PARTICIPANT STATISTICS CELL IDENTIFICATION THIRD QUADRIMESTER 2017

PROFICIENCY TESTING SERVICE

AMERICAN ASSOCIATION OF BIOANALYSTS

205 West Levee St. w Brownsville, TX 78520-5596800-234-5315 w 281-436-5357 w Fax 713-781-5008

Q3-2017 - PBSC donor

This patient is a healthy individual who has volunteered to be a peripheral blood stem cell (PBSC) donor for a patient being treated for leukemia. This donor is not related to the patient. She was identified from a bone marrow donor registry.Established in 1986 as a nonprofit organization, The National Marrow Donor Program (NMDP) supports the largest registry in the United States. When a volunteer donor registers with the NMDP, their HLA and contact information is sent to the NMDP, which stores it in their computers. To date, this registry, appropriately named, Be The Match, has 16 million US based registry members. Additionally they have access to 225,000 preserved cord blood units. They have facilitated 80,000 transplants and this number is increasing by over 6,000 every year.

Hematopoietic ("blood-forming") cells are non-embryonic stem cells that can multiply and differentiate into red blood cells, white blood cells, and platelets. These are what are actually transfused during the procedure referred to as bone marrow transplantation or hematopoietic cell transplantation (HCT). Patients with life-threatening disorders such as leukemia, lymphoma, aplastic anemia, as well as certain immune system and metabolic disorders are the most frequent recipients of hematopoietic stem cell transplantation. Hematopoietic cells can come from bone marrow, the circulating blood or umbilical cord blood.

Bone marrow is extracted from the donor's pelvic bones while the donor is under general or local anesthesia. Peripheral blood stem cells (PBSC) are collected, via apheresis, from the donor's blood after five days of taking a drug that causes hematopoietic cells in the bone marrow to move into the circulating blood. In both cases, recovery is usually swift and donors typically have fully restored marrow and blood cell counts in under two weeks. Cord blood cells are obtained from the umbilical cord and placenta of a newborn baby after the cord is clamped and cut as in a normal delivery. The cord blood is then stored frozen in a bank until needed for a transplant.

To increase the likelihood of successful engraftment, the donor tissue must be as closely matched as possible to the recipient. Matching is defined as having the same Humans Leukocyte Antigens (HLA). There is a group of genes present in all animals called the major histocompatibility complex (MHC). In humans the MHC is called human leukocyte antigen (HLA). They are proteins located on the surface of WBCs and other tissues in the body. They are one of the tools of the normal human immune system uses to identify foreign invaders such as bacteria viruses and parasites. The more HLA antigens shared between a recipient and donor, the better the potential outcome of the transplantation.

Finding an HLA-matched donor can be difficult because there are so many different HLA antigens, or proteins. Statistically there are billions of different HLA types possible. Because there is a 1-in-4 chance of being identical to a sibling, the best potential for an HLA match is within the family. Unfortunately, only about 30% of patients with diseases treatable with hematopoietic cell transplantation can find a suitably HLA matched donor among their family members.

The other 70% of patients must be matched to an unrelated hematopoietic cell donor. The odds that two random individuals are HLA matched exceeds one in 20,000. This is where large registries of unrelated donors are so important, a registry's success depends on a large number of volunteer donors. Physicians look for donor material on behalf of a patient by submitting the patient’s HLA tissue type to the NMDP, which then searches its computerized database for matching donor (marrow or PBSC) or cord blood units. Depending on the results of the search, the transplant physician evaluating the patient decides whether to use an adult donor's marrow, PBSC, or cord blood for a particular patient.

If the NMDP finds a match with a donor, the potential donor is educated regarding the specifics of the donation process and then NMDP request they donate. If the potential donor wishes to proceed, they are further screened with a thorough medical exam including an extensive battery of blood tests designed to clear them of infectious diseases.

Historically bone marrow collection had been the primary source of donor tissue. Over the last decade PBSC has replaced bone marrow as the predominant means by which donor stem cells are collected. PBSC are collected from the blood through a process known as apheresis. For five days prior to apheresis, the donor receives daily subcutaneous injections of granulocyte-colony stimulating factor. Functionally, both a cytokine and hormone, granulocyte-colony stimulating factor is a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream. In our case study, the donor patient received filgrastim, a pharmaceutical analog of naturally occurring G-CSF.

The peripheral blood picture for this patient is typical for someone receiving G-CSF treatment. The WBC count is elevated, there are some immature granulocytes present, and a few granulocytes demonstrate toxic granulation or Dohle bodies. These light gray-blue cytoplasmic inclusions are composed of agglutinated ribosomes. Dohle bodies, toxic granulation, and cytoplasmic basophilia are markers for inflammation and active granulocytopoiesis. Several days of this medication serves to mobilize stem cells from the donor's bone marrow into their peripheral circulation. This increases the numbers of circulating peripheral stem cells, which typically peak after 5 days of filgrastim. On day 5, the donor’s CD34+ stem cell levels are measured using flow cytometry. Provided that the stem cell counts are sufficiently high, apheresis is then performed. The purpose of this stepwise process is to insure that the recipient receives as many stem cells as possible. The higher the number of stem cells the recipient receives, the greater the chance of a successful engraftment.

During the apheresis, the donor's blood is withdrawn through a sterile needle in one arm and is circulated though a machine which removes the stem cells and returns the remaining blood and plasma back to the donor through another needle inserted into the opposite arm. PBSC harvest apheresis is a safe procedure. However, a few side effects have been reported in some donors including some associated with filgrastim administration (local reactions, pain, flulike symptoms), complications associated with placement of a central venous catheter when peripheral access is inadequate (infection, bleeding, pneumothorax), and/or (3) problems with apheresis (bleeding secondary to anticoagulation, hypocalcemia due to ACD). In addition, the NMDP has an ongoing study addressing the theoretic concern of whether filgrastim causes an increase in risk of leukemia in donors.

17Q3 Immunohematology Page 1 of 3

Cell Identification

Specimen 1 Specimen 2 Specimen 3 Specimen 4 Specimen 5

Result No. Flag Result No. Flag Result No. Flag Result No. Flag Result No. Flag

Lymphocyte, normal 153 Hypersegmentated Neutrophil 266 Erythrocyte, normal RBC 230 Eosinophil, any stage 270 PMN with Dohle Bodies 220

Lymphocyte, reactive 46 *** Segmented Neutrophil (PMN, poly) 6 *** Hypochromic 21 *** Abnormal Granulocyte, would refer 1 *** Immature Neutrophil 20 ***

Lymphocyte; atypical, Downey, variant 19 *** Erythrocyte, normal RBC 16 PMN with Toxic Granulation/Vacuolization 1 *** Segmented Neutrophil (PMN, poly) 15

Abnormal Lymphocyte, would refer 15 *** Macrocytic 2 *** PMN with Toxic Granulation/Vacuolization 6 ***

Myelocyte 10 *** Eosinophil, any stage 1 *** PMN with bacterial inclusion 3 ***

Promyelocyte 8 *** Abnormal, would refer 2 ***

Monocyte, any stage 4 *** Abnormal Granulocyte, would refer 1 ***

Myeloblast 3 *** Myeloblast 1 ***

Lymphocyte, abnormal/atypical 3 *** Auer Rods (myeloblast) 1 ***

Abnormal, would refer 2 *** Hypersegmentated Neutrophil 1 ***

Immature WBC, would refer 2 *** PMN with Degenerated Nucleus (pyknotic PMN) 1 ***

Abnormal Granulocyte, would refer 2 ***

Monocyte, normal/any stage 1 ***

Nucleated RBC, any stage 1 ***

Metamyelocyte 1 ***

Hypersegmentated Neutrophil 1 ***

PMN with Toxic Granulation/Vacuolization 1 ***

Total Population 272 Total Population 272 Total Population 270 Total Population 272 Total Population 271

Intended result: Lymph, normal Intended result: Hypersegmented PMN Intended result: Normal RBC Intended result: Eosinophl Intended result: PMN with Dohle Body

18 of 22 Referee Laboratories correctly identified

the intended result of 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.

The recipient receives the collected donor tissue intravenously. The transfused hematopoietic stem cells then migrate to the recipient's bone marrow, a process known as stem cell homing, where the transplanted cells override the previous bone marrow. Engraftment of the stem cells occurs when the donated cells begin producing new blood cells. Depending on the type of transplant and the disease being treated, engraftment usually occurs between 15 and 30 days post transplant. Engraftment can be delayed because of infection, medications, recurrent disease, low donated stem cell count, or graft failure.

Another extremely serious potential complication is Graft-versus-host disease (GVHD). GVHD occurs when the donor's immune system reacts against the recipient's tissue. As opposed to an organ transplant where the patient's immune system will attempt to reject only the transplanted organ, in GVHD the new or transplanted immune system can attack the entire patient and all of his or her organs. This is because the new cells do not recognize the tissues and organs of the recipient's body as self. Over time and with the help of medicines to suppress the new immune system, it will begin to accept its new body and stop attacking it. The most common sites for GVHD are GI tract, liver, skin, and lungs. After HSCT it can take months, even years, for the entire immune system to fully recover.

Hematopoietic stem cell transplantation remains a dangerous procedure with many possible complications; it is reserved for patients with life-threatening diseases. As with any procedure, in bone marrow transplant the prognosis and long-term survival can vary greatly from person to person. New methods to improve treatment and to decrease complications and side effects of a HSCT are continually being discovered. As survival following the procedure has increased, its use has expanded beyond cancer to autoimmune diseases and hereditary skeletal dysplasias; notably malignant infantile osteopetrosis and mucopolysaccharidosis.

17Q3 Immunohematology Page 2 of 3

Cell Identification - Educational Challenge

Specimen 1 No. Specimen 2 No.Platelet Clumping 185 Blast, undifferentiated 62Abnormal Platelet, would refer 4 Lymphocyte, reactive 49Platelet, normal 2 Lymphocyte; atypical, Downey, variant 20Segmented Neutrophil (PMN, poly) 1 Immature WBC, would refer 12Lymphocyte, reactive 1 Abnormal Lymphocyte, would refer 12Megakaryocyte 1 Myeloblast 11

Abnormal, would refer 8Lymphocyte, abnormal/atypical 5Promyelocyte 3Monocyte, normal 2Metamyelocyte 2Lymphocyte, normal 3Immature Neutrophil 1Monocyte, any stage 1Platelet Satellitosis around PMN 1

Total Population: 207 Total Population: 207Intended result: Blast, undifferentiated

*To see the original fullsized immages, please sign on to your data entry sheet at http://www.aab-pts.org/

Intended result: Platelet Clumping

Sample 17Q3 - Clinical Discussion

Normal:

History: A 34-year-old woman is seen by here primary care physician for diarrhea associated with abdominal cramping. For the past two months she has been experiencing uncomfortable abdominal bloating after eating, followed by

cramping in her lower abdomen. Diarrhea stools may or may not occur. Although the bloating occurs after eating, the diarrhea “just comes on suddenly” and is having an impact on her quality of life as she is apprehensive about being

far from a restroom. She is otherwise healthy and in good physical condition. CBC results: WBC 6.1, Hgb 13.1 g/dL, Hct 42.4%, Plts 265,000/µL. Would you refer the slide for a pathologist’s review?

Hodgkin Lymphoma (Left-Shift):

A 56-year-old man is seen by his physician with complaints of increasing fatigue and night sweats. He has lost 10 pounds in the past month, stating that he doesn’t have an appetite; he denies dieting or excessive exercise. He is

employed as a math teacher at a local high school. His family history is significant for congestive heart failure in his mother and diabetes in his father and one brother. On physical examination, two enlarged lymph nodes are palpated in

his right axillae; there is no enlargement of the spleen or liver on palpation. CBC results: WBC 5.2 x 103/µL, Hgb 10.0 g/dL, Hct 29.8%, Plts 399,000/µL. Would you refer the slide for a pathologist’s review?

17Q3 Immunohematology Page 3 of 3