Professional Documents
Culture Documents
A. ATHANASIUS KIRCHER
- Using a microscope; Plague victims (Black Plague - Yersinia pestis) (1646)
- Scrutinium pestis - Little worms or Animalcules in the Blood (1658)
- Proposed Hygienic Measures: Isolation, Quarantine, Burning Clothes (Worn by the infected), Wearing Face
Masks
B. ANTON VAN LEEUWENHOEK
- Father of Microbiology
- Associated with Giardia lamblia
C. GUILIO BIZZOZERO
- Discovered the function of Platelets
- Labeled them as Petite Plaques
- Also discovered Helicobacter pylori
D. JAMES HOMER WRIGHT
- Discovered that Megakaryocyte is the Origin of Platelets (Platelets are fragments of Megakaryocytes)
- Modification of Romanowsky Stain (Polychromatic - Acid Dye and Basic Dye) (1902)
-Massachusetts General Hospital - Pathology Laboratory
E. PAUL EHRLICH
- Classified Leukocytes
- Discovered Salvarsan (Treatment for Syphilis)
HEMATOLOGY
Haima (Blood) + Logos (Study)
- Study of Blood
BLOOD
- A Nutritive Fluid
- Take part in the Physiologic & Pathologic Activities in the Body
2 Types of Blood
A. Venous Blood - Dark Red in color
B. Arterial Blood - Bright Red in color
FUNCTIONS
- Respiration (Transport of O2 and CO2)
- Nutrition
- Excretion
- Homeostasis and Protection
- Thermoregulation
CHARACTERISTICS
- Red in color
- Thick and Viscous
- Slightly Alkaline (pH 7.35-7.45)
- Specific Gravity (1.045-1.065)
- 7-8% of the Total Body Weight
- Total Volume: Males:5-6L; Female:4-5L
COMPOSITION
LIQUID - Plasma or Serum (Coagulated)
A. Water (91.5%)
B. Others(1.5%)
- Electrolytes
- Non-Protein Nitrogen
- Hormones
- Enzymes
- Food Materials
SOLID - Cellular Components
A. Red Blood Cells
- 45% of Blood Volume
- 4.5-6.5 x 10^12/L (M/mL)
B. White Blood Cells
- 4-11 x 10^9/L (T/mL)
- Granulocytes and Agranulocytes
C. Platelets
- 150-350 x 10^9 (T/mL)
EXAMPLES:
- Anisocytosis - patient's red blood cells are of unequal size
- Poikilocytosis patients red blood cells are of different shapes
- Anemia deficiency of red blood cells or haemoglobin in blood
- Aplasia failure of organ or tissue to develop properly or to function normally
- Dysmyelopoiesis abnormal cell production of the bone marrow
- Panmyelosis abnormal increase of bone marrow cells
Degree of Hazard:
4 Extreme Hazard
3 Serious Hazard
2 Moderate Hazard
1 Slight Hazard
0 No/Minimal Hazard
D. ELECTRICAL HAZARDS
- Electrical shock, burns, fire, or explosion
- Maintenance is important
A. Grounded (3-pronged plug 1970)
B. Avoid use of extension cords (CLSI Clinicsl Laboratory Standard Institute)
C. Operate with dry hands
E. MECHANICAL HAZARDS
- Improper use, storage, or disposal of glassware, sharp instruments, compressed gases, or equipment
A. Use caution to prevent unnecessary or accidental breakage of glasswares
B. Sharps or broken glasses dispose in puncture proof containers
C. Use common sense in storing glasswares
- Centrifuge: Balanced, Lid must NEVER be opened until rotor has completely stopped
B. HEMATOCRIT
- Method: HCT
- Measured: Packed Cell Volume (PCV)
- Reference method for calibrating blood count systems (Can use sample of normal healthy individual as a
secondary standard and perform HCT to calibrate the blood count system)
- Rough guide to accuracy of Hemoglobin measurement
- Used in the circulation of Red Blood Cell Indices
- Rule of 3:
Hemoglobin x 3 = Hematocrit +/- 3
(+/- 3 is the range)
Red Blood Cell x 3 = Hemoglobin
- Used on when patient is:
-Normochromic: normal color of Red Blood Cells
- Normocytic: normal size of Red Blood Cells
Microhematocrit Principle:
- A small amount of whole blood is centrifuged to determine maximum packing of Erythrocytes expressed
as PCV or HCT
- After centrifugation of Capillary Tube: Plasma, Buffy Coat (WBC & PLT), RBC
- No air should be present because erroneous results
- Seal should be facing the rubber gasket, balanced
Reference Values:
Male: 0.47 L/L (+/- 0.07)
R: 40-54%
Female: 0.42 L/L (+/- 0.05)
R: 35-49%
C. RED CELL INDICES
- Morphologic classification of anemia
- Used in Quality Control
- Calculated from Hemoglobin, Hematocrit, Red Blood Cell Count
(Microcytic, Normocytic,
Macrocytic)
Reference Value: 80-100 fL
(No terminologies)
Reference Value: 28-32 pg
(Hyperchromic, Normochromic,
Hypochromic)
Reference Value: 32-36 g/dL
- Central pallor (Lightly Stained Area of RBC)
- Bigger central pallor = Low MCHC
F. RETICULOCYTE COUNT
- Juvenile Red Blood Cells demonstrated by supravital stains (presence of Reticulin Network)
- Stain = Brilliant Cresyl Blue
- Reflects Erythropoietic Activity of Bone Marrow (compensated anemia)
- Relative Count (%):
Adult: 0.5-1.5%
Neonates: 1.5-6.5%
- Absolute Count (x10^9/L)
= 50 x 10^9/L
H. PLATELET COUNT
- Phase-contrast microscope
- Difficult to count
- Sample: Whole Blood with EDTA
- Diluent: 1% Ammonium Oxalate
I. PLATELET ESTIMATE
- Number of Platelets per !0 Oil-Immersion-Objective Fields
- Under 100 x Oil Immersion
- Average number of platelet field x 20,000 approximately platelet count per mm^3
- Provided that there are 200 RBCs/ Oil Immersion Field
AverageNum berofPlatelets / field
- Anemia/Erythrocytosis
TotalRBCCo unt
SPECIMEN COLLECTION BY 200 RBCs / field
VENIPUNCTURE
- Syringe Technique
- Evacuated Tube System (Black - w/ Backflow)
- Butterfly
VENIPUNCTURE
- Most common technique to obtain blood
- Requires ample skill to ensure accurate results and preservation of their vein
Steps:
- Correct patient identification (Most critical step)
- Laboratory Request (Compare information)
- No isolation restriction
- Reassure the patient (Dont deceive)
- Position the patient
- Assemble the supplies (Accesibility; Hemoconcentrated- Upright)
- Selection of the site (Median Cubital, Cephalic, Basilic)
Points:
- Avoid areas with Hematoma, Burns, Scars/Edema, IV Line, Mastectomy Site (Lymphostasis)
- Tourniquet: 3-4 inches above the site
- Clean site
- IV Site/ Medlock - 10 minutes (Sample never drawn on the same side as IV)
- Transfusion - may collect blood - drawn on the opposite side
- Hemodialysis shunt/ Fistula - avoided (Ask for the status) -
- Hemodialysis - may draw blood but distal - atleast 4 inches below the shunt)
- Mastectomy - collected in the other side (Lymphostasis - may cause erroneous result because blood may not
be filtered)
- Hematoma, burned (prone to infection), or Scarred areas are avoided
Remember:
- Each phlebotomist is allowed only allowed 2 attempts
- Inspect needle and vacuum
- Release tourniquet before the needle
- Remove needle and apply pressure (Do not allow the patient to bend the arm because it may cause reopening
of the puncture wound and may cause hemorrhage)
- Discard needle
- Label specimen (No pre-labelling)
1. Name
2. Date
3. Time
4. Age
5. Sex
6. Initials of Phlebotomist
7. Information required by the institution
- Specimen transport must be properly handled and timely transport must be observes
Source of Error (p.17 Steininger Table 2-5)
A. Errors in Venipuncture Preparation
1. Improper patient identification
2. Failure to check patient adherence to dietary restrictions
3. Failure to calm patient prior to blood collection
4. Use of improper equipment and supplies
5. Inappropriate method of blood collection
B. Errors in Venipuncture Procedure
1. Failure to dry the site completely after cleansing with alcohol
2. Inserting needle bevel side down
3. Use of needle that is too small causing hemolysis of specimen
4. Venipuncture in an unacceptable area
5. Prolonged tourniquet application
6. Wrong order of tube draw
7. Failure to mix blood collected in additive-containing tubes immediate
8. Pulling back on syringe plunger too forcefully
9. Failure to release tourniquet prior to needle withdrawal
A. Errors after Venipuncture Completion
1. Failure to apply pressure immediately to venipuncture site
2. Vigorous shaking of anticoagulated blood specimen
3. Forcing blood through a syringe needle in tube
4. Mislabeling of tubes
5. Failure to label appropriate specimens with infectious disease precaution
6. Failure to put date, time, and initials on requisition
7. Slow transport of specimens to laboratory
ORDER OF DRAW
1. Blood Gases
- Heparin (green)
- Unstable
- Longer exposure to air (erroneous result)
2. Slides
- Can also get from EDTA tube
3. EDTA
- Ethylenediaminetetraacetic Acid
4. Other Tubes with Anti-Coagulants
- Gray / Green (Not for blood gas)
SKIN PUNCTURE
- Done in infants and particularly newborns (thermoregulated baby - venipuncture)
- Can cause hospital induced anemia (aggressive collection of blood) every mL of blood counts in newborns
- Put patient at risk for Osteomyelitis - puncture not more than 2.4 mm
- Safer than venipuncture
- Puncture site should be warm (warm towel)
- Sites: 3rd and 4th finger, big toes, heels, last resort: earlobe (inadequate blood sample)
- For adults because of obesity, burns, extremely fragile or small veins
- Blood is a mixture of capillary, venous, and arterial blood with interstitial and intracellular fluid (good
reflection of ciculatory system)
- It is important to wipe the first drop of blood (tissue fluid promotes clotting)
- A good puncture requires no forcing or hard squeezing
SOURCES OF ERROR
- Hemolysis (tissue fluid and not blood - plasma reddish tinge color - centrifuged)
- Failure to dry the site
- Failure to wipe 1st drop of blood
- Vigorous massaging/ milking the area
- accidental capturing of bubbles
ANTICOAGULANTS
1. EDTA
- Ethylenediamineteraacetic Acid
- Disodium salt - Versene
- Tripotassium salt - Sequestrene
- Optimum concentration: 1.5 mg/mL of blood
- Chelates calcium (Ca needed for activating coagulation cascade - formation of clots)
- Blood smear = 2 hours of blood collected (morphology is still intact)
- Not used for coagulation studies because will not preserve Factor 5 (need for coagulation cascade)
- Increase EDTA
- Red cell shrinkage
- Falsely decrease: HCT and ESR (no rouleaux formation)
K2 Plastic Spray-dried
K3 Glass Liquid
3. HEPARIN
- Acid Mucopolysaccharide
- In vivo
- Inhibits thrombin
- Optimal concentration: 15-20 U/mL of blood
- For blood gas analysis (arterial puncture)
- For Osmotic Fragility Test
- Specimen: Defribinated blood (using glass marbles/ glass beads/ metal paper clips - removing fibrin)
- Can cause morphologic distortion of PLT and WBC not used for blood smear (bluish background)
- Not used for coagulation studies but used for Platelet Retention Test
B. HEPATIC PERIOD
- 4th to 5th or 5th and 6th week of generation
- Definitive Morphologic Hematopoiesis
- Presence of erythroblasts + lymphoid
**Erythroblasts > More Unidentifiable
**Lymphoid cells > Definitive Hematopoiesis
- Fetal Liver: Primary Erythroid Organ (Extravascular)
**Fetal Liver is the PRIMARY SITE for hepatic period
- Spleen
-Involved solely in lymphopoiesis
- Starting of megakaryocyte production
**Spleen is the largest lymphoid organ
- Fetal Hemoglobin (HbF) = 2 Alpha and 2 Gamma
- Hb A1 and Hb A are also detected
**Hb A1 = 2 alpha, 2 beta
**Hb A2 = 2 alpha, 2 delta
**Hb A1 major hemoglobin found in adults
**Extramedullary meaning outside the bone marrow > adult > hematopoiesis confined in bone marrow
Hepatic Period Quick Facts
- Site: Liver, Spleen, Thymus, Lymph Nodes
- Starts at: 4th to 5th, 5th to 6th weeks of gestation up to 1-2 weeks of birth
- Cells produced: smaller erythroblasts,
- Megakaryocytes 6th week, Granulocytes 7th week,
- Platelets in circulation 8-9th week,WBC in circulation 11th week
- Globin chains produced: alpha and gamma
- Hemoglobin: Fetal Hemoglobin (Hemoglobin F)
Organs Develop of Hematopoiesis
- Spleen, Thymus and Lymph Nodes
**Thymus First organ to be developed in fetal
**Mesoblastic Mesodermal Cells
**Mesenchymal cells migrate core of skeleton/bone forming the cavity (bone marrow)
C. MYELOID/MEDULLARY PERIOD
**Medullary seen in inner part of bone marrow
- 5th month of gestation
- Developing bone marrow cavity (long bones) - inner part of bone marrow
- EPO (erythropoiesis), G-CSF (Granulocyte Colony Stimulating Factor), GM-CSF (Granulocyte-Monocyte
Colony Stimulating Factor), HbF , HbA2 & Hb A1
- 4th year of life = marrow replaced by fats (Hematopoietically inactive marrow)
**Shaft of long bones (Fat Disposition)
Myeloid Period Quick Facts
- Site: Bone Marrow
- Starts at: 5th Month of Gestation until death
- Primary starts at: 24th week until birth
- After birth:
- 4 years old: formation of fatty reserves
- 18 years old: only hematopoietic sites are:
Sites of Red Marrow
- Skull (Frontal Bone), Ribs, Vertebrae, Proximal Ends of Bones, Clavicle, Sternum, and Pelvic Region - Iliac
crest (Safest and Most Accesible site for BM Biopsy)
**M:E (Myeloid:Erythroid) Ratio
**Normal = 2:1 4:1
>2 (2 Myeloid Progenitor) :1 (1 Lymphoid Progenitor)
Examples:
- Average = 3:1
- Infection = 6:1
- Leukemia = 25:1
(Myleoid Hyperplasia, Myeloid Hypoplasia, Erythroid Hyperplasia & Erythroid Hypoplasia)
ADULT HEMATOPOIETIC TISSUE
- Bone Marrow, Spleen, Liver, Thymus, Lymph Nodes
MARROW
2 Types of Bone Marrow
- Red Marrow
- Yellow Marrow
Red Marrow
- Hematopoietically active
- Flat bones & proximal ends of long bones
- composed of extravascular cords that contain all of the following blood cell lineages, stem cells, adventitial
cells and macrophages
**Proximal ends because yellow marrow is in shaft
Yellow Marrow
- Hematopoietically inactive
- Adipocytes
- Normally 50% red marrow, 50% yellow marrow
LIVER
- Major site in Hepatic Hematopoiesis (fetus)
- Hematopoietic Functions (Adults)
- Synthesis of various transport CHON
- Storage of Minerals and Vitamins
- Bilirubin Conjugation
- Bilirubin transportation
Pathophysiology
A. Porphyrias
- When Hb is abnormally metabolized
- Enzymatic deficiencies
- Accumulation of intermediary porphyrias
Cutaneous porphyrias
- Makes patient sensitive to light which causes pain.
B. Bilirubin Conjugation
- Severe H.A. & RBC Dysplasias
- Fe ++ storage
Storage Diseases:
1. Monocytes / Macrophages (Kupffer cells)
2. Enzymatic deficiencies
3. Hepatomegaly w/ liver dysfunction
C. Extramedullary Hematopoiesis
- Bone Marrow failure
SPLEEN
- Largest lymphoid organ
- Vital but not essential for life
**removal wont put your life in danger
Functions
- Indiscriminate filter of blood
- Sequesters senescent red cells
- IgM synthesis
- Storage for platelets (1/3)
- Culling
- Pitting
3 regions:
A. White Pulp
- Contains aggregates of lymphocytes, macrophages and dendritic cells
B. Red Pulp
- Culling
- Pitting
-Sequesters approximately 30 % of the total platelet count
C. Marginal Zone
- Surrounds the white pulp; contains blood vessels macrophages and specialized B cells
**Culling - Totally destroys senescent RBCs
**Pitting - RBC has present inclusions
**Spleen removes inclusions
Pathophysiology
A. Hemolysis
- Increased environmental stress
- Due to small openings by inter endothelial junction
B. Splenomegaly
- Enlarged and Palpable
C. Hypersplenism
- Enlarged leading to Pancytopenia
**Pancytopenia all cells decreased
- Caused commonly by congestive splenomegaly secondary to liver cirrhosis and portal hypertension
LYMPH NODES
- Bean shaped Member of lymphatic system occuring in groups or in chains and are located superficially or deep
Functions
- Formation of new lymphocytes
- Processing of specific Immunoglobulins
- Filter particulate matter, debris, bacteria entering via lymph
Pathophysiology
A. Adenitis
- Infection of Lymph Node (ex. Plucking)
- Infiltration by Malignant Cells = metastasis
THYMUS
- Atrophies with age
- Efficient, well-developed at birth
Pathophysiology
A. Non-development (gestation)
- Lack of T-lymphocytes
- Uncontrollable infections
B. Death
- Thymic disturbance (adults)
- No effect
Stem Cell Theories
A. Pluripotential Stem Cell
- One cell gives rise the varied blood
cells (E, L, M)
- Widely accepted theory
B. Committed Stem Cells
- Give rise to descendants or progeny cells that eventually become restricted to a specific cell line development
**Progeny from erythrocyte, it can be a platelet, etc.
Characteristics/Fates of Stem Cells
- Capable of self renewal (to original state)
- Give rise to differentiated progeny
- Able to reconstitute the hematopoietic system of an irradiated host
- Apoptosis programmed cell death
**Premature death cause specific cell line to be decreased
Growth Factors
**Prevents hematopoietic precursor cells from dying by inhibiting apoptosis
- Helps increase in number, survival and prolong life span of stem cell
1. Phytohemagluttinin
2. Cyclophosphamide
3. Concanavalin A
INHIBITOR Hydroxyurea
Cytokines (Hematopoietic Growth Factors)
- Group of specific Glycoprotein
- Regulates the proliferation, differentiation, and maturation of hematopoietic precursor cells
- Inlude interleukins, lymphokines, monokines, chemokines, interferon and colony stimulating factors
Colony Stimulating Factors (CSFs)
- Produced by many cells
- High specificity to target cells
**Target cells has receptors for CSF
- Active at low concentrations
- (eg: G-CSF)- primary target is granulocyte
- Can also have synergistic effect (IL-3 and G-CSF for Megakaryocyte colony stimulation)
Interleukins
- Numbered in order of identification
Characteristics
- CHON that exhibit multiple biologic activities
- Synergistic interactions with other cytokines and growth factors
- Interacting systems with amplification potential
- Effective at very low concentrations
- [Refer to Page 72-73 of Hema Book 3rd Ed by RODAK for lists of interleukin with sources, target site and their
roles]
Leukopoiesis
Major categories: Myelopoiesis and Lymphopoiesis
Myelopoiesis
- GM-CSF, G-CSF, M-CSF including IL-3, IL-5,IL-11
- IL-3-multilineage: Stimulating Granulocytes, Monocytes, Megakaryocytes, and Erythroid Cells
Lymphopoiesis
- B-cell
- B-cell growth factor
- T-cell
- IL-2
Megakaryocytopoiesis
- Platelets are fragments of Megakaryocytes (Involved in Hemostasis and Thrombus Development)
- Earlier influences include GM-CSF, IL-3, IL-6, IL-11, kit ligand and TPO
- Stimulating hormonal factor is TPO which is mainly produced by the liver
- Meg-CSF a factor that significantly increases CFU-Meg colony formation
Lymphopoiesis
- Production of T and B lymphocytes
- T Cells mature in the thymus gland then distributed to the peripheral tissues cellular immunity
- T lymphocytes may be classified under
the influence of Phytohemagluttinin, and in vivo under the influence of IL-2 and IL-7
- B Cells originate and mature in the BM then migrate to the lymph nodes then circulation and tissues
- They mature into plasma cells responsible for producing antibodies Humoral immunity
- In vitro, B cells proliferate under the influence of bacterial Lipopolysaccharide and Dextran Sulfate
Blood Cell Maturation Time from Stem Survival Time in the
Cells (hours) Circulation (days)