Table of contents :

An examination of the blood smear (or film) may be requested by physicians or initiated by laboratory staff. With the development of sophisticated automated blood-cell analyzers, the proportion of blood-count samples that require a blood smear has steadily diminished and in many clinical settings is now < 10-15%. Nevertheless, the blood smear remains a crucial diagnostic aid. The proportion of requests for a complete blood count that generate a blood smear is determined by local policies and sometimes by financial and regulatory as well as medical considerations. For maximal information to be derived from a blood smear, the examination should be performed by an experienced and skilled person, either a laboratory scientist or a medically qualified hematologist or pathologist. In Europe, only laboratory-trained staff members generally "read" a blood smear, whereas in the USA, physicians have often done this. Increasingly, regulatory controls limit the role of physicians who are not laboratory-certified. Nevertheless, it is important for physicians to know what pathologists or laboratory hematologists are looking for and should be looking for in a smear. In comparison with the procedure for an automated blood count, the examination of a blood smear is a labor-intensive and therefore relatively expensive investigation and must be used judiciously. A physician-initiated request for a blood smear is usually a response to perceived clinical features or to an abnormality shown in a previous CBC. A laboratory-initiated request for a blood smear is usually the result of an abnormality in the CBC or a response to "flags" produced by an automated instrument. Less often, it is a response to clinical details given with the request for a CBC when the physician has not specifically requested examination of a smear. For example, a laboratory might have a policy of always examining a blood smear if the clinical details indicate lymphadenopathy or splenomegaly. The International Society for Laboratory Hematology (ISLH) has published consensus criteria for the laboratory-initiated review of blood smears on the basis of the results of the automated blood count. The indications for smear review differ according to the age and sex of the patient, whether the request is an initial or a subsequent one, and whether there has been a clinically significant change from a previous validated result (referred to as a failed delta check). All laboratories should have a protocol for the examination of a laboratory-initiated blood smear, which can reasonably be based on the criteria of the International Society for Laboratory Hematology. Regulatory groups should permit the examination of a blood smear when such protocols indicate that it is necessary. There are numerous valid reasons for a clinician to request a blood smear, and these differ somewhat from the reasons why laboratory workers initiate a blood-smear examination. Sometimes it is possible for a definitive diagnosis to be made from a blood smear. Clinical indications for examination of a blood smear :

• anemia, unexplained jaundice, or both : in patients with anemia, physician-initiated examinations of blood smears are usually performed in response to clinical features or to a previously abnormal CBC. The presence of unexplained jaundice, particularly if unconjugated hyperbilirubinemia is also present, is an additional reason for a blood-smear examination. Laboratory-initiated examinations of blood smears for patients with anemia are usually the result of a laboratory policy according to which a blood smear is ordered whenever the hemoglobin concentration is unexpectedly low. This policy should be encouraged, since the consideration of the blood smear and the red-cell indices is a logical first step in the investigation of any unexplained anemia^ref. Initiating a smear as a reflex test also means that a further blood sample does not have to be taken for this purpose. Modern automated instruments impart valuable information about the nature of anemia. They provide not only a red-cell count, MCV, MCH, and the MCHC but also newer variables that give information that previously could be derived only from a blood smear. These variables usually include the RDW, which correlates on a blood smear with anisocytosis, and they may also include the hemoglobin-distribution width and the percentages of hypochromic and hyperchromic cells, which correlate with anisochromasia, hypochromia, and hyperchromia. A variety of histograms and scatterplots give a visual representation of red-cell characteristics. It may be possible to detect increased numbers of hyperchromic cells (spherocytes or irregularly contracted cells), small hyperchromic cells (microspherocytes), hypochromic microcytic cells, large normochromic cells (normally hemoglobinized macrocytes), and hypochromic macrocytes (either reticulocytes or dysplastic red cells). Despite this wealth of information, there are still morphologic abnormalities that are critical in the differential diagnosis of anemia and that can be determined only from a blood smear. Particularly important is the detection of variations in cell shape and of red-cell inclusions, such as Howell–Jolly bodies, Pappenheimer bodies, and basophilic stippling or punctate basophilia.
• hemolytic anemia : red-cell shape is of considerable diagnostic importance. Some types of hemolytic anemia yield such a distinctive blood smear that the smear is often sufficient for diagnosis. This is true of hereditary elliptocytosis (which is only infrequently associated with anemia; numerous elliptocytes and smaller numbers of ovalocytes), hereditary pyropoikilocytosis (striking poikilocytosis, with elliptocytes, ovalocytes, and fragments), and Southeast Asian ovalocytosis (moderate poikilocytosis, with the poikilocytes including several macro-ovalocytes). The presence of spherocytes is not diagnostically specific, since this may result from hereditary spherocytosis (numerous spherocytes (hyperchromatic cells with a regular outline)), or immune hemolytic anemia. Nevertheless, consideration of the clinical features, together with the results of a direct antiglobulin test, in patients with spherocytes will generally indicate the correct diagnosis. Microangiopathic hemolytic anemia resulting from cyclosporine therapy shows numerous red-cell fragments. Microspherocytes may be present in low numbers in patients with a spherocytic hemolytic anemia but are also characteristic of burns and of microangiopathic hemolytic anemia. The detection of a microangiopathic hemolytic anemia is of considerable clinical significance, since this type of anemia may indicate pregnancy-associated hypertension, disseminated cancer, chronic disseminated intravascular coagulation, the hemolytic–uremic syndrome, or thrombotic thrombocytopenic purpura; the latter 2 conditions both require urgent diagnosis so that appropriate management can be initiated. In microangiopathic hemolytic anemia, examination of the blood smear is also important to validate the platelet count, since red-cell fragments and platelets may be of similar size. Most automated instruments cannot make this distinction. A minority of automated instruments that measure both the size and the refractive index of small particles in the blood sample can make this distinction and can be used to exclude red-cell fragmentation; however, although the fragment "flag" on such instruments is sensitive, it is not specific. Hence, a blood smear is still advised for validation^ref. Blood-smear features similar to those seen in microangiopathic hemolytic anemia are also a feature of mechanical hemolytic anemia, such as that associated with a leaking prosthetic valve, and provide important evidence of this cause of hemolytic anemia. A blood smear is particularly important in the diagnosis of acute hemolysis induced by oxidant damage. The characteristic feature is the presence of keratocytes, or "bite" cells, "blister" cells, and irregularly contracted cells; the latter must be distinguished from spherocytes because of the quite different diagnostic significance. These irregularly contracted cells share with spherocytes the lack of central pallor but differ in that they have an irregular outline. Oxidant-induced hemolysis is most often seen in glucose-6-phosphate dehydrogenase (G6PD) deficiency but can also occur with other defects in the pentose shunt or in glutathione synthesis and when oxidant exposure overwhelms normal protective mechanisms. Oxidant damage may be exogenous, as in exposure to oxidant chemicals or drugs (most often dapsone), or endogenous, as in Wilson's disease^ref. G6PD deficiency affects millions of persons worldwide. A blood smear is important for the diagnosis of this condition, for 2 reasons. First, it is available far more rapidly than are the results of a G6PD assay and, when considered together with the patient's ethnic origin and clinical history, permits a provisional diagnosis. Second, a blood smear can suggest the diagnosis of G6PD deficiency even if a G6PD assay is normal. Normal G6PD activity may be found after acute hemolysis in G6PD-deficient persons in 2 situations. First, men of African or African-American ancestry who are hemizygous for the A– alloenzyme (which is present at normal levels in reticulocytes) can have normal G6PD levels because the reticulocyte count is high after hemolysis. Second, female carriers — for example, those who are hemizygous for the common Mediterranean variant of G6PD — can have a normal assay result after an acute hemolytic episode because the abnormal cells have lysed preferentially, leaving mainly the cells expressing the normal allele in the circulation. In both of these circumstances, the observation of a typical blood smear in an appropriate clinical setting is an indication to repeat the assay once the acute hemolytic episode is over. Other features may aid in the differential diagnosis of hemolytic anemia. For example, the presence of red-cell agglutinates usually indicates the presence of a cold agglutinin, and erythrophagocytosis is often a feature of paroxysmal cold hemoglobinuria
• macrocytic anemia :
• the blood smear is of great importance in the differential diagnosis of macrocytic anemias. For patients in whom there is a deficiency of vitamin B₁₂ or folic acid, the blood smear shows not only anisocytosis and macrocytes but also oval macrocytes and hypersegmented neutrophils. When the anemia is more severe, there may be marked poikilocytosis, with teardrop poikilocytes and red-cell fragments. Although these deficiency states are now usually recognized on the basis of assays of vitamin B₁₂ and folic acid, the blood smear remains important for two reasons. First, it permits a speedy provisional diagnosis, and initiation of appropriate treatment in severely anemic patients while assay results are pending. Second, occasionally there are patients with a clinically significant vitamin B₁₂ deficiency despite a normal assay result. This discrepancy occurs because much of the vitamin B₁₂ that is measured in the assay is bound to haptocorrin, whereas the functional vitamin B₁₂, which is bound to transcobalamin, contributes much less to the assay of total B₁₂. Similarly, acute folic acid deficiency sometimes develops in patients even though the total red-cell folate level remains normal. The observation of a blood smear that is typical of megaloblastic anemia despite normal assays is an indication that further investigation and a trial of treatment are needed. Liver disease and excess ethanol consumption are common causes of macrocytosis, with the blood smear usually showing round rather than oval macrocytes and lacking hypersegmented neutrophils; target cells and stomatocytes may also be present.
• in elderly patients, the myelodysplastic syndromes are an important cause of macrocytosis. Blood-smear features that may point to the diagnosis include anisocytosis, poikilocytosis, macrocytes, stomatocytes, hypogranular or hypolobulated neutrophils, blast cells, giant or hypogranular platelets, Pappenheimer bodies, and the presence of a minor population of hypochromic microcytic cells, leading to a dimorphic smear
• macrocytic anemia resulting from congenital dyserythropoietic anemia also yields a characteristic blood smear, with striking poikilocytosis. When macrocytosis is the result of hemolysis or recent blood loss, the blood smear shows polychromasia, which results from an increased reticulocyte count.
• microcytic anemia : the blood smear is generally less important. Red-cell indices and serum ferritin levels, sometimes supplemented by markers of inflammation, that are interpreted in the context of clinical features, permit the diagnosis of the majority of cases. However, it is important to note that the presence of Pappenheimer bodies and red-cell dimorphism in the sideroblastic anemias and of prominent basophilic stippling in cases of lead poisoning and in some types of thalassemia is diagnostically significant.
• hemoglobinopathy (sickle cell disease-dactylitis or sudden splenic enlargement and pallor in a young child or, in an older child or adult limb, abdominal or chest pain) and thalassemia: a blood smear is useful in the diagnosis and differential diagnosis of sickle cell disease, particularly if there is an urgent need for diagnosis and if the results of hemoglobin electrophoresis or high-performance liquid chromatography are not instantly available. Patients with sickle cell anemia (in which there is homozygosity for hemoglobin S) have anemia, but those with compound heterozygosity for hemoglobin S and hemoglobin C may have a normal hemoglobin level, and the condition thus may be confused with sickle cell trait if a blood smear is not examined. Consideration of the blood-smear features, of the hemoglobin level, and of the results of a sickle cell solubility test usually permits an accurate diagnosis^{ref1, ref2}. The blood smear of a compound heterozygote usually shows target cells, irregularly contracted cells, and boat-shaped cells but few classic sickle cells; typical hemoglobin SC poikilocytes (formed only when hemoglobin S and hemoglobin C are both present) are often seen. Sometimes the blood smear of a compound heterozygote shows only target cells and irregularly contracted cells and cannot be distinguished from the smear in hemoglobin C homozygosity; a positive sickle cell solubility test permits these conditions to be distinguished in an emergency situation (e.g., preoperatively). A blood smear is also important in the diagnosis of an unstable hemoglobin, with irregularly contracted cells and macrocytosis being characteristic of this condition; sometimes there is coexisting thrombocytopenia.
• thrombocytopenia(e.g. petechiae, abnormal bruising) or neutropenia(e.g. unexplained or severe infection) : falsely low platelet counts may be the result of small clots, platelet clumping, platelet satellitism, or abnormally large platelets. Fibrin strands indicate that thrombocytopenia is likely to be factitious. Underlying causes that may be revealed by the blood smear include the May–Hegglin anomaly,microangiopathic thrombopathies, and leukemias and lymphomas.
• thrombocytosis: high platelet counts should be confirmed microscopically with a blood smear; falsely high counts may be the result of other particles (red-cell fragments, fragments of leukemic cells, or fungi (e.g. Candida glabrata)) being counted as platelets^{ref1, ref2, ref3, ref4}. Also look for evidence of a myeloproliferative disorder, such as giant platelets, or an increase in the basophil count; the latter is not reliably detected by automated counters. A sudden, unexpected improvement in the platelet count also should be confirmed by blood-smear examination, since such an improvement may be factitious^ref
• lymphoma or other lymphoproliferative disorder (LPD)-lymphadenopathy, splenomegaly, enlarged thymus or bone marrow failure : blood smears must always be examined when there is unexplained leukocytosis, lymphocytosis, or monocytosisor when the flagging system of an automated instrument suggests the presence of blast cells. Depending on the instrument and the practice of the local laboratory, a flag for atypical or variant lymphocytes may also be an indication for examination of a blood smear, since this flag is sometimes indicative of the presence of blast cells. Low rather than high counts likewise are an indication for a smear, since they may be indicative of aplastic anemia, acute leukemia, hairy-cell leukemia, or infiltration of nonhematopoietic malignant cells into the bone marrow. The role of the blood smear in the diagnosis of leukemia and lymphoma is to suggest a likely diagnosis or range of diagnoses, to indicate which additional tests should be performed, and to provide a morphologic context without which immunophenotyping and other sophisticated investigations cannot be interpreted. For 2 conditions, Burkitt's lymphoma(basophilic vacuolated lymphoma cells) and acute promyelocytic leukemia(bilobed leukemic promyelocytes), a blood smear is of particular importance because it facilitates rapid diagnosis and specific treatment.
• myeloproliferative disorder (MPD)-splenomegaly, plethora, itching, weight loss
• suspicion of disseminated intravascular coagulation (DIC)
• acute or recent onset renal failureor unexplained renal enlargement, particularly in a child
• on retinal examination, hemorrhages, exudates, signs of hyperviscosity
• bacterialor parasiticdisease that can be diagnosed from a blood smear
• disseminated non-hematopoietic cancer-weight loss, malaise, bone pain
• general ill health, often with malaise and fever, suggesting infectious mononucleosisor other viral infection or inflammatory or malignant disease
• probable factitious results : members of the laboratory staff should always initiate a blood smear if an automated instrument produces a highly improbable result. Such results may be factitious, resulting from the accidental freezing or heating of the blood, from hyperlipidemia, or from the presence of cold agglutinins, a cryoglobulin, bacteria, or fungi. Factitious results also may stem from unusual characteristics of the blood cells or the plasma, such as a pseudo-neutropenia caused by a myeloperoxidase deficiency that occurs when the automated instrument employs a peroxidase reaction for the identification of neutrophils, eosinophils, and monocytes. Falsely low counts also may result from neutrophil or platelet clumping or from platelet satellitism.

RBC inclusions
• Howell-Jolly bodies : smooth, round purple staining remnants of nuclear chromatin due to incomplete nucleus expulsion

Aetiology :
• hemolytic anemia
• after splenectomy
• megaloblastic anemia (multiple)
They can be removed in spleen by pitting and culling

• chromatin dust : small red granules, smaller than Howell's bodies, sometimes seen at the periphery of stained erythrocytes
• Cabot's ring bodies : red-purple staining threadlike filaments in the shape of a ring or figure 8 in stained red blood cell, often in association with basophilic stippling. They may also appear as granules in a linear array rather than as complete rings. Cabot rings are thought to be microtubules from a mitotic spindle or remnants of the nuclear membrane.

Aetiology :
• severe anemia including megaloblastic anemia
• leukemia
• lead poisoning.
They are distinguished from the ring forms of Plasmodium by their larger size and by the absence of a red chromatin mass

• Heinz-Ehrlich bodies or granules : coccoid inclusion bodies resulting from oxidative injury to and precipitation of hemoglobin

Aetiology :
• Hb H
• Hb Köln
• enzyme deficiencies (G6PD)
• a-thalassemia
• lead poisoning
Refractile in fresh blood smears, they are not visible when stained with Romanowsky dyes but may be stained supravitally, e.g. with methylene blue.

• Pappenheimer bodies : very fine dark violet staining (basophilic), separated or connected granules present in the cytoplasm of the erythrocyte, particularly often near the periphery of the red cell. They must be confirmed with an iron stain. Probably these are the equivalent to nonhaeme iron granules of siderocytes.


Aetiology :
• sideroblastic anemia
• megaloblastic anemia
• ethanol
• following splenectomy
• some hemoglobinopathies
• sickle cell anemia (SCA)
• siderosomes : ferritin and hemosiderin granules
• hectoid bodies : sickle cell anemia (SCA)
• protozoal inclusions : Giemsa is the stain of choice for blood parasites
• babesial inclusions (Babesia spp.) : both thick and thin blood smears should be prepared. At least 200 to 300 oil immersion fields should be examined before the smears are considered negative (use the 100X oil immersion objective). The parasites appear as pleomorphic, ringlike structures (ring forms). They can resemble the early forms of malarial parasites, particularly Plasmodium falciparum(but falciparum rings are rarely extracellular, babesia parasites tend to be much more pleomorphic and may be 5-6 rings per cell vs. commonly 2 in falciparum cases). The classic arrangement of the 4 rings (Maltese cross) is not always seen. One negative set of blood smears does not rule out a Babesia infection


• leishmanial inclusions from Leishmania spp.


• plasmodial inclusions
• Maurer spots from Plasmodium falciparum


• Schüffner's dots or granules from Plasmodium vivax (arrow). Malaria male microgametes (arrowhead) are produced from the gametocytes by exflagellation, which takes place in the mosquito immediately after the blood meal. Exflagellation is induced by a temperature decrease and by an increase in pH, which occur on passage from the human bloodstream to the mosquito digestive tract. Microgametes are rarely seen in human blood and seem to be produced in vitro when laboratory conditions favor exflagellation during the preparation of blood smears; familiarity with their appearance may prevent diagnostic confusion with organisms such as trypanosomes and spirochetes^ref


• Ziemann granules : from Plasmodium malariae
• granules from Plasmodium knowlesi



• abnormally dried RBCs


• xerocyte : an erythrocyte that is dehydrated and has decreased cations due to abnormal permeability of the membrane that allows leakage of potassium ions and water out of the cell

Aetiology : hereditary xerocytosis
• poikilocyte : an abnormally shaped erythrocyte, such as a
• crenation / crenulation : the notched appearance of an erythrocyte caused by its shrinkage after suspension in a hypertonic solution, creating a burr cell or erythrocyte / crenated erythrocyte, crenocyte, and echinocyte : a spiculed erythrocyte that has multiple small projections evenly spaced over the cell circumference

Aetiology :
• uremia
• gastric carcinoma
• bleeding peptic ulcer

• acanthocytes (spur cells) : red cells with finger-like (thorny) projections due to an increased ratio of free cholesterol to phospholipid in the erythrocyte membranes. These acanthocytes undergo rapid splenic destruction and consequently have a shortened survival. Recent studies have indicated that alcoholic iron overload may be associated with spur cell anaemia rather than hereditary haemochromatosis. Patients usually need frequent blood transfusions and the prognosis is extremely poor. Liver transplantation, which improves hepatic function and resolves spur cell anaemia, has been the most effective treatment.


Aetiology :
• abetalipoproteinemia
• spur cell anaemia (acanthocytosis), a rare acquired haemolytic anaemia observed mainly in the end stages of alcoholic liver cirrhosis^ref
• after splenectomy
• McLeod syndrome
• drepanocyte (sickle cell)


Aetiology :
• sickle cell anemia (SCA)
• elliptocytes are red blood cells that are oval or cigar shaped


Aetiology :
• various anemias
• found in large amounts in hereditary elliptocytosis.
• codocyte / target cell or erythrocyte / Mexican hat cell or erythrocyte / leptocyte (?) are abnormally thin erythrocytes that when stained show a dark central color spot in the area of pallor and a peripheral ring of hemoglobin, separated by a pale unstained ring containing less hemoglobin, resembling a target


Aetiology :
• congenital and acquired anemias
• certain hemoglobinopathies
• liver disease, especially obstructive jaundice
• other disorders
• post-splenectomystate
• many hemolytic anemias, especially sickle cell anemia (SCA), HbC disease, and thalassemia
• dacryocyte / teardrop cell : an abnormal erythrocyte shaped like a teardrop


Aetiology :
• myelofibrosis with myeloid metaplasia and other myeloproliferative disorders
• pernicious anemia
• thalassemia
• some hemolytic anemias
• helmet cell


• schistocytes / keratocytes (bite cells) are red blood cell fragments that result from membrane damage encountered during passage through vessels


Aetiology :
• hemolytic anemias caused by
• microangiopathic hemolytic anemia (TTP and HUS)
• physical agents
• homozygous thalassemia
• severe burns
• uremia
• disseminated intravascular coagulation (DIC)
• spherocytes are almost spherical in shape. They are not biconcave like a normal red blood cell and do not have the central area of pallor which a normal red cell shows.


• microspherocytes
• hereditary spherocytosis
• burns
• microangiopathic hemolytic anemia

Aetiology :
• hemolytic anemia
• Rh deficiency syndrome
• ABO hemolytic disease of the newborn (HDN)
• hereditary spherocytosis
In some hereditary red cell enzyme deficiencies, the spherocytes may have many fine needle-like projections on the surface of the cell
• stomatocytes : an abnormal erythrocyte in which a slit-like, rectangular or mouthlike area replaces the normal circle of pallor, usually due to intracellular edema. These cells have lost the indentation on one side


Aetiology : stomatocytosis / hydrocytosis
• liver disease
• Rh deficiency syndrome
• b-sitosterolemia
• electrolyte imbalance
• cardiovascular disease
• hereditary stomatocytosis
• poikiloblast : an abnormally shaped erythroblast.
• asynclitism / dyserythropoiesis : defective development of erythrocytes, such as ...
• poikilocytosis / poikilocythemia : presence of poikilocytes in the blood
• pyropoikilocytosis : presence in the blood of numerous bizarre-shaped heat-sensitive poikilocytes.

Aetiology : hereditary pyropoikilocytosis
• size :
• macrocyte : MCV > 100 fL (Ø = 9-12 µm)

Aetiology :
• megaloblastic anemia (MCV > 120 fL). When associated with vitamin B₁₂or folic acid deficiency, the macrocytes may appear slightly oval in shape
• aplastic anemia (also normochromic, anisopoikilocytosis with dacryocytosis, leukoerythroblastosis)
• erythroblastopenia (also normochromic)
• 5q syndrome
• refractory anaemia with ringed sideroblasts (hypochromic microcytes usually constitute only a minor population of cells but their presence is a clue to the correct diagnosis)
• liver disease/chronic ethanol abuse

• normocyte : 81 < MCV < 99 fL

Aetiology :
• normal
• anemia of chronic disease (ACD) (also normochromic)
• myelodysplastic syndromes (MDS) (also normochromic, leukocytosis with relative neutropenia, and thrombocytopenia)
• liver disease/uremia
• hypometabolic anemia (hypothyroidism, hypoadrenalism, protein deficiency)
• acute bleeding
• hemolysis
• microcytes : MCV < 80 fL (Ø < 6 mm)
• microcythemia / microcytosis : a condition in which the erythrocytes are smaller than normal
• micronormoblast : an abnormal red cell precursor in which there has been defective hemoglobin synthesis, characterized by a narrow rim of cytoplasm and an overdeveloped pyknotic nucleus
Aetiology :
• anemia of chronic disease (ACD) (also hypochromic)
• impaired Hb synthesis (also hypochromic, increase in [soluble CD71 / TfR]_blood and decrease in [reticulocytes]_blood)
• sideropenic anemia (also anisopoikilocytosis); > 30% of cases of iron-deficiency anemia will be misdiagnosed if physicians rely on MCV and MCHC only
• sideroblastic anemia
• thalassemias
• hemoglobin Lepore trait
• hemoglobin E trait or homozygous
• hemoglobin H disease
• combination of above (double heterozygotes)
• anisocytosis / anisopoikilocytosis : presence in the blood of erythrocytes with excessive variation in size (RDW > )
• myelofibrosis with myeloid metaplasia
• aplastic anemia
• essential pernicious anemia
• sideropenic anemia
• thalassemia (also hypochromia and target cells)
• hemoglobin concentration :
• hypochromia : MCHC < 32 g / dL
• leptocyte (hypochromic microcytic red cell)


• achromocyte / demilune body / achromic erythrocyte : a crescent-shaped red cell artifact that stains more faintly than intact red cells
• ghost or shadow cell / erythroclast : a degenerating or fragmented erythrocyte with no hemoglobin
Aetiology :
• anemia of chronic disease (ACD) (also microcytic)
• impaired Hb synthesis (also microcytic, increase in [soluble CD71 / TfR]_blood and reticulocytopenia)
• sideropenic anemia (also anisopoikilocytosis); > 30% of cases of iron-deficiency anemia will be misdiagnosed if physicians rely on MCV and MCHC only
• PNH
• sideroblastic anemia
• thalassemias
• normochromia : MCHC = 33-36 g / dL

Aetiology :
• normal
• aplastic anemia (also macrocytic, anisopoikilocytosis with dacryocytosis, leukoerythroblastosis)
• erythroblastopenia (also macrocytic)
• myelodysplastic syndromes (MDS)(also normocytic, leukocytosiswith relative neutropenia, and thrombocytopenia)
• hyperchromia / polychromasia : MCHC> 36 g / dL


• anisochromasia : hemoglobin-distribution width >
• rouleaux formation occurs when RBCs form stacks or rolls

Aetiology :
• artifact (such as a result of not preparing the blood smear soon enough after placing the blood on the slide)
• hyperfibrinogenemia
• plasma cell dyscrasias (PCD)

• erythroblastosis / erythroblastemia : the presence in the peripheral blood of abnormally large numbers of erythroblasts (nucleated red blood cells (NRBCs))

Aetiology :
• previous studies have reported an association between acute and chronic hypoxia and presence of fetal heart rate accelerations. In vitro placental NRBC counts correlate with umbilical cord NRBCs and suggest that antenatal evaluation of fetal NRBCs could be achieved by placental FNAB^ref.
• reticulocytosis : an increase in the number of reticulocytes in the peripheral blood.
• reticulocyte response : increase in the formation of reticulocytes in response to a bone marrow stimulus, such as that provided by administration of a hematinic.
• reticulocytopenia : a decrease in the number of reticulocytes in the blood.
• basophilia : the reaction of immature erythrocytes to basic dyes so that they become stippled
• basophilia punctata / punctate basophilia / basophilic stippling : basophilia in which erythrocytes are blue and stippled with round, dark-blue granules on smears stained with supra vital stains such as brilliant cresyl blue, representing precipitated ribosomes and mitochondria. many course blue-purple staining granules within the red blood cell


Aetiology :
• lead
• poisoning with certain other heavy metals
• exposure to some drugs
• severe burns
• ethanol
• anemias with impaired hemoglobin synthesis
• megaloblastic anemia
• refractory anemia
• severe bacterial infection, septicemia
Laboratory examinations : stippled cell (an erythrocyte containing granules of varying size and shape, taking a basic or bluish stain with Wright's stain, as in punctate basophilia)
• diffuse basophilia : basophilia in which erythrocytes are blue-gray and not stippled.
• anemias (HGB < 13 g/dL or HCT < 39% in males; HGB < 12 g/dL or HCT < 35% in females)

Lower limits of normal of hemoglobin concentration of the blood of adult men and women as assessed by various sources :

Source	Men, g/dL	Women, g/dL	Percent normal below cutoff	Effect of race
WHO (Blanc B, Finch CA, Hallberg L, et al. Nutritional anaemias. Report of a WHO Scientific Group. WHO Tech Rep Ser. 1968;405: 1-40)	13	12	Not provided	Not provided
Jandl (Jandl JH. Blood. Boston, MA: Little, Brown and Company; 1996)	14.2	12.2	2.5	Discussed
Williams (Beutler E, Lichtman MA, Coller BS, Kipps TJ, Seligsohn U. Williams Hematology. New York, NY: McGraw-Hill; 2001)	14.0	12.3	2.5	Not provided
Wintrobe (Lee GR, Foerster J, Lukens J, Paraskevas F, Greer JP, Rodgers GM. Wintrobe's Clinical Hematology. Baltimore, MD: Williams and Wilkins; 1998)	13.2	11.6	Not provided	Not provided
Rapaport (Rapaport SI. Introduction to Hematology. Philadelphia, PA: JB Lippincott Company; 1987)	14	12	Not provided	Not provided
Goyette (Goyette RE. Hematology. A Comprehensive Guide to the Diagnosis and Treatment of Blood Disorders. Los Angeles, CA: Practice Management Information Corporation (PMIC); 1997)	13.2	11.7	5	Blacks' hemoglobin 0.5 g/dL lower
Tietz (Tietz NW. Clinical Guide to Laboratory Tests. Philadelphia, PA: WB Saunders Co; 1995)	13.2	11.7	Not provided	Not provided
Hoffman et al (Hoffman R, Benz EJ Jr, Shattil SJ, Furie B. Hematology: Basic Principles and Practice. New York, NY: Churchill-Livingstone; 2004)	13.5	12.0	2.5	Not provided

Mean hemoglobin (g/dL) by sex and age in white and black subjects :

	Scripps-Kaiser (collected in the San Diego area between 1998 and 2002)ref1, ref2			NHANES III (the third US National Health and Nutrition Examination Survey database)
	No.	Mean Hgb	SD	No.	Mean Hgb	SD
White men, y
20-29	250	15.43	0.77	332	15.51	0.96
30-39	894	15.24	0.85	389	15.29	0.95
40-49	2219	15.20	0.89	379	15.20	0.99
50-59	3346	15.10	0.93	356	15.17	1.01
60-69	3073	15.02	0.95	364	14.94	1.04
70-79	1976	14.75	1.07	315	14.70	1.15
80+	466	14.36	1.20	255	14.29	1.31
White women, y
20-29	240	13.41	0.80	322	13.57	0.88
30-39	809	13.59	0.86	402	13.66	0.86
40-49	1917	13.58	0.86	321	13.58	0.94
50-59	2829	13.62	0.86	312	13.76	0.94
60-69	3006	13.63	0.88	320	13.66	1.04
70-79	2051	13.66	0.94	421	13.68	0.97
80+	427	13.54	0.95	342	13.44	1.21
Black men, y
20-29	27	14.83	0.76	403	14.84	0.96
30-39	87	14.76	1.02	394	14.61	1.18
40-49	163	14.58	1.06	290	14.50	1.07
50-59	157	14.46	1.08	166	14.25	1.27
60-69	103	14.27	0.98	137	14.19	1.02
70+	32	14.44	1.06	98	13.75	1.15
Black women, y
20-29	17	13.26	0.75	321	12.78	0.92
30-39	68	12.97	0.93	339	12.77	1.11
40-49	120	12.88	0.91	244	12.87	1.05
50-59	135	12.96	0.89	146	13.07	0.95
60-69	90	13.03	0.91	161	12.89	1.01
70+	30	12.93	0.88	135	12.65	1.11

Proposed lower limits of normal for hemoglobin concentration of the blood for white and black adults :

Group	Hemoglobin, g/dL
White men, y
20-59	13.7
60+	13.2
White women, y
20-49	12.2
50+	12.2
Black men, y
20-59	12.9
60+	12.7
Black women, y
20-49	11.5
50+	11.5

Based on Scripps-Kaiser data for the 5th percentiles given in Table 2. NHANES data are considered to be confirmatory.
The ethnic origin of the individuals designated as white in these 2 databases is probably very similar. White Americans are a genetic mixture of persons from various parts of Europe and the Middle East. Data regarding ancestral origin are available in the Scripps Kaiser database but not in NHANES-III, so that direct comparison is not possible. Among black Americans there are varying degrees of admixture with non-African ancestry. The NHANES database attempts to reach a nationwide balance. It is likely that more of the black population of San Diego originates from the Northeast and the Midwest; therefore, San Diego may have greater white admixture than the NHANES sample. However, this has not been documented by the study of genetic markers in either data set. The NHANES-III database has recently been "mined" for hematologic values. The data in these compilations are valuable, but unfortunately the hemoglobin values in one report are derived from the entire population, not corrected for subjects with disease states that may be associated with anemia (Hollowell JG, Van Assendelft OW, Gunter EW, Lewis BG, Najjar M, Pfeiffer C. Hematological and iron-related analytes: reference data for persons aged 1 year and over: United States, 1988-94. Vital Health Stat. 2005;11: 1-156). The other report uses exclusion criteria that are so draconian that less than 40% of the subjects remain in  the data set^ref. Moreover, data derived from white subjects are not provided. In these and many other previous analyses of hematologic data the subjects have been divided into age in deciles. This type of analysis is shown in Table 2. Because there are virtually no differences in hemoglobin values of men in the age range of 20 to 59 or in women aged 20 through 49 in our data or in the analysis of the NHANES-III hemoglobin values,12 we have used these age ranges representing younger adults, and the ages beyond these ranges to represent older adults. In any analysis, elimination of subjects from consideration is constrained by the data that are available. Both the Scripps-Kaiser and the NHANES databases included transferrin saturation and serum ferritin levels, and we were therefore able to exclude subjects with a transferrin saturation of less than 16% or a serum ferritin level less than 10 µg/L. This serves to eliminate most subjects with iron deficiency anemia and also many of those with the anemia of chronic inflammation, in which the transferrin saturation is also usually low. We were also able to determine whether excessive alcohol intake, diabetes mellitus, renal failure, or inflammatory markers (C-reactive protein in NHANES, erythrocyte sedimentation rate in Scripps-Kaiser, elevated serum ferritin, or leukocytosis) affected the hemoglobin concentrations in these populations. None of these had an effect in the younger subjects, but in older subjects a serum creatinine of more than 106 µM (1.4 mg/dL) and elevated inflammatory markers (ESR > 30 in women and ESR > 20 in men or CRP > 1 mg/dL) were associated with significantly lower mean hemoglobin values. Older subjects with elevated creatinine and ESR or CRP were thus excluded from our calculations. Pregnant women were also excluded. Overall, the percentage of subjects excluded from the analysis ranged from 10% to 30%, except for black subjects in NHANES, in which the exclusion rate was approximately 50% for older men and 40% for women. The mean and 1.65 standard deviation below the mean of the hemoglobin values of the white subjects are shown in Figure 2; this provides the hemoglobin concentration below which only 5% of normal subjects in the population will be found, given the Gaussian distribution of hemoglobin values. Table 3 presents the lower limit of normal for white and black men and women. These data are presented to indicate the 2.5th and the 5th percentile as the actual percentile in the data set and as the predicted value based on the assumption that the hemoglobin values have a Gaussian distribution, as assumed in Figure 2. This assumption is clearly valid, as shown by the excellent correspondence between the predicted cutoff and the actual cutoff based on the number of cases found. The results obtained from the 2 databases are in very good agreement, but there are some minor differences that reach statistical significance. In particular, the mean hemoglobin of older men is slightly higher in the Scripps-Kaiser data than in the NHANES data. The latter difference in Table 3 is clearly an artifact resulting from the consolidation of all subjects older than 60 years into one group. As shown in Table 2, there are a higher proportion of men > 80 years in the NHANES survey than in the Scripps-Kaiser data, and there is a slight but well-documented decrease in the mean hemoglobin concentration of each decile among men (Hollowell JG, Van Assendelft OW, Gunter EW, Lewis BG, Najjar M, Pfeiffer C. Hematological and iron-related analytes: reference data for persons aged 1 year and over: United States, 1988-94. Vital Health Stat. 2005;11: 1-156). Some minor discrepancies could also be due to differences in ethnic origin within populations studied. This would obviously be true with the population designated "black," because it is well known that the genes of individuals classified as African American could be anywhere between 10% and 90% of European origin, with most of the remaining genome being of African origin. As pointed out earlier, differences in the degree of the European admixture could well account for the small differences in hemoglobin concentrations documented in the 2 data sets. This difference in genetic composition could also extend to the population designated "white": we have previously shown that there is a difference of about 2.8 g/L (0.28 g/dL) in the average hemoglobin concentration of a white subject with northern versus southern European ancestry^ref. This differential between northern and southern Europeans is maintained when the exclusion criteria used here are applied (data not shown). How is it that the WHO standard, so widely used in epidemiologic studies, is so seriously flawed? In fairness to the Expert Committee, a group of distinguished hematologists and nutritionists, it is clear that it was not their intent to set up a hallowed standard for all to follow. The values defining anemia are given in a small table and are presented without decimal points, clearly as approximations to be used within the context of the nutritional studies that were the principal topic of the meeting. The few references that are given to document this choice of numbers are, in general, surveys of the relatively small number of subjects, without documentation of methodology, and without efforts being made to remove patients with anemia in calculating the values. To further put these studies into the context of the times (Beutler E. Hemoglobin level and red blood cell count findings in normal women. JAMA. 1958; 167: 1551), the dilution of blood, sometimes obtained by finger puncture^ref, was carried out with pipettes that were often inaccurate. Cyanmethemoglobin standards were not widely available, and calibration required oxygenation of a blood sample to serve as a standard with measurement of the amount of oxygen released by acid in a van Slyke apparatus. Needless to say, this procedure was not always carried out, and, as a consequence, results were often unreliable. In view of these difficulties, perhaps we should marvel not only at the fact that the WHO standard is still used but also that it was not further from the mark. What, then, are reasonable benchmarks for anemia for a clinician to use today, based on today's improved laboratory practice and the data that we have been able to marshal from the Scripps-Kaiser and the NHANES study? The suggested lower limits of normal are summarized in Table 4. Based mostly on the larger Scripps-Kaiser database, but confirmed by the NHANES data, it would seem that a hemoglobin concentration below 137 g/L (13.7 g/dL) in a white man aged between 20 and 60 years would have only an approximately 5% chance of being a normal value. For older men, this hemoglobin value would be 132 g/L (13.2 g/dL). The corresponding value for women of all ages would be 122 g/L (12.2 g/dL). As shown in Table 1 and in a number of previous studies^{ref1, ref2},17,18 the lower limit of normal of hemoglobin concentrations of African Americans are appreciably lower. Although some of this difference is a result of the high frequency of -thalassemia in this population, other, as-yet-unidentified genes are involved, and because the gene frequency for -thalassemia is extraordinarily high in the black population, and the diagnosis is not readily made, we judge it best not to censor such cases in determining the normal range for the black population
The mean and -1.65 standard deviations of the hemoglobin values of the white subjects in the Scripps-Kaiser and NHANES-III databases after applying the criteria described in the text. The bottom of the error bars represents the hemoglobin value below which only 5% of normal values would be predicted to be present :

Lower limits of normal for hemoglobin concentration of the blood in g/dL of younger (age 20-59 for men; 20-49 for women) and older white and black adults :

	Scripps-Kaiser					NHANES-III
	No.	2.5% actual	2.5% normal distribution	5% actual	5% normal distribution	No.	2.5% actual	2.5% normal distribution	5% actual	5% normal distribution
White men, y
20-59	6709	13.4	13.4	13.7	13.7	1456	13.4	13.4	13.8	13.7
60+	5515	12.8	12.8	13.2	13.2	934	12.2	12.4	12.8	12.7
White women, y
20-49	2966	11.9	11.9	12.2	12.2	1045	12.0	11.9	12.2	12.1
50+	8313	11.9	11.9	12.2	12.2	1395	11.5	11.6	12.0	11.9
Black men, y
20-59	434	12.6	12.5	12.9	12.9	1253	12.3	12.4	12.8	12.8
60+	135	—	12.4	—	12.7	235	11.4	11.9	11.8	12.2
Black women, y
20-49	205	11.2	11.2	11.5	11.5	904	10.9	10.8	11.3	11.1
50+	255	11.2	11.2	11.5	11.5	442	11.0	10.9	11.3	11.2

Classification :
• according to MCV :
• < 80 fL (Ø < 6 mm) : microcytic anemia
• iron deficiency anemia
• PNH
• thalassemia
• myelodysplastic syndromes (MDS)
• hemoglobin Lepore trait
• hemoglobin E trait
• hemoglobin H disease
• combination of above (double heterozygotes)
• homozygous hemoglobin E
• sideroblastic anemia
• anemia of chronic disease
• 81 < x < 99 fL : normocytic anemia
• acute or chronic inflammation
• hypometabolic anemia (hypothyroidism, hypoadrenalism, sustained starvation)
• liver disease/uremia
• protein deficiency
• acute bleeding
• hemolysis
• > 100 fL (Ø > 9 mm)^ref : macrocytic anemia
• 5q syndrome
• megaloblastic anemia : MCV may exceed 150 fl. The RBCs vary considerably in size and shape. The macrocytes tend to be oval. Serum vitamin B12 determination remains the best test for unmasking vitamin B12 deficiency. It should be ordered in conjunction with serum and red cell folate determinations. When vitamin B₁₂ deficiency has been established, a Schilling test or plasma uptake test is indicated to pinpoint the cause.
• reticulocytosis : MCV < 110 fl; high reticulocyte count
• hemolytic anemia other than PNH (increase in [LDH₁ and LDH₂]_serum, [soluble CD71 / TfR]_plasma and [reticulocytes]_blood)
• uremia
• liver disease: abnormal liver function test, mild and uniform macrocytosis. The RBCs are round
• copper deficiency(associated with neutropenia, normal platelet count, and elevated serum ferritin and EPO levels)^ref
• Fanconi anemia (FA) or syndrome
• Shwachman-Diamond syndrome (SDS)
• dyskeratosis congenita (DC)
• according to MCHC :
• < 32 g / dL : hypochromic anemia
• 33 < x < 36 g / dL : normochromic anemia
• no hyperchromic anemia exists !
Compensation mechanisms : metabolic acidosis and hypercapnia => increase in 2,3-BPG
Symptoms & signs : severity directly relates to
• anemization rate and is mainly due to mechanisms that tend to compensate hypoxia :
• peripheral ischaemia (hemometakinesis in favour of vital organs) => pallor, tachycardia => hyperdynamic circulation=> functional heart murmurs when in clinostasis (disappearing in orthostasis, when venous blood return decreases), dizziness, diaphoresis, tachypnea, fatigue, amaurosis.
• pale palmar crests : HGB< 8 g/dl
• pale lip mucosae : HGB < 8-10 g/dl (conjunctival mucosae are normal)
• hyperdynamic ventricular output, tachycardia, systolic ejection murmur over pulmonary area, systodiastolic venous murmurs over neck disappearing after mild pression upstream of jugular vein (differential diagnosis with carotid artery stenosis > 75%)
• increased [EPO]_blood (except in chronic renal failure !) => aspecific stimulation on hematopoiesis, causing thrombocytosisand leukocytosis(except in hypoproliferative anemias !)
• jaundice
• splenomegaly
• lymphadenomegaly
• petechias
• concurrent cardiovascular diseases (IHD, CVD, lower limb obliterating arteriopathy)
Laboratory examinations :
• measure ABP in orthostatism and clinostatism
• reticulocyte index
Aetiology :
• hypoproliferative anemias : decrease in [soluble CD71 / TfR]_blood and [reticulocytes]_blood
• impaired medullary space
• myelophthisis / myelophthisic anemia / secondary myelofibrosis (normocytic and normochromic)
• impaired DNA synthesis
• impaired HSCs
• aplastic anemia (AA) / pure red cell aplasia (PRCA) : severe normochromic, normocytic anemia, reticulocytosis, and erythroblastopenia in bone marrow that produces the other cellular elements in a normal way (for general physicians) (normochromic and macrocytic, anisopoikilocytosis with dacryocytosis, leukoerythroblastosis). As the genetic basis and molecular pathogenesis of many of the inherited marrow failure syndromes are identified, it has become clear that some of these disorders may present as bone marrow failure or myelodysplasia (MDS) as well as epithelial malignancies well into adulthood. Distinguishing inherited aplastic anemia from acquired is not simply an esoteric exercise. The particular diagnosis will have an impact on proper management strategies and therapeutic approaches.
• congenital hypoplastic anemia (65%) : many inherited marrow failure syndromes may result in pancytopenia

disease		signs  (nonhematopoietic)	screening  tests	biomarkers
disorders frequently associated with multilineage bone marrow failure. Both are associated with a high risk of MDS/AML, and, when adults with these disorders are diagnosed, they commonly present with either hematopoietic (MDS/AML) or epithelial neoplasms. Because the initial diagnosis of both of these disorders has been made in individuals in their 5th and 6th decades of life, they must be considered even in adults with any one of the following: subtle but characteristic physical anomalies, hematologic cytopenias, unexplained macrocytosis, MDS/AML, or squamous cell cancer even in the absence of severe pancytopenia or a positive family history	Fanconi anemia (FA) or syndrome Estren-Dameshek variant	café-au-lait spots, skeletal  anomalies (thumb and radius),  short stature, microcephaly	DEB or MMC  sensitivity  (chromosomal breakage)	increased HbF;  macrocytosis
	dyskeratosis congenita (DC)	nail dystrophy, macular  or reticular hypopigmentation, mucosal leukoplakia	genetic	increased HbF; macrocytosis
inherited diseases associated with failure of single hematopoietic lineages : only occasionally evolve to hypoplastic bone marrows and pancytopenia. For this reason only rare patients may be ascertained in the later pancytopenic stages. Clonal evolution to MDS and AML has been described in both DBA and SDS. They rarely present in adulthood and most often begin with a failure of a single hematopoietic lineage.	neutropenia (e.g., Shwachman-Diamond syndrome (SDS))	exocrine pancreatic insufficiency, metaphyseal  dysostosis, short stature,  hepatic dysfunction	genetic  serum trypsinogen  & isoamylase	increased HbF; macrocytosis
	anemia (Diamond-Blackfan anemia (DBA))	congenital anomalies (thumb), short stature, cardiac  (ventricular or atrial septal defects)	genetic	increased HbF; increased RBC, increased eADA

• physiologic anemia is a common and normal finding in newborn infants
• anemia of prematurity : in preterm infants requiring transfusions of RBCs for, transfusion with 20 mL/kg RBCs produces a significantly greater increase in hemoglobin and hematocrit levels than does a transfusion with 10 mL/kg, without any detrimental effects on pulmonary function.
• Zinsser-Engman-Cole syndrome / congenital dyskeratosis
• Chediak-Higashi syndrome (CHS)
• Kostmann syndrome(severe congenital neutropenia), in which pancytopenia occurs only very rarely
• congenital amegakaryocytic thrombocytopenia
• congenital dyserythropoietic anemia
• Dubowitz and Seckel syndromes
• Pearson syndrome
• cartilage-hair hypoplasia or reticular dysgenesis
For more information regarding these entities: Alter BP. Inherited bone marrow failure syndromes. In: Nathan DG, Orkin SH, Ginsburg D, Look AT, eds. Hematology of Infancy and Childhood. Philadelphia: W.B. Saunders Co.; 2003:280–365
• acquired AA (35%) : pancytopenia and a hypocellular bone marrow

Epidemiology : preferentially affects young adults < 30 years and individuals aged > 60 years^ref.
Aetiology :
• idiopathic (50%)
• acute form that is self-limited :
• drugs :
• chloramphenicol
• quinacrine
• tolbutamide
• chlorpropamide
• phenylbutazone
• Au salts
• acetazolamide
• acetylsalicylic acid
• chlordiazepoxide
• chlorpheniramine
• chlorothiazide
• chlorpromazine
• diphenylidantoine / phenytoin / phenantoine
• mepazine
• pe