2956_Ch04_287-366 30/01/14 4:25 PM Page 287

4
SECTION

Hematology
Review
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Blood Cells Hematology Review 288

CELL FUNCTION SITE OF PRODUCTION

Erythrocytes O2 transport Bone marrow
Granulocytes Defense against bacterial infection Bone marrow
Lymphocytes Cellular & humoral immunity Lymphoid tissue
Platelets Coagulation Bone marrow
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Comparison of Conventional and SI Units Hematology Review 289

for Adult Reference Ranges
CONVENTIONAL UNITS SI UNITS
3
WBC 4.5–11.5 × 10 /L 4.5–11.5 × 109/L
RBC Male: 4.6–6 × 106/L Male: 4.6–6 × 1012/L
Female: 4–5.4 × 106/L Female: 4–5.4 × 1012/L
HGB Male: 14–18 g/dL Male: 140–180 g/L
Female: 12–15 g/dL Female: 120–150 g/L
HCT Male: 40%–54% Male: 0.40–0.54 L/L
Female: 35%–49% Female: 0.35–0.49 L/L
MCV 80–100 fL 80–100 fL
MCH 27–31 pg 27–31 pg
MCHC 32%–36% 32–36 g/dL
3
PLT 150–450 × 10 /L 150–450 × 109/L

SI = Syst‘ me International d’Unit”s (international system of units).

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Reference Ranges for Red Cell Parameters Hematology Review 290

BIRTH 1–2 MO 1–3 YR 8–13 YR ADULT COMMENTS

RBCs (× 1012/L) 4.10–6.10 3.4–5 3.4–5.2 4–5.4 M: 4.6–6
F: 4–5.4
HGB (g/dL) 16.5–21.5 10.6–16.4 9.6–15.6 12–15 M: 14–18 Preterm infants: About 1 g
F: 12–15 lower than full-term
HCT (%) 48–68 32–50 38–48 35–49 M: 40–54
F: 35–49
MCV (fL) 95–125 83–107 78–94 80–94 80–100 Macrocytes 1st 5 days. MCV
higher in preterm infants
RDW (%) 14.2–19.9 11.4–14.5 11.5–14.5 11.5–14.5
Retic (%) 1.5–5.8 0.8–2.8 0.5–1.5 0.5–1.5 0.5–1.5 Newborns: ↑ polychromasia
NRBCs (/100 WBCs) 2–24 0 0 0 0 Preterm infants: Up to 25 for
>1 wk

Note trends and comparisons to adult values.

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Reference Ranges for Leukocytes and Hematology Review 291

Platelets
BIRTH 1–2 MO 1–3 YR 8–13 YR ADULT COMMENTS
WBCs (× 109/L) 9–37 6–18 5.5–17.5 4.5–13.5 4.5–11.5
Segs (%) 37–67 20–40 22–46 23–53 50–70
Bands (%) 3–11 0–5 0–5 0–5 0–5 Newborns: Occasional metas &
myelos. More immature grans in
preterm infants.
Lymphs (%) 18–38 42–72 37–73 23–53 18–42 Newborns: A few benign
immature B cells may be seen
(“baby” or “kiddie” lymphs).
PLT (× 109/L) 150–450 150–450 150–450 150–450 150–450 Newborns: Variation in size &
shape.

Note trends and comparisons to adult values.

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Hematopoietic Cell Differentiation Hematology Review 292

Pluripotential Hematopoietic Stem Cell

(HSC)

Multipotential Progenitor Cell

(MPP)

Common Myeloid Progenitor Common Lymphoid Progenitor

(CMP) (CLP)

Granulocyte Erythrocyte Monocyte Megakaryocyte T-lymph NK cell B-lymph

Note that this algorithm does not show all differentiation steps.
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Erythropoiesis Hematology Review 293

PERIOD SITE(S) COMMENTS

1–2 mo of gestation Yolk sac & aorta-gonads- Primitive erythroblasts. Embryonic hemoglobin (Gower I, Gower II,
mesonephros (AGM) region Portland).
3–6 mo of gestation Liver, spleen Liver is primary site.
7 mo of gestation—age 4 yr Bone marrow All marrow is active.
Adult Bone marrow Only active sites are pelvis, vertebrae, ribs, sternum, skull. Shafts of long
bones filled with fat. Fatty marrow may be reactivated to compensate
for anemia. Liver & spleen may be reactivated (extramedullary
hematopoiesis) if bone marrow fails to keep up with demand.
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Changes During Cell Maturation Hematology Review 294

CHARACTERISTIC CHANGE(S) WITH MATURATION

Size Becomes smaller.
N:C ratio Becomes smaller.
Cytoplasm Less basophilic due to loss of RNA. Granulocytes produce granules. Erythrocytes become pink due to Hgb
production.
Nucleus Becomes smaller. Nuclear chromatin condenses. Nucleoli disappear. In granulocytic series, nucleus indents,
then segments. In erythrocytic series, nucleus is extruded.

N:C ratio = nucleus to cytoplasm ratio

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Erythrocytic Developmental Series Hematology Review 295

RUBRIBLAST TERMINOLOGY NORMOBLAST TERMINOLOGY KEY CHARACTERISTICS

Rubriblast Pronormoblast 14–24 m. N:C ratio 8:1. Royal blue cytoplasm. Fine chromatin.
1–2 nucleoli. Normally confined to bone marrow.
Prorubricyte Basophilic normoblast 12–17 m. N:C ratio 6:1. Chromatin is coarser with slightly
visible parachromatin. Nucleoli usually not visible. Normally
confined to bone marrow.
Rubricyte Polychromatophilic normoblast 10–15 m. N:C ratio 4:1. Cytoplasm is polychromatophilic due
to hemoglobin production. Chromatin is clumped with distinct
areas of parachromatin. Last stage to divide. Normally confined
to bone marrow.
Metarubricyte Orthochromic normoblast 8–12 m. N:C ratio 1:2. Nucleus is pyknotic. Last nucleated stage.
Normally confined to bone marrow.
Reticulocyte Polychromatophilic erythrocyte 7–10 m. No nucleus. Cytoplasm is diffusely basophilic (bluish
tinge). Reticulum seen with supravital stain. 0.5%–1.5% of
RBCs in adult peripheral blood.
Mature erythrocyte Mature erythrocyte 7–8 m. Biconcave disk. Reddish-pink cytoplasm with area of
central pallor 1/3 diameter of cell.
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Asynchronous Erythropoiesis Hematology Review 296

TYPE CAUSE EXPLANATION CHARACTERISTICS EXAMPLE

Megaloblastic Vitamin B12 or folic Nucleus lags behind cytoplasm Oval macrocytes Pernicious anemia
acid deficiency in maturation. Cells grow larger
without dividing.
Iron deficiency Iron deficiency Cytoplasm lags behind nucleus Microcytic, hypochromic Iron deficiency anemia
in maturation due to inadequate RBCs
iron for hgb synthesis.
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Hemoglobin Hematology Review 297

HEMOGLOBIN MOLECULAR STRUCTURE ADULT REFERENCE VALUE NEWBORN REFERENCE VALUE

A 2 α , 2 β chains >95% 20%
A2 2 α , 2 δ chains 1.5%–3.7% <1%
F 2 α , 2 γ chains <2% 50%–85%
S Valine substituted for glutamic 0 0
acid in 6th position of β chain
C Lysine substituted for glutamic 0 0
acid in 6th position of β chain
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Hemoglobin Electrophoresis Hematology Review 298

Cellulose Acetate pH 8.6

Cathode (–) Anode (+)

A2
S F A
C

Origin “Crawl” “Slow” “Fast” “Accelerated ”

Citrate Agar pH 6.2

Cathode (–) Anode (+)

A
F S C
A2

Origin
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Hemoglobin Derivatives Hematology Review 299

NORMAL % OF
HEMOGLOBIN CAUSE EFFECT TOTAL HGB OTHER
Methemoglobin Iron oxidized to ferric (Fe3+) Can’t bind O2. Cyanosis, ≤ 1% Heinz bodies. Treat with
state. Usually acquired from possibly death. methylene blue.
exposure to oxidants. Rarely
inherited.
Sulfhemoglobin Sulfur bound to heme. O2 affinity 1/100th 0 Can’t be converted back to
Acquired from exposure to normal. Cyanosis. normal hemoglobin. Not
drugs & chemicals. detected in cyanmethemo-
globin method.
Carboxyhemoglobin Carbon monoxide bound ↓ O2 to tissues. Can <1 % Affinity of hgb for CO is
to heme. be fatal. 200× greater than for O2.
Skin turns cherry red.

Hemoglobin derivatives are quantitated by differential spectrophotometry.

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RBC Morphology Hematology Review 300

ABNORMALITY DESCRIPTION SIGNIFICANCE

Size
Anisocytosis Variation in size. Seen in many anemias.
Macrocytes RBCs >9 µm. Megaloblastic anemias, liver disease, reticulocytosis. Normal
in newborns.
Microcytes RBCs <6 µm. Iron deficiency anemia, thalassemia, anemia of chronic
infections.
Shape
Poikilocytosis Variation in shape. Seen in many anemias.
Elliptocytes/ovalocytes Oval or pencil/cigar shaped. Membrane defect. Hereditary ovalocytosis, various anemias.
Crenated RBCs Round cell with knobby, uniform Osmotic imbalance. If seen in most cells in thin part of smear,
projections. don’t report. Probably artifact due to excess anticoagulant or
slow drying.
Burr cells (echinocytes) Round cell with evenly spaced blunt or Membrane defect. Uremia, pyruvate kinase deficiency. May
pointed projections. be drying artifact. A few can be present in healthy individuals.
Acanthocytes (spur cells) Small, dense cells with irregularly Membrane defect. Severe liver disease, abetalipoproteinemia.
spaced projections of varying length.

continued...
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RBC Morphology continued Hematology Review 301

ABNORMALITY DESCRIPTION SIGNIFICANCE

Schistocytes RBC fragments RBCs split by fibrin strands. Microangeopathic hemolytic
anemias (DIC, TTP, HUS), prosthetic heart valves.
Sickle cells (drepanocytes) Crescent, S or C shaped, boat shaped, Sickle cell anemia.
oat shaped
Hemoglobin C crystals Blunt, 6-sided, dark-staining projection. Hemoglobin C disease.
“Bar of gold.” “Washington monument”
Hemoglobin SC crystals Glove-like intracellular crystals Hemoglobin SC disease.
Teardrops (dacryocytes) Teardrop shaped Myelofibrosis, thalassemia & other anemias.
Staining
Hypochromia Central pallor >1/3 cell diameter Iron deficiency anemia, thalassemia.
Anisochromia Mixture of normochromic & Dimorphic anemia, post-transfusion.
hypochromic RBCs
Polychromasia Bluish-gray color Young RBCs. Retics with supravital stain. Sign of active
erythropoiesis. 1%–2% in normal adult. ↑ with acute
blood loss, hemolytic anemia, following treatment for iron
deficiency or pernicious anemia.

continued...
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RBC Morphology continued Hematology Review 302

ABNORMALITY DESCRIPTION SIGNIFICANCE

Target cells (codocytes) Bull’s-eye, “Mexican hat cell” Hemoglobinopathies, thalassemia, liver disease. May be
artifact if observed in only 1 part of smear.
Stomatocytes RBC with slit-like central pallor Hereditary stomatocytosis, hereditary spherocytosis, tha-
lassemia, alcoholic cirrhosis, Rh null disease. May be artifact
in parts of smear that are too thin or too thick.
Spherocytes Small, dark-staining RBCs without Membrane defect. Hereditary spherocytosis, autoantibodies,
central pallor burns, hemoglobinopathies, hemolysis, ABO HDN, incompat-
ible blood tf, tf of stored blood. A few are normal due to
aging of RBCs.
Arrangement
Rouleaux RBCs resemble stack of coins Serum protein abnormality; e.g., ↑ globulins or fibrinogen.
Seen in multiple myeloma & macroglobulinemia. May be
artifact due to delay in spreading drop of blood or smear
that’s too thick.
Agglutination RBCs in irregular clumps Autoantibodies, cold autoagglutinin

DIC = disseminated intravascular coagulation

TTP = thrombotic thrombocytopenic purpura
HUS = hemolytic uremic syndrome
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RBC Inclusions Hematology Review 303

INCLUSION STAIN DESCRIPTION EXPLANATION SIGNIFICANCE CONDITIONS

Basophilic Wright’s & new Multiple, irregular Aggregation of RNA Coarse: exposure Exposure to lead,
stippling methylene blue purple inclusions (ribosomes) to lead. Fine: accelerated or abnor-
evenly distributed young RBC mal hemoglobin
in cell synthesis, thalassemia
Howell-Jolly Wright’s & new Round, purple, Nuclear remnants Usually pitted by Postsplenectomy,
bodies methylene blue 1–2 µm in (DNA) spleen. Seen with thalassemia, hemolytic
diameter. Usually accelerated or & megaloblastic
only 1 per cell abnormal anemias, sickle cell
erythropoiesis anemia
Cabot rings Wright’s Reddish purple May be part of mitotic Rapid blood regen- Megaloblastic anemia,
rings or figure-8s spindle, remnant of eration, abnormal thalassemia,
microtubules, or erythropoiesis postsplenectomy
fragment of nuclear
membrane
Pappenheimer Wright’s (siderotic Small purplish blue Iron particles Faulty iron Sideroblastic anemias,
bodies granules with granules. Vary in utilization postsplenectomy,
Prussian blue stain) size, shape, #. thalassemia, sickle
Usually in clusters cell anemia,
at periphery hemochromatosis

continued...
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RBC Inclusions continued Hematology Review 304

INCLUSION STAIN DESCRIPTION EXPLANATION SIGNIFICANCE CONDITIONS

Siderotic Prussian blue Blue granules of Aggregates of iron Faulty iron Sideroblastic anemias,
granules varying size & particles utilization in postsplenectomy,
shape hgb synthesis thalassemia, sickle
cell anemia,
hemochromatosis
Reticulocytes New methylene Blue-staining Residual RNA >2% = ↑ Hemolytic anemia,
blue (polychromasia network (ribosomes) erythropoiesis blood loss, following
on Wright’s stain) <0.1% = ↓ treatment for iron defi-
erythropoiesis ciency or megaloblastic
anemia
Heinz bodies Supravital stain, Round blue Precipitated, Normal during G6PD deficiencies,
e.g., crystal violet, inclusions, varying oxidized, denatured aging but pitted by unstable hemoglobins,
brilliant cresyl blue, sizes, close to cell hemoglobin spleen chemical injury to
methylene blue membrane. May RBCs, drug-induced
be >1 hemolytic anemia
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Staining of RBC Inclusions Hematology Review 305

INCLUSION WRIGHT’S STAIN NEW METHYLENE BLUE STAIN PRUSSIAN BLUE STAIN
Reticulum Cell appears polychromatophilic Yes No
Howell-Jolly bodies Yes Yes No
Pappenheimer bodies Yes Yes Yes
Siderotic granules Yes, but called Pappenheimer bodies Yes Yes
Heinz bodies No Yes No
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Erythrocyte Indices Hematology Review 306

INDEX DEFINITION MANUAL CALCULATION REFERENCE RANGES COMMENTS

MCV = HCT (%) × 1210

Mean corpuscular Average volume of RBC 80–100 fL Used to classify anemias.
volume (MCV) RBCs ( × 10 / L) Normal MCV = normocytic.
MCV >100 = macrocytic.
MCV <80 = microcytic. MCV
is an average. Combination
of microcytes & macrocytes
may result in normal MCV.
Mean corpuscular Average weight of hgb HGB (g/dL) × 10 27–31 pg Varies in proportion to MCV.
MCH =
hemoglobin (MCH) in individual RBCs RBC ( × 1012 / L)

MCHC = HGB (g/dL) × 100

Mean corpuscular Average concentration 32–36 g/dL RBCs with normal MCHC =
hemoglobin of hgb per dL of RBCs HCT (%) normochromic (area of cen-
concentration tral pallor 1/3 diameter of
(MCHC) cell). MCHC ↓ in hypochromic
cells (↑central pallor). 50%
of hereditary spherocytosis
patients have MCHC ≥ 36.
MCHC >37 may indicate
problem with specimen
(hyperlipidemia, cold
agglutinins) or instrument.
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Hemoglobinopathy Versus Thalassemia Hematology Review 307

HEMOGLOBINOPATHY THALASSEMIA
Abnormality Qualitative abnormality. Abnormality in amino acid Quantitative abnormality. Amino acid sequence of globin
sequence of globin chain, not in amount of globin chains is normal, but underproduction of 1 or more globin
produced. chains.
Examples Sickle cell anemia & trait, hemoglobin C disease & trait. β -thalassemia major & minor.

Note: Some hematologists refer to all qualitative & quantitative hemoglobin abnormalities as hemoglobinopathies.
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Normocytic Anemias Hematology Review 308

HEMOGLOBIN
ANEMIA ETIOLOGY BLOOD SMEAR ELECTROPHORESIS OTHER
Sickle cell Inheritance of sickle cell Aniso, poik, sickle cells, ≥ 80% S, 1%–20% Hgb S polymerizes under ↓ O2
anemia (SS) gene from both parents. target cells, nRBCs, F, normal A2, no A & ↓ blood pH. Disease not
Valine substituted for HJ bodies, basophilic evident in newborn because
glutamic acid in 6th stippling, siderotic gran- of ↑Hgb F. Pos solubility test.
position of β chain. ules, polychromasia. Retics 10%–20%. May have
↑ WBC with shift to left &
↑ PLT. Moderate to severe
anemia.
Sickle cell Inheritance of sickle cell Occasional target cells. No 50%–65% A, No anemia. Pos solubility test.
trait (AS) gene from 1 parent. sickle cells unless hypoxic. 35%–45% S, Important to Dx for genetic
normal F, N to counseling.
slightly ↑ A2
Hemoglobin C Inheritance of gene for Many target cells, folded >90 C, <7% F, no A Mild to moderate anemia.
disease (CC) Hgb C from both parents. cells, occasional Hgb C
Lysine substituted for crystals.
glutamic acid in 6th
position of β chain.

continued...
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Normocytic Anemias continued Hematology Review 309

HEMOGLOBIN
ANEMIA ETIOLOGY BLOOD SMEAR ELECTROPHORESIS OTHER
Hemoglobin C Inheritance of gene for Many target cells. 60%–70% A,
trait (AC) Hgb C from 1 parent. 30%–40% C
SC disease (SC) Inheritance of 1 sickle cell Many target cells. Folded & >S than C, normal Pos solubility test. Mild to
gene & 1 Hgb C gene. boat-shaped cells, occa- to 7% F, no A moderate anemia.
sional SC crystals (finger-like
projections, “Washington
Monument” crystals).
Hereditary Defect of cell membrane. Spherocytes, polychromasia. Normal MCHC usually >36 g/dL.
spherocytosis ↑ retics, ↑ osmotic fragility.
Autoimmune Autoantibodies. Polychromasia, sphero- Normal ↑ retics, ↑ indirect bili,
hemolytic anemia cytes, nRBCs. ↓ haptoglobin, pos DAT.

N = normal, nRBCs = nucleated red blood cells, DAT = direct antiglobulin test.
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Macrocytic Anemias Hematology Review 310

HEMOGLOBIN
ANEMIA ETIOLOGY BLOOD SMEAR ELECTROPHORESIS OTHER
Megaloblastic
Folate deficiency Nutritional deficiency, ↑ cell Oval macrocytes, Normal Pancytopenia, ↑ LD
replication (e.g., hemolytic Howell-Jolly bodies,
anemias, myeloproliferative dis- hypersegmentation,
eases, pregnancy), malabsorp- aniso, poik
tion, drug inhibition. Deficiency
impairs DNA synthesis.
Vitamin B12 Nutritional deficiency, malab- Same Normal Same. Pernicious anemia
deficiency sorption, impaired utilization, is most common type.
parasites. Deficiency impairs Autoimmune disease.
DNA synthesis. Gastric atrophy leads to
↓ intrinsic factor needed
for B12 absorption
Nonmegaloblastic Alcoholism, liver disease, Round macrocytes, no Normal WBC & PLT normal
↑ erythropoiesis. hypersegmentation
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Microcytic, Hypochromic Anemias Hematology Review 311

ANEMIA ETIOLOGY BLOOD SMEAR OTHER

Iron deficiency Insufficient iron for hgb Aniso, poik, hypochromic Most common anemia.
anemia (IDA) synthesis. microcytes
Sideroblastic anemia Enzymatic defect in heme Dual population of RBCs RBC indices usually normal.
synthesis. (normocytic & microcytic), Ringed sideroblasts in marrow.
Pappenheimer bodies,
basophilic stippling
β –thalassemia major ↓ β -chain production. Marked aniso & poik, Homozygous. Little or no Hgb A,
hypochromic microcytes, 95%–98% F, 2%–5% A2.
target cells, ovalocytes, Severe anemia. MCV <67 fL.
nRBCs, basophilic stippling
β –thalassemia minor ↓ β -chain production. Aniso, poik, hypochromic Heterozygous. >90%–95% Hgb A,
microcytes, target cells, 3.5%–7% A2, 2%–5% F.
basophilic stippling Mild anemia.
Anemia of chronic Acute phase reactants (e.g., 60%–70% of cases have Associated with chronic infections &
inflammation* hepcidin) affect iron absorp- normocytic normochromic inflammation, malignancies, autoim-
tion & release. Iron in bone RBCs; 30%–40% microcytic mune diseases. 2nd most common
marrow macrophages is not hypochromic anemia after IDA. Most common
released to developing RBCs. anemia in hospitalized pts.

*Formerly known as anemia of chronic disease. More often normocytic normochromic but included here because must be considered in differential Dx of microcytic anemia.
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Differentiation of Microcytic Hematology Review 312

Hypochromic Anemias
ANEMIA RBCS RDW SERUM IRON TIBC SERUM FERRITIN HGB A2
Iron deficiency anemia ↓ ↑ ↓ ↑ ↓ N
Sideroblastic anemia ↓ ↑ ↑ N ↑ N
β -thalassemia minor ↑ N N N N ↑
Anemia of chronic inflammation ↓ N ↓ ↓ ↑ N

TIBC = total iron binding capacity.

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Acute Versus Chronic Blood Loss Hematology Review 313

ACUTE BLOOD LOSS CHRONIC BLOOD LOSS

Definition Rapid loss of >20% blood volume. Loss of small amounts of blood over extended period of time
RBCs Normocytic, normochromic. May be transient Microcytic, hypochromic (due to iron deficiency)
macrocytosis when ↑ retics reach circulation.
WBCs ↑ (up to 35 × 109/L) with shift to left for about Normal
2–4 days.
Retics ↑ in 3–5 days. Peak around 10 days. Normal or slightly ↑
HGB/HCT Steady during 1st few hr due to vasoconstriction & ↓
other compensatory mechanisms. Can be 48–72 hr
before full extent of hemorrhage is evident (after
fluid from extravascular spaces moves into circulation
to expand volume).
Other Immediate fall in PLT, followed by ↑ within 1 hr. ↓ serum iron & ferritin
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Granulocytic Maturation Hematology Review 314

STAGE KEY CHARACTERISTICS
Myeloblast 15–20 m. Small amount of dark blue cytoplasm. Usually no granules. Nucleus has delicate chromatin with nucleoli.
Promyelocyte 12–24 m. Similar to myeloblast but has primary (nonspecific) granules.
Myelocyte 10–18 m. Secondary (specific) granules (eosinophilic, basophilic, or neutrophilic). Last stage to divide.
Metamyelocyte 10–18 m. Nucleus begins to indent.
Band 10–16 m. Nuclear indentation is more than half.
Segmented neutrophil 10–16 m. 2–5 nuclear lobes connected by thin strands of chromatin.
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Normal Leukocytes of the Peripheral Blood Hematology Review 315

ADULT REFERENCE ADULT REFERENCE

RANGE: RANGE:
CELL SIZE NUCLEUS CYTOPLASM RELATIVE (%) ABSOLUTE (× 109/L)
Segmented 10–16 m Segmented. 2–5 lobes Pinkish tan with 50–70 2.4–7.5
neutrophil connected by thread-like neutrophilic granules
filament of chromatin.
Band 10–16 m Horseshoe shaped. Parallel Pinkish tan with 2–6 0.1–0.6
sides with visible chromatin in neutrophilic granules
between. No filament.
Eosinophil 10–16 m Band shaped or segmented Large red granules 0–4 0–0.4
into 2 lobes.
Basophil 10–16 m Usually difficult to see Dark purple granules 0–2 0–0.2
because of overlying granules.
Monocyte 12–18 m Round, horseshoe shaped, or Gray-blue with indistinct 2–9 0.1–0.9
lobulated. Convoluted. Loose pink granules. Vacuoles.
strands of chromatin. Occasional pseudopods
Lymphocyte 7–15 m Round or oval. Dense blocks of Sparse to abundant. Sky 20–44 1.2–3.4
chromatin. Indistinct chromatin/ blue. May contain a few
parachromatin separation. azurophilic granules

Note: Automated analyzers do not differentiate between bands and segs.

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Leukocyte Abnormalities Hematology Review 316

ABNORMALITY DESCRIPTION SIGNIFICANCE

Shift to the left Presence of immature granulocytes in peripheral blood Bacterial infection, inflammation.
Toxic granulation Dark-staining granules in cytoplasm of neutrophils Infection, inflammation.
Döhle bodies Light blue patches in cytoplasm of neutrophils Infection, burns.
Vacuolization Phagocytic vacuoles in cytoplasm of neutrophils Septicemia, drugs, toxins, radiation.
Hypersegmentation >5 % of segs with 5-lobed nuclei or any with >5 lobes One of 1st signs of pernicious anemia.
Pelger-Huët anomaly Most neutrophils have round or bilobed nuclei Inherited disorder. No clinical effect. May be
misinterpreted as shift to left.
Auer rods Red needles in cytoplasm of leukemic myeloblasts & Rules out lymphocytic leukemia. Seen in up to
occasionally promyelocytes & monoblasts 60% of patients with AML. From abnormal fu-
sion of primary granules.
Variant lymphocytes 1 or more of following: large size, elongated or indented Viral infections (e.g., IM, CMV).
(atypical or reactive) nucleus, immature chromatin, ↑ parachromatin,
nucleoli, ↑ cytoplasm, dark blue or very pale cytoplasm,
peripheral basophilia, scalloped edges due to indenta-
tion by adjacent RBCs, frothy appearance, many
azurophilic granules

IM = infectious mononucleosis, CMV = cytomegalovirus.

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Quantitative Abnormalities of Leukocytes Hematology Review 317

ABNORMALITY ASSOCIATIONS
Neutrophilia Bacterial infection, inflammation, hemorrhage, hemolysis, stress
Neutropenia Acute infection, antibodies, drugs, chemicals, radiation
Lymphocytosis IM, CMV, whooping cough, acute infectious lymphocytosis
Monocytosis Convalescence from viral infections, chronic infections, TB, subacute bacterial endocarditis, parasitic infec-
tions, rickettsial infections
Eosinophilia Allergies, skin diseases, parasitic infections, CML
Basophilia Chronic myelogenous leukemia, polycythemia vera
2956_Ch04_287-366 30/01/14 4:26 PM Page 318

Hematopoietic Neoplasms* Hematology Review 318

DISORDER EXPLANATION EXAMPLE(S) OTHER

Myeloproliferative Premalignant hematopoietic stem Polycythemia vera, Usually in older adults. Caused by
neoplasms (MPN) or cell disorders involving overproduc- chronic myelogenous mutations in hematopoietic stem
myeloproliferative tion of 1 or more myeloid (nonlym- leukemia, essential cells. Primarily chronic but can
disorders (MPD) phocytic) cell lines. Bone marrow & thrombocythemia, transform into acute leukemia.
peripheral blood show ↑ RBCs, gran- primary myelofibrosis Splenomegaly, extramedullary
ulocytes, &/or platelets, with 1 cell hematopoiesis common.
line usually predominate. Normal
maturation & morphology.
Myelodysplastic Premalignant hematopoietic stem Refractory anemia, More common in elderly. May be due
syndromes (MDS) cell disorders involving ineffective refractory neutrope- to exposure to chemicals, radiation,
hematopoiesis in 1 or more myeloid nia, refractory chemotherapy, viral infections. Can
cell lines. Hypercellular bone thrombocytopenia transform into acute leukemia.
marrow with maturation abnormali-
ties (dysplasias). Peripheral blood
cytopenias (↓ counts) & morpho-
logic abnormalities.
Myelodysplastic/ Premalignant neoplasms with both Chronic myelomono-
myeloproliferative myeloproliferative & myelodysplastic cytic leukemia (CMML)
disorders (MDS/MPN) features.

continued...
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Hematopoietic Neoplasms* continued Hematology Review 319

DISORDER EXPLANATION EXAMPLE(S) OTHER

Leukemia Malignant neoplasms involving Acute lymphoblastic Classified as acute or chronic, &
unregulated proliferation of leukemia (ALL), lymphoid or myelogenous. See
hematopoietic stem cells. Abnormal chronic lymphocytic “Common Leukemias” card for
cells in bone marrow & peripheral leukemia (CLL) more detail.
blood.
Lymphoma Malignant neoplasm of lymphoid Hodgkin lymphoma, Solid tumors. Classified as B or T cell.
cells in lymphatic tissues or lymph non-Hodgkin Can spread to bone marrow, then
nodes. lymphoma malignant cells can be present in
peripheral blood (leukemic phase).

*Neoplasm = new growth; unregulated growth of a single transformed cell; may be benign or malignant. A benign neoplasm can progress to a malignant neoplasm.
2956_Ch04_287-366 30/01/14 4:26 PM Page 320

Classification of Hematopoietic Neoplasms Hematology Review 320

FRENCH-AMERICAN-BRITISH (FAB) WORLD HEALTH ORGANIZATION (WHO), 2008

Criteria Morphology, cytochemistry, immunophenotyping Morphology, cytochemistry, immunophenotyping,
cytogenetics, clinical features
Major groups Myeloproliferative disorders (MPD) Myeloproliferative neoplasms (MPN)
Myeloplastic syndromes (MDS) Myeloid & lymphoid neoplasms associated with eosinophilia
Acute leukemias (AL) & abnormalities of PDGFRA, PDGFRB, or FGFR1*
Myelodysplastic/myeloproliferative neoplasms (MDS/MPN)
Myelodysplastic syndromes (MDS)
Acute myeloid leukemia (AML) & related neoplasms
Acute leukemias of ambiguous lineage
B-lymphoblastic leukemia/lymphoma
T-lymphoblastic leukemia/lymphoma
Criteria for ≥ 30% blasts ≥ 20% blasts
diagnosis of AML
Use 1st system. Still used by some but being Widely used
replaced by WHO.

*PDGFRA, PDGFRB, & FGFR1 are genes that code for production of platelet-derived growth factor receptor (alpha & beta types) & fibroblast growth factor receptor 1.
Abnormalities in these genes are a factor in selection of drug therapy.
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Acute Versus Chronic Leukemia Hematology Review 321

ACUTE CHRONIC
Age All ages, with peaks in 1st decade & after 50 yr Adults
Onset Sudden Insidious
Median survival time, untreated Weeks to months Months to years
WBC ↑, N, or ↓ ↑ (may be >50,000)
Differential Blasts usually present More mature cells
Anemia Mild to severe Mild
Platelets Mild to severe ↓ Usually N
Other Usually lymphoid in children, myeloid in adults Myeloid mostly in young to middle-aged, lym-
phoid in older adults. Most go into blast crisis
Methods used to diagnose Peripheral blood smear, bone marrow Same but less use of cytochemical stains
examination, cytochemical stains,
immunophenotyping, cytogenetics,
molecular genetics
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Common Leukemias Hematology Review 322

TYPE OTHER NAMES WBC (× 109/L) BLOOD SMEAR OTHER

Acute myeloid Acute myelocytic, Usually 5–30 ≥ 20% blasts. May have Auer Most common type in children
leukemia (AML) acute myeloge- but can range rods, pseudo-Pelger-Huët <1 yr & adults. Rare in older
nous, acute from 1–200 cells, Howell-Jolly bodies, children & teens. ↑ uric acid &
nonlymphocytic Pappenheimer bodies, LD from ↑ cell turnover.
leukemia (ANLL) basophilic stippling, nRBCs,
hypogranular or giant PLT.
Acute lymphoblastic Acute lymphocytic ↑ in 50% of Small, homogeneous blasts in Peak incidence 2–5 yr. Smaller
leukemia (ALL) patients. Can children; larger, heteroge- peak in elderly. ↑ uric acid &
be N or ↓ neous blasts in adults. Many LD. Spreads to central nervous
do not have circulating blasts. system. Immunophenotyping
to determine lineage (T or B).
Cytogenetics & molecular
analysis for prognosis.
Chronic myelogenous Chronic granulo- Usually >100 All stages of granulocytic mat- Most common MPD. Most
leukemia (CML) cytic, chronic uration. Segs & myelocytes common after age 55 yr.
myeloid predominant. ↑ eos & basos. Philadelphia (Ph) chromo-
Pseudo-Pelger-Huët cells some. ↓ LAP. Eventually
(hyposegmentation of becomes AML or ALL.
neutrophil nuclei), NRBCs,
abnormal PLT may be seen.

continued...
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Common Leukemias continued Hematology Review 323

TYPE OTHER NAMES WBC (× 109/L) BLOOD SMEAR OTHER

Chronic lymphocytic 30–200 80%–90% small, mature- Most common type of
leukemia (CLL) looking lymphs. May have leukemia in older adults.
hypercondensed chromatin & Proliferation of B lymphs.
light-staining parachromatin
(“soccer ball appearance”),
few prolymphocytes.
Smudge cells.
2956_Ch04_287-366 30/01/14 4:26 PM Page 324

Cytochemical Stains for Differentiation Hematology Review 324

of Acute Leukemia
STAIN AML ALL
Myeloperoxidase Pos Neg
Sudan black Pos Neg
Napthol AS-D chloroacetate esterase (specific esterase) Pos Neg
Periodic acid–Schiff (PAS) Neg or diffusely pos Pos (coarse granular or block-like)
2956_Ch04_287-366 30/01/14 4:26 PM Page 325

Leukemoid Reaction Versus Chronic Hematology Review 325

Myelogenous Leukemia
LEUKEMOID REACTION CML
WBC count High High
Peripheral blood smear Shift to left (blasts rare), toxic Shift to left with blasts, eosinophilia,
granulation, Döhle bodies basophilia
Leukocyte alkaline phosphatase (LAP) High Low
Ph chromosome Neg Pos
2956_Ch04_287-366 30/01/14 4:26 PM Page 326

Plasma Cell Disorders Hematology Review 326

DISORDER KEY CHARACTERISTICS

Multiple myeloma Malignant plasma cells in marrow. Normocytic, normochromic anemia. Rouleaux on blood smear.
↑ ESR due to ↑ globulins. M spike on serum protein electrophoresis (monoclonal gammopathy).
May have Bence Jones proteinuria. Lytic bone disease.
Plasma cell leukemia Form of multiple myeloma. Plasma cells in peripheral blood. Pancytopenia. Rouleaux. Monoclonal
gammopathy.
Waldenström’s macroglobulinemia Malignant lymphocyte–plasma cell proliferative disorder. Monoclonal gammopathy due to ↑ IgM.
Rare plasmacytoid lymphocytes or plasma cells on peripheral smear. Rouleaux. May have Bence
Jones proteinuria & cryoglobulins.

ESR = erythrocyte sedimentation rate.

2956_Ch04_287-366 30/01/14 4:26 PM Page 327

Manual Hematology Procedures Hematology Review 327

TEST PURPOSE METHOD COMMENTS

Manual WBC Differential Dx CSF loaded into Neubauer hemacytome- Acetic acid can be used to lyse RBCs, if
count, CSF of meningitis ter. WBCs counted in all 9 squares of necessary. Disposable 1-piece hemacytome-
each side under 10× . ters available. Most labs perform counts on
hematology analyzers today. Manual counts
are no longer performed on blood.
Microhematocrit Screening for Microhematocrit tubes centrifuged at Values may be slightly higher than calcu-
(packed cell anemia 10,000-15,000 rpm for 5 min. % of total lated values from automated analyzers.
volume, PCV) volume occupied by RBCs determined.
Reticulocyte count Assess rate of Blood smear stained with new Miller ocular can be used to facilitate count-
erythropoiesis methylene blue. 1,000 RBCs counted. ing. Adult reference range = 0.5%–1.5%.
% containing reticulum determined. ↑ with ↑ erythropoiesis, e.g., blood loss,
hemolytic anemia, following treatment of
pernicious or iron deficiency anemia. Most
retic counts are performed on automated
analyzers today.
Erythrocyte Screen for Whole blood added to Westergren tube Nonspecific. CRP preferred. Reference
sedimentation inflammation & placed in vertical rack. Height of RBC ranges: males 0–15 mm/hr; females
rate (ESR) column read after 1 hr. 0–20. ↑ with inflammation. Automated
methods available with results in <60 min.

continued...
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Manual Hematology Procedures continued Hematology Review 328

TEST PURPOSE METHOD COMMENTS

Tube solubility Screening for Blood mixed with reducing agent, e.g., Not specific for Hgb S. Doesn’t differentiate
screening test for Hgb S sodium dithionite. Hgb S is insoluble, SS from AS. Follow up with hgb elec-
Hemoglobin S produces turbid solution that obscures trophoresis.
black lines behind tube.
Osmotic fragility Dx of hereditary Blood added to serial dilutions of NaCl & ↑ in hereditary spherocytosis. ↓ with target
spherocytosis incubated. Amount of hemolysis deter- cells, sickle cell anemia, iron deficiency ane-
mined by reading absorbance of super- mia, thalassemia.
natant from each tube.
Donath-Landsteiner Dx of paroxys- Blood collected in 2 clot tubes. Tube 1 Rare autoimmune hemolytic anemia due to
(DL) test mal cold incubated at 4°C, then 37°C. Tube 2 in- biphasic antibody (autoanti-P) that binds
hemoglobinuria cubated at 37°C only. Pos = hemolysis in complement to RBCs in capillaries at <20°C
Tube 1, none in Tube 2. & elutes off at 37°C. Complement remains
attached & lyses cells.

CRP = C-reactive protein.

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Changes in Blood at Room Temperature Hematology Review 329

PARAMETER CHANGE
MCV ↑ due to RBC swelling
HCT ↑ due to ↑ MCV
MCHC ↓ due to ↑ HCT
ESR ↓ (swollen RBCs don’t rouleaux)
Osmotic fragility ↑
WBC ↓
WBC morphology Necrobiotic cells, karyorrhexis (nuclear disintegration), degranulation, vacuolization
2956_Ch04_287-366 30/01/14 4:26 PM Page 330

Methods of Automated Cell Counting Hematology Review 330

& Differentiation
METHOD PRINCIPLE APPLICATION
Electrical impedance Low-voltage direct current (DC) resistance. ↑ resistance (impedance) Cell counting & sizing
(Coulter principle) when nonconductive particles suspended in electrically conductive diluent
pass through aperture. Height of pulses indicates cell volume, # pulses
indicates count.
Radio frequency (RF) High-frequency electromagnetic probe measures conductivity. Change in WBC differential
RF signal provides information about nucleus-to-cytoplasm ratio, nuclear
density, granularity.
Optical light scattering Hydrodynamically focused stream of cells passes through quartz flow cell Cell counting & sizing,
(flow cytometry) past light source (tungsten halogen lamp or laser light). Scattered light is WBC differential
measured at different angles. Provides information about cell volume &
complexity, e.g., granularity.
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Graphic Representations of Cell Populations Hematology Review 331

GRAPH INFORMATION USE

Histogram Size distribution graph that plots cell size (x axis) RBC, WBC, & PLT
vs. relative number (y axis). Size thresholds
separate cell populations.
Relative number

Lymphocytes

Mononuclear cells
Granulocytes

50 100 200 300 400

Femtoliters

(From Ciesla B. Hematology in Practice,

2nd ed. Philadelphia: FA Davis; 2012:323.)

continued...
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Graphic Representations of Cell Hematology Review 332

Populations continued
GRAPH INFORMATION USE
Scatterplot or cytogram Cells are plotted based on 2 characteristics, WBC differential
e.g., volume vs. light scatter. Separates cells
into distinct populations.

(From Ciesla B. Hematology in Practice, 2nd ed.

Philadelphia: FA Davis; 2012:329.)
2956_Ch04_287-366 30/01/14 4:26 PM Page 333

Technologies Used in Automated Hematology Review 333

Hematology Analyzers
MANUFACTURER TECHNOLOGIES
Impedance instruments
Beckman Coulter Cell counting & sizing: electrical impedance
(LH Series) WBC differential: VCS technology

PARAMETER MEASUREMENT INFORMATION

Volume Direct current (DC) impedance Cell volume
Conductivity (opacity) Radiofrequency (RF) Cell size & internal structure
Scatter Light scatter as cells pass through Cell surface structure & cellular
laser beam granularity
Sysmex Impedance, radiofrequency, absorption spectrophotometry, & flow cytometry with fluorescent dyes
(X-Series)
Abbott Impedance, fluorescence staining, flow cytometry, multiangle polarized scatter separation (MAPSS)
(CELL-DYN)
Light-scattering instruments
Siemens Light scattering & cytochemical analysis
(Advia)
2956_Ch04_287-366 30/01/14 4:26 PM Page 334

Automated CBC Hematology Review 334

PARAMETER VARIOUS METHODS USED

Cell counts Impedance
Light scatter
WBC differential VCS technology (volume, conductivity, scatter)
Fluorescent flow cytometry & light scatter
MAPSS technology (multiangle polarized scatter separation)
Cytochemistry (peroxidase) & optical flow cytometry
HGB Cyanmethemoglobin method
Modified cyanide-free cyanmethemoglobin method
Sodium lauryl sulphate (SLS-hgb) method
HCT Calculated from RBC & MCV
Cumulative pulse heights detection
MCV Mean of RBC volume histogram
Calculated from HCT & RBC
MCH Calculated from HGB & RBC
MCHC Calculated from HGB & HCT

continued...
2956_Ch04_287-366 30/01/14 4:26 PM Page 335

Automated CBC continued Hematology Review 335

PARAMETER VARIOUS METHODS USED

RDW CV of RBC histogram
Retics Staining with new methylene blue; VCS technology
Staining with auramine O; fluorescence detection
Staining with fluorescent dye; light scatter & fluorescence detection
Staining with oxazine-750; optical scatter & absorbance
2956_Ch04_287-366 30/01/14 4:26 PM Page 336

QA/QC for Automated Hematology Hematology Review 336

Analyzers
BEFORE IMPLEMENTATION AFTER IMPLEMENTATION
Verification of accuracy & precision Periodic calibration with stabilized whole blood calibrators
Linearity studies to verify analytic measurement range Periodic calibration verification
Correlation studies to compare new method to current method Analysis of at least 2 levels of control material each day of testing
Instrument maintenance
Participation in proficiency testing program
2956_Ch04_287-366 30/01/14 4:26 PM Page 337

Flow Cytometry Hematology Review 337

Principle Measurement of physical, antigenic, functional properties of cells suspended in fluid

Measurements Fluorescence: Cells stained with antibodies conjugated to specific fluorochrome pass 1 by 1 in front of laser light
source. Electrons of fluorochrome raised to higher energy state; emit light of specific wavelength as they return
to ground state. Emitted light detected by photodetectors for specific wavelengths.
Forward scatter (FS): Photodetector in line with laser beam measures forward scatter (FS). Proportional to
volume or size.
Side scatter (SS): Photodetector to side measures side scatter (SS). Reflects surface complexity & internal structures.
FS, SS, fluorescence displayed simultaneously on screen. Cell populations with similar characteristics form clusters
on dot plot. Specific populations can be selected with cursor (gating).
Applications Immunophenotyping: Differentiating cells on basis of surface & cytoplasmic markers. Can determine lineage &
maturity of cells in hematologic malignancies. Useful for Dx, follow-up, & prognosis. Certain immunopheno-
types associated with specific cytogenetic abnormalities.
Dx & monitoring of immunodeficiencies
Dx of paroxysmal nocturnal hemoglobinuria
Enumeration of stem cells
Quantitation of fetal hemoglobin
2956_Ch04_287-366 30/01/14 4:26 PM Page 338

Hematology Calculations Hematology Review 338

FORMULA EXAMPLE CALCULATION

retics per1,000RBCs What is the retic count if reticulum is observed in 15 of 1,000 RBCs?
Retic % = 10
Retic % = 15 = 1.5
10
Reticulocyte % using Miller Disc = What is the retic count if 60 retics are counted in square A & 300 RBCs are counted in
square B?
retics insquare A × 100
RBCs insquareB × 9 Retic % = 60 × 100 = 2.2
300 × 9
Absolute retic count (ARC) (× 109/L) = What is the absolute retic count if the retic count is 2% & the RBC is
5.2 × 1012/L?
retic% × RBC (10 /L) × 1,000
12

ARC = 2 × 5.2 × 1 000 = 104 × 109/L

100 ,
100
Corrected retic count (CRC) = What is the corrected retic count if the uncorrected retic count is 5% & the HCT is 36%?

retic % × HCT(%) CRC = 5 × 36 = 4%

45 45

continued...
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Hematology Calculations continued Hematology Review 339

FORMULA EXAMPLE CALCULATION

Retic production index (RPI) = What is the RPI if the corrected retic is 5% & the HCT is 35%? (maturation time cor-
rection factor for HCT of 35% is 1.5)?
correctedretic count
maturation time correction factor* RPI = 5 = 3.3%
1.5
Calculate the MCV if the RBC is 3 × 1012/L, the HGB is 6 g/dL, & the HCT is 20%.
MCV = HCT (%)12× 10
RBC (10 /L)
MCV = 20 × 10 = 66.7 fL
3
HGB (g/dL) × 10 Calculate the MCH if the RBC is 3 × 1012/L, the HGB is 6 gm/dL, & the HCT is 20%.
MCH =
RBC (1012/L)
MCH = 6 × 10 = 20 pg.
3

MCHC = HGB (g/dL) × 100 Calculate the MCHC if the RBC is 3 × 1012/L, the HGB is 6 g/dL, & the HCT is 20%.
HCT (%)
MCHC = 6 × 100 = 30%
20
*The maturation time correction factor is based on the patient’s HCT & is obtained from a maturation timetable.

continued...
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Hematology Calculations continued Hematology Review 340

FORMULA EXAMPLE CALCULATION

Rules of Three: What should the HGB be if the RBC is 4.1 × 1012/L?
RBC × 3 = HGB ± 0.5 HGB = 4.1 × 3 = 12.3 ± 0.5 = 11.8–12.8 g/dL
HGB × 3 = HCT ± 3%
What should the HCT be if HGB is 12.3 g/dL?
HCT = 12.3 × 3 = 36.9 ± 3 = 33.9%–39.9%
Manual Cell Count: Calculate the CSF WBC count if 18 WBCs are counted in 9 mm2 on 1 side of a
Neubauer hemacytometer, using undiluted CSF.
Cells/mm3 (µL) = # cells counted × depth factor
(always 10) × reciprocal of dilution × reciprocal of WBCs/µL = 18 × 10 × 1 × 1/9 = 20
area counted (mm2)
Absolute WBC = total WBC × relative count Calculate the absolute lymphocyte count if the total WBC is 10 × 109/L & there are
(% from differential) 70% lymphocytes.
Absolute count = 10 × 0.70 = 7 × 109/L
Corrected WBC = The automated hematology analyzer reports a WBC of 30 × 109/L. The technologist
counts 115 NRBCs per 100 WBCs while performing the differential. What is the cor-
uncorrected WBC × 100 rected WBC?
100 + NRBCs per100 WBCs
Corrected WBC = 30 × 100 = 14 × 109/L
100 +115
2956_Ch04_287-366 30/01/14 4:26 PM Page 341

Overview of Hemostasis Hematology Review 341

Primary hemostasis • Vasoconstriction

• Platelet adhesion
• Platelet aggregation to form primary hemostatic plug at injury site
Secondary hemostasis • Interaction of coag factors to produce fibrin (secondary hemostatic plug)
• Fibrin stabilization by factor XIII
Fibrinolysis • Release of tissue plasminogen activator
• Conversion of plasminogen to plasmin
• Conversion of fibrin to fibrin degradation products
2956_Ch04_287-366 30/01/14 4:26 PM Page 342

Quantitative Platelet Disorders Hematology Review 342

DISORDER EXPLANATION CLINICAL MANIFESTATIONS LAB TESTS

Thrombocytopenia ↓ production (e.g., aplastic ane- <30 × 10 /L: petechiae, menorrha-
9
PLT <150 × 109/L
mia, myelodysplastic syn- gia, spontaneous bruising.
dromes), ↑ destruction (e.g.,
immune thrombocytopenic pur- <10 × 109/L: severe spontaneous
pura, drugs, DIC, mechanical bleeding
destruction by artificial heart
valves), splenic sequestration,
massive transfusion (dilution
effect)
Primary thrombocytosis Unregulated production of Thrombosis or hemorrhage PLT usually >1,000 × 109/L.
megakaryocytes in bone Platelet aggregation may
marrow, e.g., essential thrombo- be abnormal
cythemia, other myeloprolifera-
tive disorders
Secondary or reactive ↑ PLT due to another condition, Thrombosis or hemorrhage PLT >450 × 109/L but
thrombocytosis e.g., hemorrhage, surgery, infrequent usually <1,000 × 109/L
splenectomy
2956_Ch04_287-366 30/01/14 4:26 PM Page 343

Qualitative Platelet Disorders Hematology Review 343

DISORDER EXPLANATION LABORATORY TESTS

Inherited
Bernard-Soulier syndrome Lack of functional glycoprotein (GP) Ib/IX/V on Giant plts with dense granulation. ↑ closure
plt surface prevents interaction with VWF. time (PFA). Abnormal aggregation with
Abnormal plt adhesion to collagen. ristocetin.
Glanzmann’s thrombasthenia Deficiency or abnormality of plt membrane GP ↑ closure time (PFA). Abnormal aggregation
IIb/IIIa. Fibrinogen can’t attach to plt surface & with ADP, collagen, epinephrine.
initiate plt aggregation.
δ -storage pool disorder Dense granule deficiency. Lack of ADP release. Abnormal secondary aggregation with ADP &
epinephrine.
Acquired Functional plt disorders occur with chronic Abnormal plt aggregation.
renal failure, myeloproliferative disorders, car-
diopulmonary bypass, use of aspirin & other
drugs. Mechanisms vary.

All can result in serious bleeding.

VWF = Von Willebrand factor.
2956_Ch04_287-366 30/01/14 4:26 PM Page 344

Tests of Platelet Function Hematology Review 344

TEST METHOD CLINICAL SIGNIFICANCE

Platelet aggregation Aggregating agent (e.g., ADP, collagen, risto- Abnormal curves with plt dysfunctions such as
cetin, epinephrine) added to plt suspension. von Willebrand disease, Bernard-Soulier syn-
As plts aggregate, ↑ in light transmittance. Plt drome, plt storage pool defects, idiopathic
aggregation curves generated (time vs. % thrombocytopenia purpura, drugs.
transmittance).
Platelet function assay (PFA) Citrated whole blood drawn through capillary Screening test for qualitative plt defects. Re-
tubes coated with ADP/collagen or epineph- places bleeding time. Von Willebrand disease:
rine/collagen. Plts adhere & aggregate when prolonged with collagen/ADP & collagen/
exposed to collagen. Closure time = length of epinephrine. Defects related to drugs (e.g.,
time for plts to form platelet plug & close aspirin): normal with collagen/ADP, prolonged
aperture of capillary tube. with collagen/epinephrine.
VWF:Ag Immunologic tests (e.g., EIA) using mono- VWF connects plts to collagen. ↓ in von
clonal antibodies to VWF. Willebrand disease, so plts don’t function
normally.
2956_Ch04_287-366 30/01/14 4:26 PM Page 345

Coagulation Factors Hematology Review 345

NAME PATHWAY INHERITED DEFICIENCY OTHER

I Fibrinogen I, E, C Rare Converted to fibrin by thrombin.
II Prothrombin I, E, C Rare Precursor of thrombin.
III Tissue factor (TF) E Phospholipid released from
injured vessel wall. Not normally
in blood.
IV Ca2+ I, E, C Bound by anticoagulant sodium
citrate. In assays using citrated
plasma, must be supplied by
reagents.
V Labile factor (proaccelerin) I, E, C Rare Deteriorates rapidly.
VII Stable factor (proconvertin) E Rare
VIII Antihemophilic factor I Common (hemophilia A) Circulates in association with von
Willebrand factor (VWF). VIII:
C = coagulant portion. Extremely
labile.

continued...
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Coagulation Factors continued Hematology Review 346

NAME PATHWAY* INHERITED DEFICIENCY OTHER

IX Christmas factor (plasma thromboplas- I Common (hemophilia B).
tin component)
X Stuart factor I, E,C Rare.
XI Plasma thromboplastin antecedent I Rare (hemophilia C).
May or may not cause
bleeding.
XII Hageman factor (contact factor) I No bleeding. Glass activation factor. Not part of
in vivo coagulation.
XIII Fibrin stabilizing factor I, E, C Rare. Stabilizes fibrin clot.
Poor wound healing.
HMWK High molecular weight kininogen I Rare. Not part of in vivo coagulation.
(Fitzgerald factor) No bleeding.
PK Prekallikrein (Fletcher factor) I No bleeding. Not part of in vivo coagulation.

*I = intrinsic, E = extrinsic, C = common

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Functional Classification of Hematology Review 347

Coagulation Factors
EXPLANATION FACTORS
Substrate Substance changed by an enzyme. Fibrinogen
Cofactor Protein that accelerates enzymatic reactions. No enzymatic V, VIII (V is cofactor for Xa; VIII is cofactor for IXa)
activity of its own.
Enzyme Protein that catalyzes a change in specific substrate. Serine proteases: thrombin (IIa), VIIa, IXa, Xa, XIa,
Secreted in inactive form (proenzyme, zymogen). Must be XIIa, prekallikrein
activated to function.
Transglutaminase: XIIIa

Letter “a” following Roman numeral indicates activated form of enzyme.

2956_Ch04_287-366 30/01/14 4:26 PM Page 348

Summary of Coagulation Factors Hematology Review 348

FACTORS EXPLANATION
Contact group PK, HMWK, XII, XI Factors involved in initiation of intrinsic pathway.
Prothrombin group II, VII, IX, X Vitamin K–dependent factors.
Fibrinogen group I, V, VIII, XIII Factors acted on by thrombin (V, VIII, & XIII are acti-
vated; I is converted to fibrin). All are high molecular
weight proteins.
Factors in extrinsic pathway TF, VII
Factors in intrinsic pathway PK, HMWK, XII, XI, IX, VIII
Factors in common pathway X, V, II, I
Extrinsic tenase complex VIIa/TF Acts on X.
Intrinsic tenase complex IXa/VIIIa Acts on X.
Prothrombinase complex Xa/Va Acts on prothrombin.
Factor VIII complex VIII:C & von Willebrand factor (VWF) VIII:C is the procoagulant; VWF is the carrier protein.
Produced in liver All

continued...
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Summary of Coagulation Factors continued Hematology Review 349

FACTORS EXPLANATION
Require vitamin K for synthesis II, VII, IX, X
Affected by Coumadin (warfarin) II, VII, IX, X All that require vitamin K. Warfarin is a vitamin K
antagonist.
Consumed during clotting I, II, V, VIII, XIII Not present in serum.
Labile factors V, VIII
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Coagulation Theories Hematology Review 350

CASCADE MODEL* CELL-BASED OR PHYSIOLOGICAL MODEL

Overview Focuses on role of coagulation factors. Sees coagulation Focuses on role of receptors for coagulation factors on surface
as chain rxn in which each coag factor is converted to of tissue factor (TF)–bearing cells (e.g., fibroblast or monocyte)
active form by preceding factor. Intrinsic & extrinsic & platelets. Sees coagulation as 3 overlapping phases that
pathways converge on common pathway. begin with small amount of thrombin formation on surface of
TF-bearing cells, followed by large-scale thrombin production
on platelet surface.
Steps Extrinsic pathway (TF, factor VII): Initiation (on surface of TF-bearing cell):
• TF from injured blood vessel wall activates factor VII. • Break in vessel wall exposes extravascular TF-bearing cell to
• TF:VIIa activate factor X. plasma.
• Factor VII binds to TF on cell membrane.
• TF:VIIa activates factors IX & X.
• Factor Xa combines with factor Va.
• Xa:Va generates small amount of thrombin, but no fibrin
formed at this point.
Intrinsic pathway (factors XII, XI, IX, VIII): Amplification:
• Factor XII activated by exposure to collagen. • Thrombin & collagen activate platelets.
• Factor XIIa, HMWK, & PK activate factor XI. • Platelets release factor V from granules.
• Factor XIa activates factor IX. • Thrombin activates factors V, VIII, & XI.
• IXa:VIIIa activates factor X. • Factor XIa supplements activation of factor IX.

continued...
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Coagulation Theories continued Hematology Review 351

CASCADE MODEL* CELL-BASED OR PHYSIOLOGICAL MODEL

Common pathway (factors X, V, II, I): Propagation (on surface of activated platelet):
• Xa:Va converts prothrombin (II) to thrombin (IIa). • Factor Xa binds to factor VIIIa on platelet.
• Thrombin cleaves fibrinogen (I) into fibrin & acti- • IXa:VIIIa activates factor X.
vates factor XIII to stabilize clot. • Xa:Va converts prothrombin (II) to thrombin (IIa).
• Thrombin cleaves fibrinogen (I) into fibrin & activates factor
XIII to stabilize clot.
Comments “Classic” theory. Explains in vitro coag (PT & APTT To compare models, think of extrinsic pathway as occurring on
tests) & helps ID factor deficiencies, but doesn’t fit cur- TF-bearing cell & intrinsic pathway (without factor XII, HMPK,
rent understanding of coag in vivo. Pathways don’t PK) on platelet surface. (Factor XII, HMPK, PK aren’t needed in
operate independently. vivo because factor XI is activated by thrombin produced in
initiation phase.)

*For simplicity, Ca2+ & platelet factor 3 not shown.

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Cell-Based Model of Hemostasis Hematology Review 352

X
VI•Ia II Free vWF
1. Initiation TF Xa• (Small amount
Va
of thrombin)
VIIIa
IIa •
vWF
TF
Tissue-factor bearing XI
VII•a cell (fibroblast) VIIIa
IX

X XIa Platelet
Va

IXa II 2. Amplification
IXa•
VIIIa (From Harmening DM. Clinical
IX Hematology and Fundamentals
Xa•

XIa IIa of Hemostasis, 5th ed. Philadel-

Va

(Large amounts phia: FA Davis; 2009:569.)

of thrombin)

3. Propagation
Activated platelet
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Prothombin Time (PT) and Activated Partial Hematology Review 353

Thromboplastin Time (APTT)
PT APTT
Purpose To detect deficiencies in extrinsic & common pathways To detect deficiencies in intrinsic & common pathways & to
& to monitor coumadin (warfarin) therapy. monitor unfractionated heparin therapy.
Reagent(s) Thromboplastin reagent (thromboplastin, Activated partial thromboplastin reagent (phospholipid,
phospholipid, Ca2+). activator), CaCl2.
Prolonged Coumadin therapy; deficiency of VII, X, V, II, or I; Heparin therapy; deficiency of HMWK, PK, XII, XI, IX, VIII, X, V,
results circulating inhibitors. II, or I; circulating inhibitors.
Other Report INR (international normalized ratio). INR =
[patient PT/mean normal PT]ISI. ISI = international
sensitivity index. Supplied by manufacturer. Be sure to
use ISI for current lot of thromboplastin & analyzer
being used. Therapeutic range for most situations is
2–3. For patients with artificial heart valves, 2.5–3.5.
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Interpretation of PT/APTT Hematology Review 354

PT APTT POSSIBLE DEFICIENCY

Prolonged Normal VII
Normal Prolonged HMWK, PK, XII, XI, IX, VIII
Prolonged Prolonged X, V, II, I
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Other Coagulation Tests Hematology Review 355

TEST DISCUSSION
Mixing studies Follow up to abnormal PT or APTT. Test is repeated on 1:1 mixture of pt plasma & normal plasma. If pt has factor defi-
ciency, time will be corrected because normal plasma supplies missing factor. If time is not corrected, an inhibitor is
present, e.g., antibody or anticoagulant.
Activated clotting Whole blood clotting method using point-of-care analyzer. Used in cardiac care units & during cardiac surgery to
time (ACT) monitor heparin.
Thrombin Measures time required for thrombin to convert fibrinogen to fibrin. Prolonged with hypo- or dysfibrinogenemia,
time (TT) heparin, FDPs.
Reptilase time Similar to TT except uses reptilase (snake venom enzyme) instead of thrombin. Prolonged results with afibrinogenemia &
most congenital dysfibrinogenemias. Variable results with hypofibrinogenemia. Not affected by presence of heparin.
Fibrinogen Estimation of fibrinogen level by modified TT. Thrombin added to dilutions of pt plasma. Results obtained from
calibration curve prepared from testing dilutions of a fibrinogen standard. Normal: 200–400 mg/dL.
Factor assays % of factor activity determined by amount of correction of PT or APTT when dilutions of pt plasma are added to
factor-deficient plasma.
Factor XIII Pt’s platelet-rich plasma mixed with CaCl2. Clot placed in urea or monochloroacetic acid & incubated at 37°C. With XIII
screening test deficiency, clot dissolves within 24 hr.
Anti-Factor Xa Test to monitor therapy with low molecular weight heparin. Can also be used instead of APTT to monitor therapy with
assay unfractionated heparin. Pt plasma added to excess factor Xa & substrate specific for factor Xa. Heparin in sample forms
complex with AT & inhibits factor Xa. Residual factor Xa cleaves substrate to produce colored product whose intensity
is inversely proportional to concentration of heparin.
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Most Common Inherited Coagulation Hematology Review 356

Disorders
DISEASE DEFICIENCY CLINICAL FINDINGS LABORATORY FINDINGS OTHER
Von Willebrand VWF Mucocutaneous bleed- PLT: N Most common inherited
disease ing ranging from mild Closure time (PFA): N or ↑ bleeding disorder. Autoso-
to severe Platelet aggregation: abnormal mal dominant. Both sexes
with ristocetin affected. Plts can’t adhere
PT: N to collagen to form plt plug.
APTT: N or ↑ Lab results vary.
Factor VIII: N or ↓
VWF:Ag: ↓
Hemophilia A Factor VIII Varies from asympto- PLT: N 2nd most common inher-
matic to crippling bleed- PT: N ited bleeding disorder.
ing into joints, muscles, APTT: ↑ Sex-linked recessive. Occurs
& fatal intracranial Factor VIII: ↓ primarily in males. Mothers
hemorrhage are carriers.
Hemophilia B Factor IX Same as hemophilia A PLT: N Sex-linked recessive.
(Christmas PT: N
disease) APTT: ↑
Factor IX: ↓
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Acquired Factor Deficiencies Hematology Review 357

CONDITION EXPLANATION
Liver disease Coagulation proteins are synthesized in liver.
Vitamin K deficiency Vitamin K is needed for synthesis of II, VII, IX, X.
Disseminated intravascular coagulation (DIC) Uncontrolled formation & lysis of fibrin in blood vessels. Fibrinogen, II, V, VIII, XIII, & plts
are consumed.
Primary fibrinolysis (fibrinogenolysis) Plasminogen activated to plasmin, degrades fibrinogen, V, VIII, XIII. No fibrin formation.
Acquired inhibitors (circulating anticoagulants) Antibodies against coagulation factors. Inhibitors to VIII & IX are most common & usually
in pts who have received replacement therapy for hemophilia A or B. Occasionally associ-
ated with other diseases or in normal individuals.
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Tests of Fibrinolytic System Hematology Review 358

TEST EXPLANATION METHOD(S) CLINICAL SIGNIFICANCE

D-dimer Fragment that results from Latex agglutination using Marker for DIC. Also pos with deep vein
lysis of fibrin by plasmin monoclonal antibodies against thrombosis, pulmonary embolism, & after
D-dimer, ELISA lytic therapy. Neg in primary fibrinolysis.
Fibrin(ogen) Product of action of plasmin Latex agglutination using Sign of ↑ fibrinolytic activity. Doesn’t
degradation on fibrin or fibrinogen antibodies against FDP differentiate between fibrin degradation
products (FDP) products & fibrinogen degradation products.
Present in DIC, primary fibrinolysis, deep vein
thrombosis, pulmonary embolism, & after
lytic therapy.
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Disseminated Intravascular Coagulation vs. Hematology Review 359

Primary Fibrinolysis
DIC PRIMARY FIBRINOLYSIS
PT Prolonged Prolonged
APTT Prolonged Prolonged
Fibrinogen ↓ ↓
Platelets ↓ Normal
FSP Present Present
D-Dimer Pos Neg
RBC morphology Schistocytes Normal
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Tests to Assess Risk of Thrombosis Hematology Review 360

TEST SIGNIFICANCE ASSAYS

Antithrombin (AT, formerly Plasma inhibitor that neutralizes all serine proteases, Chromogenic substrate assay, immunologic
antithrombin III) including thrombin. Deficiencies associated with assay, nephelometry.
↑ risk of thrombosis.
Protein C Coagulation inhibitor. Inactivates Va & VIIIa. Deficien- Immunologic assay, chromogenic substrate
cies associated with ↑ risk of thrombosis. assay, clot based assay.
Protein S Cofactor for protein C. Clotting assay, immunologic assay.
Factor V Leiden Most common cause of hereditary activated protein C APC resistance assay is most frequent screening
resistance (APC). Mutation that makes V resistant to test. Patient plasma diluted in V-deficient
activity of activated protein C. ↑ risk of thrombosis. plasma. Activated protein C added. APTT or dilute
Russell viper venom time (dRVVT) performed.
Abnormals must be confirmed by molecular test-
ing (e.g., PCR, restriction fragment length
polymorphism).
Lupus anticoagulants Risk factor for thrombosis & recurrent spontaneous Detected by unexplained prolongation of APTT
abortion. Acquired antiphospholipid antibodies that that isn’t corrected by addition of equal volume
interact with phospholipid in APTT reagent & prolong of normal plasma. No definitive assay.
time. In vitro phenomenon. Patient doesn’t have factor
deficiency or bleeding. Present in patients with lupus,
other autoimmune diseases, neoplasms, infections,
drugs. Also present in some normal individuals.
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Anticoagulant (Antithrombotic) Therapy Hematology Review 361

UNFRACTIONATED HEPARIN LOW MOLECULAR WEIGHT

COUMADIN (WARFARIN) (UFH) HEPARIN (LMWH)
Administration Oral IV Subcutaneous
Action Vitamin K antagonist Catalyzes inhibition of thrombin, Catalyzes inhibition of Xa by AT
Xa, & IXa by AT
Effect Slow acting Immediate Immediate
Duration Long Short Longer than UFH; shorter than warfarin
Test(s) for PT APTT, ACT (point of care), Monitoring usually not required. If
monitoring anti–factor Xa needed, anti–factor Xa should be used.
Other Decreases production of II, Requires AT to be effective APTT is insensitive to LMWH
VII, IX, X
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Coagulation Instrumentation Hematology Review 362

ENDPOINT DETECTION PRINCIPLE

Mechanical Change in electrical conductivity between 2 probes or change in movement of steel ball when clot forms
Photo-optical ↓ in light transmittance as fibrin forms
Chromogenic ↑ in light absorbance at 405 nm as para-nitroaniline (pNA) is cleaved from synthetic substrate by coag
enzyme
Immunologic ↑ in light absorbance as latex particles coated with specific antibody are agglutinated by antigen

Some analyzers have multiple detection methods.

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Examples of Criteria for Specimen Rejection Hematology Review 363

in Coagulation Testing
• Improper labeling

• Delay in delivery to lab

• Exposure to extremes of temperature

• Tube <90% full

• Specimen clotted

• Specimen hemolyzed
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Sources of Error in Coagulation Testing Hematology Review 364

ERROR COMMENT
Incorrect anticoagulant 3.2% sodium citrate should be used. Labile factors are preserved better.
Drawing coagulation tube Contamination with other anticoagulants can interfere.
after other anticoagulant tubes
Probing to find vein Tissue thromboplastin activates coagulation & ↓ times.
Incorrect ratio of blood to Need 9:1 blood to anticoagulant ratio. Tubes <90% full will have longer times.
anticoagulant
Failure to mix anticoagulant with blood Blood will clot.
Polycythemia HCT >55% leads to longer times. Anticoagulant must be reduced.
Heparin contamination from Will prolong times. Lines must be flushed with saline, first 5 mL drawn discarded.
catheter or heparin lock
Hemolysis Hemolyzed RBCs may activate clotting factors. Hemolysis may interfere with photometric
reading.
Lipemia May interfere with optical methods. Test by mechanical method.

continued...
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Sources of Error in Coagulation Hematology Review 365

Testing continued
ERROR COMMENT
Improper storage of specimen Should be stored in vertical position at RT with stopper on to prevent change in pH.
Specimens for PT must be tested within 24 hr of collection, APTT within 4 hr. (If APTT is
for monitoring heparin, must be centrifuged within 1 hr of collection.)
Improper storage or reconstitution Run normal & abnormal controls every 8 hr & with each change of reagents to verify system
of reagents performance.
Equipment malfunction, e.g., temperature, Run normal & abnormal controls every 8 hr & with each change of reagents to verify system
timer, detector, volumes dispensed performance.