hemii chapter 14 part 2 spring 2016..huda

7/24/2019 HemII Chapter 14 Part 2 Spring 2016..Huda

1/56

Leukocytes: The Granulocytic and

Monocytic Series

Part II


2/56

Learning objectives:

Upon completion of this lecture series the learner should be

able to competently discuss:The differentiation and maturation of the granulocyte cellseries in detail

The differentiation and maturation of the monocyte-macrophage cell series in detail

The function of granulocytes and monocytes

Methods of assessment of granulocytes and monocytes in aclinical setting (absolute counts, differential counts, morphology,staining patterns, etc.)

Chapter 14The Leukocytes (part 2)

(monocytes and lymphocytes)


3/56


4/56

Monocyte-macrophage cell series

Types of Macrophages: the name depending of the tissue

where they are located

1. Histiocytes in connective tissue

2. Kupffer Cells in liver

3. Osteoclastsin bone

4. Microglialin nervous system

These cells plus the reticular cells of primary and secondary

lymphoid organs (thymus, spleen, etc.)mononuclear

phagocytes (Mononuclear cells are distributed throughout the

body)

PMNs as the major phagocytes (segmented neutrophils) remain in

circulation unless recruited to sites of injury / infection.


5/56

Maturation of monocytes

Lineage: CFU-GMCFU-Mmonoblastpromonocytemonocyte

2-3 mitoticdivisions in 2 days

(to bring number of differentiating cells up)

This developmental pathway in BM occurs under the influence of severalgrowth factors; chief among them is the M-CSF

Monocytes are mostly released into circulation within 12-14 hrs after

maturation in BMNo large reserve of cells in BM maturation/storingcompartment as do the granulocytes

In circulation, monocytes locate into circulation & marginating pool @ a

ratio of 1:3.5


6/56

Maturation of monocytes

Monoblast:

(morphologically indistinguishable

from myeloblast by light microscopy

Except for highly experienced hematologist )

Size: 14 to 20 m

N/C ratio: High

Nucleus: Oval, round, or folded

Chromatin: finely dispersed not clumped

(lacy-like), light blue purple

Nucleoli: several

Cytoplasm: agranular, blue gray.


7/56


8/56

Promonocyte

Size: 12 to 20 m

N/C ratio: Moderate (N in size)

Nucleus: Irregular and folded

Chromatin: Coarser thanmonoblast

Nucleoli: May be present

Cytoplasm: abundant, blue gray.

Granules: Azurophilic may

present


9/56

Monocyte

Size: 12 to 20 m (Largest mature cells in

the periphery)

Nucleus: Folded, horseshoe,

bean-shaped

Chromatin: Loose and linear,

lacy pattern when compared with

granulocytes

Nucleoli: rarely are present

Cytoplasm: blue gray, ground glass

appearance, ; exhibit irregular outline showing

pseudopodes and vacuoles

Granules: Fine, dust-like and membrane-bound granules


10/56


11/56


12/56


13/56

Monoblast: Moderate amount of gray-blue cytoplasm. The

centrally located nucleus is round with a fine chromatin

pattern.


14/56

Promonocyte: Abundant gray blue cytoplasm with mildly irregular outline & few

granules, minor vaculation, nucleus is folded and clumped.


15/56

Monocyte: Abundant vacuolated gray blue cytoplasm with irregular outline and few

granules. Nucleus is kidney beanshaped and clumped.


16/56

Summary of granulocyte & monocyte functions

Granulocytes and monocytes represent a significant part of the

innate arm of the immune system participate in variousimmune mechanisms

Eosinophils

Primary function is against helminthes (parasites). Eosinophilia may occur in allergic conditions and with

parasitic infections, and with chronic inflammation.

Basophils:

Mediators of the inflammatory response, particularly in

hypersensitivity reactionsRelease of inflammatory

mediators (histamine) during allergic reactions is mediated

by basophils and mast cells (in the presence of IgE)


17/56

Mature neutrophils (phagocytes)

Express cell surface markers that correlate with their functional state:

Receptors for adhesion: CD11b, CD62L, CD49d, CD54

IgG receptors {FcRI(CD64), FcRIIA(CD32)and FcRIII(CD16) } CD32 (down-regulates IgG production by B cells)

Anaphylatoxin (C3a, C4a and C5a) receptors eg CD88(C5a

peptide)

TLRs

Macrophages (phagocytes)

Variably express CD14, CD16, CD45, IgG receptors, TLRs, etc.

different functional subsets. Example:

CD14+CD16-= classical monocytesexcellent

phagocytes but poor producers of inflammatorycytokines

CD14lowCD16+= nonclassical monocytesgood APCs
https://en.wikipedia.org/wiki/CD32https://en.wikipedia.org/wiki/CD32https://en.wikipedia.org/wiki/CD32


18/56

Summary of granulocyte & monocyte functions (cont.)

Neutrophils and macrophages carry out phagocytosisas well

as antigen processing and presentation (so as to engage

adaptive immunity)

Dendritic cells (DCs), which arise from monocytes andexpress multiple TLRs and T cell co-stimulatory molecules

like CD80/CD86, CD40 etc., are excellent APCs especially to

Th cells.

Release of inflammatory mediators (cytokines): neutrophils,

monocytes/macrophages, & DCs


19/56

The acute inflammatory response

Injury/ infectionpathogen-related antigens (e.g. LPS,

proteins, etc.) trigger complement activation and releaseof inflammatory mediators (cytokines like TNF-& IL-

1, IL-8, etc.)

Activation of complement

release of C3b (coats pathogen to initiate MAC

formation and/or to facilitate phagocytosis)

release of chemo-attractants like C5b.

C3b, C4a and C5b trigger degranulationof mast cells

and release of histaminewhich along with TF and other

mediators induce muscle contraction and vasodilatation


20/56

Pathogen-related antigens plus mediators up-regulate

expression of homing markers like sialyl-Lewisx on

neutrophils on in circulation.

P-selectin+ (CD62P+) on vascular endothelium cells

recognize and bind sialyl-Lewisx neutrophils arrest at

site of infection.

Neutrophils up-regulate expression of certain integrins(e.g. CD11b) on their surface and bind with VCAM,

ICAM on vascular endothelium cells further adhere to

vascular endothelium and

Neutrophils exit circulation by diapedesis through P-

CAM on endothelial cells and neutrophils.

On site, neutrophils start phagocytosing (eliminating)

C3b-coated pathogens.


21/56


22/56


23/56


24/56

Phagocytosis


25/56

Di ti


26/56

Digestion:

1)The action of the oxygen-dependent myeloperoxidase-mediated system,

hydrogen peroxide, and an oxidizable cofactor serve as major factors in the actual

killing of bacteria within the vacuole.

2) Other oxygen-independent systems, such as alterations in pH, lysozymes,

lactoferrin, and the granular cationic proteins, also participate in the bactericidal

process.


27/56

Antigen processing and presentation


28/56

Methods of Assessment forleukocytes

Normally only mature cells are released into the peripheral blood.Mature cells may also remain stored in BM.

Increase / decrease in WBC count may be caused by an alteration

of all WBC cell lines, but more commonly results from an

alteration of only one type of WBC Hence, a differential countis important.

From the differential and the WBC count, the absolute values for

each type of WBC can be calculated (relative differential (%) x

total WBC count).

Most variations in the leukocyte count are due to increases or

decreases in the number of neutrophils since, by percentage,

they are the most numerous.


29/56

Reference ranges of granulocytes and monocytes

Total number and range vary with age, gender, and raceconsult

appropriate ref. values for interpretation of results.

Examples of WBC value shifting:

Blackshave lower total leukocyte count as average neutrophilcounts are lower than in whites

Smoking slightly elevates percentage of neutrophils

Eosinophils exhibit hormone-related daily fluctuations

(diurnal) (high at night\sleeping time).

Count and distribution also varies with health status of the

host; most significant in clinical practice.


30/56

Generally, leukocyte concentration in blood is as follows :

1. Total WBC count

At birth: 9-30 X 109/L

In children (average): 8 X 109/L

In adults: 3.5-11.0 X 109/L

2. Differential count (adults)

Segmented neutrophils: 40-75%

Neutrophils-bands: 0-3%

Eosinophils: 1-6% Basophils:0.5-1% (rarest type)

Lymphocytes: 25-40% (up to 60% in children)

Monocytes: 2-8%


31/56

Fl i i l L k C


32/56

Fluctuations in total Leukocyte Count

Increases:

Acute inflammation

Pregnancy

Strenuous exercise

Decreases:

Sepsis (infection-related systemic inflammation) causessignificant tissue damage)

Immunosuppressive agents (steroids, antineoplastic agents, HRT,etc)

I. Absolute Cell Value =

Total Leukocyte count X Percentage of Cell Type

Example: Patient Data

Total Leukocyte Count = 15.0 X 109/L

Differential blood smear results: Bands: 12%, segmented neutrophils 80%,

lymphocytes 8%?


33/56

II. Differential Blood Smear Evaluation

Observe abnormal cells

Observe percentage of each type ofleukocytes

Observe presence of band forms; normal

range of band forms in adults: 0-3% Observe Shift to the left: increased band forms

with the presence of metamyelocyte and

myelocytes.


34/56

Shift to Left in granulocytes = increased percentage of immature/total

leukocytes in cases of infection , inflammation, cancers and certain types

of leukemia, etc.


35/56

III. Assessment of Eosinophils and Basophiles

When % of eosinophils increase, absolute count is recommended

because:

Eosinophil count rises during parasitic worm infections

(roundworm, hookworm), rheumatoid arthritis, inflammatory

bowel disease, autoimmune diseases, .

Eosinophils may increase due to Hodgkins lymphoma, lung

and stomach cancer; any neoplams (cancer) or leukemias

can increase the eosinophil count.

Allergies (hay fever, asthma, ..) can increase eosinophils.

Basophiles are the least numerous of granulocytes in circulation.

Normally, 1-2 basophiles are seen on differential. Increased % is

seen in conditions such as chronic myelogenous leukemia and

polycythemia vera.


36/56

IV. Leukocyte Alkaline Phosphatase Test:

This special test, which measures the amount of the ALP

in test cells, is performed to differentiate malignant

disorders [e.g. chronic granulocytic leukemia] from the

leukemoid reaction [leukocytosis exceeding 50,000

WBC/mm3with a significant increase in early

neutrophil precursors] as seen in severe infections.

* High readings = leukomoid reaction


37/56

LAP is found within white blood cells, WBC levels ofLAP can help in the diagnosis of certain conditions.

Higher levels: are seen in polycythemia vera (PV),

essential thrombocytosis (ET), primary myelofibrosis(PM), and the leukemoid reaction.

Lower levels : are found in chronic myelogenous

leukemia(CML), paroxysmal nocturnal

hemoglobinuria (PNH) and acute myelogenous

leukaemia (AML).

LAP Test

N t hili H t ti


38/56

Neutrophilic Hypersegmentation

Index

Mature segmented neutrophils have from two to five nuclear

lobes (segments).

Counting the number of lobes can be performed to determine

the neutrophilic hypersegmentation index (NHI). The NHI is clinically useful in vitamin B12 deficiency (pernicious

anemia) and folic acid diagnosis.


39/56

1. Lobe average: This is determined by counting the number

of lobes in a number of neutrophils. The reference value is 2.5 to 3.3.

2. Percentage of neutrophils with five or more lobes to the totalneutrophils.

3. Hypersegmentation index. To calculate this index,

Number of neutrophils with 5 or more lobes 100

Number of neutrophils with 4 lobes

Values greater than 16.9are considered to indicate

hypersegmentation.

This method is considered to be the most sensitive method.


40/56


41/56

WBCs

Histogram


42/56

WBCsHistogra3-PartDifferentiation

WBC Hi t

LYSE R t ff t WBC


43/56

WBCs Histogram

0 m

3-PartDifferentiation

2 4 6 8 10 12 14 16 18 20 22

Lymphocytes

Monocytes

Eosinophils

BasophilsNeutrophils

Before adding LyseReagent

12 20

712

11 16

9 1410

15

Cell diameter

in m

LYSEReagent effect on WBCs

WBC Hi tLYSE ff


44/56

WBCs Histogram

100200 300

fL


After adding LyseReagent


Lymph

Lyse reagent causes

WBCs to shrink to a

defined volume

according to their

cell type.

WBCs are presented as

3 separate populations

in the histogram.

WBC Hi tLYSE R ff WBC


45/56

WBCs Histogram

100200 300

fL

3-PartDifferentiationAfter adding LyseReagent


Lymph

Mono

Eosino

Baso

Lyse reagent causes

WBCs to shrink to a

defined volume

according to their

cell type.


3 separate populationsin the histogram.

WBCs HistogramLYSE R t ff t WBC


46/56

WBCs Histogram

100200 300

fL




Lyse reagent causes

WBCs to shrink to a

defined volume

according to their

cell type.Lymph

Mono

Eosino

Baso

Neutrophils


3 separate populationsin the histogram.

WBCs HistogramLYSE Reagent effect on WBCs


47/56

WBCs Histogram

100200 300

fL




Lymph

Mono

Eosino

Baso

Neutrophils

Cells will be shown in a histogram according to their size.

Lymphocytes

Monocytes

Eosinophils

Basophils

Neutrophils120 250fL

80 140fL

70 130fL

60 120fL

3080fL

WBCs Hi tWBCs counted between


48/56

WBCs Histogram

100 200 300 fL

LD


WL

=LD Lower Discriminator

WL WBCsLower discriminator

[ Flexible] fluctuates between 30

60 fL

2outer

discriminators

WBCs counted between

WBCs Histogram



49/56

WBCs Histogram

100 200 300

LD


WL

UD

WU

=UD Upper DiscriminatorWU WBCsUpper discriminator

[ Fixed ] at 300fL

2outer

discriminators


fL

WBCs Histogram


50/56

WBCs Histogram

100 200 300 fL

LD UD1 T2


WL WU

WBCs histogram consists of 2

Troughs

T : 78114 fL

T2: > 150 fL

[ Detected by 2innerdiscriminators]

[ Valleys ]

WBCs Histogram


51/56

WBCs Histogram

100 200 300 fL

L

DU

D1T

2


WL WU

Peakbetween LDand T1

represents small cells


Troughs

i.e. Lymphocytes


[ Valleys ]

WBCs HistogramWBCs histogram consists of 2


52/56

Cs Histogram

100 200 300 fL

L

DU

D1T

2


WL WU


Troughs

Metamyelo

cytes

Peakbetween T1 and T2 includes

Eosinophils

Monocytes

Basophils Blast cells

Promyelocytes

Myelocytes


[ Valleys ]

WBCs Histogram


53/56

WBCs Histogram

100 200 300 fL

L

DU

D1T

2


WL WU


Troughs

Peakafter T2represe

Neutrophils


[ Valleys ]

WBCs Histogram


54/56

WBCs Histogram

100 200

L

DU

D1T

2


WL WU

Important Notes

The distribution curveshould be within the

discriminators, starts and

endsat the base line.

WBCs Histogram


55/56

WBCs Histogram

100 200

L

DU

D1T

2


WL WU

Important Notes

The volume of the platelet is

usually between 8 - 12 fl,therefore the LD at the WBC

Histogram seperates the

WBCs from the

platelets.(Platelets were not

counted).

The WBC-channel shows only

WBCsand platelets(RBCs are

lysed)

The LD is flexiblewhile UD is

fixed.

The distribution curve should

be within the discriminators,

starts and ends at the base

line.

At 300 fL

3060 fL


56/56

hemii chapter 14 part 2 spring 2016..huda

Documents