Screening for hemoglobin disorders: Tosoh G7 thalassemia mode Henri Brinkman, Frans Verheul General Clinical Laboratory (AKL) Ysselland Hospital Capelle aan den Yssel The Netherlands Stockholm, October 2005
Capelle aan den Yssel Rotterdam YSSELLAND HOSPITAL
The General Clinical Laboratory AKL includes: Clinical Chemistry - Chemistry - Hematology - Blood transfusion - Immunochemistry Laboratory for Endocrinological Chemistry Laboratory for Neonatal Screening Pharmaceutical Laboratory Medical Microbiological Laboratory Nuclear Diagnostics
Tosoh G7: - Separation: : reversed phase cation exchange - Detection: colorimetric (415( 510 nm, LED) - Variant mode, HbA1c: 2 minutes per run - 6 fractions: : HbA1a, HbA1b, HbF,, l-hba1c, l s-hbs HbA1c, HbA0-2-point calibration (DCCT oro IFCC) - Thalassemia mode: 7.3 minutes per run - Detection possible of HbF,, HbA2, HbS, HbC and HbD - No daily maintenance - Primary ry tube (cap piercing) - STAT possibillity - Bi-direction directional coupling to LIS is possible - Can be connected to a sample transport system (LAS)
The 2003 study
Aim of the 2003 study Introduction of a rapid and analytically reliable HbA1c test Possibility of detecting abnormal hemoglobins (e.g. thalassemias and hemoglobinopathies)
AKL HbA1c in % 14 13 12 11 10 9 8 7 6 Correlation on with a Dutch reference laboratory Regression: Passing & Bablok (SKB) HbA1c Correlation: Pearson SKB vs Menarini HA8160 y = 1.0019x + 0.0385 R 2 = 0.9890 n = 101 SKB vs TOSOH G7 y = 0.9444x + 0.3278 R 2 = 0.9824 n = 101 SKB vs Bayer DCA2000+ y = 1.0357x - 0.2982 R 2 = 0.9708 n = 100 5 4 4 5 6 7 8 9 10 11 12 13 14 SKB HbA1c in % SKB: Streekziekenhuis Koningin Beatrix, Winterswijk, The Netherlands SKB vs (Menarini HA8140) y = 0.9000x + 0.6600 R 2 = 0.9498 n = 100
Precision Tosoh G7 Bio-Rad Lyphochek Diabetes Control Levels 1 & 2 Intra assay n=10 Mean SD %VC Mean SD %VC HbA1c 5,59 0,032 0,6 10,5 0,048 0,5 Inter assay n=10 Mean SD %VC Mean SD %VC HbA1c 5,56 0,052 0,9 10,4 0,063 0,6
The 2004-2005 2005 study
Aim of the 2004-2005 2005 study Can the Tosoh G7 Variant mode be used for screening on thalassemia s and hemoglobinopathies? Can the Tosoh G7 Thalassemia mode be used for identifying thalassemia s and hemoglobinopathies and therefore be used in stead of Hb-electrophoresis?
Variant and ThalassemiT halassemia mode: : known samples Samples with known abnormal hemoglobins * were primarily analyzed in the Variant mode. Q1: Are these abnormal hemoglobins detected in this mode? All abnormal samples were then analyzed in the Thalassemia mode. Q2: Are thalassemias and hemoglobinopathies recognized and correctly identified in the Thalassemia mode? a1 All abnormal samples were detected in the Variant mode a2 All thalassemias and hemoglobinopathies were recognized and correctly identified in the Thalassemia mode * 33 of these samples were donated by Dutch colleagues
Variant and ThalassemiT halassemia mode: our patients HbA1c requests and Hb electrophoresis requests were primarily analyzed in the Variant mode HbA1c requests indicating abnormal hemoglobins and Hb electrophoresis requests were then analyzed in the Thalassemia mode Are thalassemias and hemoglobinopathies recognized and correctly identified in the Thalassemia mode?
Stability of controls Storage of the Tosoh F&A2 control set showed the following results for CV: Level 1 Level 2 (HbF: 1.7 %, HbA2: 3.0 %) (HbF: 7.0 %, HbA2: 7.3 %) CV% at 4 C (n=10) HbF 7.7 % HbF 1.1 % HbA2 11.6 % HbA2 3.1 % HbA0 3.8 % HbA0 1.1 % CV% at -20 C (n=7) HbF 8.2 % HbF 0.5 % HbA2 10.8 % HbA2 8.1 % HbA0 0.3 % HbA0 0.7 % CV% at -80 C (n=10) HbF 6.0 % HbF 1.4 % HbA2 4.9 % HbA2 5.7 % HbA0 1.7 % HbA0 0.7 %
Normal chromatograms Variant mode Thalassemia mode Reference values (150 apparently healthy adult volunteers) F A2 < 2.0 % 2.0 3.5 %
Variant mode V0 (D?) V1 (S?) V2 (C?)
Thalassemia mode HbAD HbAS HbAC
Variant mode a thalassemi alassemia Thalassemia mode
Variant mode ß thalassemia Thalassemia mode
Hb AJ Baltimore Variant mode Thalassemia mode
Hb SE Variant mode Thalassemia mode
Some more examples (thalassemia( mode) SC EE CC
Always check the complete printout! AS + - α/- α AS AS? AS? Why? Hb-Savaria Always confirm a positive S-peak!
Therapeutic drugs can cause mis-interpretation! interpretation! SS SS? S-Beta O S-Beta 0? SS + td
What did we find with Tosoh G7 thalassemia mode ß thalassemia α thalassemia AS SS S-Beta 0 AC CC SC AD AE EE SE HbD Punjab AJ F n-, A2 (max 8.3 %) A0fl, A2fl S-peak, A0fl, A2 S-peak, A0flfl, F, A2 S-peak, A0flfl, F, A2 C-peak, A0fl, A2 n- C-peak, A0flfl F, S-peak, C-peak, A0flfl D-peak, A0fl, A2 F, A0flfl, A2 F, A0flfl, A2 S-peak, A0flfl, A2 Extra peak, A0fl Extra peak, A0flfl, A2
S-peaks 30-45%: probably HbAS (sicklecell trait) 25-35%: probably HbAS + alpha thalassemia > 50% + HbF <10%: probably HbSS (sicklecell disease) > 50% + HbA flfl + HbF>10% probably HbSS (sicklecell disease) + beta thalassemia
Conclusions Switching from Variant mode to thalassemia mode is simple Although the Variant mode can detect most Hb disorders, thalassemia mode is recommended for this purpose Most common abnormal hemoglobins can be positively identified in the Thalassemia mode, therefore the G7 can be used in stead of Hb-electrophoresis All printouts (percentages and chromatograms) have to be examined thoroughly before validation of results
So the Tosoh G7 in thalassemia mode can be used for first line screening for hemoglobin disorders.
2005 2006 study
Aim of the 2005-2006 2006 study Can the thalassemia mode be used for neonatal screening (dried blood spots on filter paper, DBS)? Can the thalassemia mode identify the sickle cell disorders in neonatal DBS as advised by the Dutch Health Council?
Preliminary results: adult EDTA WB vs. filter paper EDTA WB (adult) DBS (adult) 10 healthy adult volunteers donated blood samples: EDTA WB sample DBS sample from finger prick No significant difference was found between the HbF, HbA0 and HbA2 results.
Preliminary results: neonatal EDTA WB vs. filter paper EDTA WB (newborn) DBS (newborn) Thalassemia mode Until now good correlation is found between EDTA whole blood and filter paper results. Unfortunately (?) no abnormal screening samples have been found yet.
Preliminary results: dried blood spots 12 newborns, DBS on day 4 7 Measured HbA0% Measured HbF% Total HbF% F Mean 15.7 54.3 70.6 P00 SD 7.2 7.0 7.9 %CV 46 13 11
Future The use of the Tosoh G7 for neonatal screening for hemoglobinopathies (using DBS) is still under research, but results so far are promising
General Clinical Laboratory Ysselland Hospital Capelle aan den Yssel The Netherlands