identifying immune cells / lab 2 (LR 1)

- stain and observe human blood cells
- identify the erythrocytes and lymphocytes

pluripotent hematopoietic stem cells in the bone marrow

hemocytoblast

- myeloid lineage
- lymphoid lineage

- megakaryocyte
- erythrocyte
- mast cell
- myeloblast

- thrombocytes (platelets)

- basophil
- neutrophil
- eosinophil
- monocyte

- macrophage
- dendritic cell

- natural killer (NK) cell (large granular lymphocyte)
- small lymphocyte

- T lymphocyte
- B lymphocyte

plasma cell

- granulocyte
- nucleus: 2-5 lobes
- cytoplasm: pale pink with fine granules
- function: engulfs and destroys bacteria

- granulocyte
- nucleus: usually 2 lobes
- cytoplasm: large red-orange granules
- function: fights parasites; involved in allergies

- granulocyte
- nucleus: often hidden by granules
- cytoplasm: large dark blue-purple granules
- function: releases histamine during allergic and inflammatory responses

- agranulocyte
- nucleus: large, round, dark purple
- cytoplasm: thin rim of light blue
- function: B cells make antibodies; T cells coordinate immune responses or kill infected cells

by having antigen receptors

thymus

bone marrow

- agranulocyte
- nucleus: kidney-shaped or folded
- cytoplasm: abundant gray-blue
- function: performs phagocytosis and can develop into macrophages or dendritic cells

white blood cells (leukocytes) that do not have prominent, visible GRANULES in their cytoplasm when examined using a standard Wight-Giemsa stain

- RBCs: pink, round, no nucleus; transport oxygen
- Platelets: tiny purple framents; helps blood clot

- innate immune cell
- phagocyte
- nucleus: large, oval or kidney shaped
- cytoplasm: abundant, often containing vacuoles
- function: engulfs pathogens and dead cells, releases cytokines, and presents antigens to T cells

- innate immune cell
- antigen-presenting cell
- nucleus: oval or irregularly shaped
- cytoplasm: has long, branching projections
- function: captures antigens and presents them to T cells, helping activate adaptive immunity

- innate immune cell
- granule-rich
- nucleus: round or oval, often hidden by granules
- cytoplasm: filled with large purple-blue granules in appropriate stains
- function: releases histamine and other mediators during allergic reactions and inflammation; also contributes to defense against parasites

- innate lymphoid cell
- nucleus: round or slightly indented
- cytoplasm: pale blue with possible purple granules
- function: kills virus-infected and cancer cells by releasing perforin and granzymes; also prduces interferon-gamma

- Wright-Giemesa
- Complete Blood Count (CBC) with differential
- Flow Cytometry
- Immunofluorescence microscopy
- Immunocytochemistry (ICC)
- Mass cytometry (CyTOF)
- Single-cell RNA sequencing

what it identifies: five major WBC types
main advantage: inexpensive, easy to perform

what it identifies: number and proportion of WBC types
main advantage: quantitative clinical results

what it identifies: specific immune cell populations using CD markers
main advantage: highly precise cell characterization

what it identifies: cells expressing specific immune markers
main advantage: shows marker expression and location

what it identifies: specific immune proteins on or within cells
main advantage: visual identification using antibodies

what it identifies: many immune markers simultaneously
main advantage: deep immune profiling

what it identifies: immune populations based on gene expression
main advantage: reveals cell diversity and activation states

- a blood smear is stained with Wright or Wright-Giemsa stain. Different cellular structures take up different colors, allowing immune cells to be distinguished by their morphology
* important limitation: microscopy can distinguish the five major leukocyte types, but generally cannot distinguish between B cells, T cells, and NK cells from one another because they have similar appearances

branch of medicine & science that studies blood, blood-forming organs (such as bone marrow), and blood disorders

- an automated HEMATOLOGY ANALYZER counts blood cells and catagorizes white blood cells based on properties (ex: size, light scattering, electrical impedance, or cytochemical signals, depending on the instrument
- reports:
* total white blood cell count
* neutrophil, lymphocyte, monocyte, eosinophil, and basophil percentages
* absolute counts of these populations
- a CBC is useful for detecting changes in circulating immune-cell numbers, but it does NOT ordinarily identify detailed T cell and B cell subsets

- identifies immune cells using FLUORESCENTLY LABELED ANTIBODIES that bind to specific surface or intracellular proteins

CD45

CD3

CD3+, CD4+

CD3+, CD8+

CD19+, CD20+

CD3-, CD56+, and/or CD16+

CD14, CD16, HLA-DR

CD4+, CD25^highCD127^low, FOXP3 confirmation

fluorescent antibodies are used to label specific cellular proteins, and cells are visualized unde a fluorescence miroscope

immunocytochemistry uses antibodies to detect specific proteins in cells
detection can use an enzyme-generated colored product, such as brown DAB staining, which can be viewed under a bright field microscope
it can identify cells expressing markers such as CD3, CD20, or CD68

- mass cytometry (CyTOF)
- single-cell RNA sequencing (scRNA-seq)

uses metal-labeled antibodies and mass spectrometry to measure many immune-cell markers simultaneously. It is particularly useful for studying heterogeneous immune populations

measures gene expression in individual cells to identify known cell types, discover rare populations, and study activation states
cell identity is inferred from gene-expression patterns and is often confirmed using additional methods

1) prepare the slide
- prepare the human blood smear on a staining tray, with the smear facing upward
2) Add wright stain
- add 9 drops wright stain to cover the blood smear
- wait 3 minutes
3) add buffer
- add 9 drops of wright stain buffer
- avoid splashing
- wait 90 seconds
- tilt the slide and drain the liquid into the designated staining tray
- add another 9 drops of buffer
4) rinse
- gently rinse the slide with distilled water
- do NOT use a strong steam of water
5) dry
- allow excess water to drain
- carefully blot the slide using bibuous paper without rubbing the blood smear
- make sure the slide is completely dry
6) observe under the microscope
- start with the 10x objective to locate the smear
- switch to 40x to locate white blood cells