Lab 8 – Blood Cells and Hemopoiesis Blood Cells and ... · Blood Cells and Hemopoiesis SEM of a...

Blood Cells and Hemopoiesis

SEM of a neutrophil (purple) ingesting S. aureus bacteria (yellow). NIAID.

Lab 8 – Blood Cells and HemopoiesisIUSM – 2016

I. IntroductionII. Learning ObjectivesIII. KeywordsIV. Slides

A. Blood Cells1. Erythrocytes (Red Blood Cells)2. Leukocytes (White Blood Cells)

a. Granulocytes (PMNs)i. Neutrophilsii. Eosinophilsiii. Basophils

b. Agranulocytes (Mononuclear)i. Lymphocytesii. Monocytes

3. Thrombocytes (Platelets)B. Bone Marrow

1. General structure2. Cells

a. Megakaryocytesb. Hemopoietic cells

i. Erythroid precursorsii. Myeloid precursors

V. Summary

Blood

1. Blood is a specialized type of fluid connectivetissue that provides the body’s tissues withnutrition, oxygen, and waste removal and servesas a means of transportation for the activity ofother body systems (e.g., carrying hormonesfrom source to target for the endocrine system).

2. It consists of plasma (liquid ECM of blood) andformed elements (cells and platelets).

3. The “formed elements” of blood derive fromhematopoietic stem cells located in the red bonemarrow of flat bones in adults.

4. Blood cells can be classified as red blood cells(about 45% of blood) and white blood cells(about 1% of blood) based upon their grossappearance upon centrifugation.

5. White blood cells (leukocytes) can be furtherclassified as granulocytes (neutrophils, basophils,eosinophils) or agranulocytes (monocytes andlymphocytes) based upon the presence ofabsence of cytoplasmic secretory granules.










V. Summary

Classifying Blood Cells

Cells of the blood can be classified using several different schema depending upon whatrelationship between the sets is considered important or to be emphasized:

1. Gross appearance: upon centrifugation, blood cells tend to separate into a red-coloredband and a white-colored band (“buffy coat”); this gross distinction allows for cells to beconsidered either red blood cells (RBCs) or white blood cells (WBCs).

2. Location: blood cells can be categorized based upon where they are generally found inthe body, such as in the peripheral vasculature or only in the bone marrow, or more specificlocations, such as for lymphocytes.

4. Microscopic appearance: microscopic examination of blood cells allows categorizationbased upon particular structures, such as the presence of cytoplasmic specific granules(i.e., granulocytes vs agranulocytes), organelle morphology (e.g., polymorphonuclear ormononuclear nucleus), or based upon staining characteristics (i.e., neutrophil, eosinophil,and basophil).

5. Lineage: blood cells can be categorized depending upon their lineage relationship withother blood cells; all blood cells derive from hematopoietic stem cells (HSCs) but soon splitinto branching lineages based upon common progenitor cells (i.e., myeloid vs. lymphoid).










V. Summary

Identifying “Formed Elements”

Since some “blood cells” are not actually cells when functional, the term “formed elements” isoften used to describe the “cellular” content of blood. When evaluating the formed elementsof blood, there are several characteristics to keep in mind when attempting to identify thetype of “cell”:

1. Location the sample was taken from (if known)

2. Size of the element

3. Nucleus to cytoplasm volume ratio (N:C)

4. Number and shape of nuclei

5. Degree of chromatin condensation

6. Presence or absence of nucleoli

7. Cytoplasmic staining

8. Presence and staining of cytoplasmic granules










V. Summary

Learning Objectives

1. Understand that blood is a connective tissue with “formed elements”(cells and platelets) in a fluid matrix (plasma).

2. Understand the morphology and function of red blood cells(erythrocytes) and the role of the spectrin membrane skeleton inmaintaining their biconcave shape and flexibility.

3. Understand the relative numbers of the various types of white bloodcells in the blood of normal adults and know the major functions ofeach type.

4. Understand that all leukocytes display cell motility and function insecretion, phagocytosis, etc. primarily in the ECM of tissues aftermigrating across the blood vessel wall (diapedesis).

5. Be able to recognize and find neutrophils, eosinophils, basophils,monocytes, and platelets in a blood smear.










V. Summary

Learning Objectives (cont.)

6. Understand and recognize the compartments and tissues in bloodmarrow.

7. Understand the development sequences and recognize theintermediate cells in the formation of red blood cells and the threetypes of granulocytes.

8. Be able to recognize megakaryocytes and understand how plateletsare formed and released.










V. Summary

Keywords

BasophilBone marrowEosinophilErythrocytesHemopoiesisHemopoietic cordLeukocytesLymphocyteMegakaryocyteMetamyelocytesMonocyteMyelocytesNeutrophil

NormoblastsPlateletsProerythroblastsRed blood cellsRed marrowReticulocytesSinusoidsStab cellsStromal cellsThrombocytesWhite blood cellsYellow marrow










V. Summary










V. Summary

Slide 35a (NW): Blood Smear

Slide 23 (464): Blood Smear, Wright’s

zoom in to identify leukocytes (white blood cells) amongst all the erythrocytes (red blood cells)

Slide 10: Blood Smear, Wright’s

notice the difference in staining of the RBCs onthe three blood smear slides; use the colorationof the acidophilic RBCs as a reference inidentifying other cell types (e.g., eosinophils)

https://vmicro.iusm.iu.edu/virtual_h/10_2_bl_5.html

http://medsci.indiana.edu/a464/virtual/464_23_5.html


Diameter 6-8 µm

N:C --

Nucleus --

Chromatin --

Nucleoli --

Cytoplasm Eosinophilic

erythrocytes (red blood cells) are the most numerous type of cell found in blood; before leaving the bonemarrow where they are formed, they lose their nucleus and most organelles and become filled withhemoglobin, giving them acidophilic cytoplasm; they circulate for about 120 days and are essential fortransport of carbon dioxide and oxygen around the body; they serve as a useful size reference for other cells

Slide 10: Blood Smear, Wright’s stainLab 8 – Blood Cells and Hemopoiesis

IUSM – 2016









V. Summary


Blood Smear, Wright’s stain

% of WBCs 40-75%

Diameter 10-15 µm

N:C 1:3

Nucleus Multi-lobed in a variety of shapes

Chromatin Condensed

Nucleoli No

Cytoplasm Non-distinct staining of granules

neutrophils (polymorphonuclear neutrophils, polymorphs, or segmented neutrophils) are the mostabundant type of leukocyte with approximately 1011 leaving the bone marrow each day; they circulate for 6-10 hours before exiting into the tissues where they live for 1-2 days; like most mature leukocytes, they havecondensed chromatin and few cytoplasmic organelles; they are phagocytic and the first-responders at sitesof inflammation to destroy damaged tissue and bacteria










V. Summary


a (NW): Blood Smear, Wright’s stain

band cells are the final stage of all granulocyte development before becoming mature cells but are usuallyonly seen for neutrophils; they generally have a characteristic “horseshoe”-shaped nucleus, instead of thesegmented (“string of sausages”) nucleus of mature neutrophils; they are normally only seen in lownumbers in the peripheral circulation but may become more numerous during inflammation or infection;this increase is referred to as a “left-shift” (etymology of the term is uncertain) or bandemia

% of WBCs < 5 %

Diameter 10-15 µm

N:C 1:3

Nucleus “Horseshoe”

Chromatin Condensed

Nucleoli No

Cytoplasm Non-distinct staining










V. Summary

https://vmicro.iusm.iu.edu/virtual_h_nw/nw_hist_1_35a.html


% of WBCs 1-6%

Diameter 12-17 µm

N:C 1:3

Nucleus Bi-lobed

Chromatin Condensed

Nucleoli No

Cytoplasm Large, acidophilic granules that stain the same color as surrounding RBCs

eosinophils are granulocytes with intensely-staining eosinophilic (acidophilic) cytoplasmic granules; theycirculate for a few hours before exiting into tissues; eosinophils are important in killing parasitic worms(helminths) and modulating inflammation










V. Summary


% of WBCs < 1%

Diameter 14-16 µm

N:C 1:3

Nucleus Bi-lobed (if seen)

Chromatin Condensed

Nucleoli No

Cytoplasm Large, basophilic granules that often obscure everything else in the cell

basophils are granulocytes and the rarest type of circulating leukocyte; they appear similar to mast cells(only in tissues) with a bi-lobed nucleus that is usually obscured by the numerous, large, strongly-basophilicgranules that fill the cytoplasm; basophils play a role in parasitic infections and are part of the inflammatoryresponse (contain histamine and heparin); a neutrophil can be seen in the lower left of the slide

Slide 23 (464): Blood Smear, Wright’s stainLab 8 – Blood Cells and Hemopoiesis

IUSM – 2016









V. Summary



% of WBCs 20-50%

Diameter 6-12 µm (90%)15-20 µm (10%)

N:C 4:1 (small cells)2:1 (large cells)

Nucleus Large, spherical

Chromatin Condensed

Nucleoli No (until activated)

Cytoplasm Minimal and basophilic; Golgi is usually visible

lymphocytes are agranulocytes generally similar in size to RBCs but have a very large, round nucleus whichfills most of the cell, leaving little cytoplasm with few organelles; the cells function as part of the adaptiveimmune system but specific subtypes (B and T cells) are not distinguishable in light microscopy (majorityare T cells, however); activated lymphocytes may enlarge and appear similar to monocytes but are stilldistinguishable by their nuclei










V. Summary



% of WBCs 2-10%

Diameter 15-20 µm

N:C 3:1

Nucleus Large and indented or kidney-shaped

Chromatin Condensed

Nucleoli Maybe

Cytoplasm “Dishwater”appearance with fine purple/pink granules

monocytes are agranulocytes and the largest leukocytes; they are macrophage-precursor cells thatcirculate in blood for about a day before migrating into tissues and becoming macrophages; they may beconfused with large lymphocytes (two small lymphocytes are seen below), but the distinctive shape of therespective nuclei should help to differentiate them










V. Summary



Diameter 1-3 µm

N:C --

Nucleus --

Chromatin --

Nucleoli --

Cytoplasm Basophilic

Granulocyte? --

platelets are small cellular fragments derived from megakaryocytes in the bone marrow; when in contactwith collagen (exposed CT), platelets adhere and degranulate, triggering the formation of a blood clot;around 1011 platelets are produced each day in a healthy adult, and they survive in the circulation for 7-10days










V. Summary


Bone, H&E

yellow(er) bone marrow(less hematopoietically active)

by adulthood, 85% adipose tissuefound in medullary cavity (trabecular bone) of long

bones

Slide 34: Bone, H&E

red bone marrow(more hematopoietically active)

by adulthood, 40% adipose tissuefound in flat bones and trabecular bone near

epiphysis of long bones










V. Summary

https://vmicro.iusm.iu.edu/virtual_h/msci_104_5.html

https://vmicro.iusm.iu.edu/virtual_h/34_bl_5.html










V. Summary

Slide 20a (464): Bone Marrow

Slide 77 (464): Bone Marrow, Giemsa

Slide 12 (464): Decalcified Bone

look at additional bone marrow slides tocompare the amount of adipose andhemopoietic cells seen

http://medsci.indiana.edu/a464/virtual/464_20a_5.html



trabecula of bone

hemopoietic cordwith clusters of blood cell precursors and megakaryocytes

“spaces” are adipocytes

Slide 153: Bone Marrow, Needle Biopsy, H&ELab 8 – Blood Cells and Hemopoiesis

IUSM – 2016









V. Summary


a (464): Bone Marrow, H&E

sinusoids

adipocyte

hemopoietic cord










V. Summary

http://medsci.indiana.edu/a464/virtual/464_20a_5.html

Bone, H&Eendosteum lining inner surface of trabecular bone

sinusoidfull of RBCs

hemopoietic cord

macrophage

stromal cell

endothelial celllining sinusoid

in red bone marrow, the stroma consists of reticular connective tissue cradling collections of hemopoieticcells called hemopoietic cords; within the stroma, there are macrophages and specialized fibroblast-like cellscalled stromal cells (reticular or adventitial cells); the cords are situated between specialized blood vesselscalled sinusoids, lined by simple squamous epithelium (endothelium), which blood cells must enter into inorder to leave the bone marrow and enter the peripheral circulation










V. Summary


Bone Marrow, Needle Biopsy, H&E

megakaryocyte

megakaryocytes are very large cells (50-70µm) with a single, multi-lobed nucleus from endomitosis(up to 48N); they are generally found located adjacent to sinusoids in the bone marrow where they budoff platelets, releasing them into the sinusoids to enter the peripheral blood; each megakaryocyteproduces between 1000-3000 platelets










V. Summary


Developing Blood Cells:Erythropoiesis and Granulopoieisis

Identifying hemopoietic (hematopoietic) cells in bone marrow samples can be very challenging: there are lots of celltypes, lots of terms, and lots of ambiguity. It is certainly not expected to be able to identify all the cells observable on a slide(at least not for this course), so focus should be on understanding the overall processes of erythropoiesis (red blood celldifferentiation) and granulopoiesis (granulocyte differentiation) and appreciating the respective readily identifiable cellstages and the defining characteristics for each.

Be sure to look at lots of cells on each slide to gain a comparative perspective amongst the cells. Every bone marrow smearlooks different. Find easily identifiable cells first (e.g., band cells) and use them as a reference when identifying other cells.Early cell types (e.g., proerythroblasts vs myeloblasts) are very difficult to differentiate, especially without better slides andhigher magnifications, so focus on identifying the later cell stages as highlighted in the lab guide.

Terminology Understanding, instead of just memorizing, the cell names is key to identifying the cells and retaining the material(hopefully a goal). A few ideas to keep in mind:

1. As seen in other cell lineages (e.g., osteoblasts and osteocytes), “blasts” precede “cytes”. For example, myeloblastsoccur before myelocytes (i.e., myelocytes are more differentiated).

2. The term myeloid (Gr. “bone marrow”) is used generally to refer to granulocyte precursor cells. This usage reflects anhistoric misunderstanding of hemopoiesis, but the terminology remains and is seen in the naming of granulocyteprecursor cells, e.g., myelocytes. The normal ratio of myeloid cell to erythroid cells (M:E) is about 3:1.

3. Erythroid terminology: the cells are named primarily based upon the staining of their cytoplasm. As the cellsdifferentiate, rER is lost and hemoglobin is gained, shifting the cytoplasm from basophilic to acidophilic. Keeping thisis mind should help in remembering the names and the order of the cell types.

4. Myeloid terminology: based upon the appearance of secondary granules, the myelocyte stage is the first stage whichis recognizable to a specific granulocyte lineage (e.g., an eosinophilic myelocyte looks different than a neutrophilicmyelocyte). Myelocytes therefore serves as the “center” of the terminology. Cells that precede the myelocyte stage arepromyelocytes (Gr. “before”), and cells that follow the myelocyte stage are metamyelocytes (Gr. “after”).










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V. Summary

Slide 127: Bone Marrow Smear

Slide 39b (NW): Bone Marrow Smear

Slide 39a (NW): Bone Marrow Smear

hemopoietic cells can be seen in the bonemarrow slides previously studied or on thebone marrow blood smear slides seen here

how do these blood smear slides appear differentfrom the peripheral blood smear slides seenearlier?


https://vmicro.iusm.iu.edu/virtual_h_nw/nw_hist_1_39b.html

https://vmicro.iusm.iu.edu/virtual_h_nw/nw_hist_1_39a.html

(OrEb) Orthochromatophilic erythroblast (normoblast)• small cell with mainly acidophilic cytoplasm (hemoglobin)• orthochromatic = “right color” = typical cytoplasm color of RBC• highly-condensed nucleus, soon to be ejected from cell

(ProEb) Proerythroblasts (earliest recognizable precursor)• large cell with sparse, basophilic cytoplasm (lack hemoglobin)• large, intensely-staining granular nucleus with one or more

nucleoli• thin Golgi adjacent to nucleus

(BaEb) Basophilic erythroblast• smaller than proerythroblast with decreasing N:C ratio• more abundant basophilic cytoplasm due to abundance of rER

for synthesizing hemoglobin• condensing nucleus and chromatin with no clear nucleoli

(PolEb) Polychromatophilic erythroblast• reduced cell volume• polychromatic = basophilic (rER) and acidophilic (hemoglobin)

cytoplasm• nucleus continuing to condense with clumped chromatin

Reticulocyte (not seen)• nucleus has been extruded• cytoplasm still contains small amount of rER but difficult to see• reticulocyte = specialized stains cause ribosomes to clump and

form reticular networkImage adapted from: Wheater’s Functional Histology, 5 ed.

ProEb

PolEb

BaEb

OrEb

As the RBC differentiation pathway advances, there is: (1) adecrease in overall size of the cell and the nucleus (chromatincondenses), (2) a decrease in N:C ratio, (3) a loss of organelles (andbasophilia), and (4) an increase in hemoglobin (and eosinophilia).










V. Summary

(Mye) Myelocyte (first cell unique for each granulocyte type)• secondary (specific) granules (pink/lilac for neutrophils)• more abundant cytoplasm; N:C ~2:1• nucleus becomes ovoid and eccentrically-placed• coarse chromatin and nucleoli become indistinct

(Meta) Metamyelocyte (different granulocyte types are easily seen)• nucleus is indented (“kidney-bean” shaped)• cytoplasm becomes less basophilic (continued loss of rER)• secondary granules are prominent; very few primary granules

Band cell (stab cell)• nucleus typically has “horseshoe” appearance• indentation of nucleus is > ½ the diameter of the nucleus• nucleus has uniform width and is not yet segmented

As the granulocyte differentiation pathway advances, there is: (1)a decrease in N:C ratio, (2) condensation of the nucleus,eventually becoming indented and finally segmented, (3) theappearance of non-specific and then specific cytoplasmicgranules, (4) a loss of organelles (and cytoplasmic basophilia).

(Pro) Promyelocyte• large, azurophilic (purple) primary (non-specific) granules• largest cell size of the series (15-24µm)• decreasing N:C ratio with basophilic cytoplasm• nucleus becoming heterochromatic with less prominent nucleoli

Myeloblast (not seen)• 14-20µm cell with high N:C ratio (i.e., not much cytoplasm)• euchromatic nucleus with generally 3-5 prominent nucleoli• deeply-basophilic cytoplasm lacking granules; Golgi is visible

Myeloid precursors of neutrophil granulocytes.

Mye

Meta

Band

Pro










V. Summary

Common Confusion:Band vs. Monocyte

Band cell

Band cells: final precursor cells of mature granulocytes butgenerally only readily seen for neutrophils; seen mostly inbone marrow; once nucleus becomes segmented, no longerconsidered a band

Look for: (1) 10-12µm diameter; (2) small, elongatednucleus with N:C ratio of 1:2; (3) nucleus has a “horseshoe”shape; (4) granular cytoplasm

Monocytes: largest type of leukocyte; they are circulatingprecursors of macrophages

Look for: (1) 20µm diameter; (2) larger nucleus with N:Cratio of ~3:1; (3) agranular, blue-gray cytoplasm(“dishwater” cytoplasm); (4) small “holes” (vacuoles) ofunstained areas may be seen in the cytoplasm

Monocyte










V. Summary

Common Confusion:Large Lymphocyte vs. Monocyte

Large Lymphocyte

Large lymphocytes: usually immunoblasts that haveenlarged after activation from antigen stimulation; someincrease in the size of the nucleus occurs, but most of thesize increase is in the cytoplasm, resulting in a decreased N:Cratio; plasma cells are not normally seen in the blood

Look for: (1) nucleus is round or ovoid, but not generallyindented; (2) more abundant cytoplasm with a variable N:Cratio, but ~2:1 is common; (3) chromatin is usually fine withvisible nucleoli; (4) cytoplasm is light blue and may containazurophilic granules; (5) cell borders may be distorted bysurrounding RBCs

Monocytes: largest type of leukocyte; they are circulatingprecursors of macrophages and are generally more commonthan large lymphocytes

Look for: (1) nucleus is indented; (2) N:C ratio of ~3:1; (3)coarser chromatin without nucleoli; (4) agranular, blue-graycytoplasm (“dishwater” cytoplasm); (5) small “holes”(vacuoles) of unstained areas may be seen in the cytoplasm

Monocyte










V. Summary

Megakaryocyte

Megakaryocytes: large cells in bone marrow that give riseto thrombocytes (platelets) in peripheral blood

Look for: (1) 20-60µm diameter; (2) large, single nucleusmay be irregularly lobulated, but lobes are connected andnot separated by cytoplasm; (3) generally located in bonemarrow adjacent to sinusoids, not immediately adjacent tobone; (4) finely-granular cytoplasm

Osteoclasts: very large cells responsible for boneresorption; they are derive from the fusion of mononuclearhemopoietic precursor cells

Look for: (1) 50-150µm diameter; (2) multinucleated –nuclei are usually round and distinctly separate, average of4-8 per cell; (3) generally located adjacent to bone,especially in Howship’s lacunae; (4) granular cytoplasm

Osteoclast

Common Confusion:Megakaryocyte vs. Osteoclast










V. Summary

Summary

1. Blood is a specialized type of connective tissue with a liquid extracellular matrix (ECM) calledplasma; the cellular elements of blood are referred to as formed elements and consist of red bloodcells (erythrocytes), which are non-nucleated cells that contain hemoglobin, and white blood cells(leukocytes), which are nucleated cells that lack hemoglobin.

2. Leukocytes are traditionally divided into granulocytes (have cytoplasmic granules that affect thestaining of the cell), which include neutrophils, eosinophils, and basophils, and agranulocytes(generally lack readily discernable cytoplasmic granules), which include lymphocytes andmonocytes.

3. The prevalence of leukocytes in the blood is often remembered by the perhaps misguidedmnemonic: never let monkeys eat bananas (for neutrophils, lymphocytes, monocytes, eosinophils,and basophils); the relative percentages can be remembered approximately by the “6/3 rule”: 60%for neutrophils, 30% for lymphocytes, 6% for monocytes, 3% for eosinophils, and 1% (remainder)for basophils.

4. The principal functional site of leukocytes is outside the blood in tissues; in loose CT, they are eitherpermanent or wandering cells and generally appear different than as seen on blood smears; see Lab5 – Connective Tissue for examples.

5. Hemopoietically-active bone marrow is referred to as red bone marrow; in adults it is primarilylocated within the cancellous bone of flat bones (such as the bones of the skull, sternum, vertebrae,and pelvis). Erythroid precursors and granulocyte precursors are generally recognizable based uponthe characteristic appearance and changes of their nuclei and cytoplasm during differentiation.










V. Summary

Characteristic Appearance

Structure Electron Microscopy Light Microscopy (H&E stain)

Neutrophil

Lymphocyte

Monocyte

Eosinophil

Basophil

Orthochromatophilicerythroblast

Reticulocyte

Promyelocyte

Myelocyte

Band cell

Appearance of Blood Cells in Electron and Light MicroscopyLab 8 – Blood Cells and Hemopoiesis

IUSM – 2016









V. Summary

How does the shape and appearance ofthe nuclei of erythroid precursorschange in comparison to the nuclei ofmyeloid precursors?

Why are mature granulocytes alsoreferred to as polymorphonuclearleukocytes? What types of leukocytesare mononuclear?

How does the cytoplasm of erythroidprecursors change in comparison tothe cytoplasm of myeloid precursors?

If viewing a blood smear, what cluescan be used to determine if it is fromperipheral blood or bone marrow?

Why do hemopoietic cells experience adecreasing N:C and loss of nucleoli asthey differentiate?

How are eosinophils and basophilsdifferentiated in electron micrographs?

The principal functional location ofwhich formed elements is in the blood?










V. Summary

Questions about Blood Cells and Hemopoiesis

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