My Hematology I Notes

Topics

• Blood components
• Anticoagulants
• Phlebotomy
• Hemoglobin
• Hematocrit
• Blood Cell Count 
• Blood smearing (peripheral blood film) 
• RBC index 
• Staining

001 Blood and Hematopoiesis

Objective

• To learn the concept of hematology 
• To study the blood and its component 
• To know the process of blood formation (hematopoiesis) 

1. Introduction

Study of blood forming tissues and circulating blood components

2. Blood 

Blood is a complex and unique fluid of variable composition circulating through the heart, arteries, capillaries, and veins, known as the vascular system of the body. 

It is a tissue in which cellular constituents are suspended in a liquid medium performing specialized functions. 

It is composed of 2 main parts. 

Blood cells, the particles suspended in plasma, making up approximately 45% of total blood volume

• Erythrocytes
• Leukocytes
• Thrombocytes

Plasma, the fluid portion of the blood, which consists primarily of water in which are dissolved proteins and many inorganic and organic substances carried by the blood to and from the tissues. It makes up 55% of the blood. 

3. Cellular and fluid elements

Plasma is the clear yellowish fluid obtained upon separating whole blood into its solid and liquid components. 

Blood is heavier than plasma

Water 91~92%

Plasma proteins 6~7%

• liver 
• albumin, globulin, fibrinogen, and prothrombin
• Function 
• maintenance of water balance 
• imparts a viscosity to the blood 
• sources of antibodies 
• blood clotting
• maintain acid-base balance

Blood sugar 

• glucose 
• source of energy 

Lipids

LDL -bad 

HDL

• neutral fats, cholesterol, and phospholipids 
• supply energy or stored as fat 

Inorganic salts

• Calcium ions and sodium ions 
• Salt ions (chlorides, bicarbonates, phosphates, sulfates) 
• Electrolytes (sodium, calcium, potassium, and magnesium) 
• Iodides and iron 

Special plasma substances

• hormones 
• enzymes
• clotting elements 
• antibodies
• urea
• uric acid 
• creatinine 

Gases 

• oxygen 
• carbon dioxide
• nitrogen

Plasma is used for blood banking and hematology

5. Hematopoiesis 

Proliferation of progenitor (originate) cells (in bone marrow), maintained by stem cells and differentiation into all the mature cellular components of blood

6. Sites of Hematopoiesis

Yolk sac

Liver and spleen

Bone marrow

Expansion can occur during increased need for cell production 

Fetus 

0~2 m yolk sac 

2~7 m liver and spleen

5~9 bone marrow

Infants

Bone marrow 

Adults 

Vertebrae, ribs, sternum, skull, sacrum and pelvis, proximal ends of femur

7. Distribution of active marrow

Pleuripotent stem cells

Proliferation, Self renewal, Differentiation

3 month of lifespan 

Myeloid stem cells, Lymphoid stem cells

Generation of different types of blood cells 

• Self-renewal 
• Have the intrinsic capacity for self-reproduction but are low in number and divide infrequently
• Multipotentiality 
• Capacity to generate cells of all the hematopoietic lineages 
• Differentiation 
• Myeloid or lymphoid lineage 
• Differentiate into 5 major of blood cellular components

8. Characteristic of stem cells

9. Progenitor cells

Encompasses from immediate progeny of stem cells to cells committed to one differentiation lineage 

• myeloblast to promyelocyte to myelocyte to metamyelocyte to band neutrophil 

Progenitor cells are responsible for initiating the large amount of cell proliferation required to maintain blood cell production

Late progenitor cells eventually restricted 

10. Mature cells 

Mature cell production takes place from the more developmentally-restricted progenitors 

Neutrophil, basophil, erythrocytes, platelet 

11. Hematopoiesis

Hematopoietic Growth Factors (HGFs)

• Regulate proliferation, differentiation and maturation of hematopoietic progenitor cells 
• Influence the commitment of progenitors to specific lineages
• Affect the function and survival of mature blood cells

Erythropoiesis

Production of erythrocytes is regulated by erythropoietin which is produced by cortex in the kidney

Needs Fe2+, vitamin B12, folate and other elements for development 

Odour: Un

Size: 6~8u in diameter

Shape: round, biconcave 

Content: hemoglobin

Lifespan: 120 days 

Normal morphology: known as normocytic 

Leukopoiesis

Granulopoiesis (granulocytic series)

Granulocytes

Regulated by hematopoietic growth factor called G-CSF (Granulocyte-colony stimulating factor) and interleukin or cytokine (IL-3, IL-5) 

Precursor

Myeloblast

Promyelocytes

Myelocytes

Metamyelocytes

Band form (stab form) 

Neutrophils

3~5 lobes 

40~75%

C/G pinkish purple

N purplish

Non specific defence against bacteria and fungal

Destruction of bacteria and release chemicals that kill or inhibit the growth of bacteria 

The granules in the neutrophil are lysosomes

Neutrophils play a crucial role in combating bacterial infections, their primary function being phagocytosis

The neutrophils at these sites engulf and destroy invading bacteria, and then die; their dead and lysed bodies constitute the bulk of pus at sites of suppurating wounds

Eosinophils

2 lobes (bilobes) 

1~6%

C/G red orange

N purplish

Allergy, Parasite inflammation

Increase in hypersensitivity reactions and to mechanism acting against larval states of parasitic infections 

Eosinophils are much less common than neutrophils

They are the same size or even slightly larger (10 to 14 microns) 

Cytoplasmic granules are strongly eosinophilic, staining a bright orange pink color

Typically the nucleus is bi-lobed, not multi-lobed as in the neutrophils

The eosinophil's granules contain lytic enzymes, but they are much larger than those of neutrophils

The granules stain pinkish red in Wright's smears 

Basophils

Multilobe 

C/G blue black

N dark purplish blue 

Hypersensitivity reaction

Allergy reaction

secrete histamine which increase the blood flow via dilating blood vessel

secrete heparin that promotes mobility of the WBC by preventing clotting 

They have pale nucleus that is usually hidden by granules

They are about the same size as neutrophils, and the nucleus is usually bi-lobed of S shaped

The cytoplasmic granules are coarse, and strongly basophilic

Monopoiesis (monocytic-macrophage series)

Process of production of monocyte and macrophages

Regulated by hematopoietic growth factor called GM-CSF which produced by stromal cells and IL-6

Progenitor stem cells

Monoblast

Promonocyte 

Monocyte

Blood circulation

Agranulocytes 

2~10% 

16~20 m (larger than lymphocytes) 

C: bluish grey

N: dark purple/dark blue 

Remove and process aged red cells and debris 

They function in differentiating into macrophages, which are large phagocytic cells and digest pathogens, dead neutrophils, and the debris of dead cells

The nucleus varies in form, and may be kidney shaped, bean shaped or classically horseshoe shaped, with a deep indentation

The cytoplasm tends to be abundant and relatively clear

Macrophages

Tissues 

Agranulocytes

Kidney, bean, horseshoe shaped 

C bluish gray

N dark purple/blue

Remove

Digest 

Lymphopoiesis

Regulated by hematopoietic growth factor called G-CSF and Interleukin/Cytokine IL 12357

Primary lymphoid organs

Bone marrow

Thymus

Secondary lymphoid organs

Lymph nodes

Spleen

Lymphoid tissues 

Lymphocytes

Agranulocytes 

20~50%

6~15 m

C: sky or dusky blue

N: dark purple/blue

N>C 

Humoral immunity

Cell mediated immunity

Lymphocytes function in destroying cancer cells, cells infected by viruses, and foreign invading cells. In addition, they present antigens to activate other cells of the immune system

Lymphocytes can be identified in smears by their very high ratio of nucleus to cytoplasm

The nucleus is very densely stained, and usually round although it may have a very slightly indentation

The cytoplasm is a thin band to one side of the nucleus, and is stained a dusky blue color

B lymphocytes

T lymphocytes

NK cells

Thrombopoiesis

Regulated by thromopoietin which are largely made in the liver 

Megakaryoblasts

Primary hemostasis

Lifespan 7~10 days

Production, fragmentation of cytoplasm undergoes endomitotic division/synchronous nuclear replication

150, 000~400,000 mm3

1,5~3.0 m

Purplish color

Coagulation/arrested bleeding 

002 Anticoagulant

Objective

• To know the various types of anticoagulants presence in the vascular tubes 
• To describe the types of anticoagulants

1. Definition

Anticoagulant is an additive substance that added into the blood collection tube 

2. Vacutainer 

specifically designed for venipuncture

Container containing coagulant/non additive tubes 

Container containing anticoagulant

Red: plain tube, coagulant for clotting

Blue: clotting factors

Purple: EDTA 

Green: Heparin, cardiovascular

3. Characteristics of anticoagulants

must not alter the size of red blood cell 

must not cause hemolysis

minimize platelet aggregation

minimize disruption of the staining and morphology of leukocytes

readily soluble in blood

4. EDTA

EDTA refers to the chelating/sequester agent with the formula (HO2CCH2)2NCH2CH2N(CH2CO2H)2

EDTA binds to metals via 4 carbohydrate and 2 amino groups

2 forms of EDTA anticoagulants are used: tripotassium salt (K3EDTA) and disodium salt (Na2EDTA)

The potassium salts (liquid or dry powder) are used in commercial tubes because they are more soluble 

Mode of action

forms insoluble calcium salts by chelation process

0.5~2.0 mg EDTA per mL will preserve blood excellently for at least 6 hours 

refrigeration will extend the preservation to 24 hours 

Uses 

blood smear for cell morphology studies 

perform blood cell count, plasma protein, fibrinogen, glucose, Coomb's Test 

preserves the staining and morphological characteristics of leukocytes

Disadvantages

excessive concentrations cause shrinkage of RBC and erroneous PCV, MCV, MCHC results

fill the vacutainer with required quantity of blood 

hemoglobin concentration is not affected

interferes with blood chemistry tests 

falsely decreases alkaline phosphatase by binding magnesium ion 

interferes with Jaffe reaction for creatinine test 

alters sodium, potassium and calcium concentrations in plasma 

Heparin

antithromboplastin or antithrombin

available in a liquid or dry form as sodium, calcium, ammonium and lithium salts 

each of these will interfere with determinations of their respective ions in the plasma

Mode of action

interferes with the formation and/or activity of thrombin and the activity of clotting factors IX, X, XI, XII

the optimum concentration is 0.1~0.2 mg/mL of blood

excessive heparin does not alter RBC volume

Uses 

Troponin T/I

blood pH and blood gas (artery) analysis for acid base balance

special trace element studies and some cytology

Disadvantages

clumping of leukocytes

interfere with the staining of leukocytes 

the most expensive of the anticoagulants 

blood will clot in 8~12 hours because clotting is only delayed and not prevented 

not suitable for agglutination tests, coagulation studies (prothrombin time tests) or plasma fibrinogen determination

Sodium citrate (light blue) 

Mode of action

combines with calcium to form an insoluble salt of calcium citrate 

Uses 

Thrombin Prothrombin

AVTT

platelet function and morphology studies

standard concentration is 1 part 3.8% NaC solution to 9 parts of blood and suitable for blood coagulation assay

1 part of NaC mixed with 4 parts of blood is recommended for ESR

Disadvantages

interferes with many chemical tests

used alone it preserves blood for only a few hours

tendency to shrink cells

because of a 10% dilution of blood, Na-citrate is generally not used for CBC

4 Sodium Fluoride+ 5 Potassium Oxalate (grey) 

Mode of action

NaF inhibits the glycolytic enzymes responsible for the breakdown of glucose

at room temperature, about 10% of the glucose is lost per hour from an untreated sample

potassium oxalate is the primary anticoagulant, as NaF has a poor anticoagulant effect 

optimum concentration is 1 mg of the mixture per 1 mL of blood

Uses 

Glucose testing

Disadvantages

poisonous

inhibit of urease and glycolytic enzymes which may interfere with urea and glucose determinations that employ enzyme activity 

Types of anticoagulant

003 Phlebotomy

1. Definition 

Phlebotomy is the process of making incision in a vein with a needle

venipuncture

phlebotomist

2. Blood specimen collection and processing

acquiring a quality lab

test result for any patient is the specimen collection procedure 

blood specimens are obtained through capillary skin puncture (finger, toe, heel), arterial or venous sampling

Capillary/dermal/skin puncture, earlobe stick, finger stick, heel stick (infants) 

Venous puncture (direct puncture of vein)

3. Venipuncture

Venipuncture is the process obtaining blood samples from veins for lab testing. It is probably the most common procedure in the medical field

perform diagnosis

collect blood for later use should the patient's condition requires transfusion

remove blood that was found with excessive levels of erythrocytes or iron 

4. Venipuncture Procedure Steps 

Explain the procedure and purpose for the patient

Assess the patient's disposition

Position the patient sitting or lying (never allow the patient to sit upright on a high stool or standing due to the possibility of syncope) 

Check the requisition form for requested tests, patient information, and any special requirements

Label according to the requisition form

Select a suitable site for venipuncture

Prepare the equipment, the patient and the puncture site 

Perform the venipuncture

Collect the sample in the appropriate container

While the tube fills, remove the tourniquet

Label the collection tubes at the drawing area 

5. Order of draw

blood collection tubes must be drawn in a specific order to avoid cross-contamination of additives between tubes

a vacutainer tube is a sterile glass or plastic tube with a closure that is evacuated to create a vacuum inside the tube facilitating the draw of a predetermined volume of liquid 

6. Unacceptable specimens

There is a patient name or file number discrepancy between specimen label and request form

There is no patient name or other unique identifier on specimen

Specimen is too old when received 

There is apparently no specimen in container

The expiration date of the transport medium has been exceeded 

7. Avoid performing a venipuncture on 

Arm on side of mastectomy

If drawn here, the test results could be inaccurate because of lymph edema 

Scarred or burned areas

Performing a venipuncture at these sites is more difficult due to the scar tissue

Arm in which blood is being transfusion I IV cannula

The fluid in the IV could dilute the specimen

A hematoma is an abnormal collection of blood outside of a blood vessel 

It occurs because the wall of a blood vessel wall, artery, vein or capillary, has been damaged and blood has leaked into tissues 

Edematous (swelling by fluid retention) should be avoid because the accumulated fluid could alter test results 

8. New vein finder devices 

9. New device for infant blood collection

automatic sample mixing reduces repeat collections due to clots and hemolysis

collect even the smallest drops from the puncture site with no squeezing, scoping, or stopping to mix the sample blood immediately

003 Hemoglobin

1. Introduction

The hemoglobin (Hb) is red globular proteins, which has a molecular weight of about 68,000 kDa and comprise almost one third of the weight of a red blood cell. 

Heme and Globin gives red color

Carry oxygen to the tissues and return carbon dioxide to the lungs 

Gaseous exchange achieved by red cells containing specialized protein (hemoglobin) 

Each red cell contains approximately 280 millions of Hb molecules 

2. Hemoglobin synthesis

65% of hemoglobin is synthesized in the erythroblast

35% at the reticulocyte stage 

Heme synthesis occurs largely in the mitochondria of immature RBC

Globin synthesis occurs in the polyribosomes in the cytosol of immature RBC

Hemoglobin molecule is a tetramer consisting of a 4 polypeptide chains, known as globins, which are usually 

• 2 alpha chains 141 amino acid long
• 2 beta chains 146 amino acid long

Ferrous iron 

Each molecule of heme combines with a globin chain

A tetramer of 4 globin chains, each with its own heme group is formed to make up a hemoglobin molecule 

3. Globin synthesis

8 functional globin which are coded by 2 clusters of genes expressed under genetic control 

a and z globin genes cluster is located on the short arm of  chromosome 16

b, d, g and e globin on chromosome 11

4. Ontogeny of globin synthesis

Globin synthesis is first detected in the primitive erythroid precursors of the yolk sac at about 3 weeks of gestation

Embryonic: 

Gower I (z2e2), Portland (z2g2), Gower II (a2e2) 

Fetal (ag): 

HbF (a2g2)

HbA (a2b2)

Normal adult blood also contains small quantities of 2 other hemoglobins, Hb-F and Hb-A2

These also contain a chains but with g and d chains respectively instead of b

The major switch from fetal to adult hemoglobin occurs 3~6 months after birth

5. Heme synthesis

Heme synthesis starts with the condensation of glycine and succinyl CoA to form d-aminolevulinic acid synthase enzyme (d-ALA)

Vitamin B6 is a coenzyme for this reaction

2 molecules of d-ALA condense to form a porphobilinogen (PBG)

4 PBG condense to form a uroporphyrinogen III

UPG III is then converted to coproporphyrinogen 

CPG then changes to protoporphyrin which ultimately combines with iron in the ferrous state (Fe2+) to form heme

Iron is brought to the developing red cells by a carrier protein (transferrin) which attaches to special binding sites on the surface of these cells 

Transferrin releases iron and returns back to circulation

6. Hemoglobin structure

polypeptide chain (coiled)

iron molecules (silver ball) 

heme (gold surrounding iron) 

7. Hemoglobin catabolism (Normal red cell destruction)

Red cell destruction usually occurs after a mean life span of 120 days (3 months)

Removed by extravascularly by macrophages of the reticuloendothelial system RES, specially in bone marrow but also in the liver and spleen

The breakdown of red cells results in: 

iron for recirculation via plasma transferrin to marrow erythroblasts 

protoporphyrin which is broken down to bilirubin

globins which ae converted to amino acids 

bilirubin circulates to the liver where it is conjugated to glucuronides which are excreted into the gut via bile and converted to sterobilinogen and stercobilin (excreted in faeces) 

sterobilinogen and stercobilin are partly reabsorbed and excreted in urine as urobilinogen and urobilin 

005 Hematocrit 

Greek "haiman": blood and krite "to separate blood"

1. Hematocrit and erythrocyte sedimentation rate ESR (black)

Packed Cell Volume or Erythrocyte Volume Fraction 

Hematocrit is the proportion or percentage of blood volume that is occupied by RBC

Principle

A decrease in the number or size of RBC and amount of space occupied, resulting in lower hematocrit

2. Hematocrit measurement

Blood obtained using microcapillary or transferred from anticoagulant tube to the capillary itself

Centrifugation

Fluid element and cellular element is obtained 

Cell element is measured 

PCV (%)

4. Hematocrit method 

Capillary tube coated with anticoagulant (heparinized tube) or without anticoagulant (non-heparinized tube)

Specimen 

Finger puncture 

Vein puncture 

Sealing of one end of the capillary tube (plastercine)

Centrifuge for 3~5 minutes at high speed (5000 rpm) 

Measure packed cells height 

Measure total height of entire specimen 

The height of the red cell column is measured as a percent of the total blood column

The higher the column of red cells, the higher the hematocrit

5. Normal range

Adult female (42%): 37~47% (0.37~0.47 L/L)

Adult male (47%): 42~52% (0.42~0.52 L/L)

6. Main functions are to diagnose

Anemia (plasma more) 

Polycythemia

Hemodilution

Hemoconcentration

reduce the volume of plasma water causing a relative increase in RBCs, which concentrates the RBCs

7. Sources of error

Mechanical error

• Interval between centrifugation and measurement 
• Centrifugation time too short and speed too low
• Forcing centrifuge to stop 
• Inadequate mixing 
• Incomplete sealing of the capillary 
• Bubbles in hematocrit 

Biological error

• Clotting of sample
• Inadequate specimen 
• Inclusion trapped plasma 
• Hemolysis of sample
• Measurement of buffy coat 

8. ESR

Background 

Blood is prevented from clotting by using blood collected in citrate anticoagulant 

allow to settle and sedimentation of the erythrocyte will occur

Sedimentation: erythrocytes clump/aggregate together in a column-like manner (rouleaux formation)

Changes are related to alterations in the plasma proteins 

Invented by Polish physician, Edmund Faustyn Biernacki (1866~1911)

Principle

measures the rate of settling of erythrocytes in a diluted human plasma 

• Plasma protein composition 
• Concentration of erythrocytes
• Number and size of erythrocytes
• Certain technical and mechanical factors

ESR value is measuring the distance from surface meniscus to the top of the erythrocyte sedimented in a special tube 

placed perpendicular in a rack for 1 hour 

the rate of fall of RBCs is the size of the falling particles 

the larger the particles, the faster it falls

In normal blood, RBCs tend to remain separate from one another because they are negatively charged (zeta potential) 

In many pathological conditions, the phenomenon of Rouleaux formation is caused by the alterations of the erythrocyte surface due to change by plasma protein

IDE 

Based on

Inflammatory + necrotic processes 

Alteration in blood proteins 

Resulting in aggregation of RBCs

RBCs heavier and likely to fall rapidly in special vertical tube 

Cells settle faster 

Higher ESR 

9. Stages of cell sedimentation

Initial period of aggregation 

rouleaux formation + sedimentation

relatively slow

lasts about 10 minutes 

Fast settling stage 

settling rate is constant 

most settling occurs at this stage 

last about 1 hours 

Packing stage 

Sedimentation occurs at slowest rate 

RBCs sediment to the bottom of the tube 

10. ESR method 

Westegren method 

Westegren tube

Westegren rack

Timer

Trisodium citrate anticoagulant 3.1%

Specimen 1 anticoagulant:4 blood

Principle

anticoagulated blood is placed in Westegren tube 

thoroughly mix blood specimen 

fill the tube to marked 0 (top)

left vertically on the rack for 1 hour at room temperature 

read the upper level of red cells exactly after 1 hour 

record blood cells in mm unit

ESR result 

< 50 years 

Male: 0~15 mm/h

Female: 0~20 mm/h

> 50 years 

Male: 0~20 mm/h

Female: 0~30 mm/h

Wintrobe method

11. Clinical significance

Increased ESR 

• pregnancy due to increased fibrinogen and hemodilution
• anemia/multiple myeloma (rouleaux formation)
• mestruation
• inflammation as certain proteins cause RBC to stick together and fall more quickly than normal 
• macrocytosis as smaller surface volume ratio settle more quickly 

Decreased ESR 

• polycythemia due to increased RBC tend to cause a marked slowing of sedimentation
• spherocytosis or microcytosis 
• afibrinogenemia 

12. Sources of error

False increased results (HIGH) 

• increased room temperature 
• tube stand for > 1 hour 
• positioning of the tube 

False decreased results (FALL)

• anticoagulated blood > 2 hours old 
• improper concentration of anticoagulant-whole blood ratio
• refrigerated blood or decreased room temperature 
• tube stands < 1 hours 

006 Blood smear and staining

1. Blood film 

A blood film or peripheral blood smear is a thin layer of blood smeared on a microscope slide

2. Aim of blood smear 

investigate hematological problems (blood disorders) 

occasionally to look for parasites within blood (malaria and filaria) 

examination of thin BF to investigate and manage anemia, infections, and conditions which produce changes in appearance of blood cells and differential WBC count

report can provide rapidly and at low cost

useful info about patient

Prepare, Fixation, Stain

3. Thin blood film 

prepared from anticoagulated blood obtained by venipuncture/from free flowing finger prick blood by:

4. Thick blood film 

detecting blood parasites such as malaria and microfilaria 

Place a large drop 

Spread it in a circular area of 1.5 cm with stick or end of another glass slide 

Dry it 

Staining

6. Fixation of blood smear

preserve the morphology

must be fixed ASAP after drying

prevent contact with water before fixation complete 

Methyl alcohol (methanol) 

Ethyl alcohol (absolute alcohol)

Methylated spirit (95% ethanol) must not be used as it contains water 

place films in a covered staining jar or tray containing alcohol for 2~3 min 

in humid climates, it may be necessary to replace methanol 2~3 times per day

old portion can be used for storing clean slides

Leishman stain contains methanol

7. Staining of blood smear

Romanowsky stains are universally employed for staining. All Romanowsky combinations have 2 essential ingredients (methylene blue and eosin or azure)

Methylene blue is the basic dye and has affinity for acidic component of cell (nucleus) and eosin/azure is acidic dye and has affinity for basic component (cytoplasm) 

Most Romanowsky prepared in methyl alcohol so that they can combine fixation and staining 

Leishman stain

Giemsa stain 

Wright's stain 

Field stain (thick film)

J.S. stain 

8. Qualities of a good blood film 

• not cover the entire surface of slide
• have smooth and even appearance
• free from waves and holes 
• not have irregular tail 

9. The thickness of the spread 

• Angle of spreader slide 
• Size of blood drop 
• Speed of spreading

Notes:

• If the hematocrit increased, the angle of the spreader slide should be decreased

10. Parts of a thin blood film

• Head: the portion of blood film near the drop of blood
• Body: the main part of the blood film
• Tail: the tapering end of the blood film

11. Common cause of a poor blood smear

• Drop of blood too large or too small
• Spreader slide pushed across the slide in a jerky manner
• Failure in keep the entire edge of the spreader slide against the slide while making the error 
• Failure in keep the spreader slide at 30 angle with slide
• Failure to push the spreader slide completely across the slide
• Irregular spread with ridges and long tail: edges of spreader dirty or chipped; dusty slide
• Holes in film: contaminated with fat or grease and air bubbles
• Cellular degenerative changes: delay in fixing inadequate fixing time or methanol contaminated with water

12. Biologic cause of a poor smear

• Cold agglutinin: RBCs will clump together
• Warm the blood at 37°C for 5 minutes, and then remake the smear
• Lipemia: holes will appear in the smear
• Rouleaux: RBCs will form into stacks resembling coins  

007 RBC indices *anemia 

1. RBC indices and RBC count are used to diagnose  anemia

Low RBC count or abnormal RBC indices may indicate presence of anemia 

Measurement of erythrocytes

• Quantitative measurements of erythrocytes erythrocytes indices or red cell absolute value 
• Define cell size, number and concentration of Hb within the cell

2. Mean corpuscular volume (MCV)

most often used index 

categorize RBC by size 

Normocytic anemias, normal MCV

Microcytic, decreased MCV

Macrocytic, increased MCV

stained RBC with high MCV is larger 

measures average volume or size 

Normal range

Male: 80~98 fL

Female: 96~108

MCV values are higher in newborn and infants

patient's packed cell volume (L/L)/erythrocyte count (×10¹²/L) 

femtolitre (fL)= 10¹⁵L

3. Mean corpuscular hemoglobin (MCH)

picogram

The average weight of hemoglobin

Normal range: 27~32 pg

MCH direct proportional to amount and size of hemoglobin 

Hemoglobin (g/L)/erythrocyte count

4. MCHC

Normal range: 32~36% (g/dL)

average concentration of hemoglobin

normochromic

hypochromic

physical limit in amount, no hyperchromic

normal RBCs stain pinkish red with a paler area 

hypochromic lighter in color with a larger pale area, has low MCHC

Anemias depend on MCHC index 

Hemoglobin (g/dL)/patient's PCV (L/L) 

Microcytic hypochromic anemia: 

5. Significance 

normocytic normochromic anemia often caused by acute blood loss

microcytic, hypochromic anemia often caused by iron deficiency 

macrocytic anemia most often caused by vitamin B12 deficiency (due to pernicious anemia) and folic acid deficiency 

Abnormal erythrocyte indices help to classify anemia 

Diagnosis must based on clinical history, physical examination...

008 Blood cell counts

Hemocytometer

counting chamber 

Consist of heavy glass slide 

9 large 1mm squares

16 small square

RBC count

Whole blood is anticoagulated with EDTA, then dilute the blood 1:200 with 0.85% sodium chloride (normal saline) 

Normal saline have salts, concentration similar with RBC, isotonic

Observe under microscope ×10 then ×40

1/400 mm²

5×16=80 boxes 

Total RBC in 5 boxes×the dilution factor×10⁴

Volume (area×depth)

Dilution factor: 200

Reference range 

Male: 4.5~6.5×10¹² /L

Female: 3.8~5.8

Infant: 4~6

WBC 

1:20 

Power ×10 

Four squares

Dilution factor: 20 

Normal values

Platelets

Decreased erythrocyte

Causes anemia

Anemia condition may caused by excessive blood loss or blood destruction, bone narrow failure, 

Polycythemia

Bone marrow disorder

Malignancy

Leucopenia

Hepatitis

Cirrhosis

Leukocytosis

Appendicitis

Ulcer 

Viral infection

Leukemia

Measure coagulation period

Thrombocytopenia

Petechiae or Purpura

Thrombocytosis

Benign/reactive etiologies