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on the evidence of spontaneous hemolysis after incubation of sterile debrinated blood at 37°C for 48h in the presence of two parallel tubes containing glucose and ATP.In spherocytosis and various enzyme deciency conditions, hemolysis is corrected by both additions; however, the test is not very sensitive and specic.

Various hemolytic conditions show the presence of Heinz

bodies. These are bodies of denatured hemoglobin adherent to the erythrocyte membrane. Causal conditions include glucose- 6-phosphate dehydrogenase (G6PD) deciency, the presence of unstable hemoglobins, some hemolytic thalassemias, and some chemical agents. Heinz bodies are visible microscopically after staining with supravital staining (e.g., methyl violet). In enzymatic deciency conditions, their presence is accentuated by incubation with invitro oxidizing agents (e.g., acetylphenyl hydrazine).

The glutathione stability test evaluates the residual reduc-

ing capacity of the erythrocyte under conditions of oxidative stress. Glutathione is mainly present in the erythrocyte in its reduced form (GSH), at concentrations in the reference range of 5.3–6.8μmol/g Hb. Under normal conditions, these values vary little, even under oxidative stress (e.g., with acetylphenyl hydrazine). In enzyme deciencies, particularly G6PD deciency, GSH falls to values near zero.

Other laboratory tests generally used in hemolytic condi-

tions include measurement of intrinsic enzyme activities of erythrocytes, assessment of hemoglobin balance when unstable hemoglobins are suspected, and Ham’s test for paroxysmal nocturnal hemoglobinuria (PNH), a membrane structure disorder, performed to reveal increased sensitivity of cells to complement lysis.

The diagnostic approach to hemolytic anemias involves

the establishment of two conditions, the rst of which is preliminary: (1) demonstration of the presence of the hemolytic state and (2) determination of the specic cause of hemolysis. In the differential diagnosis of hemolytic conditions, it is important to consider conditions that may mimic the presence of hemolysis (Table15.14).

Once the presence of hemolysis has been demonstrated,

the process of identifying the specic cause must begin with an accurate medical history, a microscopic examination of

Table 15.14 Conditions mistaken for hemolytic state

Associated with anemia and reticulocytosis Hemorrhage Deciency of Fe, folate, or vitamin B12in the recovery phase Recovery after bone marrow deciency (e.g., poisoning) Associated with anemia and acholuric jaundice Ineffective erythropoiesis (intramedullary hemolysis) Intracavitary or intratissutal hemorrhages Acholuric jaundice without anemia Bone marrow invasion (myelobrosis, metastasis) Myoglobinuria

the blood smear, and antiglobulin testing. Based on the data obtained, patients can be distinguished into ve groups:

1. Patients with exposure to hemolytic agents.

2. Patients with positive antiglobulin tests.

3. Patients with a negative antiglobulin test are likely to have spherocytosis.

4. Patients with morphological abnormalities (ellipsocytes, schistocytes, ovalocytes, etc.).

5. Patients with a negative antiglobulin test and normal morphology, on which to proceed with investigations on erythrocyte enzymes, hemoglobin balance, PNH tests, etc.

Hereditary Spherocytosis

Hereditary spherocytosis (HS) is a form of hereditary hemolytic anemia common in Northern Europe and is inherited in an autosomal dominant pattern due to alterations of a gene on chromosome 8, in which about a quarter of the cases are clinically normal at diagnosis and of which sporadic cases due to new mutations are described. The disease is due to defects in the expression of erythrocyte membrane proteins that cause structural changes. The mechanism of sphericization in spherocytosis is caused by a spectrin deciency, either primary or secondary to an ankyrin deciency. This results in reduced cytoskeleton density, failure of the cytoskeleton to bind to band 3 (structural membrane protein), and subsequent instability of the lipid bilayer. Thus, lipid loss in the form of vesicles is observed both in vitro and in vivo. A simi- lar effect is caused by the reduction of band 3. Further causes of spherocytosis are due to protein 4.2 deciency, in which altered binding of spectrin via ankyrin and band 3 to the membrane protein CD47 is observed, and defective spectrinbinding protein 4.1. The various defects cause alterations in membrane properties that are reected in erythrocyte metabolism (Table15.15). Increased erythrocyte fragility is

Table 15.15

spherocytosis

Defect of erythrocyte membrane skeleton

Altered properties of membrane

Altered metabolism cell phone

Erythrocyte abnormalities inherent in hereditary

Spectrin deciency Protein 4.2 deciency Spectrin–protein 4.1 bond defective Loss of surface area Lipid alterations Altered calcium content Altered membrane proteins Increase of membrane catalase and/

or Hb Altered protein phosphorylation Protein aggregation defective Increased sodium permeability Increased glycolysis Increased turnover of ATP Decreased transport of phosphoenolpyruvate (PEP)

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G. C. Guidi

directly related to spectrin deciency, which can range from 30 to 80% of normal, and the severity of hemolysis. The chronic hemolysis that usually accompanies spherocytosis requires splenectomy in the most serious cases. The efcacy of the treatment appears to be related to the extent of spectrin deciency so that with a deciency >70%, a practically complete reduction of chronic hemolysis is usually observed. The laboratory data characteristics of hereditary spherocytosis are shown in Table15.16. The diagnosis and judgment of the severity of hereditary spherocytosis are based on the integration of clinical and laboratory data. Sometimes anamnestic and family reports, as well as splenectomy, are useful (Table 15.17). A consistent presence of spherocytes is not always detectable; indeed sometimes, they are scarce and therefore do not represent a safe indicator. In addition, there are other conditions that can cause spherocyte formation, such as immunohemolytic anemias, certain enzymatic decits of the erythrocyte and trauma. Although not very sensi-

Table 15.16 Tests and laboratory data in pre and postsplenectomy hereditary spherocytosis

Presplenectomy

Modest anemia often compensated MCHC increased due to erythrocyte dehydration Normal/variable MCV and MCH RDW mostly increased Reticulocytosis (5–20%) Polychromatophilia and anisocytosis Spherocytes (∅6.2–7.0μm, thickness 2.2–3.4μm) in number from 1% to 30% Normoblasts and normal reticulocytes

Postsplenectomy

Hb increases RDW returns to normal Reduced microcytes Hyperchromic cells increased

Table 15.17

Hemoglobin (g/L) Reticulocytes (%) Bilirubin (mg/L) Splenectomy Usually not

Severity classication of hereditary spherocytosis

Mild Moderate Serious 110–150 80–120 60–80

3–6 >6 >10

17–34 >34 >51

necessary

Erythrocyte osmotic fragility curve with hemolysis starting from 0.5 to

0.7g/dL of NaCl with tails Increase in fragility after incubation at 37°C for 48h Corrected hemolysis by glucose and ATP Increase of unconjugated bilirubin Decrease of haptoglobin Coomb’s test negative Modest neutrophilia-platelet disease Medullary hyperplasia (megaloblastosis)

Hb increased to normal-high values Normal 2,3-DPG P

normal-high

Reticulocytosis remains moderate Normal-high bilirubin Erythrocyte osmotic fragility curve loses tails

Indicated during school age, before of puberty

Necessary (delay up to 6years of age if possible)

tive, an increase in self-hemolysis and its correction with glucose and ATP are indices with good diagnostic value. To have diagnostic certainty, it is necessary to analyze the membrane protein content by separation procedures (SDS-PAGE electrophoresis, HPLC+MS). An alternative procedure is ow cytometry of intact red blood cells labeled with eosin5′-maleimide, which binds to band 3.

Rare forms of morphological abnormalities are represented by hereditary elliptocytosis and ovalocytosis. These are a heterogeneous group of conditions in which the suggested, but not exclusive, diagnostic criterion is the presence of at least 25% abnormal (elliptical/oval) erythrocytes under the microscope. These forms are also due to genetic mutations affecting the expression of membrane cytoskeleton proteins.

Glucose-6-Phosphate Dehydrogenase (G6PD) Deciency

G6PD is an enzyme widely distributed in cells of all organs. In erythrocytes, it catalyzes the rst reaction of the pentose phosphate pathway, which performs an essential function in oxidoreductive balance, as it is the only source of hydride ions (one proton and two electrons) for the reduction of NADP+ to NADPH and thus for the reduction of glutathione and the elimination of hydrogen peroxide (H

) (Fig.15.25).

2O2

The gene encoding the enzyme is in the telomeric region of the long arm of the X chromosome. More than 300 variants of the enzyme are known, of which four are the most common, with different properties (Table 15.18). Since the hereditary transmission is linked to the X chromosome, males are hemizygous for the relevant gene, so in them, there are two possibilities of expression, normal or decient G6PD.In females, since there are two X chromosomes, there are three possibilities of expression: normal, heterozygous, and homozygous (in populations with a high frequency of deciency). Heterozygous females have mosaicism due to random inactivation of the X chromosome. Therefore, there can also be conditions of complete inactivation and decit similar to that of the male, but these conditions are rarer as usually, nonheterozygous females do not manifest severe decits as the male. G6PD variants are classied according to their activity values compared to those of the normal contextual population. Specically, class I variants cause congenital nonspherocytic hemolytic anemia (activity <10% of normal); class II variants exhibit severe deciency but not hemolytic anemia (activity <10% of normal); class III variants exhibit mild to moderate deciency (activity 10–60% of normal); class IV variants exhibit very mild deciency (activity 60–100% of normal); and class V variants are very rare and exhibit increased enzyme activity compared to normal, over 150%. Although many individuals with deciency are asymptomatic throughout life, acute hemolytic crises can be observed unpredictably because of oxidative stress, which

AT P ADP

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Glucokinase

Glucose

2O2

ydrogen

peroxide)

+ O

Glutathione

peroxidase

GSH

(reduced

glutathione)

GSSG

(oxidized

glutathione)

(nicotinamide adenine

dinucleotide phosphate)

Glutathione

reductase

(reduced NADP

NADP

NADPH

+ CO

Fig. 15.25 Metabolism of the red blood cell with the cycle based on G6PD, 6PGD and glutathione oxidation-reduction by NADP+/NADPH

(glucose-6phosphate)

)

(6-phospho-

gluconate)

Glyceraldehyde-3-P

+ H+ + NADPH

nal reduction of H with permission)

Glucose-6-phosphate

G-6-P F-6-P

Glucose-6-phosphate

6-P-G

isomerase

70% Embden-

Meyerhof pathway

pathway

dehydrogenase

30% pentose phosphates

to H2O. (Copyright EDISES 2021. Reproduced

2O2

(fructose-6-

phosphate)

2 Pyruvic acid +

+ 2ATP + 2 NADH + 2H

and glutathione-peroxidase/glutathione-reductase highlighted, with a

Table 15.18

Common variants of glucose-6-phosphate

dehydrogenase

Activities, characteristics, and

Variant Gd

structure Diffusion

Normal activity

All world populations

Half-life 62days

Normal activity Half-life 13days Aspartic acid→Asparagine

376

G→A

A-

8–20% of normal activity Methionine→Valina Aspartic acid→Asparagine

202

A→G

376

G.→A

Med

Activity <5% of normal Half-life 13days Highly unstable Phenylalanine→Serine

563

T→C

Africa (the most common)

126

Africa

126

Mediterranean (Greece, Sardinia, etc.), Iran, Iraq,

188

India, Pakistan

can be caused by drugs, infections, ingestion, or simple contact with broad beans, especially in Mediterranean forms. The precise mechanism by which the crises are triggered is not known, and they appear to be due to increased sensitivity to oxidative damage. Lists of substances that are capable of triggering seizures have been prepared and can be found on several websites, which are continuously updated: http://

www.cych.org.tw/pharm/MIMS%20Summary%20 TableG6PD.pdf and http://www.g6pd.org/it/G6PDDeciency it/SafeUnsafe- it/DaEvitare_ISS- it.aspx.

During a crisis, fatigue, back pain, anemia, jaundice with increased unconjugated bilirubin, reticulocytosis, increased LDH, the appearance of Heinz bodies, hemoglobinemia, hemoglobinuria, and other symptoms common to acute hemolytic manifestations are generally observed. A G6PD deciency may be revealed at birth in the form of neonatal hemolytic jaundice, which should be considered in the differential diagnosis. To prevent such occurrences, in many countries, screening for G6PD deciency is part of neonatal prevention programs. Seizures are more characteristic of

Med

enzyme variants, while chronic hemolysis with chronic

Gd jaundice and cholelithiasis are observed more in GdA variants. In Africa and the Mediterranean, the selection of the decient gene has been operated by malaria, particularly the one due to Plasmodium falciparum, as the parasite entering the erythrocytes of a subject with deciency is in an unfavorable metabolic environment, with limited ability to replicate.

The laboratory diagnosis of G6PD deciency is based on the evaluation of enzymatic activity by quantitative spectrophotometric analysis of NADPH produced by NADP+ in a physiological pH system having as substrate glucose-6phosphate to which is added hemolysate of the sample to be examined. There are standardized commercial methods based on a procedure initially recommended by the World Health Organization (WHO). Screening tests are also available, which can make use of samples collected on bibulous paper. Since the activity of the enzyme in reticulocytes is

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much higher than the average of red blood cells (the half-life of the enzyme is 62days), it is necessary to interpret very carefully the results obtained from subjects with reticulocytosis, as can happen at the end of a hemolytic crisis because the values obtained could simulate a normal condition.

Unstable Hemoglobins

Historically, the name refers to the property of some Hb to precipitate when incubated at 50 °C for 1 h, unlike HbA, which remained stable. Subsequently, new tests were developed to assess stability, such as incubation at higher temperatures (up to 65°C) and kinetic assessment of stability. One test that has proven to be very valid for assessing the stability of Hb is the isopropanol test, which, however, should be considered parallel to and not a substitute for traditional heat tests.

The instability is caused by structural changes that allow water (H2O) access to the heme (hydrophobic) pocket, resulting in the development of free radicals, denaturation, and precipitation of the molecule.

Typically, it is the introduction of proline or glycine into an Hb helix that disrupts the secondary structure and causes instability and precipitation with the detachment of the heme. Structural abnormalities can be close to the heme or in the contact area between globin and heme or be due to insertion or deletion (Fig.15.26). The α, β, and γ globins are affected.

Of the approximately 250 mutations that affect globin chains and can reduce the stability of the molecule, roughly half are important because of the clinical consequences of the variant. Furthermore, since instability is caused in only a portion of the variant hemoglobin molecules, few Hbs are detectable by traditional methods. Mutations can cause four conditions:

• Instability detectable only in vitro, without clinical-

hematological consequences.

• The destruction of the most unstable Hb within minutes/

hours of synthesis causes ineffective erythropoiesis and

thalassemia-like syndrome. They should be identied by

molecular biology methods.

• The intermediate condition between the two previous

ones, with a variant present in erythrocytes where it pre-

cipitates under conditions of oxidative stress.

• Dominant phenotypic characteristics due to de novo

mutations.

From a clinical point of view, they are present at varying degrees depending on the type of variant:

• Congenital nonspherocytic hemolytic anemia with sple-

nomegaly and cholelithiasis (bilirubin stones)

• Hemolytic anemia with Heinz bodies, with sensitivity to

oxidizing drugs such as sulfonamides

G. C. Guidi

β98

β92

β88

β141

β98 Val → Met (Koln) (FG5) Ala (Djelfa) Glu (Mainz)

β92 His → Gln (St. Etienne) (F8) Asn (Redondo) Pro (Newcastle)

β88 Leu → Pro (Santa Anna) (F4) Arg (Boras)

β141 Leu → Arg (Olmsted) (H19)

β42 Phe → Ser (Hammersmith) (CD1) Lue (Bucuresti)

Fig. 15.26 Molecular abnormalities causing hemoglobin instability. Next to each type of abnormality are the names of some better-known unstable hemoglobins. (Copyright EDISES 2021. Reproduced with permission)

β42

β63

β45 Phe → Cys (Arta) (CD4)

β63 His → Arg (Zurich) (E7) Pro (Bicetre)

β28 His → Gln (St. Louis) (B10) Pro (Genoa)

β45

β28

• Modest/slight anemia with reticulocytosis disproportionate to the level of circulating hemoglobin

• Peripheral hematological picture similar to thalassemia with hypochromic erythrocytes

• Increased methemoglobin formation

Among the most known variants, Hb Koln (β 98

Val→Met) has been described in different groups and geographical locations; Hb Hasharon (α 47 Asp→His) is predominantly described among Ashkenazi Jews where it causes neonatal hemolysis; unstable γ variants, such as Hb Poole (γ 130 Trp→ Gly), are associated with hemolysis in the rst months of life and then disappear as the synthesis of γ chains decreases within the rst year of life; on the contrary, children with unstable β variants appear normal at birth, with progressive hemolysis developing during the rst year of life as a result of increased synthesis of β chains. Unstable α variants differ depending on whether the affected gene is α1 or

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Table 15.19

First name Mutation Mechanism Clinical expression Observation Hb Koln

Hb Hammersmith

Hb Genoa

Hb Medicine Lake

Hb Aghia Sophia

Hb Constant Spring

α2, which encodes for higher levels of mRNA.Consequently, a mutation in the α2 gene has a stronger clinical impact than that in α1, resulting in classic unstable Hb syndrome and syndrome with thalassemia-like phenotype with intramedullary Hb destruction. Some forms of HbH (β4) are due to the

Main unstable hemoglobins

β98 (FG5) Val→Met

β42 (CD1) Phe→Ser

β28 (B10) Leu→Pro

β98 (FG5) Val→Met + β32 (B14) Leu→Gln

α62 (E11) Val→0 (α1)

α142 Term→Gln (+31 residues) (α2)

Heme contact, heme detachment, increased O

Heme contact, heme shift to the deoxy form, decreased O formation

Internal, α-helix disruption

Two mutations are associated in the same gene, each causing instability. The hyper-unstable β-chain variant is destroyed in erythroid precursors Deletion in frame of a residue in the middle of a helix with alteration of the heme pocket structure

Elongated unstable C-terminal α-chain Determine α+- thalassemia, severe

afnity

afnity, hemicrome

Medium to severe chronic hemolytic anemia

Very severe hemolytic anemia with Heinz bodies

Thalassemia major-like syndrome Single case described

Determine α+-thalassemia

hemolytic anemia, and HbH disease when associated with α1 thalassemia (with deletion −)

Frequent all over the world

Very rare

Rare, in a few families

Interaction with a deletion– MED in a patient with HbH disease Frequent in southeast Asia

tic. Folate and vitamin B12 are closely involved in DNA synthesis through the methylation of deoxyuridine monophosphate to deoxythymidine monophosphate.

• Anemias from other causes, in which the marrow is normoblastic.

unstable α gene. These forms, not being of thalassemic nature, should be considered during genetic counseling. The main unstable hemoglobins are shown in Table15.19.

The diagnosis of unstable hemoglobin should be made

based on the following:

Hyporegenerative Macrocytic Anemias/ Megaloblastic Anemias

They are due to vitamin B12 and folate deciency. Absorption of vitamin B12 occurs through the ileum, facilitated by Castle intrinsic factor (IF), which is secreted by the parietal cells of

• Regenerative hemolytic anemia with increased reticulocytosis.

• Heinz bodies (not always pathognomonic as they are also

the stomach; underlying the anemia is an autoimmune mechanism that leads to loss of intrinsic factor secretory capacity.

The causes of megaloblastic anemia are as follows: present in erythroenzymopathies and following the intake of certain drugs).

• Anisocytosis, basophilic punctuation, and macrocytosis with reduced MCH.

• Separation procedures (electrophoresis, HPLC). However, they do not always allow identifying variants unless they are mutations causing altered HPLC mobility and/or electrophoretic migration.

• The presence of hemichromes, semi-Hb, or free α-chains, may indicate the presence of unstable β-chain.

• Combinations of methods and use of HPLC combined with MS.

1. Folate deciency:

• Diet (elderly patients and alcoholics)

• Malabsorption (ethylism, coeliac disease, tropical sprue, Crohn’s disease, and jejunal resection)

• Increased demand (pregnancy, prematurity, chronic hemolytic anemia, inammatory diseases, and neoplasms)

• Drug interference (antiepileptics, folate antagonists such as methotrexate, trimethoprim, sulfasalazine, and oral contraceptives)

• Renal loss (congestive heart failure and dialysis)

• Hereditary abnormalities of folate absorption and metabolism (absence of enteric folate receptors)

2. Vitamin B12 deciency:

Macrocytic Anemias

• Reduced intake and release of vitamin B12 by diet

• Inadequate secretion of intrinsic factors, which can be

Two types can be distinguished according to the causes:

distinguished in:

– Classic pernicious anemia, an autoimmune condi-

• Vitamin B12 (cobalamin) or folate deciency causes macrocytic anemia, in which the marrow is megaloblas-

tion with antibodies directed against the parietal cells of the stomach; in the affected patient there is

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G. C. Guidi

autoimmune gastritis or atrophy of the parietal cells of the gastric mucosa, with achlorhydria and reduced secretion or absence of intrinsic factor. This deciency limits vitamin B12 absorption. The lack of IF can be congenital (rare) or acquired on an autoimmune basis with the presence of highly specic pernicious anemia intrinsic factor antibodies (50–70% of cases). Anti-intrinsic factor antibodies have been found in some patients with Graves’ disease and in insulin-dependent diabetes. They are of two types: type I antibodies, which block the binding of vitamin B12 to the IF, and type II antibodies, which block the binding of the B12-IF complex to the ileal receptor. Antiparietal cell antibodies have been reported in 50–90% of patients with perni-

cious anemia. – Total or partial gastrectomy. – Congenital intrinsic factor deciency or abnormal

intrinsic factor.

3. Diseases of the ileum:

• Ileal resection

• Crohn’s disease

• Chronic tropical sprue

• Other acquired diseases

4. Different utilization of vitamin B12:

• Abnormal intestinal ora (diverticula of the jejunum, excluded intestinal loop with stagnation of bacteria).

• Parasitic infestation by Diphyllobothrium latum (botryocephalus).

5. Defective release of vitamin B12:

• Pancreatic insufciency due to lack of trypsin which detaches vitamin B12 from its complex.

• Zollinger–Ellison syndrome: acidication of the small intestine delays the absorption of vitamin B12.

6. Hereditary abnormalities of vitamin B12 transport or its intracellular metabolism.

The causes of vitamin B12 deciency listed at points 3–6

occur in the presence of a normal intrinsic factor.

Causes ofMacrocytosis withNormoblastic Erythropoiesis

Some patients present with macrocytosis with MCV up to 110–112fL independent of vitamin B12 and folate. Causes are as follows:

• Chronic alcohol abuse

• Myelodysplastic syndromes

• Hypothyroidism

• Chronic lung disease with hypoxia

• Heavy smokers

• Chronic liver diseases

• Chronic hemolytic anemia

• Therapy with antiepileptic drugs

• Myeloma (some cases)

• Hypoplastic anemia

In some physiological conditions, such as infants and women with physiological pregnancies, macrocytosis could be observed.

The laboratory diagnosis of megaloblastic anemia is based on the following ndings:

• Normochromic macrocytic anemia (MCV>102fL; rang-

ing from 100 to 160fL). Macrocytosis may not be as evi-

dent if iron deciency anemia, thalassemic trait, or

intercurrent infections coexist. It is possible that even in

the presence of vitamin B12 deciency, the hematocrit

value may be normal; hemoglobin and erythrocyte count

are both signicantly reduced, and MCH is increased

with normal MCHC.It should be kept in mind that MCHC

represents the ratio of the mean mass of hemoglobin con-

tained in each erythrocyte to the mean mass of the eryth-

rocyte itself. This ratio tends to be rather conservative in

many hematologic disorders, ranging between 32% and

35%. As mentioned above, the most well-known condi-

tion in which MCHC increases is hereditary spherocyto-

sis. In this condition, MCHC is higher because the

erythrocyte membrane mass decreases and erythrocytes

accumulate excess H

O due to altered extrusion

mechanisms.

• Low reticulocyte count, unless a hemolytic component is

present.

• Possible decrease of leukocytes and platelets.

• Hypersegmented neutrophils; a plurilobed nucleus with

5–6 lobes is always indicative of megaloblastic anemia.

Other data that may be altered are those related to possible ineffective bone marrow erythropoiesis and hemolysis, in particular:

• Indirect bilirubin: increased

• Sideremia: increased

• Ferritin: increased

• LDH: increased

• Haptoglobin: decreased or absent.

Erythrocytes show variations in shape (macro-ovalocytes) and volume. They contain within them basophilic inclusions and nuclear remnants (Cabot rings and Howell–Jolly bodies).

The bone marrow aspirate is rich in megaloblasts and hyperplastic cells, with a decreased ratio of myeloid and erythroid series. Obvious alterations are present in the precursors of all cell lines, but the erythroid line is especially affected. Widespread basophilia of the precursors gives the

15 Hematological Diagnostics

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megaloblasts a blue May-Grünwald-Giemsa (blue marrow) coloration. The nuclear chromatin is scattered and appears poorly stained, with fenestrated aspects typical of megaloblastic anemia. The presence of ringed sideroblasts in the marrow, as well as hypersideremia and hyperferritinemia, indicates decreased iron utilization.

Platelet Alterations

The study of platelet alterations has beneted from considerable progress in recent decades, mainly due to important acquisitions in physiopathology, new knowledge in molecular biology, and the development of innovative technologies applied to instruments that have progressively spread in laboratories. The starting point, however, is the complete CBC with microscopic observation of the peripheral blood smear. From these initial tests, it is possible to obtain information on the diagnosis of two main platelet diseases: immune thrombocytopenic purpura, also known as Werlhof’s disease, and thrombotic thrombocytopenic purpura (TTP). In addition, immunoglobulin measurements are sometimes required in pediatric patients to rule out common variable immunodeciency as a cause of thrombotic thrombocytopenic purpura. Careful observation of the peripheral smear is essential in a patient with thrombocytopenia. It can rule out forms of pseudothrombocytopenia, such as those due to platelet satellites, in which platelets adhere to neutrophils, causing a false reduction in count on CBC (Fig. 15.27). Moreover, giant platelets could be found in patients with immune thrombocytopenic purpura; in this case, they are not specic markers of the condition but rather a sign of the stimulus exerted by thrombopoietin on the bone marrow due to thrombocytopenia (Fig.15.28).

In thrombotic thrombocytopenic purpura, fragmentation

of red blood cells (schistocytes) due to peripheral microangi-

Fig. 15.27 Microscopic image of a blood smear from a patient with pseudothrombocytopenia from satellite

191

Fig. 15.28 Giant platelet in peripheral smear. Normal-sized platelets on the right are observed for comparison

Fig. 15.29 Giant platelet in a patient with Bernard–Soulier syndrome

opathy, as well as thrombocytopenia, could be observed. The minimum criteria for the diagnosis of thrombotic thrombocytopenic purpura are thrombocytopenia and microangiopathic hemolytic anemia without apparent etiology. In the complete form, neurological abnormalities, high fever, and acute renal failure occur. A smear examination shows thrombocytopenia and the presence of schistocytes. In addition, increased LDH and reticulocytosis are observed. Signs of intravascular coagulation are absent or only slightly increased in patients with TTP.

Rare congenital thrombocytopenia, known as Bernard– Soulier syndrome, which causes easy nose and gum bleeding as well as gastrointestinal bleeding, can also be suspected based on the examination of the peripheral smear (Fig.15.29) when thrombocytopenia is accompanied by the presence of giant platelets. This syndrome is due to a molecular defect in which the glycoprotein complex of the platelet membrane, gp Ib-gp V-gp IX is missing, with a consequent inability to bind to von Willebrand factor (VWF) and therefore to adhere to the platelet. The genes involved are GP1BA, GP1BB, and GP9, which encode for gp Ibα, gp Ibβ, and gp IX subunits. The bleeding time is prolonged. A careful smear examination is essential to exclude thrombotic thrombocytopenic

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purpura and rare cases of acute leukemia that initially present with thrombocytopenia.

Antiplatelet antibodies responsible for auto-immune thrombocytopenia (ITP) do not induce complementmediated lysis. Patients with autoimmune thrombocytopenia have larger platelets than normal ones, with increased membrane surface area and an increased number of Fc receptors than normal platelets. For these reasons, any increase in platelet-related immunoglobulins (antiplatelet antibodies) is not useful for diagnostic purposes because they are increased in almost all conditions associated with thrombocytopenia. This limits the value of the antiplatelet antibody test in the diagnosis of immune thrombocytopenic purpura.

One form of acquired thrombocytopenia that often comes rst to the attention of the laboratory is heparin-induced thrombocytopenia. This is a particular alteration usually observed in samples from surgical (but also medical) patients who have received some form of heparin for prophylactic/ therapeutic purposes, although the highest frequency is observed with unfractionated heparin and can affect up to 5% of patients. The cause is due to the exposure of a particular masked epitope that occurs in some individuals because of the binding that normally takes place between heparin and PF4. The generation of antibodies, in response to this epitope exposure, proceeds with the formation of circulating immune complexes that activate platelets through binding to the Fc receptor. Platelet activation continues with further release of PF4, and the phenomenon tends to amplify, causing thrombocytopenia due to the aggregation of additional platelets and hypercoagulability that can result in thrombosis. Heparin-induced thrombocytopenia appears approximately 5–10days after the initiation of drug administration. A rapidonset form of heparin thrombocytopenia is anamnestically observed in patients who had previously received heparin without developing thrombocytopenia. The immediate measure is to discontinue heparin administration. The risk is much lower with fractionated low molecular weight heparins. The risk is absent when using one of the direct anti-FXa anticoagulants (e.g., rivaroxaban) for the same indications.

A rare form of spurious thrombocytopenia that usually comes rst to the attention of the laboratory is that resulting from samples collected in EDTA.This phenomenon is due to the presence of EDTA-dependent antiplatelet antibodies that are activated at low temperatures. They cause agglutination of platelets and, therefore, a lowering of count values in automated instruments. It is suspected when unexplained thrombocytopenia, without clinical signs (absence of petechiae, normal bleeding time), is observed in outpatients. In order to ascertain whether EDTA is the cause of thrombocytopenia, it is necessary to take a second sample with a differ-

ent anticoagulant (e.g., sodium citrate) and perform the count again, which will give a normal value if positive.

Bleeding time is the most widely used test for assessing primary hemostasis. However, despite its theoretical usefulness, this test is highly operator-dependent and not recommended as a screening test in the diagnostic routine for disorders of hemostasis. Bleeding time is measured by measuring at 30-s intervals the time required to observe the cessation of bleeding following a 1-mm-deep, 1-cm-long supercial cut on the volar surface of the forearm made by a standard cutter. Under these conditions, cessation of bleeding results from the formation of a primary hemostatic plug. There is a fairly linear correlation between bleeding time and platelet count between 10,000/μL and 100,000/μL.Bleeding time is prolonged with platelet counts below 75,000/μL, although this does not provide an explanation as to why the platelet count is low. In patients with thrombocytopenia, bleeding time should not be performed as it has no diagnostic signicance. In alterations of secondary hemostasis related to coagulation mechanisms (e.g., in patients with hemophilia A or B), the bleeding time is always normal.

Evaluation ofPlatelet Function InVitro withPFA-100

The Platelet Function Analyzer 100 (PFA100) is an automated benchtop instrument that evaluates primary hemostasis in vitro in a shear-stressed blood sample. The PFA-100 uses disposable test cartridges, each containing a nitrocellulose membrane impregnated with collagen plus ADP (Col/ADP) or collagen plus epinephrine (Col/Epi). A sample of citrated blood (0.8mL) is placed in a cup of the instrument and aspirated through the membrane opening. Shear stress occurs when platelets pass through Col/ADP or Col/Epi, which act as activation agonists, aggregate and progressively reduce until the ow through the membrane is stopped. This represents the endpoint, expressed as the time of closure. The formation of platelet aggregates depends on the following:

• The binding of von Willebrand factor to the collagen-

coated nitrocellulose membrane

• Platelet adhesion to von Willebrand factor via activation

of the gp Ib receptor

• Platelet aggregation is mediated by the interaction of gp

IIb/IIIa with von Willebrand factor and brinogen

Normal closure times range from 77 to 133s for the Col/ADP membrane and from 98 to 185s for the Col/Epi

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membrane. The PFA-100 has been tested in patients with bleeding disorders. The closure time using the Col/Epi cartridge is abnormal in patients with congenital defects of platelet function and von Willebrand disease after aspirin intake, whereas the closure time using the Col/ ADP cartridge is abnormal mainly in patients with von Willebrand disease and in congenital diseases. Aspirin prolongs the closing time in 94% of cases with the Col/ Epi cartridge and only in 27% of cases with the Col/ADP cartridge. Glanzmann thrombasthenia, Bernard–Soulier syndrome, and most mild forms of von Willebrand disease are associated with prolonged closing time with both cartridges, whereas a storage pool defect and giant platelet thrombocytopathy have prolonged closing time only with the Col/Epi cartridge.

The advantages of this instrument include simplicity and reproducibility. The PFA-100 has been reported to have a coefcient of variation of less than 10%. It may be useful for determining global platelet function and assessing the efcacy of antiplatelet therapy.

Platelet aggregation is measured by turbidimetric methods. When platelets aggregate, the platelet-rich plasma suspension becomes clearer and allows more light to be transmitted. The degree of aggregation is determined by measuring the progression over time, usually for 5min, of the increase in light transmission and by reporting the aggregation curve, which evaluates the reversibility/irreversibility of aggregation (primary vs. secondary), the shape change, that is, the change in platelet shape upon stimulation with agonists such as ADP and collagen, and the maximum amplitude of the aggregation wave.

Small doses of ADP (<1μmol) induce a reversible form of platelet aggregation (primary wave), unaccompanied by thromboxane synthesis and intraplatelet ADP release. However, with increasing doses of ADP, sufcient stimulation of platelets occurs with intraplatelet ADP release and thromboxane A2 synthesis by rapid metabolism of arachidonic acid (released from platelet membrane phospholipids) by the cyclo-oxygenase enzyme; the nal consequence is a more pronounced and irreversible aggregation wave (secondary wave). A secondary wave of aggregation is also caused by collagen and directly by arachidonic acid, which is used as an agonist. Ristocetin causes platelet aggregation by inducing von Willebrand factor to bind to the gp Ib protein complex on the platelet.

Platelet aggregation tests are useful in distinguishing various disorders of platelet function. They are also particularly used in the differential diagnosis of von Willebrand disease, in which ristocetin-induced platelet aggregation is defective.