SOP FOR THALASSEMIA

SCREENING
 SCREENING FOR HAEMOGLOBINOPATHIES
ADOLESCENT COLLEGE/HIGHER
PREGNANT
GIRLS/WOMEN SEC SCHOOL COMMUNITY
WOMEN
ATTENDING STUDENTS
(GA<18 Weeks)
HOSPITALS

EDUCATION/AWARENESS/CONSENT

SCREENING CASCADE
NEGATIVE POSITIVE
[CBC, HPLC] SCREENING
 METHODOLOGY

• Complete Blood Count:

• Fully automated or semi automated analyzers can be used
• They should be calibrated regularly and controls should be run daily and
monitored.
• Haemoglobin Analysis
• Cation exchange high performance liquid chromatography (HPLC) - method of
choice for quantification of HbA2, HbF and for detection and quantitation of the
Hb variants
• Calibration should be done daily and response factor monitored
• Retention time of HbA2 peak in the calibrator should elute at a standard time
(Refer kit insert). The column temperature needs to be adjusted sometimes to
ensure this
• Normal and abnormal controls should be included in each run
 RESULT INTERPRETATION

MCV <78fL
Normal Range MCH <27pg β-Thal Carrier
Elevated HbA2 (>3.5%)
Hb Genetic
Men -14-16g/dL Normal or Low MCV
Borderline HbA2 Diagnosis
Women- 12-14g/dL
Pregnant women > 11g/dL (3.3-3.7%) Spouse
Screening β-Thal Carrier
MCV > 80fL Low MCV (<78fL)
Normal HbA2 Iron def
MCH >27pg
High RDW
HbF < 1.5% MCV <78fL
MCH <25pg
Normal HbA2 Alpha Thalassemia
HbA2 < 3.5%
High RBC Count

Normal MCV and

Normal HbA2 NORMAL
 RESULT INTERPRETATION

Variant haemoglobins of clinical relevance are haemoglobins S,E, Lepore, D-Punjab

Normal/Low MCV
S-Window Heterozygous Sickle
(27-41%)

Normal/Low MCV
S-Window >50%
Homozygous Sickle
HbF> 10%
HbA2 <3.5%

Low MCV
S-Window >50%
Sickle-β Thalassemia
HbF> 10%
HbA2 >3.5%
 RESULT INTERPRETATION

Variant haemoglobins of clinical relevance are haemoglobins S,E, Lepore, D-Punjab

Normal/Borderline MCV
Heterozygous HbE Hb Lepore carrier
HbA2/E -25-32%
MCV- Borderline
HbA2 >10% but less than 20%
Retention time- less than
Low MCV HbA2
HbF< 10% Homozygous HbE
HbA2/E>80%

Low MCV
HbF> 10%
HbE-β
Thalassemia
HbA2/E > 50%
 RESULT INTERPRETATION

Normal/Borderline MCV
MCH <27pg
Heterozygous delta
beta thalassemia
HbF >5 -25%

Normal MCV, MCH

HbF >25-35%
Heterozygous
HPFH

REFERENCES
• Indian J Hum Genet. 2014 Apr-Jun; 20(2): 101–119
• British Journal of Haematology, 2010, 149, 35–49
• Prevention of Thalassaemias and Other Haemoglobin Disorders: Volume 2: Laboratory Protocols [Internet]. 2nd edition. 2012.
• British Journal of Haematology, 2014, 166, 607-611
 PRENATAL DIAGNOSIS
PREGNANT
WOMEN β-THALASSEMIA CARRIER
(GA<18 Weeks) GENETIC COUNSELLING AND
GENETIC
PRENATAL DIAGNOSIS
DIAGNOSIS
β-THALASSEMIA CARRIER
SPOUSE

NORMAL

Chorionic Villus HOMOZYGOUS

Sample COMPOUND HETEROZYGOUS

(MATERNAL CONTAMINATION TO BE RULED OUT BY VNTR ANALYSIS)

HETEROZYGOUS
(MATERNAL CONTAMINATION TO BE RULED OUT BY VNTR ANALYSIS IF IT
CARRIES THE MOTHER’S MUTATION)
 NEWBORN SCREENING OF HAEMOGLOBINOPATHIES
The purpose of newborn hemoglobinopathy screening is primarily to detect sickle cell
disease.

Methodology Available
1. The Bio-Rad VARIANT™nbs Sickle Cell Program
The VARIANTnbs Sickle Cell Program utilizes the principles of cation-exchange high-performance liquid
chromatography (HPLC). Eluates of dried blood spot specimens in microplates are maintained at 9±2°C in the
system’s Newborn Automatic Sampler (VNAS). Each specimen is sequentially injected into the analysis stream
and then separated by the analytical cartridge.

2. HemotypeSCTM
HemotypeSC is a competitive lateral flow immunoassay incorporating monoclonal antibodies for determination
of the presence of hemoglobins A, S and C providing rapid detection of hemoglobin phenotypes HbAA, HbSS,
HbSC, HbCC, HbAS, and HbAC.
 NEWBORN SCREENING OF HAEMOGLOBINOPATHIES
INTERPRETATION OF RESULTS
Bio-Rad VARIANT™nbs Sickle Cell Program
• All QC requirements must be met to accept a run.
• The VARIANTnbs identifies hemoglobins by the comparison of peak retention times with the peak retention
time of a known hemoglobin analyzed on the system. Known hemoglobins elute in retention time “windows”
that have been previously established.
• Total area must be between 750,000 and 6,300,000 microvolt•second.

Normal Neonate Sickle cell Trait

 NEWBORN SCREENING OF HAEMOGLOBINOPATHIES
INTERPRETATION OF RESULTS
. HemotypeSCTM