PROFICIENCY TESTING SERVICE AMERICAN ASSOCIATION OF BIOANALYSTS 205 West Levee St.  Brownsville, TX 78520-5596 800-234-5315  281-436-5357  Fax 713-781-5008

PARTICIPANT STATISTICS CELL IDENTIFICATION THIRD QUADRIMESTER 2015

Q3‐2015 IDA‐Hypersegmentation‐ Thrombocytosis This case exhibits abnormalities in all three cell lines: RBCs, WBCs and platelets.

This patient lives with his mother and baby sister in public housing, where there are few public transportation options. The family receives food assistance but, because they live in an area termed an “urban food desert”, meals, for both the mother and her 3 year old son, consist primarily of fast foods or processed packaged items stocked in nearby, small corner stores. Other than the infant, whose primary nutritional source is baby formula, these circumstances make it very difficult for the mother and son to regularly obtain nutritionally adequate meals.

The CBC reveals that this child is anemic and has thrombocytosis. In this laboratory, both these abnormalities reflex to a blood smear review. Moderate hypochromic, microcytic numbers of RBCs were seen, several hypersegmented PMNs were noted and the elevated platelet count, including some clumping, was confirmed microscopically.

The presence on the blood smear of moderate hypochromic, microcytic RBCs suggests that he could be iron deficient. His socioeconomic history make the most likely differential diagnosis iron deficiency anemia (IDA ), secondary to inadequate dietary iron intake. But, since children affected by lead poisoning can also show a normocytic to microcytic, hypochromic blood picture, the age and the living conditions of this patient prompted the physician to also request testing for blood lead levels.( Note: a common finding in lead poisoning, , was not noted on the blood smear). In addition, a serum ferritin was performed and results confirmed the presumptive diagnosis of iron deficiency anemia.

Individuals are at high risk for inadequate dietary iron intake when, either by choice, as is the case with vegans and vegetarians who do not replace meat with other iron‐rich foods, or circumstances, when only a poor or restricted diet low in fruits, vegetables, and meat is available, such as appears to be the case for this patient. There are many causes of anemia, but iron deficiency anemia is the most common type of anemia The World Health Organization regards iron deficiency as the top nutritional disorder in the world. A 2008 World Health Organization report makes the case for as many as 30 percent of people in the world having anemia due to prolonged iron deficiency

Pregnant women, who are severely iron deficient, frequently give birth prematurely and /or deliver low birth weight babies. To prevent this from happening, most pregnant women take iron supplements as part of their prenatal care. Infants and children severely deficient in iron may experience a delay in their growth and development. They may also be more susceptible to infections. In this case, during her recent pregnancy, the mother had been receiving iron supplementation as a component of her prenatal vitamins. The baby was getting sufficient iron in her formula. It was only the rapidly growing 3 year old who was receiving no iron supplementation to his, presumably, iron poor diet.

This patient also exhibited hypersegmented neutrophils on his peripheral blood smear. After neutrophils move from the bone marrow into the blood, the number of segments in the nucleus of a neutrophil will increase as the cell continues to mature. Normal neutrophilic nuclei have three or four lobes, also called segments. Neutrophils having nuclei with six or more lobes are described as being hypersegmented. These hypersegmented neutrophils are most frequently considered to be markers for the caused by either folate or vitamin B12 deficiencies. Although less widely associated with this finding, hypersegmented neutrophils are also often seen in patients, especially children, with IDA. Data, in one well known study of pediatric subjects with IDA, found over 80% of the subjects had documented hypersegmented neutrophils seen on their blood smears. In contrast, hypersegmented neutrophils were seen on less than 10% of the blood smears from of non‐IDA pediatric control group.

Investigators initially assumed the hypersegmented neutrophils observed in iron deficiency anemia was associated with either a concurrent vitamin B12 or folate deficiency. However, when the blood smears, from groups of IDA patients who did not have either of these nutritional deficiencies were evaluated, hypersegmented neutrophils were often still present. Further evidence of the relationship between IDA and hypersegmented neutrophils is the observation that when the IDA is corrected with iron supplementation treatment, hypersegmented neutrophils are no longer observed. Still, although the relationship between the two is documented, it is not yet clear what biologic mechanism(s) is responsible for the phenomenon. With this information in mind, it is best to not automatically assume that the presence of hypersegmented neutrophils on the blood smear automatically means the patient is either folate or vitamin B12 deficient. In addition to the low hematocrit and hemoglobin, the CBC also revealed an elevated platelet n count. As previously mentioned, in this particular laboratory, if certain abnormalities were detected on the initial CBC/Diff request for a patient, the QA procedure automatically included a smear review. Since this was the initial CBC/Diff ordered for this patient, his low Hct/Hgb and high platelet count triggered this additional review. The elevated platelet count, including some clumping, was confirmed microscopically

Thrombocytosis, defined as a platelet count above the normal range of 150‐400 X 109/L, may be either primary thrombocytosis or secondary (reactive) thrombocytosis. Primary thrombocytosis results from abnormally regulated platelet production. Examples of these types of disorders include polycythemia, CLL, and TTP.

Secondary thrombocytosis, also referred to a reactive thrombocytosis, is a benign process. This patient has developed secondary (reactive) thrombocytosis probably as a result of his iron deficiency anemia. Platelets are acute‐phase reactants and will increase in response to various stimuli, including, but not limited to, the following, systemic infections, inflammatory conditions, tumors, allergic reactions, bleeding, chronic kidney failure or another kidney disorders, post splenectomy, exercise, heart attack, hemolytic anemia, pancreatitis and burns. Secondary (reactive) thrombocytosis can also occur as a response to exposure to certain medications, for example, epinephrine (Epi‐pen), Retin‐A, Vincristine and heparin sodium.

Secondary (reactive) thrombocytosis is a relatively common condition. No age, gender, other than women who are pregnant, or race predilection exists for secondary thrombocytosis. In addition to those previously noted, other conditions associated with secondary thrombocytosis include trauma, hemorrhage, blood loss and, as is the case for this patient, iron deficiency anemia. Cases of thrombocytosis for which there is no clear clinical cause are termed, idiopathic thrombocytosis. Treatment of secondary (reactive) thrombocytosis focuses on resolving the underlying cause. In general, no treatment is indicated to directly reduce the platelet count. However, to minimize the chance of a stroke, patients with platelet counts in excess of 1,000 X 109/L, usually are advised to

Specimen 1 Specimen 2 Specimen 3 Specimen 4 Specimen 5 Code ‐ Result No. Flag Code ‐ Result No. Flag Code ‐ Result No. Flag Code ‐ Result No. Flag Code ‐ Result No. Flag 631 ‐ Hypochromic 350 671 ‐ Hypersegmentated Neutroph 324 692 ‐ Platelet Clumping 353 680 ‐ Lymphocyte, normal 331 684 ‐ Monocyte, any stage 343 632 ‐ Macrocytic 2 *** 667 ‐ Segmented Neutrophil (PMN, 20 *** 690 ‐ Platelet, normal 4 *** 681 ‐ Lymphocyte; atypical, Downey, v 8 *** 682 ‐ Lymphocyte, reactive 5 *** 633 ‐ Microcytic 2 *** 661 ‐ Eosinophil, any stage 5 *** 693 ‐ Platelet Satellitosis around 1 *** 682 ‐ Lymphocyte, reactive 8 *** 605 ‐ Monocyte, normal 3 610 ‐ Abnormal, would refer 1 674 ‐ PMN with / 4 *** Total Population 357 610 ‐ Abnormal, would refer 2 *** 610 ‐ Abnormal, would refer 1 *** 629 ‐ Dimorphic RBC 1 *** 660 ‐ Basophil, any stage 2 *** Intended result: Platelet Clumping 687 ‐ Megakaryoblast 2 *** 662 ‐ Metamyelocyte 1 *** 630 ‐ Elliptocyte/Ovalocyte 1 *** 610 ‐ Abnormal, would refer 1 613 ‐ Abnormal Lymphocyte, would re 1 *** 674 ‐ PMN with Toxic Granulation/Vac 1 *** 641 ‐ Target Cell () 1 *** 631 ‐ Hypochromic 1 *** 646 ‐ Nucleated RBC, any stage 1 *** 680 ‐ Lymphocyte, normal 1 *** Total Population 357 673 ‐ PMN with Dohle Bodies 1 *** 664 ‐ Myelocyte 1 *** 681 ‐ Lymph; atyp, Downey, variant 1 *** Intended result: Hypochromic Total Population 358 671 ‐ Hypersegmentated Neutrophil 1 *** Total Population 356 Intended result: Hpersegmented Neut 678 ‐ Blast, undifferentiated 1 *** Intended result: Monocyte, any stage 683 ‐ Hairy Cell 1 *** 685 ‐ , any stage 1 *** Total Population 358 Intended result: Lymphocyte, normal

Correct responses are defined as those reflecting agreement among 80% or more of all participants or referees. Unacceptable responses are indicated by "***" on the Flagging line of each specimen. SECOND QUADRIMESTER 2015

EDUCATIONAL CHALLENGES

Specimen 1 No. Specimen 2 No. 674 ‐ PMN wToxic Gran/Vacuol 97 646 ‐ Nucleated RBC, any stage 181 672 ‐ PMN with bactl inclusion 33 611 ‐ Immature RBC, would refer 4 616 ‐ Abnl Gran, would refer 26 680 ‐ Lymphocyte, normal 4 661 ‐ Eosinophil, any stage 10 672 ‐ PMN with bacterial inclusion 2 667 ‐ Seg Neut (PMN, poly) 5 674 ‐ PMN with Toxic Granulation/Vacuolization 2 673 ‐ PMN with Dohle Bodies 5 681 ‐ Lymphocyte; atypical, Downey, variant 2 610 ‐ Abnormal, would refer 4 682 ‐ Lymphocyte, reactive 2 660 ‐ Basophil, any stage 4 603 ‐ Segmented Neutrophil (PMN, poly) 1 676 ‐ PMN Pelger‐Huet Nucleus 3 683 ‐ Hairy Cell 1 602 ‐ Eosinophil, any stage 2 691 ‐ Platelet, giant 1 675 ‐ PMN with Degen Nucleus 2 Total Population: 0 611 ‐ Imme RBC, would refer 1 Intended result: Nucleated RBC 618 ‐ Bacteria 1 646 ‐ Nucleated RBC, any stage 1 650 ‐ Basophilic Stippling 1 685 ‐ Plasma Cell, any stage 1 Total Population: 196 Intended result: Giant granules

Sample 15Q3‐1 & Sample 15Q3‐2: A 5‐year‐old child is brought to the Emergency Department with a fever of 104.2F. On physical examination he is noted to have very fair skin with scattered ecchymoses, silvery‐blond hair, and light blue‐gray eyes. Ocular examination shows nystagmus, reduced vision, and marked sensitivity to bright light. He is unable to walk without stumbling and has an unsteady gait. His parents state he has frequent nosebleeds and episodes of pneumonia. CBC results: WBC 7,400/µL, Hgb 12.1 g/dL, Hct 38.5%, Plts 56,000/µL. Identify the indicated cells.

The automated CBC values show mild to moderate thrombocytopenia. Review of the peripheral blood smear shows a significant amount of background debris and staining. There is mild to moderate aniso‐ and with , polychromasia, and mild formation. The WBC distribution is significant for an increased number of immature cells. Most striking is the presence of large inclusions in many of the neutrophils. The cells to be identified in Specimen 1 are neutrophils containing these inclusions. These are not bacterial inclusions or vacuoles, but rather giant granules. Although vacuoles are seen in Cell #3, they are not apparent in Cells #1 and #2, whereas the granules are prominent. These granules can be confused with bacteria within the neutrophil cytoplasm and correlation with the clinical history is important. In addition, the granules vary in size from cell to cell; intracellular bacteria tend to be of a consistent size and shape. The cells to be identified in Specimen 2 are nucleated red blood cells (NRBC). This child has Chediak‐Higashi Syndrome. Chediak‐Higashi Syndrome (CHS) is a very rare genetic disorder resulting in partial albinism and abnormalities of the immune and nervous systems. There are fewer than 500 reported cases worldwide; all races are affected. There are two forms of CHS: the classic form is present at birth or within a couple of months of birth; the late‐onset form occurs later in childhood or early adulthood. The mean age of onset is 5.8 years. The classic form of CHS is more severe in terms of pigmentation changes and recurrent infections; however, individuals with the late‐onset form are more at risk for profound neurological problems, including weakness and decreased sensation in the arms and legs (peripheral neuropathy), and decreased intellectual functioning.

CHS is an autosomal recessive inherited disorder and occurs due to a defect in a specific gene, called the LYST gene. Both parents must pass the defective gene to a child in order for CHS to occur. These heterozygous carriers of the gene are not affected and typically show no signs or symptoms of CHS. The LYST gene is responsible for making a protein that enables the body to transport various materials to lysosomes. Lysosomes are present within many cells of the body and work as “recycling centers”, producing enzymes that kill bacteria, break down various toxins, and remove debris within cells. The defective LYST gene results in abnormal lysosomal function – cellular lysosomes become extremely large and interfere with functioning of the other organelles in the cells. Neutrophils, lymphocytes (particularly natural killer cells) and macrophages are no longer able to adequately sequester and kill bacteria, resulting in repeated, severe infections of the lungs, skin, and mucous membranes. Children are susceptible to infection by gram‐positive and gram‐negative bacteria, fungi, and viruses. The most common agent causing infections is Staphylococcus aureus, however, Haemophilus influenza, Streptococcus pyogenes, and Streptococcus pneumoniae have also been seen.

Cell ID ‐ Page 2 of 3 SECOND QUADRIMESTER 2015

Melanin, the pigment that gives color to the skin, hair, and eyes, is housed in cells known as melanosomes, which produce and distribute the melanin to these structures. The LYST gene defect also affects the melanosomes, with trapping and subsequent destruction of melanin within giant melanosomes, rather than allowing it to be released. In CHS, this results in what is termed oculocutaneous albinism – abnormal pigmentation of the skin, hair, and eyes. Children with CHS have very light‐colored hair with a silvery or metallic sheen, light‐colored (usually blue) eyes, and a pale or gray skin tone with patchy areas of nonpigmented skin. These children also have several vision problems, including increased sensitivity to light (photophobia), blurring of edges around objects, and involuntary eye movements (nystagmus).

On the peripheral blood smear, WBCs contain “giant ”. During neutrophil maturation, normal granules progressively fuse together to form prominent mega‐granules. Consequently, the number of true, functional granules is reduced. In neutrophils, these large granules stain a pale slate‐gray to gray‐green to dark red‐purple and are peroxidase‐positive, indicating they represent fusion of primary granules. There are often increased numbers of autophagic valuoles. Although the neutrophils may have relatively normal phagocytic abilities, they display defective chemotaxis and degranulation, and delayed killing of bacteria The giant granules are present in the cytoplasm of essentially all cells which contain granules, including monocytes and lymphocytes. Neutropenia and anemia are common; hence nucleated red blood cells can be seen. Many individuals with CHS also have coagulation abnormalities. Abnormal lysosome‐like structures have been found within platelets as well as a deficiency of platelet dense‐granules, leading to prolonged bleeding times, easy bruising and abnormal bleeding.

Diagnosis of CHS is established by demonstrating characteristic eosinophilic peroxidase‐positive giant granules in leukocytes, either in the bone marrow or the peripheral blood. Rarely, giant granules (pseudo‐Chediak‐Higashi granules) may be seen in the WBCs of patients with acute leukemias (AML, in particular) and chronic myelodysplastic disorders. These granules are less

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