MEDICAL/SCIENTIFIC AFFAIRS GUIDELINE

Xpert® BCR-ABL Ultra (CE-IVD, US-IVD)

Testing of Bone Marrow Samples

Disclaimer: This document, developed by Cepheid’s Medical and Scientific Affairs

Oncology staff, is provided as a courtesy to customers to provide guidance on queries
regarding the dilution protocol for testing bone marrow samples. Cepheid does not
endorse the use of Xpert BCR-ABL Ultra tests for other purposes not included in the
product labeling, but recognizes that there are situations where patient’s bone marrow
samples may be used for monitoring. The guideline below, references the package inserts
of Xpert BCR-ABL Ultra (CE-IVD, US-IVD) and results of studies published in peer-reviewed
scientific journals that have been independently validated by the study authors. If you
choose to use the Xpert BCR-ABL Ultra test for any indication other than stated in the
package insert (“off-label” indications), it is your laboratory’s responsibility to validate the
test in accordance with federal, state/province, and local laws.*

Background

This guideline summarizes the current on-label information for using EDTA – whole blood samples
and off-label information for using bone marrow samples with the Xpert BCR-ABL Ultra assay. In
cases with invalid results due to excess ABL or BCR-ABL transcripts (RNA) in the sample, which are
often seen in cases where bone marrow is used or whole blood with high white blood cell count, the
package insert advises a retest procedure provided in Section 18.2 [Retest Procedure for ERROR
(Code 2008) or INVALID (Type 2)].1,2

The monitoring of BCR-ABL transcript levels in the peripheral blood of patients using real-time
quantitative PCR (RT-qPCR) is standard of care in the management of Chronic Myeloid Leukemia
(CML). Xpert BCR-ABL Ultra monitors the input RNA quality and quantity based on the cycle
threshold (Ct) of the control gene ABL, which is highly correlated to the input white blood cell
(WBC) number in EDTA whole blood (WB). Using a lysate prepared from 4 mL WB, resulting in
an effective WB input volume of 600 μL, Xpert BCR-ABL Ultra reproducibly achieves molecular
response (MR) sensitivity to 4.5 logs below baseline (BCR-ABL1/ABL1 0.003% (IS) MR4.52) per the
WHO International Scale (IS)3,4. There are, however, clinical situations where the total RNA from high
numbers of WBCs circulating in the patient’s blood or in bone marrow (BM) samples can overload the
Xpert BCR-ABL Ultra cartridge and other quantitative BCR-ABL assays, requiring subsequent dilution
of the sample and retesting.

IVD. In Vitro Diagnostic Medical Device. May not be available in all countries.
This document and all guidelines may only be distributed by Medical/Scientific Affairs in response to an unsolicited request.

A better way.
Intended Use of Xpert BCR-ABL Ultra as Stated in the Package Insert

Xpert BCR-ABL Ultra is available in the following versions (depending on your location):
• GXBCRABL-10 (CE-IVD)1
• GXBCRABL-US-10 (US-IVD)2

The Xpert BCR-ABL Ultra test is an in vitro diagnostic test for the quantitation of BCR-ABL1 and ABL1
mRNA transcripts in peripheral blood specimens of diagnosed t(9;22) positive Chronic Myeloid Leukemia
(CML) patients expressing BCR-ABL1 fusion transcripts type e13a2 and/or e14a2 [p210]. The test utilizes
automated, quantitative, real-time reverse transcription polymerase chain reaction (RT-qPCR). The
Xpert BCR-ABL Ultra test is intended to measure BCR-ABL1 to ABL1 percent ratios on the International
Scale (IS), and is also expressed as a log molecular reduction (MR value) from a baseline of 100% (IS), in
t(9;22) positive CML patients during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs).

The test does not differentiate between e13a2/b2a2 or e14a2/b3a2 fusion transcripts and does not
monitor other rare fusion transcripts resulting from t(9;22). This test is not intended for the diagnosis
of CML.

The Xpert BCR-ABL Ultra test is intended for use only on the Cepheid GeneXpert® system and the
GeneXpert Infinity system.

Methods (see Table 1)

• For blood specimens with normal white blood cell (WBC) count (4.5 to 11.0 × 109/L), use the
recommended 4 mL whole blood to prepare the lysate for testing following the package
insert instructions.
• For bone marrow samples, a starting volume of 50 µL bone marrow might be considered to increase
the likelihood of valid assay results (ABL1 and BCR-ABL1 Ct in the valid range). See Figure 1 below.
- Customers can follow the 50 µL retest procedure per the package insert (section 18.2, Retest
Procedure for ERROR (Code 2008) or INVALID (Type 2).
Figure 1 Bone Marrow Preparation Procedure

This document and all guidelines may only be distributed by Medical/Scientific Affairs in response to an unsolicited request.

Xpert® BCR-ABL Ultra Testing Bone Marrow Samples | PAGE 2

 • For blood specimens with WBC > 30 million cells/mL and expected very high BCR-ABL mRNA
expression, even lower starting volumes of 2-20 µL whole blood may be required to achieve valid
ABL1 and BCR-ABL1 Ct results.
• Also, for blood specimens with very high white blood cell counts (above 200 million cells/mL) a further
dilution or much smaller sample volume might be needed.
- In cases where a very low volume of whole blood is to be used, consider the option to make a
pre-dilution before taking the final aliquot for the lysate.
- For example, to use a whole blood volume of 2 µL, the following procedure could be considered:
Mix 50 µL whole blood sample with 450 µL of 1X phosphate-buffered saline (PBS), then use 20 µL
of the diluted whole blood, equivalent to 2 µL of original whole blood.
• Usually, a very high WBC count will make the first lysate very viscous, “gluey,” and difficult to pipet.
Samples with these qualities need further treatment to improve the sample quality and to achieve a
more even suspension and target analyte distribution.
- If you observe a very viscous first lysate, warm the first lysate to room temperature (RT), add 4 mL
molecular grade water or 1X PBS, add 100 μL of proteinase K, then add 2.5 mL of Lysis Reagent,
swirl to mix 10 times in 5 seconds, vortex for 30 seconds, incubate at RT for 5 minutes, vortex
again for 30 seconds and incubate at RT for 10 minutes. Then use 10 uL in the assay.

Note: If you dilute the sample or use a smaller amount, the result reported by the software is the result.
You do not need to multiply the result by the dilution.

TABLE 1. RECOMMENDED WHOLE BLOOD INPUT VOLUME RELATIVE TO THE WHITE BLOOD CELL COUNTS
(FROM MAHAFFEY ET AL3)
White Blood Cell Count X
(million/mL whle blood)

X ≤ 30 30 < X ≤ 200 X > 200

Starting WB volume 4 mL 50 μL 10 μL
Input Whole Initial Testing
Actual WB input
Blood (WB) 600 μL* 50 μL 10 μL
volume in 2nd lysate
or Bone
Marrow When repeat testing is needed Starting WB volume 10 μL - 50 μL 2 μL^- 10 μL 2 μL^
(BM) Volume due to an initial INVALID call
with the ABL CT < 8 Actual WB input
10 μL - 50 μL 2 μL^ - 10 μL 2 μL^
volume in 2nd lysate
* Calculated WB input volume using the 1st lysate prepared from 4 mL whole blood.
^ Calcualated WB input volume using 10X pre-diluted WB with 1X PBS.

Conclusion

The bone marrow protocols described in this guideline have not been validated and integrated into the
Xpert BCR‑ABL˛Ultra package insert. Therefore, these procedures represent off-label use of the
test and require validation in the end-user’s laboratory.

This document and all guidelines may only be distributed by Medical/Scientific Affairs in response to an unsolicited request.

Xpert® BCR-ABL Ultra Testing Bone Marrow Samples | PAGE 3

Questions

For any questions or additional information related to this guideline, please contact:
Medical/Scientific Affairs, Oncology at MedSci.Oncology@cepheid.com.

For general technical support questions, please contact:

Technical Support at techsupport@cepheid.com or support@cepheideurope.com

REFERENCES
1 Xpert BCR-ABL Ultra Package Insert. Sunnyvale. (CE-IVDI) 302-0742 Current Version.

2 Xpert BCR-ABL Ultra Package Insert. Sunnyvale. (US-IVD) 302-0738 Current Version.

3 Mahaffey V, et al. Development of Sample Preparation Procedure for Testing Specimens with High White Blood Cell Counts for
Xpert BCR-ABL Ultra*. Poster presented at 20th Annual John Goldman Conference on Chronic Myeloid Leukemia: Biology and Therapy.
2018 Sept 13-16. Miami, FL, USA.

4 Ferrand C, et al. Xpert BCR-ABL Ultra, a High Sensitivity Assay with a Limit of Detection Reaching MR4.5 and Below on an International
Reporting Scale. Poster presentated at 59th ASH Annual Meeting. 2017 Dec 9-12. Atlanta, GA, USA.

This document and all guidelines may only be distributed by Medical/Scientific Affairs in response to an unsolicited request.

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