incorrect diagnosis. HbA_1c can be unreliable in certain clinical scenarios, as expanded upon below.

Q: In what circumstances is HbA_1c unreliable?
Any pathophysiological process leading to reduced erythrocyte lifespan has the potential to lead to a falsely low reported HbA_1c concentration. This includes chronic and haemolytic anaemia, renal anaemia with use of erythropoietin, acute blood loss and recent transfusion. General recommendations are to not perform HbA_1c testing within 3 months of a blood transfusion. In addition to this, chronic liver disease, dialysis and chronic malaria can lead to a falsely low HbA_1c. Conversely, iron-deficiency anaemia may cause a falsely high HbA_1c, possibly due to altered glycation rates (Weykamp, 2013). However, if haemoglobin and mean cell volume are normal in someone previously treated for iron-deficiency anaemia or vitamin B12 deficiency, HbA_1c should be reliable.

In some cases where HbA_1c is potentially unreliable, there are solutions to consider. When HbA_1c cannot be measured reliably due to the presence of a haemoglobin variant, it may be possible to analyse a sample via an alternative method which may be able to provide a valid result and this should be discussed with the local biochemistry laboratory as appropriate. For cases where this is not possible, it may be necessary to use standard capillary blood glucose techniques alone to assess glycaemic control. Other options are available, such as fructosamine or glycated albumin, but there are interpretive issues that limit their usefulness and it is unclear how they relate to the development of long-term complications. Whether alcoholism can cause a falsely high HbA_1c is under debate, with conflicting reports on its effect on HbA_1c testing. The mechanism at work is not currently known, nor is it clear if it is a genuine effect rather than a confounding factor, such as background nutritional status.

Q: When should HbA_1c not be used to diagnose diabetes?
HbA_1c should not be used to diagnose gestational diabetes. In this situation OGTT should be used in accordance with NICE guidelines (2015). If there has been a rapid onset of symptoms, then HbA_1c may not be able to detect the change until several weeks later and, as such, it should not be used as it may delay diagnosis. Such situations could include suspected type 1 diabetes, suspected steroid-induced diabetes and diabetes potentially arising from pancreatitis or following pancreatic surgery.

HbA_1c gives an indication of “average” blood glucose levels over a period of 2–3 months and, therefore, it is generally not appropriate to repeat the test more frequently than this.

Q: When is it appropriate to perform an HbA_1c test earlier?
There will be times when more frequent requesting of HbA_1c may be indicated. For instance, tight glycaemic control is advised for people with diabetes who are considering pregnancy. Recent NICE guidelines (2015) suggest monthly requesting of HbA_1c in the preconception period.

Q: In the event of a normal thyroxine (T4) but raised thyroid-stimulating hormone how would you monitor, and how frequently, for potential hypothyroidism?
Thyroid function tests which demonstrate  increased thyroid-stimulating hormone (TSH) with an FT4 that falls within the reference range are suggestive of subclinical hypothyroidism. There is no consistent evidence from the literature that an raised TSH, which is <10 mU/L, is associated with symptoms of hypothyroidism, or secondary biochemical effects, such as hyperlipidaemia, or cardiac dysfunction. For patients where the TSH concentration is >10 mU/L with a normal FT4, there is evidence of greatly increased risk of progression to overt hypothyroidism, and also deterioration in hyperlipidaemia (Association for Clinical Biochemistry, 2006). Thus if the TSH concentration is >10 mU/L then treatment with thyroxine replacement is advised in most cases. When the TSH concentration is raised but <10 mU/L, treatment is usually not recommended unless the patient has a goitre or is planning pregnancy.

Patients with subclinical hypothyroidism and a TSH <10 mU/L require monitoring to assess for progression of thyroid disease. It is advised that such individuals should have a repeat TSH and FT4 after 3–6 months along with measurement of thyroid peroxidase (TPO) antibody. Those with a persistent raised TSH, which remains <10 mU/L, who are TPO antibody positive should have repeat thyroid function tests in 1 year. Those who are TPO antibody negative should have repeat thyroid function tests (TFTs) in 3 years. Those with worsening symptoms may require testing sooner as appropriate. Patients whose TSH has increased to >10 mU/L should be considered for thyroxine replacement therapy (Association for Clinical Biochemistry, 2006).

Q: When and how is chronic kidney disease diagnosed and classified?
Chronic kidney disease (CKD) is an all-encompassing term for describing a heterogeneous collection of disorders which result in abnormal renal function or structure. CKD can be detected and assessed using routine laboratory tests. Renal function can be assessed by measuring or estimating glomerular filtration rate (GFR); formal measurement of GFR, such as by using radioactive isotopes is cumbersome and resource intensive, so commonly it is estimated from serum creatinine and other parameters such as age, sex, ethnic origin and body size. Several methods are available for estimating GFR, including the Modification of Diet in Renal Disease (MDRD) study group equation and the CKD Epidemiology Collaboration equation (CKD-EPI). The MDRD equation is reasonably accurate when the estimated (e)GFR is <60 mL/min/1.73 m² but is significantly limited by imprecision when the eGFR is higher, and consequently is often not reported when at such levels (Stevens et al, 2007). The CKD-EPI equation is less susceptible to bias at higher levels and can be used to report eGFR results >60 mL/min/1.73 m², and is recommended for use in estimating GFR in the recent NICE guidance (2014a) on CKD.

CKD can be diagnosed on the basis of the presence of renal damage, such as albuminuria, or decreased renal function such as an eGFR <60 mL/min/1.73 m², present for at least 3 months irrespective of the underlying aetiology. The eGFR can then be used to classify CKD into a number of distinct stages:

• Stage 1: ≥90 mL/min/1.73 m²
• Stage 2: 60–89 mL/min/1.73 m²
• Stage 3a: 45–59 mL/min/1.73 m²
• Stage 3b: 30–44 mL/min/1.73 m²
• Stage 4: 15–29 mL/min/1.73 m²
• Stage 5: <15 mL/min/1.73 m² 

It is important to note that for individuals with an eGFR ≥60 mL/min/1.73 m² (i.e. stages 1 and 2), CKD cannot be diagnosed unless there is co-existing evidence of renal damage, such as albuminuria.

eGFR can be used in combination with urine albumin–creatinine ratio (ACR) from an untimed spot urine to further classify patients with CKD by stratifying them into various risk groups. This is demonstrated in Table 1, adapted from NICE (2014a) guidance. This enables a consistent approach to diagnosis, staging and risk stratification in CKD.

Q: When should you use a cystatin C-based estimate of GFR for diagnosis of CKD (and is it currently available)?
Cystatin C is a proteinase inhibitor and has a low molecular weight. It is freely filtered at the glomerulus, almost completely reabsorbed and undergoes no tubular secretion. It is produced at a constant rate from all nucleated cells in the body. Its serum concentration is largely determined by glomerular filtration and as such it is considered an endogenous marker of GFR. Cystatin C-based GFR estimation is considered to have some advantages over creatinine as its non-GFR determinants seem to be less affected by muscle-wasting and race. There is also some data to suggest that it is more predictive of subsequent cardiovascular mortality and morbidity (Shlipak et al, 2005).

Current NICE (2014a) guidance advises consideration of cystatin C-based eGFR for initial diagnosis of CKD in people with a creatinine-based eGFR of 45–59 mL/min/1.73 m² who also have an ACR of <3 mg/mmol. In this situation, cystatin C-based eGFR can confirm or exclude a diagnosis of CKD. If the eGFR as determined using cystatin C is >60 mL/min/1.73 m² then the diagnosis can be excluded at that time.

There are issues with the use of cystatin C, however. It is considerably more expensive than creatinine and has poor general availability, being offered currently by just three laboratories in the UK. There are also significant issues with assay standardisation, and in addition to this, there is no clear consensus regarding the best equation to use to estimate GFR based on the serum level. It could also be argued that in those situations where an accurate GFR estimation is required, where one might consider using cystatin C, then the best course of action may be to formally measure GFR using nuclear medicine techniques. For these reasons, cystatin C-based GFR estimation is rarely undertaken in routine clinical practice despite the recommendations in the recent NICE (2014a) guidance.

Q: How frequently should people with CKD undergo monitoring of eGFR and ACR?
It is important to remember that CKD represents a broad spectrum of disease, ranging from rapidly progressive disease leading to end-stage renal failure within months, to those with little to no progression in disease stage over many years of follow-up. It is recommended that all patients with a formal diagnosis of CKD should have regular monitoring to assess for the possibility of disease progression. Table 2 outlines the monitoring schedule as advised by NICE (2014a).

Q: When should individuals with CKD be referred to a renal physician?
Recommendations from NICE emphasise the need for an open dialogue with the patient, taking into account their wishes and also any comorbidities when considering who to refer for specialist management. People with a diagnosis of CKD should be considered for referral to a renal physician in the following situations:

• eGFR <30 mL/min/1.73 m² with or without diabetes.
• ACR >70 mg/mmol unless known to be associated with diabetes.
• ACR >30 mg/mmol along with haematuria.
• A decrease in eGFR of 25% or more with a change in GFR stage or sustained decrease in GFR of 15 mL/min/1.73 m² or more within 12 months.
• Known or suspected rare or genetic cause of CKD or suspected renal artery stenosis.
• Poorly controlled hypertension despite use of four or more agents at therapeutic doses.

Q: Cholesterol is often raised at the time diabetes is diagnosed or when glycaemic control is very poor – should the cholesterol levels be treated based on these findings or rechecked when glycaemic control improves?
The key issue in this situation is being clear regarding the rationale for treatment. It is not uncommon for individuals with newly diagnosed type 2 diabetes to have co-existent severe hypertriglyceridaemia as a result of their underlying insulin resistance (Subramanian and Chait, 2012). Significant hypertriglyceridaemia will result in a total cholesterol concentration that is often greatly elevated and not representative of the individual’s baseline lipid status. As the patient has their diabetes treated and glycaemic control improves, the hypertriglyceridaemia will generally improve and their total cholesterol will reduce. There is no definitive evidence that pharmacological treatment of hypertriglyceridaemia in itself has a beneficial impact on cardiovascular risk (Miller et al, 2011). However, it is advisable that if the triglyceride concentration remains >10 mmol/L after treatment of diabetes has been optimised, treatment with a fibrate should be considered to lower triglycerides due to the risk of acute pancreatitis (Berglund et al, 2012).

Once lipid levels have stabilised, a formal cardiovascular risk assessment using QRISK2 can take place and this will inform the decision to prescribe a statin. Current NICE (2014b) guidance is that all individuals with a calculated cardiovascular risk greater than 10% should be considered for statin treatment. It is worth considering that cholesterol forms just one part of a patient’s overall cardiovascular risk package and close attention should be paid to all modifiable risk factors.

Q: Is non-HDL-cholesterol a more reliable measure than LDL-cholesterol?
LDL-cholesterol is not generally measured directly in routine laboratory testing; instead it is calculated using the Friedewald equation (Warnick et al, 1990):

• LDL-cholesterol=total cholesterol − HDL-cholesterol − (triglycerides/2.2)

The equation has very well-recognised limitations, and is known to be inaccurate in cases of moderate hypertriglyceridaemia, where there are chylomicrons present, and in people with type 3 hyperlipidaemia (Matas et al, 1994). These issues introduce the potential for misclassification of patients on the basis of their cardiovascular risk. This is particularly of concern for people with diabetes as they are more likely to have abnormal triglycerides. Direct LDL-cholesterol assays have been developed which would be theoretically preferable, but analytical issues prevent them being used routinely. However, it is important to consider that LDL-cholesterol remains crucial for the diagnosis of familial hypercholesterolaemia, which is a relatively common monogenic inherited disorder of LDL metabolism.

Non-HDL-cholesterol is the difference between total cholesterol concentration and HDL-cholesterol concentration, and thus represents an estimate of the cholesterol present in atherogenic particles such as IDL, LDL, VLDL and Lp(a). With regards to its efficacy as a cardiovascular risk biomarker, there is clear evidence of the superiority of non-HDL cholesterol over LDL-cholesterol (van Deventer et al, 2011). Some clinical trials go as far as to say that non-HDL-cholesterol has equivalent vascular event prediction capabilities as measurement of apolipoprotein B or LDL particle number, both of which are significantly more expensive tests and not routinely available (Blaha et al, 2008). However, much of the established evidence base refers to lipid status in terms of total cholesterol and LDL-cholesterol and this should be considered when interpreting lipid results in an individual.

Glossary:
HDL=high-density lipoprotein
IDL=intermediate-density lipoprotein
LDL=low-density lipoprotein
Lp(a)=lipoprotein-a
VLDL=very low-density lipoprotein