Rita Steen

Differences in the distribution of CD34 epitopes on normal haemopoietic progenitor cells and leukaemic blast cells

The CD34 molecule expressed on haemopoietic progenitor cells contains a large number of epitopes whose expression may be related to the maturation or function of the cells. Monoclonal antibodies specific for different epitopes have been reported to detect different numbers of CD34+ leukaemic blast cells. We wanted to confirm this observation and study whether parallel findings could be observed for normal haemopoietic progenitor cells. The cells were immunophenotyped by flow cytometry with a series of monoclonal antibodies reactive with different CD34 epitopes. Class III epitopes (resistant to enzymatic cleavage with neuraminidase, chymopapain and a glycoprotease from Pasteurella haemolytica) showed a broader distribution on normal haemopoietic progenitor cells and leukaemic blast cells than class I epitopes (sensitive to cleavage with all three enzymes) and class II epitopes (sensitive to degradation with glycoprotease and chymopapain, and resistant to neuraminidase). The subpopulation of normal progenitor cells which exclusively expressed class III epitopes had flow cytometric characteristics compatible with mature myeloid progenitor cells, whereas class I, II and III epitopes were equally expressed on cells enriched for immature subsets. No discordant epitope expression could be observed for the more immature leukaemias (AML‐M0/1) and a higher percentage of the more mature leukaemic blast cells (AML‐M3 and AML‐M4/5) expressed class III epitopes compared to the percentage expressing class I and II epitopes.

Haemopoietic progenitor cell differentiation: flow cytometric assessment in bone marrow and thymus

Summary. We have recently shown that expression of any of the lineage‐associated molecules CD2, CD7, CD10, CD19 or CD33 does not ensure lineage‐commitment of CD34+ progenitor cells. Further, normal progenitor cells and leukaemic blast cells have been shown to coexpress molecules associated with more than one haemopoietic lineage. Five‐dimensional flow cytometric analysis of normal bone marrow cells was exploited to investigate the hypothesis of a developmental stage in haemopoiesis comprising CD34+ cells coexpressing CD2, CD5, CD7, CD10, CD19 and CD33 or any combination of these molecules. We report on a subpopulation of CD34+ bone marrow cells constituting < 5% of the CD34+ cells and characterized by extensive coexpression of several molecules associated with the B lymphoid, T lymphoid and myeloid lineages. There is every probability that some cells display the CD34+ CD2+ CD5+ CD7+ CD10+ CD19+ CD33+ phenotype. Studies on postnatal thymocytes suggest that this may be the phenotype or one of a few phenotypes of a candidate thymus‐seeding progenitor cell population. Finally, our findings that CD34+ as well as CD34+ CD5+ thymocytes can be driven into non‐T‐lymphoid differentiation by cytokines, support the notion that the thymus is seeded by uncommitted progenitors.

Thrombopoietin Combined with Early-Acting Growth Factors Effectively Expands Human Hematopoietic Progenitor Cells In Vitro

Thrombopoietin (TPO) is established as a powerful stimulant of megakaryocyte differentiation and platelet production both in vivo and in vitro. In preparation for future transplantation of ex vivo expanded CD34+ hematopoietic progenitor cells (HPCs), we have examined the in vitro effect of TPO on cultures of HPC when combined with other early‐acting hematopoietic growth factors (GFs) in an attempt to decrease post‐transplant thrombocytopenia and accelerate engraftment. By adding TPO to all possible combinations of GM‐CSF, IL‐3, and c‐kit ligand (CKL) in a suspension culture system, we found a significant increase in both relative and absolute numbers of cells in cultures containing TPO of the megakaryocytic lineage and CD34+ cells after 14 days of culture.

CD34 Molecule Epitope Distribution on Cells of Haematopoietic Origin

The CD34 molecule belongs to the mucin membrane molecule family and is expressed on virtually all normal haematopoietic progenitor cells (HPC). Due to its heavy glycosylation, several different epitopes exist on the molecule. Based on the sensitivity of the glycosylated molecule to degradation with a glycoprotease from Pasteurella haemolytica and neuraminidase, three classes of epitopes have been identified. The class I and II epitopes are probably related to the glycosylated part of the molecule while class III epitopes are core protein related. It has been known for some time that CD34 class I epitopes are absent on CD34 molecules expressed on high endothelial venules. Here we review recent observations that expression of both class I and II epitopes, but not class III epitopes, is impaired on mature myeloid CD34-pos. HPC while no diverse class epitope expression was observed on immature HPC. In addition, cells from patients with CD34-pos. acute myeloid leukaemia of FAB classification M4-M5, i.e., leukaemic blast cells of relatively mature morphologic phenotype, also express less class I and II epitopes than class III epitopes. It therefore seems that HPC maturation and class I and II epitope deprivation are concomitant events and that CD34 class I and II epitopes are lost prior to downregulation of the CD34 molecule per se. The biological significance of this observation is discussed as well as the need to carefully select CD34-specific monoclonal antibodies for research and clinical purposes.

Characteristics of haemopoietic progenitor cells related to CD34 epitope class expression

Abstract: Haemopoietic progenitor cells (HPCs) express the CD34 molecule, a heavily glycosylated transmembrane protein displaying three main classes of epitopes. The CD34 epitope class expression may vary between different subsets of HPCs. The aim of this study was to characterise the subsets of HPCs expressing CD34 class II and III epitopes. The cells were studied for coexpression of activation‐, lineage‐ and adhesion‐associated molecules, and their clonogenic ability and morphological features were examined.

Thymic stromal cells support differentiation of natural killer cells from CD34+ bone marrow cells in vitro

Abstract:  Natural killer (NK) cells are CD3− CD56+ lymphocytes characterized by exhibiting non‐MHC restricted cytotoxicity. A developmental relationship between NK cells and T lymphocytes has been proposed, and, moreover, the thymus has been shown to contain NK cell precursors. In this study we utilized an in vitro assay, devised to study T‐lymphocyte development from bone marrow progenitors, to investigate the ability of thymic stromal cells to support generation of NK cells from CD34+ bone marrow cells. CD34+ cells purified from healthy adults were seeded on adherent thymic stromal cells. The cells emerging after culture were phenotypically characterized by flow cytometry. We show that lymphocytes expressing the phenotypical characteristics of NK cells were generated from CD34+ bone marrow cells, and that these cells represented 1% of the cells recovered from the cultures. Furthermore, this was accomplished without supplement of exogenous interleukin 2 which is required for NK cell differentiation in bone marrow cultures.

Lowered reference limits for hCG improve follow-up of patients with hCG-producing tumors

BACKGROUND Human Chorionic Gonadotropin (hCG) is produced by germ cell tumors, but can also be elevated in benign conditions such as primary hypogonadism, where hCG is produced by the pituitary gland. In our experience, the reference limits for hCG (Elecsys hCG+β-assay, Roche Diagnostics), were unnecessarily high and did not reflect levels encountered in clinical practice. We wanted to establish new reference limits to increase the clinical utility of the hCG-assay. METHODS We analysed hCG in serum samples from a healthy adult population and in a cohort of testicular cancer survivors. The gonadotropins LH and FSH were measured in the cohort and in a selection of the reference population to assess gonadal function. RESULTS We found low hCG levels for all men and women <45years (97.5 percentiles 0.1 and 0.2IU/L, respectively) from the healthy population (n=795) having normal FSH and LH. Due to assay limitations, we suggest a common reference limit of <0.3IU/L. For the age group ≥45, the 97.5 percentiles in the healthy population were 0.5IU/L for men and 6.0IU/L for women. In all subjects from both the reference population and the cohort (n=732), hCG levels exceeding the reference limit could be fully explained by reduced gonadal function indicated by elevated LH and FSH levels. CONCLUSION The Elecsys hCG+β-assay should have lower reference limits than recommended by the manufacturer, with important implications for tumor follow-up. Elevated hCG is rare with intact gonadal function, both in a normal population and among survivors of testicular cancer, and should lead to further investigations when encountered in clinical practice.