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HEMATOLOGY FUNDAMENTALS SERVICE TRAINING COURSE BASIC HEMATOLOGY BASIC HEMATOLOGY

OBJECTIVE
Given the Basic Hematology course materials: Training Guide The books titled Hematology: Principles and Procedures by Barbara A. Brown and BLOOD by Dennis W. Ross The Johns Hopkins Development o Blood !ells cell maturation chart.

Read, study, review, and be able to answer the Hematology Basic nowledge !heck "uestions. The knowledge check will be considered completed when the student can achieve a minimum overall score o# $%&.

KNOWLEDGE CHECK
'ou will be given a nowledge !heck and asked to answer the "uestions using all the course materials provided.

MODULE RESOURCES
To !omplete this module you will need: Books: Hematology "undamentals #ervice $raining !ourse $raining %uide H&'A$OLO%(: Principles and Procedures by Barbara A. Brown BLOOD by Dennis W. Ross

Johns Hopkins Development o Blood !ells cell maturation chart

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BASIC HEMATOLOGY

What to Do
Read and study all the course materials provided. (hen you #eel con#ident that you know the material, ask your )nstructor #or the )nowledge !*ec+. !omplete the )nowledge !*ec+ and have your )nstructor check it and review any mistakes with you. *lthough it is recommended that you read and study all the course materials, i# at any time you #eel con#ident you know the material well enough to complete the nowledge !heck, #eel #ree to do so.

Hematology Fundamentals Service Training Course 04/11/2001

HEMATOLOGY FUNDAMENTALS SERVICE TRAINING COURSE BASIC HEMATOLOGY BASIC HEMATOLOGY Introdu t!on
This session is intended to provide you with a basic introduction to Hematology. !ell structure, cell maturation, methods #or measuring components o# blood samples and common terms used in hematology will be presented. How Beckman !oulter instrumentation is used in the hematology setting will also be addressed.

Wh" Stud" H#$ato%o&"'


To communicate more e##ectively with the end user o# our instrumentation To gain an understanding o# the material being tested on our e"uipment To work sa#ely and e##iciently with blood To understand troubleshooting on a system #rom your customer+s point o# view To establish the relevance #or this type o# testing and why it is necessary #or good patient care

What !( B%ood'
The #luid that circulates through the heart, lungs, arteries, veins and capillaries carrying o,ygen and nourishment to the tissues and carrying away carbon dio,ide and waste products produced by the tissues. * diagnostic tool #or the clinician to assess patient status. -ince blood is e,posed to virtually all tissues in the body, it becomes the .barometer. o# the condition o# the body. )n normal healthy individuals, the blood contains a normal number o# blood cells. )#, however, there is an abnormal or disease process, the blood typically re#lects a change #rom normal by either

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BASIC HEMATOLOGY

raising or lowering the number o# blood cells and their relative proportions. )t important to study blood to assist in screening or identi#ying a normal versus an abnormal condition o# the body.

Co$)o(!t!on o* B%ood
Blood is comprised o# appro,imately //& plasma and 0/& cellular components.

+LASMA ,,-

(ater $%& 1utrients !lotting 2rotiens *ntibodies Hormones -alts (astes

Red Blood !ells CELLS .,- (hite Blood !ells 2latelets

Figure 1

Composition of Blood

2lasma is comprised o# $%& water and 3%& salts, nutrients, hormones, antibodies, clotting proteins and waste products. The cellular components are red blood cells, white blood cells and platelets.

Who Stud!#( What A/out B%ood'


!hemistry 4epartment: !oagulation 4epartment: 5low !ytometry 4epartment: Hematology 4epartment:

Hematology Fundamentals Service Training Course 04/11/2001

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The three cellular components are red blood cells, white blood cells and platelets. Plasma and the three cellular components comprise the four major components of blood. Normally for every white blood cell, there are 1000 red blood cells and 20 platelets.

Figure 2

Formed lements of Blood

Hematology Fundamentals Service Training Course 04/11/2001

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Ba(! C#%% Stru tur#


*ny cell consists o# 6 basic parts:

Figure 3

Basic Cell Structure

C#%% M#$/ran# * semipermeable separation between the various internal cellular components, the organelles, and the surrounding environment. *ntigens are bound to the sur#ace o# cell membranes. 7aintains the cellular integrity o# the interior o# the cell by controlling the passage o# materials in and out o# the cell. C"to)%a($ !ontained within the cell membrane.

!omposed o# a variety o# small cellular structures called organelles which are the #unctional units o# the cell. -maller organelles can only be seen with an electron microscope. 8arger organelles can be seen on stained blood preparations using a light microscope. These larger organelles give the cytoplasm a characteristic .look. that can help identi#y the cell.

"

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Nu %#u( 5unctions as the control center o# the cell and is essential #or long9term survival. !ontrols the cell division process to produce an e,act replicate o# the cell. 7any cells retain the ability to divide and replicate themselves throughout the li#e o# the cell: #or e,ample, skin, gut, lining o# the mouth, etc. *s a blood cell matures, the nucleus either decreases in relative si;e or is e,truded <pushed out o# the cell= and, as a result, the blood cell loses its ability to divide and replicate. 8ymphocytes are the only blood cells that continue to replicate themselves even a#ter maturity. )n hematology, identi#ication o# a cell and its maturity is typically based on: !ell -i;e 1uclear appearance Granularity

B%ood C#%% Maturat!on Chara t#r!(t! (


O0#ra%% C#%% S!1# >verall cell si;e is usually compared with the si;e o# a mature red cell. *s a cell matures, it usually becomes smaller in si;e.

Nu %#ar to C"to)%a($! Rat!o 1:! or the amount o# space occupied by the nucleus in relationship to the space occupied by the cytoplasm. 1ucleus o# an immature cell tends to be round or oval and is very large in proportion to the rest o# the cell. *s the cell matures, the nucleus decreases in relative si;e and may take on various shapes. -ome cells loose their nucleus entirely. 7ature red blood cells and platelets are the two cell types in the circulating blood that do not have a nucleus. #

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Nu %#ar Chara t#r!(t! ( !hromatin pattern, nuclear shape and the presence or absence o# nucleoli are important nuclear #eatures that can aid in the identi#ication o# cell type.
Chro$at!n

2hysical basis o# heredity. 8oose and #ine in most immature cells. !lumped with a dark appearance in more mature cells.
Nu %#o%!

5ound only in immature cells.


Sha)#

?ither round or oval in young cells. )n cells that retain their nucleus as they mature, nuclear shapes become very distinctive #or particular cell types.

Wh#r# B%ood C#%%( ar# +rodu #d


Re er to t*e Development o Blood !ells c*art )n an adult, blood cells are #ormed in the bone marrow.

The nucleus o# the immature cell is typically round or oval and is very large in proportion to the rest o# the cell. *s the cell matures, the nucleus decreases in relative si;e and may take on various shapes. -ome cells loose their nucleus entirely. The nucleus is the initiator o# cell division. 1otice that the nucleus o# a mature blood cell is very coarse and condensed. This is because the nucleus o# most mature blood cells is no longer #unctioning in cell division. !ells are produced, mature and take on the appearance and #unction o# a mature cell in the bone marrow. *s cells mature, they usually become smaller in si;e.

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*s cells mature in structure and #unction, they are released into the blood circulating through the veins and arteries. This circulating blood is commonly re#erred to as the peripheral blood. *n immature cell is not capable o# carrying out the speci#ic #unctions o# a mature cell. Because mature cells have a speci#ic role and #unction when circulating in the peripheral blood, only mature cells should be circulating in the peripheral blood. The presence o# immature cells in the peripheral blood typically indicates a problem.

R#d B%ood C#%%(


Red blood cells are also re#erred to as erythrocytes or RB!s. The nucleus is completely e,truded #rom the cell by maturity. 7ature RB!s have no nucleus or remnants o# one. Their biconcave disk shape allows #or more sur#ace area, there#ore more o,ygen carrying capacity.

Figure 4

%ed Blood Cell

Their #le,ibility <or ability to de#orm= also allows the RB!s to s"uee;e through small capillaries to the tissues. The "uantity o# RB!s may be e,pressed as a red blood cell count or as a ratio o# the volume o# red cells to the volume o# whole blood.
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BASIC HEMATOLOGY

The "uantity o# red cells is e,pressed as an R ! count. The ratio o# the volume o# red cells to volume o# whole blood is de#ined as the hematocrit "#ct$.

Figure !

Hematocrit

The Reference Ran%e "also referred to as &Normal Ran%e&$ for an R ! count for males is '.( to ).1 million cells per *+ "microliter$ of whole blood. The Reference Ran%e for an R ! count for females is '.2 to ,.' million cells per *+ of whole blood. The Reference Ran%e for hematocrit "#ct$ for males is '2- to ,2-. The Reference Ran%e for hematocrit "#ct$ for females is .(- to '(-.

H#$o&%o/!n
The portion o# the RB! that transports o,ygen #rom the lungs to the tissues and carbon dio,ide #rom the tissues to the lungs, is a protein called hemo%lobin. <*emo @ blood : globin @ protein= The red cell membrane serves as a retaining barrier #or the hemoglobin molecule but is also permeable to permit o,ygen and carbon dio,ide to pass #reely. 10
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Normally, the #emo%lobin "#%b$ value is appro/imately three times the R ! count " R ! / . 0 #%b $. The Reference Ran%e for #%b for a male is 1'112 %3d+ "%rams per deciliter$. The Reference Ran%e for #%b for a female is 1211) %3d+. Normally, the #emo%lobin "#%b$ value times three e4uals the #ematocrit "#ct$ appro/imately " #%b / . 0 #ct $.

RBC Ind! #(
)n Hematology, indices <pronounced: nAdi ss= re#ers to calculated values used #or describing red cell properties. There are three RB! )ndices 7!B, 7!H, 7!H! The indices remain constant in a stable patient population, there#ore, the laboratory can use the indices to monitor instrument per#ormance. This #orms the basis o# CB *nalysis. MCV 5ean !orpuscular 6olume "5!6$, which describes the volume o# the average red cell in a given sample o# blood, is one o# the RB! indices. The calculation for 5!6 is Hct 9999999999 , 3% RB!

The Reference Ran%e for 5!6 for males is 2( 7 ( f+ "femtoliters$8 for females 90 7 9 f+. 5icrocytic <micro @ small : cytic @ cell= red cells decrease the 7!B result while macrocytic <macro @ large : cytic @ cell= red cells increase the 7!B result. )# an 7!B #or a male or #emale is 3D% #8, the red cell population is considered macrocytic: however, an 7!B o# $E #8 is considered macrocytic only #or a male. * variation in the si;es o# red cells is called anisocytosis <an @ without : iso @ same : cyto @ cell : osis @ condition o#=. )n other words, a condition o# .unlike. cells.

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ACTIVITY !onsider the e##ect o# the diluent on the si;e o# the RB!. 3. )# the RB! si;e is responsive to its supporting media, what would be the outcome i# red cells are suspended in waterF

D.

(hat would be the outcome i# red cells are suspended in a highly concentrated salt solutionF

6.

(hat do you think the saline concentration in !oulter+s diluting #luid <)->T>1<r= )))= isF

ACTIVITY ANSWER KEY 3. )n e##ort to e"uilibrate the internal and e,ternal environment, the RB!s tend to take in water #rom the suspending media, swell and burst. The opposite e##ect would be observed. The .water. #rom inside the cell will pass through the membrane into the suspending media thereby shrinking the RB!. )n this case, the RB! volume <7!B= would be decreased. !>G8T?R )->T>1 ))) diluent has been #ormulated to best mimic plasma to keep cells in their near9native state.

D.

6.

MCH 5ean !orpuscular #emo%lobin "5!#$ describes the average weight o# hemoglobin in the red blood cell. Hgb The calculation for 5!# is 99999999999 , 3% RB!

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7!H is directly proportional to the si;e o# the red blood cell and the concentration o# hemoglobin in the cell. The Reference Ran%e for 5!# is 2( to .1 p% "pico%rams or **% 3 micro1micro%rams$.

MCHC 5ean !orpuscular #emo%lobin !oncentration "5!#!$ describes the average concentration o# hemoglobin in the red blood cells. 7!H! gives the ratio o# the weight o# the hemoglobin to the volume o# the red blood cell e,pressed in a percentage. 7!H! is e,pressed as an average concentration by dividing the hemoglobin value by the hematocrit and multiplying by 3%%. Hgb 9999999999 , 3%% Hct The Reference Ran%e for 5!#! for males and females is .. to .(-. The calculation #or 7!H! is >ne o# the best parameters #or indicating either instrument mal#unction or an improper or unusual specimen. !alculation is dependent on all measured RB! parameters HHgb and Hct <which is based on the RB! and 7!B=I. *ny problem with the Hgb, RB! or 7!B is o#ten re#lected by an abnormal 7!H!. -pecimens with 7!H!s outside the normal range should there#ore be suspected. *n 7!H! above 6E& should never occur. *n 7!H! should never #all below DD& even when hypochromia is present.

)# the #re"uency o# either high or low 7!H!s increases, an instrument problem is most likely present.

RBC E0a%uat!on
The hematology technologist evaluates red cells by their #orm, shape and appearance as observed through a microscope.

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'orp*o is a pre#i, that means #orm, shape and appearance: logy is a su##i, meaning the study o#. There#ore, the study o# red cell si;e, shape and appearance is red cell morpholo%y. The presence o# a variety o# shapes o# red cells is called poi:ilocytosis.

ACTIVITY
3. Biew odachromes o# the normal and abnormal RB!s and classi#y as 1ormochromic, Hypochromic, 1ormocytic, 7acrocytic, 7icrocytic, *nisocytosis or 2oikilocytosis. 4escribe what the #ollowing terms indicate about the RB!:

D.

1ormochromic JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ Hypochromic JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ 1ormocytic JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ 7acrocytic JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ 7icrocytic JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ *nisocytosis JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ 2oikilocytosisJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

ACTIVITY ANSWER KEY


1ormochromic Hypochromic 1ormocytic 7acrocytic 7icrocytic 14 RB!s having normal color K 7!H normal RB!s having less than normal color K 7!H decreased RB!s having a normal si;e K 7!B normal RB!s larger than normal K 7!B increased RB!s smaller than normal K 7!B decreased
Hematology Fundamentals Service Training Course 04/11/2001

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*nisocytosis 2oikilocytosis

RB!s varying in si;e RB!s varying in shape

LECTURE 2 DISCUSSION R#t! u%o "t#(


* reticulocyte is the last stage be#ore a red cell is considered mature. * reticulocyte, when stained with (right -tain, has a bluish cast to it and is slightly larger than a normal mature RB!. Reticulocytes have residual R1*, which when stained with a supravital stain <a stain that stains the cells while they are still living= becomes visually evident when viewed microscopically. 1ew 7ethylene Blue is an e,ample o# a commonly used supravital stain. ; normal reticulocyte count is considered to be appro/imately 0.( 1 2.2- of a normal R ! count.

+%at#%#t(
Platelets may also be re#erred to as Thrombocytes or Plts

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BASIC HEMATOLOGY

Figure "

Formation of 'latelets

The normal li#e span o# a platelet is 3% days L 3 day

Re er to t*e Development o Blood !ells c*art. Locate t*e 'ega+aryoblast on t*e ar rig*t and top o t*e page. ,otice as t*e cell matures- small pieces o t*e cytoplasm .bud. o /platelets0. The clotting process is an e,tremely comple, system.

!oagulation instruments can test #or various components in this system. )t is important to be aware that platelets play a signi#icant role in the clotting process. The Reference Ran%e for a Platelet count is about 1,0,000 1 '00,000 cells3*+. -ince platelets are consumed in the clotting process, clotted blood specimens or poorly collected #ingerstick samples have a low platelet count. )t is unacceptable to use clotted blood samples #or hematology studies. 2latelets appear in the peripheral blood as small, disc9 shaped cellular #ragments about D to 0 microns in diameter. RB!s are lar%er than platelets.

1"

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Wh!t# B%ood C#%%(


<hite lood !ells are also re#erred to as +eu:ocytes or < !s The normal li#e span o# a (B! is 33 days in the bone marrow to mature then hours to years in the tissues. The Reference Ran%e for a < ! count is ',000 to 10,000 cells3*+. 8eukocytes can be classi#ied or di##erentiated in a number o# ways. The most common is morphologically <#orm, shape and appearance when looked at through a microscope=.

Hematology Fundamentals Service Training Course 04/11/2001

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BASIC HEMATOLOGY

T")#( o* WBC(
7ononuclear 8ymphocytes 7onocytes 2olymorphonuclear Granulocytes 9 9 1eutrophils ?osinophils

Basophils

Figure #

()ite Blood Cells

ACTIVITY
1$ Re er to t*e Development o Blood !ell c*art.
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>bserve the di##erent coloration o# 1eutrophils compared to Basophils compared to ?osinophils. >bserve the granulocytes #rom the immature stem cell to the more mature cell. 1otice the nucleus change #rom a large round nucleus in the immature cells to a .multi9lobed. nucleus in the mature cell.

LECTURE 2 DISCUSSION Mononu %#ar L#u3o "t#(


L"$)ho "t#( +ymphocytes are produced in the bone marrow and migrate to the lymph tissues <lymph glands= where they become involved with the body+s immune #unction. These cells can be subclassi#ied by use o# .cell sur#ace markers. as T cells, B cells or 1atural iller <1 = cells. !lassi#ying lymphocytes is very important in the diagnosis and management o# *)4- patients. 4etermining cell sur#ace markers can be described as .#ingerprinting. the lymphocytes. This type o# testing uses antibodies manu#actured against certain antigens on the lymphocyte cell membrane <see 5igure 6=. The antigens, in this case, are proteins attached to the lymphocyte cell membrane. *ntibodies are special proteins that attach to only one speci#ic type o# antigen . . . like a lock and key=. !ertain kinds o# lymphocytes have speci#ic kinds o# antigens, so i# we use a known type o# antibody and it attaches to the antigen on the cell, we can identi#y the type o# lymphocyte it isM * flow cytometry instrument can per#orm this special type o# lymphocyte subclassi#ication.

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BASIC HEMATOLOGY

Mono "t#( 5onocytes are produced in bone marrow but they migrate to body tissues to serve as scavenger cells that .eat. #oreign particles and digest them.

+o%"$or)honu %#ar L#u3o "t#(


Granu%o "t#( =ranulocytes are polymorphonuclear cells meaning the nucleus has more than one lobe. 4escribed by the granules in their cytoplasm and their staining characteristics when stained with (rightsKGiemsa stain. Neutrophils 9 granules stain .neutrally. and appears as a slight pink color in the cytoplasm >osinophils 9 granules stain a reddish K orange color asophils 9 granules stain dark bluish9black Granulocyte #unctions di##er in relation to their granule content. 1eutrophils 9 granules contain digestive en;ymes to destroy bacteria ?osinophils 9 granules contain histamine and en;ymes and are involved in the latter stages o# in#lammation, allergic reactions and parasitic reactions Basophils 9 granules contain histamine, heparin and heparin like substances: although the #unction o# basophils has been debated, they do seem to be associated with allergic reactions

WBC D!**#r#nt!a% Ana%"(!(


(B! di##erential analysis, which is one o# the #undamental analyses o# hematology, is a diagnostic tool that can act as a pointer to aid the physician in the diagnosis andKor monitoring o# a multitude o# disease states. )n the di##erential blood count procedure, leukocytes are identi#ied and manually enumerated #rom their morphological appearance on stained blood smears or by automated instruments that use a combination o# cellular characteristics and histochemical reactions.

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R#%at!0# Nu$/#r( 4-5 4i##erential results are generally reported in percentages which means the enumeration o# a particular type o# white blood cell is actually relative to all the other cell types that are present. )n other words, a lymphocyte percentage o# 0%& means that in the total number o# white blood cells counted <3%%&=, 0%& o# those cells were lymphocytes while the other N%& o# the cells were some other type o# white cell. Relative numbers e,pected in normal circulating blood: 1eutrophils 8ymphocytes 7onocytes ?osinophils Basophils A/(o%ut# Nu$/#r( 465 *nother way to report di##erential results. /%& to O/& D%& to 0/& 6& to 33& 3& to 6& %& to 3&

)ndividual absolute numbers are computed based on the total (B! count and the individual di##erential percentage <&=. The availability o# absolute numbers helps resolve the di##iculties sometimes posed by the use o# percentages alone. 5or e,ample, i# the laboratory+s (B! normal range lies between /,%%% and 3%,%%% and the lymphocyte range is between D%& and 0%& o# the total count, then the normal absolute lymphocyte count should be between 3,%%% and 0,%%%KP8.

Patient ; 8ymphocyte & 8ymphocyte Q (B! K P8 $%& 36,/%% 3/,%%%

Patient $%& 6,N%% 0,%%%

8ymph& , total (B! @ absolute number o# lymphs

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2atient * and B both show a relative lymphocytosis o# $%&. However, only 2atient * has an absolute lymphocytosis with a lymphocyte count o# 36,/%%KP8. 2atient B, on the other hand, has a normal absolute lymphocyte count o# 6,N%%KP8. )n other words, the patient may have a relative increase in lymphocytes not because they are producing more but because they lack other cells types such as neutrophils. >n the other hand, an absolute increase in lymphocytes means there is an actual increase in the production o# lymphocytes.

T#r$!no%o&"
The su##i, penia means a severe decrease: there#ore, leukopenia indicates a decrease in the number o# (B!s. The su##i, cytosis means an increase o : there#ore, leukocytosis indicates an increase in the number o# (B!s. =ranulocytosis indicates there is an increase in granulocytes: lymphocytosis, an increase in lymphocytes. +ymphopenia indicates a decrease in lymphocytes: %ranulopenia, a decrease in granulocytes. Because o# the relative relationship among the white cells, granulopenia is generally accompanied by lymphocytosis while granulocytosis is generally accompanied by a lymphopenia.

La/orator" M#a(ur#$#nt o* B%ood


S)# !$#n Co%%# t!on * Technologist or Technician uses a peripheral blood specimen to study the blood cells in vitro <outside the body=. -pecimens are typically collected by a phlebotomist who is speci#ically trained in blood collection. The phlebotomist may obtain the specimen #rom a vein <venipuncture= or #rom capillaries <skin puncture o# the #inger, heel or ear lobe=. * venipuncture collected in an evacuated collection tube is the pre#erred specimen. The normal inclination o# blood is to clot once released #rom the natural environment o# the body . . . this is nature+s way o# protecting us #rom bleeding.

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)# a patient has blood collected into an empty tube, the platelets and special clotting proteins cause the sample to clot. *ll cell types become enmeshed in the clot. The straw colored #luid that is e,pressed #rom the clot is called serum. -erum is typically used in chemistry studies in the laboratory. )# blood is drawn into a tube containing a special chemical additive to prevent clotting, the blood remains in a #luid state. This chemical additive is called anticoa%ulant <to prevent coagulation or clotting=. )# anticoagulated is allowed to sit without mi,ing #or several hours , the clear #luid that appears in the upper portion o# the tube is the plasma. Ant! oa&u%ant( *nticoagulants may be dry9powder #orm or li"uid.

There are a variety o# anticoagulants used and can be identi#ied by the color o# the tube stopper. )n hematology, the anticoagulant o# choice is 6?4T* <ethylenediamine tetra acetic acid= and is #ound in a purple or lavender stoppered tube. !alcium is a re"uired component to the clotting process. ?4T* has the ability to bind up calcium in the blood. This process o# binding up calcium is commonly re#erred to as .chelating calcium.. The bottom line . . . no calcium, no clottingM 7ost anticoagulants <!itrate, >,alates= work by the same mechanism as ?4T* <binding calcium to prevent clotting.= Heparin acts as an antagonist to the normal clotting reactions o# the clotting cascade. The choice o# anticoagulant in Hematology is determined by what mi,es "uickly and e##iciently with the blood and also what maintains the cells in their near native state. Beckman !oulter recommends 6?4T* as the optimal anticoagulant because all testing and studies done on our instrumentation was per#ormed using 6?4T* samples. 6?4T* is cited in our instrument manuals as the pre#erred anticoagulant: however, the maRor manu#acturers o# blood
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specimen tubes are in the process o# converting #rom glass to plastic tubes containing the anticoagulant D?4T*. There#ore, in the near #uture, testing and claims made by Beckman !oulter )nc. will cite D?4T* as the pre#erred anticoagulant.

ACTIVITY
>bserve the variety o# tubes available. 1otice the presenceKabsenceKvolume o# anticoagulant associated with the color stoppered tube <B*!GT*)1?R and H?7>G*R4 by Becton94ickinson as well as B?1>J?!T by Terumo=. >bserve the corresponding blood #illed tubes <D o# each: mi, only one=. *ssociate the e##ects o# the presenceKabsence o# anticoagulant and observe the di##erence between serum and plasma.
TUBE 2 ANTICOAGULANT SUMMARY
Tu/# Co%or Sto))#r Ant! oa&u%ant D#)art$#nt Wh#r# Co$$on%" U(!n& C)emistry Blood Ban+ Hematology Flo3 Cytometry Coagulation C)emistry Flo3 Cytometry Blood Ban+ C)emistry

%ed ,avender Blue 4reen 5ello3 4ray

*one -T. /*a2 0 12 0 132 Sodium Citrate Heparin .C- /.cid Citrate -e6trose2 'otassium 76alate8 Sodium Fluoride

LECTURE 2 DISCUSSION S)# !$#n Co%%# t!on D#0! #(


To increase sa#ety #or laboratory personnel, manu#acturers are producing stoppers di##erent #rom the traditional rubber stopper in an e##ort to minimi;e aerosoli;ation and splattering o# blood when removing the stopper. <H?7>G*R4 and Terumo tubes are e,amples.=

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5or children, babies and di##icult patients, microcollection devices allow the #inger, heel or earlobe to be pricked and blood collected manually. e aware of the potential for clottin% in this type of specimen.

S)# !$#n Co%%# t!on +r# aut!on(


Blood tubes must be properly #illed to assure the appropriate blood to anticoagulant ratio is maintained. (hen blood is drawn with a syringe, it must be trans#erred to a blood specimen tube. (hen this trans#er occurs, it is very easy to either over#ill or under#ill the specimen tube. E**# t( o* O0#r*!%%!n& a S)# !$#n Tu/# >ver#illing overcomes the #unction o# the additive K anticoagulant. >ver#illed tubes are not properly anticoagulated and, as a result, microclots can #orm. ;ny clottin% in a specimen tube ma:es it an unacceptable sample for hematolo%y and analysis on any ec:man !oulter #ematolo%y ?nstrument. The specimen must be redrawn.

E**# t( o* Und#r*!%%!n& a S)# !$#n Tu/# Gnder#illed specimen tubes have an e,cess o# anticoagulant. ?,cess anticoagulant may cause cells to crenate and shrink.

Sa$)%# Hand%!n&
Baries depending on the type o# tube and the testing plans #or the sample. 4epartment re"uirements K storage re"uirements: !hemistry !oagulation

Hematology Fundamentals Service Training Course 04/11/2001

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BASIC HEMATOLOGY

5low !ytometry Hematology 7i,ing Re"uirements

?"uilibration cells go through in response to the anticoagulated environment. < shrink K swell = @ey Points +aboratory results from any instrument are only as %ood as the specimen they come from. Never overloo: the inte%rity of the sample"s$ bein% run throu%h the system.

CBC7 th# Co$)%#t# B%ood Count


>ne o# the most #re"uently re"uested laboratory tests. 2atient results are considered against a .1ormal Range.. 1ormal Range is established in the laboratory by taking a sampling o# population to determine the average number and "uantity o# each !B! parameter. >nce the average is established, a range is mathematically determined by calculating a D-4 <-tandard 4eviation= #rom the mean o# the collected data. >n printed tickets #rom !oulter <and in any Hematology te,t=, normal ranges are given but are generic. !B! typically includes:

* (B! di##erential includes:

!linical utility o# a !B!:

2"

Hematology Fundamentals Service Training Course 04/11/2001

BASIC HEMATOLOGY

M#thod( o* +#r*or$!n& a CBC


7anual, semi9automated and automated methods are available #or per#orming a !B!.

Manua% M#thod( 8ocate the procedures #or manual blood cell counts starting on page E$ o# your te,tbook, H&'A$OLO%(: Principles and Procedures by Barbara Brown. H#$o&%o/!n M#a(ur#$#nt (hen a speci#ied volume o# red blood cells is e,posed to a chemical <lytic a%ent= that destroys the red cell membrane, hemoglobin is released. The released hemoglobin reacts with the cyanide in the reagent and is converted to a stable pigment. The color o# the .lysed. solution can be measured spectrophotometrically. By comparison with standard solutions, a value #or hemoglobin is calculated. 2rocedures #or a manual hemoglobin determination starts on page E6 o# your te,tbook, H&'A$OLO%(: Principles and Procedures by Barbara Brown. H#$ato r!t M#a(ur#$#nt (hen an anticoagulated whole blood sample is placed in a straw like glass tube and centrifu%ed, the ratio o# volume o# RB!s to volume o# whole blood can be determined. This is de#ined as the hematocrit. The procedure #or a manual hematocrit determination begins on page E/ o# your te,tbook, H&'A$OLO%(: Principles and Procedures by Barbara Brown. Ca% u%at!on o* th# RBC Ind! #( RB! )ndices can be calculated manually by inserting the RB!, Hgb andKor Hct results into the #ollowing e"uations: 7!B
Hematology Fundamentals Service Training Course 04/11/2001

@ < Hct K RB! = ,

3% 2#

BASIC HEMATOLOGY

7!H

@ < Hgb K RB! = , ,

3% 3%%

7!H! @ < Hgb K Hct =

WBC D!**#r#nt!a%
Manua% M! ro( o)! M#thod The procedure #or a manual di##erential count starts on page 3%D o# your te,tbook, H&'A$OLO%(: Principles and Procedures by Barbara Brown. )ndicate what the #ollowing terms re#er to in terms o# the associated parameter and whether high or low: Hypochromic 7acrocytic 8eukocytosis *nemia JJJJJJJJJ parameter would be JJJJJJJJJ. JJJJJJJJJ parameter would be JJJJJJJJJ. JJJJJJJJJ parameter would be JJJJJJJJJ. JJJJJJJJJ parameter would be JJJJJJJJJ.

Thrombocytosis JJJJJJJJJ parameter would be JJJJJJJJJ.

ACTIVITY ANSWER KEY


Hypochromic 7acrocytic 8eukocytosis *nemia Thrombocytosis 7!H parameter would be 8>(. 7!B parameter would be H)GH. (B! parameter would be H)GH. RB! parameter would be 8>(. 28*T?8?T parameter would be H)GH.

2$

Hematology Fundamentals Service Training Course 04/11/2001

BASIC HEMATOLOGY

LECTURE 2 DISCUSSION
7ost laboratories no longer per#orm their daily !B!s using the manual methods to measure (B!s, RB!s, Hgb, Hct, platelets and calculate the RB! indices.

ACTIVITY
3. 8ist potential sources o# error when per#orming a manual !B!:

JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ D. Based on your knowledge o# !>G8T?R automated hematology systems, list the advantages o# an automated !B!:

JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ

Hematology Fundamentals Service Training Course 04/11/2001

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BASIC HEMATOLOGY

ACTIVITY ANSWER KEY


3. *nswers may vary: 4ilutions may be made improperly or with incorrect reagents 4ilutions are incorrectly charged on the hemacytometer )nconsistent methods o# counting cells on the hemacytometer #rom tech to tech 5atigue in counting Hematocrit sample may be drawn up into the microhematocrit tube incorrectly yielding bubbles )mproper centri#ugation o# the sample )nconsistent reading o# the results #rom tech to tech Hemoglobin calibration is incorrect Reagent e,pired -ample dilution error (B! 4i##erential inconsistently read #rom tech to tech <very subRective=: poor "uality o# wedge smear andKor staining 1o subRectivity, better precision, accuracy, consistent pipetting and dilution preparation, consistent counting K measuring method.

D.

LECTURE 2 DISCUSSION Th# La/orator"


D#)art$#nt( * typical hospital laboratory consists o# many departments. Hematology, !hemistry, Blood Bank, Grinalysis, !oagulation, 5low !ytometry, etc. L! #n(!n& A&#n !#( 30 *-!2 <*merican -ociety o# !linical 2athologists=
Hematology Fundamentals Service Training Course 04/11/2001

BASIC HEMATOLOGY

*-!8- <*merican -ociety o# !linical 8aboratory -cience= #ormerly *-7T <*merican -ociety o# 7edical Technologists= 1!*, 1ational !erti#ication *gency #or 7edical 8aboratory 2ersonnel )-!8T, )nternational -ociety #or !linical 8aboratory Technology H?( <Health, ?ducation and (el#are=

+#r(onn#% Titles associated with di##erent laboratorians: 7T 7edical Technologist 0 year degree including 3D months o# clinical training K licensed 7edical 8aboratory Technician D years o# college credit including 3D months training in an approved hospital9related school K licensed !erti#ied 8aboratory *ssistant 3D months training in an approved hospital related school K licensed )ndividual responsible #or collecting blood samples #or the laboratory

78T

!8*

2hlebotomist

K#" O)#rator *s a !oulter representative, it is imperative that you communicate with the appropriate person about the instrument you are working on. The person who generally knows the most about the operation o# the instrument and its per#ormance is re#erred to as the ey >perator. 7ost o# the time, this person has attended a !ustomer training course at the ?ducation !enter. *lthough we recogni;e that the -upervisor or the 8aboratory 4irector andKor 2athologist have an interest in the !>G8T?R instrument and its operation, you should always direct your communication initially with the .key operator..

Hematology Fundamentals Service Training Course 04/11/2001

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BASIC HEMATOLOGY

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Hematology Fundamentals Service Training Course 04/11/2001

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