TITLE

Hematology Fundamentals Service Training course

BASIC hematologyBASIC hematologyBASIC 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 of Blood Cells cell maturation chart.

Read, study, review, and be able to answer the Hematology Basic Knowledge Check questions. The knowledge check will be considered completed when the student can achieve a minimum overall score of 90%.

Knowledge Check

You will be given a Knowledge Check and asked to answer the questions using all the course materials provided.

Module Resources

To Complete this module you will need:

Books:

Hematology Fundamentals Service Training Course Training Guide HEMATOLOGY: Principles and Procedures by Barbara A. Brown BLOOD by Dennis W. Ross Johns Hopkins Development of Blood Cells cell maturation chart

What to Do

Read and study all the course materials provided. When you feel confident that you know the material, ask your Instructor for the Knowledge Check. Complete the Knowledge Check and have your Instructor check it and review any mistakes with you.

Although it is recommended that you read and study all the course materials, if at any time you feel confident you know the material well enough to complete the Knowledge Check, feel free to do so.

Basic Hematology

Introduction

This session is intended to provide you with a basic introduction to Hematology. Cell structure, cell maturation, methods for measuring components of blood samples and common terms used in hematology will be presented. How Beckman Coulter instrumentation is used in the hematology setting will also be addressed.

Why Study Hematology?

To communicate more effectively with the end user of our instrumentation

To gain an understanding of the material being tested on our equipment

To work safely and efficiently with blood

To understand troubleshooting on a system from your customer's point of view

To establish the relevance for this type of testing and why it is necessary for good patient care

What is Blood?

The fluid that circulates through the heart, lungs, arteries, veins and capillaries carrying oxygen and nourishment to the tissues and carrying away carbon dioxide and waste products produced by the tissues.

A diagnostic tool for the clinician to assess patient status.

Since blood is exposed to virtually all tissues in the body, it becomes the "barometer" of the condition of the body.

In normal healthy individuals, the blood contains a normal number of blood cells.

If, however, there is an abnormal or disease process, the blood typically reflects a change from normal by either raising or lowering the number of blood cells and their relative proportions.

It important to study blood to assist in screening or identifying a normal versus an abnormal condition of the body.

Composition of Blood

Blood is comprised of approximately 55% plasma and 45% cellular components.

Water 90%Nutrients

PLASMA 55%Clotting Protiens

Antibodies

Hormones

Salts

Wastes

Red Blood Cells

CELLS 45%White Blood Cells

Platelets

Figure 1 Composition of Blood

Plasma is comprised of 90% water and 10% salts, nutrients, hormones, antibodies, clotting proteins and waste products.

The cellular components are red blood cells, white blood cells and platelets.

Who Studies What About Blood?

Chemistry Department:

Coagulation Department:

Flow Cytometry Department:

Hematology Department:

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 Elements of Blood

Basic Cell Structure

Any cell consists of 3 basic parts:

Figure 3 Basic Cell Structure

Cell Membrane

A semipermeable separation between the various internal cellular components, the organelles, and the surrounding environment.

Antigens are bound to the surface of cell membranes.

Maintains the cellular integrity of the interior of the cell by controlling the passage of materials in and out of the cell.

Cytoplasm

Contained within the cell membrane.

Composed of a variety of small cellular structures called organelles which are the functional units of the cell.

Smaller organelles can only be seen with an electron microscope.

Larger organelles can be seen on stained blood preparations using a light microscope. These larger organelles give the cytoplasm a characteristic "look" that can help identify the cell.

Nucleus

Functions as the control center of the cell and is essential for long-term survival.

Controls the cell division process to produce an exact replicate of the cell.

Many cells retain the ability to divide and replicate themselves throughout the life of the cell; for example, skin, gut, lining of the mouth, etc.

As a blood cell matures, the nucleus either decreases in relative size or is extruded (pushed out of the cell) and, as a result, the blood cell loses its ability to divide and replicate.

Lymphocytes are the only blood cells that continue to replicate themselves even after maturity.

In hematology, identification of a cell and its maturity is typically based on:

Cell Size

Nuclear appearance

Granularity

Blood Cell Maturation Characteristics

Overall Cell Size

Overall cell size is usually compared with the size of a mature red cell.

As a cell matures, it usually becomes smaller in size.

Nuclear to Cytoplasmic Ratio

N:C or the amount of space occupied by the nucleus in relationship to the space occupied by the cytoplasm.

Nucleus of an immature cell tends to be round or oval and is very large in proportion to the rest of the cell.

As the cell matures, the nucleus decreases in relative size and may take on various shapes.

Some cells loose their nucleus entirely. Mature red blood cells and platelets are the two cell types in the circulating blood that do not have a nucleus.

Nuclear Characteristics

Chromatin pattern, nuclear shape and the presence or absence of nucleoli are important nuclear features that can aid in the identification of cell type.

Chromatin

Physical basis of heredity.

Loose and fine in most immature cells.

Clumped with a dark appearance in more mature cells.

Nucleoli

Found only in immature cells.

Shape

Either round or oval in young cells.

In cells that retain their nucleus as they mature, nuclear shapes become very distinctive for particular cell types.

Where Blood Cells are Produced

Refer to the Development of Blood Cells chart

In an adult, blood cells are formed in the bone marrow.

The nucleus of the immature cell is typically round or oval and is very large in proportion to the rest of the cell.

As the cell matures, the nucleus decreases in relative size and may take on various shapes.

Some cells loose their nucleus entirely.

The nucleus is the initiator of cell division.

Notice that the nucleus of a mature blood cell is very coarse and condensed.

This is because the nucleus of most mature blood cells is no longer functioning in cell division.

Cells are produced, mature and take on the appearance and function of a mature cell in the bone marrow.

As cells mature, they usually become smaller in size.

As cells mature in structure and function, they are released into the blood circulating through the veins and arteries.

This circulating blood is commonly referred to as the peripheral blood.

An immature cell is not capable of carrying out the specific functions of a mature cell.

Because mature cells have a specific role and function when circulating in the peripheral blood, only mature cells should be circulating in the peripheral blood.

The presence of immature cells in the peripheral blood typically indicates a problem.

Red Blood Cells

Red blood cells are also referred to as erythrocytes or RBCs.

The nucleus is completely extruded from the cell by maturity. Mature RBCs have no nucleus or remnants of one.

Their biconcave disk shape allows for more surface area, therefore more oxygen carrying capacity.

Figure 4 Red Blood Cell

Their flexibility (or ability to deform) also allows the RBCs to squeeze through small capillaries to the tissues.

The quantity of RBCs may be expressed as a red blood cell count or as a ratio of the volume of red cells to the volume of whole blood.

The quantity of red cells is expressed as an RBC count.

The ratio of the volume of red cells to volume of whole blood is defined as the hematocrit (Hct).

Figure 5 Hematocrit

The Reference Range (also referred to as "Normal Range") for an RBC count for males is 4.7 to 6.1 million cells per L (microliter) of whole blood.

The Reference Range for an RBC count for females is 4.2 to 5.4 million cells per L of whole blood.

The Reference Range for hematocrit (Hct) for males is 42% to 52%.

The Reference Range for hematocrit (Hct) for females is 37% to 47%.

Hemoglobin

The portion of the RBC that transports oxygen from the lungs to the tissues and carbon dioxide from the tissues to the lungs, is a protein called hemoglobin.

(hemo = blood ; globin = protein)

The red cell membrane serves as a retaining barrier for the hemoglobin molecule but is also permeable to permit oxygen and carbon dioxide to pass freely.

Normally, the Hemoglobin (Hgb) value is approximately three times the RBC count ( RBC x 3 = Hgb ).

The Reference Range for Hgb for a male is 14-18 g/dL (grams per deciliter).

The Reference Range for Hgb for a female is 12-16 g/dL.

Normally, the Hemoglobin (Hgb) value times three equals the Hematocrit (Hct) approximately ( Hgb x 3 = Hct ).

RBC Indices

In Hematology, indices (pronounced: ndi ss) refers to calculated values used for describing red cell properties.

There are three RBC Indices MCV, MCH, MCHC

The indices remain constant in a stable patient population, therefore, the laboratory can use the indices to monitor instrument performance. This forms the basis of XB Analysis.

MCV

Mean Corpuscular Volume (MCV), which describes the volume of the average red cell in a given sample of blood, is one of the RBC indices.

Hct

The calculation for MCV is---------- x 10

RBC

The Reference Range for MCV for males is 87 + 7 fL (femtoliters); for females 90 + 9 fL.

Microcytic (micro = small ; cytic = cell) red cells decrease the MCV result while macrocytic (macro = large ; cytic = cell) red cells increase the MCV result.

If an MCV for a male or female is 120 fL, the red cell population is considered macrocytic; however, an MCV of 98 fL is considered macrocytic only for a male.

A variation in the sizes of red cells is called anisocytosis

(an = without ; iso = same ; cyto = cell ; osis = condition of).

In other words, a condition of "unlike" cells.

ACTIVITY

Consider the effect of the diluent on the size of the RBC.

1. If the RBC size is responsive to its supporting media, what would be the outcome if red cells are suspended in water?

2. What would be the outcome if red cells are suspended in a highly concentrated salt solution?

3. What do you think the saline concentration in Coulter's diluting fluid (ISOTON(r) III) is?

ACTIVITY ANSWER KEY

4. In effort to equilibrate the internal and external environment, the RBCs tend to take in water from the suspending media, swell and burst.

5. The opposite effect would be observed. The "water" from inside the cell will pass through the membrane into the suspending media thereby shrinking the RBC. In this case, the RBC volume (MCV) would be decreased.

6. COULTER ISOTON III diluent has been formulated to best mimic plasma to keep cells in their near-native state.

MCH

Mean Corpuscular Hemoglobin (MCH) describes the average weight of hemoglobin in the red blood cell.

Hgb

The calculation for MCH is ----------- x 10

RBC

MCH is directly proportional to the size of the red blood cell and the concentration of hemoglobin in the cell.

The Reference Range for MCH is 27 to 31 pg

(picograms or g / micro-micrograms).

MCHC

Mean Corpuscular Hemoglobin Concentration (MCHC) describes the average concentration of hemoglobin in the red blood cells.

MCHC gives the ratio of the weight of the hemoglobin to the volume of the red blood cell expressed in a percentage.

MCHC is expressed as an average concentration by dividing the hemoglobin value by the hematocrit and multiplying by 100.

Hgb

The calculation for MCHC is ---------- x 100

Hct

The Reference Range for MCHC for males and females is 33 to 37%.

One of the best parameters for indicating either instrument malfunction or an improper or unusual specimen.

Calculation is dependent on all measured RBC parameters [Hgb and Hct (which is based on the RBC and MCV)].

Any problem with the Hgb, RBC or MCV is often reflected by an abnormal MCHC.

Specimens with MCHCs outside the normal range should therefore be suspected.

An MCHC above 38% should never occur.

An MCHC should never fall below 22% even when hypochromia is present.

If the frequency of either high or low MCHCs increases, an instrument problem is most likely present.

RBC Evaluation

The hematology technologist evaluates red cells by their form, shape and appearance as observed through a microscope.

Morpho is a prefix that means form, shape and appearance; logy is a suffix meaning the study of. Therefore, the study of red cell size, shape and appearance is red cell morphology.

The presence of a variety of shapes of red cells is called poikilocytosis.

ACTIVITY

7. View Kodachromes of the normal and abnormal RBCs and classify as Normochromic, Hypochromic, Normocytic, Macrocytic, Microcytic, Anisocytosis or Poikilocytosis.

8. Describe what the following terms indicate about the RBC:

Normochromic ______________________________________

Hypochromic _______________________________________

Normocytic ________________________________________

Macrocytic _________________________________________

Microcytic _________________________________________

Anisocytosis _______________________________________

Poikilocytosis_______________________________________

ACTIVITY ANSWER KEY

Normochromic

RBCs having normal color / MCH normal

HypochromicRBCs having less than normal color / MCH decreased

Normocytic

RBCs having a normal size / MCV normal

Macrocytic

RBCs larger than normal / MCV increased

Microcytic

RBCs smaller than normal / MCV decreased

Anisocytosis

RBCs varying in size

Poikilocytosis

RBCs varying in shape

LECTURE / DISCUSSION

Reticulocytes

A reticulocyte is the last stage before a red cell is considered mature.

A reticulocyte, when stained with Wright Stain, has a bluish cast to it and is slightly larger than a normal mature RBC.

Reticulocytes have residual RNA, which when stained with a supravital stain (a stain that stains the cells while they are still living) becomes visually evident when viewed microscopically.

New Methylene Blue is an example of a commonly used supravital stain.

A normal reticulocyte count is considered to be approximately 0.7 - 2.8% of a normal RBC count.

Platelets

Platelets may also be referred to as Thrombocytes or Plts

Figure 6 Formation of Platelets

The normal life span of a platelet is 10 days + 1 day

Refer to the Development of Blood Cells chart. Locate the Megakaryoblast on the far right and top of the page. Notice as the cell matures, small pieces of the cytoplasm "bud" off (platelets).

The clotting process is an extremely complex system.

Coagulation instruments can test for various components in this system. It is important to be aware that platelets play a significant role in the clotting process.

The Reference Range for a Platelet count is about 150,000 - 400,000 cells/L.

Since platelets are consumed in the clotting process, clotted blood specimens or poorly collected fingerstick samples have a low platelet count. It is unacceptable to use clotted blood samples for hematology studies.

Platelets appear in the peripheral blood as small, disc-shaped cellular fragments about 2 to 4 microns in diameter. RBCs are larger than platelets.

White Blood Cells

White Blood Cells are also referred to as Leukocytes or WBCs The normal life span of a WBC is 11 days in the bone marrow to mature then hours to years in the tissues.

The Reference Range for a WBC count is 4,000 to 10,000 cells/L.

Leukocytes can be classified or differentiated in a number of ways. The most common is morphologically (form, shape and appearance when looked at through a microscope).

Types of WBCs

Mononuclear

Lymphocytes

Monocytes

Polymorphonuclear

Granulocytes

-Neutrophils

-Eosinophils

Basophils

Figure 7 White Blood Cells

ACTIVITY

Refer to the Development of Blood Cell chart.

Observe the different coloration of Neutrophils compared to Basophils compared to Eosinophils.

Observe the granulocytes from the immature stem cell to the more mature cell.

Notice the nucleus change from a large round nucleus in the immature cells to a "multi-lobed" nucleus in the mature cell.

LECTURE / DISCUSSION

Mononuclear Leukocytes

Lymphocytes

Lymphocytes are produced in the bone marrow and migrate to the lymph tissues (lymph glands) where they become involved with the body's immune function.

These cells can be subclassified by use of "cell surface markers" as T cells, B cells or Natural Killer (NK) cells.

Classifying lymphocytes is very important in the diagnosis and management of AIDS patients.

Determining cell surface markers can be described as "fingerprinting" the lymphocytes. This type of testing uses antibodies manufactured against certain antigens on the lymphocyte cell membrane (see Figure 3).

The antigens, in this case, are proteins attached to the lymphocyte cell membrane.

Antibodies are special proteins that attach to only one specific type of antigen . . . like a lock and key).

Certain kinds of lymphocytes have specific kinds of antigens, so if we use a known type of antibody and it attaches to the antigen on the cell, we can identify the type of lymphocyte it is!

A flow cytometry instrument can perform this special type of lymphocyte subclassification.

Monocytes

Monocytes are produced in bone marrow but they migrate to body tissues to serve as scavenger cells that "eat" foreign particles and digest them.

Polymorphonuclear Leukocytes

Granulocytes

Granulocytes are polymorphonuclear cells meaning the nucleus has more than one lobe.

Described by the granules in their cytoplasm and their staining characteristics when stained with Wrights/Giemsa stain.

Neutrophils - granules stain "neutrally" and appears as a slight pink color in the cytoplasm

Eosinophils - granules stain a reddish / orange color

Basophils - granules stain dark bluish-black

Granulocyte functions differ in relation to their granule content.

Neutrophils - granules contain digestive enzymes to destroy bacteria

Eosinophils - granules contain histamine and enzymes and are involved in the latter stages of inflammation, allergic reactions and parasitic reactions

Basophils - granules contain histamine, heparin and heparin like substances; although the function of basophils has been debated, they do seem to be associated with allergic reactions

WBC Differential Analysis

WBC differential analysis, which is one of the fundamental analyses of hematology, is a diagnostic tool that can act as a pointer to aid the physician in the diagnosis and/or monitoring of a multitude of disease states.

In the differential blood count procedure, leukocytes are identified and manually enumerated from their morphological appearance on stained blood smears or by automated instruments that use a combination of cellular characteristics and histochemical reactions.

Relative Numbers (%)

Differential results are generally reported in percentages which means the enumeration of a particular type of white blood cell is actually relative to all the other cell types that are present.

In other words, a lymphocyte percentage of 40% means that in the total number of white blood cells counted (100%), 40% of those cells were lymphocytes while the other 60% of the cells were some other type of white cell.

Relative numbers expected in normal circulating blood:

Neutrophils50% to 75%

Lymphocytes20% to 45%

Monocytes3% to 11%

Eosinophils1% to 3%

Basophils0% to 1%

Absolute Numbers (#)

Another way to report differential results.

Individual absolute numbers are computed based on the total WBC count and the individual differential percentage (%).

The availability of absolute numbers helps resolve the difficulties sometimes posed by the use of percentages alone.

For example, if the laboratory's WBC normal range lies between 5,000 and 10,000 and the lymphocyte range is between 20% and 40% of the total count, then the normal absolute lymphocyte count should be between 1,000 and 4,000/L.

Patient APatient B

Lymphocyte %90%90%

Lymphocyte #13,5003,600

WBC / L15,0004,000

Lymph% x total WBC = absolute number of lymphs

Patient A and B both show a relative lymphocytosis of 90%. However, only Patient A has an absolute lymphocytosis with a lymphocyte count of 13,500/L. Patient B, on the other hand, has a normal absolute lymphocyte count of 3,600/L.

In 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. On the other hand, an absolute increase in lymphocytes means there is an actual increase in the production of lymphocytes.

Terminology

The suffix penia means a severe decrease; therefore, leukopenia indicates a decrease in the number of WBCs.

The suffix cytosis means an increase of; therefore, leukocytosis indicates an increase in the number of WBCs.

Granulocytosis indicates there is an increase in granulocytes; lymphocytosis, an increase in lymphocytes.

Lymphopenia indicates a decrease in lymphocytes; granulopenia, a decrease in granulocytes.

Because of the relative relationship among the white cells, granulopenia is generally accompanied by lymphocytosis while granulocytosis is generally accompanied by a lymphopenia.

Laboratory Measurement of Blood

Specimen Collection

A Technologist or Technician uses a peripheral blood specimen to study the blood cells in vitro (outside the body).

Specimens are typically collected by a phlebotomist who is specifically trained in blood collection.

The phlebotomist may obtain the specimen from a vein (venipuncture) or from capillaries (skin puncture of the finger, heel or ear lobe).

A venipuncture collected in an evacuated collection tube is the preferred specimen.

The normal inclination of blood is to clot once released from the natural environment of the body . . . this is nature's way of protecting us from bleeding.

If a patient has blood collected into an empty tube, the platelets and special clotting proteins cause the sample to clot.

All cell types become enmeshed in the clot.

The straw colored fluid that is expressed from the clot is called serum.

Serum is typically used in chemistry studies in the laboratory.

If blood is drawn into a tube containing a special chemical additive to prevent clotting, the blood remains in a fluid state.

This chemical additive is called anticoagulant (to prevent coagulation or clotting).

If anticoagulated is allowed to sit without mixing for several hours , the clear fluid that appears in the upper portion of the tube is the plasma.

Anticoagulants

Anticoagulants may be dry-powder form or liquid.

There are a variety of anticoagulants used and can be identified by the color of the tube stopper.

In hematology, the anticoagulant of choice is K3EDTA (ethylenediamine tetra acetic acid) and is found in a purple or lavender stoppered tube.

Calcium is a required component to the clotting process. EDTA has the ability to bind up calcium in the blood. This process of binding up calcium is commonly referred to as "chelating calcium." The bottom line . . . no calcium, no clotting!

Most anticoagulants (Citrate, Oxalates) work by the same mechanism as EDTA (binding calcium to prevent clotting.)

Heparin acts as an antagonist to the normal clotting reactions of the clotting cascade.

The choice of anticoagulant in Hematology is determined by what mixes quickly and efficiently with the blood and also what maintains the cells in their near native state.

Beckman Coulter recommends K3EDTA as the optimal anticoagulant because all testing and studies done on our instrumentation was performed using K3EDTA samples.

K3EDTA is cited in our instrument manuals as the preferred anticoagulant; however, the major manufacturers of blood specimen tubes are in the process of converting from glass to plastic tubes containing the anticoagulant K2EDTA. Therefore, in the near future, testing and claims made by Beckman Coulter Inc. will cite K2EDTA as the preferred anticoagulant.

ACTIVITY

Observe the variety of tubes available. Notice the presence/absence/volume of anticoagulant associated with the color stoppered tube (VACUTAINER and HEMOGARD by Becton-Dickinson as well as VENOJECT by Terumo).

Observe the corresponding blood filled tubes (2 of each; mix only one). Associate the effects of the presence/absence of anticoagulant and observe the difference between serum and plasma.

TUBE / ANTICOAGULANT SUMMARY

Tube Color StopperAnticoagulantDepartment Where Commonly Using

RedNoneChemistry

Blood Bank

LavenderEDTA (Na2 , K2 , K3)Hematology

Flow Cytometry

BlueSodium CitrateCoagulation

GreenHeparinChemistry

YellowACD (Acid Citrate Dextrose)Flow Cytometry Blood Bank

GrayPotassium Oxalate-Sodium FluorideChemistry

LECTURE / DISCUSSION

Specimen Collection Devices

To increase safety for laboratory personnel, manufacturers are producing stoppers different from the traditional rubber stopper in an effort to minimize aerosolization and splattering of blood when removing the stopper. (HEMOGARD and Terumo tubes are examples.)

For children, babies and difficult patients, microcollection devices allow the finger, heel or earlobe to be pricked and blood collected manually.

Be aware of the potential for clotting in this type of specimen.

Specimen Collection Precautions

Blood tubes must be properly filled to assure the appropriate blood to anticoagulant ratio is maintained.

When blood is drawn with a syringe, it must be transferred to a blood specimen tube. When this transfer occurs, it is very easy to either overfill or underfill the specimen tube.

Effects of Overfilling a Specimen Tube

Overfilling overcomes the function of the additive / anticoagulant.

Overfilled tubes are not properly anticoagulated and, as a result, microclots can form.

Any clotting in a specimen tube makes it an unacceptable sample for hematology and analysis on any Beckman Coulter Hematology Instrument. The specimen must be redrawn.

Effects of Underfilling a Specimen Tube

Underfilled specimen tubes have an excess of anticoagulant.

Excess anticoagulant may cause cells to crenate and shrink.

Sample Handling

Varies depending on the type of tube and the testing plans for the sample.

Department requirements / storage requirements:

Chemistry

Coagulation

Flow Cytometry

Hematology

Mixing Requirements

Equilibration cells go through in response to the anticoagulated environment. ( shrink / swell )

Key Points

Laboratory results from any instrument are only as good as the specimen they come from.

Never overlook the integrity of the sample(s) being run through the system.

CBC, the Complete Blood Count

One of the most frequently requested laboratory tests.

Patient results are considered against a "Normal Range."

Normal Range is established in the laboratory by taking a sampling of population to determine the average number and quantity of each CBC parameter.

Once the average is established, a range is mathematically determined by calculating a 2SD (Standard Deviation) from the mean of the collected data.

On printed tickets from Coulter (and in any Hematology text), normal ranges are given but are generic.

CBC typically includes:

A WBC differential includes:

Clinical utility of a CBC:

Methods of Performing a CBC

Manual, semi-automated and automated methods are available for performing a CBC.

Manual Methods

Locate the procedures for manual blood cell counts starting on page 89 of your textbook, HEMATOLOGY: Principles and Procedures by Barbara Brown.

Hemoglobin Measurement

When a specified volume of red blood cells is exposed to a chemical (lytic agent) 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 of the "lysed" solution can be measured spectrophotometrically.

By comparison with standard solutions, a value for hemoglobin is calculated.

Procedures for a manual hemoglobin determination starts on page 83 of your textbook, HEMATOLOGY: Principles and Procedures by Barbara Brown.

Hematocrit Measurement

When an anticoagulated whole blood sample is placed in a straw like glass tube and centrifuged, the ratio of volume of RBCs to volume of whole blood can be determined. This is defined as the hematocrit.

The procedure for a manual hematocrit determination begins on page 85 of your textbook, HEMATOLOGY: Principles and Procedures by Barbara Brown.

Calculation of the RBC Indices

RBC Indices can be calculated manually by inserting the RBC, Hgb and/or Hct results into the following equations:

MCV=( Hct / RBC )x10

MCH=( Hgb / RBC )x10

MCHC=( Hgb / Hct )x100

WBC Differential

Manual Microscopic Method

The procedure for a manual differential count starts on page 102 of your textbook, HEMATOLOGY: Principles and Procedures by Barbara Brown.

Indicate what the following terms refer to in terms of the associated parameter and whether high or low:

Hypochromic_________ parameter would be _________.

Macrocytic_________ parameter would be _________.

Leukocytosis_________ parameter would be _________.

Anemia_________ parameter would be _________.

Thrombocytosis_________ parameter would be _________.

ACTIVITY ANSWER KEY

HypochromicMCH parameter would be LOW.

MacrocyticMCV parameter would be HIGH.

LeukocytosisWBC parameter would be HIGH.

AnemiaRBC parameter would be LOW.

ThrombocytosisPLATELET parameter would be HIGH.

LECTURE / DISCUSSION

Most laboratories no longer perform their daily CBCs using the manual methods to measure WBCs, RBCs, Hgb, Hct, platelets and calculate the RBC indices.

ACTIVITY

9. List potential sources of error when performing a manual CBC:

__________________________________________________

__________________________________________________

__________________________________________________

__________________________________________________

10. Based on your knowledge of COULTER automated hematology systems, list the advantages of an automated CBC:

__________________________________________________

__________________________________________________

__________________________________________________

__________________________________________________

ACTIVITY ANSWER KEY

11. Answers may vary:

Dilutions may be made improperly or with incorrect reagents

Dilutions are incorrectly charged on the hemacytometer

Inconsistent methods of counting cells on the hemacytometer from tech to tech

Fatigue in counting

Hematocrit sample may be drawn up into the microhematocrit tube incorrectly yielding bubbles

Improper centrifugation of the sample

Inconsistent reading of the results from tech to tech

Hemoglobin calibration is incorrect

Reagent expired

Sample dilution error

WBC Differential inconsistently read from tech to tech (very subjective); poor quality of wedge smear and/or staining

12. No subjectivity, better precision, accuracy, consistent pipetting and dilution preparation, consistent counting / measuring method.

LECTURE / DISCUSSION

The Laboratory

Departments

A typical hospital laboratory consists of many departments.

Hematology, Chemistry, Blood Bank, Urinalysis, Coagulation, Flow Cytometry, etc.

Licensing Agencies

ASCP (American Society of Clinical Pathologists)

ASCLS (American Society of Clinical Laboratory Science) formerly ASMT (American Society of Medical Technologists)

NCA, National Certification Agency for Medical Laboratory Personnel

ISCLT, International Society for Clinical Laboratory Technology

HEW (Health, Education and Welfare)

Personnel

Titles associated with different laboratorians:

MTMedical Technologist 4 year degree including 12 months of clinical training / licensed

MLTMedical Laboratory Technician 2 years of college credit including 12 months training in an approved hospital-related school / licensed

CLACertified Laboratory Assistant 12 months training in an approved hospital related school / licensed

PhlebotomistIndividual responsible for collecting blood samples for the laboratory

Key Operator

As a Coulter 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 of the instrument and its performance is referred to as the Key Operator. Most of the time, this person has attended a Customer training course at the Education Center.

Although we recognize that the Supervisor or the Laboratory Director and/or Pathologist have an interest in the COULTER instrument and its operation, you should always direct your communication initially with the "key operator".

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