challenges in interpreting abnormal hemoglobin study- the key is to correlate with patient age, ethnicity,RBC indices & morphology findings. Two tier approach for correct characterization of abnormal hemoglobins of HPLC &/or capillary electrophoresis.

1. Interpretation of AbnormalInterpretation of Abnormal
Hemoglobin studyHemoglobin study
Dr Rajesh V BendreDr Rajesh V Bendre
MD(path, DNB(path),DPBMD(path, DNB(path),DPB
MumbaiMumbai

2. Normal Hemoglobin StructureNormal Hemoglobin Structure
 Hemoglobin A is aHemoglobin A is a
tetramer composed of 4tetramer composed of 4
subunits:subunits:
– 22αα and 2and 2ββ
 Each subunit has aEach subunit has a
porphyrin ring whichporphyrin ring which
holds an iron molecule.holds an iron molecule.
– This is the bindingThis is the binding
site of oxygensite of oxygen
Hemoglobin tetramer

3. Other Hemoglobins in normalOther Hemoglobins in normal
adultsadults
HemoglobiHemoglobi
nn
StructureStructure %%
AA αα22 ββ22 92%92%
AA22 αα22 δδ22 2.5%2.5%
AA1C1C αα22 ((ββ-N-glucose)-N-glucose) 3%3%
FF αα22 γγ22 <1%<1%
Gower-1Gower-1 ζζ22 εε22 0*0*
Gower-2Gower-2 αα22 εε22 0*0*
PortlandPortland ζζ22 γγ22 0*0*
* Indicates early embryonic form not seen in adults

4. Other Hemoglobins in normalOther Hemoglobins in normal
adultsadults
 HbAHbA22::
– Decreased in iron deficiency, alpha-thalassemiaDecreased in iron deficiency, alpha-thalassemia
– Elevated in megaloblastic anemia, hyperthyroidism,Elevated in megaloblastic anemia, hyperthyroidism,
Beta-thalessemiaBeta-thalessemia
 HbF:HbF:
– Elevated in HPFH, Sickle cell anemia (preferentialElevated in HPFH, Sickle cell anemia (preferential
survival of RBCs because HgF inhibits sickling), Betasurvival of RBCs because HgF inhibits sickling), Beta
thalessemia majorthalessemia major
– Normal levels in Beta-thalassemia minorNormal levels in Beta-thalassemia minor
– Normal or mildly elevated in congenital hemolyticNormal or mildly elevated in congenital hemolytic
anemiaanemia
– Marked elevation in juvenile CML (up to 70%)Marked elevation in juvenile CML (up to 70%)

5. Hemoglobin AbnormalitiesHemoglobin Abnormalities
 There are 3 main categories of inheritedThere are 3 main categories of inherited
Hemoglobin abnormalities:Hemoglobin abnormalities:
– Structural or qualitative: The amino acid sequenceThe amino acid sequence
is altered because of incorrect DNA codeis altered because of incorrect DNA code
(Hemoglobinopathy).(Hemoglobinopathy).
– Quantitative: Production of one or more globin chainsProduction of one or more globin chains
is reduced or absent (Thalassemia).is reduced or absent (Thalassemia).
– Hereditary persistence of Fetal HemoglobinHereditary persistence of Fetal Hemoglobin
(HPFH):(HPFH): Complete or partial failure ofComplete or partial failure of γγ globin to switchglobin to switch
toto ββ globin.globin.

6. Abnormal HemoglobinAbnormal Hemoglobin
 Reasons to suspect a hemoglobinReasons to suspect a hemoglobin
disorder:disorder:
– Patient presents with suspicious history orPatient presents with suspicious history or
physical examphysical exam
– Laboratory tests: Microcytic hypochromicLaboratory tests: Microcytic hypochromic
RBCs, hemolytic anemiaRBCs, hemolytic anemia
– Screening test abnormality (primarily inScreening test abnormality (primarily in
neonates)neonates)

7. Laboratory Methods to evaluateLaboratory Methods to evaluate
HemoglobinHemoglobin
 Red cell morphologies:Red cell morphologies:
– HbS: Sickle cellsHbS: Sickle cells
– HbC: Target cells, crystals after splenectomyHbC: Target cells, crystals after splenectomy
– Thalassemias: Microcystosis, target cells, basophilicThalassemias: Microcystosis, target cells, basophilic
stipplingstippling

8. Laboratory Methods to evaluateLaboratory Methods to evaluate
HemoglobinHemoglobin
 Solubility test :Solubility test :
– Test to identify HbS. HbSTest to identify HbS. HbS
is relatively insolubleis relatively insoluble
compared to othercompared to other
Hemoglobins.Hemoglobins.
– Add reducing agentAdd reducing agent
– HbS will precipitate formingHbS will precipitate forming
and opaque solutionand opaque solution
compared with the clearcompared with the clear
pink solution seen in HbSpink solution seen in HbS
is not present.is not present.

9. Laboratory Methods to evaluateLaboratory Methods to evaluate
HemoglobinHemoglobin

10. Laboratory Methods to evaluateLaboratory Methods to evaluate
HemoglobinHemoglobin
 Electrophoresis:Electrophoresis:
– Alkaline (Cellulose Acetate) pH 8.6:Alkaline (Cellulose Acetate) pH 8.6:
 All Hemoglobin molecules have a negative charge, and migrate towards the anodeAll Hemoglobin molecules have a negative charge, and migrate towards the anode
proportional to their net negative charge.proportional to their net negative charge.
– Amino acid substitutions in hemoglobin variants alter net charge and mobility.Amino acid substitutions in hemoglobin variants alter net charge and mobility.
– Acid (Citrate agar) pH 6.2:Acid (Citrate agar) pH 6.2:
 Hemoglobin molecules separate based on charge differencesHemoglobin molecules separate based on charge differences andand their ability totheir ability to
combine with the agar.combine with the agar.
– Used to differentiate Hemoglobin variants that migrate together on theUsed to differentiate Hemoglobin variants that migrate together on the
cellulose gel (i.e. HbS from HbD and HbG, HbC from HbE).cellulose gel (i.e. HbS from HbD and HbG, HbC from HbE).

11. Laboratory Methods to evaluateLaboratory Methods to evaluate
HemoglobinHemoglobin
 High-Performance LiquidHigh-Performance Liquid
Chromatography (HPLC):Chromatography (HPLC):
– Weak cation exchange column.Weak cation exchange column.
The ionic strength of the elutingThe ionic strength of the eluting
solution is gradually increasedsolution is gradually increased
and causes the variousand causes the various
Hemoglobin molecules to have aHemoglobin molecules to have a
particular retention time.particular retention time.
 Amino acid substitutions willAmino acid substitutions will
alter the retention timealter the retention time
relative to HbA.relative to HbA.
 There is some analogyThere is some analogy
between retention time andbetween retention time and
pattern on alkalinepattern on alkaline
electrophoresis.electrophoresis.

12. Laboratory Methods to evaluateLaboratory Methods to evaluate
HemoglobinHemoglobin

13. Most common HemoglobinMost common Hemoglobin
abnormalitiesabnormalities
 ThalassemiasThalassemias
– AlphaAlpha
– BetaBeta
 HemoglobinopathiesHemoglobinopathies
– HbS trait; diseaseHbS trait; disease
– HbC trait; diseaseHbC trait; disease
– HbEHbE
– Hereditary Persistence of Hemoglobin FHereditary Persistence of Hemoglobin F
(HPHF)(HPHF)

14. Approach for ReportingApproach for Reporting
Reporting Protocol for Abnormal Hemoglobin study by HPLC
(Beta2 variant Programme-Biorad)-
Although a cut off of >4.0% of HbA2 is recommended for identifying b-
thalassemia carriers, each laboratory needs to establish their own normal
ranges.
Also some cases of silent b-thalassemia can have HbA2 between 3.6 to 3.9%
Blood transfusion within 3 months will have dilutional effect & false low HbA2
& Hb F
Coexisting Iron deficiency tends to decrease Hb A2 & cause microcytosis
Coexisting Folate & B12 deficiency tend to falsely elevate Hb A2 levels
Some hemoglobins co-elute in the Hb A2 window like Hb E, Hb D Iran, Hb G
Copenhagen, Hb Lepore and Hb Osu Christianbourg making it impossible to
quantitate HbA2 in the presence of these hemoglobins

15.  HPLC assigned windows & hemoglobin variants may require capillary
electrophoresis for correct categorization
 During interpreting capillary electrophoresis- HbA2 always elutes at
240, all other hemoglobins to be interpreted with reference to Hb A2.
Approach for ReportingApproach for Reporting
P1
(0.63 –
0.85)
F
(0.98 –
1.20)
P2
(1.24
-1.40)
P3
(1.40 –
1.90)
A0
(1.40-
1.90)
A2
(1.90 -3.90)
D
(3.90- 4.30)
S
(4.30-
4.70)
Unknown
(4.70-
4.90)
C
(4.90 –
5.30)
No Hb
variant.
However,
part elution
of tetramers
Hb barts, Hb
H in alpha
thalassemia,
Hb F1
(acetylated
form) in
neonates,
high
bilirubin
HbF > 10%
which is not
age
appropriate
Can have
other Hb
variants
requiring
DNA studies
HbA1C
elutes
here,
however
>7%
needs to
rule out
diabetic
status
Denotes the
status of
sample
integrity,
>10 % can
have other
Hb variants
like Hb J-
alpha chain
variants
Hb A elutes
here. Co-
eluting Hb
variants are
Hb Koln
(peak in
initial upward
slope +
secondary
peak at
4.90), Hb
twin peaks
(hump on
downward
slope)
Hb A2> 11%
can have other
Hb variants
like
HB E,
Hb Lepore
(hump on
downward
slope),
Hb D Iran,
Hb G Honolulu
Hb D Punjab,
Hb Korle-bu,
Hb G
philadelphia
Hb S,
Hb Q
thailand
Hb
hasharon,
Hb O arab
Hb C,
Hb G
sriraj,
Hb E
saskatoon

16. Hb A2
Levels
Hb F Levels
(always
correlate
with Age)
Microcytic
hypochromic
anaemia with
or without
NRBC
Remark
Always correlation with Age, H/o transfusion
within past 3 months & Patient Ethnic Origin
is necessary
3.5 – 3.8
3.6 – 7.5
6 – 8
3.6 – 11
Upto 3
Less than
3
1 – 5
2 – 10
20 – 50
10 – 90
80-100
1 - 3
Present(+)
Present(+)
Present (++)
Present (+++)
Present (+++)
Present (++)
B-thalassemia carrier (advised DNA studies)
B- thalassemia trait
B-thalassemia intermedia or Delta Beta thalassemia
trait
B-thalassemia Syndrome(?major / ?intermedia)
(advised family studies )
Delta Beta thalassemia disease(advised family
studies & DNA studies)
Alpha thalassemia trait/Hb H disease (advised family
studies & DNA studies)
Approach for ReportingApproach for Reporting

17. Hb A2
Levels
Hb F
Levels
Type of
anaemia
Remark
3.5-
3.8
Upto
3.2
1 – 2
20 - 60
Normocytic
normochromic
Advised DNA studies to rule out Silent/ carrier
BTT
Hereditary persistence of Hb F (HPFH-
Deletional / Non-Deletional variants))
3.5 – 4 2 -10 Macrocytic Advised Vitamin B12 & Folate studies & repeat
HPLC after treatment of hematinics to rule out
BTT
3.6 –
7.5
2-10 Microcytic (RDW-
>20)
BTT & advised Iron studies
• In India- common hemoglobinopathies seen with geographic distribution (Gujarat,
Rajasthan, Maharashtra- Betathalassemia, sickle cell)(Pujab,Haryana-Hb D) (west
Bengal,North East-Hb E). Hb C is extremely rare.
Approach for ReportingApproach for Reporting

18. Case 1Case 1
 47 year old female47 year old female
presents with apresents with a
history of peptic ulcerhistory of peptic ulcer
disease, H. Pylori andisease, H. Pylori an
anemia.anemia.
Labs:Labs:
 Hgb: 10.2Hgb: 10.2
 Hct: 30.9Hct: 30.9
 MCV: 96.4MCV: 96.4
 B12: 338B12: 338
 Iron: 122Iron: 122
 Ferritin: 304.5Ferritin: 304.5
 IBC: 226IBC: 226

19. Case 1Case 1
HbF: 1.3%
HbA2: 4.1%Sickle solubility test POSITIVE

20. Case 1Case 1
 Hemoglobin S/C disease:Hemoglobin S/C disease:
– Second most commonSecond most common
hemoglobin variant inhemoglobin variant in
Africans; 1 in 1000 births ofAfricans; 1 in 1000 births of
African AmericansAfrican Americans
– Relatively benign condition;Relatively benign condition;
Milder disease than SickleMilder disease than Sickle
cell disease. Patients havecell disease. Patients have
normal growth andnormal growth and
developmentdevelopment
– Do not see the classic sickleDo not see the classic sickle
cellscells
– Peripheral smear revealsPeripheral smear reveals
anisocytosis, target cells,anisocytosis, target cells,
poikilocytosis,poikilocytosis,
polychromasiapolychromasia
 Hemoglobin S/C disease:Hemoglobin S/C disease:
– Most patients have moderateMost patients have moderate
splenomegaly with manysplenomegaly with many
having autosplenectomy,having autosplenectomy,
usually older age than withusually older age than with
Sickle cell diseaseSickle cell disease
– May have veno-occlusiveMay have veno-occlusive
disease, but less commondisease, but less common
and less severe than in sickleand less severe than in sickle
cell diseasecell disease
– May have aseptic necrosis ofMay have aseptic necrosis of
bone with osteomyelitisbone with osteomyelitis
– ~50% HbS: 50% HbC; rarely~50% HbS: 50% HbC; rarely
is HbF >2%is HbF >2%

21. Case 2Case 2
 A 45 year old GermanA 45 year old German
man who isman who is
asymptomatic is seenasymptomatic is seen
for microcytosis.for microcytosis.
 Peripheral smearPeripheral smear
shows microcytosis,shows microcytosis,
hypochromia, targethypochromia, target
cells, basophiliccells, basophilic
stippling,stippling,
polychromasiapolychromasia
Labs:Labs:
 Hgb: 11.8Hgb: 11.8
 Hct: 37.5Hct: 37.5
 MCV: 65.9MCV: 65.9
 Iron: 119Iron: 119
 Ferritin: 506Ferritin: 506
 IBC: 275IBC: 275
 Fe Sat: 43%Fe Sat: 43%

22. Case 2Case 2
HbF: 1.6%* HbA2: 5.1%

23. Case 2Case 2
 Beta Thalassemia Minor:Beta Thalassemia Minor:
– The thalassemia seen mostThe thalassemia seen most
commonly is caucasians (primarilycommonly is caucasians (primarily
Mediterranean descent)Mediterranean descent)
– Beta thalassemia minor is loss ofBeta thalassemia minor is loss of
one of two genes for Beta globin onone of two genes for Beta globin on
chromosome 11chromosome 11
– Patients generally asymptomaticPatients generally asymptomatic
– May have mild microcytic anemiaMay have mild microcytic anemia
(MCV: 60-70; Hgb: 10-13) with a(MCV: 60-70; Hgb: 10-13) with a
normal or slightly increased RBCnormal or slightly increased RBC
countcount
– The peripheral smear will showThe peripheral smear will show
target cells and basophilic stipplingtarget cells and basophilic stippling
– See increased HbASee increased HbA22 in the range ofin the range of
5-9% with normal HbF5-9% with normal HbF
– Thalassemia found most commonlyThalassemia found most commonly
in caucasiansin caucasians
– See mild microcytosisSee mild microcytosis
 Beta Thalassemia Minor:Beta Thalassemia Minor:
 Primary indication is a slightlyPrimary indication is a slightly
elevated HbAelevated HbA22 detected by HPLCdetected by HPLC
(usually around 4-7%, up to 10%)(usually around 4-7%, up to 10%)
typically without elevation of HbFtypically without elevation of HbF
 Diagnosis may be obscured inDiagnosis may be obscured in
concomitant iron deficiency presentconcomitant iron deficiency present
because Beta-thalassemia causes anbecause Beta-thalassemia causes an
increase in HbAincrease in HbA22 while iron deficiencywhile iron deficiency
causes a decrease in HbAcauses a decrease in HbA22. Both. Both
create a microcytosis.create a microcytosis.
– May see a anemia that partiallyMay see a anemia that partially
responds to iron therapyresponds to iron therapy
– Always want to look at ironAlways want to look at iron
studies when interpretingstudies when interpreting
hemoglobin electrophoresis;hemoglobin electrophoresis;
usually wait to diagnose untilusually wait to diagnose until
nutritional deficiencies have firstnutritional deficiencies have first
been corrected.been corrected.

24. Case 2Case 2
 Beta Thalassemia Major:Beta Thalassemia Major:
– Homozygous double gene deletion with no BetaHomozygous double gene deletion with no Beta
globin productionglobin production
– Presents with lethal anemia, jaundice, splenomegaly,Presents with lethal anemia, jaundice, splenomegaly,
growth retardation, bone malformations, deathgrowth retardation, bone malformations, death
– Severe hypochromic, microcytic anemia with verySevere hypochromic, microcytic anemia with very
bizarre cellsbizarre cells
– HbAHbA22 is not increasedis not increased
– HgF is at nearly 100%HgF is at nearly 100%
– Abundant intra-erythrocyte precipitation of alphaAbundant intra-erythrocyte precipitation of alpha
monomers that are insolublemonomers that are insoluble

25. Case 3Case 3
 47 year old African47 year old African
American femaleAmerican female
presents to the ERpresents to the ER
with drug intoxicationwith drug intoxication
and marked anemia.and marked anemia.
She is unable toShe is unable to
provide any adequateprovide any adequate
history to thehistory to the
clinicians.clinicians.
Labs:Labs:
 Hgb: 5.9Hgb: 5.9
 Hct: 17.8MCV: 97.1Hct: 17.8MCV: 97.1
 RDW: 20.9RDW: 20.9
 Iron: 83Iron: 83
 Ferritin: 394.3Ferritin: 394.3
 IBC: 144IBC: 144
 Fe Sat: 58%Fe Sat: 58%

26. Case 3Case 3
Sickledex is POSITIVE; Peripheral smear with 2+ sickle cells
HbF: 1.0%; HbA: 38.7%; HbA2: 4.4%; HbS: 56.1%

27. Case 3Case 3
 Sickle cell anemia:Sickle cell anemia:
– In sickle cell trait, usuallyIn sickle cell trait, usually
see HbS concentrations ofsee HbS concentrations of
35 to 45% of total35 to 45% of total
Hemoglobin because theHemoglobin because the
HbS has a slower rate ofHbS has a slower rate of
synthesis than HbAsynthesis than HbA
 If HbS is less than 33%,If HbS is less than 33%,
start thinking about S-start thinking about S-
alpha-thalassemiaalpha-thalassemia
 If HbS is greater thanIf HbS is greater than
50%, worry about S-50%, worry about S-
Beta-thalassemia orBeta-thalassemia or
Sickle cell disease withSickle cell disease with
transfusiontransfusion
 Sickle cell anemia:Sickle cell anemia:
– This patient was transfusedThis patient was transfused
with two units of RBCs beforewith two units of RBCs before
the HPLC was performed.the HPLC was performed.
– It is important to know theIt is important to know the
appropriate ratios of HbS:appropriate ratios of HbS:
HbA expected. If the patientHbA expected. If the patient
does not fit,does not fit, alwaysalways look at thelook at the
transfusion history.transfusion history.
 If concerned aboutIf concerned about
overlying Beta-overlying Beta-
thalassemia, repeat HPLCthalassemia, repeat HPLC
after four months of mostafter four months of most
recent transfusionrecent transfusion

28. Case 3Case 3
HbAHbA HbSHbS HbAHbA22 HbFHbF
Hb ASHb AS 55-6055-60 40-4540-45 2-32-3 <1<1
Hb SSHb SS 00 90-9590-95 2-32-3 5-105-10
Hb S-Hb S-αα-thal-thal 7575 2525 2-32-3 <1<1
Hb S-Hb S- ββ thal majorthal major 00 90-9590-95 Inc.Inc. 5-105-10
Hb S-Hb S- ββ thal minorthal minor 5-305-30 60-9060-90 Inc.Inc. 5-105-10
Hb S HPFHHb S HPFH 00 70-8070-80 2-32-3 20-3020-30
Hb SCHb SC 00 5050 2-32-3 <1<1
Expected ratios

29. Case 4Case 4
 31 year old healthy31 year old healthy
female, pregnant withfemale, pregnant with
moderate target cellsmoderate target cells
detected on routinedetected on routine
peripheral smearperipheral smear
Labs:Labs:
 Hgb: 15.0Hgb: 15.0
 Hct: 42.5Hct: 42.5
 MCV: 87.8MCV: 87.8
 MCH: 31.0MCH: 31.0
 RDW: 12.6RDW: 12.6

30. Case 4Case 4
HbF: 0.6%; HbA2: 2.9%; HbA: 56.3%

31. Case 4Case 4
 Hemoglobin C trait:Hemoglobin C trait:
– Hemoglobin C trait (Heterozygotes) are clinically andHemoglobin C trait (Heterozygotes) are clinically and
hematologically wellhematologically well
– Moderate target cells seen on peripheral smearModerate target cells seen on peripheral smear
– HbA and HbC in a 60:40 ratio on HPLCHbA and HbC in a 60:40 ratio on HPLC
– 2% of African Americans have HbC trait2% of African Americans have HbC trait
– Homozygotes have mild hemolytic disease,Homozygotes have mild hemolytic disease,
cholelithiasis and occasional aplastic crisis.cholelithiasis and occasional aplastic crisis.
 See reduced MCV with increased MCHCSee reduced MCV with increased MCHC
– Intracellular HbC crystals, block-like structures mayIntracellular HbC crystals, block-like structures may
be seen and are pathognomonic of HbC.be seen and are pathognomonic of HbC.

33. Thank YouThank You