Importance of CD34 in Screening Tests for Hematolymphoid Neoplasms: ClearLLab LS Shows the Way
Badri Narayan Natarajan1, Neha Girish1, Xiangyang Dong2, Jin Zhang2, Lilly Lopez2, Diana B Careaga3 Bharathi Ravi1, Pooja R Dalvi1, Jorge Quintana2.
1Research & Development, Bangalore Development Centre, Beckman Coulter, Inc. Bangalore, India.
2Research & Development, Beckman Coulter, Inc. Miami, FL, USA.
3Clinical Research, Beckman Coulter, Inc. Miami, FL, USA.
Abstract
Introduction:
Identification of dim CD45 blasts, together with the exclusion of normal residual cells that follow systematic lineage maturation, is crucial in the study of hematopoietic cell differentiation. ClearLLab LS (Lymphoid Screen) is a dried, unitized, multicolor screening reagent designed for the identification of hematolymphoid neoplasms. One of the unique features of this reagent is the presence of CD34 antibody, with the goal of enhancing blast-cell identification in supplementation to using CD45 expression alone. CD34 as a backbone marker, in addition to CD19, CD3 , CD10 and others, was selected in the panel due to its ameliorative expression in a significant number of acute leukemias of any lineage. In this study, we analyzed the efficacy of using the CD34 marker in the context of the ClearLLab LS reagent in order to support microscopy-based diagnoses by the lymphoid screening tube.
Method:
A total of 405 clinical specimens from peripheral blood, bone marrow and lymph node samples were collected across Asia, Europe and North American geographies, and were screened with the ClearLLab LS reagent. Data was analyzed to look at the CD34 phenotype and percentage expression of CD34. The usefulness of CD34 in identifying blast cells was analyzed in the context of other markers and clinical diagnoses. The relative frequency of CD34 expression, together with other markers, such as CD10, CD5, CD19 and CD56, in Acute B, T, and Myeloid leukemias, was assessed in all specimens and demonstrated that CD34 in the ClearLLab LS reagent resolves blast cells from normal maturing populations in acute leukemias of any lineage. In addition, evaluation of the patterns of expression for the CD10 and CD34 markers in B-ALL specimens demonstrated a very clear, spatially resolvable, partial-expression pattern for both CD10 and CD34.
Results:
Our results demonstrate that the presence of CD34 in the ClearLLab LS screening tube improves the identification of blast cells with very clear resolution, and works well with the existing backbone markers to improve immunophenotyping of hematological malignancies from both normal hematogones and CD34-negative mature lymphoid neoplasms.
Materials & Methods
The ClearLLab LS panel is designed to screen acute and chronic hematolymphoid neoplasms (Figure 1). Biological specimens were processed using a pre-wash-lyse-wash-stain-wash protocol for optimized performance with the dry reagent. Multicolor compensation setup was performed with the ClearLLab Compensation kit.
Figure 1: ClearLLab LS panel design
A total of 405 clinical specimens from bone marrow, peripheral blood and lymph node were analyzed at sites across India, Germany, Canada and the USA. Of the 405 clinical samples, 74 specimens were CD34+ lymphoid precursor neoplasms, which are either B and T cell precursor acute lymphoblastic leukemia/lymphoma (BCP-ALL and T-ALL), or acute myeloid leukemia (AML) and related myeloid precursor neoplasms. We present different cases of acute leukemia cases screened with ClearLLab LS reagent for co-expression of CD34 with backbone markers specific for B, T and myeloid lineages. Though ClearLLab LS reagent is intended for use as screening tube, the importance of CD34 in assessing primary clinical information about myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) are also shown below.
B Acute Lymphoid Leukemia (B ALL)
43 cases of B Acute Leukemia samples were tested with ClearLLab LS reagent on either whole blood or bone marrow with CD45 dim blasts greater than 75% of the total leucocytes. Co-expression of CD34 with CD10 was common among these cases and a clear resolution was achieved between these two markers as shown in the figure below:
A
B
C
D
Figure A displays strong CD34 expression on a portion of CD19+ CD45 dim blasts. Figure B represents very high expression of CD34 and CD10 on CD19+ blasts. Figure C represents asymmetrical distribution of CD34 and CD10 on CD19+ blasts and figure D represents CD10 dim or negative blasts that are strong CD34 and CD19 positive. The above representations are from different specimen screened with ClearLLab LS reagents for B ALL.
T Acute Lymphoid Leukemia (T ALL)
Six cases of T Acute Leukemia samples were tested with ClearLLab LS reagent on either whole blood or bone marrow with CD45 dim blasts greater than 70% of the total leucocytes. Co-expression of CD34 with CD5 was common among these cases with surface CD3 staining dim or negative. A clear resolution was achieved between these two markers as shown in the figure below:
A
B
C
Figure A displays strong CD34 expression on CD5+ CD45 dim blasts. Figure B represents high expression of CD34 on CD5+ blasts that are surface CD3 negative. Figure C represents loss of CD4 and CD8 antigens on the T cells implying DN stage of malignancy. The above representations are from T ALL specimen screened with ClearLLab LS reagents for T ALL.
Acute Myeloid Leukemia (AML)
17 cases of Acute Myeloid Leukemia samples of different stages (AML M0 – M4) were tested with ClearLLab LS reagent on either whole blood or bone marrow with CD45 dim blasts greater than 63% of the total leucocytes. Strong expression of CD34 unaided by any of the backbone markers in the lymphoid lineage were observed. A clear resolution was achieved between these two markers as shown in the figure below:
A
B
Figure A displays CD45 dim AML blasts which show differential expression of CD45 and maturation of these blasts as they lose CD34 and gain CD45 during development. Figure B represents pattern of loss of CD34 and gain of CD45 antigens. None of the lymphoid phenotype expression markers in the ClearLLab LS panel were seen in these cases.
Myelodysplastic Syndromes and Myeloproliferative Neoplasms (MDS - MPN)
12 cases of Myelodysplastic Syndrome, Myeloproliferative Neoplasms samples were tested with ClearLLab LS reagent on either whole blood or bone marrow with CD45 dim blasts greater than 63% of the total leucocytes. Strong expression of CD34 unaided by any of the backbone markers in the lymphoid lineage were observed. A clear resolution was achieved between these two markers as shown in the figure below:
A
B
C
Figure A and B display CD34 bright expression together with CD10 on dim CD45+ cells. This is representation of MPN in lymphatic accelerated phase and differential diagnosis in blast phase. The blasts are seen in bone marrow and whole blood that are positive for CD19, CD10 and CD34. Figure C represents an example of MDS, where CD34 expression on CD45 dim blasts and negative for all lymphoid markers are absent.
Conclusion
The consistent and versatile performance of the ClearLLab LS reagent demonstrates the advantage of using a standardized product compared to lab-developed tests (LDTs) for screening of hematological neoplasia on the Navios Flow Cytometer to:
- Identify major blood and bone marrow cell populations
- Detect aberrant antigen expression on B cells (CD5, CD10)
- Establish B cell Clonality status
- Identify B cell maturation status
- Identify aberrant expression of antigens on T and NK cells
The specific choice of CD34 and combinations in the ClearLLab LS reagent together with backbone markers help address the clinical indications and provide sufficient comprehensive identification of all major categories of hematopoietic cell populations in both normal and neoplastic states.
*ClearLLab LS (Lymphoid Screen) is CE-IVD marked under Regulation (EU) 2017/746. For In Vitro Diagnostic Use. The Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries.
