Matrices Flashcards
What is the sample matrix and how do matrix effects affect analysis?
The sample matrix refers to all components of the sample apart from the analyte being measured. Different matrices place different demands on the test and the person analysing, eg calprotectin in blood and faeces.
Matrix effects can be defined as the combined effects of all components of the sample, other than the analyte, on the measurement. They affect the way that analysis is conducted as something in the matrix may interfere with the test, this can affect the accuracy, reliability and quality of results.
What is the composition of blood?
Serum / Plasma: 55-65%
- Proteinaceous fluid containing Immunoglobulins, clotting factors, enzymes, enzyme inhibitors, electrolytes, hormones
- Serum = clotted, no anticoagulant
- Plasma = anticoagulated (usually Heparin, but may also use EDTA, etc.) before centrifugation
White cell/platelets: Buffy coat
- WBC ~ small amounts but can have a disproportionate influence
Packed RBCs: 35-45%
- RBC ~ ½ total volume of whole blood
DEPENDING WHAT YOU ARE MEASURING EVERYTHING ELSE IS PART OF THE MATRIX AND WILL BE CONTRIBUTING MATRIX EFFECTS
What is the composition of urine?
Mostly water (>95%), depends on diet, time of day, climate, physical activity and body size
Electrolytes, excreted from kidneys into urine
Nitrogenous compounds, products of protein degradation (proteins too if the patient has kidney damage and therefore proteinuria)
Phosphates
Drug metabolites, if a patient is on drugs cleared by the kidneys
Constituents vary throughout the course of the day, eg most concentrated in morning
DEPENDING WHAT YOU ARE MEASURING EVERYTHING ELSE IS PART OF THE MATRIX AND WILL BE CONTRIBUTING MATRIX EFFECTS
What is the composition of faeces?
Complex mixture of food residues, secreta of the GI tract, desquamated cells of the GI wall and constituents of the intestinal flora (mainly from colon) so is therefore different in everybody.
Exact composition is very inconsistent, and depends on diet, intestinal luminal function, water intake, infection etc
Highly variable water content (from ~30% to ~95%)
Hyperosmolal (solutes expressed per Kg) compared to blood (typically 280 – 500 mOsmol/kg, where as blood is <280)
Acidic relative to blood (pH ≤ 6 usually, but not much below this)
Also contains electrolytes and nitrogenous compounds
DEPENDING WHAT YOU ARE MEASURING EVERYTHING ELSE IS PART OF THE MATRIX AND WILL BE CONTRIBUTING MATRIX EFFECTS
What is the composition of CSF?
Secretory product of the ventricles, choroid plexus and structures of the cerebral subarachnoid space
Slightly hyperosmolal compared with blood (~290 to 324 mOsmol/kg) as it is a means of maintaining hydration of the brain
Levels of typical components such as urea, creatinine, sodium and potassium are similar to those found in blood or plasma, there should be no cells at all, for example seeing white blood cells here is a sign of serious disease.
Very low total protein levels (200 to 400 mg/L), in plasma is 50 - 80 000.
Requires a lumber puncture so may see haemoglobin or bilirubin if subarachnoid haemorrhage has occurred or if previous lumbar puncture has been performed.
DEPENDING WHAT YOU ARE MEASURING EVERYTHING ELSE IS PART OF THE MATRIX AND WILL BE CONTRIBUTING MATRIX EFFECTS
What is a dried blood spot and why could it be advantageous?
Production of dried blood spots involves allowing a finger prick to drip then drying the blood spot and punch out the dried spot. This allows you to make assumptions about the diameter and use this to interpret diagnosis.
Particularly useful for patients with low blood volume such as the elderly and babies as it is less invasive, less painful and requires a lower sample volume.
The sample is also stable and amenable to field studies.
However the relationship between spot punched disc and blood volume can be variable. If the spot is of poor quality (too small or multi spotted) it can be difficult to measure effective volume
There is also query interference filter paper fibres and other manufacturing components and the calibration is obviously more challenging.
Why should calibrators be made up to be as similar to the matrix as possible?
Calibrators should be made up with a diluent as close in composition as possible to the matrix in which you want to measure this is a zero-standard diluent.
Synthetic matrix matched calibrators exist for urine and saliva, but not for plasma, blood or faeces.
It is unfavourable to use an aqueous calibrator - SHOULD NEVER USE WATER.
If there is no synthetic matrix and it is not possible to perform standard additions then the next best option is to make ‘stripped’ plasma using e.g. Charcoal; Antibodies etc.
In this case calibrators are diluted in 145 mmol/L saline (typical conc of sodium, etc in the blood) containing 1 or 2% Albumin (less than you may encounter but referenced widely in the literature).
If no other options are available saline can be used as a zero calibrator.
What is an acceptable alternative if zero-standard diluents are not available for calibration?
Use the method of Standard Additions if no zero-standard diluent is available. This involves measuring the signal intensity for each concentration of standard added in order to produce a calibration curve.
Why is urine less favourable than blood as a measurement?
Though blood is still not ideal it is more convenient than Tissue Biopsies(due to ethics). It travels round the entire body therefore is representative of all the tissues at that given snapshot. In this way it can indicate changes in the tissues. It is a large assumption but it reflects all tissues smoothing out the peaks and troughs - not tissue specific.
However, compared to urine it is preferential. A 24 hour urine sample can be difficult to retrieve in children and results in lots of contamination caused by opening and closing the collection vessel. Furthermore, it has temperature dependent solubility. Additionally often the preservatives used are dangerous, for example HCl is used when checking for neuroblastomas in children. Copper is measured in potential Wilson’s disease patients, however since copper is everywhere it can easily lead to contamination. Also there is a lot of fluctuation depending on the time of the day and several stages of cleanup can be required in cases such as drug analysis.
List five examples of analytical issues that can occur due to the matrix.
It can be necessary to perform pre-analytical clear up.
Certain analytes can only be measured if other species are present. For example, co-factors (eg ALT as a marker for liver function requires vit B6 and zinc in excess), activators, inhibitors (some analytes exist in free and bound form - for total would need to unbind using inhibitors)
Post-mortem bloods require clean-up as all red cells are haemolysed leaving tar-like blood that is difficult to extract, instead it can be more advantageous to use vitreous humour from the eye or CSF.
During analysis one must be mindful of specific intrinsic interactions: Autoantibodies, heterophilics - when a patient keeps small rodents that were used to generate the monoclonal antibodies etc.
Similar there can be interference during analysis that affect results, for example chromatic Interference: serum indices
The matrix itself can present issues with analysis, for example lipaemia (cloudiness of blood), haemolysis (intense redness from broken red blood cells) and icterus (high levels or bilirubin in jaundice patients).
Temperature, pressure and haematocrit can cause particular issues in POCT, arterial blood gasses - extremes cant be measured) - particularly causing issues in POCT
Interactions of analytes with blood (in vitro as this can also happen in the tube once the blood is drawn) or interference of drugs during testing (eg paracetamol in glucose measurements)
Dietary additives - in vitro interference eg herbal remedies high in heavy metal trace elements. This is often seen in T3 - people buy this active form of thyroxin for weight loss.
What is the qualitative post-column infusion method of assessing matrix effects in liquid chromatography mass spectrometry (LCMS)?
A constant flow of analyte solution is introduced into the mobile phase post column. This results in a raised background signal. The
A blank sample extract is then injected into the LC-MS system using the LC method under test
The sample components elute from the column and enter the MS source. If any of the eluting components interfere with the ionisation of the analyte the background signal will be disrupted
Using this technique the size and spread (across the chromatogram) of the matrix effect is visually represented which allows one to determine whether adjusting the LC method will be sufficient to avoid the matrix effect or whether a better sample clean-up is required.
What is the quantitative post-extraction spike method of assessing matrix effects in liquid chromatography mass spectrometry (LCMS)?
The MS responses of two samples are compared; one sample is in the matrix under test, e.g. plasma, the other in a matrix considered to be clean, e.g. water
Using this technique, if there is a matrix effect, the signals measured from the sample and the clean control will be different and can be quantified
It would seem an easy approach to spike analyte into both matrices and then extract them, but it is possible that the recovery of analyte would be different for each, giving a skewed result.
The way around this is to extract the samples first and then add the analyte at the final stage
Using both techniques together is recommended
