BASIC EXAMINATION OF BLOOD

Mila Amor V. Reyes, MD, FPSP | August 29, 2020

Trans by: Dela Cruz, Fernando, Nava, Tamacay

OUTLINE Hemoglobin Concentration Determination

1. Hemiglobincyanide (HiCN or cyanmetHb)
I. Hematology B. Red Blood Cells
A. Hemoglobin • Method of choice
C. White Blood Cells
B. Hematocrit D. Platelets • Principle: K ferriCN oxidizes Hb to Hi, and KCN provides CN-
C. Blood Cell Counting to form HiCN
III. Erythrocyte
II. Blood Film Examination Sedimentation Rate • Drabkin's reagent
A. Staining of Blood IV. References • It measures most forms of hemoglobin except SHb
Films 2. OxyHb Method
• Only OxyHb is determined
I. HEMATOLOGY
3. Chemical Method
• Hematology – study of blood cells and coagulation
Errors in Hemoglobinometry
• CBC: Hemoglobin (Hb) concentration, Hematocrit (Hct), RBC
count and indices, WBC count, platelet count, and differential • Errors inherent in the sample
count • Errors inherent in the method
• Anticoagulant of choice: Ethylenediaminetetraacetic acid • Errors inherent in the equipment
(EDTA) • Operator’s errors
A. HEMOGLOBIN B. HEMATOCRIT
• Transport O2 and CO2 • Hematocrit – packed RBC volume
• Normal values: • Ratio of the volume of RBC to that of the whole blood
→ Male: 136–172 g/L or 13.6–17.2 g/dL • Normal values:
→ Female: 120–150 g/L or 12–15 g/dL → Male: 0.39–0.49 or 39–49%
→ Female: 0.33–0.43 or 33–43%
Types of Hemoglobin
I. Physiologic Hemoglobin Measurement
A. OxyHb – HbO2 (bright-red), seen in arteries • Directly by centrifugation
B. Reduced Hb – HbCO2, carbaminoHB (dark-red), seen in → Wintrobe macromethod
veins ▪ Thick-walled glass tube with a uniform internal bore and a
II. Hemoglobin derivatives flattened bottom
• Loses its capacity to bind and carry O2 ▪ No longer used
A. Methemoglobin/Hemiglobin (Hi)—Fe+++ → Capillary micromethod
▪ Heparinized capillary tubes (red color)
• Chocolate-brown
▪ After centrifugation, compared to a reader
• NV = 1.5% of total Hb
• Indirect
• Ferrous (Fe2+) to Ferric3+ → MCV x RBC count
→ Reducing systems within RBCs: NADH-cytochrome b5
reductase, ascorbic acid, reduced glutathione, and Sources of Error
NADPH-metHb reductase • Centrifugation
• Methemoglobinemia (condition) → Adequate duration and speed
→ Hereditary – NADH-cytochrome b5-reductase • Sample
deficiency and HbM with increased tendency toward → Posture, muscular activity, prolonged tourniquet stasis,
oxidation and decreased susceptibility of excess EDTA
methemoglobin to reduce back to hemoglobin • Technical errors
→ Acquired – exposure to oxidizing drugs and chemicals— → Failure to mix blood adequately
nitrites, nitrates, chlorates, and quinones → Improper interpretation
B. Sulfhemoglobin C. BLOOD CELL COUNTING
• Mauve-lavender • Normal values:
• Irreversible → RBC Count
• NV = <1% of total Hb ▪ Male: 4.3–5.9 x 106/mm3 or
• Mixture of oxidized and denatured form of hemoglobin and ▪ Male: 4.3–5.9 x 106/µL (conventional) or
precipitates as Heinz bodies ▪ Male: 4.3–5.9 x 1012/L (SI units)
• Sulfur is incorporated into the heme rings ▪ Female: 3.5–5.0 x 106/mm3 or x 106/µL or x 1012/L
C. Carboxyhemoglobin—HbCO → WBC Count
• Cherry-red ▪ 4.5–11.0 x 109/L
▪ 4,500–11, 000/µL or mm3
• NV = 0.5% of total Hb
→ Platelet Count
• Hemoglobin can combine with CO with an affinity of 210x
▪ 150–450 x 109/L
greater than that for O2, shifting the O2 dissociation curve
▪ 150,000–450,000/µL or mm3
to the left
• Endogenous CO production – degradation of heme to 3 Steps in Cell Counting Procedure
bilirubin, increased in hemolytic anemia 1. Dilution of blood
• Exogenous sources – gasoline motors, illuminating gas, 2. Sampling the diluted suspension into a measured volume
gas heaters, defective stoves, smoking 3. Counting the cells in that volume
• Acute and chronic CO poisoning RBC Counts
• Cyanosis will occur at 5g HbCO/dL, 1.5g Hi/dL, and 0.5 • Manual Method
SHb/dL of blood. → Hemocytometer

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 → As a check on the validity of electronic methods for
calibration
→ As a check on the validity of electronic counts in patients with
profound leukopenia or thrombocytopenia
→ For blood specimens with platelet counting interference (i.e.,
very microcytic RBC)
→ As a back-up method
→ For counting cells in CSF
→ n-RBC

Corrected WBC Count = Total WBC Count x 100_

Fig 1. Hemocytometer. [Google] 100 + n-RBC/100WBC
• Unopette System
→ Sources of Error:
→ Combination of microcapillary tube with a plastic vial ▪ Errors due to the nature of the sample
containing a premeasured volume of diluent ▪ Operator’s errors
▪ Errors due to equipment
▪ Inherent or field errors
▪ Presence of n-RBCs
• Electronic Counting
→ Same as that for RBCs, except that RBCs are lysed before
counting
Platelet Counts
• Hemocytometer Method
→ Phase contrast microscopy, Reese-Ecker method

Platelet Count/µL = No. of cells counted x dilution x 250

Fig 2. Unopette system. [Google] no. of squares counted
• Semi-automatic Methods
→ Sources of Error:
→ Instruments for precise diluting, which both aspirate the ▪ Platelets are small and must be distinguished from debris
sample and wash it out with the diluent ▪ Tendency of platelets to adhere to glass, foreign body, and
• Electronic Counting Methods to one another
→ Electrical Impedance – cell passing through an aperture • Electronic Counting
through which current is flowing cause changes in electrical → Electrical impedance, and light scattering
resistance that are counted as voltage pulses. Reticulocyte Counts
▪ e.g. Coulter counter, Sysmex • Reticulocytes
→ Light scattering – electro-optical analyzers, a light-sensitive → Immature, non-nucleated RBCs that contain RNA
detector measures light scattering, the size of the pulse → Produced by bone marrow and matures into erythrocyte in the
detected is proportional to the size of the particle (RBC, WBC, circulation within 24 hours.
Platelets) • Reticulocyte count – measure of actual bone marrow response
RBC Indices to anemia
• Useful in the morphologic characterization of anemias • Normal values:
• Mean Cell Volume (MCV) → Adults: 0.5–1.5%
→ Volume of the average RBC → Newborn: 2.5–6.5%
→ Normal value: 76–100 fL or µm3 • Manual method – blood is incubated in new methylene blue
(NMB) or brilliant cresyl blue (BCB) (supravital stains) – RNA is
MCV = Hct x 1000_ precipitated as a dye-ribonucleoprotein (dye-RNP) complex;
RBC count /µL appears as a dark blue network (reticulum) or granules
• Percentage of reticulocyte is determined in at least 1000 RBCs
• Mean Corpuscular Hemoglobin (MCH) Corrected Reticulocyte % = Uncorrected retic % x Patient’s Hct_
→ Content (weight) of hemoglobin of the average RBC 45
→ Normal value: 27–33 pg

MCH = Hb (g/L)__ Absolute reticulocyte count = % reticulocyte x RBC count

RBC count/µL
→ Normal value: 10–75x109/L or 10,000 – 75,000/mm3
• Mean Corpuscular Hemoglobin Concentration (MCHC) →
→ Average concentration of hemoglobin in a given volume of • Reticulocyte Production Index (RPI) – generally accepted as
PRBC reflective of the actual bone marrow response
→ Normal value: 330–370 g/L or 33–37 g/dL
RPI = Corrected reticulocyte %
Correction Factor
MCHC = Hb (g/dL)_
Hct Table 1. Corresponding correction factor according to Hct %
Hct % CF
WBC Counts 35-40 1.5
• Hemocytometer Method 25-35 2.0
→ Only occasionally used 15-25 2.5
10-15 3.0

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• Flow cytometry – fluorescent dyes (e.g., acridine orange or • RBCs – circular, biconcave discs of nearly uniform size, 6-8 μ in
thioflavin T) bind RNA in reticulocyte diameter, the center 1/3 is paler
• Automated methods – thiazole orange (Beckton-Dickinson Color
FACScan), auramine-O (Sysmex), oxazine 750 (Miles • The color of RBC depends on its hemoglobin content
Technicon)
• Normochromic – normal intensity of staining
II. BLOOD FILM EXAMINATION • Hypochromic – central pale area becomes larger, decrease
hemoglobin content, MCH and MCHC are decreased (e.g., IDA,
• Preparing a smear
thalassemia)
→ Wedge method (2-slide method) – convenient, easy
• Hyperchromic – less or no central pallor (e.g., HS)
handling
• Anisochromia – dimorphic anemia, presence of hypochromic
→ Coverglass method – uniform distribution of cells
and normochromic cells in the same field (e.g., sideroblastic
→ Automatic spinner method – centrifuge method, producing anemia)
a uniform blood film, in which cells are separated in monolayer
and randomly distributed Polychromatophilia or Polychromasia
• Sources of error • Blue-gray tint to the RBCs – affinity of hemoglobin for acid
→ Mechanical errors (due to variations in collecting the blood stains and affinity of RNA for basic stains
samples, inadequate mixing, irregularities in distribution • Polychromatophilic RBCs – Wright stain, reticulocytes – BCB or
depending on the type and quality of the blood films, and poor NMB
staining) • Therefore, increase polychromasia implies reticulocytosis
→ Errors in cell identification (which depend on the judgment Size
and experience of the observer) • Normocytes – 6–8μ in diameter
• Microcytes – decreased MCV
• Macrocytes – increased MCV
• Anisocytosis – variation in size
Shape
• Poikilocytosis – variation in shape
• Elliptocytes/ovalocytes
→ Hereditary elliptocytosis, IDA, myelofibrosis with myeloid
metaplasia, megaloblastic anemias, sickle cell anemia
• Spherocytes
Fig 3. Blood film preparation for hematologic examination using the 2-slide
→ Hereditary spherocytosis (HS), Autoimmune hemolytic
method. [http://mt-lectures.blogspot.com/2017/08/lecture-13-morphological-examination-of.html] anemia (AIHA), Microangiopathic hemolytic anemia (MAHA)
• Target cells (codocytes)
A. STAINING OF BLOOD FILMS
→ Obstructive jaundice, following splenectomy, thalassemia,
• Romanowsky stains: Giemsa and Wright stains (thiazine HbC disease
methylene blue and eosin stains)
• Schistocytes, helmet cells, bite cells
→ RBCs – pink
→ Indicate the presence of hemolysis, AIHA, MAHA,
→ Nuclei of WBCs – purple megaloblastic anemia
→ Neutrophil granules – tan to pink • Echinocytes (crenated cells) – artifact
→ Eosinophil granules – red-orange • Acanthocytes – abetalipoproteinemia, liver disease
→ Basophil granules – dark purple • Sickle cells (drepanocytes) – sickle cell anemia
→ Platelet granules – dark lilac
→ Lymphocyte cytoplasm – light blue Structure
→ Monocyte cytoplasm – faint blue-gray • Basophilic stippling (punctuate basophilia)
→ Bacteria – blue → Presence of irregular basophilic granules, fine to coarse,
→ Malarial parasites – sky-blue cytoplasm and red-purple within the RBCs, usually multiple
chromatin → Pb poisoning, megaloblastic anemia
• Hema-Tek automated slide stainer • Siderocytes
→ RBCs with inorganic Fe-containing granules (Prussian Blue
B. RED BLOOD CELLS stain)
→ Called Pappenheimer bodies with Wright stain
→ Following splenectomy
• Howell-Jolly bodies
→ Smooth, round remnants of nuclear chromatin
→ Single HJ – megaloblastic anemia, HA, after splenectomy
→ Multiple HJ – megaloblastic anemia
• Cabot rings
→ Ring-shaped, figure of 8, or loop-shaped structures, stain red
or reddish purple with Wright stain
→ Probably microtubules remaining from mitotic spindle and
interpreted as evidence of abnormal erythropoiesis
• Malarial stippling
→ Fine granules in RBCs that harbor P. vivax (Schuffner's dots)
→ Stain purplish red with Wright stain
• Rouleaux formation
→ Alignment of RBCs resembling stack of coins; occur with
elevated fibrinogen or globulins and promote an increase ESR
Fig 4. Red blood cell morphology. → Marked in paraproteinemia
[https://theartofmed.wordpress.com/2015/09/05/morphological-abnormalities-of-red-blood-cells/] • Agglutination
→ Clumping of RBCs, may occur with cold agglutinins

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• n-RBCs (normoblasts) • Atypical lymphocytes and lymphoblast
→ Precursor of non-nucleated mature RBCs, normally present → Indicates transformation of lymphoid cells due to antigenic
only in the bone marrow stimulation
▪ Stages of maturation: pronormoblast, basophilic • Plasma cells
normoblast, polychromatophilic normoblast, → Abundant blue cytoplasm, eccentric round nucleus with
orthochromatophilic normoblast, reticulocytes, chromatin arranged radially (spoke of a wheel), a well-defined
erythrocytes clear Golgi zone adjacent to the nucleus; not normally present
− Normally, pinaka-immature release is reticulocyte but in in the blood
the presence of severe anemia, polychromatophilic • Two types of lymphocytes:
normoblast is released [Doc Amor] 1. T cell – mediated immunity; can kill antigen
→ n-RBCs that might appear in the blood in disease are 2. B cell – humoral immunity [Doc Amor]
polychromatophilic normoblasts, their presence usually Automated Differential Leukocyte Counting Machines
denotes an extreme demand on the marrow, extramedullary
1. Flow system
hematopoiesis, or marrow replacement
• Cell size is determined by measurement of light scattering or
→ Increased in HDN, thalassemia major
conductivity
→ Present normally in the blood of the fetus and of the very
• Cell characteristics are determined by measurement of light
young infants
absorption of either unstained or stained cells, or fluorescence
• Leukoerythroblastotic reaction of cellular constituents after staining with fluorescent dyes
→ Presence of normoblasts and immature neutrophils in the → e.g., Technicon H600 system
blood; often indicates space-occupying lesion of the marrow: 2. Pattern recognition or digital image processing system
myelofibrosis with myeloid metaplasia, metastatic CA,
• Identifies stained cells
leukemias, multiple myeloma, Gaucher’s disease
• Optical images are recorded by a television camera analyzed
C. WHITE BLOOD CELLS by the computer, and converted to digital form
• Cellular characteristics are compared with a memory bank; if
the pattern “fits” that of a normal cell type, it is identified as
such, otherwise, it is classified as unknown
• Usually, classified manually if the automated system can’t
determine the type of cell
→ e.g., Coulter Diff 3, Geometric Data Hematrak, Abbott
ADC 500
D. PLATELETS
• Round or oval, 2–4 μ in diameter
• Platelet estimate: if platelet count is normal, about 1 platelet/10–
Fig 5. Morphology of WBCs (and NRBCs) in peripheral smear. 30 RBCs or 7–20 platelets/OIF—platelet is adequate
[https://www.bioscience.com.pk/topics/hemotology/item/800-white-blood-cells-morphology]
• Estimated by peripheral blood smear
• Differential WBC count
→ 100 cells are usually counted, expressed as % III. ERYTHROCYTE SEDIMENTATION RATE (ESR)
• Absolute count = % x WBC count, (x 109/L) • Length of fall of the top of the column of RBCs in a given interval
→ Normal value (WBCs normally present in the blood) • Accelerated ESR: increased fibrinogen and globulins which
Table 2. Differential and absolute count of WBCs promotes rouleaux formation, macrocytosis
Diff count % Absolute count* • Prolonged ESR: albumin, lecithin, poikilocytosis which hinders
Neutrophils 50–70 2.5–7.0 rouleaux formation; due to hyperproteinemia
(segmented, 2-5) • Sedimentation rate is directly proportional to the weight of the cell
Neutrophils, band 2–6 0.1–0.6 aggregate and inversely proportional to the surface area;
or stab microcytes sediment slower than macrocytes
Lymphocytes 20–40 1.0–0.4 Stages in ESR
Monocytes 2–8 0.1–0.8
• Initial 10 minutes – little sedimentation as rouleaux forms
Eosinophils 1–3 0.05–0.3
• About 40 minutes – settling occurs at a constant rate
Basophils 0–1 0.0–0.1
*Absolute count: x 109/L
• Final 10 minutes – sedimentation slows as cells pack at the
bottom of the tube
PMNs
Table 3. Normal values (Westergren method)
• Granules are specific (2/3) and azurophilic (1/3) Men Women
• Shift to the left—increased bands or immature forms Below age 50 15 mm/hr 20 mm/hr
• Stages of maturation: myeloblast, promyelocyte, myelocyte, Above age 50 20 30
metamyelocyte, band or stab, PMN Above age 85 30 42
• The band or stab can already be released by the bone marrow
normally but in cases of severe infection (kailangan ng maraming Methods
WBC) the bone marrow can release metamyelocyte [Doc Amor] • Westergren method—Westergren tube, standard method
• Smaller than monocytes and eosinophils, larger than basophils • Zeta-sedimentation ratio
and lymphocytes → Zetafuge that spins capillary tubes in vertical position in four
Eosinophils 45-second cycles
→ ZSR=Hct/Zct, expressed in %
• Bilobed nucleus; increased in allergy, parasitism
→ NV = 41–54%
Monocytes • Micro-ESR method
• Nucleus is single, partially lobulated, deeply indented, → 0.2 mL of blood to fill a plastic disposable tube 230-mm long
horseshoe-shaped, round or oval, stain less densely, may with 1-mm internal bore, useful for pediatric patients
transform into macrophages outside the circulation
IV. REFERENCES
Lymphocytes
Lecture of Dr. Reyes
• Nucleus is about the size of RBC; nucleus is single, round or oval,
stains dark blue
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