Inclusion Information and Courses from MediaLab, Inc.

Anemia is moderate.RBC count is increased.Hb is moderately decreased.MCV is decreased. MCHC is decreased.RDW is increased.Red Blood Cell morphology shows slight hypochromic microcytosis with codocytes, schizocytes, and basophilic stippling.Reticulocytes are moderately increased.Hb electrophoresis demonstrates abnormal patterns in both adults and neonates.Adults:HbA decreasedHbA2 decreasedHbF normal to decreasedHb H -2-40% (beta chain tetramers)Neonates: 10-40% Bart's (gamma chain tetramers)Hb H inclusions are frequently seen.Bone marrow demonstrates erythroid hyperplasia.

Anemia is absent.RBC count is within normal limits.Hb is within normal limits.MCV is normal to slightly decrease.MCHC is normal to slightly decrease.RDW is within normal limits.Red Blood Cell morphology is normal.Reticulocytes are within normal limits.Hb electrophoresis demonstrates a normal pattern in adults:Hb A - 97-98%Hb A2 - 1-2.5% Hb F - < 1%. Neonates have 1-2% Bart's Hemoglobin (gamma chain tetramers).Hb H inclusions are rarely seen.Bone marrow is normal.

RNA present in reticulocytes will appear as darker inclusions, similar to how they appear with New Methylene Blue stain. These RNA inclusions are fewer in number than Hemoglogin H bodies, and are only seen in reticulocytes.

Pappenheimer bodies, while visible on a Wright's stained smear, should be Perls' Prussian blue stain, which is specific for iron. Pappenheimer bodies are seen in certain types of anemia characterized by an increase in the storage of iron, such as sideroblastic anemia and thallassemia. These inclusions are also seen in the peripheral blood following a splenectomy. In a healthy person with a normal spleen, Pappenheimer bodies are destroyed before the erythrocytes enter the peripheral circulation.

To verify that red cell inclusions contain iron, it is necessary to use an iron stain, such as Perl's Prussian blue. The iron-containing granules are called siderotic granules. A mature erythrocyte containing siderotic granules is referred to as a siderocyte, while an immature (nucleated) erythrocyte containing siderotic granules is known as a sideroblast. A Pappenheimer body is a siderotic granule which is visible on Wright stain. All Pappenheimer bodies are Prussian blue positive, but not all siderotic granules are visible on Wright's stain as Pappenheimer bodies.

The appearance, composition and associated physiology is specific for each type of inclusion. Identification and quantification of these inclusions is important because their presence may indicate an abnormality in the red cell system. Each of the inclusions listed above can be seen in more than one condition. There are erythrocyte inclusions specific to disorders which cannot be seen with either Wright-Giemsa stain or Perls' Prussian blue iron stain.

Erythrocyte inclusion is a term used to describe structures that may be present in red cells. The inclusions discussed in this course are: reticulocyte - Wright-Giemsa stain (polychromasia)basophilic stipplingHowell-Jolly bodiesPappenheimer bodies (Wright-Giemsa stain)siderotic granules (iron stain)Cabot rings

A ten-year-old boy came to a physician's attention because of recent jaundice and icteric sclerae. The immediate laboratory work revealed: Hct 24%(normal 36%-47%), MCV 79.5 fl (normal 78-95fl),RDW 13%(normal 11.5-15.0%). His blood smear findings are reflected in these photomicrographs. Note particularly the spherocytes in the upper picture. Some resemble a half-blister with the other half of the cell containing solidly-staining hemoglobin. These are called eccentrocytes. When present, they should trigger a search for red cell hereditary G-6PD deficiency and the oxidant that triggered hemolysis. These morphological findings are only clues; specific testing for G-6PD deficiency should be performed. The blue arrows in the upper photomicrograph are directed toward solid-staining spherocytes in which the cell membrane is beaded by inclusions wrapped within the cell membrane, suggesting the remains of denatured hemoglobin. Included on the smear is a target cell, several acanthocytes, a smudge cell, and a few schistocytes. The lower photomicrograph is supravital staining of affected red blood cells, verifying the presence of Heinz bodies. This disorder was first recognized during the Korean war in 10% of black American soldiers given the antimalarial drug primiquine.

Hemoblobin H disease follows deletions of 3 of the 4 alpha globulin chains. Beta chains, unable to bind with insufficient numbers of alpha chains, form beta chain tetramers, or HbH.These beta chain tetramers appear as numerous dot size inclusions in erythrocyte cytoplasm, best seen in supravital brilliant cresyl blue stains (lower photograph).The most common molecular defect in alpha thalassemia is DELETION, not MUTATION; whereas, in beta thalassemia, the molecular defect is MUTATION.Leptocytes, as illustrated in the upper photograph,(lepto, derived from a Greek word meaning thin, fine, or slight), are characteristic of HbH disease. They have thinner cell membranes than the cells we recognize as target cells. They stain more lightly than normal erythrocytes and their centers are almost colorless.Subtle changes perhaps, but worth keeping in mind

Laboratory data must be presented to clinicians in a user friendly way to promote effective decision making. Databases must be designed to provide clear information that leads quickly to the best patient care outcome. We continue learning how to collect and retrieve laboratory data from our machines, but we are not always in tune to how entry and retrieval of data is geared to and, more directly, influences patient care outcomes. Examples of blood cell abnormalities on a peripheral blood smear that may immediately direct the physician to a specific diagnosis are: (1) presence of target cells as found in thalassemia or hemoglobinopathies and target cells in liver disease, particularly with obstructive jaundice; (2) burr cells as a signal of chronic renal disease and uremia; and (3)atypical neutrophil inclusions relating to genetic disorders. Critical appraisal of such observations could add valuable clues for a diagnosis. Laboratory professionals must establish a set of principles for orderly observation of blood cell morphology, have a clear vision of the applications of their work, and understand the potential clinical implications of their reports and interpretations. Emphasis on values and relevance focuses on patient care outcomes and their dependency on prompt availability of results and contextual interpretations.

The cytoplasmic morphological changes included in this exercise are:Dohle bodiesMay-Hegglin bodiesAuer rodsVacuolesAbnormal granulationA general term used to describe these changes is white cell inclusions.

May-Hegglin bodies stain blue and appear to have a more definite shape than Dohle bodies. When examined under electron microscopy, they appear to be aggregates of thread-like structures in a crystal-like arrangement. May-Hegglin inclusions are RNA material believed to be derived from the endoplasmic reticulum. May-Hegglin bodies can be seen in monocytes and platelets as well as in all mature granulocytes. The platelets in May-Hegglin anomaly are very bizarre in appearance and thrombocytopenia is usually noted. When examining a slide that has bizarre platelets and blue-appearing bodies in the cytoplasm, thought should be given to the possibility of the May-Hegglin anomaly.

Several additional familial and congenital disorders associated with atypical inclusions in WBCs are now recorded. These individual syndromes carry the following names: Fechtner, Alport, Epstein, Sebastian, and Paris-Trousseau.Fechtner syndrome( Peterson etal,Blood 65:397-406,1985)was described with 8 family members spanning 4 generations presenting with varying degrees of nephritis, deafness,and congenital cataracts. The syndrome is likely a variant of Alport syndrome with the addition of leukocyte inclusions and macrocytothemia. Several more cases involving other families have been reported. The inclusions resemble toxic Doehle bodies or those of the May-Hegglin anomaly by light microscopy, but are ultrastructurally unique.Alport syndrome in itself is autosomal dominant, X-linked , hereditary and characterized by sensorineural deafness and hereditary nephritis. It is believed to result from abnormal glycopeptide synthesis in renal basement membranes. Recurrent hematuria and slowly progressive renal insufficiency are clinical findings. Cataracts and platelet abnormalities may be added features.Epstein syndrome is essentially Alport syndrome with the addition of macrothrombocytopenia (Seri, et al. Hum Genet 110:182-186, 2002). Neutrophil inclusions are absent in this disorder; neutrophilic inclusions are considered part of the Fechtner syndrome. The Sebastian platelet syndrome is a variant of hereditary macrothrombocytopenia combined with neutrophil inclusions that differ from Doehle bodies, but are similar to those inclusions in Fechtner syndrome. (Greinacher, et al, Blut 61:282-288, 1990).Paris-Trousseau syndrome includes large platelets containing giant alpha granules identifiable in the peripheral blood.(Breton-Gorius, Blood 85:1805,1995)

The presence of atypical inclusions within the cytoplasm of neutrophils and other leukocytes should lead to a clinical investigation of the setting for these findings.Atypical neutrophil inclusions may be seen in the following disorders: Chediak-Higashi syndrome, May-Hegglin anomaly, Alder-Reilly anomaly, Fechtner , Sebastian, Epstein and Alport-like syndromes and in infectious and toxic conditions (in the form of Doehle bodies).Although a specific entity may not be evident from examination of the peripheral blood alone, it is important that hematology technologists include a comment reporting on the presence of these inclusions or granules. A clinical investigation with further hematologic and genetic studies may then appropriately be considered.Many of the disorders with atypical neutrophil cytoplasmic granules are also associated with platelet abnormalities, particularly giant platelets (lower photograph).Therefore, when atypical granules are recognized, scanning of the peripheral blood smear for atypical platelets may be revealing. These observations serve as readily identifiable markers for acquired and genetic human maladies, and as a guide for unraveling the reasons for a patient's suffering and impaired health.

Illustrated in the upper photograph is a poorly defined cytoplasmic inclusion somewhat resembling a Doehle body. Note, however, that this inclusion is well defined and there is no evidence of toxic granulation in the cytoplasm.When Doehle-like bodies are identified, May-Hegglin anomaly should be considered in the differential diagnosis even though this entity is rare.The May-Hegglin anomaly is an inherited dominant condition in which large 2 - 5 um, basophilic and pyronophilic inclusions are present in granulocytes, including neutrophils, eosinophils, basophils, and monocytes.Similar to Doehle bodies, the May-Hegglin inclusions also are composed of RNA, probably derived from the rough endoplasmic reticulum. May-Hegglin anomaly includes giant platelets containing few fine granules (lower photograph).Sometimes the platelets have bizarre shapes and variable sizes. Variable degrees of thrombocytopenia complicated by mild bleeding problems and purpura may accompany the aberrant platelets.