Hematology Lecture

Front 1

Hematology

Back 1

• The study of blood
  
• We talk about blood, different elements of blood & disorders of blood
• Two common test types: CBC and coagulation tests

Front 2

Blood Forming Tissues/Organs

Back 2

• Bone Marrow - most blood cells come from bone marrow, plays the biggest role

• The thymus, spleen, liver and LNs play a secondary role.
• They can have bigger roles in development & kick in if something wrong with the bone marrow

Front 3

Volume of Blood in Average Person

Back 3

• 4-6 L
• ~8% of total body weight

Front 4

What should the pH of blood be?

Back 4

• 7.35-7.45

Front 5

Blood: Plasma & Formed Elements

Back 5

• Blood is about 55% plasma and 45% formed elements
  
  

• Plasma contains water, electrolytes, proteins, hormones, nutrients, and respiratory gases
  
  

• Formed elements are RBCs, WBCs, and platelets

• RBCs function to transport O2 and nutrients

• Platelets maintain hemostasis and inflammation

Front 6

What are the 5 types of White Blood Cells

Back 6

• Neutrophils - fight infections, involved in inflammation, phagocytosis
• Eosinophils - Allergy response, parasitic infections, asthma
• Basophils - immune response, have histamine in granules, inflammation
• Lymphocytes - Immune (cellular & humoral)
• Monocytes - phagocytosis

Front 7

Coulter Principle

*be familiar with for CSMLS*

Back 7

• Electronic method for counting and sizing particles
  
• "low-voltage direct current resistance"
• Based on principle of electrical impedance (the fact that cells are poor electrical conductors & will interrupt a current flow)
  
• Cells suspended in a conductive diluent are pulled through the aperture and impede the current.
  
• The resistance creates a pulse which is sensed and counted by the instrument as a particle
  
• The length (height) of resistance (impedance) is directly related to the size (volume) of the particle and the pulses will help generate histograms
  
• There are thresholds (electronically set size limits). These thresholds exclude unwanted particles (debris) from analysis
  
• The only particles analyzed are those above the threshold
  

*Remember impedence has to do with volume

*Our lab is strictly based on impedance – fancier machines use VCS technology for cell counting and sorting

Front 8

Coulter Machines

Back 8

• Coulter machines in our lab have two baths – a WBC bath & a RBC bath, and within each bath there are two electrodes suspended in a conductive diluent
  
• Most common diluent is isoton – buffered saline.
  
• One external electrode (–) and one internal electrode (+) – both required for electricial current
  
• (+) electrode enclosed in non–conductive casing, (–) electrode suspended in diluent
  

Front 9

RBC Bath (Coulter Machines)

Back 9

• In this bath red cells and platelets are counted by the impedance principle
  
• Thresholds set to electronically distinguish between red cells and platelets (particle that is 2–20fl = platelet, if it is >36 fl = red cell)
  
• Thresholds are electronically set size limits to exclude unwanted particles (i.e. debris)
• Red cells categorized by size. Raw data histogram is created.
  
• Y–axis = # of red cells, X–axis = average volume in femtolitres (fl)
  
• MCV = average size of all cells. Derived from red cell histogram
  

36 fl = threshold, peak on histogram is is at ~ 90fl therefore MCV is ~90fl

Hint 9

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Front 10

WBC Bath (Coulter Machines)

Back 10

• White cells counted based in impedance principle
  
• Hemoglobin measured based on cyanmethemolglobin principle
  
  
• Lytic reagent enters WBC bath and lyses the red cells releasing hemoglobin, and the reagent contains postassium ferricyande and postassium cyanide, which gives us cyanmethemoglobin (read at 525 nm)
  
• For WBCs any particle in dilution larger than 35 fl counted as a white cell. They are also sorted according to size producing a raw data histogram.
  
• Red cells do not interfere with this count bc of the lytic agent & platelets fall well under the threshold
  
  3 populations of white cells are derived from the histogram:
  
• Lymphocytes – mature & variant lymphs
  
• Large mononuclear cells – monocytes, blast cells, promyelocytes, prolymphocytes, promonocytes, myelocytes, and plasma cells
  
• Granulocytes – segmented and banded neutrophils & eosinophils
  

*Our machines do 3 part diff (lymphocytes, monocytes, granulocytes), newer/bigger machines do 5 part diff (lymphocytes, monocytes, neutrophils, eosinophils, basophils), some even do a 7 part diff.

Front 11

Volume, Conductivity & Light Scatter (VCS) Technology

Back 11

• Many Coulter instruments use this type of technology
  
• RBCs, WBCs & PLTs are still counted using impedance principles, but classification and differentiation of white cells is much more specific and sensitive
  
  
• Uses 3 technologies (volume, conductivity & light scatter)
  
• Each of the technologies are used simultaneously and differentiate cells into neutrophils, eosinophils, basophils, monocytes, and lymphocytes (5 part diff)
  
• All 3 technologies are applied simultaneously to each of the cells as they pass through the flow cell. Thousands are measured in seconds.
  
• A histogram and scatterplot are determined
  

Hint 11

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Front 12

Volume

Back 12

• Volume differentiation is done through the impedance principle like already discussed
  
• Direct current impedance
• Low frequency current to measure volume

Front 13

Conductivity

Back 13

• High frequency current passing through cell walls to interior
  
  
• Conductivity is based on the principle that cell membranes are permeable to high frequency electromagnetic energy, and that cell walls act as conductors when exposed to a high frequency current, such as radio frequency
• Once the high frequency current has penetrated the cell membrane its path is interrupted by the intracellular components of the white cell wall.
  
• Characteristics of the white cells internal physical & chemical content will interrupt the frequency and be detected by the instrumentEx. things like granularity, nuclear composition, chemical composition and N:C ration will each affect the frequency
  

Front 14

Light Scatter

Back 14

• White cells passed in front of a beam of laser light, and are differentiated based on the amount of light that they scatter
  
• Light scatter provides information about the cells shape, structure and internal granularity.
  
• Ex. the more granules present & the coarser they are the more light will be scatteredEach of the
  

Front 15

RDW

Back 15

• Obtained from red cell distribution data
  
• Is the coefficient of variation of the red cell volume distribution
  
• Analyzers computer disregards both extremes of the distribution curve (like outliers in statistical calculations) and then calculates the CV of the size of cells in the remainder of the curve [(SD of RBC Volume x 100)/Mean MCV]
  
• If the analyzer detects a wide population of sizes it will flag the RDW
  

Front 16

MCV

Back 16

Mean Corpuscular Volume

Average volume of red cells
Calculated from hematocrit and RBC count

Front 17

What kind of things do automated machines measure?

Back 17

• WBC count and subpopulations
• RBC count, hgb, hct & indices
• Plt count

Front 18

hgb

Back 18

hemoglobin

Front 19

hct

Back 19

hematocrit

Front 20

What are some things that cause problems in automated machines

Back 20

• Bubbles
  
• Clots/agglutination (biggest issue)
  
• Debris (usually falls below the threshold, doesn't cause issues but can)

Front 21

Flow Cytometry

Back 21

Forward Scatter

• 180 degrees from light source
• Determines volume/density

Side Scatter

• 90 degrees from light source
• Determines cellular contents

Can use fluorochrome dyes (immunological markers)

Front 22

Histograms/Scattergrams

Back 22

• Graphically show the size and distribution of cells

• Must always compare the histogram to numbers we get to be sure that they correspond

• Many machines do a scatter plot rather than a histogram. More dots= more concentrated

Hint 22

Front 23

Coulter QC

Back 23

• Remember to run the prime blood first
• Remember to trash any QC that you re-run due to flags
• Remember mono on high QC, may get * flag - this is ok (only high level)
• Westgard rules

Front 24

12S

Back 24

• One data point plus or minus 2 SD from the mean
• Sensitive to random error

Front 25

13S

Back 25

• One data point plus or minus 3 SD from the mean
• Sensitive to random mean

Front 26

8X

Back 26

• 8 or more on one side of the mean, with no requirements on distance from the mean

Front 27

7T

Back 27

• 7 more data points trending in one particular way (towards or away from the mean)

Front 28

Hematopoiesis

Back 28

• The dynamic processes of the production and maturation of the formed elements of blood
• It is a complex network/system that is able to respond to stimuli such as an increase in required elements
• Blood cells are the progeny of a hematopoietic stem cell
• Generally hematopoiesis is sustained in a steady state as production of mature cells equals blood cell removal

Front 29

Things that can cause an increase in hematopoiesis

Back 29

• Bleeding
• Low oxygen conditions (need more RBCs to transport as much oxygen as possible) ex. when at high altitudes
• During an infection
  
  
• Typically cell production is specific (ex. in infection we will produce white cells, in low O2 conditions we produce red cells)

• When there is increased demand for blood cells, active hematopoiesis may again occur in the spleen, liver, and other tissues as a compensatory mechanism known as extramedullary hematopoiesis
  

Front 30

Origin of Hematopoiesis

Back 30

• Begins in the yolk sac of the fetus during the first weeks of embryonic development -> continues throughout life
• Constantly occurring - balance of production and destruction
• Blasts -> differentiation -> maturation -> cyte (in peripheral blood)
• Blasts = most immature cells
  
  
  
• In the newborn infant hematopoiesis occurs in the liver and spleen, it stops, and in adults it occurs entirely in the bone marrow
• Gradual decrease of active sites
  
  
  

Front 31

Back 31

Front 32

Apoptosis

Back 32

Programmed cell death

Front 33

Medullary Hematopoiesis

Back 33

Medullary Hematopoiesis

• Blood cell production in bone marrow
• Most blood cell production in bone marrow

Front 34

Extramedullary Hematopoiesis

Back 34

Extramedullary Hematopoiesis

• Blood cell production outside the bone marrow (i.e. liver, spleen etc.)
• Occurs when BM can't keep up with demands, or BM is not functioning well
  

Front 35

Medullary Space

Back 35

• Spaces or cavities between the trabeculae of bone where bone marrow is formed

Front 36

Hematopoietic Marrow

Back 36

Hematopoietic Marrow

• Actively producing blood cells
• Red marrow

Front 37

Non-hematopoietic Marrow

Back 37

Non-hematopoietic Marrow

• Not actively producing cells - mostly fat
• Yellow marrow

Front 38

2 Pools of Bone Marrow Hematopoiesis

Back 38

Bone marrow hematopoietic activity can be divided into 2 pools: the stem cell pool and the bone marrow pool, with eventual release of mature cells into the peripheral blood

Stem Cell Pool

• Typically all stem cells
• Can differentiate into almost anything
• Morphologically unidentifiable multipotential stem cells (MSCs) & unipotential committed stem cells reside here

Bone Marrow Pool

• Can be further differentiated into two pools
• Proliferating & maturing cells pool - cells that are proliferating and maturing
• Storage pool - stored cells that are fully mature or almost and ready to head into circulation

Front 39

2 Pools in Peripheral Blood

Back 39

• There are also two separate granulocytic pools in the peripheral blood:
• 30% of platelets stored in the spleen.
• Neutrophils that line the walls of blood vessels are sometimes referred to as the marginating pool
• Circulation
• **100% of red cells in functional pool. None in storage.**
• 70% of platelets in circulation

Front 40

Hematopoietic System

Back 40

• Where undifferentiated cells divide, mature, and differentiate

Front 41

Hematopoietic Stem Cells

Back 41

Hematopoietic Stem Cells

• Duplicate during cell division
• Hematopoietic stem cells are pluripotential (can differentiate into different cells)
• They undergo continuous self-replication and proliferation
• They differentiate into committed progenitor cells of lymphoid and myeloid lineages
• Progenitor cells are cells that are dedicated to one certain lineage, but still not sure what they want to be

Front 42

Hematopoietic Stem Cells

Back 42

• Pluripotential Stem Cells (PSCs) differentiate into multipotential stem cells (MSCs)
• Multipotential stem cells belong to a specific lineage
• There are multiple cell types within a lineage
• Multipotential stem cells differentiate into committed stem cells (progenitor cells).
• Progenitor cells are committed to becoming one specific cell

Front 43

Hematopoiesis

Back 43

• Do not have to memorize, but have idea of what is going on
• GEMM = Granulocyte Erythrocyte Monocyte Megakaryocyte
• BFU-E = Burst-Forming Unit Erythrocyte

Front 44

Multipotential Stem Cell Lineages

Back 44

-There are 2 lineages for all blood cells

Myeloid Cell Lineages

• Can distinguish these under microscope
• Granulocytes
• Monocytes
• Erythrocytes
• Platelets

Lymphoid Cell Lineages

• Cannot distinguish under a microscope
• T-lymphocytes
• B-lymphocytes

Front 45

Cytokines

Back 45

• Glycoproteins
• Control the formation, development, and differentiation of blood cells
• Secreted by a variety of cells
• Many are not lineage specific
• Interact synergistically with each other

Front 46

What are the two main types of cytokines?

Back 46

Growth factors

• Colony stimulating factors (CSFs)
  
• Produced by a variety of cells
• ex. EPO, G-CSF, GM-CSF, M-CSF, SCF

Interleukins (IL's)

• Many are produced by T-lymphocytes

Front 47

Common Maturation Features

**very important**test**

Back 47

These things happen in all of the cell lines:

• Decrease in cell volume (size)
• Loss of nucleoli (pale staining section in nucleus)
• Nuclear chromatin gets courser/condenses (only see parachromatin in mature cells)
• Nucleus gets smaller
• Cytoplasm less blue colour (less RNA)

Front 48

Erythropoiesis

Back 48

• Production & maturation of red cells
• Red blood cells produced in the bone marrow
• Stimulated by erythropoietin (EPO)
• Most immature red cell is (pronormoblast)
• From pronormoblast to erythrocyte it takes 3-5 days for this maturation, and there are 3-5 cell divisions
• 14-16 red blood cells are formed from 1 pronormoblast
• As the cell matures, you get more and more hemoglobin accumulated, and the cell takes on more of a pinkish hue.

Front 49

Erythropoietin (EPO)

Back 49

• Glycoprotein hormone
• Heat stable (most hormones are heat labile)
• Produced in the kidney (small amount in the liver), therefore kidney disease tends to cause anemia
• Athletes - blood doping with EPO - stimulates body to produce more red cells so they can carry more O2. Dangerous because high hematocrit could cause clotting & stroke.
• Main function of EPO - stimulate release of reticulocytes (retics)
• Reticulocytes are immature RBCs capable of oxygen transport. They continue to mature in circulation as they transport oxygen

Front 50

When is EPO produced?

Back 50

• Hypoxia (low oxygen) stimulates the kidneys to produced EPO

• EPO travels to the bone marrow and stimulates production of RBCs
  
  

Other factors stimulate EPO production too:

• TSH, GH, ACTH
• Androgens & estrogens
• High altitudes
• Diet

Front 51

Stages of Maturation - Erythrocytes

Back 51

1. Pronormoblast
2. Basophilic normoblast
3. Polychromatic normoblast
4. Orthochromatic normoblast
5. Reticulocyte
6. Mature Red Cell

Front 52

Pronormoblast

Back 52

Pronormoblast

• RBC progenitor - committed to red cell lineage
• Most immature
• Actively dividing
• Basophilic cytoplasm (RNA)
• 0-2 nucleoli
• Fine chromatin
• N:C ration 8:1 to 6:1 (a lot of nucleus, very little cytoplasm)
• Has a nucleus
• Percent in BM = 0-1.5%

1. Pronormoblast
2. Basophilic normoblast
3. Polychromatic normoblast
4. Orthochromatic normoblast
5. Reticulocyte
6. Mature Red Cell
   

Front 53

Basophilic Normoblast

Back 53

Basophilic Normoblast

• Smaller than pronormoblast
• Basophilic cytoplasm
• 0-1 nucleoli
• Chromatin slightly condensed
• N:C ratio 6:1 to 4:1 - a little less cytoplasm than in previous stage
• Has a nucleus
• Percent in BM = 1-5%

1. Pronormoblast
2. Basophilic normoblast
3. Polychromatic normoblast
4. Orthochromatic normoblast
5. Reticulocyte
6. Mature Red Cell
   

Front 54

Polychromatic Normoblast

Back 54

Polychromatic Normoblast

• Smaller than basophilic normoblast
• Cytoplasm is starting to look pinkish (because we are starting to accumulate hemoglobin)
• Hemoglobin synthesized right from the beginning, but is really starting to accumulate now
• Last stage for mitosis
• N:C ratio 4:1 to 2:1
• Still has a nucleus
• Percent in BM = 5-30%

1. Pronormoblast
2. Basophilic normoblast
3. Polychromatic normoblast
4. Orthochromatic normoblast
5. Reticulocyte
6. Mature Red Cell
   

Front 55

Orthochromatic Normoblast

Back 55

Orthochromatic Normoblast

• Cytoplasm is pink
• Looks like an RBC, but with a nucleus
• Very dense dark staining nucleus (nucleus is degenerating/dying & o longer active)
• Last stage with a nucleus
• Not dividing (pyknotic)
• N:C ratio 1:1 to 1:2 (almost 50/50 ratio of nucleus to cytoplasm)
• Percent in BM = 5-10%

1. Pronormoblast
2. Basophilic normoblast
3. Polychromatic normoblast
4. Orthochromatic normoblast
5. Reticulocyte
6. Mature Red Cell
   

Front 56

Reticulocyte

Back 56

• Immediate precursor to mature RBC
• Release stimulated by EPO
• No nucleus
• Contains fragments of RNA
• Immature RBCs - capable of O2 transport
• Retics continue to mature in circulation as they transport oxygen
• Small amount of retics in blood s normal
• If we have low RBCs in bone marrow we will release retics to keep up with demand. Not necessarily something bad going on, just means something is up, maybe the person is sick
• The worse you are the more immature the released cells will be - this is bad
• Almost a red cell, but still a bit bigger than a mature red cell
• Need a retic stain (supravital stain) to definitively ID reticulocytes.
• Retic stain in this image. Reticulocytes under wright stain on Hint slide

1. Pronormoblast
2. Basophilic normoblast
3. Polychromatic normoblast
4. Orthochromatic normoblast
5. Reticulocyte
6. Mature Red Cell
   

Hint 56

• Reticulocytes cannot be definitively ID'd under Wright's stain
• Need a retic stain
• Cannot call it a reticulocyte if you haven't done a retic stain, can only call it a polychromatic cell (might be a retic, but it might not be)
• Reticulocytes have certain look under Wright stain

Front 57

Mature Red Cell (Erythrocyte)

Back 57

• Mature RBC
• Lifespan ~100-120 days
• Size 6-8 microns in diameter
• Also known as a discocyte
• 100% of mature RBCs are in the functional pool in peripheral blood *test*

The big picture in image on this slide & hint slide

Hint 57

Front 58

Common Maturation Features

**test**

Back 58

• Decrease in cell volume (size)
• Loss of nucleoli
• Nuclear chromatin gets courser/condenses
• Nucleus gets smaller
• Cytoplasm less blue colour (less RNA)

Front 59

Leukopoiesis

Back 59

• Form of hematopoiesis where white blood cells are formed in the bone marrow

Leukopoiesis

• Myelopoiesis (myelocyte cell line) - Neutrophils/eosinophils/basophils
• Monopoiesis (monocytic cell line) - monocytes/macrophages
• Lymphopoiesis (lymphocytic cell line) - B & T lymphocytes

Megakaryocytopoiesis (Megakaryocytic cell line) - Platelets

Front 60

Myelopoiesis

Back 60

• AKA Granulocytopoiesis
• Myelocytic cell line (granulocytic)
• Follows the common maturation characteristics, plus nucleus segments
• Granules are produced (primary and then secondary)

Front 61

Myelopoiesis: Primary Granules vs. Secondary Granules

Back 61

Primary Granules

• Not specific to the granulocyte that it is going to become
• Looks the same in neutrophils, eosinophils, and basophils
• Big chunky granules, can see individual granules

Secondary Granules

• Specific to the different cells
• Can ID by stain & viewing under microscope

Front 62

Stages of Myelopoiesis

Back 62

1. Myeloblast
   
2. Promyelocyte
3. Myelocyte
4. Metamyelocyte
5. Band Cell
6. Mature form (neutrophil/eosinophil/basophil)

*Myeloblast & Promyelocyte look the same whether or not it is going to be neutrophil, eosinophil or basophil. Both have primary granules

*Myelocyte is the first one where we can start to differentiate between eosinophil/basophil/neutrophil

Front 63

Myeloblast

Back 63

• Most immature
• Progenitor cell (committed to this line), earliest cell we can def. say is in myelo line, but don't know yet if neutrophil/eosinophil/basophil
• Percent in B = 0-2 %
• No cytoplasmic granules yet (no primary or secondary granules)
• N:C ratio = 7:1 to 5:1, much more nucleus than cytoplasm
• 1-3 nucleoli
• Size: 10-20 micrometers - very large cells

1. Myeloblast
   
2. Promyelocyte
   
3. Myelocyte
   
4. Metamyelocyte
   
5. Band Cell
   
6. Mature form (neutrophil/eosinophil/basophil)
   

Front 64

Promyelocyte

Back 64

• Start to develop primary granules
• Size 12-24 micrometers
• Percent in BM = 1-4%
• N:C ~5:1 to 3:1
• May still see nucleoli
• Called primary granules or non-specific granules
• Cytoplasm is less blue

1. Myeloblast
   
2. Promyelocyte
   
3. Myelocyte
   
4. Metamyelocyte
   
5. Band Cell
   
6. Mature form (neutrophil/eosinophil/basophil)
   

Front 65

Myelocyte

Back 65

• More cytoplasm, less nucleus
• Cytoplasm less blue, slight pinkish hue
• Specific or secondary granules (harder to see)
• Percent in BM = 5-20%
• N:C ratio: ~2:1
  
  
• Left image = neutrophil. Has slightly lighter area near top right that helps ID it . Most we find will be neutrophils, just because there are so many in blood compared to eosinophils/basophil
• Middle image = eosinophil
• Right image = basophil

1. Myeloblast
   
2. Promyelocyte
   
3. Myelocyte
   
4. Metamyelocyte
   
5. Band Cell
   
6. Mature form (neutrophil/eosinophil/basophil)
   

Front 66

Metamyelocyte

Back 66

• Nucleus starts to indent
  
• Noticeable condensing of chromatin
• Percent in BM: 5-20%
• N:C ~1:1

1. Myeloblast
   
2. Promyelocyte
   
3. Myelocyte
   
4. Metamyelocyte
   
5. Band Cell
   
6. Mature form (neutrophil/eosinophil/basophil)
   

Front 67

Band Cell

Back 67

Band Neutrophils

• Nucleus pinched in more
• "Horseshoe shaped"
• Percent in BM: 10-35%
• N:C ~1:1
• Starting to look a lot more like neutrophil
• If the nucelus is more than halfway in, it is a band neutrophil, if it is less than halfway in it is a metamyelocyte. If you are unsure, always err on the side of mature

1. Myeloblast
   
2. Promyelocyte
   
3. Myelocyte
   
4. Metamyelocyte
   
5. Band Cell
   
6. Mature form (neutrophil/eosinophil/basophil)
   

Front 68

Is this a band neutrophil or a metamyelocyte?

Back 68

• Metamyelocyte
• Nucleus is less than half-way in

Front 69

Mature Form (Neutrophil/Eosinophil/Basophil)

Back 69

Basophil

• Granules dark blue (almost blackish/purple)
  
• Segmented nucleus - usually can't see
• Large granules
• Circulate in the peripheral blood (PB) and move to the tissue
• Tissue Basophils: Also called Mast cells. Have a structure & function similar to blood basophils. Note the shape of the nucleus

Eosinophils

• Granules more coarse than neutrophils, but less coarse and smaller than basophils
• Distinct orange/pinkish granules
• Tissue Eosinophils - have the granules typical of eosinophils & have a nucleus that is generally not segmented. Don't have a fancy name like mast cells, just called tissue eosinophils

Neutrophils

• Fine granules
• Pinkish Salmon colour
• Strands connecting lobes - chromatin
• 2-5 nuclear lobes (3 is the most common number). If they have more or less than 2-5 nuclear lobes they have special names
• Tissue Neutrophils are large marrow cells with lots of cytoplasm. They have irregular, blunt pseudopods, and are easily indented by surrounding cells. They are not phagocytic in tissue, and are infrequent in normal marrow. Don't see in peripheral blood.

1. Myeloblast
   
2. Promyelocyte
   
3. Myelocyte
   
4. Metamyelocyte
   
5. Band Cell
   
6. Mature form (neutrophil/eosinophil/basophil)
   

Front 70

Tissue Migration & Diapedesis

Back 70

• 50% of granulocytes in functional pool & 50% in marginating pool

• Cells migrate to tissue - diapedesis

• Chemoattractants/chemotaxis cause diapedesis

• Neutrophils last ~7 hours in the tissue

• Eosinophils last ~12 days in tissue

• Basophils last a few hours in tissue

Front 71

Back 71

Myelopoiesis

• Myeloblast -> mature form
  
• Typically don't want to see all of these stages in the patients blood. Could indicate leukemia or other diseases.
• If we see pros/blasts in blood this is BAD
• If we have a lot of immature leukemic cells, they tend to stick/clump together

Should we see it in blood?

• Myeloblast - no
• Promyelocyte - no
• Myelocyte - don't want to see, but may if person has bad infection
• Metamyelocyte - don't want to see, but may if person has bad infection
• Band Cells - See normally in low numbers, when numbers increase, it means person is trying to fight something

Front 72

Monocytic Cell Line (Monopoiesis) & Stages

Back 72

• Still part of myeloid lineage
• Exception #1 to our common characteristics rule - the size stays relatively the same throughout the maturation process
• N:C does decrease

Stages

1. Monoblast
2. Promonocyte
3. Monocyte
4. Macrophage

Front 73

Monoblast

Back 73

• N:C ~4:1
• Cytoplasm very basophilic - non-granular
• Monoblast & myeloblast (1st stage of myelopoiesis) look very similar. Won't have to distinguish, just know it is a blast

Has a nucleoli

1. Monoblast
   
2. Promonocyte
   
3. Monocyte
   
4. Macrophage
   

Front 74

Promonocyte

Back 74

• Indented or folded nucleus
• Nucleoli
• N:C ~4:1 to 2:1
• Not easy to ID

1. Monoblast
   
2. Promonocyte
   
3. Monocyte
   
4. Macrophage
   

Front 75

Monocyte

Back 75

• N:C ~2 or 1:1
• Irregular nucleus
• Big phagocytes - see lots of vacuoles
• Fairly easy to ID
• Large irregular nucleus
• Larges normal cell in peripheral blood

1. Monoblast
   
2. Promonocyte
   
3. Monocyte
   
4. Macrophage
   

Front 76

Macrophage

Back 76

• Transformation (like continuation of cell line) to macrophage state in tissue
• Last 12-14 hours in circulation as monocytes
• Can last years in tissue as macrophages
• Image on left - carbon particles in macrophage from smoke/pollution

1. Monoblast
   
2. Promonocyte
   
3. Monocyte
   
4. Macrophage

Front 77

Lymphocytic Cell Line (Lymphopoiesis)

Back 77

• Follows the common maturation characteristics

The Stages:

1. Lymphoblast
2. Prolymphocyte
3. Lymphocyte

Front 78

Lymphoblasts

Back 78

• Usually have a large round nucleus - this helps to ID
• Chromatin is thin, loose, and evenly stained
• N:C ratio ~7:1 to 4:1
• Look a lot like myeloblasts/monoblasts. Just be able to ID as a blast

The Stages:

1. Lymphoblast
   
2. Prolymphocyte
   
3. Lymphocyte
   

Front 79

Prolymphocyte/Lymphocyte

Back 79

• Mature form comes in different sizes (small, medium, large)
• N:C ratio will vary
• Reported as lymphocytes only (can't distinguish as B or T)
• Population varies with age
• Xerophilic granules - reddish staining, sometimes seen

Front 80

Plasma Cells

Back 80

• End of the B lymph lineage
• In peripheral blood? NO - would be bad
• Perinuclear zone (cytoplasmic region just around the nucleus)
• Secretory vesicles - periphery
• May have 1 or more vacuoles

Front 81

Megakaryocytic Cell Line (Megakaryocytopoiesis)

Back 81

• AKA called Thrombopoiesis or Megakaryopoiesis
• Break the rules! Cells get larger as they mature
• Nucleus gets larger & multilobulated
• Cytoplasm becomes granular

Stages:

1. Megakaryoblast
2. Promegakaryocyte
3. Megakaryocyte
4. (budding megakaryocyte) - not a true stage
5. Platelet

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Megakaryoblast

Back 82

• Very large
• One cell can make millions of platelets
• Very little cytoplasm (very basophilic)
• Round or slightly oval nucleus
• N:C ~5:1 to 3:1
  
  

Stages:

1. Megakaryoblast
   
2. Promegakaryocyte
   
3. Megakaryocyte
   
4. (Budding Megakaryocyte)
   
5. Platelet
   

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Promegakaryocyte

Back 83

• More cytoplasm & more granular
• Note size - getting larger
• Has lobes
• N:C ~3:1 to 1:1

Stages:

1. Megakaryoblast
   
2. Promegakaryocyte
   
3. Megakaryocyte
   
4. (budding megakaryocyte)
   
5. Platelet
   

Front 84

Megakaryocyte

Back 84

• Lots of cytoplasm
  
• Lots of granules
• N:C ~1:1 to 1:2
• Endomitosis - this is when it starts to "explode" and we get platelets
• Start to get segmenting nucleus

Stages:

1. Megakaryoblast
   
2. Promegakaryocyte
   
3. Megakaryocyte
   
4. (budding megakaryocyte)
   
5. Platelet
   

Front 85

Budding Megakaryocyte

Back 85

• Not really distinguished as a separate stage

• Membrane ruptures

• Cytoplasm fragments

• Start to get platelets

Stages:

1. Megakaryoblast
   
2. Promegakaryocyte
   
3. Megakaryocyte
   
4. (budding megakaryocyte)
   
5. Platelet
   

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Platelet

Back 86

• Not cells - cytoplasm fragments
• Migrate to peripheral blood
• Lifespan of 9-12 days

Stages:

1. Megakaryoblast
   
2. Promegakaryocyte
   
3. Megakaryocyte
   
4. (budding megakaryocyte)
   
5. Platelet
   

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Back 87

All the phases together

Front 88

Smear Technique

Back 88

Factors affecting good wedge smears

• Speed of spreading - if it is too slow we get poor leukocyte distribution. Quicker and faster gives you thicker and longer smear.
• Angle of spreader slide - affects blood viscosity. Lower angle = thicker and shorter, higher angle = thinner and longer
• Size of the blood drop - too bigs = too much blood, too small = no matter our angle it won't be enough

Front 89

Smear Errors

Back 89

• Scrapy at end - caused by spreader slide with chip or jagged edge
• Ridges - caused by unequal pressure & unsteady hands
• Short - caused by spreading too quickly
• Really short - didn't use enough blood
• Thin smear - caused by not waiting long enough for blood to spread on slide before starting to spread
• Holes - Debris on slides. This could be caused by handling slides. Should only touch frosted part. Can also be caused be very lipemic blood.
• Uneven at end of smear - caused by uneven pressure
• Streak down middle - waiting too long after putting blood on slide before spreading

Front 90

3 Main Elements of a Manual Differential

Back 90

• Erythrocytes - Morphology
• Leukocytes - 100 cell diff. (some places do 200 cell diff.)
• Platelets - Estimates and morphology (make sure that your estimate matches the machines estimate)

Front 91

Absolute Count vs. Relative Count

Back 91

Relative Count

• The number of each type of cell you counted in 100 WBCs
• Always in decimal form (ex. if you counted 30, report as 0.30)
• All of the relative counts (in decimal form) for each of the cell types we count should add up to 1.

Absolute Count

• Takes into account the patients total WBC count
• Be sure you have appropriate units (usually x10^9/L)
• Absolute count = Relative Count x WBC Count (from CBC)
• All of the absolute counts should add up to equal the total WBC count

Ex. If you counted 35 lymphocytes in your manual differential and the patient had a total white count of 6.5x10^9/L what is the absolute count?

0.35 x 6.5 = 2.3 x10^9/L

Machines count the 100 cells (usually count more than 100) and calculate the absolute values for us.

Front 92

What are the steps in doing a Diff?

Back 92

Low Power Scan (10x)

• Check the quality of the stain, smear, and distribution of the cells/clumping
• Gives us an overall picture and allows us to assess the smear
• Look for cell distribution and platelet clumping
• Look at sides and feathered edge to observe for malignant cells

WBC Estimate (40x)

• Manual estimate - compare to the value the analyzer got
• WBC estimate = (average # of WBCs in 10 different fields) x 2

High Power (100x)

• At 100x we do the differential, the platelet estimate, and look at RBC morphology
  
• When doing a manual differential the platelet count is estimated and compared to the machine count.
• When doing a platelet estimate we not any unusual platelets such as giant platelets or abnormal forms
• Platelet estimate = (average number of platelets in 10 diff. fields) x 20
  OR
• Plt estimate = (total # of plts in 10 fields) x 2
• 
  

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Back 93

• Arrows are pointing towards normal platelets

Front 94

Back 94

Normal Neutrophil

• 3 segments with thin filaments between them
• Should only see 3-5 lobes
• Slightly pink cytoplasm
• Can see fine granulation in nucleus

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Back 95

Band Cell

• Less mature form of a neutrophil

Neutrophil

• Can't see the filaments on this neutrophil (folded in on self)
• If unsure of ID always lean on the side of more mature
• Neutrophils can phagocytose bacteria

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Back 96

Band Cell

• Very thick
• Not segmented

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Back 97

Picture on the front = Eosinophil

Image on left = Eosinophil

Image on right = neutrophil

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Back 98

Basophil

• Very large granules (can see individual granules)
• Granules are water soluble (can wash out while staining)
• Granules are unevenly distributed

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Back 99

Lymphocyte

• Often see xerophilic granules
• Cytoplasm is sky blue but not cloudy
• Small and large variety (small on front, large on this side)

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Back 100

Monocytes

• Fair amount of cytoplasm
• "Twisted"/convoluted cytoplasm
• Cytoplasm lighter coloured/"lacy"
  
• Can't see granules but looks grainy ("ground glass")
• Very large, especially compared to the red blood cells

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Back 101

Cell on left = lymphocyte

Cell on right = monocyte

Large lymphocytes & monocytes look very similar

Front 102

Large Lymphocytes vs. Monocytes

Back 102

Large Lymphocytes

• Nucleus is oval, round, indented/stretched. It stains a deep purplish-blue colour. It has clumped chromatin particularly at periphery, and is is very dense
• The cytoplasm of large lymphs is sky blue. It has a clear and nongranular background. Azurophilic granules are prominent and easy to distinguish if present.

Monocytes

• The nucleus of monocytes is oval, round, indented or convoluted. It stains a pale purplish blue, and has fine, lacy, spongy chromatin.
• The cytoplasm of monocytes is more of a blue grey colour, and is more cloudy/opaque. It has a "ground glass appearance" (fine granules). Frequently vacuoles can be seen. Any azurophilic granules will be hard to distinguish.

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Back 103

Monocyte

• Note the vacuoles
• Indented/convoluted nucleus
• Pale purple nucleus
• bluish-grey cytoplasm

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Back 104

Band Cell

• Compared to monocyte has darker nucleus
• More obvious parachromatin (unstaining whiter area)
• Cytoplasm more of a pinkish colour than the bluish grey of monocyte cytoplasm

The blueish looking cell is a giant platelet

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Back 105

Basophil

Front 106

Back 106

Eosinophil

• Sometimes they explode when we make the smear - delicate cell

Front 107

Back 107

Platelets clumping

• If we have platelet clumping typically see a lot of clumping throughout the smear

Front 108

EDTA Sample

Back 108

• EDTA can only be kept at room temperature for 5 hours max.
  
• If it is kept at room temperature for more than 5 hours we can get blood cell artifacts
• EDTA prevents platelets from clumping on glass slide
• Some circumstances require different anticoagulant such as platelet satellitosis & EDTA induced Platelet Clumping

Front 109

Platelet Satellitosis

Back 109

• Condition that patient has that causes their platelets to adhere to neutrophils
• Doesn't normally cause problems in patient, just makes it hard to do a platelet estimate

Front 110

EDTA Induced Platelet Clumping

Back 110

• Some patients have platelets that clump in EDTA
• Can cause erroneously high white cell count & bad platelet estimate
• Retake blood with sodium citrate to prevent this

Front 111

How would we correct for platelet clumping or platelet satellitosis?

Back 111

We can correct for clumping or satellitosis by:

• Recollecting the sample, but this time in Sodium Citrate
• For the WBC and platelet count we use the recollected citrate sample
• For the other CBC parameters we use the original EDTA sample that was collected

Front 112

Back 112

Platelet Satellitosis

Front 113

Finger or Heel Sample

Back 113

• If we can't get blood sometimes we do finger or heel punctures
• Some platelet clumping may occur with these samples
• EDTA microtainers are used to collect
• Slides are usuaqwlly made right at the bedside. The quicker we get it done, the better

Front 114

RBC Structure

Back 114

Biconcave Disk

• Natural shape
• Provides flexibility
• Increases surface area - more efficient for gas exchange
• Normocytic RBC = RBC that is normal in size
• Normochromic RBC = RBC that is normal in hemoglobin content

Front 115

3 Parts of RBC

Back 115

3 Parts of RBC

• Membrane
  
• Internal Stroma
  
• Cell Contents
  

*All 3 of these parts are crucial to normal RBC survival and function

Front 116

RBC Membrane

Back 116

RBC Membrane

• Intact & flexible (deformable)
• Mature RBCs don't have a nucleus. This has benefits, but also drawbacks (no cellular machinery therefore no limited ability to repair self)
• Macrophages clean up senescent RBCs
• Has a phospholipid bilayer
• Semipermeable
• Integral proteins embedded
• Fluid mosaic
• *54% proteins, 40% lipid, 8% carbs* (Know these ratios)

Front 117

Normal Cells vs. Sickle Cells

Back 117

-Normal red cells on top

-Sickled cells on the bottom

• Sickled cells do not live as long - spleen removes them, it's job is to remove deformed cells
• Can cause clogging of arteries/veins

Front 118

RBC Membrane: Lipid Layer - 2 main groups of phospholipids/2 lipid layers

Back 118

External Layer - Choline Phospholipids

• Choline phospholipids are primarily on the external lipid layer - readily accessible to the external environment
• Because of their outward arrangement they may represent controlling points in the major pathways of lipid renewal, because there is an exchange between plasma fatty acids and RBC membrane
• 2 main choline phospholipids are phosphatidyl choline & sphingomyelin

Internal Layer - Amino Phospholipids

• Aminophospholipids are primarily on the internal surface of the membrane
• 3 main aminophospholipids are: phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol
• The precise arrangement of these is important
• If you find aminophospholipids on the outside of the cell - not good - can start coagulation cascade

*The asymetrical arrangement of these lipids (aminophospholipids & choline phospholipids) allows selective movement in and out of the membrane

Front 119

Other Lipids in the RBC Membrane

Back 119

Glycolipids

• Most in outer layer
• Important interactions with glycoproteins

Cholesterol

• On both sides of the bilayer
• ~25% of membrane lipids
• 1:1 ratio with phospholipids
• Constant exchange between plasma and membrane cholesterol - this exchange is affected by changes in overall body lipids and lipid transport

Front 120

When Membrane Cholesterol is altered what do we see?

Back 120

Too Much Cholesterol

• Caused by lipid imbalance
• Membranes containing excess cholesterol are more viscous and less fluidic. These leads to a decrease in deformability and RBC survivability
  
• Altered membrane structure - causes target cells and acanthocytes
  

Front 121

Target Cells

Back 121

• Accumulation of too much membrane cholesterol

• Usually very serious issue

• Left is normal cells, right is target cells

• Decreased survival rate because excess lipid makes the cell less deformable

Front 122

Acanthocytes

Back 122

• Also caused by too much cholesterol
• Irregular projections (spicules)
• Increased rigidity
• Decreased motility
• Decreased survival rate because excess lipid makes the cell less deformable
• Clog up in arteries
• Get destroyed by spleen - can function normally, but the spleen functions to remove damaged cells so it wants to remove them.
• Don't mix up with crenated cells - spikes are more regular and evenly spaced in crenated cells than we see in these cells.

Front 123

Two Main Types of RBC Membrane Proteins

Back 123

Integral Proteins

• Span lipid bilayer and are exposed on the surface
• Most carry red cell antigens and are receptors or transport proteins
• Glycophorins are the main RBC glycoprotiens (~20% of the total membrane protein)
• Glyophorin molecules contains ~60% carbohydrate
• Glyocophorin accounts for most of the membrane sialic acid which gives the red cells a slight (-) charge. This helps them repel each other so that they can circulate easier.

Peripheral Proteins

• Important for the integrity of the red blood cell
• Form a lattice network
• 3 important peripheral proteins (*that we need to know*) are Spectrin, Actin, and Ankyrin
• Spectrin - main peripheral protein. Needs to undergo phosphorylation to do its job properly.
• Actin - forms lattice network with spectrin
• These 3 proteins are also called Band 4.1, 4.6, and 4.9 (named for where they form bands in electrophoresis)
• Adducin & Tropomyocin are also 2 peripheral membrane proteins

Front 124

Deformability

Back 124

RBC Deformability is critical for:

• RBC survival as the cell travels through the microvasculature
• Oxygen delivery

Things that affect deformability:

• Loss of ATP (energy) levels leads to a decrease in the phosphorylation of spectrin.
  
• This leads to a loss of membrane deformability, and increase in membrane rigidity

• Increase in deposition of membrane calcium also results, causing an increase in membrane rigidity and loss of membrane pliability

• When cells that have decreased deformability pass through the spleen they may be damaged or removed - sinusoidal orifaces of spleen designed to remove aged, damaged, and less deformable RBCs.

Front 125

Spherocytes & Helmet cells

Back 125

• Spherocytes - cells with reduced surface to volume ratio, No central pallor, dark/deep staining. Spleen or macrophage tried to destroy RBC but it survived & repaired self forming a spherocyte.
  
• "Bite Cells" - cells in which the removal of a portion of membrane has left a permanent indentation in the remaining cell membrane. RBCs have very limited ability to repair self.
  
• Survival time of these forms of cells is shortened

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Back 126

Spherocyte

Front 127

Back 127

Bite Cell/Helmet Cell

Front 128

RBC Membrane Permeabillity

Back 128

• Freely permeable to water and anions
• Relatively impermeable to cations
• The permeability of the RBC helps with water volume and homeostasis

Front 129

Isotonic Solution

Back 129

• Cells in solution with a [solute] similar to the [solute] inside the cell
• No net flow of water either way

Front 130

Hypertonic solution

Back 130

• Cells placed in a solution with a [solute] greater than the [solute] inside the cell (ex. concentrated salt solution)
  
• Water flows out of the cell
• Cells shrink
• Crenation

Front 131

Hypotonic Solution

Back 131

• Cells placed in a solution with a [solute] less than the [solute] inside the cell (ex. distilled water)

• Water flows into the cell

• Cells swell and burst

Front 132

Membrane integrity depends on:

Back 132

Membrane integrity depends on:

• ATP
• Intra and extracellular concentration
• Proteins
• Lipids, phospholipids & cholesterol

Front 133

Back 133

• Cells on left are normal RBCs - normal RBCs are mostly hemoglobin
  
• The cells on the right are hypochromic - they have very little hemoglobin
• The larger the central pallor, the less hemoglobin is in the cell

Front 134

Blasts

Back 134

• Earliest morphologically recognizable WBC precursors
• Myeloblast = committed to produce granulocyte
• Lymphoblast = committed to produce lymphocytes
• We expect to see very little cytoplasm & a very dark staining nucleus, possibly with a paler staining region (nucleolus)
• If we see a nucleolus it is a hint we are looking at something immature
• Nucleus has fine chromatin pattern (not coarse and condensed like in mature cells)

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Back 135

Myeloblast

• Generally round or ovoid
• Generally round or ovoid nucleus
• Variable size (10-20 um)
• Nucleus may be indented slightly
• Chromatin is finely distributed with distinct parachromatin
• One or more nucleoli may be seen
• Cytoplasm is blue and there is a scant to moderate amount
• N:C ratio is 7:1 to 5:1
• Often have no granules, but some blasts (type II) may contain a few distinct azurophilic granules)
• May see Auer rods (clumps of azurophilic granular material that form elongated needles seen in the cytoplasm of leukemic blasts)
• If found in peripheral blood could mean indicate many diseases: AML, MDS, CMPD (may), G-CSF, GM-CSF therapy, Leukamoid reactions

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Back 136

Promyelocyte

• Round or ovoid with round or ovoid nucleus
• Often larger than blast (12-24 um)
• N:C ratio 5:1 to 3:1 (don't have a lot of cytoplasm, and the cytoplasm that is there is still dark staining)
• Fine dispersed chromatin with obvious parachromatin
• One or more nucleoli
• Can't distinguish baso/eo/neutro
• Start to see primary azurophilic granules in cytoplasm... usually overlie the nucleus too
• If found in peripheral blood could mean indicate many diseases: AML, MDS, CMPD (may), G-CSF, GM-CSF therapy, Leukamoid reactions (same as blast)
  

Front 137

Back 137

Myelocyte

• 10-20 um
• Round or ovoid with round or ovoid nucleus
• Nucleus may be slightly indented or flattened on one side & may be central or eccentrc
• Variable amount of chromatin clumping
• In a large myelocyte, may see one nucleoli, but generally will not see nucleoli
• Cytoplasm is abundant, bluish-pink or clear, and often a few coarse primary azurophilic granules, and numerous secondary granules are present
• Could see these in blood if a patient has a severe infection without it being too alarming.
• Cytoplasm starts to take on a pinkish hue because of primary granules coming in
• Can distinguish between neutro/eo/baso (but usually report as eo/baso and make a note that some immatures were noted. We mostly just worry about reporting immature neutropihils because there are so few baso/eo's in the blood anyways
• If Found in the peripheral blood, indicative of these diseases: LR, CMPD, Myelodysplasia, same diseases as mentioned previously for blasts (AML, MDS, CMPD (may), G-CSF, GM-CSF therapy, Leukamoid reactions).

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Back 138

Metamyelocyte

• Indented a little but not enough to be a band (Indentation is less than half-way in)
• Round to oval shaped
• Variable sized (10-18 um)
• Usually indented or kidney shaped nucleus
• Chromatin clumped
• No nucleolus
• Lots of pink to bluish-pink cytoplasm with lilac coloured secondary graules
• A few course azurophilic granules may be seen
• If we see eo/baso versions just call eo/baso and note on req that we saw some immature forms
• If found in peripheral blood: left shift of nucleus, LR, CMPD, and rest the same as myelos

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Back 139

Band Cells

• Round to ovoid
• 10-20 um in diameter
• Nucleus even further indented - shape of band or sausage
• Chromatin even more coarse and clumped and then meta
• No nucleolus
• Abundant clear or pink staining cytoplasm - filled with secondary granules
• May see a few azurophilic granules

IF found in Peripheral blood

• Small amount in RB is normal

If increased in PB

• Left shift
• Inflammation, infection
• LR, CMPD, AML, MDS, CMPD (may), G-CSF, GM-CSF therapy, Leukamoid reactions

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Back 140

Lymphoblasts

• Round or ovoid
• 9-20 um
• Round or ovoid nucleus - usually centrally placed
• Nucleus may be slightly indented, clefted, folded, or irregular
• Chromatin is coarsely granular with distinct parachromatin
• 1 or more nuclei can be seen
• Scant to moderate cytoplasm, stains blue, usually no granules (rarely few pink azurophilic)
• N:C 6:1 to 4:1

If found in PB

• ALL
• Lymphoid blast crisis in CML
• May see occasional one in newborn, especially if premature

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Back 141

Prolymphocytes

• Round or ovoid
• 12-20 um
• Nucleus usually round or ovoid and centrally placed
• Chromatin condensed - absent or indistinct parachromatin
• Typically single, prominent nucleolus
• Moderate amount of blue cytoplasm
• N:C 5:1 to 3:1
• Difficult to ID

If found in PB

• CLL
• Prolymphatic transformation of CLL
• PLL

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Back 142

Monoblasts

• Round or ovoid
• 12-24 um
• Round, ovoid or slightly indented nucleus with one or more nucleoli
• Fine chromatin
• Abundant grey cytoplasm - may see pink azurophilic granules - often see vacuoles
• N:C 6:1 to 4:1

When found in PB

• Acute monoblastic leukemia
• Acute monocytic leukemia
• Acute myelomonocytic leukemia
• CML and other chronic myeloproliferative disorders

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Back 143

Promonocytes

• Morphological features between monocyte & monoblast
• May see a nucleolus
• Chromatin is coarsely or finely granular
• Abundant grey-blue cytoplasm
• May see azurophilic granules