2. Introduction.Introduction.
►Haemoglobinopathies are ‘inheritedHaemoglobinopathies are ‘inherited
abnormalities of globin chain synthesis’abnormalities of globin chain synthesis’ (B.(B.
Bain 2007)Bain 2007)..
►Encompass a clinical spectrum fromEncompass a clinical spectrum from
asymptomatic findings on blood film toasymptomatic findings on blood film to
death in utero.death in utero.
►Wide spread distribution throughout theWide spread distribution throughout the
world and increasing prevalence inworld and increasing prevalence in
multicultural Australia.multicultural Australia.
3. Hemoglobin Structure.Hemoglobin Structure.
►Hemoglobin transports oxygen to theHemoglobin transports oxygen to the
tissues.tissues.
►Each RBC contains hemoglobin.Each RBC contains hemoglobin.
►A normal hemoglobin molecule consists of:A normal hemoglobin molecule consists of:
Four globin chains (2 alpha, 2 beta).Four globin chains (2 alpha, 2 beta).
Each globin chain has an iron containing hemeEach globin chain has an iron containing heme
molecule.molecule.
The iron in the heme molecule binds to oxygen.The iron in the heme molecule binds to oxygen.
4.
5. Genetics.Genetics.
►In the first 8 weeks of embryonic life theIn the first 8 weeks of embryonic life the
predominant forms of hemoglobin are:predominant forms of hemoglobin are:
Hb Gower 1 (Hb Gower 1 (ζζ22εε22).).
Hb Gower 2 (Hb Gower 2 (αα22εε22).).
Hb Portland 1 (Hb Portland 1 (ζζ22γγ22).).
►By the 12By the 12thth
week embryonic hemoglobin isweek embryonic hemoglobin is
replaced by Hb F (replaced by Hb F (αα22γγ22) which represents 70) which represents 70
– 100% of hemoglobin in fetal life.– 100% of hemoglobin in fetal life.
6. Genetics (2).Genetics (2).
► Adult hemoglobin Hb A (Adult hemoglobin Hb A (αα22ββ22) detectable from) detectable from
16/40, replaces Hb F as predominant hemoglobin16/40, replaces Hb F as predominant hemoglobin
by 6/12 after birth, up to 30% of Hb in fetal life.by 6/12 after birth, up to 30% of Hb in fetal life.
► Hemoglobin HbA2 (Hemoglobin HbA2 (αα22ΔΔ22) is present in utero but) is present in utero but
only very minor in normal adults.only very minor in normal adults.
► In normal adults 96 – 98% of hemoglobin is HbA,In normal adults 96 – 98% of hemoglobin is HbA,
Hb A2 (2 – 3%) and HbF (<1%) constitute a minorHb A2 (2 – 3%) and HbF (<1%) constitute a minor
component of the total hemoglobin.component of the total hemoglobin.
10. Geography.Geography.
►Commonest genetic defect world wide withCommonest genetic defect world wide with
an estimated 269 million carriers.an estimated 269 million carriers.
►90 million carriers in South East Asia, 8590 million carriers in South East Asia, 85
million in Sub Saraharan Africa, and 48million in Sub Saraharan Africa, and 48
million in the West Pacific region.million in the West Pacific region.
►Distributed across South East Asia in a lineDistributed across South East Asia in a line
stretching from Southern China down thestretching from Southern China down the
Malaysian Peninsula to Indonesian islands.Malaysian Peninsula to Indonesian islands.
11. Geography (2).Geography (2).
►Also distributed across the Mediterranean,Also distributed across the Mediterranean,
Middle East, and Indian Subcontinent.Middle East, and Indian Subcontinent.
►The distribution of the defect is thought toThe distribution of the defect is thought to
be due to partial protection for carriers frombe due to partial protection for carriers from
Plasmodium Falciparum Malaria.Plasmodium Falciparum Malaria.
15. Haemoglobinopathy Classification.Haemoglobinopathy Classification.
►Thalassaemia:Thalassaemia:
Primary abnormality is a reduced rate ofPrimary abnormality is a reduced rate of
synthesis of globin chains.synthesis of globin chains.
Defined by imbalanceDefined by imbalance αβαβ ratio.ratio.
Traditionally though not invariably microcyticTraditionally though not invariably microcytic
hypochromic anemia.hypochromic anemia.
►Variant hemoglobin's in which there is aVariant hemoglobin's in which there is a
structural abnormality in the globin chain.structural abnormality in the globin chain.
18. Alpha Thalassaemia ClassificationAlpha Thalassaemia Classification
(2).(2).
►αα trait due to deletion of one or two of thetrait due to deletion of one or two of the
four alpha genes, asymptomatic (egfour alpha genes, asymptomatic (eg
-- αα// α αα α, --/, --/ α αα α, -, - αα/-/- αα).).
►Hemoglobin H disease is the lack of three ofHemoglobin H disease is the lack of three of
the fourthe four αα genes resulting in alphagenes resulting in alpha
thalassaemia major.thalassaemia major.
►Hemoglobin Bart’s Hydrops Fetalis resultsHemoglobin Bart’s Hydrops Fetalis results
from absence of all fourfrom absence of all four αα genes,genes,
incompatible with post natal life.incompatible with post natal life.
19. Beta Thalassemia Classification.Beta Thalassemia Classification.
►ββ thal major is homozygosity or compoundthal major is homozygosity or compound
heterozygosity resulting in severeheterozygosity resulting in severe
phenotype.phenotype.
►ββ thal minor or trait is heterozygosity withthal minor or trait is heterozygosity with
asymptomatic phenotype.asymptomatic phenotype.
►ββ thal intermedia is an intermediatethal intermedia is an intermediate
phenotype produced by a variety ofphenotype produced by a variety of
genotypes.genotypes.
20. Beta Thalassemia Classification (2).Beta Thalassemia Classification (2).
►ββ 0 syndromes are characterized by the0 syndromes are characterized by the
affected gene producing no beta chain.affected gene producing no beta chain.
►ββ + syndromes are characterized by the+ syndromes are characterized by the
abnormal gene producing beta chains at aabnormal gene producing beta chains at a
reduced rate.reduced rate.
►Usually due to point mutations.Usually due to point mutations.
21. Variant Hemoglobin Classification.Variant Hemoglobin Classification.
►The variant haemoglobins are disorders ofThe variant haemoglobins are disorders of
globin chain synthesis.globin chain synthesis.
►NormalNormal αβαβ ratio so most have normal MCVratio so most have normal MCV
and MCH.and MCH.
►There are over 1000 mutations associatedThere are over 1000 mutations associated
with the haemoglobinopathies most of whichwith the haemoglobinopathies most of which
will produce variants.will produce variants.
22. Variant Hemoglobin Classification.Variant Hemoglobin Classification.
►Initially recognized forms were classifiedInitially recognized forms were classified
alphabetically (Hb C, D, E), subsequentalphabetically (Hb C, D, E), subsequent
naming after the location of discovery.naming after the location of discovery.
►The most common forms in AustraliaThe most common forms in Australia
include Hb S, Hb E, Hb Constant Springinclude Hb S, Hb E, Hb Constant Spring
and Hb C.and Hb C.
►Usually caused by point mutations.Usually caused by point mutations.
23. FBC.FBC.
►Thalassaemias are typically microcytic andThalassaemias are typically microcytic and
hypochromic anemia's.hypochromic anemia's.
►Thalassemia causes a uniform microcytosisThalassemia causes a uniform microcytosis
without increase in RDW (cf iron deficiency).without increase in RDW (cf iron deficiency).
►Hb H andHb H and ΔβΔβ thal however can cause anthal however can cause an
increased RDW.increased RDW.
24. FBC (2).FBC (2).
►RBC often increased in thal but decreasedRBC often increased in thal but decreased
in iron deficiency and AOCD.in iron deficiency and AOCD.
►Hb typically normal in thal minor butHb typically normal in thal minor but
decreased in intermedia and majordecreased in intermedia and major
syndromes.syndromes.
►MCV is the most valuable parameter inMCV is the most valuable parameter in
predicting thal.predicting thal.
27. Iron Studies.Iron Studies.
►Except in the urgent situation of pregnancyExcept in the urgent situation of pregnancy
iron deficiency should always be excludediron deficiency should always be excluded
and treated prior to work up for thal.and treated prior to work up for thal.
►MCV and MCH are influenced by ironMCV and MCH are influenced by iron
deficiency.deficiency.
►Hb A2 can be lowered by iron deficiencyHb A2 can be lowered by iron deficiency
falsely normal results if tested when ironfalsely normal results if tested when iron
deficient (false negatives).deficient (false negatives).
28. Table 1: Laboratory features in different clinical states.
Iron
Deficiency
Chronic
Disease
Iron
Overload
Thalassemia
Haemoglobin N or ↓ ↓ N N or ↓
Serum Fe ↓ ↓ ↑ ↑ or N
Transferrin
Receptor
↑ ↓ or N ↓ N
Transferrin Sat. ↓ ↓ ↑ N
Ferritin ↓ ↑or N ↑ ↑ or N
MCV ↓ ↓ or N N ↓
Marrow Fe ↓ ↑ ↑ ↑
29. Hb H Inclusions.Hb H Inclusions.
►Hb H is an insoluble tetramer consisting ofHb H is an insoluble tetramer consisting of
four beta globin chains, due to a lack offour beta globin chains, due to a lack of
alpha chains in alpha thal major.alpha chains in alpha thal major.
►Oxidation of these tetramers provokesOxidation of these tetramers provokes
precipitation which can be visualizedprecipitation which can be visualized
microscopically as ‘golf ball’ inclusions.microscopically as ‘golf ball’ inclusions.
►Oxidation can be precipitated by oxidativeOxidation can be precipitated by oxidative
dyes (although there is significant batch todyes (although there is significant batch to
batch variability making controls essential).batch variability making controls essential).
30. Hb H Inclusions (2).Hb H Inclusions (2).
►In Hb H disease 30 – 100% of RBCSIn Hb H disease 30 – 100% of RBCS
contain Hb H inclusions.contain Hb H inclusions.
►In alpha thal minor there is one cell with HbIn alpha thal minor there is one cell with Hb
H inclusions per 1000 – 10,000 RBCS.H inclusions per 1000 – 10,000 RBCS.
►Other nucleic acid and protein precipitatesOther nucleic acid and protein precipitates
also stain (without ‘golf ball’ pattern).also stain (without ‘golf ball’ pattern).
31. Hb H inclusions (3).Hb H inclusions (3).
►When there is a reticulocytosis a rare Hb HWhen there is a reticulocytosis a rare Hb H
inclusion may be missed – operatorinclusion may be missed – operator
experience crucial.experience crucial.
►Detection of Hb H inclusions points to anDetection of Hb H inclusions points to an
alpha chain mutation and narrows thealpha chain mutation and narrows the
amount of DNA analysis required.amount of DNA analysis required.
►False negatives problematic, even with 2False negatives problematic, even with 2
alpha gene deletions no inclusions may bealpha gene deletions no inclusions may be
seen after several minutes of searching.seen after several minutes of searching.
32.
33. Kliehauer Test For Hb F.Kliehauer Test For Hb F.
►Used to detect the presence of Hb F (fetalUsed to detect the presence of Hb F (fetal
hemoglobin).hemoglobin).
►RBCS on a slide are stained to detect theRBCS on a slide are stained to detect the
presence of Hb F.presence of Hb F.
►Can distinguish hetrocellular HbF fromCan distinguish hetrocellular HbF from
pancellular HbF seen in HPFH.pancellular HbF seen in HPFH.
34. Kleihauer Test For Hb F (2).Kleihauer Test For Hb F (2).
►Rarely done and difficult to interpret andRarely done and difficult to interpret and
standardize due to significant variabilitystandardize due to significant variability
between observers.between observers.
►Confirms maternal blood contamination withConfirms maternal blood contamination with
fetal blood in cases of fetomaternalfetal blood in cases of fetomaternal
hemorrhage, with D mismatch.hemorrhage, with D mismatch.
►Flow cytometry is now the primary tool forFlow cytometry is now the primary tool for
investigation of fetal haemoglobins ininvestigation of fetal haemoglobins in
Australia.Australia.
35.
36. Electrophoresis Principle.Electrophoresis Principle.
►Separation of haemoglobins withSeparation of haemoglobins with
electrophoresis at pH 8.4 (alkaline) and pHelectrophoresis at pH 8.4 (alkaline) and pH
6.2 (acid).6.2 (acid).
►Scanning allows quantification of theScanning allows quantification of the
hemoglobin present, bands are seen byhemoglobin present, bands are seen by
staining.staining.
►At alkaline pH Hb C, E, A2 and O migrateAt alkaline pH Hb C, E, A2 and O migrate
together to form a single band, Hb S, D andtogether to form a single band, Hb S, D and
G also co migrate.G also co migrate.
37. Electrophoresis Principle (2).Electrophoresis Principle (2).
►At acid pH Hb C separates from E and OAt acid pH Hb C separates from E and O
and Hb S separates from D and G.and Hb S separates from D and G.
►Hb E and O cannot be separated byHb E and O cannot be separated by
electrophoresis neither can Hb D and G.electrophoresis neither can Hb D and G.
38. (1) Normal (2) New born (3) Hb C trait [A-C] (4) Hb SC disease [S-C] (5) Sickle cell
disease [S-S], (6) Sickle cell trait [A – S] (7) New born (8) Normal.
39. Electrophoresis Interpretation.Electrophoresis Interpretation.
HbA2 rangeHbA2 range InterpretationInterpretation
> 7.0 %> 7.0 % Rare, repeat to verify test.Rare, repeat to verify test.
Exclude a structural variant.Exclude a structural variant.
Can be due to rareCan be due to rare ββ thal mutations.thal mutations.
3.8 – 7.0 %3.8 – 7.0 % Beta thal trait or unstable Haemoglobin.Beta thal trait or unstable Haemoglobin.
3.4 – 3.7 %3.4 – 3.7 % Fe deficiency inFe deficiency in ββ thal trait;thal trait; ΔΔ chain variant withchain variant with ββ thal trait.thal trait.
Interaction ofInteraction of αα andand ββ thal traits; rarethal traits; rare ββ thal mutations.thal mutations.
HbS making measurement inaccurate; interaction ofHbS making measurement inaccurate; interaction of αα - Hb S.- Hb S.
2.0 – 3.3 %2.0 – 3.3 % Normal.Normal.
ΔΔ andand ββ thal (but HbF should be elevated); alpha thal trait.thal (but HbF should be elevated); alpha thal trait.
Rare cases ofRare cases of ββ thal trait coexisting with eitherthal trait coexisting with either ΔΔ oror αα thal trait.thal trait.
< 2.0 %< 2.0 % ΔΔ ββ thal (but HbF should be elevated).thal (but HbF should be elevated).
Alpha thal trait; Hb H disease;Alpha thal trait; Hb H disease; ΔΔ variant or delta Thalassemia.variant or delta Thalassemia.
Iron deficiency.Iron deficiency.
40. Electrophoresis Strengths.Electrophoresis Strengths.
►Commercial, widely available, rapidCommercial, widely available, rapid
methods used for many years.methods used for many years.
►Gives an estimate of HbA2 level.Gives an estimate of HbA2 level.
►Identifies some variant haemoglobins whichIdentifies some variant haemoglobins which
are well characterized.are well characterized.
42. Electrophoresis Disadvantages (2).Electrophoresis Disadvantages (2).
►Coefficient of variation (CV) 33.6% for gel
electrophoresis (mean HbA2 concentration
2.41%).
►Column chromatography has CV 14.6%
(mean HbA2 3.21%) and HPLC has CV
4.3% (mean HbA2 3.47%).
► An imprecise test in comparison to other
tests now available.
43. Isoelectric Focusing.Isoelectric Focusing.
►Equilibrium process in which Hb migrates in
a pH gradient to a position of 0 net charge
can be used to separate and quantify Hb.
►Excellent resolution allowing precise and
accurate Hb quantification.
►Labor-intensive and time-consuming.
44. Isoelectric Focusing (2).Isoelectric Focusing (2).
►The migration order is the same as with
alkaline electrophoreses however HbC and
E separate as do HbO and S and HbD and
G.
►Hb A and F are also clearly separated.
►Both more accurate and more precise than
standard electrophoresis.
45. Capillary Isoelectric Focusing.Capillary Isoelectric Focusing.
►Hybrid technique combining capillary
electrophoresis sensitivity with automated
sampling and data acquisition of HPLC.
►Established role in the detection and
quantification of Hb variants.
►Separation of Hb in this method is related to
the isoelectric point of the Hb, and this may
enhance inter laboratory reproducibility.
46. Capillary Isoelectric Focusing (2).Capillary Isoelectric Focusing (2).
►Used to quantify Hb variants, HbA2 and
HbF.
►Quantitative and qualitative Hb variant
results from CIEF and cation exchange
chromatography are highly correlated.
►CIEF gives slightly better resolution of the
unusual variants HbC Harlem and HbD
Punjab compared to chromatography.
47. HPLC Principle.HPLC Principle.
► Cation-exchange HPLC can be preformed on an
automated instrument that can quantify Hb A2, Hb
F, Hb A, Hb S, and Hb C.
► Studies show equivalence or superiority over
electrophoresis in terms of identification of variant
haemoglobins and quantification of HbA2 level.
► Negatively charged carboxyl molecules bound to
silica make up the cartridge matrix.
48. HPLC Principle (2).HPLC Principle (2).
►Positively charge molecules (salt and
hemoglobin) bind to the carboxyl groups.
►Haemoglobin molecules are bound and
displaced by increasing salt concentration.
►Haemoglobin variants separate out due to
variation in charge.
49.
50.
51.
52.
53.
54. HPLC Disadvantages.HPLC Disadvantages.
► Hbs may co-elute or may elute before instrument
peak integration.
► HbE, HbOsu Christianbourg, and HbG
Copenhagen co-elute with Hb A2, making
quantification impossible when these variants
present.
► The measurement of Hb A2 is complicated in
individuals with Hb S because the Hb A2 is falsely
increased by the presence of Hb S adducts.
55. HPLC Disadvantages (2).HPLC Disadvantages (2).
► Capillary zone Electrophoretic method can be
used to quantify Hb A2 in the presence of Hb S by
eliminating interference from these adducts.
► Interference can also be eliminated by the use of
micro anion-exchange column methodology.
► Integration errors can result in false decreases in
the values obtained, although this can be
minimized by applying known corrections.
56. HPLC Disadvantages (3).HPLC Disadvantages (3).
►Haemoglobinopathies cause inappropriately
high HbA1c (HbNiigata, HbSherwood
Forest, HbRambam, HbRaleigh).
►The presence of a structural Hb variant may
adversely affect the measurement of
HbA1C.
57. HPLC Strengths.HPLC Strengths.
► Method of choice for screening for Hb variants; for
quantification of HbA2 + HbF concentrations and
mandatory in neonatal screening.
► Quicker and more sensitive than standard
techniques for detecting HbF (in diagnosis of
HPFH and monitoring sickle cell anemia).
► Indeed alkaline gel electrophoresis cannot detect
HbF in healthy adults or those with marginally
increased Hb F.
58. HPLC Strengths (2).HPLC Strengths (2).
►Can be used to characterize rare
haemoglobinopathies not well detected with
other methods (HbRambam).
►Established role in the diagnosis of
thalassaemia and haemoglobinopathies,
including with cord blood samples.
59. HPLC Strengths (3).HPLC Strengths (3).
►HPLC should be the primary method for
detecting variant hemoglobin's and
simultaneous quantitation of HbA2 and HbF.
►This replaces three separate methods:
Haemoglobin electrophoresis.
Quantitation of HbA2.
Quantification of HbF).
60.
61. DNA Analysis.DNA Analysis.
► Indicated when the hemoglobinopathy not
confirmed by other methods or when the
underlying mutation important to management.
► Other techniques lead to a presumptive
identification of the hemoglobinopathy only.
► For genetic counseling defining the particular
mutation or deletion is often required – this is
achieved by a variety of molecular techniques.
62. DNA Analysis (2).DNA Analysis (2).
► DNA from WBCs, amniocytes, or chorionic tissue
may be utilized for diagnosis of various α and β
globin chain abnormalities.
► Southern blot hybridization using restriction
enzymes digesting labeled complementary probes
define deletional mutations in α and rare β thal.
► PCR amplifies globin genes and utilizes allele
specific primers to detect known globin chain
mutations eg HbS, E, D, O + several β thal.
63. DNA Analysis (3).DNA Analysis (3).
►PCR can be used to detect unknown
mutations.
►Aims to separate amplified DNA on gels or
with HPLC on the principle that different
amino acids migrate differently.
►3 primary methods – mutation analysis,
DNA scanning and DNA sequencing.
64. Mutation Analysis.Mutation Analysis.
►DNA testing for thal is tailored to prevalent
local mutations and suggested mutations on
the basis of preliminary testing.
►Based on PCR which provides rapid,
accurate identification of multiple single
point mutations.
65. Mutation Analysis (2).Mutation Analysis (2).
►In HK there are 15 common non deletional
alpha gene defects and 23 common beta
thal mutations.
►HK mutations involve single base mutations
and can be simultaneously tested for by
printing relevant oligonucleoties onto slides.
►This allows for mass screening.
66. DNA Scanning.DNA Scanning.
► Useful when screening large DNA segments or
exons for nucleotide changes is necessary due to
the possibility of many different mutations.
► Can dissect a gene into discrete fragments eg 500
bp in size allowing mutations to be found in large
genes.
► Uses techniques such as SSCP (single stranded
conformation polymorphism) and DGGE
(denaturing gradient electrophoresis).
67. DNA Scanning (2).DNA Scanning (2).
► Denaturing HPLC is most common and based on
different melting temps of hetroduplexes and
homoduplexes which can be separated by EP.
► Any putative mutations must be confirmed by DNA
sequencing to distinguish mutations from neutral
polymorphisms.
► Hemoglobin genes are relatively small (1.6 kb with
3 exons) and DNA sequencing is increasingly
accessible hence scanning is less frequently
used.
68. DNA Sequencing.DNA Sequencing.
► DNA sequencing is now standard practice for
looking for mutations in the beta and alpha globin
genes.
► Indicated if mutations are not detectable with the
preliminary screening and in difficult cases eg N
HbA2 beta thal or silent beta thalassaemia.
► Difficult cases best delineated by direct gene
sequencing because a number of causative
mutations result in the observed phenotype.
69. DNA Sequencing (2).DNA Sequencing (2).
►In beta thalassaemia there can be normal
HbA2 so if the mutation absolutely needs to
be excluded, DNA sequencing is preferred.
►Entire sequence of both α genes and most
of the β globin gene can be sequenced with
four primer sets.
►Dye primers or fluorescently labeled M13
primers are used to initiate elongation.
70. DNA Sequencing (3).DNA Sequencing (3).
► In heterozygous thal DNA sequence will show a
mutated gene and normal nucleotide base easy
to miss the mutated gene.
► Overcome by sequencing forward and reverse
strands but still necessary to visually inspect the
sequence tedious and source of error.
► Becoming cheaper and more accessible, as
software is developed to assist in sequence
analysis.
71. DNA sequencing trace (a) forward primer, (b) reverse primer. In the reverse primer it is
clear on visual inspection that there is a point mutation with both G and C being present.
The forward sequence, when it has been magnified, shows a small “blip” representing
the mutant C base under the normal G sequence.
72. Sickle Solubility Tests.Sickle Solubility Tests.
► Works on principle that HbS is insoluble inWorks on principle that HbS is insoluble in
deoxygenated state forming crystals that refractdeoxygenated state forming crystals that refract
light and cause the solution to be turbid.light and cause the solution to be turbid.
► Detects HbS at conc.Detects HbS at conc. >> 20% and differentiates20% and differentiates
HbD and G which migrate with Hb S on celluloseHbD and G which migrate with Hb S on cellulose
acetate electrophoresis at alkaline pH.acetate electrophoresis at alkaline pH.
► Positive results are also obtained on samplesPositive results are also obtained on samples
containing both HbS and beta globulin mutations.containing both HbS and beta globulin mutations.
73. Sickle Solubility Tests (2).Sickle Solubility Tests (2).
►False positives can occur in leukocytosis,False positives can occur in leukocytosis,
hyperprotienemia and unstable hemoglobinhyperprotienemia and unstable hemoglobin
states.states.
►False negatives can occur in patients withFalse negatives can occur in patients with
anemia or if outdated buffer is used and inanemia or if outdated buffer is used and in
infants less than 6 months.infants less than 6 months.
►All results must be confirmed by the moreAll results must be confirmed by the more
accurate HbEP or HPLC.accurate HbEP or HPLC.
74.
75. Immunoassay For VariantImmunoassay For Variant
Hemoglobin.Hemoglobin.
►Kits are available for the immunoassay ofKits are available for the immunoassay of
HbS, C, E and A.HbS, C, E and A.
►Detect the appropriate hemoglobin down toDetect the appropriate hemoglobin down to
5 – 10%.5 – 10%.
►These cards however can be unreliable withThese cards however can be unreliable with
intermittent failure of the method.intermittent failure of the method.
76.
77.
78. Screening And PreventionScreening And Prevention
Programs.Programs.
► UK guidelines: “selective testing of parents forUK guidelines: “selective testing of parents for
haemoglobinopathies can be done if thehaemoglobinopathies can be done if the
percentage of patients at risk is low”percentage of patients at risk is low”
► However this policy is reliant on reliableHowever this policy is reliant on reliable
information regarding ethnic origin being available.information regarding ethnic origin being available.
► It is not always possible to predict prenatally whichIt is not always possible to predict prenatally which
fetus will have beta thal major and which will havefetus will have beta thal major and which will have
beta thal intermedia.beta thal intermedia.
79. Arguments In Favor Of UniversalArguments In Favor Of Universal
Antenatal Screening.Antenatal Screening.
►Gets around the problem of inaccurateGets around the problem of inaccurate
ethnic histories.ethnic histories.
►Minimizes the chance of a child being bornMinimizes the chance of a child being born
with a major phenotype.with a major phenotype.
►Picks up those missed due to normalPicks up those missed due to normal
indices and normal HbA2.indices and normal HbA2.
80. Arguments Against UniversalArguments Against Universal
Antenatal Screening.Antenatal Screening.
►Cost efficacy.Cost efficacy.
►Once a woman is pregnant the only way ofOnce a woman is pregnant the only way of
preventing the outcome is abortion.preventing the outcome is abortion.
►Low true positive rate in our community,Low true positive rate in our community,
false positives may cause unnecessaryfalse positives may cause unnecessary
anxiety.anxiety.
81. What We Do At Alfred.What We Do At Alfred.
► Patients selected for HPLC based on:Patients selected for HPLC based on:
Physician request eg family history, microcytic,Physician request eg family history, microcytic,
hypochromic indices.hypochromic indices.
Antenatal screening clinic:Antenatal screening clinic:
► Lab registrars review ethnicity, MCV, MCH, MCHC, film, Hb andLab registrars review ethnicity, MCV, MCH, MCHC, film, Hb and
Fer for all antenatal patients referred.Fer for all antenatal patients referred.
► High risk patients and their partners are referred for HPLC.High risk patients and their partners are referred for HPLC.
► Depending on the findings of HPLC samples mayDepending on the findings of HPLC samples may
undergo further DNA analysis to characterize theundergo further DNA analysis to characterize the
mutation.mutation.
► Currently approximately 30 – 40 samples testedCurrently approximately 30 – 40 samples tested
per month in this way.per month in this way.
82. Future Directions.Future Directions.
► The FBE/ film and ethnicity are central toThe FBE/ film and ethnicity are central to
screening however as the community ethnicscreening however as the community ethnic
profile changes more people will be screened.profile changes more people will be screened.
► The UK recently moved to universal perinatalThe UK recently moved to universal perinatal
screening in this scenario HPLC is the only way toscreening in this scenario HPLC is the only way to
provide accurate and efficient screening.provide accurate and efficient screening.
► If an abnormal hemoglobin is deemed probableIf an abnormal hemoglobin is deemed probable
further characterization of the genetic defect withfurther characterization of the genetic defect with
DNA techniques can be implemented.DNA techniques can be implemented.
83. Conclusions.Conclusions.
► Hemoglobinopathy is an important cause ofHemoglobinopathy is an important cause of
disease world wide with significant implications fordisease world wide with significant implications for
genetic counseling.genetic counseling.
► An increasing number of individuals are at risk andAn increasing number of individuals are at risk and
ethnic history is unreliable, prompting moves toethnic history is unreliable, prompting moves to
universal antenatal haemoglobinopathy screening.universal antenatal haemoglobinopathy screening.
► Increasing number of tests being preformedIncreasing number of tests being preformed
necessitates the use of fast, accurate and efficientnecessitates the use of fast, accurate and efficient
HPLC with DNA analysis for further clarification.HPLC with DNA analysis for further clarification.
