A Concise Guide on Routine Blood Collection and Commonly Used Tests for Hematology
Phlebotomy – the art and science of collecting blood
Venipuncture (via ETS / syringe)
Ideal site for collection: Antecubital fossa – (“ante” for opposite and “cubital” for elbow)
a. Basilic vein – inner portion of the arm
b. Cephalic – edge of the outer portion of the arm
c. Median cubital – preferred; well-anchored in tissue and does not roll
(Review procedural steps and the order of draw for ETS and syringe; review anticoagulants of tubes
and the mechanisms of each)
Gauge most commonly used: 20 g (for adults = 19, 20, 21,22; for pediatric/geriatric/HTE = 25); Length
of needle = 1-1.5 inches
Capillary / Skin Puncture
Ideal for: Infants (less than six months of age), pediatric patients if small amount of blood is needed;
HTE (hard to extract) adults due to IV infusions, burns, extreme obesity, etc.
Recommended to warm up the site up to 42 degrees Celsius to improve blood flow.
Concern: May include tissue (interstitial fluid) juice that may affect test results (may be avoided by
discarding first drop of blood after pricking); May also give higher WBC results and lower RBC, Hgb,
Hct and Platelet count.
Sites of collection:
For infants less than one year – medial or lateral portion of the plantar surface of the foot (depth: not
more than 2.0 mm to prevent osteomyelitis)
For patients older than one year – third / fourth finger most commonly used (last resort – second finger
but avoid this and the fifth finger as much as possible because these contain more nerve endings; avoid
thumb due to callous). For finger puncture, inoculate perpendicular to the fingerprint to allow the
formation of a ball-form drop of blood.
(Review procedural steps and the order of draw for capillary puncture)
Anticoagulant used for Hematology and Coagulant studies in general: EDTA, Heparin and Sodium
citrate* (For citrate: conc. of 0.109 M = 3.2% is 1:9 anticoagulant:blood ratio; 0.129 M = 3.8% 1:4)
(Review on sources of error / complications related to blood collection)
Manual Cell Count (RBC, WBC, Platelet) –done to double check abnormal CBC results.
Pipets Used:
Thoma RBC Pipet
Color of bead
RED
Markings
0.5, 1.0, 101
Volume of the bulb
100
Dilutional range*
Can range from 1:100 -1:1000
To get dilution factor: dilution = amount of sample / total volume
Thoma WBC Pipet
BLUE / WHITE
0.5, 1.0, 11
10
Can range from 1:10 – 1:100
i.e. For RBC: Amount of blood aspirated up to 0.5 mL mark; diluent aspirated up to 101 mark (100 mL
the 1 mL or 3-4 drops is discarded prior to charging to counting chamber; can be excess diluent)
0.5 / 100 = 1/200 or 1:200 (same concept applies with WBC; specimen up to 0.5mL; diluent-11mark)
Diluting fluid used for manual cell counting:
RBC diluting fluid – must be isotonic to avoid lysis of RBC
e.g. NSS – most commonly used (ideal for excessive rouleux and autoagglutinating RBCs); FormolCitrate (Dacie’s Fluid) – the best
WBC diluting fluid – ideally hypotonic; purpose is to lyse the red cells (note: cannot lyse young,
nucleated red blood cells if present. After obtaining total WBC count, differential count of white blood
cells and counting of NRBCs in a stained peripheral blood smear must be done. Formula of WBC
correction must be applied afterwards)
e.g. 2-3% glacial acetic acid with Gentian violet – commonly used; Turk’s solution – glacial acetic
acid and methyl violet – enhances nuclear definition
Platelet diluting fluid – must preserve platelet integrity
e.g. Tocantin’s method: rees-ecker diluent uses citrate formaldehyde buffer with brilliant cresyl blue
and viewed under light microscopy; Guy and Leake’s method – uses crystal violet sodium citrate and
formalin and viewed under light microscopy; For better viewing: Use 1% ammonium oxalate diluent
for phase-contrast microscopy
Neubauer Counting Chamber: Manual Red cell count (red squares); White blood cell count (blue
squares); Platelet cell count (green squares)
One Neubauer = two chambers on each side; each counted and then the average of the total is taken
and represents the total cell counted.
Total area = 3mm x 3mm = 9mm2
Depth = 0.1 mm or 1/10
Total volume = 3 mm x 3mm x 0.1
mm = 0.9 mm3
Area of the large square (for WBC
count) = 1 mm2
Area of the smaller central square
(where platelets and red cells are
counted; composed of 25 smaller
squares) = 1mm/25 = 0.04 mm2
Formula:
No. of cells counted x dilution factor / area x depth
Area of the smallest square (inside
one of the smaller square used for
RBC counting; composed of 16
smallest squares) = 0.04 / 16 =
0.0025 mm2
= /uL or mm3 or x 106 (conventional unit); multiply with
0.000001 to get x 1012 / L (SI unit)
e.g. 25 WBC cells counted in all four large corner squares using dilution of 1:20
25 (cells) x 20 (dilution) / 4 (because 1 large square is 1 mm2, so times 4 = 4) x 0.1 = 1250 uL
= 1.25 x 1012 / L
Review on the procedure of using counting chamber; cell estimate and interpretation – i.e. for platelet
count 0-49,000 uL is MARKEDLY DECREASED)
Blood film analysis (aka Peripheral Blood Smear / PBS)
2 glass slide method – commonly used for PBS preparation
Drop of blood = 2-3 mm; distance of drop of blood from frosted end of slide (1cm); angle of spreader
estimated around 25 – 45 degrees
Factors that can affect blood smear thickness:
unstained
P – Pressure (lesser = thicker; more = thinner)
A – Angle (lesser = thinner; more = thicker)
S – Size of drop of blood (lesser = thinner; more = thicker)
S – Speed (lesser = thinner; more = thicker)
Counting of cells can be done using:
stained
Cross-sectional/crenellation method – counted in consecutive fields from one side to the other end
Longitudinal – counted in consecutive fields from the tail to the head of the smear
Battlement method – begins near the tail on a horizontal edge: count three consecutive horizontal edge
fields then count two fields up/towards the center then count two field horizontally then count two
field vertically again to the center; etc.
Romanowsky stains – group of stains most commonly used; polychromatic stain
Mainly contains Wright’s and Giemsa: Wright-Giemsa stain
pH: 6.4-6.8 (Alkaline) consists of methanol (fixative); methylene blue (primary stain); Sorenson’s
phosphate buffer; and eosin (secondary stain)
Supravital stains – commonly used to demonstrate reticulocytes (can also be used for Heinz Bodies)
e.g. Brilliant Cresyl Blue (sodium citrate + NaCl) and New Methylene Blue (sodium oxalate + NaCl)
Note: WBC differential count in PBS
-
Goal is to classify 100 leukocytes under OIO ad report the relative count of each (percent);
REMEMBER: if more than 5 NRBC’s are present for adults (10 for babies) per 100 WBCs
perform WBC correction: Formula = Total WBC count (from machine) x 100 / NRBCs + 100
(the machine may count NRBCs as WBCs due to their nucleus)
Reticulocyte count in blood film
-
Formula: No. of reticulocytes counted (in 10 OIO fields) / 1000 red blood cells x 100 = %
If patients have anemia, reticulocyte count must be corrected:
Corrected Retic Count = %Retics x Hematocrit (%) / 45
For the shift correction and to get the Reticulocyte Production Index (RPI)
RPI = Corrected Retic Count / Maturation time
Hct
15% (plus/minus 5)
25% (plus/minus 5)
35% (plus/minus 5)
45% (plus/minus 5)
Maturation time
2.5
2.0
1.5
1.0
-
Note: Miller’s disk can be used for reticulocyte count (Formula: Retics counted x 100 / RBC
counted x 9)
Manual Platelet count using PBS
Easy method: no. of platelets counted in ten fields x 10
Manual Hemoglobin Determination
Acid Hematin Method
-
Uses Sahli’s hemoglobin pipet nnd tube; diluent: HCl (converts hemoglobin to acid
hematin) followed by dilution with distilled water – drop by drop until color matches the
standard block.
Cyanmethemoglobin method (Aka Hemiglobincyanide / HCN)
Reagent: Modified (Detergent modified) Drabkin’s Reagent
pH = 7.0-7.4
Contains: Potassium ferricyanide – converts hemoglobin into methemoglobin
Potassium cyanide – converts methemoglobin into cyanmethemoglobin
Dihydrogen Potassium Phosphate (original drabkin’s uses sodium bicarbonate) – shortens
original conversion ime from 10-15 minutes to just 3 minutes
Detergent used (such as Sterox or Triton X) enhances lysis of red cells to free hemoglobin
For analysis: Spectrophotometer is used – measured at 560 nm (can detect all forms of hemoglobin
except sulfhemoglobin)
Hemoglobin F (Kleihauer-Betke method)
Count dense-staining Hgb F cells and the number of ghost cells containing Hgb A to obtain
percentage.
1. It is used to detect the presence of fetal cells in the maternal circulation during problem pregnancies
because Hgb F in fetal cells resists acid elution.
2. It differentiates hereditary persistence of fetal hemoglobin from other conditions associated with
high Hgb F levels.
3. Normal newborns have 70-90% Hgb F levels.
Solubility Test for Hemoglobin S (Sickle Cell identification)
1. Hemoglobin S is insoluble when combined with a reducing agent (sodium dithionite).
2. Hgb S will crystallize and give a turbid appearance to the solution.
3. The test will not differentiate homozygous from heterozygous conditions containing Hgb S.
4. Follow up a positive solubility test with hemoglobin electrophoresis.
Hemoglobin Electrophoresis
1. Procedure for the identification of normal and abnormal hemoglobins
2. Methodology is based on net negative charges, which cause hemoglobins to migrate from the
negative (cathode) region toward the positive (anode) region. The distance a particular hemoglobin
molecule migrates is due to its net electrical charge.
3. Two types of electrophoresis: Cellulose acetate at pH 8.6 and citrate agar at pH 6.2
4. Migration of hemoglobin is dependent on net negative charge and buffer pH.
-
Manual Hematocrit Determination
Hct is also known as packed red cell volume (Conventional: L/L; SI Unit: %)
Adam’s Microhematocrit
Tube: Capillary tube – filled up at least ¾’s; centrifuged and compared with standard
READ AT START OF PACKED RED
CELL. MAKE SURE THE END IS
ALIGN WITH THE “0” mark.
(Review on RBC indices – MCV, MCH, MCHC and RDW)
-
Erythrocyte Sedimentation Rate (ESR)
Measure of the degree of settling of RBCs in plasma for 60 minutes
Non-specific indicator of inflammation
Stages: 1. Lag (1st 10 mins), 2. Decantation (Next 40 mins – sedimentation is at constant rate),
3. Last 10 minutes – sedimentation slows down
(Review difference between Westergren and Wintrobe methods)
Special Procedures: Use of Cytochemical Stains for Diagnosis of Hematologic Disorders
1. Myeloperoxidase (MPO)
a. Cells of the granulocytic series and to a lesser degree the monocytic series contain the enzyme
peroxidase in their granules that is detected by this stain. Auer rods also stain positive; lymphocytic
cells are negative for this stain.
b. Used to differentiate blasts of acute myelogenous leukemias (AMLs) from acute lymphoblastic
leukemias (ALLs)
2. Sudan black B
a. Stains phospholipids and lipoproteins
b. Granulocytic cells and Auer rods stain positive (blue-black granulation); lymphocytic cells are
negative for Sudan black B (reaction parallels MPO).
c. Used to differentiate blasts of AML from ALL
3. Esterases
a. Specific esterase stain (naphthol AS-D chloroacetate esterase stain)
1) Detects esterase enzyme present in primary granules of granulocytic cells; monocytic cells negative
for this stain
b. Nonspecific esterase stains (alpha-naphthyl acetate and alpha-naphthyl butyrate)
1) Detects esterase enzyme present in monocytic cells; granulocytic cells negative for these stains
c. The esterase stains may be useful in distinguishing acute leukemias that are of myeloid origin (FAB
Ml, M2, M3, M4) from those leukemias that are primarily cells of monocytic origin (FAB M5).
4. Periodic acid-Schiff (PAS)
a. PAS stains intracellular glycogen bright pink.
b. Immature lymphoid cells, malignant erythroblasts, and megakaryocytic cells stain positive with this
stain; myeloblasts and normal erythrocytic cells are negative with this stain.
c. Useful in diagnosis of erythroleukemia (FAB M6) and acute lymphoblastic leukemia
5. Leukocyte alkaline phosphatase (LAP)
a. Detects alkaline phosphatase enzyme activity in primary granules of neutrophils
b. A positive stain will show dark precipitate when alkaline phosphatase activity is present; color is
dependent on dye used.
c. Used to differentiate chronic myelogenous leukemia (CML) from a neutrophilic leukemoid reaction
(NLR)
LAP score:
1) 100 neutrophils are graded on a scale from 0 to 4+ based on stain intensity and size of granules.
Results are added together.
2) Reference range is 13-130.
e. Clinical significance
1) Decreased LAP score: CML, paryoxysmal nocturnal hemoglobinuria
2) Normal LAP score: CML in remission or with infection, Hodgkin lymphoma in remission,
secondary polycythemia
3) Increased LAP score: Neutrophilic leukemoid reaction, polycythemia vera, CML in blast crisis, late
trimester pregnancy
6. Tartrate-resistant acid phosphatase stain (TRAP)
a. Almost all blood cells contain the acid phosphatase enzyme and show positivity with acid
phosphatase stain. Once tartrate is added, staining is inhibited in most cells.
b. Only hairy cells from hairy cell leukemia are resistant to inhibition with tartrate and continue to
stain positive; all other cells stain negative.
7. Perl's Prussian blue stain
a. Free iron precipitates into small blue/green granules in mature erythrocytes; cells are called
siderocytes. Iron inclusions are called siderotic granules or Pappenheimer bodies when visible with
Wright's stain.
b. Sideroblasts are nucleated RBCs in bone marrow that contain iron granules. These are normal.
Ringed sideroblasts contain iron that encircles the nucleus. These are abnormal.
c. Increased percentage of siderocytes is seen in severe hemolytic anemias (e.g., beta-thalassemia
major), iron overload, sideroblastic anemia, and post-splenectomy; ringed sideroblasts are seen in
bone marrow of myelodysplastic syndrome (refractory anemia with ringed sideroblasts [RARS]) and
sideroblastic anemias.
Automated Procedures
Electrical impedance
a. Cells pass through an aperture with an electrical current flowing through simultaneously. Cells do
not conduct current but rather they change electrical resistance, which is then counted as voltage
pulses.
b. The number of pulses generated is proportional to the number of cells present; amplitude of the
pulse generated is proportional to the size of the cell.
c. Sample is diluted in isotonic conductive solution that preserves cell shape and characteristics.
1) Dilutions used are dependent on instrument/methodology used.
2) Platelets are counted simultaneously with RBCs.
3) Sample for counting WBCs is mixed with reagent to lyse RBCs.
(Utilizes cyanmethemoglobin method for hemoglobin determination)
Light scattering optical method
a. Uses a flow cytometer with laser to measure light scattering properties of cells
1) Forward angle light scatter measures cell size.
2) Side angle light scatter provides information on cell granularity and lobularity.
3) Number of pulses generated is proportional to the number of cells present.
b. Dilutions used are dependent on instrument/methodology used.
Note: Automated cell count errors that may affect accuracy of result
a. WBC counts exceeding instrument linearity limits result in increased cell turbidity and may falsely
increase the hemoglobin, MCH, and MCHC.
b. Glucose over 600 mg/dL (hyperosmolarity) may increase the MCV and hematocrit and decrease the
MCHC.
c. Cold agglutinins increase the MCV, MCH, and MCHC and decrease the RBC count and
hematocrit.
d. Lipemia increases the hemoglobin, MCH, and MCHC.
e. Repeat the analysis if:
1) Rule of three (shown below) not coinciding - (remember: can only be used for normocytic samples)
(especially MCHC >37 g/dL)
a) RBC X 3 = Hgb
b) RBC X 9 = Hct
c) Hgb X 3 = Hct
2) Any result outside linearity limits established by manufacturer (dilute into linearity range)
Flow Cytometry
1. Principle: Cells in a suspension of buffered solution are labeled with one to several fluorescent
compounds. This cell suspension is run under high pressure and in a single, narrow stream through a
laser, causing excitation of the fluorescent compound(s) and resulting in the emission of light energy.
This energy is detected by a photomultiplier tube and is subsequently converted into computerized
data, which upon analysis provides information regarding number, size, and cellular composition of
the population assayed.
Histograms and Scatterplots
1. A histogram utilizes impedance technology, and it is a representation of cell number versus one
measured property, usually cell size. It is used for WBCs, RBCs, and platelets.
a. WBC histogram
1) 35^-50 fL is the reference size range for WBCs.
2) 1st peak: 35-90 fL is the range for lymphocytes.
3) 2nd peak: 90-160 fL is the range for mononuclear cells (monocytes,
reactive lymphocytes, and immature WBCs).
4) 3rd peak: 160^-50 fL is the range for granulocytes.
Abnormal WBC histogram
1) Population before 35 fL may indicate nucleated RBCs (nRBCs), giant or clumped platelets.
2) Peak overlap at 90 fL may indicate reactive lymphocytes or blast cells.
3) Peak overlap at 160 fL may indicate an increase in bands, immature neutrophils, eosinophils, or
basophils.
4) Population after 450 fL may indicate a high granulocyte count.
b. RBC histogram
1) 36 fL and above is the reference size range for RBCs.
2) A normal RBC histogram will show a single peak between 70 and 110 fL that will correlate with
the MCV.
Abnormal RBC histogram
1) Two peaks indicate a dimorphic erythrocyte population.
2) Increased curve width will correlate with an increased RDW (anisocytosis).
3) Shift to the right indicates an increased MCV (macrocytic).
4) Shift to the left indicates a decreased MCV (microcytic).
c. Platelet histogram
1) 2-20 fL is the reference size range for platelets.
2) Lower region interference (<2 fL) indicates electrical interference; upper region interference (>20
fL) indicates microcytic RBCs or schistocytes, giant or clumped platelets.
2. A scatterplot/scattergram is a two-dimensional representation of two or more cell properties or
characteristics plotted against each other (e.g., size versus granularity or lobularity).
(Note: Review the tests and principles for coagulation studies discussed last semester – go back to
your compiled notes i.e. PT, APTT etc.)