Shenzhen Lifotronic Technology Co., Ltd.

H9
β-thalassemia
Clinical Application
Report

H9 β-thalassemia Clinical Application Report

Intended Use.....................................................................................................................................................................................................................................................1

Summary and Explanation ofthe Test..........................................................................................................................................................................................1

Test Components..........................................................................................................................................................................................................................................4

Specimen Collection and Handling...................................................................................................................................................................................................6

Procedures.........................................................................................................................................................................................................................................................6

Procedural Notes.........................................................................................................................................................................................................................................10

Limitations of the Procedure.................................................................................................................................................................................................................10

Interpretation of Results............................................................................................................................................................................................................................11

Expected Values..........................................................................................................................................................................................................................................11

Performance Characteristics.................................................................................................................................................................................................................13

H9 β-thalassemia Clinical Application Report

Intended Use
The Lifotronic Hemoglobin Analyzer H9: β-thalassemia Analysis Mode is intended for IN VITRO
DIAGNOSTIC(IVD) USE ONLY for the separation and area percent determinations of
Hemoglobin A1c (HbA1c),Hemoglobin A2 (HbA2) and Hemoglobin F (HbF) and as an aid in the
presumptive identification of abnormal human hemoglobins in whole blood using ion-exchange
high performance liquid chromatography (HPLC).

β-thalassemia reagents,column and software are intended only for use on the Lifotronic
Hemoglobin Analyzer H9: β-thalassemia Analysis Mode.

In the β-thalassemia Analysis Mode H9 uses non-porous cation exchange high performance
liquid chromatography (HPLC) and microcomputer technology to:

1) Quantitatively measure HbA1c,HbA2 and HbF as a percentage of the total amount of

hemoglobin present in the sample compared to known standards;

2) Yield useful qualitative information regarding the detection and presumptive identification of
abnormal hemoglobins by comparing the relative retention times.

Summary and Explanation of the Test

Blood from normal adults contains mostly hemoglobin A. There is a measurable amount of
Hemoglobin A1c,Hemoglobin A2 and Hemoglobin F, but these percentages are generally not
in excess of approximately 6.5% for HbA1c,3% for HbA2 and 2% for HbF.

If HbA1c percentage exceeds 6.5%,the risk of diabetes will increase dramatically.

When elevations of HbA2 and HbF are found concurrently, these findings may be suggestive
of a condition called β-thalassemia though HbA2 at normal levels is also found in this
condition.

Thalassemias are a group of inherited anemias characterized by defects in the synthesis of

one or more of the globin chain subunitsof the hemoglobin molecule.

Individual syndromes are named according to the globin chain whose synthesis is adversely
affected. In β-thalassemia the β-globin synthesis is absent or reduced.

Existing in the heterozygous state, an individual would be said to have β-thalassemia minor
or β-thalassemia trait.

In the homozygous state, a serious disease condition called β-thalassemia major exists in
which affected individuals may not reach maturity unless they are aggressively and
continuously treated for anemia, iron- overload, jaundice and other severe complications of
this affliction.

Identification of carriers of β-thalassemia is crucial since the children born of two people
both carrying the β-thalassemia trait have a 25% chance of having β-thalassemia major.

Presumptive qualitative identification of abnormal hemoglobins is accomplished by

comparing the retention times of unknown peaks with those of the most common variants:
HbE, HbD, HbS, HbC.

H9 β-thalassemia Clinical Application Report

 Figure 1
Regional Thalassemia and Variants Distribution Diagram

Retention time “windows” set up for each of these entities are designated “HbE”, “HbD”, “HbS”,
and “HbC”. Any hemoglobin peak falling within one of these “windows” is designated as that
“window” title.

It is possible for many different hemoglobin variants to elute within each of these designated
retention time intervals.

H9 β-thalassemia Clinical Application Report

The analyzer creates a chromatogram showing changes in absorbance versus retention time
for each peak fraction as shown in the following examples.
SN Peak Name
1 HbA1a
2 HbA1b
3 HbF
4 LA1c
5 HbA1c
6 P3
7 P4
8 HbA0
9 HbA2
10 HbE
11 HbD
12 HbS
13 HbC

Table 1
H9 Hemoglobin Fraction Software Windows

Figure 2
Typical β-thalassemia and Variants Chromatogram

H9 β-thalassemia Clinical Application Report

 Figure 3
Printed Reports of E/EE/D/S/C Variants

H9 β-thalassemia Clinical Application Report

 Figure 4

Printed Report of J/D/β-thalassemia/F Variants

H9 β-thalassemia Clinical Application Report

Test Conditions

The following test conditions for the H9 β-thalassemia Analysis Mode:

SN Item Content
H9
1 Machine

System Ver:V01.01.00
MCU1 Ver: V01.01.07
2 Software Version
MCU2 Ver: V01.00.01
UI Ver: C0
IA2B19100255(India)
2 SN
IA2B18500206(Nepal)
Reagent
3 H9XT19001
Lot Number
I02BXT19600005(India)
4 Column SN
I02BXT19600007(Nepal)
Lifotronic HPLC Standard Laboratory
5 Location Primegen Healthcare Chennai India
Niramaya Diagnostics Pvt.Ltd Nepal
6 Test Mode Thalassemia
7 Calibrator Lot H9XT19001
8 QC Lot H9XT19001
9 Manufactured Date 2019/6/21
10 Expired Date 2020/6/20
11 Volume 100ul
10/July-10/August 2019(China)
12 Test Date 26-29/June/2019(India)
5-8/July/2019(Nepal)

H9 β-thalassemia Clinical Application Report

Specimen Collection and Handling
Collect whole blood specimens in vacuum collection tubes containing EDTA and mix thoroughly.

Specimens may be stored up to seven days at 2-8°C before analysis. Specimens may
be stored up to 24 hours at 25°C before analysis.

The minimum volume required for analysis is 1 mL of whole blood when sampling from a primary
tube. Performing a dilution in a sample cup requires ratio 10(whole blood):1500(lyse)μL of .H9 will
consume 750ul diluted blood each time.

For specimens from patients with grossly abnormal hematocrits, appropriate manual dilutions
can be made with the Lyse. Dilute these specimens so that the Total Area reported is within
the range of 50,000–170,000.

Procedures

Reagent and Column Preparation and Installation

The H9 β-thalassemia column, Reagents are provided ready to use. All reagents must be at
room temperature before use.

Installing Reagents

1. Select Service interface.

2. Remove the empty container.

3. Remove caps of the new reagents, leaving the caps in place.

4. Carefully place the capped A/B/C reagents bottles on the rack of instrument,lyse should
be beside the machine L port.

5. Securely tighten reagent caps.

6. Select Reset A/B/C/L,then select System Cleaning.

H9 β-thalassemia Clinical Application Report

Install the H9 β- thalassemia column

1. Select Service interface.

2. Unscrew column fittings and remove used column.

3. Remove protective plugs from new column. Do not discard the plugs, as they are
needed for storage.Verify that the column master lot matches the reagent lots.

4. Check flow direction - the arrow on the column should point to the left.

5. Slide the inlet tubing until it extends ¼ inch passed each end fitting. Connect column on
the right side.

6. Select System Maintenance screen, press H.P.Pump to start pumping buffer. When
reagent flows from the left side of the column, press H.P.Pump again to stop pumping.

7. Connect column on the left side.

8. Press H.P.Pump to start pumping and check for leaks. Confirm that the pressure is
approximately 4-12 MPa and that no fluid is leaking at the column connections.

9. When pressure reaches a steady state, press H.P.Pump again to stop pumping.

10. Return to Service interface. Press COLUMN RESET to set column count back to zero if it
is a new column. For reinstalling a previously used column the H9 will store the injection
count so there is no need to reset the counter.

11. Before calibrating the newly installed column, run one whole blood sample at least 3
times to prime the column.Use whole blood primer(100uL(whole blood):1500ul(lyse)) to
activate the column if possible.

12. Calibrate the H9 system using only Lifotronic F/A1c/A2 Calibrator. Run two levels of
quality control material.

H9 β-thalassemia Clinical Application Report

Calibrator Preparation

1. Remove caps from the calibrator vials.

2. Reconstitute the F/A1c/A2 Calibrators with 100ul Deionized Water according to the
package insert instructions.

3. Ti g h t e n cap and mix thoroughly by gentle swirling. Store reconstituted calibrators

upright at 2-8°C for up to seven days.

Calibration Procedure

Each laboratory must monitor QC results according to good laboratory practices to determine
when to recalibrate. The analyzer has a dual analyte single-point automatic calibration function
for F/A1c/ A2.

Calibration should always be performed when:

1. Control values are out of range.

2. After column replacement.
3. After major analyzer maintenance or repair.

To perform the calibration:

1. Verify that there is sufficient volume of reagents.Replace if necessary.

2. Enter lot number and target values of F/A1c/A2 calibrator.

3. Follow the package instructions to reconstitute the calibrator and then make a working
dilution of the reconstituted calibrator with Deionized Water to obtain a Total Area of
50,000-170,000.

4. Run a sample at least 3 times to check the repeatibility.

5. Select Auto Calibration,place rack with the calibrator in position 1&2. Place an empty
rack on the loader to signal the end of the run.

6. Press the START key to begin the calibration. The analyzer samples the calibrator one
time. The analyzer uses two measurements to calculate factors K and offset B. Following a
successful calibration, patient and control samples will be calculated using the new factors
and offset

Calibration Acceptability Criteria

When the calibration procedure is completed, the analyzer automatically accepts or rejects the
calibration results. If the calibration is unsuccessful, recalibration will be required. A Calibration
Failed message appears and the run aborts if:

The Factor K of HbF%,HbA1c% or HbA2% exceeds 0.7- 1.4 range or Offset B exceeds
-2 to +3 range.

H9 β-thalassemia Clinical Application Report

Assay Procedure

1. Verify that there is sufficient volume of reagents. Replace if necessary.

2. Mix each specimen by gentle inversion.

3. Place specimen tubes in the rack. Place an empty rack on the loader to signal the end of the
run.

4. Press the START key to begin the procedure.

Quality Control

In order to monitor and evaluate the accuracy and precision of the analytical performance, Lifotronic
recommends that commercially available control specimens be assayed daily and after column
replacement.

If the value of one or more control specimens is out of the acceptable range, recalibrate the
system and rerun the controls before reporting patient results.

Follow standard quality control procedures in accordance with the strictest regulatory agency
under which the laboratory operates.

Controls should be diluted with lyse to obtain a Total Area within the range of 50,000-170,000
on the chromatogram.

H9 β-thalassemia Clinical Application Report

Procedural Notes
1. Each master reagent buffer lot number supplied by Lifotronic is performance matched to
the supplied H9 β -thalassemia columns. Following any announced change in
supplied columns, contact Lifotronic to determine suitability of existing reagents.

2. Reagents must be at room temperature prior to use.

3. When replacing the column, run at least three whole blood samples to prime the column
then calibrate the system before running quality control materials.

4. Replace the filter element if pressure is greater than 12 MPa or after 400 injections.

5. At least once a day check the waste container to ensure that there is enough space
remaining to accommodate a run. If necessary, empty the container and add about one
liter of fresh 5% sodium hypochlorite solution (household bleach).

Limitations of the Procedure

Dilution studies demonstrate that the assay is linear from a Total Area of 50,000 to 170,000.
Do not report results for any sample where the Total Area is not within this above stated range.

A result of HbA2 within the normal reference interval does not preclude a diagnosis of β-
thalassemia.

If HbA2 results exceed 10%, DO NOT REPORT out the HbA2 result.The presence of some
hemoglobin with a similar charge should be suspected in such cases.

Confirm the identity of the hemoglobin(s) by alternative methods since the peak identified as HbA2
may contain either a single variant or a variant with concurrent HbA2.

HbA2 results may be physiologically decreased in iron deficiency anemia.

Results of serum iron, TIBC, RBC morphology, hemoglobin, hematocrit, and free erythrocyte
protoporphyrin as well as family history will aid in the differential diagnosis.

For diagnostic purposes, the results obtained from this assay should be used in conjunction with
other data (for example, results of other tests, age of patient, ethnicity, and clinical pathophysiology).

For the positive confirmation of any particular hemoglobin variant, globin chain analysis in
conjunction with iso-electric focusing and PCR will be required.

H9 β-thalassemia Clinical Application Report

 Figure 5
β-thalassemia/Variants Clinical Process

If there is a peak in the S window, verification with a sickle solubility test must be performed. A
Sickle Solubility test should be performed on all presumptive HbS samples without exception
before the results are released as presumptive HbS.

Interpretation of Results
An unusual chromatogram can result from either an abnormal sample, a procedural error, an
instrument malfunction or a sample-handling problem. In general, review and question any
chromatogram with the following characteristics:

1. The Total Area reported is less than 50,000 or greater than 170,000.
2. The HbA2 concentration exceeds 10%.
3. T he baseline of the chromatogram is shifting and peak separation is not good(not
sharp and symmetrical).
4. There appears to be no HbA0.

If a result displays any of these characteristics, repeat the sample.

If the repeated sample also displays the unusual characteristics, then it is appropriate to evaluate
whether the unusual result is due to an abnormal sample, a procedural error, an instrument
malfunction or a sample-handling problem. Criteria 2 and 3 above may not affect qualitative results
for presumptive identification of abnormal hemoglobins but will certainly affect quantitative results
for the hemoglobin affected.

Important! Do not report any quantitative result on chromatograms that display the
characteristics listed above. The presence of any of these criteria may indicate that there is a
problem or the sample may be inappropriate for measurement by this method. Qualitative results
should be carefully reviewed by knowledgeable personnel. Call Lifotronic Technical Support.

H9 β-thalassemia Clinical Application Report

Expected Values
The adult upper limit of normal cited in the literature for HbF is typically 1-2%.

The adult normal reference range for HbA1c 4.0-6.5%.

The adult normal reference range for HbA2 is typically listed as 2-3%, though there is no exact
boundary between normal and abnormal values.

Each result must be interpreted carefully considering other clinical and laboratory findings as well as
patient and family history.

Values between 3.5% and 8.0% have been regarded as being indicative of β-thalassemia trait as a
general rule, however, it is possible to have β-thalassemia and display a normal level of HbA2.

As stated previously in this Report, a value of HbA2 in excess of 10% is an indication of the
presence of other hemoglobin entities migrating in the same window as the HbA2.

Each laboratory should determine a reference interval that corresponds to the characteristics of
the population being tested.

As a preliminary guideline, Table 2 lists published reference intervals for some patient conditions.

Patient State HbA2 Result HbF Result

Heterozygous β-thalassemia 4 – 9% 1 – 5%
Homozygous β-thalassemia Normal or Increased 80 – 100%
Heterozygous HPFH < 1.5% 10 – 20%
Homozygous HPFH None Seen 100%
Table 2 Expected Values for HbA2 and HbF
HPFH is Hereditary Persistence of Fetal Hemoglobin

Studies using the Lifotronic Hemoglobin Analyzer H9: β-thalassemia

Analysis Mode to analyze 137 different adult patient whole blood samples from apparently
healthy individuals with no known hemoglobin variants indicate the following normal reference
intervals:

H9 β-thalassemia Clinical Application Report

HbF by Nonparametric (NCCLS C28-A): 0.5% - 1.9 %
Mean: 1.25%
SD: 0.3479
Median: 1.20
Range: 0.6 – 2.4%

HbA1c by Nonparametric (NCCLS C28-A): 4.0% - 6.1 %

Mean: 5.46%
SD: 0.3938
Median: 5.50
Range: 4.4 – 6.5%
H9 β-thalassemia Clinical Application Report
HbA2 by Nonparametric (NCCLS C28-A) 1.7% - 2.9 %
Mean: 2.35%
SD: 0.3122
Median: 2.40
Range: 1.4 – 3.0%

H9 β-thalassemia Clinical Application Report

Performance Characteristics

Accuracy

Dilution / Linearity

Packed red blood cells from a whole blood specimen were diluted with Lyse and assayed. The
study demonstrates that the HbF,HbA1c and HbA2 results on the Lifotronic Hemoglobin Analyzer H9:
β-thalassemia Analysis Mode are linear in samples with Total Area values from 50,000 to at least
170,000.

SN HbF HbA1c HbA2 TOTAL AREA

1 7.1 5.7 4.1 39922
2 7.2 5.8 4.2 58298
3 7.1 5.9 4.3 70711
4 7.1 5.9 4.2 80654
5 7.1 5.9 4.2 101275
6 7.2 5.8 4.3 109198
7 7.1 5.9 4.3 121342
8 7.1 5.9 4.3 135045
9 7.1 5.9 4.3 156215
10 7.2 5.9 4.4 172210

Table 3 Dilution/linearity
High HbF,HbA1c and HbA2

TOTALAREA

H9 β-thalassemia Clinical Application Report

Comparative Analysis Quantitative HbA2
Patient specimens were analyzed with LifotronicHemoglobin Analyzer H9: β-thalassemia Analysis
Mode and D10 Dual Program. Approximately one-half the samples had HbA2 within the normal
reference interval. The remainder showed elevated results. The comparison yielded the following
correlation statistics when the two methods were compared using linear regression analysis:

Slope: 0.9185
Y-Intercept: -0.0107
Correlation Coefficient R2: 0.9826
Range of Samples (%): 1.6 – 6.5%
n: 53

Comparative Analysis Quantitative HbF

Patient specimens were analyzed with the LifotronicHemoglobin Analyzer H9: β-thalassemia
Analysis Mode and D10 Dual Program. The comparison yielded the following correlation statistics
between these two machines.

Slope: 0.8772
Y-Intercept: 0.2407
Correlation Coefficient R2: 0.9960
Range of Samples (%): 0.4 – 24.7%
n: 53

H9 β-thalassemia Clinical Application Report

Comparative Analysis Quantitative HbA1c
Patient specimens were analyzed with the LifotronicHemoglobin Analyzer H9: β-thalassemia
Analysis Mode and G8. The comparison yielded the following correlation statistics between these
two machines.

Slope: 0.9690
Y-Intercept: 0.1647
Correlation Coefficient R2: 0.9923
Range of Samples (%): 4.5 – 15.3%
n: 42

Comparative Qualitative Analysis Abnormal Hemoglobins

Patient whole blood specimens collected with EDTA and submitted to the laboratory at two clinical
trial sites for hemoglobin electrophoresis/HPLC studies were analyzed using the LifotronicHemoglobin
Analyzer H9: β-thalassemia Analysis Mode.

Results from the H9 and a variety of commercially available electrophoretic/HPLC assays in routine
lab use for the qualitative identification of abnormal hemoglobins were compared.

The remaining samples were from patients with a variety of hemoglobinopathies.Table 4 shows the
results from different methods. A total of 383 samples were compared.

All samples determined to have Hemoglobin HbF,HbA1c,HbA0,HbA2,“HbE”,“HbD”, “HbS” and

“HbC” by electrophoresis/HPLC were detected within the appropriate “windows” on the Lifotronic
Hemoglobin Analyzer H9: β-thalassemia Analysis Mode.

In addition, the H9 detected several specimens with abnormal hemoglobins in which the
chromatograph showed an unidentified peak.

Hemoglobin electrophoresis/HPLC also showed peaks not consistent with the usual patterns and
classified as “unknown” on these same samples.

The asterisks (*) in the following Table 4 indicate either abnormal hemoglobins or hemoglobins
found in concentrations exceeding the normal reference interval.

H9 β-thalassemia Clinical Application Report

H9 Presumptive Result Electrophoresis/HPLC Result Number of Analyses
HbA/HbS* HbA/HbS* 27
HbA/HbS*/HbA2* HbA/HbS*/HbA2* 17
HbF*/HbS* HbF*/HbS* 5
HbF*/HbSS* HbF*/HbSS* 1
HbA/HbD* HbA/HbD* 7
HbA/HbC* HbA/HbC* 1
HbA/HbCS* HbA/HbCS* 1
HbA/HbE* HbA/HbE* 5
HbEE*/HbA HbEE*/HbA 1
HbA/HbE*/HbA2* HbA/HbE*/HbA2* 1
HbA/HbA1a(10.6)* HbA/Hb Barts* 1
HbA/HbA2*/HbF* HbA/HbA2*/HbF* 5

HbA/HbA2*/HbF HbA/HbA2*/HbF 38

HbF*/HbA/HbA2 HbF*/HbA/HbA2 2

HbA/HbF* HbA/HbF* 5

Normal HbA/HbF/HbA2 Normal HbA/HbF/HbA2 238

HbA1c* HbA1c* 24

TOTAL COMPARISONS 379

Table 4
Qualitative Comparison of Hemoglobin in Samples

H9 β-thalassemia Clinical Application Report

 Precision
Intra-assay

The intra-assay (within run) precision coefficient of variation was evaluated in a commercial control
and a whole blood specimen for HbF,HbA1c and HbA2.

Within-Run Precision of QC Within-Run Precision of whole blood

HbF HbA1c HbA2 HbF HbA1c HbA2
N 20 20 20 N 20 20 20
SD 0.0410 0.0598 0.0562 SD 0.0834 0.0639 0.0616
MEAN 2.02 9.74 2.60 MEAN 6.13 4.98 4.52
CV 2.03% 0.61% 2.16% CV 1.36% 1.28% 1.36%

Between day

The between day precision coefficient of variation was evaluated using Lifotronic control and whole
blood samples for HbF,HbA1c and HbA2 with testing over 20 days.

Between day Precision of QC Between day Precision of whole blood

HbF HbA1c HbA2 HbF HbA1c HbA2
N 20 20 20 N 20 20 20
SD 0.0760 0.1801 0.0826 SD 0.1575 0.1273 0.1653
MEAN 2.97 9.53 2.60 MEAN 7.13 5.42 5.51
CV 2.56% 1.89% 3.18% CV 2.21% 2.35% 3.00%

H9 β-thalassemia Clinical Application Report