Chemical pathology VI - Interferences in lab test

Question 1

Define interference

Answer 1

Effect of a substance present in the sample that alters the correct value of the result,

expressed as concentration or activity for an analyte

Question 2

Effects of interference in lab tests?

Answer 2

False results > misinterpretation > wrong treatment

Question 3

Hb concentration is estimated by which index?

Main source of interference of Hb analysis?
How to prevent?

Answer 3

Hemolysis index

In vitro hemolysis = main source of interference
» release intracellular LDH, K, Mg to extracellular fluid, specific levels at specific Hb conc.

Hemolysis occurs in sample collection/ transport:
- careful handling

Question 4

3 ways high lipid causes chemical analysis interference?

Answer 4

Chylomicrons cause turbidity, light scattering and volume displacement

Partitioning of non-polar compounds

Question 5

Explain how pseudohyponatremia is linked to high serum lipid or protein?

Answer 5

Pseudohyponatremia caused by hyperlipidemia and hyperproteinaemia

lipids and proteins in blood are barely soluble and precipitates > these lipids and proteins in high amount can occupy space in the plasma

If 10mL of plasma is extracted and high lipid and protein occupies space (e.g. 2mL of space), then the remaining 8mL contains a lower mass of Na than anticipated in 10mL, even though the concentration of Na is the same

Hyperproteinaemia e.g. multiple myeloma&raquo_space; lots of protein in antibodies
Hyperlipidemia e.g. excess chylomicrons

Question 6

Difference between direct and indirect ISE?

Answer 6

Direct and indirect ion selective electrodes

Direct ISE = direct measurement of serum and plasma ion (Na)

Indirect ISE = measure serum and plasma ion After dilution. Ion level is lower because serum lipid or protein occupies significant space before dilution

Question 7

3 ways bilirubin causes chemical analysis interference?

Answer 7

• Anti-oxidant&raquo_space; interferes with peroxidase catalyzed reactions (e.g. glucose, cholesterol, uric acids)
• Absorption spectrum with multiple peaks (major at 421nm)&raquo_space; interfere with other chemical spectrums
• Direct chemical reaction with some reactants

Question 8

2 ways that paraproteins cause chemical analysis interference?

Answer 8

Paraprotein = monoclonal Ab

1) Increases serum viscosity and affects sampling
2) Binds enzymes and forms macro-molecules (e.g. bind to creatine kinase)

Question 9

Cephalosporin interference.

• What condition?
• What test?
• Consequence?

Answer 9

Cephalosporin can be mistaken as Creatinine in Jaffe test ( colorimetric method used to determine creatinine levels in blood and urine)

e.g. Cephalosporin given to treat renal infection&raquo_space; increase creatinine reading might be false indicator of increased GFR and aggravated infection&raquo_space; False alarm

Question 10

Glucose oxidase method.

- Name one chemical that causes interference?

Answer 10

Ascorbic acid&raquo_space; Negative interference on glucose oxidase method

Question 11

5 ways to tackle lab test interferences? ***

Answer 11

1) Delta check: compare current and previous results, half-life and biological changes of reactants and samples
2) Check lab test incompatible with clinical conditions
3) Check Contradictory results/ unusual reaction rates
4) Choose another method with no interference (e.g. Change Jaffe reaction to enzymatic reaction in cephalosporin- creatinine interference)
5) Specific methods to block interference (e.g. block HAMA, PEG precipitation to filter macro-prolactin)

Question 12

Interference from time of reaction?

Answer 12

Reactions have variable rates

Always allow time to reach steady state

Question 13

Immunoassay interferences in a double antibody sandwich assay?

Answer 13

1) Interfering or cross-reacting substances:
- bind analyte
- Bind labeled detector antibody
- Bind capture antibody (fixed)
- bridge 2 antibodies without analyte

2) Heterophilic antibody:
- bridge 2 antibodies without analyte
- Bind capture antibody
- Bind labeled detector antibody

3) Poor antibody reactions:
- 2 antibodies bridge without analyte
- Labeled detector antibody fail to bind analyte

Question 14

Explain how cross-reactivity occurs.

Give one example

Answer 14

Antibodies recognize and bind to cross reactant and original analyte, both share similar structure

e.g. Digoxin immunoassay kits can cross react with digoxin-like immunoreactive substances

Question 15

What interferences can autoanalyte antibodies produce?

Answer 15

AutoAb that recognizes and binds to target analytes

Cause interference in non-immunoassays and immunoassays:

• e.g. Bind to prolactin/insulin/thyroglobulin and form large Ig complex
• e.g. Bind to enzymes and aggregates to disable enzyme metabolism

Question 16

Cause of macroprolactinemia?

Answer 16

Prolactin polymerize and aggregates with IgG (autoantibodies) and makes Macroprolactin&raquo_space; false reading of serum hyperprolactinemia without any symptoms

anti-PRL autoantibodies.

10–25% in patients with hyperprolactinemia and 3.7% in general population

Question 17

D/dx macroprolactinemia and genuine hyperprolactinemia

Answer 17

• Subfractionation with PEG&raquo_space; distinguish true hyperprolactinemia *****
• Dopamine agonist treatment
• Presence of adrenal incidentaloma in normal people without hyperprolactinemia

Question 18

3 ways that antibodies can cause chemical test interference?

Answer 18

1) Heterophilic Ab&raquo_space; Attacks reagent Ab
(e. g. Human Against Animal Ab HAMA)

2) High-dose Hook effect
3) Aberrant binding with other serum proteins: paraprotein, complement, lyzozymes…etc

Question 19

Define high dose hook effect

Answer 19

Very high analyte level results in paradoxically low result

Overloaded analytes cause conformational change to Ag-Ab complexes and dissociation, giving abnormally low reading after the maximum equivalence zone

Question 20

Explain one major source of interference with POCT glucometers.

Answer 20

Peritoneal dialysis: solution contains Icodextrin

Icodextrin metabolized in systemic circulation into glucose polymers (mainly maltose)&raquo_space;> Interferes with GDH-PQQ based Glucometer

> > > Falsely high blood glucose levels

Question 21

2 methods for POCT glucometers?

Name one interference for each.

Answer 21

Glucose oxidase-based system (interfered by ascorbic acid)

GDH-PQQ based method (interfered by Icodextrin in peritoneal dialysis)

Question 22

Explain one major interference in Digoxin immunoasay?

Answer 22

Generation of Digoxin-like immuno-reactive substances (DLIS, protein bound) in volume expansion conditions

> > DLIS cross react with anti-digoxin antibodies
Falsely high serum digoxin concentration

Question 23

List 4 conditions that generate DLIS and gives falsely high serum digoxin readings?

UELP

Answer 23

Volume-expanded conditions

Uremia
Essential hypertension
Liver disease
Pre-eclampsia

Question 24

How to overcome DLIS interference of Digoxin immunoassay?

Answer 24

Analyze Protein-free ultrafiltrate

Because DLIS are strongly protein-bound