Hemolysis, lipemia , Icterus

1. INTERFERENCES IN CLINICAL
CHEMISTRY ANALYSIS
(Hemolysis, lipemia , Icterus)
Raffat Al Telbani
MSc. Biological Sciences

2.  Interference occurs when a substance or process
falsely alters an assay result.
 These altered results may lead to repeat tests,
incorrect diagnoses, and treatments with potentially
unfavorable outcomes for the patient.
2

3.  Interferences are classified as:
 Endogenous
 Exogenous
3

4.  Endogenous interference originates from
substances found naturally in the patient sample.
 Hemolysis
 Bilirubin
 Lipids
 Paraproteins
 Excessive analyte concentration
4

5.  Exogenous interference results from
substances not naturally found in the patient’s
specimen.
 Drugs
 Poisons
 Herbal products
 IV fluids
 Substances used as therapy (Antibodies)
 Collection tube components
(Anticoagulant)
 Test sample additives (Preservatives)
5

6.  Hemolysis is the release of hemoglobin and other
intracellular components of erythrocytes and
other blood cells to the surrounding plasma
following damage of the cell membrane.
 Hemolysis occurs in about 3% of all specimens
received in the laboratory and accounts for 40%
to 70% of all unsuitable specimens.
 Hemolysis may occur either in vivo or in vitro.
6

7.  In vivo hemolysis can originate from:
7
 Inherited hemolytic anemia
 Defect in hemoglobin production
(Thalassemia, Sikckle cell disease)
 Defect in RBC membrane
(Spherocytosis, Elliptocytosis, PNH)
 Defect RBC metabolism
(G6PD, Pyruvate kinase deficiency)

8. 8
 Acquired hemolytic anemia
 Immune mediated causes
(Autoimmune hemolytic anemia)
(Alloimmune hemolytic anemia)
(Drug induced immune hemolytic anemia)
 Non-immune mediated causes
(Drugs) (Toxins) (Mechanical)
(Microangiopathic hemolytic anemia)
(Infections)
 In vivo hemolysis can originate from:

9.  Usually less than 2% of all the specimens with
hemolysis may be due to in vivo hemolysis.
 In vivo hemolysis does not depend on the
technique of the healthcare provider and it is thus
virtually unavoidable and cannot be resolved.
9

10.  In vitro hemolysis can occur beginning at the
patient’s bedside and continue through sample
handling, processing and storage.
 Leading causes of in vitro hemolysis:
 Patient dependent
 Operator dependent
 Device dependent
 Handling of the specimen
 Transport of the specimen
 Sample processing
 Sample storage
10

11. 11
 Differentiating between in vivo and in vitro
hemolysis is vitally important to patient
management
In vitro hemolysisIn vivo hemolysis
Haptoglobin↓ Haptoglobin
↑ AST
↑ Unconjugated
Bilirubin
↑ K↑Reticulocytes
↑ LDHLDH↑

12. 12
Gross AppearanceFree HemoglobinClassification
Yellow≤5 mg/dLNo hemolysis
yellow to Pink25-50 mg/dLSlightly hemolysis
Pink to Red50-200 mg/dLModerate hemolysis
Red to brown200 mg/dL≤Sever hemolysis

13.  Additive: Several constituents are normally present in large
amounts within the red blood cells. When the red blood cells
burst these substances will be released and their values will
become falsely elevated (LDH, K, AST).
13
E/P RatioPlasmaErythrocytesSubstance
16036058000LDH, U/L
22.84.4100.0Potassium, mmol/L
2025500AST, U/L
73.022.0APs, U/L
530150ALT, U/L

14.  Dilutional: release of intracellular fluid into the
surrounding extracellular area, the extracellular fluid
becomes more dilute and analytes will appear falsely
low (Na, Cl, Ca).
14
E/P RatioPlasmaErythrocytesSubstance
0.110.01.0Calcium, mg/dL
0.11140.016.0Sodium, mmol/L
0.5104.052.0Chloride, mmol/L

15.  Chemical: by hemolysis there is several substances in
the solution that may cross-reacting with the analyte.
 Adenylate kinase (CK and CKMB)
 Pseudoperoxidase activity of hemoglobin (Bilirubin)
 Heme group (Iron)
15

16.  Spectral: Hemoglobin shows strong absorbance
at 415 nm and have two peaks at 540 and 570
nm. Therefore it may greatly interfere with
measurement at these wavelengths.
16

17. 17
Hemolyzed
Sample
Quantification of Hemolysis
Clinically or analytically
Significant
Clinically or analytically
Insignificant
Perform analyses and report
result without warnings
Analyses unaffected
by hemolysis
Do not perform analyses and
ask for an additional Sample
Analyses Affected
by Hemolysis
perform analyses and report
result with warnings
If the recollected specimen is also hemolyzed,
perform analyses and report result with warnings

18. 18
Test to be
suppressed
Free
Hemoglobin
Degree of
Hemolysis
• LDH
• Potassium
• AST
25-50 mg/dLSlightly
• CK & CKMB
• ALP
• ALT
50-200 mg/dLModerate
Virtually all test200 mg/dL≤Sever
 The amount of hemolysis needed to affect a test is
dependent on the test being performed.

19. 19
 Lipemia is the presence of a fine emulsion of
fatty substance in the blood.
 It is characterized by turbidity of samples which
may range from slightly opaque through
transparent, turbid to milky appearance.

20. 20

21. 21
VLDLChylomicrons LDL HDL
VLDL
L
VLDL
M
VLDL
S
70 – 1000 nm
20 – 26 nm 6 – 12.5 nm
60 – 200 nm 35 – 60nm 27 – 35 nm

22.  The overall frequency of lipemic samples ranges
from 0.5-2.5 %, depending on the type of hospital
and proportion of inpatient and outpatient samples.
22

23. 23
 Pathophysiological conditions
 Acute pancreatitis
 Alcoholism
 Chronic kidney failure
 Hypothyreosis
 Multiple myeloma
 Primary biliary cirrhosis
 Diabetes mellitus

24. 24
 Preanalatical factors
 Improper time of sampling
 Medications

25.  Visual detection
 Serum / plasma
TG over 300 mg/dL
 Full blood
TG over 1000 mg/dL
25

26.  Triglyceride measurement: Measurement
of triglyceride concentration may be
assessment of degree of lipemia.
26

27. 27
 Spectral: Lipeuimia interferes by scattering the
light and disturbing the transmission of light
through the reaction mixture.

28. 28
 Volume displacement: An increased
concentration of lipids results in serum volume with
decreased water content

29. 29
 Non-homogeneity:
Hydrophobic
(lipid) phase
Falsely
Hydrophilic
analytesHydrophilic
(lipid) phase
Falsely
Hydrophobic
analytes

30. 30
 Centrifugation
 Ultracentrifugation:
~ 90,000 rpm.
 High speed centrifugation:
~ 13,000 rpm
 centrifugation:
~ 3000-5000 rpm

31. 31
 Extraction
 Extraction of lipoproteins into hydrophobic
phase
 Polyethylene glycol
 Cyclodextrin
 Lipoclear

32. 32
 Sample dilution
 Sample can be diluted only enough to remove the
turbidity interference (2 or 3 fold).

33. 33
 Icterus is the yellowish discoloration of skin
and mucous membrane and body fluids,
resulting from increased level of bilirubin in
the plasma (Hyperbilirubinemia).

35. 35
 Pre-hepatic (Hemolytic)
 Hepatic (Hepatocellular)
 Post-hepatic
(Cholestatic)

36. 36
 Spectral interference: Bilirubin absorbs
strongly between 340 nm and 500 nm
wavelengths

37. 37
 Chemical interference:
 Reducing substance
 Dye binding
 Destruction of reaction intermediate

38. 38

39. 39