CLINICAL HEMATOLOGY  

 
     -Etymology BUFFY COATS
     -Hemato and Logus  white blood cells
     -Definition  Neutrophils (LDOC) 
       1. Blood 1. Phagocytic PROPERTIES :
        2. Composition *SPC*  Release LYSOZYMES that destroy bacteria. 
           2. 1 Serum     2. Release DEFENSIN proteins
           2. 2 Plasma  that act like ANTIBIOTICS
           2. 3 Cells and theirs word deviation 3. Release strong OXIDANTS (bleach like and strong
 Erythrocytes chemicals that destroy bacteria)
 Leukocytes 4. Release CYTOKINES that attract other Neutrophils. 
 Thrombocytes
  EOSINOPHILS (Parasitic Defense Cells)
 COMPONENTS OF BLOOD 1. Allergic response
   PLASMA  Release Anti Histamine
 Transport Mechanism  2. Slow down Inflammation caused by BASOPHILS 
 90-92% WATER
 6-7% PROTEINS BASOPHILS 
 2-3% *FECCG*   Release Histamine, Heparin, Serotonin.   
o Fats 1. Highten the INFLAMMATORY response and
o Electrolytes HYPERSENSITIVITY reaction. 
o Carbohydrates (glucose)
o Chemical messengers MONOCYTES - enter various tissues and differentiate into
o Gases (O2, CO2) PHAGOCYTIC MACROPHAGE. 

   55% PLASMA contains;  LYMPHOCYTES - T & B CELLS

1. Dissolved substances *VALDISH* 
 Vitamins GENERAL FUNCTIONS OF THE BLOOD
 Amino acids
DENR
 Lipids
 Defensive Function
 Dissolved gasses
 Excretion
 Ions
 Nutrition
 Sugar
 Respiration
 Hormones
 RRAT       
What is the role of PROTEINS?  Regulation of body temperature
 Balancing OSMOTIC PRESSURE  Regulation of Water Balance
 Balancing of water flow between the blood and  Acid-Base Balance
extracellular fluids/tissues.   Transport of hormones

What will happen if there is a DECREASE OF PLASMA PHYSICAL CHARACTERISTICS OF BLOOD
PROTEIN?   THICKER (More Viscous) than the water and flows more
1. OSMOTIC PRESSURE IS DECREASED and causes slowly
water flow out to the blood into the tissues and will result  TEMPERATURE of 100. 4 degrees F
to SWELLING.   pH 7. 4 (7. 35-7. 45) 
 8% of total body weight
 PLASMA (55% of total BLOOD)  BLOOD VOLUME 
 BUFFY COAT LEUKOCYTES AND PLATELETS (<1% of  5-6 Liters in average MALE
total blood)  4-5 Liters in average FEMALE
 ERYTHROCYTES (45% of total BLOOD)  HORMONAL NEGATIVE feedback systems maintain
constant blood volume and OSMOTIC PRESSURE. 
   COMPONENTS OF BLOOD
BLOOD VISCOSITY
1. RED BLOOD CELLS     Direct measure of the ability of BLOOD FLOW through the
 Erythrocytes  vessels. 
         - Hemoglobin - O2 bearing molecule   Screening test that measures how much 
           *COMPRISED OF 4 SUBUNITS; 1. FRICTION on the blood that causes against the vessels. 
1. GLOBIN (binds to 1 O2 molecule) 2. How hard the HEART has to work to pump blood. 
             2. HEME (iron)  3. How much OXYGEN is delivered to organs and tissues. 
             3. 100% Saturation=4 GLOBULIN SUB-UNITS carrying O2
             4. Each Gram of HEMOGLOBIN=1. 34 mL O2 VISCOSITY - property of the fluid that resists the force causing it to
      flow. 
2. RED BLOOD CELL PRODUCTION   N.V. = 1. 4 to 1. 8 
1. Erythropoiesis   Two factors that affect viscosity 
 Erythropoietin
2. Hemolysis INCREASED viscosity is seen in:
3. Sequestration 1. IgM monoclonal gammopathy
4. Laboratory analysis of red blood cells 2. Waldenstrom's Macroglobulinemia
 Red blood cell count 3. Lymphomas: IgA and IgG myeloma
 Hematocrit
 hemoglobin BLOOD VISCOSITY    
to set apart, to detach a small portion of tissue from the rest.   High blood pressure
 Elevated LDL cholesterol
    3. PLATELETS  Low  HDL 
         3. 1. Megakaryocytes  Type-II diabetes, 
 Thrombopoietin  Metabolic syndrome
         3. 2. Thrombocytopenia  Obesity
        3. 3. Thrombocytosis  Smoking
  Age SECOND POLYCYTHEMIA
 Male gender  Factors EXTERNAL to red blood cell  production HYPOXIA. 
 Sleep apnea
The PRIMARY determinants of BLOOD VISCOSITY  Certain tumors
 Hematocrit
 Red blood cell deformability SITE SELECTION FOR VENIPUNCTURE
 Red blood cell aggregation 1. Accessibility of the vein
 Plasma viscosity 2. General condition of the vein
3. Types of fluid to be infused
  FACTORS that affect blood viscosity;  Irritating: a different site will be suitable from the sites,
 PLASMA protein which can be used for non-irritating fluids. 
 TYPES of PROTEINS in the plasma. 4. Period in intravenous line is expected to e in place. 
 
VENEPUNCTURE (venopuncture or venepuncture) Long term intravenous therapy will often require a different site from
 is the process of obtaining intravenous access for the ones that can be used if a single bottle or bag of fluid will be used
purpose of intravenous therapy obtaining a sample of
venous blood FACTORS IN GOOD VENIPUNCTURE
1. Venipuncturist
ADVANTAGE of Venipuncture  motto : *PRIMUM NON NOCERE*
 Easiest and most convenient  *good poise*
 Reduces the amount or variety of apparatus  must comfort the patient  
 Allows repetition of test/follow  up test 2. Patient and his veins
 Avoid tissue juices  Comfortable position
 Proper selection of the veins
WHO PERFORM VENEPUNCTURE? 
1. Medical Laboratory Scientist, RMT MATERIALS
2. Doctors (surgeons etc.) 1. Safety needles, 22g or less
3. Paramedica 2. Butterfly needles. 21g  or less
4. Phlebotomist 3. Syringes
5. Nurses and Rad. techs (trained) 4. Blood Collection Tubes
   Vacuum tubes - draw a predetermined volume of
VENIPUNCTURE is the one of the most routinely performed invasive blood. 
procedures and is carried out for THREE REASONS;  Tubes with different additives - used for collecting
1. to obtain blood for DIAGNOSTIC PURPOSE blood specimens for specific types of tests. 
2. To monitor LEVELS OF BLOOD COMPONENTS (Lavery
& Ingram, 2005) MATERIALS needed for VENIPUNCTURE
3. For INJECTING MEDICINES or other solutions for further  Selecting the appropriate collection tube and specimen
diagnosis container types
 Tourniquets, latex-free tourniquets are available (velket,
What is the vein that is COMMONLY used?  veniket, velcro)
 Media cubital vein      Antiseptic, individually packaged 70% isopropyl alcohol
Why?  wipes
1. Accessibility:  2x2 gauze or cotton balls
 Lies at the side within the fold of the elbow  Sharps disposal container, an OSHA acceptable,
 Lies close to the surface of the skin puncture proof container marked “biohazardous”
2. No large nerve supply  Bandages or tapes

COLOR CODED VACUUM TUBES

Other Veins
 Blue- citrates
 Median Basilic
 Green - Heparin
 Median Cephalic
 Lavender- EDTA
 Antebrachial Vein in the Lower arms
 Gray - Fluoride, Lithium Oxalate
 Black -  Sodium Oxalate
OTHER SITES
1. Veins at the  Yellow and amber - for culture
 Dorsum,
ORDER OF DRAW (HENRY) YBGGLG
 Wrist, 
1. Blood culture tubes (YELLOW)
 Legs 2. Coagulation sodium citrate tube (BLUE STOPPER)
(But not in patient with DIABETES, CIRCULATORY PROBLEM, and 3. Serum tubes with or without clot activator or gel separator
HEMOGLOBINOPATHIES.) 4. Heparin tubes with or without gel (GREEN STOPPER)
5Ethylenediaminetetraacetic acid tubes (LAVENDER STOPER)
INFANT 6. Glycolytic inhibit tube (GRAY STOPPER)
1. Longitudinal sinus
2. Femoral vein ORDER OF DRAW (CAPILLARY PUNCTURE) by RODAK
1. blood gases
OTHER USES OF PHLEBOTOMY 2. Slides, unless made from the EDTA microcollection tube. 
 (incision into a vein) is also used for the  treatment of 3. EDTA microcollection tube
certain diseases such as; 4. Other microcollectiontubes with anticoagulants 
 HEMOCHROMATOSIS 5. Serum microcollection tubes. 
 PRIMARY POLYCYTHEMIA
 SECONDARY POLYCYTHEMIA COLOR CODED VACUUM TUBES (w/o Anticoagulants)
  1. Pink
 PRIMARY POLYCYTHEMIA  2. Red
 Abnormalities in RED BLOOD CELL PRODUCTION 3. Amber (for tissue culture)
cause an increase in red cell count.  4. Yellow (for blood culture)
 Yellow- Broth mixture Preserves viability Microbiology -
Black of microorganisms aerobes,
AFTER CENTRIFUGE anaerobes, fungi
 Top - Plasma or Serum Black Sodium Forms calcium salts Westergren
 Middle - Gel citrate to remove calcium Sedimentation Rate;
(buffered) requires full draw
 Bottom - blood cells w/o mixing the plasma
Orange Thrombin Quickly clots blood STAT serum
chemistries
GOLD: SERUM SEPARATING TUBE
Light Sodium Inactivates Serum lead
 It is centrifuged
Brown heparin thrombin and determination
 The blood cell sink to the bottom of the tube are
thromboplastin;
covered by a layer of the gel
contains virtually no
 The serum or plasma is left on top lead
 The gel enables the tube to be tipped upside-down and Pink Potassium Forms calcium salts Immunohematology
transported w/o the blood cells remixing with the EDTA
plasma
White Potassium Forms calcium salts Molecular/PCR and
 the Serum or Plasma is the left on Top.  EDTA DNA testing
1. RED
REQUISITION FORM (PATIENTS)
2. GOLD- a Serum Separating Tubes (SST)
1. Surname, First name, and middle initial
 contains particles that cause blood to CLOT quickly, 2. ID number
 GEL  : to separate blood cells from Serum  3. Date of irth and sex
4. Requesting physician's complete name
3. GRAY - these tubes contain FLUORIDE and OXALATE.  5. Source of specimen   
What is the ROLE of FLUORIDE?   Must  given when requesting microbiology, cytology
 It prevent glycolysis fluid analysis.,or other testing where analysis and
 Fluoride prevent enzymes in the blood from reporting  is site specific
working, so a substance such as glucose will not 6. Date and time of collection. 
be gradually used up during storage 6. Test(s) requested

4. DARK BLUE - contains the sodium salt of heparin, san LABELING THE SAMPLE
anticoagulant.   properly labeled the sample is essential so that the results
5. ORANGE - contain thrombin w/c makes the blood clot of the test MATCH THE PATIENT.the key elements in
extremely rapidly.  labeling are;
 these allows the serum to be analyzed in a shorter 1. Patient's Surname, first and middle
Time. 2. ID number
6. LIGHT YELLOW - used in HLA phenotyping.  3. NOTE ;  both of the above Must match the same on
7. PINK - similar to purple tubes (both contain EDTA) these the requisition FORM. 
are used for  ABO groupings and cross-matching    4. Date, time and initials of the phlebotomist must be
              *original PINK does not contain anticoagulant.  on the label of Each tube
‘
COLLECTION TUBES FOR PHLEBOTOMY AUTOMATED SYSTEM
Test tubes Additive Mode of Action Uses o May include labels with bar codes
Red None Blood clots, and the Chemistries, o Labeled collection tubes
serum is separated by immunology and
centrifugation serology, blood PATIENTS RELATIONS AND IDENTIFICATION
bank (cross Phlebotomist roles requires
match) o Professional
Gold None Serum separator tube Chemistry, o Courteous
(SST) contains a gel immunology and o Understanding
at the bottom to serology
-greet
separate blood from
-inform
serum in
-effective communication (both verbal and non verbal is
centrifugation
essential)
Light Plasma Anticoagulates with Chemistries
 Proper patient identification is MANDATORY
Green Separating lithium heparin;
 If patient is able to respond, ask for full name and always check
Tube (PST) Plasma is separated
the armband for confirmation
w/ lithium with PST gel at the
heparin bottom of the tube  DO NOT DRAW BLOOD if the armband is missing
Green Sodium Inactivates thrombin For lithium level,  Always THANK THE PATIENT and excuse yourself
heparin or and thromboplastin use sodium courteously when finished
lithium heparin
heparin For ammonia An Outpatient
level, use sodium 1. Must provide identification other than the verbal statement of a
or lithium heparin name
2. using the requisition for reference, ask a patient to provide
Dark Blue EDTA- Tube is designed to Trace element
additional information such as a surname or birth date.
contain no testing (zinc,
contaminating metals copper, lead,
PATIENT ‘S BILL OF RIGHTS
mercury) and
toxicology  Declared by JOINT COMMISION ON ACCREDITATION OF
HEALTHCARE ORGANIZATION (JCAHO)
Light Gray Sodium Antiglycolytic agent Glucose requires
fluoride and preserves glucose up full draw (may  Basic patient rights endorsed by JCAHO
potassium to 5 days cause hemolysis if
oxalate short draw) PATIENT’S RIGHT
Yellow ACD (acid- Complement HLA tissue  Impartial access to treatment/accommodation
citrate- inactivation typing, paternity  Considerate, respectful care
dextrose) testing, DNA  Confidentiality of all communication
studies
 14. Remove the needle from the patient's arm using a swift backward
AREAS TO AVOIDED motion.
 EXTENSIVE SCARS from burns and surgery 15. Press down on the gauze once the needle is out of the arm,
 The upper extremity on the side of a previous applying adequate pressure to avoid formation of a hematoma.
MASTECTOMY test result may be affected because of
LYMPHEDEMA SUMMARY
1. Fill out the requisition
LYMPHEDEMA 2. Equipment
 Swelling of the arms and legs caused by build up of lymphatic 3. Apply tourniquet and palpate for veins
fluid 4. Sterilize the site
5. Insert needle
 TYPES OF LYMPHEDEMA 6. Drawing the specimen
1. INHERITED (primary lymphedema) 7. Releasing the tourniquet
 Patients with inherited lymphedema were born 8. Applying pressure over the vein
lacking lymph vessels and nodes 9. Disposing needle into sharp containers
 Swelling in the foot, appear during ADOLESCENCE
2. ACQUIRED (secondary lymphedema) DISADVANTAGES OF NEEDLE AND SYRINGE BLOOD DRAWS
 Syringe are not safe because a tube transfer is necessary
 Swelling in the arms, hands, legs, and feet caused
 The specimen may be hemolyzed
by injuring the lymphatic system
 Not good for dehydrated patients
 Not good for patient with poor circulation
AREAS TO BE AVOIDED WHEN CHOOSING A SITE
 Cannot be used for blood cultures and ESR
1. HEMATOMA
2. INTRAVENOUS THERAPHY (IV) BLOOD
COMPLICATION IN VENIPUNCTURE
TRANSFUSIONS
Immediate Local Complications
o Fluid my dilute the specimen so collect from the
1.1 Hemoconcentration
opposite arm if possible 1.2 Hematoma
o Satisfactory samples can be drawn below the IV by
1.3 Failure of blood to enter the syringe
the following procedure 1.3.1 Excessive Aspiration
- Turn off the IV for atleast 2 mins before 1.3.2 Piercing the outer coat of the vein
venipuncture 1.3.3 Transfixation of the vein
- Apply the tourniquet below the IV site or 1.4 Syncope
select a vein other than the one with the IV 1.5 Bleeding
- Perform the venipuncture, draw 5ml of blood 1.6 Transmission of Diseases
and discard before drawing the specimen tube
testing Additional Consideratios:
To prevent a hematoma:
CANNULA/FISTULA/HEPARIN LOCK 1. Puncture only the uppermost wall of the vein
 Blood should not be drawn from an arm with a fistula or 2. Remove the tourniquet before removing the needle
cannula w/o consulting the attending physician 3. Use the major superficial veins
EDEMATOUS EXTREMITIES 4. Make sure the needle fully penetrates the upper most
 Tissue fluid accumulation alters tests results wall of the vein. (Partial penetration may allow blood to
leak into the soft tissue surrounding the vein by way of the
PROCEDURE FOR VEIN SELECTION needle bevel)
 Palpate and trace the path of veins with index finger
 ARTERIES PULSATE are most elastic and have thick ◦ Mix tubes with anticoagulant additives gently
wall 5-10 times
 THROMBOSED VEINS lacks resilience, feel cord-like ◦ Avoid drawing blood from a hematoma
and roll easily ◦ Avoid drawing the plunger back too forcefully,
if using a needle and syringe, and avoid frothing of the
1. If superficial veins are NOT readily apparent you can force blood sample
into the vein by massaging the arm from wrist to elbow ◦ Make sure the venipuncture site is dry
2. Tap the site with index and second finger ◦ Avoid a probing, traumatic venipuncture
3. Apply a warm, damp wash cloth to the site for 5mins.
4. Lower the extremity over the bedside to allow the veins to fill INDWELLING LINES OR CATHETERS
 Potential source of test errors
VENIPUNCTURE PROCEDURE  Most line are flushed with a solution of heparin tom reduce the
1. Approach the patient in a friendly calm manner risk of thrombosis
2. Identify the patient correctly  DISCARD a sample at least three times the volume of the
3. Properly fill out appropriate requisition forms, indicating the test line
orders.
4. Verify patients condition HEMOCONCENTRATION
 Fasting  An increased concentration of larger molecules and forced
 Dietary restrictions elements in the blood may be due to several factors.
 Medications 1. Prolonged tourniquet application (1 min)
 Timing 2. Massaging, squeezing or probing a site
 Medical treatment 3. Long-term IV therapy
5. Check for any allergies to antiseptics, adhesives or latex by 4. sclerosed or occluded veins
observing for armbands and/or asking the patient 5. Dehydration
6. POSITION THE PATIENT
7. APPLY THE TOURNIQUET 3-4 INCHES above the selective Prolonged Tourniquet Application:
puncture site ◦ Primary effect is hemoconcentration of non-
8. The patient should make a fist w/o pumping the hand filterable elements (i.e. proteins). The hydrostatic pressure
9. Select venipuncture site causes some water and filterable elements to leave the
10. Cleanse in a circular fashion, beginning at the site and working extracellular space.
outward. Allow to air dry.
◦ Significant increases can be found in total
11. Grasp the patient's arm firmly using your thumb to draw the skin
protein, aspartate aminotransferase (AST), total lipids,
taut and anchor the vein.
cholesterol, and iron
12. The needle should form a 15 to 30 degree angle
13. When the last tube to be drawn is filling, remove the tourniquet.
 ◦ Affects packed cell volume and other cellular  collecting the specimen
elements  specimen container

DRUG MONITORING DEFIBRINATION

Considerations ADVANATGES
1. Administration  RBC and WBC morphological study
2. Body distribution,  Gives a large volume of serum
3. Metabolism  Used for L.E preparation
4. Elimination that affect the drug concentration as measured METHODS
in the blood.  With the applicator sticks
Check for timing instructions for drawing the appropriate samples.  Used of Erlenmeyer with beads
 Used of Erlenmeyer with glass rod
EFFECTS OF EXERCISE
 Increase of SAFETY AND INFECTION CONTROL UNIVERSAL
 Creatinine kinase(ck) PRECAUSTION
 Aspartate aminotransferase (ast)
 Lactate dehydrogenase (ldh) PROTECT YOURSELF
 Platelet count Practice universal precautions:
 - Wear gloves and lab coats/gowns when handling
What will return to normal shortly after exercise? blood/body fluids
1. All analytes like: Lactic acid, creatinine, fatty acids, amino - Changes gloves after each patient or when contaminated
acids, proteins and enzymes except: - Wash hands frequently
 Creatine kinase (CK) - Dispose of items in appropriate containers
 Aspartate Aminotransferase - DISPOSE OF NEEDLES immediately upon removal from
 (AST) the patient’s vein. DO NOT BEND, BREAK, RECAP, OR
 Lactate dehydrogenase (LDH) RESHEATH NEEDLES to avoid accidental needle puncture
- CLEAN UP ANY BLOOD SPILL with a disinfectant such
STRESS: CAUSES as freshly 10% bleach
 Transient elevation in white blood cells (wbc)
 Elevated adrenal hormone values (cortisol and If you stick yourself with a contaminated needle:
catecholamines)  Remove your gloves and dispose
them properly.
ANXIETY: CAUSES  Squeeze puncture site to promote
 Acid-base imbalances bleeding.
 Increased lactate  Wash the area well with soap and
 Diurnal Rhythms: water.
Ex:  Record the patient's name and ID
1. Serum cortisol levels are highest in early morning but are number.
decreased in the afternoon.  Follow institution's guidelines
2. Serum iron levels is decreased during the day. regarding treatment and follow-up.
 NOTE: The use of prophylactic
POSTURE: postural changes (supine to sitting etc.) zidovudine following blood exposure to HIV has shown
Reason: Certain larger molecules are not filterable into the effectiveness (about 79%) in preventing seroconversion
tissue, therefore they are more concentrated in the blood
 Increased with changes in Position ZIDOVUDINE OR AZIDOTHYMIDINE (AZT) (also called ZDV)
 Enzymes ( Enproliic) - A nucleoside analog reverse-transcriptase inhibitor (NRTI),
 Proteins, a type of antiretroviral drug used for the treatment of
 Lipids, HIV/AIDS infection.
 Iron
PROTECT THE PATIENT
 calcium
- Place BLOOD COLLECTION EQUIPMENT away from
Other Factors: Gender, Age, Pregnancy (GAP)
patients
- PRACTICE HYGIENE for patients protection
SKIN PUNCTURE
- Always wear a clean lab coat or gown
Small quantities are required
1. Infants Depth
SKIN PRICK IN INFANTS
2. Extensive burns 2-3 mm- Adults
HEEL
3. Bed ridden 1.6 mm- infants
 is recommended location for blood collection on a new born baby
SITES Tenderfoot  pre warming the infants heel (42 C for 3-5 mins) is important to
 Ear Lobe Tenderlett obtain CAPILLARY BLOOD GAS SAMPLES
 pad of the finger Preemi etc.  greatly increase the flow of blood for collection of other
 big toe or heel in infants specimens
 NOT SO HIGH TEMP. infants skin is so thin and easily get
DISADVANTAGES OF SKIN PUNCTURE burned
 less amount  Clean the site to be punctured with an alcohol sponge.
 coagulation  Dry the cleaned area with a dry cotton sponge
 more glucose  Using a sterile blood lancet, puncture the side of the heel in the
 more leukocytes appropriate regions.
 higher RBC and Hgb  DO NOT USE the central portion of the heel
 platelet Ct tends to fall  DO NOT USE a previous puncture site.
 Wipe away the FIRST DROP OF BLOOD with dry cotton.
FINGERSTICK PROCEDUERE ILLUSTRATED  use GENTLE PRESSURE to produce a rounded drop of
 equipment blood
 proper location on finger  Fill the capillary tube(s) or micro collection device(s) as needed.
 puncture w/ lancet  When finished, ELEVATE THE HEEL, PLACE A PIECE OF
 drop of blood CLEAN, DRY COTTON ON THE PUNCTURE SITE
 wipe the first blood
  DISPOSE OF CONTAMINATED MATERIALS IN - Alkali hematin
APPROPRIATE WASTE RECEPTACLES. - Cyanmethemoglobin
 Remove your gloves and wash your hands.
FORMS OF HEMOGLOBIN
THE STUDY OF HEMOGLOBIN - Reduced Hgb
Hemoglobin - Oxyhemoglobin
 Hb or Hgb is the iron containing oxygen transport
In general, hemoglobin can be saturated with oxygen molecules
 Hemoglobin in the blood carries oxygen from the respiratory (oxyhemoglobin), or desaturated with oxygen molecules
organs (lungs) to the rest of the body (i.e. the tissues) where (deoxyhemoglobin).
it releases the oxygen to burn nutrients to provide energy and
collects the resultant carbon dioxide to bring it back to the
respiratory organs to be dispensed from the individual
 Hemoglobin has an oxygen binding capacity of 1.34 mL O2 per Oxyhemoglobin
gram of hemoglobin,which increases the total blood  Is formed during physiological respiration when oxygen
oxygen capacity seventy-fold compared to dissolved oxygen binds to the heme component of the protein hemoglobin
in blood. in red blood cells.
 This process occurs in the pulmonary capillaries adjacent
 HEMOGLOBIN consist mostly of PROTEIN SUBUNITS (the to the alveoli of the lungs. The oxygen then travels
"globin" molecules), and these proteins, in turn, are folded through the blood stream to be dropped off at cells where
chains of a large number of different amino acids called it is utilized as a terminal electron acceptor in the
polypeptides. production of ATP by the process of oxidative
 The amino acid sequence of any polypeptide created by a cell is phosphorylation.
in turn determined by the stretches of DNA called genes.
 In all proteins, it is the amino acid sequence which determines
the protein's chemical properties and function.

MUTATION IN THE GENES for the hemoglobin proteins in a species

results in HEMOGLOBIN VARIANTS

ALL THESE DISEASES PRODUCE ANEMIA

 HEMOGLOBINOPATHIES – cause a group of hereditary

disease

 SICKLE CELL DISEASE – best known hemoglobinopathy,

first human disease whose mechanism was understood
at the molecular level

 THALASSEMIAS – separate set of disease, involves

underproduction of normal and sometimes abnormal
hemoglobins, through problems and mutations in globin
gene regulation

HEMOGLOBIN (Hb)
- Is synthesized in a complex series of steps
- The HEME part is synthesized in a series of steps in the
MITOCHONDRIA AND THE CYTOSOL of immature red
blood cells
- The GLOBIN protein parts are synthesized by RIBOSIMES
in the cytosol
- Production of Hb continues in the cell throughout its early
development from the proerythroblast to the
reticulocyte in the bone marrow.)
- At this point, the nucleus is lost in the red blood cells.
- The residual ribosomal RNA allows further synthesis of Hb
until the reticulocyte loses its RNA soon
- (This hemoglobin-synthetic RNA in fact gives the reticulocyte
its reticulated appearance and name).
ABNORMAL FORMS OF HEMOGLOBIN
Hgb MOLECULE
1. CARBOXYHEMOGLOBIN (CO)
 - globin- 2 sets of polypeptide chains
- Gasoline motors
 - 4 mol. Of protoporphyrin IX - Gasoline heaters
 - 4 iron atoms in the Fe++ state - Defective gas stoves
 - 2-3 DPG (BPG) Bisphosphoglycerate mol. - Burning coal
 Resident at the center of Hgb unit - Cigars and cigarettes

PRINCIPAL CHARACTERISTICS 2. METHEMOGLOBIN

 - iron in the ferrous state - Iron is oxidized to ferric state consists of 4 polypeptide
 - contains 4 pyrolle groupings related to chains and 4 molecules of hemin
 chlorophyll
3. METHEMOGLOBINEMIA
RED BLOOD CELL - is a blood disorder in which an abnormal amount of
- contains several hundred thousand hemoglobin molecules, methemoglobin – a form of hemoglobin is produced
which transport oxygen - With methemoglobinemia, the hemoglobin can carry
- Hemoglobin is easily oxidized to oxygen but is unable to release it effectively to body
- Acid hematin tissues.
  A quick and easy bedside test for determining whether
TWO FORMS OF METHEMOGLOBINEMIA dark blood is due to methemoglobinemia is to bubble
100% oxygen in a tube that contains the dark blood.
1. GENETICS  Blood that remains dark likely does so because of the
- The first form is passed on by both parents. The presence of methemoglobin.
parents usually do not have the condition themselves,
but they carry the gene that causes the condition. OXIMETERY
- It occurs when there is a problem with an enzyme called - Another simple test (and one that is less likely to splash
cytochrome b5 reductase. potentially infectious blood) is to place 1-2 drops of
blood on white filter paper, then evaluate for color
change upon exposure to oxygen. (This test can be
accelerated by gently blowing supplemental oxygen onto
the filter paper.)
- Deoxygenated hemoglobin changes from dark red or
violet to bright red, whereas methemoglobin remains
2. HERIDITARY METHEMOGLOBINEMIA brown.
 Serum Nitrite
2 TYPES SULFHEMOGLOBINEMIA
 Type 1 (also called erythrocyte reductase
deficiency) occurs when red blood cells lack the  Is a rare condition in which there is excess
enzyme. sulfhemoglobin (SulfHb) in the blood. The pigment is a
 Type 2 (also called generalized reductase greenish derivative of hemoglobin which cannot be
deficiency) occurs when the enzyme doesn't work converted back to normal, functional hemoglobin. It
anywhere in the body. causes cyanosis even at low blood levels.
 It is a rare blood condition that occurs when a sulfur
 The second form of inherited methemoglobinemia is atom is incorporated into the hemoglobin molecule.
called hemoglobin M disease. When hydrogen sulfide (H2S) (or sulfide ions) and ferric
 It is caused by defects in the hemoglobin protein ions combine in the blood, the blood is incapable of
itself. carrying oxygen.
 Genetics: Only one parent needs to pass on the
abnormal gene for the child to inherit the disease. SULFHEMOGLOBIN
 - Non life threatening
ACQUIRED METHEMOGLOBINEMIA  - Remains in the RBCs
1. ANESTHETICS such as benzocaine  - Can’t transport O2 and combines with CO to form
2. BENZENE carboxyhemoglobin
3. ANTIBIOTICS (including dapsone and chloroquine),
Sulfonamides CAUSES
4. ANILINE DRUGS and its derivatives  sulfonamides
5. NITRITES (used as additives to prevent meat from spoiling)  Sulfur sulfasalazine
6. CHLORATES  aromatic amine drugs
7. QUINONES  severe constipation
8. Vegetables containing NITRATES (such as beets).
 bacteremia
 Enterogenous cyanosis
CHARACTERISTICS OF METHEMOGLOBIN
- Part of the inactive Hgb
TESTS FOR ABNORMAL HgB1.
- Unable to combine reversibly with O2 and CO2
- Causes a shift of the O2 dissociation curve and hinders thE
1. Spectrophotometric Method
transfer of O2 from the blood to the tissues.
 Carboxyhemoglobin 0.05 ml of blood + 10mg. Of Na2S2O4
- Normal Amount is 0.24 gm. 0.5 gm/100 ml will cause
cyanosis  Carbon monoxide is a poisonous invisible gas.
 Red blood cells (RBC) have hemoglobin. This protein has
Symptom of type 1 methemoglobinemia (erythrocyte reductase the property of wrapping the oxygen.  
deficiency)  Oxygen is transported throughout the entire body.
 Bluish coloring of the skin  Carbon monoxide replaces the oxygen in your RBCs,
converting hemoglobin into carboxyhemoglobin.
Symptoms of type 2 methemoglobinemia (generalized reductase
deficiency) Carboxyhemoglobin cannot carry oxygen. The origin of CO is the
 Developmental delay incomplete combustion on combustion motors or gas furnaces, fires
 Failure to thrive and tobacco smoke.
 Intellectual disability
 Seizures Symptoms of CO intoxication are:
 Headache.
Symptoms of Hemoglobin M disease  Nausea or vomiting 
 Bluish coloring of the skin  Irritability
 Dizziness.
Symptoms of Acquired methemoglobinemia include:
 Bluish coloring of the skin Purpose of the Carboxyhemoglobin test
 Headache  This test measures the concentration of carboxyhemoglobin in
 Fatigue blood.
 Shortness of breath  This test is used to diagnose and manage carbon
 Lack of energy monoxide poisoning due to exposition to incomplete
combustion gases (CO).
TEST FOR METHEMOGLOBIN
 Reference range values
 Specific enzyme assays (nicotinamide adenine Reference values for non-smokers   0.005 – 0.015
dinucleotide [NADH]–dependent reductase,  
cytochrome b5 reductase) may be determined, often in Reference values for smokers:
multiple cell lines (ie, platelets, granulocytes, and  1-2 packs/day        0.04 – 0.05
fibroblasts), to diagnose inherited cases.  >2 packs/day         0.08 –0.09
 Toxic levels                 >0.20
 o Potassium cyanide
 Abnormal findings o Potassium dihydrogen phosphate
- High level of Carboxyhemoglobin in blood is usual among
smokers. SOURCES OF ERRORS IN LAB TESTS
- Heavy smoking lead to bigger test values.  Technical sources of error
- 4% (0.04 are indicative of possible hemolytic anemia 1. Inadequate mixing of specimen
- 20% (0.2) represent intoxication that can lead to severe 2. pipets
damage and even to death. 3. pipeting error
4. Use of dirty, scratched, or unmatched
OLD METHODS cuvets
5. use of deteriorated reagents, oxidation of
Shaking Method the reagent
1. Normal Blood – No change in color Others
2. Methemoglobin- Chocolate brown Lipemic blood
3. Carboxyhemoglobin – Cherry Red Hypochromic cells
4. Sulfhemoglobin- greenish Presence of abnormal amount of proteins

Alkali Spot Test

1. Yellowish green- Normal Glycated hemoglobin (hemoglobin A1c, HbA1c, A1C, or Hb1c;
2. Blue green- Abnormal sometimes also HbA1c)

Katayama’s Test ( a qualitative colorimetric Test)  Is a form of hemoglobin that is measured primarily to
identify the average plasma glucose concentration
SPECIMEN COLLECTION REQUIREMENTS over prolonged periods of time.
 Collect - Whole Blood, one heparinized blood gas syringe  It is formed in a non-enzymatic glycation pathway by
 Handling - Collect specimen anaerobically. Blood gas syringe hemoglobin's exposure to plasma glucose.
should be capped with no air space. Transport immediately.
 Minimum Volume - 2.0 Ml I. Normal levels of glucose produce a normal amount of glycated
 Transport - Refrigerated (2-8°C) hemoglobin.
 Rejection Criteria - Specimens with syringe uncapped or II. As the average amount of plasma glucose INCREASES, the
with airspace, tubes with benzalkonium heparin, EDTA, citrate, fraction of glycated hemoglobin INCREASES in a predictable
oxalate, and fluoride way.
 Stability - Ambient (20-25°C): 30 minutes, Refrigerated (2- III. This serves as a marker for average blood glucose levels over the
8°C): 30 minutes, previous months prior to the measurement
 Frozen (at or below -20°C): Unacceptable
 Method: Colorimetry
IV. In diabetes mellitus, higher amounts of glycated hemoglobin,
 Reference Value <1.5% of total hemoglobin indicating poorer control of blood glucose levels, have been
associated with cardiovascular disease, nephropathy, and
 Toxic Levels - ≥1.9%
retinopathy.
V. Monitoring HbA1c in type 1 diabetic patients may improve outcomes
SYMPTOMS ARE PROPORTIONAL TO THE LEVEL OF
METHEMOGLOBIN:
PRINCIPLE
 Less than 10% methemoglobin: No symptoms
 10-20% methemoglobin: Skin discoloration only, most
notably on mucus membranes
 Glycation of proteins is a frequent occurrence, but in the case
of hemoglobin, a nonenzymatic reaction occurs between
 20-30% methemoglobin: Anxiety, headache, dyspnea on
glucose and the N-end of the beta chain.
exertion
 30-50% methemoglobin: Fatigue, confusion, dizziness,  This forms a Schiff base which is itself converted to 1-
tachypnea, palpitations deoxyfructose. This rearrangement is known as Amadori
 50-70% methemoglobin: Coma, seizures, arrhythmias, rearrangement.
acidosis  When blood glucose levels are HIGH, GLUCOSE
 Greater than 70% methemoglobin: Death MOLECULES attach to the hemoglobin in red blood cells.
 The LONGER HYPERGLYCEMIA occurs in blood, the more
 Most hemoglobin tests had to be performed in a medical glucose binds to hemoglobin in the red blood cells and the
laboratory.  HIGHER THE GLYCATED HEMOGLOBIN.
 HEMOGLOBIN TEST METER: Used at home to measure their  Glucose levels are intermittently raised in portal vessels
hemoglobin levels anytime during the day. carrying absorbed glucose to the liver for regulation.
 Passing red cells will have INCREASED GLYCATION AFTER
 The hemoglobin home test kit measures Hemoglobin, Total SUGARY DRINK OR PORRIDGE.
Cholesterol and Glucose levels.
  The HEMOGLOBLIN HOME TEST METER is a diagnostic tool  Once a hemoglobin molecule is glycated, it remains that way.
therefore, reflects the average level of glucose to which
and can be used to monitor the progress of certain medical the cell has been exposed during its life-cycle.
conditions involving blood count such as anemia.     Measuring glycated hemoglobin assesses the effectiveness
of therapy by monitoring long-term serum glucose
LIMITATION TO SAHLI’S METHOD regulation.
 Standard is not permanent
 Considerately delay in the development of the permanent  HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
color (may instances reach 20 marks) (HPLC) - The HbA1c result is calculated as a ratio to
o 10 mins. : 95% acid hematin total hemoglobin by using a chromatogram
o 20 mins. : 98% acid hematin  IMMUNOASSAY
o 1 hour : 100% acid hematin  ENZYMATIC
 Large errors have been found in sahlis pipette, recalibration  CAPILLARY ELECTROPHORESIS
is needed before using them
Point of care (e.g., doctor's office) devices use:
IMPROVING SAHLI’S METHOD  Immunoassay
 Cyanomethemoglobin absorbance reaching at 540 nm  Boronate affinity chromatography
 Blood sample treated with
o Potassium ferricyanide UNRELIABILITY OF RESULTS
  after blood loss In people acclimated to high altitudes, the concentration of 2,3-
 after surgery Bisphosphoglycerate (2,3-BPG) in the blood is increased,
 blood transfusions which allows these individuals to deliver a larger amount of
 anemia oxygen to tissues under conditions of lower oxygen tension.
 high erythrocyte This phenomenon is called a heterotropic allosteric effect.
 presence of chronic renal or liver disease; after administration
of high-dose vitamin C; or erythropoietin treatment THE NORMAL RANGE FOR HEMOGLOBIN IS
 For men, 13.5 to 17.5 grams per deciliter (135 to 175 grams
 In general, the reference range (that found in healthy per liter)
persons), is about 20–40 mmol/mol (4–5.9%)  For women, 12.0 to 15.5 grams per deciliter (120 to 155
 Higher levels of HbA1c are found in people with
grams per liter)
 Normal ranges for children vary with age and sex.
persistently elevated blood sugar, as in diabetes
mellitus. FACTORS AFFECTING HgB VALUES
 A diabetic person with good glucose control has a HbA1c level  Sex: Female – 12to 16 g/dl
that is close to or within the reference range. Male - 13 to 18 g/dl
 Age at birth – 15 to 20 g/dl
REFERENCE RANGE OF Hb 2 months - 9 to 14 g/dl
 International Diabetes Federation and American College of 10 yrs - 12 to 15 g/dl
Endocrinology recommend HbA1c values below 50 yrs - decreased
48 mmol/mol (6.5%)  Altitude - high- increased
 American Diabetes Association : recommends that the HbA1c - Low- decreased
be below 53 mmol/mol (7.0%) for most patients.  Time - AM- increased
PM- decreased
PERSISTENT ELEVATIONS OF BLOOD SUGAR AND HbA1c
 Increase the risk of long-term vascular complications of LOWER THAN NORMAL RESULTS
diabetes Causes of anemia:
 coronary disease  Iron deficiency
 heart attack  Vitamin B-12 deficiency
 stroke  Folate deficiency
 heart failure  Bleeding
 kidney failure  Cancers that affect the bone marrow, such as leukemia
 blindness  Kidney disease
 erectile dysfunction  Liver disease
 neuropathy (loss of sensation, especially in the feet),  Hypothyroidism
gangrene  Thalassemia — a genetic disorder that causes low levels
 gastroparesis (slowed emptying of the stomach) of hemoglobin and red blood cells
 Poor blood glucose control also increases the risk of short- HIGHER THAN NORMAL RESULTS
term complications of surgery such as poor wound Polycythemia vera
healing.  Lung disease
 Dehydration
LOWER THAN EXPECTED LEVELS OF HbAIc  Living at a high altitude
 Heavy smoking
1. Shortened red blood cell lifespan, such as with glucose-6-  Burns
phosphate dehydrogenase deficiency, sickle-cell disease, or any  Excessive vomiting
other condition causing premature red blood cell death.  Extreme physical exercise
2. Blood donation will result in rapid replacement of lost
RBCs with newly formed red blood cells.
TERMS IN CONNECTION TO RBC AND HgB
 Anemia
HIGHER THAN EXPECTED LEVELS OF HbAIc
 Polycythemia
With longer red blood cell lifespan
 Erythrocytosis
 Vitamin B12 or folate deficiency.  Erythremia
 Oligocythemia
PHYSIOLOGIC ERRORS IN HgB DETERMINATION  Erythron
 Lipemic blood (correction- + 6 ml Cyanmeth reagent  Hypochromia
 - High Leukocyte ct.- ( by centrifugation)  Hyperchromia
 -Hbs and Hbc (resistance of cells to hemolysis)- 1:2  Hemoglobinemia
dilution  Hemoglobinuria
 Easily ppted Hgbs. – M. myeloma
Ret-He (Reticulocyte Hemoglobin)
Waldenstrom Macroglobulinemia
Correction- New Drabkin’s  Is a direct assessment of the incorporation of iron into
Old Drabkin’s – 0.1 gm of CO3 erythrocyte hemoglobin
 RETICULOCYTES are red blood cells precursors.
FACTOR AFFECTING HgB AFFINITY TO O2  As such, Ret-He is a direct estimate of the recent  functional
1.0 Blood/body temp. - High –release O2 more readily availability of iron (2-3 days)
2.0 Blood pH (Bohr Effect) This decrease in hemoglobin's  TRADITIONAL CHEMISTRY test used for iron 
affinity for oxygen by the binding of carbon dioxide and acid is assessment ( SERUM IRON, Tsat, FERRITIN) are indirect
known as the Bohr Effect (shifts the O2-saturation curve to the meaasurement
right  As a direct measurement, Ret-He may identify iron
3.0 2-3 DPG deficiency earlier than traditional parameters. 
4.0 CO2 Levels high carbon dioxide levels is also lower in pH o Ret-He is an established parameter in the National Kidney
(more acidic). Increased affinity for carbon dioxide by the Foundation guidelines for assessing the initial iron status
venous blood is known as the Haldane effect and to assess IV iron replacement of HEMOdialysis
5.0 Amt of Hgb F patients with chronic kidney disease.
6.0 Abn Hgb variants  a LOW Ret-He is indicative of iron deficiency. 
Shift to the Rt.-O2 released readily  with a Ret-He above the normal range, a patient may not
Shift to the Lf – O2 –not released readily respond to additional iron theraphy
  THE REFERENCE RANGE FOR Ret-He is 28. 22-36. 6 pg.  METHODS OF HCT DETERMINATION Macro Methods (OLD
method)
TRANSFERRIN SATURATION (Tsat) 1. Wintrobe method - D.0 anticoagulant 
 in  percentage is a medical laboratory value.       Reading  X  10  - Hct
 It is the ratio of serum iron and total iron-binding 2. Haden's method - 1. 1% sod. Oxalate
capacity. it is the ratio of serum iron and total iron-binding      Reading  X 20 
capacity, multiplied by 100 of the TRANSFERRIN that 3. Van Allen - 1. 6% sod. Oxalate
is available to bind iron.  4. Sanford Magath Method - 1. 3% S.O
 This value tells a clinician how much serum iron are 5. Bray's Method - Heparin
actually bound. 
 For instance, a value of 15% means that the 15% of USE of EPPENDORF TUBES 
iron-binding sites of transferrin are being occupied  CENTRIFUGE blood samples in EPPENDORF tubes for
by IRON.  10 mins. 
 NORMAL reference ranges are : 
o Serum Iron : 60-170 ug/dl (10-30umol/L) HEMATOCRIT determination 
o TIBC : 240-450 ug/dl      Where :
        >H1 = Height of the RBC column
o Transferrin Saturation : 15-50% (males), 12-
        >H2 = Height of the RBC + height of the Plasma column
45% (females) 
        >Calculate Hc% (hematocrit) value
o ug/dl = micrograms per deciliter. 
    Formulae :
           H1  x  100 /H2 

HEMATOCRIT CONDITIONS 
 HEMATOCRIT (Ht or HCT)   A hematocrit of less than 15% can result in CARDIAC
 Packed Cell Volume (PCV) FAILURE
 Erythrocytes Volume Fraction (EVF)  a hematocrit of over 60% may result in SPONTANEOUS
 Volume Percentage (%) of Red Blood Cells BLOOD CUTTING 
 Volume Percentage (%) of Red Blood Cells in BLOOD. 
 It is normally about 45% for men & 40% for women.  MICRO METHOD
 Integral part of complete blood count.    Heparinized Capillary Tube - capilla attraction (7. 5 mm  X 
1. 0 or 1. 2 mm) 
THE HEMATOCRIT   Sealed with a CLAY (Seal ease; critoseal, critocaps,
 HCT - Volume of packed RED cells(VPRC) plastic in )
        >RCV at the bottom of the tube  Centrifuge in MICROCENTRIFUGE (10, 000 to 12, 000
        >No. of mm. of packed red cells/100 RPM) 
 USEFULNESS  direct reading device or with caliper 
        >1. 0 Absolute indices/constants
        >2. 0 Rough Quality Control Question: What is the correct order in a hematocrit  tube if malarial
        >3. 0 Buffy coat-rough idea of WBC CT.  paaraasites are present? 
        >4. 0 Buffy coat-smears A. It or Ring, Mature Trophozoites, Schizonts, Gametocytes
B. Gametocytes, Schizonts, Mature Tropozoites, Ring
HEMATOCRIT TUBE Forms (Immature Trophozoites)
 PLASMA C. Schizonts, Gametocytes, Mature Trophozoites, Immature
   - 55% of total blood volume Tropozoites
   - 91% water
   - 7% Blood Proteins (fibrinogen, albumin,, globulin) LYMOGRA 
   - 2% Nutrients (Amino acids, sugar, lipids)  Which is found at the HIGHEST PART of the white blood cell
   -       Hormones (erythropoietin, insulin, etc. ) layer? 
   -       Electrolytes (sodium,Potassium,calcium, etc. )  Which is found at the LOWEST PART of the white blood cell
 CELLULAR COMPONENTS layer? 
                 - 45% of Total Blood Volume
                 -BUFFY COAT - White Blood Cells (7000-9000 per mm^3 What is the most accurate manual method of determining packed
of blood blood volume & should be used whenever feasible? 
                                          - Platelets (250, 000 per mm^ of blood A. Microhematocri
                 - RED BLOOD CELLS (RBCs) - about 5, 000, 000 per B. Wintrobe Method
mm^3 of blood.  C. Haden Method
 
    USES OF BUFFY COAT NORMAL VALUES
1. marked leukopenia   Reference Values:
2. leukemia  1. at birth  …  45 to 69%
3. determination of malignant cells  2. 1 year  …   27-44%
4. Determination of L.E. Cells 3. Women …  36-48%
5. MEGALOBLASTS in pernicious anemia  4. Women …  38-42%
6. Phagocyted pathogenic microorganisms  5. Men     …   40-55%
6. Men   …     42-3-48%
LAYERS FORMED: 
 PLASMA, Buffy coat (Platelets & WBCs)  a Complete Blood Count (CBC) may how increased
 Packed cells (NRBC, Retics, RBC)  reticulocytes, a sign of increased red blood cell production, & 
 Anticoagulants ( H E D) decreased hemoglobin & hematocrit. The term "non-hereditary
spherocytosis" is occasionally used. 
 RULE OF THREE (3) - applies Normocytic Normochromic  In general,  patients with sickle cell disease have hmatocrits
Anemia that ar roughly half the normal value. 
 0  million RBC  X  3 = 9 g/dl of HgB  Patients  with Hemoglobin SC disease (where one of the beta
 0 g\dl of Hgb  X 3 = 27 vol. 5% of Hct  globin gees codes for hemoglobin S & the other for the variant,
 hemoglobin C) have higher hematocrits than those with  Dengue Shock Syndrome is a syndrome due to the
homozygous Hb SS disease.  dengue virus that tends to affect children under 10,
 The hematocrits of patients with Hb SC disease run in low-to causing pain, hemorrhage (bleeding) & circulatory
mid-thirties.  collapse (Shock)
 The hematocrit is NORMAL for the people with sickle cell    It starts abruptly with :
trait.  1. HIGH continuous fever
2. Headache 
SOURCE OF ERRORS 3. Respiratory & intestinal symptoms with sore
 The blood: anticoagulant ratio using EDTA.  throat, cough, nausea, vomiting, &
 Excessive EDTA will cause a falsely decreased hematocrit abdominal pain. 
due to shrinkage of the blood cells.   SHOCK occurs after 2 to 6 days with sudden collapse,
 Liquid anticoagulants, such as sodium citrate & heparin cool clammy extremities, weak thread pulse, & blueness
may cause falsely decreased hematocrits when th proper around the mouth (circumal cyanosis) 
blood : anticoagulant ratio is not,maintained by dilution of the  There is bleeding with easy bruising, blood spots in the
sample.  skin (petechiae), spitting up blood (hematemesis), blood
in the stool (melena), bleeding gums & nosebleeds
B-Thalassemia  (epistaxis) 
 ANEMIA is severe : Hgb 20 to 30 g/L. Hematocrit and RBC  Pneumonia & heart inflammation (myocarditis) may
count are also decreased hence the indices MCV, MCH & be present. 
MCHC are all decreased, the RDW is increased.   The mortality is 6% to 30%. most deaths occur in
 The morphology is severe hypochromic microcytic with children. Infants under a year  of age are especially at risk
marke anisocytosi & poikilocytosis.  of death. 
 It is also called Philippine or Southeast Asian
Hemorrhagic Fever.. 

  SOURCE OF ERRORS :
1. STASIS (Hemoconeentration) INCREASE OF HEMATOCRIT 
2. Loss of Blood (aacute hemorrhhage)  Dehydration, hematocrit is elevated
3. After Traansfusion (PCV, Plasma)  Capillary Leak Syndrome -  Episodic Leakage of
4. Vigorous Exercise (it depends) Plasma out of the circulatory system. 
5. Period of Recumbency   Sleep Apnea has been known to cause elevated
6. After Meals hematocrit levels. 
 Anabolic Androgenic Steroids (AAS) use can also
EFFECT OF AEROBIC EXERCISE increase the amount of RBCs &, therefore, impact the
 Aerobic exercise can alter the number of red blood cells in hematocrit, in particular the compounds like:
several ways.   boldenone &
 In general, endurance training increases the number of  oxymetholone 
red blood cells. 
 however, in some cases, exercise can also lead to their What is SLEEP APNEA? 
destruction and therefore DECREASE OF  is a type of sleep disorder characterized by pauses in
HEMATOCRIT.  breathing or instances of shallows or infrequent breathing
 The values  of hemoglobin, hematocrit, Erythrocytes, during sleep. 
Leukocytes, &Platelets increase with the changes of the  Each pause in breathing, called an Apnea, can last from at
body position from  recumbent  to sitting & to erect least ten seconds to several minutes, & may occur 5 to
position.  30 times or more an  hour. 
 the changes are due to a reversible HEMOconcentration
& redistribution of the blood.  >The Mean Corpuscular Volume (MCV) a& the Red Cell
 The GREATEST increase is of the neutrophils  which is Distribution Width (RDW)  can be quite helpful in evaluating a
probably due to mobilization of the pool of neutrophils lower-than-normal hematocrit, because it can help the clinician
which are nearest to the vascular wall, the process being determine whether blood  loss or Chronic or Acute 
parallel to the process of hemoconcentration. >Acute blood loss typically DOES NOT manifest as a change of
hematocrit, since hematocrit is simply a measure of how much of the
EFFECT OF MEALS (Research)  blood volume is made up of red blood cells. 
 The red blood cell count, hemoglobin concentration,     *MCV size of thee red cells
hematocrit level, & platelet count DECREASE 2 HOURS     *RDW iaa s relative measure of the variation in size of the red
AFTER MEAL CONSUMPTION.  cell population 
 the LYMPHOCYTES NUMBER decreased after the first & >a low hematocrit with a low CV with a high RDW suggests a
second hour following meal consmption CHRONIC IRON-DEFICIENT ANEMIA resulting in abnormal
 the number of neutrophils had increased 1 & 2 hours hemoglobin Synthesis during erythropoiesis.
AFTER FOOD INTAKE (P=.003 & P=.006, respectively) >One unit of packed rd blood cells will elevate the hematocrit by
about 3%. 
INCREASE HCT 
 DENGUE FEVER, a high hematocrit is a danger sign of an  LOW HEMATOCRIT 
increased risk of dengue Shock Syndrome. 1. INADEQUATE iron intake of infants
2. Rapid Growth of children during which the iron available
 POLYCYTHEMIA vera2 (PV), a myeloproliferative
cannot keep up with the demands for growing rdd cell
disorder in which the bone marrow produces excessive
mass. 
numbers of red blood cells, is associated with elevated
3. blood loos during menstruation 
hematocrit. 
4. pregnant women, in whom the growing fetus creates a
 CHRONIC OBSTRUCTIVE PULMONARY DISEASE 3
high demand for iron. 
(COPD) & other pulmonary conditions  associated with
5. Patients with chronic kidney disease whose kidneys no
HYPOXIA may elicit an increased productions of red blood
longer secrets sufficient level of the hormone
cells.
erythropoietin that promotes RBC proliferation. 
 This increase is mediated by the increased levels of
erythropoietin by the kidneys in response to hypoxia.  What is the role of eryhtropoietin in the BM? 
 Erythropoietin prevents the death of cells in the erythrocyte 
What is DENGUE SHOCK SYNDROME? Known as DHF   cell line in thee bone  marrow. 
  Therefore, erythropoietin allows those cells to continue to 1. lowest in newborn 
mature, exit the bone marrow and become RBCs.  2. increased in pregnancy  after 3 months
3. increased in  old age 
ESR HISTORY 
 ESR TESTING has long been used in the clinical setting..   ENTRINSIC FACTORS AFFECTING ESR
 Measurement of the erythrocyte sedimentation rate was first 1. TIME (on standing cells become sperical) 
described in 1897 by the Polish  Physician, EDMUND 2. TEMPERATURE
BIERNACKI.  3. TUBE (Diameter, Length, Position)
 In 1918, the method was refined by the Swedish 4. ANTICOAGULANTS (overanticoagulation)
Pathologist ROBERT SANNO F. & the internist ALFF 5. DILUTION
VILHELM ALBERTSSON WESTERGREN. 6. INHERENT ERRORS - Filling the tube, Reading of
 Years later, the cornerstone ESR TESTING method is still Results 
referred to as the Westergren method, & it is the
recommended ESR testing method of the International DRUGS that will increase ESR 
Committee for Standardization in the HEMATOLOGY.   Drugs :
 Reference ranges for the Westergren ESR are based on 1. Dextrain, methyldopa (aldomet)
age & sex.  2. Oral contraceptives
3. Penicillamine
ESR 4. Procainamide
 FALL of the red blood cells  5. Theophylline (asthma, COPD) 
 SUSPENSION STABILITY of the RBCs  6. Heparin
 VELOCITY of erythrocyte sedimentation.  7. Vitamin A 
 non-specific measurement used to detect & monitor an
inflammatory response to injury (acute phase response)  About ESR procedure 
 non specific marker of underlying inflammation.    ESR should be performed at room temperature. 
 ESR should not be determined at a temperature below  18
degree C because of changes in Erythrocytes membrane,
this will decrease the ESR. 
 performance of the procedure at a  temperature
ESR as INFLAMMATORY MARKER  exceeding 18 degree C will produce falsely increased
 it is often used to screen patients with fever of results. 
undetermined origin, arthritis, muscle pains, & other
vague symptoms.  STAGES OF ESR 
1. Initial Period of Aggregation (lag phase ; Rouleaux
1. ESR Testing  phase )
2. Rheumaatoid arthritis  2. Period of Fasting Settling (Rapid phase)
3. Tuberculosis 3. Final Period of Packing (Slow Phase)
4. Systemic Lupus Erythematosis
5. To diagnose & monitor giant cell arteritis & polymyalgia METHODS
rheumatica.  A. Macro Methods
6. An elevated ESR   Wintrobe & Landsberg Method - D.O.
7. Autoimmune disease 
8. Anemia Advantages
9. Infection  more accurate
10. Malignancy  more sensitive when the ESR IS LOW 
 Requires a small amount of blood. 
 TERMS 
 Simple
 ACUTE PHASE REACTANT - a substance in the blood that
increases as a response to an acute condition such as a  NO DILUTION of blood is required
infection, injury, burns etc.   HCT can be determined
 ROULEAX - the stacking of RBCs, caused by extra or  Allows correction of anemia using correction chart 
abnormal proteins in the blood that decrease the normal  Smears can be made in the Buffy coat
distance red cells maintain between each other.   Microbilirubin can be done in the PLASMA. 

FACTORS INFLUENCING ESR INTRINSC FACTORS WESTERGREN METHOD - 3. 8% TSC 

>INTRINSIC   Straight pipette 30 cm  X  2. 5 mm
1. number of RBCs   - albumin   DILUTION of blood 0. 5 + 4. 5 ml. 
2. size of RBCs        - chlolesterol
3. shape of RBCs     - lecithin MODIFIED WESTERGREN METHOD - EDTA
4. proteins                - plasma viscosity   2. 0 ml EDTA anticoagulated blood + 0. 5 ml of 3. 8%
  TSC or 0. 5 ml of NSS 
 Fibrinogen   -  33 X greater
 Alpha 1        -  18 X greater GRAPHIC & CUTLER - 3. 0% sod. Citrate
 Alpha 2        - 3 X greater  TUBE-- 0-50 mm, 0-8      0-10mm - men & women
 Gamma globulins 
 the modified westergren method uses EDTA as the
INCREASING FACTORS: (OCAF) anticoagulant instead of sodium citrate 
1. increased Oxygen  for both procedures, the results are expressed as millimeters
2. increased Cholesterol  per hour (mm/hr) 
3. Increased Alpha lobulin  while the westergren method is a SIMPLE TEST to perform
4. Increased Fibrinogen manually, minor technical problems can cause
5. Acute infection ERRONEOUS TEST results. 
 COMMON ERRORS 
DECREASING FACTORS (CALN)  Dilution caused by LIQUID Anticoagulant
1. increased Carbon dioxide (sodium citrate solution),
2. Increased Albumin   Mixing errors
3. Increased Lecithin  handling errors such as tilted tubes &
4. increased Nucleoprotein vibrations occurring during the sedimentation
period. 
PHYSIOLOGICAL VARIATION :
 2. Landau Method - 5% S.C. 
AUTOMATED SYSTEMS-WESTERGREN   *Tube -  12 cm X 1 mm in width
 Streck ESR-Auto Plus, a 10- position automated bench 3. Smith Method - 5% S.C. 
top ESR analyzer.    *1-5 mm/hr - men
 this instrument uses infrared light that accurately   *1-8 mm/hr  -women
measures  the sedimentation rate of Erythrocytes in 1. 2 ml
ESR - Vacuum tubes that are supplied separately.  INTERPRETATIONS 
1. SEX
ESR AUTOMATION (Streck ESR Auto Plus) 2. AGE : with increasing age ESR-higher
1. the vacuum tubes allow direct-draw collection 3. SPECIFICITY - non specific response
2. with mylar safety coating which provide impact 4. Differential Diagnosis 
resistance & will contain glass & blood specimens in the
event of breakage.  PHYSIOLOGICAL FACTORS 
3. Results are measured in mm/hr (modified westergren 1. Young children 
method) & are available in 30 mins ; therefore, the 2. Pregnancy
analyzer can handle 20 samples/hr.  3. Menstruation 
4. 60 years old
ADVANTAGE OF ESR AUTOMATION
1. 98% correlation with the manual modified westergren PATHOLOGICAL INTERPRETATIONS
method. 
2. The instrument features random access, a built-in INCREASED ESR
printer, sample ID capability, & a 15 mins 1. Active inflammation, infection & toxemia
PREDICTION MODE.  2. Cell or tissue damage
3. The ESR-Auto Plus incorporates 3. Severe anemia
4. Active tuberculosis & syphilis
a. quality control system for monitoring the 5. Malignancy 
laboratory's quality control program & the Data/can 6. Rheumatic, gonorrhea, & gouty arthritis  
be downloaded to a laboratory information system.  7. Collagen disease
 This quality control features of the ESR-Auto  Plus DECREASED ESR
eliminates the need to manually record daily runs. 1. Decreased FIBRINOGEN in infants
2. Polycythemia
 CONTROL  SAMPLES are stored in individual log files 3. Sickle Cell Anemia 
that hold up to 100 samples per level. Statistical reports 4. Congestive Heart Failure
are generated with the following features; standard 5. Salicylates, Cortisone, ACTH of the anterior lobe of the
deviation,  percent coefficient of variation, mean,  & pituitary gland cause a prompt decreased of the rate. 
highest & lowest result.  6. High Blood Sugar 

 Mechatronics USA LLC  (East province, RI) offers the ZSR

StaRRsed Compact, a benchtop automated ESR  measurement of compaction also called DISPERSION
analyzer  STRESS of the RBCs 
 it is inversely related to the zeta potential of the cells when
 HemaTechnologies (Lebanon, NJ) offers the ESR STAT suspended in particular plasma under consideration. 
PLUS analyzer, a second - generation automated sed  the test is a non-specific indicator of infectious disease
rate analyzer that uses  EZ-SAFE Mylar-wrapped, self- & inflammatory states, reflects a Acute phase resistant
sealing analysis tubes.  levels, screen for collagen disease, rheumatoid arthritis. 

FULLY AUTOMATED ANALYZER  INTERPRETATION :

   >No reagents required - no waste  INCREASED ;
   >direct use of primary EDTA tubes - no contact of blood 1. Infection
 therefore the EDTA tube can be used unchanged for 2. Inflammation
further analysis after rythrocyte sedimentation.  3. Tissue necrosis
 Compatible with all  the commonly used EDTA tubes in th 4. Macro globulins (increased)
market. 
 Photometric infrared (950nm) reading prevents  DECREASED :
interference caused by lipids or bilirubin in the sample.   Sickle Cell Disease 
 Spherocytosis
WINTROBE Method :  The test ESR, ZSR, &/or CRP are used in the
 The wintrobe tube is smaller in diameter than the differentiation of Anemia from iron deficiency. 
Westergren tube & only 100 mm long. EDTA
Anticoagulated blood without extra diluent is drawn into the ZETA SEDIMENTATION RATIO
tube, and the rate of fall of red blood cells is measured in 1. Dependent on the conc. Of fibrinogen & gamma globulins
millimeters after 1 hour.  2. Anticoagulant - EDTA 
 The shorter column makes this method LESS sensitive 3. ZETAFUGE - compaction/disperse of Erythrocytes 
than the Westergren  method because th maximal 4. SPECIAL TUBE -  75mm X   2-3 mm outer, 2. 0 - inner
possible abnormal value is LOWER. however, this method 5. CENTRIFUGE - 4 min or 45 sec (4 times) 
is more practical for demonstration purposes. 
 The normal sedimentation rate (WESTERGREN Method)  ZSR FORMULAE
  *MALE      -  0-15 millimeters per hour    
  *FEMALE -  0-20 millimeters per hour    - ZSR = Hct (%)  X  100   over Zct (%) 
 The sedimentation rate can be slightly more elevated in the                     =0. 45/0. 87 = 52%
elderly. 
>40-50 vol.% - Reference Value
LINSENMEIER METHOD (. 7% TSC) >51-54% - borderline
 Length of tube -  0-65 mm >55-59% - mild
 350-600min - men ; 300-600min - women >60-64%  - moderate
>65 & higher - marked increased.
MICRO METHODS
1. Hellige Vollmer or Crista Method - 5% S.C. 
*8-10 mm/hr  - children
*6-8 mm/hr    - adults