Analytical interferences

Question 1

4 mechanisms of interference by haemolysis

Answer 1

1. Additive - of intracellular substances being added to (released into) serum that are then directly measurable eg K+, PO4-, Mg 2+, LDH, AST
2. Spectral - by Hb absorbance max at 415nm but detectable 320-450nm, 540-589nm - lipase, albumin, GGT increased
3. Chemical - intracellular substances interfere/interact with analyte being measured eg CK increase due to red cell adenylate kinase release
4. Dilutional - intracellular fluid released causing eg low sodium, chloride, glucose

Question 2

Chemical interferences by haemolysis

Answer 2

Direct vs indirect
Direct - competition for a substrate (eg adenlyate kinase released from cells competes for ADP with CK), inhibition of the assay (eg pseudoperoxidase activity of Hb interfering with formation of diazonium salt in bili method)
Indirect - complexation, proteolysis (by cathepsin E) eg insulin/trop T, precipitation

Question 3

What substances within the RBC cause interference by haemolysis?

Answer 3

Hb
AST
K
Mg
LDH
Phosphate
Adenylate kinase
Proteases

Question 4

Describe the absorbance spectrum of Hb

Answer 4

Peaks at 415 and 540nm and 570nm, with significant absorbance between 320-450nm and 540-580nm

Question 5

Haemolysis causes negative interference with which analytes?

Answer 5

3 analytes
1. Bili - Hb has pseudoperoxidase activity that degrades Bili
2. Insulin and trop T - intracellular proteases break these down

Question 6

Haemolysis can cause interference in what general type of assay?

Answer 6

Assays employing NADH/NADPH (absorbance at 340nm)

Question 7

Causes of haemolysis

Answer 7

In vivo, in vitro
In vivo - extravascular, intravascular
Extravascular - haemoglobinopathies, G6PD deficiency, hereditary spherocytosis, AIHA, hypersplenism, liver disease, some infections
Intravascular - fragmentation (valvular disease/artificial valves), MAHA, immunohaemolytic anaemias (eg ABO incompatibility), envenomation, some infections, acute drug reaction with G6PD deficiency, thermal injury
In vitro - small gauge needle at phlebotomy, drawing syringe back too rapidly, over-mixing, vigorous shaking during transport, delayed separation, repeated freeze-thaw cycles, skin disinfectants

Question 8

Differentiation of in vivo and in vitro haemolysis

Answer 8

Measure K, LD and Hb on plasma and serum samples. Increased LD with normal K in both sample types suggest in vivo haemolysis. Decreased serum haptoglobin and increased free Hb in urine are the most specific findings for in vivo haemolysis. Supportive evidence from FBC, bilirubin, repeated haemolysis in different tube types over time.

Question 9

True or false: if in vivo haemolysis is suspected, the lab should not report affected results

Answer 9

FALSE. The results are a true reflection of what is going on in the patient as a result of the haemolysis and should be reported.

Question 10

Analytes that may be affected by the dilutional effect of haemolysis (ie will be falsely decreased)?

Answer 10

Albumin
Bilirubin
Na
Gluc

Question 11

Mechanisms of bilirubin interference?

Answer 11

1. Spectrophotometric - absorbance of bilirubin is between 400-540nm (peak 460nm) - interference proportional to its concentration
2. Chemical - reacts in oxidase/peroxidase reactions lowering test results (trig, gluc, uric acid, cholesterol)
3. Interfere with albumin-binding dyes
4. Jaffe reaction - Bilirubin oxidised to biliverdin causes negative interference by decreasing sample absorbance at 500nm - very assay dependent

Question 12

Which form of bilirubin causes the greatest amount of interference

Answer 12

Conjugated - a problem because most interference studies use commercially available forms of unconjugated bilirubin, and conjugated and unconjugated bilirubin may react very differently in the same assay system

Question 13

Ways to minimise bilirubin interference

Answer 13

Limited
1. Bilirubin oxidase
2. Blanking
3. Use a different method
4. Dilute (only for analytes with high concentrations)

Question 14

Define icterus

Answer 14

bilirubin concentration > 100umol/L

Question 15

define lipaemia

Answer 15

turbidity of a sample, visible to the naked eye

Question 16

Mechanisms of lipaemia interference

Answer 16

1. Spectrophotometric - lipoprotein particles cause scattering and absorption of light
2. Volume displacement/electrolyte exclusion
3. Partitioning / Sample non-homogeneity - affects analyser level sensing and sample pipetting
4. Physicochemical

Question 17

Explain partitioning interference from lipaemia

Answer 17

after centrifugation, lipids more concentrated in the upper portion of the sample and aqueous plasma more concentrated in the lower portion of the sample. Depending on where sampling occurs (how far down the probe goes), the concentration of more lipid soluble components of the plasma (eg drugs) may be higher (if shallow probing) or lower (if deep probing). A smaller number of larger lipid particles may have a greater partitioning effect, and not cause much of a difference in turbidity.

Question 18

Examples of physicochemical interference by lipaemia

Answer 18

1. Electrophoretic (alpha 1 and 2) and chromatographic separation
2. Masking antigen/ab binding sites in IA (immunoturbidimetric/nephelometric methods eg HbA1c IA)
3. Increased susceptibility to haemolysis
4. Absorption of hydrophobic reagents/reaction products

Question 19

Mitigation of lipaemic interference

Answer 19

1. Ultracentrifugation - up to 200000G. Very effective but not widely available, removes lipid soluble drugs and hormones - report these on untreated sample
2. High speed centrifugation - up to 20000G. Generally effective enough for most lab purposes, though not as effective as ultracentrifugation (reduce Tg 3-4 fold rather than 7fold)
3. Lipid clearing substances eg LipoClear, cyclodextrin, PEG, dextran sulphate, hexane) - low cost, convenient, easy to use, but may reduce proteins along with lipids

Question 20

Advantages of HIL indices

Answer 20

1. More reliable and accurate than visual inspection
2. Automated, faster TAT, more efficient
3. Objective, semi-quantitative and consistent methodology for sample assessment
4. Enhanced confidence in reported results

Question 21

Disadvantages and problems with HIL indices

Answer 21

1. Misses interferents other than HIL - paraproteins, drugs
2. False flagging due to eg artificial blood products, drugs, contrast
3. Cannot distinguish between in vivo and in vitro haemolysis
4. Method to method variation in sensitivity to HIL and reporting recommendations
5. Necessary to verify manufacturers’ claims (if interference studies have been performed according to regulations)
6. How should quality control be performed? Is there an EQA program available for HIL?
7. Cross-reactivity of HIL

Question 22

Strategies to minimise bilirubin interference with Jaffe creatinine method

Answer 22

1. Add potassium ferricyanide before picrate to oxidise all bilirubin to biliverdin
2. Rate blanking - measure the rate of colour change after addition of sodium hydroxide and before picrate addition, and subtract from reaction rate

Question 23

Coloured substances causing HIL interference

Answer 23

Rose Bengal (used in Rx melanoma)
Patent Blue V dye (used in sentinel lymph node biopsy)
EDTA (from underfilled EDTA tube)

Question 24

How can verification of manufacturer HIL claims be performed?

Answer 24

Add haemolysate, bilirubin and intralipid to samples in quantities that should cause various degrees of flagging and analyse

Question 25

How to create QC material for HIL?

Answer 25

Freeze patient pool aliquots with haemolysate/intralipid/commercial bilirubin QC material added to use for QC materials.

Question 26

Investigations for macro-complexes

Answer 26

1. Gel filtration chromatography (macros barrel through gel and come out faster ie have shorter retention time) 2. PEG precipitation