Hematology Lab Final

How many different types of WBCs are there?

5 types: neutrophils, lymphocytes, monocytes, eosinophils, and basophils

What is a buffy coat smear, and what is its purpose?

A buffy coat smear is a slide that is made from the portion of the EDTA specimen that is the middle layer of the blood that contains the WBCs and platelets. This type of blood smear is made when a patient's total WBC count is less than 1000/ul. A buffy coat smear is ONLY a tool to be used to scan a patient's blood for the types of cells that are present when the patient's total blood cell counts are extremely low. Concentration (centrifuging) of these blood cells allows the technician a good view of the blood cells present in the patient's sample, and if there are any "abnormal" cells, the technician can report this to the physician.

How are platelet estimates performed in our student laboratories?

A minimum of 8-10 "fields of view" are used. The number of platelets seen in these 8-10 fields are counted. The average number of platelets is then calculated, and multiplied by 15,000. This final number would be the "calculated" platelet estimate. The "calculated" platelet estimate would then be compared to the "actual" platelet count obtained from the hematology analyzer, and the two must compare within 15% or there is a problem.

What is a normal adult platelet count?

A normal adult platelet count is 150,000-450,000 x 109/L.

What are absolute WBC counts? Why do we calculate them?

Absolute WBC counts are calculated to report to the physician the ACTUAL number of the different types of WBCs found in a patient blood sample. As to why they are calculated: The overall general function of the WBCs is defense of the body. Each individual type of WBC has its own specific function. When a patient has some kind of clinical condition that requires a specific WBC to combat the condition, the physician is very interested in knowing if the patient has a normal number of these WBCs.

Describe a band /stab.

After a time, the metamyelocyte matures into the band/stab form of the granulocytic maturation sequence. Its nucleus can be shaped like a horseshoe, C or S = all sides of nucleus are parallel. This cell is smaller in size than the metamyelocyte and its cytoplasm is pink. The band/stab is a cell that is one stage from being considered a mature cell.

Describe a promyelocyte.

After several days in the blast stage, the cell matures to the promyelocyte stage. It is usually larger in size than the myeloblast, the nuclear chromatin may be as delicate looking as the myeloblast or may show slight clumping, nucleoli are seen but are beginning to fade, PRIMARY, undifferentiated granules are seen in the cytoplasm and on top of the nucleus.

Describe a metamyelocyte.

After the cessation of cell division capability, the myelocyte becomes a metamyelocyte. The traditional characteristic that discriminates a myelocyte from a metamyelocyte is the nucleus. The nucleus of the metamyelocyte starts to indent and takes on a "KIDNEY BEAN" shape, the chromatin continues to clump. This cell is smaller than the myelocyte. By this stage, the cytoplasm of the cell has a complete collection of primary and secondary granules and is pale blue to pink in color. Nucleoli are not visible.

Define apoptosis

Apoptosis is programmed cell death, a normal physiologic process that eliminates unwanted, abnormal, or harmful cells

When making a blood smear, why is it so important to maintain an even, gentle pressure while pushing the "pusher" slide?

Applying too much pressure to the pusher slide will cause larger cells (especially WBCs) to be pushed to the end of the smear. This will cause poor distribution of the cells, and therefore, erroneous patient results.

Explain how to perform a manual WBC estimate from a blood smear

At the high dry magnification, you would scan 8-10 fields at the featheredge, counting the number of WBCs you see in EACH field. Then you get an average of the number of WBCs/field. This average is then multiplied by 2000 to get an adequate approximation of the total WBC count. (WBCs/field x 2000 = total WBC count/ul).

What are the observations performed at the 100X (oil immersion) magnification?

At this magnification the WBC differential count; evaluation of the RBCs, WBCs and platelets; and a manual platelet estimate is performed.

What is the name of the counting technique we are going to use in our student laboratories to count WBCs on a blood smear?

Battlement technique

1. Where are erythrocytes produced?

Bone marrow

Where in the body are neutrophils produced?

Bone marrow

Could there be any reasons to modify the size of a drop of blood when making blood smears? If so, what are they?

Certain medical conditions, i.e. polycythemia, anemia, etc. could be reasons to modify the size of a drop of blood when making blood smears.

Why is a buffy coat smear NEVER used for a differential count?

Concentration (centrifuging) of these blood cells allows the technician a good view of the blood cells present in the patient's sample, and if there are any "abnormal" cells, the technician can report this to the physician. For this very reason, you NEVER, NEVER perform a differential count from a buffy coat smear. The concentration (centrifugation) of the WBCs would not be representative of the patient's condition--the cells are no longer in an "even distribution" pattern in the blood smear.

Why is the anticoagulant used in the hematology department?

EDTA does not distort the morphology of cells, so it is the best anticoagulant for hematology

What is the "unit of measure" reported for WBCs from a differential?

Each type of WBC counted is reported in %'s (% adds up to a total of 100%)

What is the normal life span of the different blood cells?

Erythrocytes (RBCs): 120 +/- 20 days life span in the peripheral bloodNeutrophils: about 10 hours life span in the peripheral blood Eosinophils: less than 8 hours life span in the peripheral blood Basophils: unknown, assume a life span like neutrophils/eosinophils Monocytes: variable, 11 hours-3 days life span in peripheral blood

Define erythropoiesis

Erythropoiesis is a glycoprotein that stimulates the production and differentiation of erythrocytes (RBCs). It is produced primarily in the kidneys, although a small amount is also produced in the liver. EPO responds to hypoxia to maintain adequate levels of erythrocytes to carry oxygen.

1. Define erythropoiesis

Erythropoiesis is defined as the proliferation and differentiation of erythrocytes

1. What is erythropoietin?

Erythropoietin is a hormone that is produced in the kidneys. Erythropoietin stimulates erythropoiesis

What is erythropoietin?

Erythropoietin is the hormone, which produces erythrocytes (RBCs).

List the kind of information that a well-made, well-stained, and carefully examined peripheral blood smear can provide to a physician, about a patient.

Examination of the peripheral blood smear will include the following: WBC/platelet estimates, enumeration of the types of WBCs present, examination for abnormalities of the morphology of all three formed elements.

Define hematopoiesis.

Hematopoiesis involves the processes of the formation, development, and specialization of all functional blood cells.

If clumping of platelets were noticed when reviewing a Wright's stained differential, what would be the proper procedure to follow to be able to report a "corrected" patient platelet count to a physician?

If on reviewing the peripheral blood smear, platelet "clumping" is seen, the actual platelet count will be grossly inaccurate. To resolve this problem and obtain an accurate platelet count, the patient would need to have a blood specimen drawn into buffered sodium citrate (light blue vacuumed tube used for coagulation) and this tube run through the hematology analyzer. The platelet count obtained from this specimen could be used as the "actual" platelet count.

1. Where is erythropoietin produced?

In the kidneys

What is the pH and purpose of the buffer used in the Wright's stain procedure?

It has a pH of approximately 6.8. Maintaining this pH is important in order to have a properly stained blood smear.

manual wedge technique:

It is the most convenient and commonly used technique. Two glass slides are used to make the smears. One slide serves as the "pusher" or "spreader" slide. The other is considered the "smear" slide. A drop of blood is placed on one end of the smear slide. The size of the drop of blood is important. The pusher slide is balanced on your hand at approximately a 30°- 45° angle. The pusher slide is pulled back into the drop of blood. The whole drop of blood is allowed to spread across the pusher slide. It is then quickly and smoothly pushed forward to the end of the smear slide, creating a wedge smear. Maintaining an even, gentle pressure while pushing the pusher slide is important.

How long does it take for a megakaryocyte to mature in the bone marrow?

It takes 4-5 days for a megakaryocyte to mature in the bone marrow.

What is leucopoiesis?

Leukopoiesis can be divided into two major categories: myelopoiesis and lymphopoiesis. Myelopoiesis involves the production and differentiation of the granulocytes (neutrophils, eosinophils and basophils) and the monocytes. Lymphopoiesis involves the production and differentiation of the lymphocytes.

Define leukopioesis

Leukopoiesis is defined as the proliferation (development) and maturation of the white blood cells (WBCs).

What is leukopoietin?

Leukopoietin is the hormone which produces leukocytes (WBCs).

Where does production of the lymphocytes primarily take place?

Lymphocytes are produced primarily in lymphoid tissue (lymph nodes and nodules, thymus and spleen); some are also produced in the bone marrow.

Why should a macroscopic evaluation be performed on all blood smears?

Macroscopic evaluation of the blood smear could indicate hematological abnormalities

Where are platelets formed?

Major platelet production takes place in the bone marrow, where the megakaryocyte takes up less than 1% of the nucleated cells of the bone marrow.

Differentiation in the hematology department means being able to tell the difference between the formed elements. How is the differentiation of the formed elements of the blood performed in the hematology department?

Manual differentiation of the formed elements (RBCs, WBCs, and platelets) is achieved using a bright field microscope and a slide of the blood (known as the differential) stained with Wright's or Wright's-Giemsa stain.

Which of the blood smear techniques listed in question # 11 is the most commonly used technique? Why do you think this is?

Manual wedge technique because it is the most convenient to use.

What is megakaryopoiesis?

Megakaryopoiesis involves the production of the thrombocytes (platelets).

List the three phases of hematopoietic development and at what point each one appears during fetal development.

Mesoblastic phase (yolk sac) The mesoblastic phase of blood cell formation and development begins at about 5 weeks gestation, and ceases at 6-8 weeks gestation. The stem cells produced are directed to the liver. Hepatic phase (liver and spleen) The liver remains the major site of hematopoiesis during fetal life, retaining activity until 1-2 weeks after birth. Medullary phase (bone marrow) At about the fifth month of fetal development, bone marrow begins to develop within the core of the bones and medullary production of blood cells begins. By the sixth month of development, the bone marrow has become the primary site of hematopoiesis.

Name the agranular leukocytes.

Monocytes and lymphocytes

What are the three basic parts of a cell?

Most cells have three basic parts: cellular (unit) membranes, cytoplasm, and a nucleus.

List the advantages/disadvantages of peripheral blood smears made from venipuncture collection.

Most specimens are Na2 (di-sodium) or K3 (tri-potassium) EDTA. This is the purple top vacutainer tube. The action of the EDTA anticoagulant is that it "chelates" (binds) the calcium in the patient's blood, so that clotting of the blood cannot take place. (anti = against, coagulant = clotting). EDTA does not distort the morphology of cells, so it is the best anticoagulant for hematology. The advantage of having an EDTA tube specimen is that a lot of blood smears can be made.

List the terminology used for the neutrophil maturation sequence.

Myeloblast, promyelocyte, myelocyte, metamyelocyte, band/stab, and mature neutrophil (segment, seg, PMN)

Name the granulocytic leukocytes.

Neutrophils, eosinophils, and basophils

1. What two criteria are used to identify RBCs in the RBC maturation sequence?

Nuclear and cytoplasmic maturation

Polychromatic normoblast/Rubricyte

Nucleus: The appearance of the chromatin pattern is quite variable at this stage of development. The condensation of chromatin has reduced the size of the cell considerably. Further condensation can reveal the appearance of chromatin, which ranges from a pattern resembling the spokes of a wheel to no pattern at all. Cytoplasm: The color staining reaction of this cell demonstrates the greatest variety, reflecting the inverse relationship of increasing production of hemoglobin and decreasing amounts of RNA. The color produced is a murky gray-blue.

Basophilic normoblast/Prorubricyte

Nucleus: The chromatin has begun to condense, revealing clumps along the periphery of the nuclear membrane and a few in the interior. The staining reaction is one of a deep purple-red. Cytoplasm: When stained, the cytoplasm is a deeper, richer blue than the blast.

Orthochromic normoblast/Metarubricyte

Nucleus: The nucleus is almost or completely pyknotic. It is incapable of DNA synthesis and is usually ejected from the cell at this stage. Cytoplasm: The cytoplasm reflects the full complement of hemoglobin production and, when stained, turns pink-orange, with a slightly bluish hue.

Pronormoblast/Rubriblast

Nucleus: The nucleus is round to oval, containing one or two nucleoli. The chromatin contains fine clumps. Cytoplasm: The cytoplasm, when stained, is intensely basophilic (blue). Blasts characteristically demonstrate small tufts of irregular cytoplasm along the periphery of the membrane.

Reticulocyte

Nucleus: none Cytoplasm: The cytoplasm can be easily compared with that of the orthochromic normoblast/metarubricyte in that the predominant pigment is that of hemoglobin. Residual RNA, in varying amounts, continues to cast a bluish hue to the cell until eventually the RNA is gone. When reticulocytes in the peripheral blood are observed with Wright's stain, a slightly larger cell is seen which demonstrates either residual RNA by diffuse polychromatophilia or basophilic stippling.

Erythrocyte

Nucleus: none Cytoplasm: The mature circulating erythrocyte is a biconcave disc measuring 6-8 microns in diameter. The RBCs main function is to deliver oxygen to the body tissues and cells. The average life span of the RBCs is 120-days. The interior of the erythrocyte contains 90% hemoglobin, and 10% water.

What is the average lifespan of a platelet?

Once a platelet is released into the peripheral blood, it has a life span of 9-12 days.

What are the granules called which are present in the promyelocyte stage?

PRIMARY, undifferentiated granules are seen in the cytoplasm

What is a manual WBC estimate? Why is it performed?

Performing a manual WBC estimate, and then comparing it to the automated analyzer result is considered an internal quality control check.

What is a manual platelet estimate? Why is one performed?

Performing a manual platelet estimate, and then comparing it to the automated analyzer result is considered an internal quality control check.

List the advantages/disadvantages of peripheral blood smears made from capillary collection.

Peripheral blood smears made from a fingerstick/capillary specimen are normally made in at the patient's bedside or in the outpatient drawing area. Disadvantages include: limited number of smears can be made from this type of specimen, platelets will clump on the slide because the platelets clotting function becomes activated when they come into contact with glass surfaces--this will make it very difficult to estimate their numbers.

Why should all blood smears, once they are made, be dried quickly?

Regardless of the technique used, all blood smears should be dried quickly to avoid artifact (artifact is distorted cell morphology that is not a part of the patient's true condition).

Which WBC cell type is dominant in the peripheral circulation of normal children? Why?

Remember children have more lymphocyte (55-75%) because their immune systems are developing. Remember, lymphocytes are involved in developing immunity. The lymphocyte is the dominant cell type seen in children (this is normal).

10. What are the granules called which are present in the myelocyte stage?

SECONDARY differentiated granules

What types of observations are made about the blood smear on low power magnification?

Technicians can check for the overall quality, color and distribution of the blood cells.

Miniprep:

The Miniprep makes wedge-type smears. This is a small, plastic device in which two glass slides can be placed along with a drop of blood. Then, with a push of a button, two glass blades will make two slides at once.

What is the anticoagulant of choice used in the hematology department?

The anticoagulant of choice used in the hematology department is EDTA (ethylenediaminetetraacetic acid).

1. What is the life span of the RBCs? Why do RBCs die?

The average life span of the RBCs is 120-days.

Describe a basophil

The basophil is characterized by the presence of large blue/black granules unevenly distributed in its cytoplasm.

What is the primary site of hematopoiesis in the adult?

The bone marrow is the major site of hematopoiesis in adults

The bone marrow primarily produces what type of blood cells?

The bone marrow produces the myeloid cells, namely the erythrocytes (RBCs), granulocytes (neutrophils, eosinophils, and basophils), monocytes and platelets.

Describe a mature neutrophil/PMN/segment ("seg").

The cell's nucleus continues to indent until thin strands of nuclear membrane form into segments or lobes, 2-5 lobes are characteristic of the nucleus

What components comprise (make up) the cellular membrane?

The cellular membrane is a fluid-like structure consisting of globular proteins floating in lipids. The lipids are made up of phospholipids and cholesterol arranged in two layers. The cellular membrane has both a hydrophilic (water-soluble) and a hydrophobic (water-insoluble) structure, which enable it to have semi-permeability capabilities.

Describe the appearance of a normal, mature, unactivated platelet.

The circulating platelet is circular to elliptical in shape, and 2-4 um in diameter.

Describe an eosinophil.

The cytoplasm of the eosinophil is colorless to pink, but is normally occluded by the presence of large red/orange granules. It is these granules that are characteristic of the eosinophil.

1. Of what is the membrane of the RBC composed?

The erythrocyte membrane consists of approximately 40% lipids, 52% proteins, and 8% carbohydrates.

What is the function of the erythrocytes (RBCs)? What do they look like on a differential?

The function of the RBCs is to carry oxygen to all parts of the body. Erythrocytes (RBCs) normally appear as reddish-pink , biconcave discs or doughnuts on a differential when stained with Wright's stain.

What is the primary function of the platelet?

The function of the platelet is in primary hemostasis (the stoppage of bleeding).

List the normal maturation sequence for platelets.

The megakaryoblast develops into the megakaryocyte, which then gives rise to the platelet.

What is the function of the monocyte?

The monocytes specific function is phagocytosis of bacteria fungi, viruses, and dead or dying cells. Once activated to perform its specific function, a monocyte is considered a macrophage.

1. What is the major function of the RBC?

The most important function of the erythrocyte is to deliver and exchange oxygen for carbon dioxide to the body.

Describe a lymphocyte

The nucleus of the lymphocyte is round or oval. The cytoplasm of the lymphocyte is a little variable in color. It ranges from a little basophilic to sky blue.

Describe a monocyte

The peripheral blood monocyte is the largest WBC found in the peripheral circulation. Monocytes generally have nuclei that are folded, twisted or indented and irregularly shaped. Its chromatin pattern is a little on the "lacy" side, and not condensed like the lymphocyte's nucleus. Occasionally, nucleoli can be seen in the nucleus. The cytoplasm is filled with minute granules that produce a blue/gray "ground glass" appearance.

How do you perform a manual platelet estimate?

The procedure for manually estimating platelets is the same as it is for manually estimating WBCs. Of course, the multiplication factor is different. I like to use 15,000. (average number of platelets/field x 15,000 = total platelet count/ul). The multiplication factor some laboratories use is either 10,000 or even 20,000. This is why I like using 15, 000--it is the middle of the road.

What is the angle of the "pusher" slide for the wedge technique when preparing a blood smear?

The pusher slide is balanced on your hand at approximately a 30°- 45° angle.

Why is the Wright's stain considered a "polychromatic" stain?

The stain is considered a "polychromatic" stain because it contains more than one type of dye (stain) allowing for the different staining of the blood cells.

What reagents are used in the Wright's (Wright's-Giemsa) stain procedure?

The two reagents in the procedure are a polychromatic stain and a buffer.

What types of evaluations are made at the 40X (high dry) magnification about the blood smear?

This is the magnification at which MOST laboratories use to perform a manual WBC estimate. The featheredge is where this calculation is performed.

coverslip technique

This technique is not routinely used for peripheral blood smears, but it is used for making bone marrow smears. The technique uses two coverslips to make the slides.

semi-automated technique:

This technique uses a spinner slide. It spins the cells to a one-layer thickness.

What is the function of thrombocytes (platelets)? What do they look like on a differential? Platelets are derived from what cell found in the bone marrow?

Thrombocytes (platelets) are involved in helping the blood to clot. Clotting of the blood is known as coagulation. On a differential they look like small blue/black to purple granular bodies, very small in comparison to the RBCs/WBCs. Platelets are not complete cells, but fragments of cells, which have chipped off from large cells called megakaryocytes. Megakaryocytes are found in the bone marrow.

What is thrombopoietin?

Thrombopoietin is the hormone that produces thrombocytes (platelets).

thrombopoietin:

Thrombopoietin is the hormone that stimulates the differentiation and maturation of the megakaryocytes.

How do you calculate an absolute WBC count?

To calculate an absolute count, you first do a 100-cell differential and find the percentages of the five types of WBCs present. Each individual WBC percentage is then multiplied by the total WBC count obtained from the hematology analyzer. This is the absolute WBC count. Each WBC type (neutrophil, lymphocyte, monocyte, eosinophil and basophil) has a normal absolute range.

Why is the size of the drop of blood so important when making blood smears?

Too large a drop creates a very long or thick smear; too small of a drop often makes a short or thin smear

What kind of patient samples can be collected for peripheral blood smear preparation?

Venipuncture, capillary and syringe/needle specimens

What is the function of the leukocytes (WBCs)? How many different types of WBCs are there? List them.

WBCs help the immune system to fight off infection or invasion of the body from parasites, bacteria, viruses, etc. There are 5 different types of WBCs: neutrophils, lymphocytes, monocytes, eosinophils, and basophils.

Describe a myelocyte.

When the cell ceases to produce primary undifferentiated granules, it begins to make SECONDARY differentiated granules that are specific for the granulocytes. Accumulation of secondary differentiated granules is characteristic of this cell. This cell is smaller in size than the promyelocyte, and its cytoplasm is less basophilic and begins to take on a faint bluish/pink hue, the nucleus may be round or oval, nuclear chromatin shows clumping, nucleoli are not normally visible.

What is the name of the most common stain used in the hematology department?

Wright's or Wright's-Giemsa stain is the name of the most common stain used in the hematology department.

What is the name of the most common stain used in the hematology department for staining blood smears?

Wright's stain or Wright's-Giemsa stain

How will the following changes in the angle of the "pusher" slide affect the making of a blood smear?

angle less than 30° : The drop of blood will create a smear that is too long angle greater than 45°: The drop of blood will create a blood smear that is too short

What is the function of the basophil?

basophils are associated with hypersensitivity reactions

What is the color of the cytoplasm of a mature monocyte?

blue/gray "ground glass" appearance

How many platelets can be produced from one megakaryocyte?

each megakaryocyte generally produces between 2000-4000 platelets.

What is the function of the eosinophil?

eosinophils are associated with defending the body against allergic reactions and parasitic infections

1. What are the two broad classifications of WBCs?

granular and agranular (nongranular)

Define hydophilic and hydrophobic.

hydrophilic (water-soluble) hydrophobic (water-insoluble)

Describe a myeloblast.

large basophilic (blue) cell cytoplasm containing NO GRANULES, nucleus is delicate looking (loose and lacy-looking chromatin), nucleoli are present, it is the earliest identifiable cell in the bone marrow. In the bone marrow proliferation pool, these cells take approximately 24-48 hours for a single cell to mature. These cells comprise less than 1% of the normal bone marrow.

What is the function of the lymphocyte?

lymphocytes are involved in defending the body against viral infections

1. Does the cytoplasm of a myeloblast contain any type of granules?

no

What are the functions of the neutrophil? How does it carry out these functions?

phagocytosis and pinocytosis

What is the purpose of staining blood smears?

to better view the blood cells and evaluate their morphology.

What units are platelets reported in?

x 109/L.

What is the abbreviation for a "complete blood count"? What measurements are included in a complete blood count?

· WBC: enumeration of WBCs · RBC: enumeration of RBCs · Hgb: measurement of oxygen carrying capacity · Hct: measurement of packed RBC volume · Platelet: enumeration of platelets · RDW (red cell distribution width): measurement of average size of RBCs · Indices: MCV, MCH, MCHC · Differential: percent of different types of WBCs

What are the approximate normal adult peripheral differential smear values for the following WBCs:

· neutrophils: 55-75% · bands/stabs: 1-10% · lymphocytes: 24-44% · monocytes: 1-10% · eosinophils: 2-5% · basophils: 0-3%

List the features of a well-made blood smear.

· runs 2/3 - 3/4 the length of a slide · very slightly rounded at feather edge · smooth without irregularities, holes or streaks in the feather edge · when the slide is held up to bright light, featheredge should have a "rainbow" look · the whole drop of blood should be used to make the smear