Flow cytometric immunophenotyping of feline bone marrow cells and haematopoietic progenitor cells using anti-human antibodies

Abstract

There is a paucity of species-specific antibodies available for feline haematopoietic conditions. The purpose of this study was to broaden the panel of antibodies available for use in the immunophenotypic characterisation of feline haematopoietic cells by testing clones of anti-human monoclonal antibodies (mAbs) on normal, neoplastic and cultured feline haematopoietic progenitors to determine cross-reactivity to feline counterparts. In this study, 24 clones of anti-human mAbs were tested on normal or neoplastic feline bone marrow and peripheral blood cells. Six of these mAbs, including anti-cluster of differentiation (CD)61, anti-CD18, anti-CD14, anti-CD235a, anti-CD41 and anti-CD29, cross-reacted with normal feline bone marrow cells, whereas anti-CD33 and anti-CD117 cross-reacted with the blast cells in the bone marrow of two cats with myelodysplastic syndrome, and anti-CD71, anti-235a, anti-41 and anti-42 cross-reacted with immature erythroid cells in a cat with erythroleukaemia. In a feline immunodeficiency virus-positive cat, bone marrow cells were labelled with anti-CD33, anti-14 and anti-45. Anti-CD18, anti-CD14, anti-CD41 and anti-CD61 also reacted with the peripheral blood cells of the healthy cats. The feline haematopoietic progenitors formed colonies in the methylcellulose-based semisolid medium with significant enrichment of colony-forming unit-granulocyte, monocyte and burst-forming unit-erythroid. A panel of six anti-feline mAbs (anti-CD21-like, anti-T lymphocytes, anti-CD172a, anti-granulocyte, anti-CD45-like and anti-CD18) and eight anti-human antibodies (anti-CD71, anti-CD33, anti-CD235a, anti-CD41, anti-CD61, anti-CD117, anti-CD38 and anti-CD34) were used for the immunophenotypic characterisation of the feline bone marrow progenitors. CD45, CD33, CD235a and CD18 were expressed by the feline haematopoietic progenitor cells, with the highest expression level for CD45.

Figure 1

Forward versus side scatter plots of bone marrow and peripheral blood cells from normal, healthy cats. Forward (FSC-h)/side scatter (SCC-H) dot plots with four cellular subsets, categorised into four gate regions (R1–R4), are shown for the bone marrow and peripheral blood

Figure 2

Reactivity of the human monoclonal antibodies anti-cluster of differentiation (CD)18, anti-CD41, anti-CD61 and anti-CD235a (glycophorin A) with feline bone marrow haematopoietic cells compared with the isotype control. The y-axis indicates the relative numbers of cells, and the x-axis indicates the fluorescence intensity (FL1-H) on a logarithmic scale. Blue line = CD markers; red line = isotype control. SSC-H = side scatter

Figure 3

Flow cytometric analysis of bone marrow cells from a cat with myelodysplastic syndrome with excess blasts. Flow cytometric histograms show the isotype control and cells with positive reactivity with some human monoclonal antibodies. Fifty-two percent of the cells are positive for cluster of differentiation (CD)18, whereas immunophenotyping with anti-CD33, anti-CD117 and anti-CD235a antibodies shows a weak reactivity. A significant degree of positive reaction to CD41 and CD61 is demonstrated. Blue line = CD markers; red line = isotype control

Figure 4

Flow cytometric analysis of bone marrow cells from a cat with erythroleukaemia. A distinct population of the neoplastic cells was gated (R1) in the forward scatter (FSC)–side scatter (SSC) plot. The histograms for cluster of differentiation (CD)71, CD235a, CD41 and CD42 expression by the cells in gate R1 are shown. Blue line = CD markers; red line = isotype control

Figure 5

Flow cytometric analysis of bone marrow cells from a cat with myelodysplastic syndrome. The histograms for cluster of differentiation (CD)45, CD14, CD33 and CD117 are shown. Blue line = CD markers; red line = isotype control

Figure 6

(A) Representative images of the feline haematopoietic progenitor cell colonies. Colony-forming unit–erythroid (CFU-E): a cell cluster containing fewer than 100 haemoglobinised erythroblasts. Burst-forming unit–erythroid (BFU-E): two small BFU-E colonies with more than 200 cells. Individual cells are characterised by a small size with a reddish colour. Colony-forming unit–granulocyte, macrophage (CFU-GM) colony: two distinct cell clusters are distinguishable. The monocyte/macrophage cells are larger, whereas the granulocyte lineage cells are smaller and more regular in shape. Colony-forming unit–macrophage (CFU-M): the colony is characterised by large, colourless cells. Colony-forming unit–granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM): mixture of erythroid clusters and granulocyte and macrophage cells. (B) Forward scatter (FSC-H) versus side scatter (SSC-H) plot along with dot plots of cluster of differentiation (CD)33 (anti-human), CD235a (anti-human) and CD45 (anti-feline) expression in the feline haematopoietic progenitor cells cultured in the methylcellulose-based medium. The vertical line is placed to the right of the cells labelled with isotype control