Download HbTyping

Guideline for The Detection of
Thalassemia :
Hemoglobin Identification
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General status
 Confirmatory testing after screening test and
prior to DNA testing
 Widely available and designated personnel
are well trained
 Set up are standard and not much vulnerable
to variation given that samples are
appropriately collected and timely processed
 Interpretation skills are needed and crucial
Current Issues Regarding Hb Typing
 I. Variation in methodology
 II. Variation in testing condition
 III. Getting correct interpretation and
communicating proper results
 IV. Dealing with unknown (and known) peaks
 V. Interpreting fetal cord blood Hb typing
I. Variation in methodology
 Cellulose acetate electrophoresis
 Isoelectric focusing
 High pressure high performance liquid
chromatography (HPLC)
 Low pressure HPLC (LPLC)
 Capillary electrophoresis
 Protein sequencer
Pros and Cons of Different Methods
 Complimentariness between each can be useful
 Can be use as a stepwise testing
 Newer methods tend to be more automated,
higher throughput and with greater separating
ability
 More than one cutoff may lead to misdiagnosis
or waste of resource
 Unit cost calculation may vary thus affecting
budget calculation
Cellulose acetate electrophoresis
Courtesy of S.Sukpanichnant
Hb E and A2 are in the same position
Isoelectric focusing
Isoelectric focusing
 Different isoelectric points for each Hb result
in varying position in a pH gradient medium
 Greater separating ability than conventional
electrophoresis and HPLC
 Set up is relatively simple but not widely
available due to labor intensiveness
High Performance Liquid Chromatography (HPLC)
• Uses cation cartridge to
absorb Hb then eluents to
release Hb from the
column while their
absorbances at 415 nm are
being measured and
retention times recorded
•The result is an electropherogram showing
different peaks and
corresponding RTs
High Pressure High
Performance Liquid
Chromatography (HPLC)
A0
Variant Hemoglobin Analyzer
Normal Hb
type
Low Pressure HPLC (LPLC)
Hb-Gold, Drew Scientifics Ltd.
Capillary electrophoresis
Using small capillary and high voltage to separate Hb while using
absorbance measurement similar to chromatography method
Hb E and Hb A2 are separated
Hb H and Bart’s are well detected
II. Variation in testing condition
 How long has the sample been collected ?
Accentuation of fast moving peaks
Attenuation of slow moving peaks
 Contamination ?
Fetal maternal contamination
Contamination during collection
Carry-over contamination
Commonly encountered peaks that can
interfere with interpretation
 Tall H-Bart’s peaks with old samples
 Low E peak with old samples
 Unreliable stutters in slow moving zone esp.
Hb CS
 Shifting of RT with new column – unrecognized
peaks
 Widened base with column too old or too new –
false reading of %
 Tends to occur more with low pressure system
III. Issues regarding getting correct
interpretation and communicating proper
results
 Who is qualified to interpret ?
 Beware of confounding - transfusion
 How should it be interpreted ?
on an individual or couple basis
 Is screening available and used when interpret ?
 Can further suggestion be made to help clinician ?
(Pro)active laboratory
 Interprete possibility of hidden alpha trait
 Should alpha thal 2 be included ?
 Couple result interpretation
“non high risk” couple
at risk only for Hb H disease (mild severity)
at risk for Hb H CS disease (variable severity)
at risk for mild beta+/Hb E disease
 If possible screening and typing should appear
in the same page of report
IV. Dealing with an unknown peak
 Repeat if possible
 If suspected to be artifact, recollection may
be indicated (low peak and normal
MCV/MCH/Hb)
 Never call Hb type based upon reference
table, although specific Hb can be suspected
 Alpha variant ¼-beta ½ rule
 When found to be a rare Hb, literature or
reference or contact can be given
Hb F or Hb F + abnormal Hb
V. Interpreting fetal typing
 Need an experienced personnel
 Always make sure parental results are
available
 Keep in mind of incorrect paternity but never
mention this in the report. If highly
suspected, it is best to just state the typing
found without further interpretation
Conclusion
 Hb typing appears not to be the weakest link
in the chain of testing currently
 The system in place will only need a more
timely specimen processing, a corroborative
couple interpretation and a proactive lab
report
 Fetal typing should be at this time reserved
for specialized lab