rcpa-qap hp-2016 review - labhaem.org · hp16-10 comments dd alpha thal + delta thal +/or...

RCPA-QAP

HP-2016 REVIEW

ANNE GILBERT

ICPMR, WESTMEAD HOSPITAL

WESTMEAD, NSW

AUSTRALIA

[email protected]

RCPA-QAP HP- BACKGROUND

Compulsory surveys for Australian and New Zealand laboratories

performing Haemoglobinopathy testing

(National Accreditation Testing Authority NATA)

Running since 1976

Current committee of 3 –

Anne Gilbert, ICPMR, Westmead Hospital, NSW, Australia

Kerryn Weekes, Monash Medical Centre, Melbourne, Vic, Australia

George Chan, Labplus, Auckland Hospital, New Zealand

RCPA-QAP HP PARTICIPANTS -102

COUNTRY NUMBER OF LABS

AUSTRALIA 34

NEW ZEALAND 8

MALAYSIA 29

HONG KONG 13

SINGAPORE 5

SOUTH AFRICA 6

INDIA 2

PHILLIPINES 1

MACAU 1

FRANCE 1

GERMANY 1

OMAN 1

RCPA-QAP HP- BACKGROUND

4 surveys pa

Freeze dried haemolysate, patient history and blood film (digital)

Evaluate Hb A2, Hb F, Hb variant (if present)

Diagnosis

Assessed on all of these parameters

HP 2016 SURVEY SAMPLES

HP16-03 Heterozygous beta thalassaemia- alpha thal not excluded.

HP16-05 Hb Etobicoke [alpha 84(F5) Ser>Arg].

HP16-08 Hb J-Baltimore [beta 16(A13) Gly>Asp].

HP16-10 Low Hb A2, microcytic

HP16-03 HISTORY

Insert

28 year old pregnant Chinese female

RCC 5.4

Hb 109

MCV 64

MCH 20

HP16-03 BLOOD FILM

HP16-03 EXPECTED FINDINGS

METHOD EXPECTED RESULT

HPLC (Bio-Rad VII) Elevated Hb A2

Cap EPG (Sebia) Elevated Hb A2

Alk EPG Increased Hb A2 band

HP16-03 BIO-RAD VII HPLC

HP16-03 SEBIA CAP EPG

HP16-03: Hb A2

METHOD CLASSIFICATION MEDIAN MEAN SD CV Number

Biorad Variant II 4.6 4.6 0.1 2.9 58

Biorad D10 4.9 4.9 0.3 5.0 11

Sebia Capillarys/ Minicap 5.1 5.1 0.1 1.8 28

ALL RESULTS 4.7 4.8 0.3 6.5 106

HP16-03: Hb F

Mostly <1%

Mostly normal interpretation.

HP16-03 DX CODES

HP16-03

Chinese have high incidence of both beta thalassaemia and alpha 0 thalassaemia.

For all beta thal patients from an alpha 0 endemic region – SEA, Greece must

mention that alpha thalassaemia cannot be excluded

Cannot presume that reduced globin chain imbalance will normalise indices.

15% (2008) to 22% (2011) to 37% in 2016 indicated that alpha thal can’t be excluded

in beta thal heteroztgotes. Still 63% don’t.

HP16-03

Ma, E.S.K.; Chan, A.Y.Y.; Ha, S.Y.; Chan, G.C.F.; Lau, Y.L.; Chan, L.C.Screening for (--SEA) α-globin gene deletion in β-thalassemia carriers and prevention of hydrops fetalis. Haematologica 2000; 85:991

HP16-05 HISTORY

Insert

24 yo pregnant female

RCC 5.0

Hb 145

MCV 88

MCH 29

HP16-05 FILM



HPLC (Bio-Rad VII) Hb variant elutes in the D window

Cap EPG (Sebia) Hb variant separates in the D zone

Alk EPG Hb variant with mobility of Hb S/D

Acid EPG Hb variant does not separate.


D window

Minor peak S window

Reduced Hb A2

16-05 CAP EPG

D window

Minor peak Z1

Reduced Hb A2

HP16-05: Hb A2



Biorad D10 2.0 2.2 0.5 21.6 11

Sebia Capillarys/Minicap 2.0 2.0 0.2 7.9 33

ALL RESULTS 1.1 1.8 0.2 9.0 107

HP16-05: Hb F

All <1%

All normal

HP16-05 DX CODES

HP16-05 Hb ETOBICOKE (a84(F5)Ser>Arg)

Serine (neutral)

Arginine (basic)

Hb Etobicoke a84(F5)Ser>Arg site

a84(F5) faces into

haem group

HP16-05 Hb ETOBICOKE (a84(F5)Ser>Arg

Rare variant – participants not expected to identify it.

F helix important in oxygenation/deoxygenation and for stabilisation of the globin chain.

Hb Etibicoke is slightly unstable with increased oxygen affinity.

This is not evident in vivo – probably due to low level of alpha variants.

b mutations at this site result in erythrocytosis and a high oxygen affinity phenotype.

HP16-08 HISTORY

36 year old female

RCC 4.4

Hb 123

MCV 89

MCH 28

HP16-08 BLOOD FILM



HPLC (Bio-Rad VII) Hb variant elutes in the P3 window

Cap EPG (Sebia) Hb variant separates in the Z12 zone

Alk EPG Hb variant with mobility of Hb J

Acid EPG Hb variant does not separate.


P3 window

Hb X1c co-elutes with Hb F

Hb F inaccurate


Hb F <1%

HP16-08: Hb F



Biorad D10 3.2 3.0 0.8 28.3 11

Sebia Capillarys/Minicap 0 0.1 0.2 161.3 32

ALL RESULTS 2.2 1.7 1.1 64.3 111

HP16-08: Hb F

Data skewed

HP16-08 Hb VARIANT

Hb J-BALTIMORE [beta 16(A13) Gly>Asp].

Glycine (neutral) Aspartic Acid (acidic)

Hb J-Baltimore [beta 16(A13) Gly>Asp] site

A13 external.

Can cope with big changes

Hb J-BALTIMORE [beta 16(A13) Gly>Asp].

Rare variant – participants not expected to identify it.

Interferes with Hb A1c quantitation

Haematologically normal.

HP16-10 HISTORY

70 year old Italian male

RCC 5.4

Hb 116

MCV 68

MCH 21

HP16-10 BLOOD FILM



HPLC (Bio-Rad VII) Reduced Hb A2

Cap EPG (Sebia) Reduced Hb A2

HP16-10: Hb A2



Biorad D10 1.9 1.9 0.1 4.7 11

Sebia Capillarys/Minicap 1.7 1.7 0.1 4.7 35

ALL RESULTS 1.8 1.8 0.1 6.2 111

HP16-10: Hb F

Mostly <1%

Mostly normal

HP16-10 DX CODES

HP16-10 COMMENTS

DD alpha thal + delta thal +/or irondeficiency – code created.

Many participant RRs did not indicate reduced Hb A2

Delta thal common in Italians and Greeks –as is beta thal

Beta thal with co-inherited delta thal may mask Hb A2 elevation.

The committee has suggested that laboratories review their reference ranges , which should be calculated based on normal patient population.

Instruments such as Bio-Rad HPLC and Sebia Capillary Electrophoresis: the lower limit for Hb A2 should not be below 2.0 and the upper limit should be less than 3.5.

Currently, only about 30% participants have reference ranges that fall within these limits, which has significant implications for diagnostic decisions.

rcpa-qap hp-2016 review - labhaem.org · hp16-10 comments dd alpha thal + delta thal +/or...

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