The immuassay handbook parte82

795© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/B978-0-08-097037-0.00065-8

HematologyKaty Evans1 ([email protected])

Zane Amenhotep1 ([email protected])

Derek Dawson2

Harry Waters2

John Ardern2

1This edition.2Previous editions.

Normal Blood FunctionBlood is a specialized fluid that plays an integral role in physiological processes. The cellular components of blood include red blood cells, white blood cells, and plate-lets. Red blood cells are responsible for the transport of oxygen from the lungs to the tissues and carbon dioxide from the tissues to the lungs. White blood cells protect the body against infection, while platelets facilitate the forma-tion of blood clots at sites of injury. Blood is also respon-sible for regulating water balance and body temperature as well as carrying hormones, absorbed food materials, useful metabolites, and waste products to the appropriate tissues and organs within the body.

The formation of the cellular components of blood occurs in the bone marrow, in a process known as hemo-poiesis. DNA synthesis in the nuclei of maturing cell pre-cursors depends upon a supply of two vitamins: vitamin B12 and folate. Vitamin B12 (also termed cyanocobalamin) is a member of the cobalamin family. However, unless a spe-cific application is referred to, the term vitamin B12 will be used throughout this chapter since this is the stable form to which all other cobalamins are converted for reagent and testing purposes. Absorption of vitamin B12 from the nor-mal diet requires the parietal cells of the gastric mucosa to secrete a carrier glycoprotein called intrinsic factor (IF). The vitamin B12/IF complex is absorbed in the terminal small bowel. Folate and iron, in contrast, are absorbed in the proximal small bowel.

The formation of red blood cells in the bone marrow is known as erythropoiesis. The term erythron refers to the total erythropoietic organ, including bone marrow and cir-culating red cells. The development of functional red blood cells depends on the production of hemoglobin, a specialist protein required for carrying oxygen within the red cell. The production of hemoglobin increases as the erythroid precur-sor cells mature. Hemoglobin consists of iron atoms in heme molecules inserted into a protein shell (globin). The struc-ture of the molecule is essential for the proper control of the attachment and release of oxygen from the iron. Most of the iron in the body is in the ci rculating hemoglobin (1mg in about 2 mL of blood), and much of the rest (500–1000 mg) is stored in the reticuloendothelial cells of the bone marrow, spleen, and liver. The hemoglobin level is maintained by uti-lization of recycled iron from the hemoglobin of dying red cells and, when necessary, by utilization of iron from the

reticuloendothelial stores. The main storage protein for iron is ferritin, and the level of ferritin in the blood gives a reli-able indication of the iron stores. In plasma, iron is bound to a carrier molecule called transferrin.

There are several different types of circulating white blood cells (leucocytes) all of which help to protect the body against infection. White blood cells may be broadly characterized as granulocytes or agranulocytes depend-ing on the presence or absence of granules in the cell cyto-plasm. Granulocytes include neutrophils (polymorphs), eosinophils, and basophils. Circulating agranulocytes include lymphocytes and monocytes. Neutrophils and monocytes play a critical role fighting against acute bacte-rial infections by engulfing pathogens in a process known as phagocytosis. Lymphocytes assist phagocytes through the development of a controlled immune response. A sig-nificant component of this immune response is the synthe-sis of specific antibodies following exposure to foreign molecules. Immunological memory ensures that re-expo-sure to foreign molecules elicits a much greater immune response. The lymphocytes originate in the lymphoid tis-sues of the bone marrow, thymus, spleen, and lymph nodes.

Hemostasis refers to the sophisticated mechanisms that have evolved to allow rapid blood clotting at sites of injury, yet maintain blood fluidity in the rest of the circulation. Hemostasis depends upon the interaction of many factors including circulating platelets and coagulation factors, the endothelium of the blood vessel wall, naturally occurring anticoagulants (e.g., protein C, protein S, antithrombin), and fibrinolysis (clot digestion). When vascular damage occurs, platelets aggregate on the blood vessel endothelium and a series of coagulation factors, known by their roman numerals, are activated. Von Willebrand factor (vWF) supports platelet interaction with the exposed endothe-lium and also acts as a carrier of factor VIII. The activation of the coagulation pathway culminates in the conversion of fibrinogen to a fibrin gel by thrombin. A complex sys-tem of activating and feedback mechanisms ensures that proteases are generated that not only activate but also degrade the coagulation factors, thereby preventing extensive clot formation. The removal of fibrin is controlled by the fibrinolytic system consisting of the proenzyme plasminogen along with its associated activators and inhibitors. The main activator of plasminogen is the tissue protease, tissue plasminogen activator (t-PA), which is activated at the same time as the coagulation sequence is started. The end point of the fibrinolytic system is plasmin, which cleaves fibrinogen and fibrin into degradation prod-ucts (FDPs) which include fibrinopeptide A and D-dimer.

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9.11

796 The Immunoassay Handbook

Plasminogen activator inhibitor-1 is produced mainly by endothelial cells and is a powerful inhibitor of t-PA.

Clinical DisordersANEMIAA shortage of red blood cells, and hence a low level of hemoglobin, results in insufficient oxygen being trans-ported to the tissues. This condition is known as anemia. The symptoms of anemia are the same, whatever its cause, and include breathlessness on exertion and fatigue.

The main causes of anemia include:

� Impaired red cell production: � Bone marrow failure resulting in decreased eryth-

ropoietic mass (e.g., aplastic anemia). � Iron deficiency resulting in impaired heme produc-

tion due to depletion of the body’s iron stores. � Vitamin B12 or folate deficiency resulting in

impaired DNA synthesis in hemopoeitic precursor cells (e.g., pernicious anemia).

� Chronic infection resulting in impaired mobiliza-tion of iron from reticuloendothelial stores for heme production.

� Hemoglobinopathies due to abnormal globin chain production or underproduction of globin chains (e.g., thalassemia).

� Increased blood loss: � Acute hemorrhage. � Chronic blood loss (e.g., low level gastrointestinal

bleed, menorrhagia). � Hemolysis:

� Increased red cell destruction within the circulation or reticuloendothelial system. There are numerous hereditary and acquired causes of hemolytic anemia.

The laboratory investigation of anemia has been facilitated by technological advances of the new generation blood count analyzers (Waters and Seal, 2001). Some instru-ments are able to determine the hemoglobin content of individual red cells. This measurement reflects the iron content of circulating red cells and provides a direct indi-cation of iron delivery to the bone marrow (Macdougall et al., 1992). It is possible to perform accurate enumeration of circulating reticulocytes (immature red blood cells) as well as to assess reticulocyte maturity by the analysis of RNA content (Brugnara, 2000). The presence and number of their precursor nucleated red cells in the circulation can also be indicated. These parameters are useful indicators of erythropoiesis, which can contribute to the differential diagnosis of the various forms of anemia.

POLYCYTHEMIAThis term describes a group of conditions that have increased red cell production (erythrocytosis) as their common essential feature (Messinezy and Pearson, 1999). In contrast to anemia, patients with polycythemia have a raised hematocrit (packed cell volume). The erythrocytosis may be primary due to an intrinsic bone marrow defect

(polycythemia vera) or secondary due to increased eryth-ropoietin (EPO) secretion. The majority of patients with primary polycythemia vera have an acquired mutation in the Janus Kinase 2 (JAK2) gene, and as such, genetic test-ing by various techniques now represents a front line test in the diagnostic work-up of unexplained erythrocytosis (Landolfi et al., 2010). The World Health Organization (WHO) has revised the diagnostic criteria of polycythemia vera to reflect the implications of mutation detection (Tefferi et al., 2008). In cases where no known genetic mutation can be identified, direct radioisotopic measure-ment of the patient’s red cell mass and plasma volume together with assay of the circulating EPO level are usually required for the differential diagnosis of polycythemia.

IRON OVERLOAD AND HEMOCHROMATOSISGenetic hemochromatosis is an inherited condition which causes the body to absorb excessive amounts of iron from the diet. It is one of the most common recessive disorders in Northern Europe. The body has no active system for excret-ing excess iron. No harm results when the iron is confined to macrophages. However, when iron is in excess, it is depos-ited in tissues including the liver, heart, and endocrine glands with resulting cirrhosis, cardiomyopathy, diabetes, arthritis, hypopituitarism, and skin pigmentation. Measurement of transferrin saturation is the “best” test for indicating poten-tial or early iron overload, and the degree of overload can be confirmed and monitored using the serum ferritin concen-tration. A diagnosis of genetic hemochromatosis should be confirmed by genetic analysis of the HFE gene (reviewed in Bacon et al., 2011). Commonly known mutations of this gene include C282Y and, to a lesser extent, H63D. In some cases it also useful to demonstrate liver iron overload with a liver biopsy. Iron overload is not confined to genetic hemo-chromatosis, it can also be secondary to a prolonged inability to use iron that has entered the body in excessive amounts. This is commonly seen in thalassemia patients who are dependent of blood transfusions to treat their anemia.

HEMOSTATIC DISORDERSFollowing vascular damage, normal hemostasis requires close interaction between the blood vessel wall, circulating platelets, and blood coagulation factors. Disorders result-ing in impaired interaction between these elements often manifest as hemorrhage. Irregularities of the vascular/platelet system may result in bleeding from mucous mem-branes and bleeding into the skin, while coagulation disor-ders may result in bleeding into joints. Thrombocytopenia, reduced platelet numbers, is common and may be associ-ated with infection or autoimmune disease.

Coagulation disorders may be inherited or acquired. Hemophilia A is the classic hereditary bleeding disorder caused by an inherited deficiency of plasma coagulation factor VIII. More commonly, coagulation disorders are due to an acquired deficiency of several factors resulting, for example, from decreased production (as in hepatic dis-ease) or due to excessive utilization as in disseminated intravascular coagulation (DIC), a not uncommon result of severe infection or tissue trauma.

797CHAPTER 9.11 Hematology

The hemostatic response to vascular injury is tightly controlled. The fibrinolytic system and other plasma pro-teins that degrade activated coagulation factors limit coag-ulation to the site of injury. Thrombosis, intravascular coagulation of the blood resulting in vessel occlusion, may occur spontaneously when these factors are lacking or faulty. Factor V Leiden is a commonly inherited variant coagulation factor that is resistant to degradation by the natural anticoagulant protein C. As such, individuals who carry the factor V Leiden variant have an increased risk of venous thrombosis.

The lupus anticoagulant is found in a variety of autoim-mune diseases and occasionally in otherwise healthy indi-viduals. Contrary to what its name suggests, its presence is associated with a thrombotic predisposition.

Clinical practice requires reliable and accurate labora-tory techniques for the enumeration of platelets and assess-ment of platelet function by aggregometry. It also requires techniques to assess the functional activity of the coagula-tion factors and tests for evidence for hypercoagulability and aberrant coagulation. In general, immunological tech-niques are of greater value in conditions of hypercoagula-bility than in bleeding disorders.

LEUKEMIA AND LYMPHOMALeukemia refers to a group of disorders characterized by the malignant proliferation of precursor (acute type) or mature (chronic type) leukocytes, resulting in an increased number of white blood cells in the circulation. Lymphoma is a malignancy of the lymphoid tissue, usually producing enlarged lymph nodes. Hodgkin’s disease is the most common form.

AnalytesVITAMIN B12 AND FOLATEThese two vitamins are considered together because of their close interaction and the identical hemopoietic changes that result from their deficiency.

The cobalamins, collectively known as vitamin B12, rep-resent a family of water-soluble molecules that share a common molecular structure consisting of two parts: a pla-nar group and a nucleotide set at right angles to it. The nucleotide consists of a 5,6-dimethyl benzimidazole and a phosphorylated sugar. The planar group is a corrin ring with a central cobalt atom from which the name cobalamin is derived. The presence of the corrin ring places these molecules under the broader classification of corrinoids, a designation shared by several analogs of cobalamin that lack the specific nucleotide. IF is required for normal cobalamin absorption. It binds only to the nucleotide of the cobalamins, whereas other binders of cobalamins can also bind other corrinoids.

As the largest and most complex of all known vitamins, cobalamin synthesis is limited to natural mechanisms and occurs exclusively in microorganisms. The various vita-mers of cobalamin differ in the functional group that is attached directly to the cobalt atom. The most common form is deoxyadenosylcobalamin, found in the liver and

most tissues. Methylcobalamin, a coenzyme form and the predominant type found in plasma, directly participates in homocysteine metabolism. Hydroxocobalamin is the form to which other forms convert on exposure to light. Vita-min B12 was first crystallized as cyanocobalamin (molecu-lar mass 1355), which does not occur naturally. However, it is the most air stable form, which has led to the abun-dance of its use as a dietary supplement. It is also the form to which all functional groups are converted in the assay. Whereas a variety of bacteria may be exploited to manu-facture naturally occurring cobalamins, pharmacological synthesis of cyanocobalamin derives from the chemical substitution of cyanide for the functional group of other forms. This reaction has also been favorably exploited, in that hydroxocobalamin has been used as a relatively safe antidote for cyanide poisoning.

Folic acid, pteroylglutamic acid (PGA, molecular mass 441), consists of a pterin that is attached to glutamic acid through a p-aminobenzoic group. In nature, it is found in reduced forms with an additional single carbon unit (e.g., methyl, methylene, and formyl) and a variable number of glutamate radicals. Folates act as coenzymes in the transfer of single carbon units in amino acid metabolism. Because of the many functional forms and the rarity of PGA in nature, the term folate is used to cover all forms. Plasma folate is a monoglutamate, whereas red cells contain pre-dominantly penta- and hexa-glutamates.

Only two reactions are known in man that require cobalamin. In one, deoxyadenosylcobalamin is required as coenzyme in the conversion of methylmalonyl coenzyme A (CoA) to succinyl CoA. With cobalamin deficiency, the serum level and the urinary excretion of methylmalonic acid increase. These changes can be used to demonstrate cobalamin deficiency though this reaction is not connected with the defect leading to the anemia. In the other reac-tion, methylation of homocysteine to methionine requires both methylcobalamin and reduced methylfolate. Reduced folates, other than the methyl form, are built up into poly-glutamates to keep the folates within the cells. 5,10-meth-ylene tetrahydrofolate polyglutamate is the enzyme form (shown as polyglutamate) required in the synthesis of thy-midylate (dTMP) for DNA (see Fig. 1).

Deficiency of either of these vitamins eventually results in anemia (or pancytopenia in the setting of severe defi-ciency) as a result of the impairment of nucleic acid metabo-lism in hemopoietic precursors in the bone marrow and consequent ineffective hemopoiesis. Delays in the normal hemopoietic maturation event of nuclear chromatin con-densation create a microscopically recognizable morpho-logic change and gave rise to the description megaloblastic anemia. Cobalamin deficiency causes a build up of methyl-folate and lack of folate polyglutamate, whereas folate defi-ciency leads to a direct lack of the latter. Synthesis of the pyrimidine is essential in man, whereas it is available from a salvage pathway in other species. Hence, the characteristic megaloblastic anemia is seen only in man. The precise cause of the demyelination of axons with resulting nerve damage that occurs with cobalamin deficiency remains unknown.

Higher animals and plants are unable to synthesize the vitamin and rely on the activity of microorganisms for their provision. The only source for man is food of animal ori-gin, and it is present in virtually all animal tissues, eggs, and


dairy products. Folates are found in most foods. The United States Institute of Medicine has published a recom-mended daily allowance (RDA) for folate of 400 µg of dietary folate equivalents (DFE) for adults (600 µg DFE for pregnant women) with a tolerable upper limit (UL) of 1000 µg from supplements or fortified foods. The RDA for vitamin B12 is 2.4 µg in adults (2.6 µg in pregnant women) with no current data to support a recommendation for a tolerable UL (Institute of Medicine, 1998). Dietary insuf-ficiency is worldwide (Dawson and Waters, 1994). Several countries have mandated folic acid supplementation of wheat or flour. The main stores of both nutrients are in the liver. Cobalamin deficiency is usually due to malabsorption or, in vegans and severe lacto-vegetarians, malnutrition.

Malabsorption of cobalamin results from:

� deficiency of IF in the gastric juice. Physiological amounts of cobalamin cannot be absorbed unless bound to IF. Pernicious anemia is due to an autoimmune gastritis in which the IF-producing parietal cells are destroyed. Resection of the stomach has the same effect;

� lack of gastric acid, achlorhydria, may prevent proper digestion of animal foods, and the vitamin may not be released to combine with IF;

� disease or resection of the terminal small bowel, the site of absorption of the cobalamin-IF complex. Crohn’s disease, ulcerative colitis, tuberculosis, and severe celiac disease are the usual causes;

� utilization of the vitamin by excessive bacterial flora in the gut, usually due to jejunal diverticulitis.

Deficiency of folate usually results from:

� malnutrition. This is by far the most common cause worldwide;

� malabsorption, due to disease of the proximal small bowel. Celiac disease nearly always causes folate deficiency;

� increased requirement, as in pregnancy and chronic hemolytic states (e.g., sickle-cell anemia).

Reference IntervalsFor vitamin B12, a reference interval of 200–900 ng/L (148–664 pmol/L) embraces most assays though some have a lower limit of 150 ng/L. Values below but within 20% of the lower end of the interval are usually considered to be in an indeterminate area (British Committee for Standards in Haematology (BCSH) 1994a,b,c).

The interval for serum folate is 2–15 µg/L (5–34 nmol/L) and for red cell folate is 160–600 µg/L (362–1360 nmol/L). For red cell folate determination, a whole blood hemoly-sate is assayed followed by calculation of the red cell folate using the hematocrit. This conversion is needed to allow for the effect of anemia. There is considerable variation in the assay results of both serum and whole blood folate by different methods. Up to ninefold differences in folate concentration, especially marked at low levels, with both serum and whole blood, have been noted (Gunter et al., 1996), and this is reflected to a certain extent in the vari-ability of the reference intervals offered by manufacturers. This may be partly due to variation in the population upon which the reference interval has been based but is also due to assay methodology, especially with regard to sample preparation. All manufacturers state that their ref-erence intervals are for guidance only and that users should determine their own. Because of these differences, the result of the assay needs to be interpreted, whether normal, indeterminate, or reduced, both for the clinician and for the external quality assurance programs.

Clinical Applications � Macrocytic anemia. Deficiency of either vitamin leads

to the production of erythrocytes with an increased mean corpuscular volume. Anemia develops later. Vita-min deficiency is associated with megaloblastic changes in the bone marrow characterized by erythro-blasts with immature nuclei relative to their cytoplasm. With severe vitamin deficiency, the leukocyte and platelet count also fall.

� Neuropathy. Damage to nerves, peripheral neuropa-thy, occurs with cobalamin deficiency. With progres-sive deficiency, damage to the spinal cord occurs (subacute combined degeneration of the cord).

� Psychiatric changes, mental impairment, and even dementia may occur with cobalamin deficiency, and depression may accompany folate deficiency.

� Infertility can occur with either deficiency. � Intestinal investigations. Because of the particular

sites for the absorption of the vitamins, assessment of the patient’s vitamin status may help in the differ-ential diagnosis and management of intestinal disorders.

� Homocysteinemia. It can be seen from Fig. 1 that deficiency of either vitamin may lead to an increase in plasma homocysteine. Folic acid can reduce the plasma level even when it is normal (Daly et al., 2002). Hyper-homocysteinemia is associated with thrombotic and vascular disease (Scott and Weir, 1996; Quinlivan et al., 2002), and assessment of the folate status may need wider application than in the investigation of blood disorders.

FIGURE 1 Interaction of cobalamin and folate in DNA synthesis.


Limitations � The serum B12 concentration depends not only upon

the amount in the stores but also upon the concentra-tion and turnover of the serum cobalamin binders, the transcobalamins. A low-serum B12 concentration may be found without tissue depletion in folate deficiency, pregnancy, and the malignant disease of the bone mar-row plasma cell myeloma. It is therefore necessary to review the results of vitamin B12 and folate assays together. In folate deficiency, the folate concentration falls much more than the B12, whereas in B12 deficiency, the low-serum B12 is usually accompanied by a normal serum folate and a red cell folate that is normal or only mildly reduced (see Table 1).

� A “falsely” normal or raised vitamin B12 concentration may occur in myeloproliferative diseases and with a marked leukocytosis.

� Methotrexate and folinic acid cross-react with folic acid and can interfere with the assay of serum folate.

� Human anti-mouse antibodies, generated by therapy or diagnostic procedures, can interfere with the immuno-logic stage of some assays, depending on the prelimi-nary treatment of the sample.

� A single serum folate measurement must be interpreted with care since the concentration can vary under the influence of physiological factors such as diet. In addi-tion, a state of negative folate balance, without tissue depletion, is common in acute illnesses, and a low serum folate may be found in 30% or more of hospital patients.

� A “falsely” low red cell folate is common in B12 defi-ciency since this is required to build up the intracellular folates.

� Young red cells have a higher content than older cells, and an increase in reticulocytes may cause the red cell concentration to be normal when there is tissue deficiency.

� A blood transfusion may give a falsely normal result. � The red cell concentration may be normal with acute

folate deficiency. This is particularly seen in intensive care units.

� A case can be made for either the serum or whole blood folate to be assayed but, because of the limitations of each, it would seem reasonable to assay both or to assay the whole blood whenever the serum folate is low. The practice of assaying whole blood and converting to red cell folate using the hematocrit without correction for the serum folate concentration can give falsely elevated results in patients with tissue depletion who have recently started folate therapy.

Assay TechnologyVitamin B12

This is measured using IF in place of the antibody used in an immunoassay.

The type of assay is referred to as competitive protein binding. Semi and fully automated non-radioisotopic sys-tems have largely superceded the original radioisotopic manual kits though the principle of the assay remains the same. In these assays, a known amount of labeled vitamin B12 is diluted with the B12 in the test sample, which is first extracted from the serum binders. A volume of the mixture is bound to the specific binding protein, IF, which is added in an amount insufficient to bind all the labeled B12. The bound is separated from the free and its label measured. The reaction signal is inversely related to the vitamin B12 concentration in the test material. Vitamin B12 radioim-munoassays (RIAs) (O’Sullivan et al., 1992) have been described but are not available commercially. In assays employing IF as the binding agent, an immunologic step is commonly used in setting up the competitive binding con-ditions whereby an anti-IF is attached to a solid phase via an anti-Ig antibody (Wallac, Bayer, Beckman). The Wal-lac AutoDelfia assay system may be taken as an example. In this, the microplate wall is coated with anti-mouse IgG. In the first incubation, anti-IF IgG binds to the coated wall. In the second, vitamin B12 labeled with tracer (europium) and that in the sample bind to added IF, and the B12-IF complexes compete for sites on the solid phase anti-IF antibody. Unbound B12 is removed by washing the plates. Enhancement solution releases the bound lanthanide ions to chelate with an organic ligand to give a fluorescent sig-nal (see Fig. 2). An advantage of this type of system is that anti-IF antibodies that may be present in the sample do not interfere with such an assay.

The extraction of vitamin B12 from its bindersThe binders can be denatured by heat at an alkaline pH or by raising the pH to 12.0 or higher. Human anti-mouse antisera are also denatured at this high pH. The denatured protein will give a varying amount of nonspecific binding (NSB) which may be excessive with sera in certain diseases such as chronic myelogenous leukemia. In solid-phase systems, this would yield a falsely low result. Dithiothrei-tol (DTT) reduces NSB. Conversion of all cobalamin to the cyano (vitamin B12) form is usually undertaken at this stage since the affinities of the coenzymes for IF differ.

Binding agentPorcine IF is used in purified form or its specificity achieved by attachment to IF antibody or addition of cobinamide, a cobalamin analog, which preferentially binds to non-IF cobalamin binders rather than to IF. The subsequent adjustment of the pH to that appropriate for the later IF-binding stage is especially important. In solid-phase systems, the IF can be carried on the wall of the reaction tube, microparticles, beads, paramagnetic parti-cles, or on a glass fiber matrix. Recombinant human IF has been produced (Fedosov et al., 2003). The key properties of this preparation were identical to those of native IF. This offers the potential for large-scale production of human IF for analytical and therapeutic purposes.

TABLE 1 Comparision of Typical Results of Vitamin B12 and Folate Assays in Vitamin B12 and Folate Deficiency

DeficiencySerum B12

VitaminFolate

Red CellFolate

Vitamin B12 ↓ ↓ (↓) N(↑) N(↓)Folate N(↓) ↓ ↓ ↓ ↓(↓)

Arrows in brackets indicate less common finding: N=normal


LabelThe label is either isotopic, CN(57Co)cobalamin, or che-miluminescent (acridinium ester or alkaline phosphatase [AP] /dioxetane phosphate), or fluorescent (AP/4-methy-lumbelliferyl phosphate or europium).

CalibratorsThe calibrators are made using cyanocobalamin and range from 0 to 2000 ng/L (1476 pmol/L). With automated sys-tems, a master rather than a full set of calibrators may be used for individual runs. The zero calibrator corrects for NSB. It is important that the protein content of the cali-brators should be as similar to that of the test sample as possible. A WHO B12 standard is available from the National Institute for Biological Standards and Control (NIBSC—see STANDARDIZATION AND CALIBRATION for address and web site). Its use should reduce differences between methods in their reference intervals. However, a multi-laboratory study showed that all methods then in use, although giving a wide scatter of values when assaying normal and pernicious anemia sera, were able to give meaningful clinical results (International Committee for Standardization in Hematology (ICSH), 1986). A method for the preparation of calibrators, approved by the Interna-tional Committee for Standardization in Hematology, has been published (Nexo et al., 1989).

Separation of free and bound B12

In semi and fully automated systems, the vitamin B12 bound to IF is separated from the free B12 by washing or magneti-zation of the solid phase. In liquid systems, charcoal or cel-lulose is used to remove unbound B12. The amount of labeled vitamin B12 that is bound is usually estimated and this is inversely related to the concentration in the sample.

Serum folateThe principle and procedure of the assay are the same as for vitamin B12. Anti-mouse IgG may be used to attach anti-folate binder IgG to a solid phase (e.g., Beckman, Wallac, Bayer). Ascorbic acid or DTT is used to maintain

the folate in the reduced, stable form. β-Lactoglobulin from cows’ milk is the binding agent commonly used and is more reliable than porcine serum. The correct pH is important at the binding stage. At pH 9.3, the affinity of folic acid and methylfolate for the binder is the same. Nonradioactive labels are in common use. The tracer in radioactive kits is 125I folic acid. Because the emissions of this and of 57Co can be readily distinguished and because the pH for folate binding is satisfactory for IF-B12 binding, dual assays for the simultaneous determination of cobala-min and folate have been popular.

CalibratorsMost methods use folic acid calibrators since methylfolate is less stable. This may cause some underestimation of the serum concentration. An International whole blood stan-dard is available from NIBSC.

Red cell folateThe affinity of the binder for folates varies with the num-ber of glutamate residues. It is necessary therefore to hemolyze the sample to release the folates and convert them to one form. For reproducible results, complete con-version to a monoglutamate is required. Adequate dilution of the red cells, a pH between 3 and 6, ascorbic acid to preserve the reduced folate and a deconjugase, provided by the plasma in the diluted whole blood sample, are needed for conversion, reduction, and preservation of the folate. Inadequate lysis and deconjugation give falsely low results. Dilutions of 1 in 20 or greater in fresh ascorbic acid solu-tion and incubation in the dark at room temperature for 30–60 min are suitable. The final ascorbic acid concentra-tion recommended by manufacturers varies from 0.2 to 1.1%. The higher concentration is the more appropriate, and kits using the weaker dilutions are generally associated with higher results. It is essential to the accuracy of the final result that the manufacturer’s instructions for hemo-lysate preparation and storage are followed fully. The hemolysate is assayed in the same way as a serum sample, correcting the result for the dilution and hematocrit of

FIGURE 2 CPB assay for vitamin B12.


the whole blood sample. With severe anemia, adjustment for the low hematocrit can contribute to imprecision.

Desirable Assay Performance CharacteristicsPrecision for within batch assay should be <5% and for between batches <10%. The best precision is required at the lower end of the reference interval, and the amount of labeled vitamin B12 added should be such that about 40–50% of it is bound at this level. Accuracy should be con-trolled by the standardization of calibrators against the vitamin B12 and whole blood folate international standards. It has been shown previously that, for vitamin B12, the mean of all methods in external proficiency programs is close to the true concentration (Dawson et al., 1987) though, with changing technology, this can no longer be presumed. Reagent stability over a reasonable period is important for the laboratory carrying out small numbers of tests.

Systems that can accept a variety of anticoagulated spec-imens as well as serum are beneficial.

With fully automated analyzers, errors are most likely to be limited to the pre- and post-analytical stages. These may be dilutional, for example in the preparation of the red cell lysate, or transcription errors. Consideration therefore needs to be given to sample labeling and tracing features. The use of barcoded labels reduces the possibility of transcription faults in addition to faster patient data input. Adequate interfacing of analyzers to a host com-puter should allow bidirectional transfer and matching of patient identification and results. Linkage to an electronic transfer system reduces the chance of error at the request receipt and result delivery stages. In addition, automation allows faster throughput of specimens and in some systems allows both continual and random access.

Types of SampleSerum is usually used for vitamin B12. Plasma from EDTA-anticoagulated blood may be used, though with liquid sys-tems the concentration may be slightly lower than in serum. Heparinized plasma and plasma containing ascor-bate and fluoride should be avoided.

Serum or plasma may be used for folate determinations. Anticoagulated blood is used for red cell folate.

Frequency of UseAssay of serum B12 and folate is common and less so for red cell folate. These together with the serum ferritin assay (below) are the most commonly used assays in hematology. This has resulted largely from the change in clinical prac-tice to a non-investigative profiling approach (Waters and Seal, 2001).

IF ANTIBODYPernicious anemia is an autoimmune disease. Circulating antibodies to IF can be detected in the majority of patients. Two types have been described. Type I (blocking anti-body) prevents the attachment of B12 to IF, and type II prevents the attachment of IF or the IF-B12 complex to the

ileal receptor. Antibodies to gastric parietal cells are found in the sera of most patients but are nonspecific.

Reference IntervalMethods are essentially qualitative, giving a positive or negative result, or at best are semiquantitative. However, they are mentioned here because of the unique relation-ship between measurement of serum B12 and detection of these autoantibodies.

Clinical ApplicationsAntibodies to IF are found in 60–75% of patients with per-nicious anemia, though not in those with the juvenile form. The incidence increases with the duration of the disease. Together with a low serum B12 concentration, they can be taken to indicate pernicious anemia. Most importantly, finding this combination obviates the need for further investigation, in particular vitamin B12 absorption studies, which are inconvenient for the patient (collection of full 24 h urine), involve administration of radioisotope (57Co oral dose) and are expensive.

Limitations � IF antibodies can rarely be found in patients with thy-

roid disease, diabetes mellitus, and myasthenia. � Serum from patients receiving vitamin B12 therapy may

give false-positive results when testing for type I antibody.

Assay TechnologyThe detection of type I antibodies is based upon the inhi-bition of vitamin B12 binding to IF by the test serum. The sera are incubated with IF immobilized on to a solid phase. The IF-cobalamin complex is then separated and 57Co-cobalamin added. If present, type I antibodies bind to the IF and reduce the radioactivity taken up by the solid phase. The results with test sera are compared with negative and positive sera provided by the manufacturers. RIA and enzyme immunoassay methods capable of detecting both type I and type II antibody (Conn, 1986; Waters et al., 1989) or type II alone (Sourial, 1988) have also been described. The recombinant IF (Fedosov et al., 2003) men-tioned above has reagent potential, either as binder or label, in the demonstration of IF antibodies.

Desirable Assay Performance CharacteristicsA balance between sensitivity and specificity has to be found. The former may be enhanced by increasing the con-centration of serum but this may lessen specificity unless prior removal or neutralization of B12 binders in the sample is done (Nimo and Carmel, 1987). However, even in the most optimized conditions, antibodies have not been found in more than 75% of patients with pernicious anemia.

The incidence of type II antibodies is at least as high as that of type I (Conn, 1986; Waters et al., 1993). The anti-bodies do not always coexist, so a system capable of detect-ing both antibodies is desirable.


Type of SampleSerum.

Frequency of UseInfrequent but increasing. Increasing difficulties are expe-rienced with carrying out differential in vivo radioactive methods for determining the patient’s ability to absorb vitamin B12. Consequently, the use of this simple, noninva-sive, diagnostic test for pernicious anemia, despite its limi-tations, has gained popularity. Addition of IF antibody detection to the repertoire of immunoassay analyzers capa-ble of measuring vitamin B12 is a logical development.

FERRITINFerritin is the main iron-storage protein, providing a reserve of iron for heme (hemoglobin) synthesis. It con-sists of a 450 kDa protein, forming a hollow shell into which iron atoms may come and go. It is found in all tis-sues, with high concentrations in the liver, spleen, and bone marrow. Ferritins from different tissues show het-erogeneity (isoferritins). Those of the liver and spleen are basic, and those of other tissues and of some tumors are acidic. In contrast to tissue ferritin, that found in serum consists of a number of glycosylated isoferritins of rela-tively low iron content. Nevertheless, the concentration of serum ferritin may be directly related to body iron stores. This was first demonstrated by Addison et al. (1972).

One-third of iron stores are made up of another protein–iron complex: hemosiderin. This is composed of aggre-gates of ferritin molecules where the iron cores have become detached from their protein shells. Hemosiderin is not water soluble and is demonstrated by cytochemical techniques. The hemoglobin concentration is maintained by utilization of recycled iron from senescent red cells and, when necessary, from the iron stores. Iron may be mobi-lized from both storage forms but is more accessible from ferritin. Iron-deficiency anemia develops when the stores are exhausted.

Clinical ApplicationsThe iron status of an individual may be assessed by a complete blood count to determine whether anemia is present and, if confirmed and there is doubt whether iron deficiency is responsible, by determining the level of iron stores. This can be done by aspirating bone marrow and staining it for hemosiderin. However, it is more practicable and less invasive to determine the serum fer-ritin concentration because this usually relates to the tis-sue stores. Each µg/L of serum ferritin is roughly equivalent to 8 mg of stored iron. Ferritin is therefore useful in detecting iron deficiency (low concentrations) and iron overload, e.g., due to hemochromatosis (high concentrations).

Reference IntervalMost results from adult males fall within an interval of 15–300 µg/L. Those from females vary more with age, with pre-menopausal concentrations being lower than postmenopausal.

Children tend to have lower concentrations than adults, and the detection of childhood iron deficiency is more difficult. The lower end of the intervals for males proposed by manu-facturers vary from 10 to 25 µg/L and for females from 3 to 17 µg/L. Nearly all manufacturers state that their reference ranges are for guidance only and that users should determine their own. Blood donation and iron supplementation need to be considered when doing this (Ledue et al., 1994). The vari-ation in reference intervals has been lessened by wider use of the WHO international standard available from NIBSC.

Limitations � Although assay results below 10 µg/L indicate iron defi-

ciency, there are a number of situations where the serum concentration does not equate so directly with the stores.

� Because ferritin is an acute-phase protein, the concen-tration can be raised in response to inflammation, and infection and figures up to 50 µg/L may be encountered in these diseases, even when there is iron deficiency.

� Concentrations above 300 µg/L suggest iron over-load, but in addition to inflammatory disorders, arti-ficially high concentrations may be encountered in acute and chronic liver disease and in malignant diseases.

� In symptomatic idiopathic hemochromatosis, concen-trations between 2000 and 4000 µg/L may be encoun-tered. However, in the early stages, the serum ferritin may be normal when evidence of iron accumulation can be observed by an increase in the serum iron con-centration and transferrin saturation. This is particu-larly important to remember when screening relatives for this condition.

� Current iron therapy gives falsely raised results. � Blood transfusion, due to the introduction of hemoglo-

bin iron, raises the serum ferritin concentration.

Assay TechnologyCompetitive binding and two-site immunometric assays using radioactive labels have been largely superseded by immunometric assays using nonradioactive labels, pri-marily based on fluorescence or luminescence. These involve sandwiching the ferritin in the sample between two layers of antibody. Solid-phase immobilized anti- ferritin antibodies react with the ferritin and are then washed. Subsequent addition of excess-labeled antibody allows binding of this tracer in direct proportion to the ferritin content of the sample. The solid phase may be the reaction tube itself, beads, glass fiber carriers, or para-magnetic particles. In these assays, the antigen–antibody complex is identified by measuring the activity of peroxi-dase or AP. Addition of substrate leads to color, fluores-cence or luminescence, proportional to the antigen concentration of the sample.

Immunometric assays are sensitive and can detect con-centrations of 0.2 µg/L ferritin. Serum proteins, in particu-lar anti-immunoglobulin antibodies, which are to be found in 10% of individuals, may inhibit the binding of ferritin to the solid phase and affect systems using labeled antibodies. This can be a source of serious error producing falsely


lowered or elevated ferritin values. Some manufacturers incorporate species immunoglobulins in the conjugate preparation to block any cross-reaction. The kit protocol should be scrutinized for information concerning cross-reaction. In practice, it is very difficult to identify samples from patients falling into this category, but cross-reaction should be considered when an unexpectedly high or low result is obtained.

In competitive binding RIAs, the ferritin in the sample competes with 125I-labeled ferritin for a limited number of antibody-binding sites. The level of radioactivity bound is inversely related to the concentration of anti-gen in the sample. After incubation, the bound and free ferritin are separated by precipitation of the antigen–antibody complex with a second antibody, an antiglobu-lin. The sensitivity of RIA is about 6–10 mg/L, which limits its use in the detection of iron deficiency. In gen-eral, competitive RIAs give lower results than immuno-metric assays.

The ferritin used as immunogen and for the calibrators should be of the same isoferritin and subunit composition as serum ferritin. Liver ferritin is almost identical and gives practically 100% cross-reactivity, whereas spleen ferritin is less so, with about 90% cross-reactivity. Some kits use monoclonal antibodies, which further enhance specificity. Standardization should be against the WHO standard.

A “high-dose hook” effect, in which high concentrations of ferritin give the same signal as much lower concentra-tions, may occur; especially in single-incubation assays. Where an assay is known to be affected in this way samples should be tested at two dilutions. However, in practice, it is sufficient for high concentration samples, or samples with results out of accord with the clinical expectation, to be repeated at a higher dilution. This does not appear to be a problem with most of the present systems.

Desirable Assay Performance CharacteristicsBecause serum ferritin assays are required to reflect an extremely wide range of concentrations, careful consider-ation of the relationship between the analytical error involved and clinical classification is important. A dose response carrying maximum sensitivity and minimum error over the range of concentrations relevant to the pur-pose for which the assay is intended is desirable. Precision, within batch and between batch, should be 5% or less.

Absence of any “high-dose hook” effect (see above) is important.

Types of SampleSerum. Anticoagulated plasma may also be used with some systems, but care must be taken and the manufacturer’s protocol carefully scrutinized for information on this.

Frequency of UseCommon and increasing due to the profiling approach mentioned above (see FREQUENCY OF USE of vitamin B12 and folate assays).

TRANSFERRINIn the plasma, iron is bound to a β-globulin, transferrin, which is indicative of the total plasma iron-binding capac-ity. One milligram of transferrin binds 1.4 mg of iron.

Reference Interval2.0–3.0 g/L (radial immunodiffusion).

Clinical ApplicationsTransferrin is normally one-third saturated with iron. In iron deficiency, the saturation is decreased and the transfer-rin concentration elevated before the appearance of anemia. The measurement of the serum iron and transferrin concen-trations, and the derivation of the percentage saturation of the latter, are of value in screening for hemochromatosis (see FERRITIN above). In the early stages of iron overload, a raised saturation of >50% in women and of >60% in men is found.

Limitations � Estrogens (pregnancy, oral contraceptives) cause an

increase in the transferrin concentration. � The transferrin concentration and its saturation are

reduced in inflammatory and malignant diseases. In hypoproteinemia, both the concentration and its satu-ration are reduced.

Assay TechnologyThe measurement of transferrin is conventionally carried out by immunological techniques such as radial immunodif-fusion and nephelometry. There is generally a good correla-tion between the results of chemical and immunological assays.

Desirable Assay Performance CharacteristicsThe usefulness of the assay is reduced by the lack of uni-formity in calibration.

Type of SampleSerum.

Frequency of UseInfrequent as an individual measurement.

TRANSFERRIN RECEPTORSThe uptake of iron by immature red cells, and many other proliferating cells, involves the binding of transferrin to spe-cific receptors on the cell surface (transferrin receptor [TfR]). Transferrin delivers its iron to these receptors. Since the major use of iron is for hemoglobin production, most TfR molecules are found on the erythroid precursor cells. During the iron transfer process, TfR molecules are cleaved from the cell surface to join a serum pool (sTfR). Having the highest concentration of TfR, erythropoietic cells are the main contributors to this pool. Assay of sTfR therefore


provides an indirect measure of total TfR and reflects either the cellular need for iron or the rate of erythropoiesis.

Reference IntervalSoluble receptor 8.7–28.1 nmol/L (Klemow et al., 1990).

Altitude and ethnic origin can affect the reference inter-val (Allen et al., 1998). These authors have reviewed cali-bration and reference intervals.

Clinical ApplicationsIn theory, the estimation of the reticulocyte count together with the sTfR concentration should give the best guide to the level of effective erythropoiesis. As iron stores are exhausted, the TfR concentration rises as the erythropoietic system responds. Unlike serum ferritin, sTfR concentrations are not influenced by inflammation or infection. sTfR assay should therefore be considered in anemic patients whose dif-ferential diagnosis includes iron deficiency and anemia of chronic disease, being raised in the former but normal or only slightly raised in the latter. Increased concentrations are also found in other causes of hyperplastic erythropoiesis (e.g., hemolytic anemia, β thalassemia, and polycythemia), whereas reduced concentrations occur in hypoplastic condi-tions (e.g., aplastic anemia, chronic renal failure, and post-transplant anemia). It has been suggested that the sTfR assay may lack specificity; however, when expressed in combina-tion with the serum ferritin concentration, the results are more informative (Punnonen et al., 1997; Means et al., 1999; Flowers and Cook, 1999; Suomimen et al., 2000).

LimitationsMalignant lymphoid disorders may markedly increase the serum concentration.

Assay TechnologyCirculating TfRs may be detected by enzyme-linked and chemiluminescence immunoassay using dual monoclonal antibodies.

Desirable Assay Performance CharacteristicsAntibodies may react differently to free and transferrin-bound receptors, giving widely differing values (Trowbridge, 1989). Convergence in antibody action would be of benefit.

Type of SampleSerum. Anticoagulated plasma may also be used with some systems, but care must be taken and the manufacturer’s protocol carefully scrutinized for information on this.

Frequency of UseInfrequent but likely to increase. As more immunoassay systems offer this assay, its application will rise, particularly where the instrument software provides the option of link-ing the sTfR and serum ferritin concentrations, thus facili-tating the adoption of an algorithm approach.

ERYTHROPOIETINMaintenance of the red cell volume is required to ensure an adequate oxygen supply to the tissues. The mechanism link-ing tissue oxygen delivery with red cell production is con-trolled mainly by EPO. EPO is a glycoprotein with a molecular mass of 30.4 kDa (Kendall, 2001). It is secreted by the liver in the early stages of fetal development and thereaf-ter 90% of production is taken up by the kidneys according to the concentration of oxygen in the blood. Lower oxygen lev-els result in increased secretion of EPO and vice versa. Under-production of EPO will therefore result in anemia (e.g., in end-stage renal failure), whereas overproduction produces erythrocytosis. The pharmacological use of recombinant human EPO (rHuEPO) in the management of patients with renal disease is now well established (Kendall, 2001).

Reference IntervalThe range encountered in normal adult subjects lies between 10 and 30 µ/mL. Babies less then 3 months old have lower EPO levels than adults. The EPO response to hypoxia is lowest in the most immature neonates. Normally a stable level is achieved by 3 months of age and through to adult-hood. No sex-related differences have been demonstrated, and levels are unaffected by the menstrual cycle. There is a diurnal variation with the highest levels at night. In preg-nancy, the level progressively increases (Kendall, 2001).

Clinical ApplicationsThe level of EPO is raised in anemia, including aplastic, iron-deficiency, and hemolytic anemias. EPO levels are depressed in patients with kidney disease, causing anemia, and low concentrations may give an early warning of kid-ney transplant rejection. Some tumors produce EPO, and in these cases, the concentration may be used as a tumor marker to monitor the effectiveness of treatment. EPO can be used to monitor AIDS patients undergoing Zidovu-dine (AZT) therapy. An increased concentration confirms that the anemia associated with AZT therapy is due to red cell hypoplasia or aplasia.

The EPO assay is useful when investigating polycythe-mia. In the primary form (polycythemia vera), overpro-duction of red blood cells occurs in the presence of EPO levels which are normal or significantly lower than in the secondary forms. The secondary polycythemias may be divided into those producing a raised EPO appropriate to the level of hypoxia (e.g., cardiac or pulmonary insuffi-ciency or high affinity hemoglobins) and those with an inappropriate rise (e.g., hypersecretion from tumors or cysts). Around 93% of primary and secondary polycythe-mias can be differentiated using measurement of EPO illustrating the value of the assay (Kendall, 2001).

Limitations � EPO levels are increased by anabolic steroids, during

pregnancy and in otherwise clinically normal persons following hemorrhage.

� EPO levels may be reduced in chronic infections, rheu-matoid arthritis, AIDS, and in the anemias associated with prematurity and hypothyroidism.


Assay TechnologyRIAs using purified human urinary EPO were developed originally. The introduction of rHuEPO obviated the need to use the native form for antibody and tracer production since results obtained from assays using the recombinant source were identical to those from established methods. Relatively few non-isotopic methods have been developed. Those which have use either monoclonal or affinity-puri-fied polyclonal anti-EPO antibody and are reported to be highly sensitive and quicker than radioassay (Kendall, 2001).

Desirable Assay Performance CharacteristicsThe assay should be standardized against one of the inter-national reference preparations available from NIBSC.

Type of SampleSerum. EDTA plasma may give slightly lower results.

Frequency of UseUsually carried out at specialist centers.

Thrombosis and HemostasisMany immunological tests, especially ELISA methods, for the determination of platelet and coagulation factors, acti-vators and inhibitors of the hemostatic system, and fibri-nolytic products are now available. The diagnosis of coagulation factor deficiencies is conventionally made by clotting tests because functional activity, which is the important measurement, and antigen concentration do not always parallel each other. An ELISA has, however, been developed for determining the functional activity of vWF. In general, immunologic techniques have been of greater value in conditions of hypercoagulability than in bleeding disorders. Those for routine use include tests for a heredi-tary thrombotic state or predisposition: proteins C and S and antithrombin and tests for evidence of DIC such as FDPs, D-dimer, and fibrinogen assays. The other tests

below are mainly for specialized centers and research. The assay technology for each is listed in Table 2.

Kits and reagents for the following assays are available from several companies, including Diagnostica Stago, Immuno, Dade, and Sysmex. It is essential to follow their instructions carefully. Because of the dynamic state of the hemostatic system, the blood sample should be taken, with-out stasis or frothing on collection, into a plastic container and it should never be taken from indwelling catheters.

A list of reference materials, including international standards, for many of the coagulation factors and the fol-lowing assays are available from NIBSC (see STANDARDIZA-TION AND CALIBRATION).

ThrombophiliaPROTEINS C AND SProteins C and S are vitamin K-dependent physiological anticoagulants produced in the liver. They act together as one of the main regulators of hemostasis by neutralizing activated coagulation factors V and VIII. Deficiency of protein C or S can be congenital or acquired. Congenital deficiency of protein C accounts for 5–10% of venous thromboses in younger persons, deficiency of protein S about half this number. Most cases are heterozygotes. Massive thromboses and skin necrosis may occur in the homozygous neonate. Factor V Leiden represents an even more common cause of heritable hypercoagulability. The variant coagulation factor is resistant to degradation by protein C, and as such, patients have an increased risk of venous thrombosis. Genetic analysis remains the preferred test for identification of the factor V Leiden variant. Screening for thrombophilia has recently been reviewed (Stegnar, 2010), and guidelines on appropriate testing are also available (Baglin et al., 2010).

Reference IntervalProtein C and S, 70–140% of average normal (protein C: 4 mg/L, protein S: 35 mg/L), by functional (coagulation) assays. Reference intervals appropriate to the local method and population should be determined. The effect of gen-der, age, and pre-analytical variables such as anticoagulant therapy (for both), pregnancy (for protein S), and oral con-traceptive use (for protein S) should be taken into account (Jennings and Cooper, 2003).

Assay TechnologyEarly immunoelectrophoretic methods have been super-seded by more sensitive ELISA and RIA techniques (Table 2) based on monoclonal antibodies.

LimitationsProtein C functional activity and antigen concentration can differ. An immunologic assay is required to identify variant proteins when the functional test gives a low result. About 60% of protein S is bound in the plasma and has to be freed before immunoassay to give the rele-vant measurement. Protein C and S levels are reduced by

TABLE 2 Immunological Tests in Hemostatic Disorders

ELISA PF4, factors VII, VIII, IX, X, vWF, proteins C, S, β-TG,fibrinopeptide A, plasminogen, tPA, D-dimer, heparin cofactorII, antithrombin

Immunodiffusion Factors VII, X, XI, prothrombin, fibrinogen, proteins C, S

Electroimmunodiffusion Factor IX, fibrinogen, vWF, proteins C, S, antithrombin

Immunoelectrophoresis Factors X, XII, XIII, fibrinogen, vWF, plasminogen, FDPs, proteins C, S

IRMA PF4, factors VIII, IX, vWFRIA β-TG, PF4, vWF, fibrinogen,

antithrombin, proteins C, SParticle agglutination β-TG, tPA, FDPs, D-dimer,

antithrombin


warfarin anticoagulant therapy (which has to be changed to heparin before assay), liver disease, DIC, and post-surgery. Additionally, these factors may be consumed in the setting of recent thrombosis. The level of protein S is about half of normal in pregnancy and about one-fifth in neonates. Individuals with thrombotic disease may have levels only just below the reference interval. As such, evaluation in the setting of recent or acute thrombosis may be uninformative or yield misleading results. Unfor-tunately, these and other thrombophilia-related assays are often overused in clinical practice. Performing these studies at an inappropriate time may lead to unnecessary repeat testing. The utility of performing a complete thrombophilia panel when there is no intent to alter clin-ical management has also been called into question. Evi-dence-based recommendations on the appropriate timing and selection of thrombophilia investigations have been proposed (Merriman and Greaves, 2006).

Desirable Assay Performance CharacteristicsInternational standards (NIBSC 86/622—protein C and 93/590—protein S) should be used to reduce differences in results from different methods.

Type of SampleCitrated plasma.

Frequency of UseCommon.

ANTITHROMBINAntithrombin (previously known as antithrombin III) is a glycoprotein which primarily inhibits thrombin but also inhibits several activated coagulation factors. Its action is enormously enhanced by heparin. Deficiency can be con-genital or acquired. Congenital deficiency accounts for about 3% of venous thrombotic disease in young persons, when a level of 40–50% of normal may be expected. The level in neonates, normally about 60–80% of the adult value, can be 30% or less in an affected individual. Two types of congeni-tal deficiency have been demonstrated. Functional activity and antigen levels are reduced in parallel in type I (quantita-tive), whereas the two levels are discrepant in type II (qualita-tive). Acquired deficiency can be caused by thrombosis, liver disease, sepsis, nephrotic syndrome, heparin therapy, and treatment with L-asparaginase (Jennings and Cooper, 2003).

Reference Interval0.125–0.39 g/L (70–130% of average normal).

LimitationsThe antigen concentration may be normal when function is reduced (type II congenital deficiency). Therefore, the functional assay is more important and must always be available. High values are seen after myocardial infarction.

Assay TechnologyAntithrombin can be measured by ELISA, RIA, electroim-munodiffusion techniques, and latex immunoassay (Table 2). Crossed immunoelectrophoresis can be used to identify molecular variants (Jennings and Cooper, 2003).

Desirable Assay Performance CharacteristicsThe assay should be standardized against an International Standard (NIBSC 93/768 [human plasma] or 96/520 [concentrate]).


Frequency of UseUncommon but increasing.

CoagulationCOAGULATION FACTORSAs mentioned above, functional activity of the clotting factors is of primary importance. However, the diver-gence between activity and antigen concentration can help in the differential diagnosis. For example, von Willebrand’s disease in which activity and antigen concentration decline in parallel (type I) or are discrepant (type II).

Reference IntervalA range of 50–200% of the reference population covers the normal range of most coagulation factors.

LimitationsFactor VIII and vWF are acute-phase proteins and may be transiently raised into the normal range by stress, exercise, pregnancy, and estrogen-containing contraceptives.

Because of the wide reference interval, it is essential to pool many normal plasmas (at least six) for the control sample or to use a commercial preparation for this purpose.

Desirable Assay Performance CharacteristicsAn international standard for vWF is available from NIBSC (00/514). NIBSC also supply standards for most other coagulation factors.


Frequency of UseFunctional assays are common; antigen concentration uncommon except in specialized centers.


FIBRINOGENFibrinogen (factor I) is a dimeric glycoprotein of molecu-lar weight 340 kDa. The end product of the coagulation cascade, thrombin, is a powerful proteolytic enzyme that transforms fibrinogen into fibrin monomers by removing amino acids from the ends of two of its chains. The cleaved products are called fibrinopeptides, and the large residual portions are fibrin monomers which combine and give a visible, strong fibrin polymer.

Reference Interval2.0–4.0 g/L.

Clinical ApplicationsHypofibrinogenemia commonly occurs as part of a wider coagulation abnormality. Deficiency or dysfibrinogen-emia (production of an abnormal form) are rare congenital events. An immunologic test is required when a functional test shows a low level; this may show a normal or even raised level in dysfibrinogenemia. The association of high levels with cardiovascular disease (Meade, 1997) may be an important reason for its estimation.

LimitationsFibrinogen is an acute-phase protein elevated by stress, infections, etc.; these factors need to be avoided when sam-ples for study of vascular disease are taken.

Immunological measurements are influenced by FDPs in patients with increased fibrinolysis, and their presence needs to be determined before interpreting the fibrinogen assay result.

Desirable Assay Performance CharacteristicsThere is still a wide variation in the reference intervals used in different laboratories. International Standards (NIBSC 98/614 [concentrate] or 96/520 [plasma]) are available and should help to resolve this problem.

Type of SampleCitrated, platelet-poor plasma.

Frequency of UseOccasional.

Evidence of Disseminated Intravascular Coagulation (dic)FIBRINOGEN/FIBRIN DEGRADATION PRODUCTSActivation of the coagulation pathway results in the gen-eration of thrombin, the enzyme that converts fibrinogen to fibrin. Cross-linked fibrin monomers are the main clot component. Fibrinolysis, the physiological mechanism for clot degradation, is activated by fibrin formation.

The fibrin clot is degraded by plasmin (the enzyme end point of fibrinolysis) resulting in the release of FDPs into the circulation. The test for FDPs is used to detect DIC in patients with hemostatic failure. Raised concentrations are also found in thrombotic diseases and with severe tissue damage (pneumonia, surgery).

Reference IntervalNormal: <10 mg/L (one in five dilution, latex agglutination).

Assay TechnologyIn the latex agglutination test, latex particles are sensitized with antibodies to FDPs D and E. A suspension is mixed on a glass slide with diluted serum, and aggregation indi-cates their presence. Serial dilution gives a semiquantita-tive result.

Type of SampleSerum. Whole blood is collected into a special tube that contains thrombin and an antifibrinolytic agent and allowed to clot at 37 °C for 30 min.


D-DIMER TESTThe D-dimer test is for fibrin derivatives containing linked D fragments, specific products of the lysis of fibrin clots by plasmin. The plasmin degradation of fibrin releases FDPs (see above), which contain D-dimers. Since the presence of D-dimer in the circulation results from fibrinolysis in response to activation of the coagula-tion system, the detection is an indirect measure of thrombin generation and subsequent clot formation. D-dimer is raised in DIC, deep vein thrombosis, and pul-monary embolism. The concentration is not influenced by fibrinogen, and the test is more specific than FDP for intravascular coagulation.

Reference IntervalNormal: <200 µg/L (latex agglutination)*.<400 µg/L (ELISA)*.

LimitationsMethods lack standardization and the local cutoff level must be determined prior to use. D-dimers are also found in pregnancy and old age and as a result of inflammation.

Desirable Assay Performance CharacteristicsThe assay must be specific, showing no cross-reaction with fibrinogen, FDPs, or fibrin monomers, otherwise an over-estimation of the D-dimer level may result.

*Cutoff levels vary and must be determined for each assay.


Assay TechnologyLatex agglutination or ELISA can be used (Table 2). Assays are based on the monoclonal antibody recognition of D-dimer epitopes. ELISA is the gold standard method but the latex agglutination test, which is similar to that for FDPs, is more popular for practical considerations includ-ing its adaptability to modern coagulation analyzers with the ability to easily run multiple coagulation tests in paral-lel from the same sample and avoidance of washing steps with decreased testing time requirements. Turbidimetry or nephelometry may be used for quantification.

Types of SampleCitrated plasma or serum (follow manufacturers’ instructions).


Immunodetection MethodsLEUKEMIA AND LYMPHOMA ANALYTESNormal and neoplastic cells may be identified by the pres-ence of antigens on the cell membrane, in the cytoplasm and by intranuclear enzymes. When the nature of a neo-plastic cell cannot be determined from morphology and cytochemistry, it is essential to employ immunopheno-typic analysis to facilitate the diagnosis of hematological malignancies (Craig and Foon, 2008; European Working Group on Clinical Cell Analysis, 1996).

Clinical ApplicationsMonoclonal antibodies that aid recognition of different types of acute leukemia and lymphoproliferative disorders are required. Becton Dickinson, Beckman Coulter, Dakocyto-mation, and Serotec provide such reagents with instructions for their use. The initial selection is usually based on those suggested by routine hematological tests, e.g., complete blood count (CBC) and blood film morphology. Wood et al. (2007) have reviewed the optimal reagents for the flow cyto-metric investigation of hematological malignancies.

A suitable panel for the investigation of acute leukemia should include cluster of differentiation (CD) reagents that allow identification of primitive cells (leukemic blasts) and assignment of cellular lineage. Investigation of lym-phoproliferative disorders requires reagents that distin-guish between B- and T-cell disease, establish clonality, and enable identification of various B- or T-cell disorders with distinct immunophenotypes. A summary of the com-mon markers employed for the investigation of hemato-logical malignancies is provided in Table 3.

Recent advances in flow cytometry technology allow leukemia patients’ response to therapy to be assessed by “minimal residual disease” (MRD) monitoring. Multipa-rameter flow cytometry is used to identify a “leukemia-associated phenotype” at diagnosis. These same markers are subsequently used to identify residual leukemic cells post treatment. MRD monitoring represents a powerful application of flow cytometry that may provide useful

prognostic information for individual patients. As such, this technology is likely to become increasingly important in the future. The use of MRD monitoring by flow cytom-etry has been extensively reviewed (Kern et al., 2010; Cam-pana and Coustan-Smith, 2004).

Assay TechnologyTwo immunologic detection techniques are used to label and identify cells of malignant hematologic diseases: immu-nofluorescence and immunoenzymatic labeling (BCSH, 1994a,b,c). Immunofluorescence, when used in conjunc-tion with flow cytometry, allows both qualitative and quan-titative assessment of cell populations within a suspension through concurrent multiparametric analysis of individual cells. Immunoenzymatic labeling is used to determine the presence or absence of antigens in the cells on glass micro-scope slide preparations from cytologic and histologic specimens. It has the added benefit of providing a concur-rent visual correlation. Several of the antigens that are most commonly assessed with either technique are not unique to a single cell type. As such, different cell subpopulations within the same suspension, cytologic preparation, or his-tologic section may have antigens in common. This lack of specificity for cell type adds to the complexity of both anal-ysis and interpretation. It further illustrates the importance of using a panel of markers to arrive at a diagnosis. Appro-priate review of the findings with each analytic method requires an individual with expertise in their interpretation. However, an important advantage of this complexity is that unique signatures of abnormal cells may be exploited, not only for subclassifying malignant cell populations but also for monitoring treatment response and disease recurrence in subsequent studies, particularly if a cell population has unique characteristics. It is important to note that correla-tion with clinical history and specimen morphology remains essential for drawing appropriate conclusions from any analysis. A brief discussion of each of these techniques and how they are used in hematology follows.

Immunofluorescence on cell suspensionsThis technique is most commonly used to examine antigen expression patterns in leukocyte populations when evaluat-ing hematologic malignancies or clonal disorders. However, applications for analysis of red blood cell antigens, platelet

TABLE 3 A Summary of the Consensus Markers Used for the Initial Immunophenotypic Investigation of Hematological Malignancies

LineageCommonly Used CD Markers

Myelomonocytic cells CD7, CD11b, CD13, CD14, CD15, CD16, CD33, CD34, CD45, CD56, CD117, HLA-DR

B cells CD5, CD10, CD19, CD20, CD45, Kappa, Lambda

T cells and natural killer cells CD2, CD3, CD4, CD5, CD7, CD8, CD45, CD56

Plasma cells CD19, CD38, CD45, CD56, CD138

Adapted from data presented in Wood et al. (2007).


glycoproteins, and bound autoantibodies and alloantibodies have also become common. Additionally, cellular fetal hemoglobin (Hb F) assessment by flow cytometry has been used as an alternative to the Kleihauer-Betke acid elution method for quantifying fetal-maternal hemorrhage (Davis et al., 1998). Such determinations assist prevention of mater-nal Rh alloimmunization, a condition with potentially dev-astating consequences for a subsequent pregnancy.

For leukocyte studies, red blood cells are lysed in order to limit assay interference, and white blood cells are incu-bated with an optimal dilution of fluorescently labeled monoclonal antibodies. The majority of antigens evalu-ated are surface membrane antigens. If cytoplasmic or nuclear antigen analysis is desired, special techniques may be used to render cell membranes permeable so that reagent antibodies may enter the cells. Multiple antibodies with different fluorescent labels may be used in a single tube. For microscopy, the cells are resuspended, mounted on a glass slide, and sealed. Alternatively, the cells are resuspended in diluent and examined by flow cytometry.

Flow cytometry using immunofluorescence labeling allows for simultaneous quantitative and multiparametric analysis of cell populations within a suspension. The parameters assessed include cell size, internal complexity (e.g., nuclear lobularity and cytoplasmic granularity), and antigenic expression pat-terns of individual cells. The technique uses multiple fluoro-chrome-conjugated monoclonal antibodies (or fluorophores) to evaluate a variety of potential antigenic targets and thereby determine a complete antigenic profile or “immunopheno-type” of a cell population. There are typically several different cell populations in any suspension, and each population can be evaluated concurrently with a single analysis. In the assay, individual cells are aligned, single file, within a laminar sheath of fluid and passed through a channel with a narrow aperture known as a flow cell. As each cell passes a common interroga-tion point, incident light strikes the cell. Light passing into each cell is scattered while strategically positioned detectors capture information such as the angle of scatter and intensity of incident light.

Morphologic characteristics of each cell are suggested by its light scattering properties including forward scatter (FSC) (indicative of cell size) and side scatter (SSC) (indic-ative of internal cellular complexity). Granulocytes, for example, have high internal complexity due to their nuclear segmentation and high granule content. Lymphocytes, by contrast, are mostly small in size, mononuclear and are generally lacking in granules. Monocytes, though compa-rable in size to granulocytes, have less granularity. These differences become readily apparent when plotting FSC against SSC on a scatter plot.

Additional distinctions can be achieved through immu-nodetection using fluorochrome-conjugated monoclonal antibodies to potential antigens of interest. In the flow cytometer, incident light striking a fluorophore results in emission of light of a different wavelength. A series of dichroic mirrors (beam splitters) and filters direct any emitted light to additional detectors that register a signal for peak emission wavelengths of a particular fluorophore.

A number of fluorochromes are available including fluo-rescein isothiocyanate, R-phycoerythrin, and allophyco-cyanin, to name just a few. The choice of fluorophores requires consideration of their excitation and emission

wavelengths. Excitation wavelengths must be matched with that of the light source of the flow cytometer. The overlap of emission spectra or peak emission wavelengths of different fluorophores used should be minimized in order to maximize the resolution for multiparametric detection. A number of commercial derivatives with enhanced performance are also available. Although mod-ern flow cytometers are capable of simultaneously assess-ing up to 10 fluorochromes (Wood, 2006) or more, achieving greater numbers introduces additional complex-ity when optimizing the resolution. As such, four- and six-color methods remain in common use. In order to evaluate additional antigens, aliquots of the suspension are split into different test tubes, each incubated with different sets of antibodies of interest. By selecting a few common anti-bodies that will be used in multiple tubes, a composite characterization of each cell population can be inferred.

The brightness of a measured reporter signal correlates with the density of antigen expression on the cell being evaluated. By comparing against isotypic (negative) con-trols on a logarithmic scale, fluorescence intensity may be interpreted as negative, dim (slightly brighter than the negative control), moderate (one decade brighter than the negative control), or bright (two decades brighter than the negative control) for each antigen assessed. The relative intensity may support a diagnosis for some malignancies with characteristic variations, e.g., dim surface immunoglo-bin expression that is typical of B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL) or bright expression of CD11c and CD20 typical of hairy cell leukemia, another B-cell malignancy. Experienced opera-tors can readily recognize these patterns.

In addition to those fluorophores that are used for anti-body conjugation, others like propidium iodide (PI), a DNA intercalating agent, may aid in determining the via-bility of the cells in the suspension. Since intact cell mem-branes would be expected to exclude this marker, viable cells should register as low fluorescence intensity. Viability is an important marker of the success of an analysis.

Measured parameters may be displayed as individual histograms but are more commonly displayed as a series of two-dimensional scatter plots using a logarithmic scale for fluorescence intensity. Scatter plots each compare two measured parameters for each cell analyzed. Thus, data points or “events” within a scatter plot reflect the analyzed cells. The analyst moves a crosshair to establish quadrants on each scatter plot that define the boundaries of positive and negative and that define populations for the software to quantify. Programs can also be set to render standard sets of scatter plots for routine studies that analysts can fine-tune with each new analysis.

Perhaps the most significant advantage of flow cytome-try is the ability to use the software for post-analytic manipulation of the views of event data in order to aid interpretation. Typically, an initial scatter plot is gener-ated using parameters that allow distinction between nor-mal leukocyte populations, e.g., FSC vs SSC. A comparison of CD45 expression vs SSC achieves even greater resolu-tion between the populations of lymphocytes (very bright CD45 and low SSC), monocytes (bright CD45 and mod-erate SSC), granulocytes (weaker CD45 and high SSC) and, importantly, blasts (weak CD45 with minimal SSC).


Recognition of abnormal patterns aids in establishing an initial impression and may dictate the additional scatter plot comparisons that would be helpful for a more thor-ough evaluation.

In the process known as gating, the analyst may select, within the initial scatter plot, groups of events for which further parameter comparisons are desired. The gated population may then be viewed exclusively in a series of two-dimensional scatter plots that compare any two parameters assessed in that tube. Useful assessments include the intensity of staining and co-expression patterns (i.e., cells that express both antigens being evaluated). By reviewing each scatter plot, the analyst can verify the pres-ence of all expected cell populations and the absence of any abnormal or unexpected cell populations.

As an example, the analyst may gate on the lymphocyte population and then evaluate T-lymphocytes by compar-ing plots of expected pan-T-cell antigens, e.g., CD3, CD2, CD5, and CD7. A comparison of CD4 and CD8 expres-sion in this population should demonstrate the presence of distinct populations of helper and regulatory T cells (CD4+) and cytotoxic T cells (CD8+) and allow determi-nation of the CD4:CD8 ratio, which is normally approxi-mately 1–2:1 in adults. This information is also useful for determining peripheral blood T-cell counts in patients with HIV as a marker of disease progression and treatment effect. The CD4:CD8 ratio may also be perturbed by other clinical conditions.

Similarly, the B-cell population can be evaluated by comparing plots of expected pan-B-cell antigens, e.g., CD19, CD20, CD22, and CD79a. A comparison of sur-face immunoglobin (kappa and lambda) expression in lym-phocytes normally demonstrates two B-cell populations, each with one of these two antigens. The ratio of kappa expressing cells to lambda expressing cells (kappa:lambda ratio) is normally 1–2:1.

Significant derangements in either of these patterns would indicate the presence of a clonally restricted cell population. Clonality is typically a feature of malignancy, though occasionally small clonal populations may be seen in reactive conditions or in conditions of undetermined clinical significance. Malignant cell populations may also show aberrant antigen expression as evidenced by the acquisition of markers that are not expressed in their nor-mal (nonmalignant) counterpart or the loss of expected normal markers. Analyzing patterns of antigen expression in a clonal population aids in further classification and with some malignancies may have prognostic significance, e.g., CD38 and/or ZAP-70 expression as negative prognostic markers for CLL.

Gated events can be quantified and compared to total events as a percentage. In this manner, a differential of the percentages of both normal and abnormal cells (as charac-terized by their respective immunophenotypes) can be readily generated. In some instances, abnormal patterns or subpopulations may not be apparent until after the initial gating. Software programs also allow a process known as back-gating whereby a subpopulation discovered after the initial gating can itself be gated and then referenced back to the original complete scatter plot using color graphics to facilitate localizing the subpopulation within the population of total events. A more detailed discussion

of the principles of this technology is covered in the chap-ter on flow cytometry (see MICROSPHERE-BASED MULTIPLEX IMMUNOASSAYS: DEVELOPMENT AND APPLICATIONS USING LUMINEX ® XMAP ® TECHNOLOGY).

The ability to determine a complete immunophenotype represents a distinct advantage over isolated microscopic examination. Several hematologic malignancies may have cytologic or histologic characteristics that render them nearly indistinguishable from other hematologic malig-nancies or, in some cases, even from reactive nonmalig-nant conditions. Additionally, the ability to quantify cell populations, particularly blasts (precursor hematopoietic cells), has clinical significance in diagnosing acute leuke-mias and in subclassifying chronic myeloid neoplasms.

Findings should always be interpreted in the context of both the clinical history and the cell morphology as seen with cytologic or histologic preparations of the specimen from which the cell suspension was derived. A working knowledge of the wide variety of characteristic immuno-phenotypic patterns that may be seen and their respective clinical implication is necessary for proper interpretation.

The use of flow cytometry for immunophenotyping also has distinct advantages over the immunoenzymatic slide-based methods (discussed next). Flow cytometry on cell suspensions is a multiparametric analysis, whereas slide-based immunoenzymatic methods are practically limited to one antigen label per slide. Flow cytometry cur-rently offers a slightly broader selection of available anti-bodies. Flow cytometry is also rapid requiring only about 2–4 h to perform. By contrast, routine histologic tissue processing and labeling typically requires 12–24 h. Immu-noenzymatic labeling is often performed after an initial morphologic assessment of a slide that has been stained with the routine hematoxylin and eosin (H&E) stain. The preliminary review of morphology allows a more directed approach for selecting appropriate immunoenzymatic labels but increases the typical turnaround time by another 12–24 h.

Both immunodetection methods are more rapid than the, often complementary, fluorescence in situ hybridiza-tion and cytogenetic analysis, which require a few days and up to 2 weeks, respectively. Each of these methods may be required for complete classification of some hematologic malignancies. However, in the setting of newly presenting acute leukemia, the ability to obtain a relatively rapid pre-liminary lineage assessment with flow cytometry can allow for critical early therapeutic determinations (Weir and Borowitz, 2001).

Immunoenzymatic labeling on slidesImmunoenzymatic labeling of cellular material on glass microscope slides allows for enhanced characterization of cells by light microscopy. Depending on whether histo-logic (tissue) material or cytologic (cellular) material is used, the terms immunohistochemical stain or immunocy-tochemical stain is used, respectively. Other descriptors include immunoperoxidase stain, if a peroxidase enzyme method is selected. The original term immunostain is less specific and may also refer to other nonenzymatic staining methods. The method is complementary to immunophe-notyping in cell suspensions. Each technique has its own advantages.


A glass microscope slide with cellular material of inter-est may be obtained from either cytologic preparations such as cell blocks or from histologic preparations obtained from paraffin-embedded tissue that has been sectioned by a microtome. Less commonly, thin smears, touch imprints, or cytocentrifuge (cytospin) specimens may be used. Slides are incubated with a monoclonal antibody to an antigen of interest. Reporting is achieved with antibody-conjugated enzymatic complexes that cleave a chromogenic substrate. The stain is evaluated by light microscopy. The most com-mon enzymes used for labeling include AP and horserad-ish peroxidase. Typical chromogen substrates include 3,3′ diaminobenzidine, which upon cleavage by peroxidase produces a dark brown precipitate that is insoluble in alco-hol, and nitro-blue tetrazolium/5-bromo-4-chloro-3′-indolyphosphate (NBT/BCIP), which produces an intense, insoluble black-purple precipitate upon cleavage by AP.

Both direct (one-step) and indirect (two-step) methods have been used. In the simple and rapid direct method, glass slides are incubated with an antibody that corre-sponds to the antigen of interest and that is conjugated to an enzymatic complex. The addition of a chromogenic substrate, upon cleavage, produces the visible color change. In the more commonly employed indirect method, an unconjugated primary antibody detects a corresponding antigen of interest and a secondary antibody is used to rec-ognize the primary antibody. The secondary antibody may be either directly conjugated to an enzyme or conjugated to a linker molecule, such as biotin. In the latter case, another molecule with affinity for the linker molecule is bound to an enzyme allowing for even greater signal amplification. With the biotin method, the enzyme com-plex could be conjugated to avidin, a tetrameric biotin-binding protein with high affinity. Other avidin-like molecules are available including streptavidin and degly-cosylated avidin (NeutrAvidin), which have the advantage of less nonspecific binding. The direct method requires labeling a primary antibody for each antigen of interest. The indirect method can avoid this requirement by using unlabeled primary antibodies raised from the same species. A species-specific secondary antibody is used for recognition.

As an example, in the immunoalkaline phosphatase anti-alkaline phosphatase (APAAP) method, the slide is first incubated with the monoclonal antibody for the antigen of interest, then with anti-mouse immunoglobulin, and finally with the immunoalkaline phosphatase anti-alkaline phosphatase (APAAP). The cell spread is developed in a solution of naphthol As-Mx phosphate, levamisole, N,N-dimethylformamide, and FAST red TR salt. A hematoxy-lin counter stain is used to enhance the visibility of negative cells and to provide contrast for the primary stain.

Positive and negative controls should be run concur-rently with the tissue of interest. Positive controls should be selected from appropriate tissue specimens that are pro-cessed in similar fashion to the tissue of interest. Several automated systems are currently available and can facilitate the staining process. However, each stain method devel-oped will require some degree of trial and error to establish an optimal staining protocol. Factors affecting optimal staining include the quality of tissue or cell procurement technique, specimen processing and preparation, fixation

methods, antibody dilution, temperature, pH, and the length of incubation for antibodies and other reagents.

When this technique is performed on sections from pre-served paraffin-embedded tissue, formalin and other fixa-tives used in the preservation process alter tissue proteins through cross-linking. Protein cross-linking may block epitopes resulting in diminished reactivity or a false-nega-tive stain. The process of antigen retrieval can uncover epitopes thereby significantly enhancing their recognition by antibodies. This restoration of immunoreactivity may be accomplished through a variety of modifications of either one of two general techniques.

Heat-induced epitope retrieval (HIER) involves the application of heat to the paraffin-embedded tissue sec-tion. This is typically performed in a buffered (acidic or basic) aqueous retrieval solution over a short period of time with several protocols calling for about 10–20 min. Buffers such as citrate, TRIS-EDTA, and EDTA have been employed, and heating devices such as microwaves, pressure cookers and steamers, water baths, and autoclaves have all been used for this purpose (Norton et al., 1994). Deparaffinization may be required for optimal results with some methods. Routinely it is desirable to avoid detergents that completely solubilize membranes since membrane antigens are the most common antigens of interest in hematopoietic tissues.

Proteolytic-induced epitope retrieval (PIER) is an alternate method that uses enzymes such as proteinase k, trypsin, chymotrypsin, pepsin, and others to disrupt bonds and uncover epitopes. However, caution is advised when optimizing reagent dilutions and selecting an incubation period since some epitopes of interest may otherwise be destroyed. Additionally, there may be consequent tissue degradation that limits microscopic evaluation.

The HIER and PIER techniques may also be combined. The necessity and success of antigen retrieval as well as the optimal method to employ will vary by the antigen of interest, the antibody used, and the tissue source.

Performing nuclear or cytoplasmic stains for immuno-cytochemistry (cytology preparations) may require mem-brane permeabilization to allow entry of reagents into the cells. This is not requisite for immunohistochemistry since cells will have been sectioned during slide preparation.

An individual with experience in interpreting immunos-tains must evaluate the pattern, intensity, and cellular loca-tion of staining (e.g., nuclear, cytoplasmic, membranous, Golgi, etc.). Correlation with morphology is also readily apparent. As with immunophenotyping on cell suspen-sions, alterations in normal patterns may indicate abnor-mality or clonality. Also similar to immunofluorescence methods, potential antigenic targets may be expressed with differing intensity in different cell populations, and this variation may be described semiquantitatively with descriptors like strongly positive, positive, moderate, weak, or negative.

Slide-based histologic immunodetection methods have an advantage over cell suspensions in that concurrent mor-phologic assessment reveals cellular relationships in con-text. Similarly, cytologic preparations allow concurrent visual assessment of the actual cells. However, immunoen-zymatic slide methods are typically limited to one or at


most two (double-stain) antigen labels on a given slide preparation. Therefore, interpretation requires a compos-ite assessment that compares expression patterns on mul-tiple different slides (one for each antigen of interest). Double stains are best performed with antigens directed at different subcellular components, e.g., nuclear vs cytoplas-mic antigens for ease of distinguishing a positive reaction and co-expression.

Whereas cell suspension-based methods require fresh viable material, slide-based methods can be performed on preserved and archival material. In some instances, even necrotic material may be acceptable, as antibodies may be capable of labeling the membranous outline of dead or dying cells. Slide methods also allow direct visual morpho-logic correlation that can provide qualification of findings or that may occasionally force the analyst to reject findings.

LimitationsImmunofluorescence on cell suspensionsA key limitation of this method is the requirement for viable and intact cells. It is imperative that viability be assessed with each analysis as significant cell damage may result in a false negative or undercounting of the abnor-mal population. This is particularly true for delays in specimen transport or analysis of aggressive or highly proliferative neoplasms. Malignancies like large B-cell lymphomas, plasma cell myelomas, and some acute leu-kemias are more susceptible to this problem. Loss of cell membrane integrity, an indicator of cell death, can be assessed with the fluorochrome PI, a DNA intercalating agent that is excluded from viable cells. Cells are gener-ally viable for up to 24 h at 4 °C except when the cell count is high. Cells from indolent malignancies like some small B-cell lymphomas may survive longer and have been detected as far out as 48–72 h post collection. Cells in cerebrospinal fluid (CSF) are more labile. The determination of whether or not to perform this analysis should be made early after sample acquisition. The use of special cell culture media may aid in prolonging viability.

Current analyzer technology allows analysis of sam-ples containing as low as 50,000 cells per sample (or less) with a minimum volume for each tube of 200–300 µL depending on the method. Some cell loss during pro-cessing is to be expected, and the required sample vol-ume should be sufficient for the preparation of multiple tubes. The typical requirement of 5 mL for whole blood or fluids is usually ample. With respect to the required concentration of antigen, the detection limit is depen-dent upon the choice of reagents, selected instrument parameters, and the thoroughness of optimization of the method. Most methods are capable of detecting cells with approximately 2000 molecules of the target antigen per cell, though amplification methods for detecting lower antigen densities have been achieved (Mavrangelos et al., 2004). The most commonly evaluated markers are normally sufficiently abundant on cell surfaces to permit detection at this level. However, studies of lower density markers may eventually yield other applications. With these considerations, the sensitivity typically allows

detection of an aberrant phenotype on as few as 1/10,000 cells or 0.01% (Borowitz, 2005).

Achieving the typical specimen requirements is usually not a problem for most evaluations. However, when run-ning specimens with a paucity of cells, such as normal CSF, blood with a very low concentration of white blood cells (leukopenia) or hemodilute bone marrow aspirates (those contaminated by peripheral blood during collec-tion), there may be insufficient diagnostic material for evaluation. As such, it may be advisable to perform an ade-quacy check by preparing a cytologic slide prior to running the assay on such specimens. This allows correlation through concurrent visual cytologic examination and can alert the analyst when a specimen may not be representa-tive or might yield a false-negative result. For specimens of limited volume for which adequate clinical history is pro-vided, selectively abridged panels of markers can be attempted and may provide limited information. Hodgkin lymphoma, a malignancy in which the diagnostic cells are few in number, cannot be excluded by this method. For this and other malignancies that are routinely underrepre-sented by flow cytometric analysis, tissue biopsy remains a mainstay of assessment.

Another significant limitation to this method is the lack of simultaneous direct morphologic correlation. So while it can be determined that an abnormal cell population is present, there is no way to view the architectural relation-ships of cells. Yet, this information may be critical to either diagnosis or prognosis. Thus, correlation with the visual examination of specimen morphology is still required. If the analysis is performed in a laboratory that is not in close proximity to where the morphologic examination will take place, it may be useful to create a slide from the specimen prior to analysis. In the absence of concurrent morpho-logic review, interpretations should reflect the need for correlation.

Suboptimal reagent performance or variation in the per-formance of different analyzers may impact analysis. Incon-sistency in gating technique may lead to poor reproducibility. Inappropriate panel selection may yield misleading results. Failure to recognize normal variation and common aber-rant patterns may also lead to misinterpretation.

Finally, the cost of analysis can still be fairly expensive depending on the number of antibodies used in the panel. Selective panels based on the cytology and clinical indica-tion can help to mitigate this cost.

Immunoenzymatic labeling on slidesImmunoenzymatic labeling methods suffer from the major limitation of assessing only a single antigen per slide prep-aration. Although double stain methods have been devel-oped, they are most practical when the antigens of interest are expressed in different parts of the cell, e.g., one nuclear and one cytoplasmic. Assessment of complete antigen expression patterns for a population of cells therefore requires the review of several slide preparations, which can make interpretation more challenging. With cytologic cell blocks and small biopsy material, there may be insufficient tissue to perform all of the stains desired. A selective panel or repeat collection may be necessary. With other cyto-logic methods, there may be only a single slide available for analysis.


Issues surrounding tissue procurement and processing also play a role in the quality of material being reviewed. Specimens that are inadvertently crushed during acquisi-tion or processing may introduce artifacts that render staining less interpretable. Antibody reactivity may vary with the type of fixative used, and fixation itself may destroy epitopes. Common fixatives encountered include formalin, glutaraldehyde, B5, Bouin’s, zinc formalin. In some cases, antibody reactivity may be enhanced with the antigen retrieval techniques described above. Despite improve-ments in antigen retrieval detection, sensitivity may remain limited and some antigens remain challenging to demon-strate with immunoenzymatic methods. With bone mar-row core biopsies, a decalcification step is required during tissue processing. Strongly acidic decal preparations used in this procedure may reduce or destroy antigenicity and thus create a weak or false-negative result (Fend et al., 2008).

Unintended reactivity may also lead to diagnostic con-fusion. Two such situations include endogenous reactivity and nonspecific binding. As an example, neutrophils con-tain the enzyme myeloperoxidase and thus may show endogenous reactivity in peroxidase-based enzymatic methods. This should not be interpreted as a positive result. They may be recognized by their morphology and the lesser intensity of the apparent stain. Endogenous AP activity may create the appearance of background staining. This effect may be reduced by pretreatment with an agent like levamisole, an AP inhibitor.

Antibody cross-reactivity resulting from partial or weak binding to sites on nonspecific proteins with structural similarity to the target antigen can result in strong back-ground staining that masks detection of target antigens. The use of monoclonal antibodies should reduce this phe-nomenon. Various blocking or quenching methods may also be employed to reduce such artifacts, with variable success.

Poor antibody potency or reduced enzymatic activity or improper dilutions may result in weak staining. Addition-ally, there may be greater subjectivity in the interpretation of staining intensity. Examination of concurrently run positive and negative controls should facilitate recognition of several of these problems. The large variety of available antibodies, commercial buffers, and other reagents neces-sitates some trial and error in order to optimize this detec-tion assay. Hsi describes a practical approach for such optimization (Hsi, 2001).

Types of SampleImmunofluorescence on cell suspensionsCell suspensions created from a variety of specimen types are acceptable. Whole blood and bone marrow aspirate samples collected in anticoagulant are common. Preserva-tive-free heparin permits other studies (e.g., cytogenetics) to be made, though EDTA may be acceptable if other studies will not be performed on the same sample. EDTA should be avoided if cytogenetic analysis will be performed on the same specimen. Other appropriate specimens include cell suspensions from fine needle aspiration biop-sies of solid tissues, body fluids (including CSF), or disag-gregated tissue from biopsy material (including bone

marrow core biopsies). The use of special cell media such as RPMI (developed at Roswell Park Memorial Institute) can help to maintain the viability of the cellular elements in the specimen, particularly for cytologic aspirates, tissue biopsies, and core bone marrow biopsies. There appears to be a lack of consensus on the optimal storage temperature for specimens. Room temperature is generally favored by many for fluids (typically stable for 24 h), though refriger-ated specimens may also be acceptable. Sample refrigera-tion is preferred for solid tissues in order to retard autolysis, particularly when adequate media to preserve viability are not readily available. However, freezing specimens is not appropriate. The variety of acceptable specimen types makes this technique equally useful in the clinical pathol-ogy laboratory and the anatomic pathology laboratory, where lymphoid malignancies may be more likely to first present.

Immunoenzymatic labeling on slidesGlass microscopy slides may be prepared from fixed histo-logic sections of paraffin-embedded tissue or cytologic cell blocks. Thin cytologic smears, touch imprints, and cyto-centrifuge (cytospin) fluid specimens may also be used, though material is generally insufficient or provides too few slides for examining multiple markers.

Frequency of UseImmunofluorescence on cell suspensionsUse is common in specialized centers and reference lab-oratories. Use is extending in general hospitals. The complexity of analysis typically requires dedicated, trained personnel for the technical components and a qualified individual with expertise in interpretation in the context of clinical history and morphology. Newer and smaller instruments have allowed migration of the method to hospital laboratories with sufficient testing volume.

Immunofluorescence labeling technology has also made its way into at least one routine hematology analyzer, though applications are limited to use as a complimentary method of platelet counting (using an antibody to CD61) and monitoring of T-cell counts with antibodies to CD4 and CD8. This appears to be practical in centers with siz-able HIV-positive patient populations that do not have the volume to justify on site complete flow cytometric immu-nophenotyping for other purposes.

Immunoenzymatic labeling on slidesVery common.

Desirable Assay Performance CharacteristicsImmunofluorescence on cell suspensionsAntibodies with the same CD number are not necessarily of equal specificity and stability.

Fluorochromes should optimally have narrow emission spectra with minimal overlap in peak emission wavelengths in order to optimize the resolution of each signal. Advice in this selection should be sought from a specialized laboratory.


Immunoenzymatic labeling on slidesOptimal assays use fixatives that do not significantly alter epitopes or for which there are successful antigen retrieval protocols. Lighter decalcification protocols can avoid the reduced antigenicity that may accompany the process. Pri-mary antibodies with good potency and specificity and enzyme complexes with good activity in the tissues being assessed should be sought. In addition to having well-titrated dilution protocols, the use of methods to limit endogenous reactivity and nonspecific binding help to ensure the optimal staining intensity.

Malarial ParasitesCLINICAL APPLICATIONSModern methods of travel and increased migration enable malaria to be detected in patients anywhere in the world. In addition to the clinical implications for the individual patient, there is a risk of transmitting malaria by blood transfusion. In the case of Plasmodium falciparum infection, the parasites should be quantified by morphological exam-ination of thick and thin blood films. This may be supple-mented by immunological, polymerase chain reaction (PCR), or fluorescence methods (BCSH, 1997).

Assay TechnologyMethods are available for direct detection of antigen or indirect detection of antibodies to malarial parasites. The most sensitive of the direct methods is based on PCR, which detects the parasite DNA. These suffer from high expense and complexity. Immunochromatographic and non-chromatographic antigen detection methods that allow both the rapid diagnosis of malaria and differentia-tion between species are also available.

Indirect methods for the detection of antimalarial antibod-ies can only be used to confirm the diagnosis since the anti-bodies are not detectable for some days following infection. Antigen-coated solid phase combined with a detection system of enzyme-labeled antigen is typical of the methods used.

Desirable Assay Performance CharacteristicsSince the introduction of a single malarial parasite to a host can lead to the disease, testing is required in both endemic and non-endemic countries. Ideally, tests need to be of high sensitivity and specificity, easy to perform and inexpensive.

Type of SampleUndiluted blood or plasma.

Frequency of UseVariable, depending on geographic location and popula-tion, and for the above reasons all laboratories should be able to detect parasites or forward the sample to a refer-ence center. It is recommended that all positive tests should be sent to a reference center for verification in all cases (BCSH, 1997).

HemoglobinopathiesNewborn and antenatal hemoglobinopathy screening programs have been established and are developing. Newborn screening is a qualitative process whereas mea-surement of various hemoglobin proportions, in particu-lar A2 and F, is required for antenatal screening. The programs are almost exclusively based on high-perfor-mance liquid chromatography and electrophoretic tech-niques. Hemoglobin F can also be measured using radial immunodiffusion (HbF QUIPlate Kit, Helena Laborato-ries) but this is used infrequently. Though not performed as frequently, the cellular content and distribution of hemoglobin F (pancellular or heterocellular) can be assessed by flow cytometry (immunofluorescence on cell suspensions) and may facilitate the diagnosis of heredi-tary persistence of fetal hemoglobin as well as distinction from other conditions for which there may be an eleva-tion in hemoglobin F (Hoyer et al., 2002). Antibodies to hemoglobin variants exist but are used mainly for research purposes.

References and Further ReadingAddison, G.M., Beamish, M.R., Hales, C.N., Hodgkins, M., Jacobs, A. and

Llewellin, P. An immunoradiometric assay for ferritin in the serum of normal subjects and patients with iron deficiency and iron overload. J. Clin. Pathol. 25, 326–329 (1972).

Allen, J., Backstrom, K., Cooper, J., Cooper, M., Detwiler, T., Essex, D.W., Fritz, R., Means, R.T., Meier, P.B., Pearlman, S., Roitman-Johnson, B. and Seligman, P.A. Measurement of soluble transferrin receptor in serum of healthy adults. Clin. Chem. 44, 35–39 (1998).

Bacon, B.R., Adams, P.C., Kowdley, K.V. and Tavill, A.S. Diagnosis and manage-ment of haemochromatosis: 2011 practice guidelines by the American associa-tion for the study of liver diseases. Hepatology 54, 328–343 (2011).

Baglin, T., Gray, E., Greaves, M., Hunt, B.J., Keeling, D., Machin, S., Mackie, I., Makris, M., Nokes, T., Perry, D., Tait, R.C., Walker, I. and Watson, H. Clinical guidelines for testing for heritable thrombophilia. Br. J. Haematol. 149, 209–220 (2010).

Bloom, A.L., Forbes, C.D., Duncan P.T. and Tuddenham, E.G.D. (eds), Haemostasis and Thrombosis, 3rd edn (Churchill Livingstone, Edinburgh, 1994).

Borowitz, M.J., Pullen, D.J., Winick, N., Martin, P., Bowman, W.P. and Camitta, B. Comparison of diagnostic and Relapse flow cytometry phenotypes in child-hood acute Lymphoblastic leukemia: implications for residual disease detec-tion: a Report from the Children’s Oncology group. Cytometry Part B (Clinical Cytometry) 68B, 18–24 (2005).

British Committee for Standards in Haematology, General Haematology Task Force. Guidelines on the investigation and diagnosis of cobalamin and folate deficiencies. Clin. Lab. Haem. 16, 101–115 (1994a).

British Committee for Standards in Haematology, General Haematology Task Force. Immunophenotyping in the diagnosis of acute leukemia. J. Clin. Pathol. 47, 777–781 (1994b).

British Committee for Standards in Haematology, General Haematology Task Force. Immunophenotyping in the diagnosis of chronic lymphoproliferative disorders. J. Clin. Pathol. 44, 871–875 (1994c).

British Committee for Standards in Haematology, General Haematology Task Force, Malaria Working Party. The laboratory diagnosis of malaria. Clin. Lab. Haem. 19, 165–170 (1997).

Brugnara, C. Reticulocyte cellular indices: a new approach in the diagnosis of ane-mias and monitoring of erythropoietic function. Crit. Rev. Clin. Lab. Sci. 37, 93–130 (2000).

Campana, D. and Coustan-Smith, E. Minimal residual disease studies by flow cytometry in acute leukemia. Acta Haematol. 112, 8–15 (2004).

Cavill, I., Macdougall, I.C., Gokal, R., Beguin, Y., Peters, T., Pippard, M., Schaefer, R. and Winearls, C. Iron management in patients on rHuEPO. Br. J. Renal. Med. 2, 6–8 (1997).

Chanarin. The Megaloblastic Anaemias 3rd edn (Blackwell Scientific Publications, Oxford, 1990).

Conn, D.A. Intrinsic factor antibody detection and quantitation. Med. Lab. Sci. 43, 48–52 (1986).

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