HEMATOLOGY NOTES

HEMATOLOGY - the study of blood-deals with the cellular elements of peripheral blood and bone marrow and components of plasma that function in coagulation and fibrinolysis.

HEMATOPOETIC SYSTEM -organ complex associated with the morphologic components of blood and its formation and function

VASCULAR SYSTEM -entire arrangement of vessels operative in circulation of all body fluids

HEMATOLOGICAL TESTS AND SPECIMEN COLLECTION:

SPECIMEN COLLECTION: PHLEBOTOMY – the act of making an incision or puncture into the blood vessel to acquire or

draw blood. CAPILLARY BLOOD – blood obtained from the end or side of the distal phalanx, in

adults; the lobe of the ear or the toe, heel or skin in the forearm in infants.- a mixture of venous, arterial and capillary blood

EQUIPMENT: i. Lancet

ii. Cotton balls/ gauzeiii. 70% Isopropyl Alcohol

PROCEDURE: cleanse the area with 70% alcohol, and then with a sterile lancet puncture the lateral aspect of the ventral part of the finger to obtain blood.

PRECAUTIONS: 1. Use lancets designed to penetrate no deeper than 2mm.2. Avoid squeezing the extremity to obtain blood since this will alter

the composition of blood3. If blood is difficult to obtain, warm the extremity or allow it to

remain in a hanging position for some time.4. When obtaining blood from infants less than 1 year of age blood

is obtained from the heel of the foot. The site chosen should be on the inside (medial) or outside (lateral) portion of the bottom of the planar surface of the foot. An imaginary line may be drawn from the middle of the big toe to the heel and a line from between the 4th and 5th toes to the heel. The area outside these two lines are considered acceptable phlebotomy site.

5. White blood cell count is increased in excessively crying patients.

ADVANTAGES:1. Obtained with ease2. Preferred site in making peripheral blood smears.3. Can be used to draw blood from a badly burned patient

DISADVANTAGES:1. Only small amounts of blood can be obtained and examinations

from the same specimen are considered invalid2. Hemolysis is frequent3. Tests on capillary blood cannot be compared with venous blood

4. Difficult to sterilize5. Difficulty in standardization6. Dilution of blood with tissue juice

VENOUS BLOOD – the specimen of choice for haematological tests. Obtained from one of the three veins in the anticubital fossa (median cubital being the primary vein chosen, the cephalic vein being the second preferred vein and the basilic vein the least chosen vein(because of the risk of puncturing the underlying nerve that is in close proximity to the vein as well as puncturing the artery.))

EQUIPMENT:i. Needle – the needle’s gauge used is determined by the

condition of the patient’s vein and volume to be drawn. The larger the gauge number the smaller the bore of the needle.

ii. Tourniquetiii. Evacuated tubes – tubes that have been designed for blood

collection purposes.iv. 70% isopropyl alcoholv. Gauze pads/ cotton balls

vi. Tape PROCEDURE: a tourniquet is applied 3-4 inches away from the cubital fossa.

Using a sterile syringe, butterfly syringe or vacutainer needle, the vein is punctured at an angle of 15 degrees to 30 degrees from the desired site and blood is drawn until desired amount.

PRECAUTIONS: 1. When the ankle vein is the only palpable vein, a doctor or nurse

must be consulted in order to continue with the venipuncture2. Areas with hematomas or scarring should be avoided since it

heightens the risk of infection3. An arm on the same side of a mastectomy must also be avoided,

since it may cause lymphostasis (obstruction of normal flow of lymph)

4. The order of draw must always be followed5. Excessive EDTA causes RBC shrinkage (HCT and ESR dec.)6. One must be aware of the different coagulants in each tube. List of

common tubes used in the laboratory: RED – no additive; clotting tube; some have silicone

coating that accelerate clotting GRAY – inert polymer barrier additive; cleaner separation

of clot and serum; used for CO2 analysis ORANGE – thrombin; accelerates clotting; stat chemistries GRAY – iodoacetate or sodium fluoride or potassium

oxalate; inhibits glycolysis; used in glucose and BUN enzymatic method (unacceptable for sodium and potassium det.)

GREEN – sodium/lithium/ammonium heparin; enhances antithrombin III; inactivates clotting factors

BROWN – sodium heparin; enhances antithrombin III; inactivates clotting factors (has minute amounts of lead)

ROYAL BLUE – none; none; used for trace elements det. LAVENDER – EDTA; binds calcium; routine hematologic

tests BLUE – sodium citrate; binds calcium; APTT, PTPA YELLOW – SPS; __; microbiological testing

ADVANTAGES:1. Repeated examinations can be done on the same specimen2. Aliquots can be drawn for future reference3. No variation from specimen taken from different veins4. Fastest method of collecting specimen from a large number of

patients5. Reduce possibility of error due to tissue juice

DISADVANTAGES:1. Requires more prep time2. Difficult to perform in infants, obese individuals, and patients in

shock3. Hemolysis leads to lowered cell counts4. Hematoma may develop (hematoma –localized mass of

extravasated blood that is relatively or completely confined in an organ, tissue or space)

5. Hemoconcentration may occur (hemoconcentration – decrease in the volume of plasma in relation to the number of blood cells.

6. Collapsing veins7. Possibility of thrombosis (thrombosis – clotting within the blood

vessel) ARTERIAL BLOOD – commonly done using the brachial, radial, and femoral

arteries. This type of specimen is mostly used for chemistry testing EQUIPMENT:

i. Syringe with needle( needle size is 1 ½ inches long)ii. Gauze/ cotton balls

iii. 70% isopropyl alcoholiv. Tape

PROCEDURE: at an angle of 90 degrees the vessel is punctured to draw arterial blood

PRECAUTIONS:1. Arterial blood is only used for special chemistry tests such as

respiratory gas exam2. After blood has been drawn, the site of puncture must be pressed

for at least 5-10mins, to make sure that bleeding within the arm of the patient has stopped.

BONE MARROW BIOPSY – taken from the bone marrow of flat bones of the sternum or iliac crest. It has a rusty-red color and has a thick fluid like consistency with varying amounts of fatty material and pale gray-white marrow fragments.

EQUIPMENT:i. Syringe with special type of needle. The preferred needle is

a simple, short, rigid needle with a 1-2 mm diameter lumen, tight fitting stylet, short bevel and sharp point with a

permanent removable guard to prevent excessive penetration.

Types of Needle:a) Vim-Silverman – used for surgical biopsy

of bone marrow when aspiration has been unsatisfactory. Needle has a cutting edge.

b) Westerman -Jensen – supplied with finger grips, an assembly stylet and an obturator that locks into position. It is however easily bent and blunted from frequent use.

c) Jamshidi biopsy aspiration needle – has a uniform external cylindrical configuration with a core of substantially constant internal diameter except for the tapered distal portion which is bevelled and has a sharp cutting edge.

ii. Anticoagulant which is usually EDTA OR HEPARINiii. Glass slides

PROCEDURE: done only by physicians and registered bone marrow therapists

PRECAUTIONS:1. Good bone marrow smears are characterized by thinness,

their nucleated cells almost touching one another, and their elements present in high proportion with visible fatty areas and concentrated marrow foci in many areas.

OTHER THINGS TO REMEMBER: PATIENT POSITION:

a) Sitting Position: preferred for venipunture since it lessens alterations in most lab tests

b) Lying Down: causes Hemodilution and can decrases the Hematocrit by an average of 8%

c) Standing Up: causes Hemoconcentration

ORDER OF DRAWa) Syringe Method: Sterile (Yellow), Coagulation (Blue), Additive

(Green, Lavender, Gray), Non-additive (Red) or SPS, Citrate, Heparin, EDTA, Fluoride, Clotting tube

b) Vacutainer Method/ Butterfly: Sterile (Yellow), Non-additive (Red), Coagulation (Blue), Additive (Green, Lavender, Gray) or SPS, Clotting tube, Citrate, Heparin, EDTA, Fluoride

c) Capillary Method: Specimen for manual counts, PBS, anticoagulant tubes with EDTA tubes first.

CITRATE/ BLUE TUBE:- CHOICE FOR COAGULATION TESTS- Preserves Factor V and Factor VIII (labile factors) better than any other

anticoagulant.- Samples anticoagulated with Citrate is more sensitive to the effects of

heparin thus preferred for monitoring heparin therapy- STANDARD RATIO: 9:1 (blood:citrate)- Comes in two forms:

a) Disodium Citrate – the ratio is 1 part of 3.8% solution to 4 parts blood. This is only useful in ESR and investigation of clotting mechanisms.

b) Trisodium Citrate – 1 part of 3.8% aqueous solution to 9 parts whole blood. Useful in blood coagulation and platelet function studies.

- this tube is used for the following important assays: =coagulation testing=fibrinogen level det.=G-6PD det.=PTT=PTPA=TT

Hematocrits with a level of 0.55 L/L or partial filling of tube may cause a falsely prolonged clotting time (PT and PTT) since the standard calcium used to recalcify plasma is inadequate to inactivate excessive citrate in addition to intiating clotting of plasma

EDTA/ LAVENDER TUBES- 1 drop of EDTA is enough to prevent coagulation of 5 ml of blood.

EDTA (ethylenediamine tetraacetate) is the anticoagulant of choice for platelet counts and platelet function tests. It also maintains good morphology of blood cells for up to 3-4 hours after blood has been drawn.

- There are three types of EDTA used:a) Versene – disodium saltb) Sequestrene – dipotassium salt, more soluble than

versene. Used in the concentration of 1.5mg/ml.c) Tripotassium salt – liquid form

- Excessive EDTA cause shrinkage of cells. Resulting in decreased Hematocrit, increased MCHC and low ESR after 3 hours at room temp.

- EDTA IS THE CHOICE COAGULANT FOR PLATELET COUNT- Can not be used for coagulation testing since is inhibits fibrinogen-

thrombin reaction and Factor V is unstable in EDTA- If blood is kept at room temp even at correct concentration of EDTA,

cells will begin to swell between 6-24 hours causing a falsely increased Hematocrit and MCV but low MCHC and ESR. (CANNOT BE OBSERVED WHEN BLOOD IS KEPT AT 4 DEGREES OR REF TEMP)

- Tests requiring the use of EDTA tubes:=CBC

=DIFF COUNT=ESR=G-6PD=HGB electrophoresis=platelet, eosinophil, and reticulocyte counts=sickle cell prep=ZSR

RED TOP TUBES=hgb det.=LE prep=serum viscosity test

DOUBLE OXALATE- Also known as ammonium potassium oxalate, balanced oxalate,

wintrobe’s solution, paul-heller solution.- Most widely used anticoagulant - Used for the PNH TEST- Not used for blood film because of rapid artefact formation and

malformation in the lymphocyte and monocytes nuclei, vacuolation in the granulocyte cytoplasm, oxalate crystal phagocytosis and red cell crenation.

HEPARIN- An antithrombic mucopolysaccharide isolated from mammalian liver or

pancreas that acts with Antithrombin III that inhibits serine proteases of coagulation

- Optimum concentration is 15-20U/mL- Anticoagulant of choice for the osmotic fragility test( because it does not

affect the size of red cells) and platelet retention test- Blood films made from heparin have a light-blue background when

stained with Wright-Giemsa stain- It is used in the concentration of 15-30 units per ml.- Not used for leukocyte and platelet counts due to cell clumping .- Can cause platelet clumping in some individuals thus is not used for

automated processes- Contraindicated for coagulation studies due to its inhibitory action against

thrombin

SEPARATION METHODS: SERUM – liquid portion of clotted blood. It does not contain the clotting factor

fibrinogen and the other labile factors.- Centrifugation accelerates the process of separation to acquire serum from

clotted blood. PROCEDURE:

- Specimen is centrifuged for about 10 mins at a relative centrifugal force(RCF) of 1000-1200 x g.

PRECAUTIONS:

1. Collection tube stoppers should not be removed prior to centrifugation.

2. In general a specimen should not be centrifuged more than once because excessive handling may cause hemolysis.

3. Blood clotting may be accelerated using thrombin tubes(orange tubes)

PLASMA – liquid portion obtained from centrifugation of anticoagulated blood.- Contains the coagulation factors fibrinogen(I), prothrombin(II), labile

factor(V), and antihemophilic factor(VIII).- Reticulocytes can be obtained from the top of the red cell coloum.- Procedure for preparation is the same as serum prep.

PREPARATION OF SPECIMEN: UNSTAINED HEMATOLOGICAL PREPARATION:

WET PREP- Used to examine the physical properties of the tissue in an unchanged

state.- Allows recognition of morphologic red cell changes, abnormal pigments,

and living parasites within red cells.o PROCEDURE:

a. IMPRINT - a microscope slide is gently brought into contact with an unfixed tissue

b. BLOOD SMEAR –1) SLIDE WEDGE – a drop of blood is placed on one

side of the slide, about 0.25 inch from the edge. With a spreader slide with an angle of 25 degrees, the blood is spread towards the end of the slide. A good smear must be about 3cm long, should not extend to the entire slide, should not have holes or waves and leukocytes must not be crowded.

2) COVERSLIP – a drop of blood is placed in the middle of a coverslip held between the thumb and the forefinger. Another coverslip is placed over the first so that the second is rotated 45 degrees from the first. With a constant motion the coverslips are pulled from opposite directions the moment the blood had spread between them. This preparation is preferred to obtain the best possible cell definition and distribution.

3) AUTOMATEDA. MINIPREP – utilizes the wedge procedure

to obtain reproducible blood smears using a precision smear at a constant angle and speed

B. CYTOCENTRIFUGE – allows concentration and spreading of cells in one operation.

STAINED PREPARATION ROMANOWSKY STAIN – composed of Methylene blue( basic fraction), eosin

dye(acidic fraction) and methanol( Mallinckodt methanol). Methanol must not contain water and can be replaced by absolute alcohol. The buffer must have a ph of 6.4-6.8

o CRITERIA FOR A GOOD STAINED SMEAR:1) Smear must be reddish-brown2) Erythrocytes are salmon pink, leukocyte nuclei should be

purple-blue and platelets should have a purple-blue or lilac cytoplasm containing red granules.

HEMATOLOGICAL TESTS:A. ROUTINE HEMATOLOGIC PROCEDURES:

I. CBC (COMPLETE BLOOD COUNT)a) Hemoglobin Determination

o PRINCIPLE: The oxygen-combining capacity of blood is directly proportional to the hemoglobin concentration rather than the red cell count.

o N.V. MALE = 13.5-18 g/dLo N.V. FEMALE = 12-16 g/dLo Most important screening test for diseases associated with

ANEMIA following treatment responseo All hemoglobin determinations are indirect because it is difficult to

crystallize and weigh hemoglobin.o The most common method used by labs is the

Cyanmethemoglobin Method ( blood is diluted into a solution of potassium ferricyanide and potassium cyanide. The potassium ferricyanide oxidizes hemoglobin to hemiglobin and potassium cyanide provide the cyanide ions to form hemiglobincyanide, which is measured at a wavelength of 540 nm) which uses the DRABKIN’S REAGENT since most forms of hemoglobin is measured.

HUFNER’S FACTOR/ OXYGEN-BINDING CAPACITY:- 1g Hgb binds 1.34 ml O2 and 1g Hgb iron binds 401 ml oxygen

thus 1g Hgb contains 0.00347g Hgb iron thus 0.347g Fe/ 100g Hgb

b) Hematocrit Determination/ Packed Cell Volumeo PRINCIPLE: The PCV is the percentage of total blood volume

occupied by packed red cells after centrifugation (10,000-15,000g) at constant speed for a constant period of time (4-5mins)

o N.V. MALE = 40 – 55%o N.V. FEMALE – 37-47%o Inc. In polycythemia, hemoconcentration, dehydrationo Dec. In anemia, pregnancy, excessive hydration

o 1 Hct = 0.34 g hgb/100 ml blood; 1 Hct = 107,000 RBC/ cumm

c) MCV/ Mean Corpuscular Volumeo Average volume occupied by an average red cell in femtoliterso N.V. = 80-100 fLo (Hct /RBC) x 10

d) MCH/ Mean Corpuscular Hemoglobino Average hemoglobin concentration o NV = 26-32 pgo (Hgb/RBC) x 10

e) MCHC/ Mean Corpuscular Hemoglobin Concentrationo Average Hgb concentration per unit volume of PCVo NV = 32-36%o (Hgb/Hct) x 100

f) White Blood Cell Counto NV = 4.5-11.0 x 10^9/Lo WBC/ cumm = # OF CELLS IN 4 SQUARES x VCF x DFo DF (20) = VOL OF FLUID IN BULB (11-1)/ VOL SAMPLE o VCF (2.5) = VOL DESIRED (1ml)/ VOL USED (4 x 0.1)o WBC/ cumm = # OF CELLS IN 4 SQUARES x 2.5 x 20

o Corrected WBC Count (5 or more NRBCs) = (ACTUAL WBC CT/ (100 + #NRBC/ 100 WBC or 1000 RBC)) X100

g) Red Blood Cell Counto NV Male = 4.6-6.2 x 10^12/Lo NV Female = 4.2-5.4 x 10^12/Lo RBC/ cumm = # OF CELLS IN 5 SQUARES x VCF x DFo DF (200) = VOL OF FLUID IN BULB (101-1)/ VOL SAMPLE o VCF (50) = VOL DESIRED (1ml)/ VOL USED (5 x 0.004)o RBC/ cumm = # OF CELLS IN 5 SQUARES x 50 x 200

h) DIFFERENTIAL COUNT WITH RBC MORPHOLOGYi. Red Blood Cell Morphology

o Normal RBC = salmon pink; 7.2-7.9 microns; biconcave; with pale area at the center

ii. Differential Counto Represents the percentage distribution of the different

leukocytes and their qualitative study in blood filmo Corticosteroid hormones cause lymphocytes and

eosinophils to disappear from circulation within 4-8 hours

o Epinephrine causes granulocytosis

o 4 techniques used in scanning:- longitudinal-crenellation-battlement-3-field meander

iii. Platelet Estimateo Represents the estimated platelet count in a blood slide

using the similar technique used in diff count except in ten fields one must count the number of platelets

o Platelets appear as small irregular lilac structures with acidophilic granules.

o NV of PLATELET COUNT: 150-450 x 10^9/L

II. ESR (ERYTHROCYTE SEDIMENTATION RATE)a. Westergren Technique

o Utilizes a 200mm column in which anticoagulated blood mixed with sodium citrate solution is allowed to settle for one hour.

o Standard Method for ESRo N.V. MALE = 0-15mm/hro N.V. FEMALE = 0-20mm/hro This test should not be delayed more than after 2 hours of sample

collectiono An angle of as little as 3 degrees can accelerate the sedimentation rate by

30%o Sedimentation rate increases as temperature increases.

B. SPECIAL HEMATOLOGICAL TESTSI. OSMOTIC FRAGILITY TEST (Sanford Method)

- PRINCIPLE: structural abnormalities and metabolic defects can make the red cell abnormally susceptible to in vitro hemolysis as in in vivo destruction.

- Utilizes 0.5% saline as diluents and 12 (8x75) tubes numbered 14-25, the number on the tube corresponds to number of drops of saline placed inside. A drop of blood is then added. The tubes are centrifuged for 1 min and initial and complete hemolysis noted.

- Complete Hemolysis is usually seen on tube 17 (NV 0.34)- Initial Hemolysis is usually seen on tube 22 (NV 0.44-0.40)- The factor used in computation is 0.02

II. MALARIAL SMEAR PREPARATIONa. THIN SMEAR – same procedure as a normal blood slide. Often

used for morphological examination and species identification of malarial parasite.

b. THICK SMEAR – a drop blood is placed on a glass slide, and carefully spread over a area measuring a dime. It is air-dried and fixed for dehemoglobinization that facilitates examination of parasites. It is used for detection of blood parasites.

III. SICKLE CELL EXAMINATION (SODIUM METABISULFITE)- PRINCIPLE: Hemoglobin is rapidly converted to reduced hemoglobin by

the reducing agent sodium metabisulfite.- Rapid and reproducible with reliable results- Sickled cells are easily distinguished from crenated cells with spiney

projections and are round.- Can not distinguish sickle cell trait from sickle cell anemia.

IV. EOSINOPHIL COUNT- The diluent consist of acetone, urea or propylene glycol- Only the eosinophils can be seen when counting under the microscope

with the hemocytometer, since they stain red.- Thorn Test is a diurnal variation of the eosinophil count seen when the

eosinophils are counted in the morning (8am), with a 20% decrease in the mid morning, and a 30% increase in the evening

V. RETICULOCYTE COUNTa. WET METHOD – using a leukocyte pipette, draw the sample up

to 0.5 mark and BCB or NMB is drawn up to the bulb, and allowed to stand for 15 mins, a smear is made from the solution. Computation: (# of retic/1000) x 100

b. DRY METHOD – 1.5% BCB is placed in 95% alcohol on a coverslip/ slide and allowed to dry, then a small drop of blood is smeared on top of thr dried film. The blood film is placed in a moist chamber up to 10 mins, then reticulocytes are counted.Computation: (# of retic/1000) x 100Corrected Retic: (Pt Hct/ 45(Normal Hct)) x reticReticulocyte Production Index: CRC/2(maturation period)Absolute Retic: (# of Retic counted/ 1000) x red cells

VI. LE PREPARATION- An LE cell is a mature leukocyte that has phagocytosed a large

homogenous mass of lyzed nuclear material- Tart Cell represents the phagocytosis of unlysed or partially lysed nuclear

protein- A positive test is diagnostic of dessiminated lupus erythematosus

HEMATOPOIESIS:

ERYTHROPOIESIS – proliferation and differentiation of red blood cells LEUKOPOIESIS – proliferation and differentiation of white blood cells THROMBOPOIESIS – proliferation and differentiation of platelets HEMOHISTIOBLAST – located in the mesenchyme, gives rise to blood cells

- 25-35 microns, large oval nucleus, one third of cytoplasm is occupied by non-specific granules

HEMOCYTOBLAST – same as hemohistioblast but has a mottled pattern nucleus

PLURIPOTENT CELL – origin of all blood cells; makes up to 10 percent of umbilical cord cells

MEDULLARY HEMATOPOIESIS – cell production within the bone marrow EXTRAMEDULLARY HEMATOPOIESIS – cell production outside the bone marrow

BLOOD AND ITS COMPONENTS:

BLOOD – the total blood volume in an adult is 5 to 6 liters- Blood accounts for 7-8% of the total body weight- Composed of two major portions: a. Plasma (liquid portion) b. Cellular

components- The specific gravity of whole blood is between 1.048 to 1.066 (1.057 in males

and 1.053 in females)- The specific gravity of serum is 1.026 to 1.031 and the red blood cells have a

specific gravity of 1.092 to 1.095- The normal blood ph is 7.35-7.45 (the range compatible with life is 6.8 to 7.8)- The total blood volume in males in terms of body weight is 75.5 ml/kg; in females

66.5 ml/kg- Blood plasma makes up 55% of blood volume (water makes up 91-92% by

weight of plasma, and plasma protein makes up 6-7% by weight of plasma)- Plasma proteins excreted by the liver exert an osmotic pressure of 25-30 mlHg,

these proteins are nondiffusible and are important in the regulation of blood volume, fluid balance, blood viscosity and blood pressure. These plasma proteins are namely:

a. SERUM ALBUMIN – (4%) responsible for maintaining the osmotic pressure in blood

b. SERUM GLOBULIN – (2.7%) is composed of three major fractions:

1. ALPHA FRACTION – associated with the transport of BILIRUBIN, lipids and steroids

2. BETA FRACTION – associated with the transport of IRON and COPPER

3. GAMMA FRACTION – associated with the production of antibodies

c. FIBRINOGEN – (0.3%) is the precursor of the fibrin clot which forms the framework of the blood clot.

- The anabolic substances in blood include inorganic materials such as sodium, potassium, acid carbonate, iodine, urea and iron, and organic substances such as amino acids, glucose, fats, cholesterol and respiratory gases.

- The catabolic substances include urea, uric acid, xanthine, creatinine, creatine, and ammonia.

CELLULAR COMPOSITION:

A. ERYTHROCYTES/ RED BLOOD CELLS/ RBC- LIFE SPAN: 120 days- Loss of nucleus is needed for function (it allows more oxygen to be accumulated)- Measures 6-8 microns in diameter with a salmon pink color in a stained smear- The main functioning unit is hemoglobin (M.W. 66000)- Main function is to transport oxygen to the tissues and to carry carbon dioxide

towards the lungs.- Also contains the enzyme carbonic anhydrase which catalyzes the

transformation of carbon dioxide to carbonic acid- Erythropoietin is an enzyme secreted by the kidneys that stimulate erythrocyte

production Erythrocyte Maturation:

1. Pronormoblast/ Rubriblast Earliest RBC measuring 20 microns 0-5 nucleoli with deep blue cytoplasm that does not contain granules and nucleus has dark areas of DNA 5:1 N:C ratio

2. Basophilic normoblast/ Prorubricyte N:C Ratio is 4:1 measuring 18 microns Has a centrally located nucleus with no nucleoli Intensely basophilic cytoplasm due to RNA activity Chromatin has a Bicycle Pattern

3. Polychromatophilic Normoblast/ Rubricyte 16 microns with a N:C ration of 1:1 Grayish cytoplasm because of hemoglobin production Start of hemoglobin production

4. Othrochromic Normoblast/ Metarubricyte Final Nucleated Stage Has pale acidophilic granules 10-12 microns with pyknotic chromatin

5. Reticulocyte Last Immature Stage No nucleus but has mitochondria and ribosomes Best indicator of Bone Marrow Function Slightly macrocytic and polychromic due to continued production

of hemoglobin6. Mature Erythrocyte

6-8 microns, biconcave Red with clearing in the center Uneven hemoglobin distribution

Hemoglobin - red conjugated protein present in red cells with the main

function of transporting oxygen- occupies 28% of the red cell mass- consists of 1 globin molecule and 4 heme molecules.

(Heme – a ferropotoporphyrin responsible for the color of the entire compound and consists of four protoporphyrin rings, each ring containing 1 iron atom in the center. It is

synthesized by nucleated red cells. The iron present is in the ferrous form)

- When fully saturated each gram of hgb holds 1.34ml of oxygen( 1g hgb=1.34ml oxygen)

- And adult contains approx. 600g of hgb (600g = 800ml)- Forms of Hemoglobin:

1) Oxyhemoglobin – Hgb + O2; Scarlet Red; easily diffusible

2) Deoxyhemoglobin – Hgb without O2 and has not carried CO2; reduced type; dark red

3) Carbaminohemoglobin – Hgb + CO24) Carboxyhemoglobin – Hgb + CO; CO has 200

times more affinity for Hgb than O2; cherry red; smoker’s hemoglobin; irreversible

5) Methemoglobin – Hgb with a ferric iron (Fe3); reversible; chocolate brown colored blood; can produce cyanosis (treatment: Methylene blue); 2 types:

a) Acquired/ Toxic MetHgb: chemical or therapeutic agents that oxidize iron

b) Primary: i. Enzymatic: recessive; deficiency in

one or more enzymes that reduces ferric iron

ii. M type: defect in the globin component; can be absorbed at 602-622nm

6) Cyanmethemoglobin – produced when ferricyanide is added to the ferrous iron of hemoglobin; most stable hemoglobin

7) Sulfhemoglobin – Hgb + sulphides; irreversible; mauvre-lavender blood after vigorous shaking for 15 mins

o Oxygen Dissociation Curve - A sigmoid shaped graph that expresses the ability to load and

unload oxygen by Hgb- The curve is dependent on: pH, pCO2, Temp, 2,3DPG (an

increase in any of the following except pH will move the curve to the right – decreased oxygen affinity)

- P50 value of the partial pressure means hemoglobin is half saturated

- The low affinity of oxygen depends on: Heterogeneous Tetramic Structure of Hgb 2,3 DPG Heme-heme interaction

- 2 Conformations:1. T (taut) – deoxygenated form; has a higher affinity

for CO2 than O2

2. R (relaxed) – oxygenated form; has a higher affinity for O2 than CO2

B. GRANULOCYTES- Motile polymorphonuclear cells exhibiting diapedesis (amoeboid activity of a

granulocyte that allows them to move from the vessel towards the tissues)- The number of lobes indicate the age of the cell- 3 Categories of Granulocytes:

a) Neutrophils:- Average size is 10-12 microns with 2-5 nuclear lobes.- Main function is to engulf (phagocytosis) foreign bodies

b) Eosinophils:- same size as the neutrophil, usually has 2 lobes connected by a

very thin chromatin thread- Exhibits coarse, large, oval granules that are acidophilic in nature.

c) Basophils:- Measures an average size of 8-10 microns, has a kideney shaped

nucleus- has deep purple granules that often obscure nuclear material.

Granulocyte Maturation:1. Meyloblast

Earliest WBC measuring 15 microns 1-2 nucleoli, 4:1 N:C ratio Comprises 1% of bone marrow Begins to produce MPO 18 hours to mature

2. Promyelocyte N:C Ratio is 3:1 measuring 20 microns Contains large non-specific cytoplasmic granules that contain

MPO Comprises 3-4% of bone marrow 24 hours to mature

3. Myelocyte 12 microns, LAST STAGE OF CELL DIVISION Contains MPO and secondary granules containing leukocyte

alkaline phosphatase 11.9 % of bone marrow nucleated cells 100 hours to mature

4. Metamyelocyte Last Mononuclear Stage Nucleus is kidney shaped with dense chromatin Has a prominent golgi apparatus (clear area located at the

indentation of the nucleus) otherwise known as hof area Comprises 18% of cells in bone marrow

5. Band Same size as a mature form (10-12 microns), Last Immature

Stage Cytoplasm is filled with neutrophilic granules N:C ratio is reversed 1:2 with sausage shaped nucleus/ band

shaped Comprises 11% of bone marrow cell, and 0-3% of peripheral cells

C. LYMPHOCYTES- Mononuclear cells that originate and differentiate from the primary lymphoid

organs (bone marrow and thymus) - Has an average diameter of 7-8 microns in the small non motile type and 12-15

microns in the large motile type - Nucleus is circular with chunky deeply basophilic chromatin which occupies

practically all the available space, but also possesses a clear blue rim of cytoplasm- Atypical lymphocytes also called Reactive Lymphocytes are produced from

viral infections, such as infectious mononucleosis that produces Downey Cells, and Turk Cells that also came from a viral infection.

Kinds of Lymphocytes:i. T cells – produced in the thymus, make up 80% of lymphocytes.

They act in cell mediated immunity, obtain information from monocytes, destroy foreign antigens, and produce cytokines. They are differentiated using the CD markers.

ii. B cells – make up 20% of lymphocytes. Identified by their surface antibodies, receptors and protein markers, when stimulated become plasma cells which produce antibodies. Plasma cells have an eccentric nucleus with a wheel chromatin pattern. B cells function in humoral immunity.

iii. LGL (Large Granular Lymphocytes) – large cells with low N:C ratio. All are positive with CD16. Function in the production of interferons, CFUs and IL2.

D. MONOCYTES- Large mononuclear cells originating from the bone marrow with an average size

of 14-19 microns - Precursor of macrophages in inflammatory conditions- Kidney-shaped nucleus- Has criss-crossing nuclear strands- Cytoplasm occasionally contains red-stained Auer Bodies (rod shaped structures

found in the cytoplasm of myeloid cells; an abnormal form of lysosomes that contain peroxidases and phosphatase)