Guideline for The Detection of Thalassemia Hemoglobin Identification

Guideline for The Detection of Thalassemia : Hemoglobin Identification นพ ชนนทร ลมวงศ

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 A 2 are in the same position

Isoelectric focusing

Isoelectric focusing Different isoelectric points for each Hb result in varying position in a p. H 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) A 0 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 A 2 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