Report on the International Society for Laboratory Hematology Survey on guidelines to support clinical hematology laboratory practice.

INTRODUCTION: Given the importance of evidence-based guidelines in health care, we surveyed the laboratory hematology community to determine their opinions on guideline development and their experience and interest in developing clinical hematology laboratory practice guidelines. METHODS: The study was conducted using an online survey, distributed to members of the International Society for Laboratory Hematology (ISLH) in 2015, with analysis of collected, anonymized responses. RESULTS: A total of 245 individuals participated. Most worked in clinical and/or research laboratories (83%) or industry (11%). 42% felt there were gaps in current guidelines. The majority (58%) recommended that ISLH engages its membership in guideline development. Participants differed in their familiarity with, and use of, different organizations' guidelines. Participants felt it was important to follow best practice recommendations on guideline development, including engagement of experts, statement about conflict of interests and how they were managed, systematic review and grading evidence for recommendations, identifying recommendations lacking evidence or consensus, and public input and peer review of the guideline. Moreover, it was considered important to provide guidelines free of charge. Industry involvement in guidelines was considered less important. CONCLUSIONS: The clinical laboratory hematology community has high expectations of laboratory practice guidelines that are consistent with recent recommendations on evidence-based guideline development.

Assembly and evaluation of an inventory of guidelines that are available to support clinical hematology laboratory practice.

INTRODUCTION: Practice guidelines provide helpful support for clinical laboratories. Our goal was to assemble an inventory of publically listed guidelines on hematology laboratory topics, to create a resource for laboratories and for assessing gaps in practice-focused guidelines. METHODS: PubMed and website searches were conducted to assemble an inventory of hematology laboratory-focused guidelines. Exclusions included annual, technical, or collaborative study reports, clinically focused guidelines, position papers, nomenclature, and calibration documents. RESULTS: Sixty-eight guidelines were identified on hematology laboratory practice topics from 12 organizations, some as joint guidelines. The median year of publication was 2010 and 15% were >10 years old. Coagulation topics had the largest numbers of guidelines, whereas some areas of practice had few guidelines. A minority of guidelines showed evidence of periodic updates, as some organizations did not remove or identify outdated guidelines. CONCLUSIONS: This inventory of current practice guidelines will encourage awareness and uptake of guideline recommendations by the worldwide hematology laboratory community, with the International Society for Laboratory Hematology facilitating ongoing updates. There is a need to encourage best guideline development practices, to ensure that hematology laboratory community has current, high-quality, and evidence-based practice guidelines that cover the full scope of hematology laboratory practice.

ICSH recommendations for the standardization of nomenclature and grading of peripheral blood cell morphological features.

These guidelines provide information on how to reliably and consistently report abnormal red blood cells, white blood cells and platelets using manual microscopy. Grading of abnormal cells, nomenclature and a brief description of the cells are provided. It is important that all countries in the world use consistent reporting of blood cells. An international group of morphology experts have decided on these guidelines using consensus opinion. For some red blood cell abnormalities, it was decided that parameters produced by the automated haematology analyser might be more accurate and less subjective than grading using microscopy or automated image analysis and laboratories might like to investigate this further. A link is provided to show examples of many of the cells discussed in this guideline.

Bone marrow evaluation for pediatric patients.

Due to the immaturity of the hematopoietic system at birth and different oxygenation and immune response needs of the growing organism, the bone marrow composition at birth and early infancy differs as compared to older children and adults. These age-related differences, while generally recognized, are not well known to the world of hematopathology. The purpose of this article is to address the current limitation of the literature by reviewing the bone marrow ontology, its composition at birth, and the changes occurring during early infancy, and to compare these findings to adults. The review also provides a useful framework for bone marrow examination in children.

Case-control study of risk factors for the development of post-transplant lymphoproliferative disease in a pediatric heart transplant cohort.

PTLD is an important complication following heart transplantation. To better define the risk factors of PTLD in children, we performed a case-control study. All pediatric cardiac transplant recipients who developed their first episode of PTLD were matched by age (+/-1 yr) and time since transplant (+/-1 yr) with those who did not. PTLD occurred in nine of 95 cardiac transplant recipients (9%), 0.3-7.8 yr following cardiac transplantation (median = 2.5 yr). Patients were 0.1-16.4 yr (median = 3.7) at transplantation. Biopsies revealed polymorphic B cell hyperplasia (three), polymorphic B cell lymphoma (one), monomorphic diffuse large cell B cell lymphoma (three) and monomorphic Burkitt's-like lymphoma (two). Patients who developed PTLD were at no greater risk of death (p = 0.31). Recipient EBV seronegativity at time of transplant (p = 0.08), EBV seroconversion (p = 0.013) and recipient CMV seronegativity (p = 0.015) were associated with the development of PTLD by conditional logistic regression; sex, race, donor age, recipient diagnosis, donor CMV seropositivity, recipient treatment for CMV infection, EBV seropositivity at the time of PTLD diagnosis, and number of rejection episodes, treated rejection episodes, and lympholytics used were not. There was no significant association between PTLD and death in our recipients. EBV seroconversion and recipient CMV seronegativity were associated with the development of PTLD.

Monosomy 7 associated with pediatric acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS): successful management by allogeneic hematopoietic stem cell transplant (HSCT).

Pediatric acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) with monosomy 7 is associated with poor disease-free survival when treated by conventional chemotherapy, immunosuppression or supportive measures. Hematopoietic stem cell transplant (HSCT) may improve outcomes; however, data to support this are limited. To better understand the curative potential of HSCT in these patients, all cases of AML and MDS with monosomy 7 treated by two transplant programs (1992 to present) were reviewed. A total of 16 patients were treated, all by allogeneic HSCT. Primary diagnoses were MDS (N = 5), therapy-related MDS (N = 3), AML (N = 5) and therapy-related AML (N = 3). In all, 11 patients (69%) survive event-free at 2 years with median follow-up of 986 days (range 330-2011 days). Toxicity caused deaths of the five nonsurviving patients, four of whom were transplanted with active leukemia. Allogeneic HSCT is effective therapy for childhood AML and MDS associated with monosomy 7, particularly for patients with AML in complete remission and MDS.

BLM, the Bloom's syndrome protein, varies during the cell cycle in its amount, distribution, and co-localization with other nuclear proteins.

BLM, the protein encoded by the gene mutated in Bloom's syndrome (BS), is a phylogenetically highly conserved DNA helicase that varies in amount and distribution in the nucleus during the cell-division cycle. It is undetectable in many cells as they emerge from mitosis but becomes abundant during G(1) and remains so throughout S, G(2), and mitosis. BLM is widely distributed throughout the nucleus but at certain times also becomes concentrated in foci that vary in number and size. It co-localizes transitorily with replication protein A (RPA) and promyelocytic leukemia protein (PML) nuclear bodies, and at times it enters the nucleolus. The observations support the hypothesis that BLM is distributed variously about the nucleus to manipulate DNA in some, very possibly several, nucleic acid transactions, when and where they take place. The specific transaction(s) remain to be identified. Although absence from the nucleus of functional BLM - the situation in BS - obviously is not lethal in the human, other helicases would appear to be unable to substitute for it completely, witness the hypermutability and hyperrecombinability of BS cells.

Transfection of BLM into cultured bloom syndrome cells reduces the sister-chromatid exchange rate toward normal.

The gene BLM, mutated in Bloom syndrome (BS), encodes the nuclear protein BLM, which when absent, as it is from most BS cells, results in genomic instability. A manifestation of this instability is an excessive rate of sister-chromatid exchange (SCE). Here we describe the effects on this abnormal cellular phenotype of stable transfection of normal BLM cDNAs into two types of BS cells, SV40-transformed fibroblasts and Epstein-Barr virus (EBV)-transformed lymphoblastoid cells. Clones of BLM-transfected fibroblasts produced normal amounts of BLM by western blot analysis and displayed a normal nuclear localization of the protein by immunofluorescence microscopy. They had a mean of 24 SCEs/46 chromosomes, in contrast to the mean of 69 SCEs in controls transfected only with the vector. BLM-transfected fibroblast clones that expressed highest levels of the BLM protein had lowest levels of SCE. The lymphoblastoid cells transfected with BLM had SCE frequencies of 22 and 42 in two separate experiments in which two different selectable markers were used, in contrast to 57 and 58 in vector-transfected cells; in this type cell, however, the BLM protein was below the level detectable by western blot analysis. These experiments prove that BLM cDNA encodes a functional protein capable of restoring to or toward normal the uniquely characteristic high-SCE phenotype of BS cells.

The DNA helicase activity of BLM is necessary for the correction of the genomic instability of bloom syndrome cells.

Bloom syndrome (BS) is a rare autosomal recessive disorder characterized by growth deficiency, immunodeficiency, genomic instability, and the early development of cancers of many types. BLM, the protein encoded by BLM, the gene mutated in BS, is localized in nuclear foci and absent from BS cells. BLM encodes a DNA helicase, and proteins from three missense alleles lack displacement activity. BLM transfected into BS cells reduces the frequency of sister chromatid exchanges and restores BLM in the nucleus. Missense alleles fail to reduce the sister chromatid exchanges in transfected BS cells or restore the normal nuclear pattern. BLM complements a phenotype of a Saccharomyces cerevisiae sgs1 top3 strain, and the missense alleles do not. This work demonstrates the importance of the enzymatic activity of BLM for its function and nuclear localization pattern.

The Ashkenazic Jewish Bloom syndrome mutation blmAsh is present in non-Jewish Americans of Spanish ancestry.

Bloom syndrome (BS) is more frequent in the Ashkenazic Jewish population than in any other. There the predominant mutation, referred to as "blmAsh," is a 6-bp deletion and 7-bp insertion at nucleotide position 2281 in the BLM cDNA. Using a convenient PCR assay, we have identified blmAsh on 58 of 60 chromosomes transmitted by Ashkenazic parents to persons with BS. In contrast, in 91 unrelated non-Ashkenazic persons with BS whom we examined, blmAsh was identified only in 5, these coming from Spanish-speaking Christian families from the southwestern United States, Mexico, or El Salvador. These data, along with haplotype analyses, show that blmAsh was independently established through a founder effect in Ashkenazic Jews and in immigrants to formerly Spanish colonies. This striking observation underscores the complexity of Jewish history and demonstrates the importance of migration and genetic drift in the formation of human populations.

A rapid method for detecting the predominant Ashkenazi Jewish mutation in the Bloom's syndrome gene.

Bloom's syndrome (BS) is a rare, autosomal recessive disease characterized by sun sensitivity, short stature, and predisposition to cancer. Although rare in the general population, BS is more common in the Ashkenazi Jewish population (German, 1993). The isolation of the gene for BS, known as BLM, has permitted the identification of mutations within the gene and the discovery that most BS individuals of Ashkenazi Jewish origin carry the identical 6-bp deletioin/7-bp insertion at position 2,281 of BLM (blmAsh). We have developed a rapid method for detecting blmAsh based on restriction enzyme digestion of a PCR product containing the mutation. blmAsh creates a restriction site within the amplified fragment allowing distinction of normal and mutant DNAs. This method has been designed for use with genomic DNA or cDNA.

Physical mapping of the bloom syndrome region by the identification of YAC and P1 clones from human chromosome 15 band q26.1.

The gene for Bloom syndrome (BLM) has been mapped to human chromosome 15 band q26.1 by homozygosity mapping. Further refinement of the location of BLM has relied upon linkage-disequilibrium mapping and somatic intragenic recombination. In combination with these mapping approaches and to identify novel DNA markers and probes for the BLM candidate region, a contiguous representation of the 2-Mb region that contains the BLM gene was generated and is presented here. YAC and P1 clones from the region have been identified and ordered by using previously available genetic markers in the region along with newly developed sequence-tagged sites from radiation-reduced hybrids, polymorphic dinucleotide repeat loci, and end sequences of YACs and P1s. A long-range restriction map of the 2-Mb region that allowed estimation of the distance between polymorphic microsatellite loci is also reported. This map and the DNA markers derived from it were instrumental in the recent identification of the BLM gene.

Bloom's syndrome. XIX. Cytogenetic and population evidence for genetic heterogeneity.

Cells with abnormally high rates of sister-chromatid exchange (SCE) are uniquely characteristic of Bloom's syndrome (BS). However, in one in five persons a minor population of cells with a low-SCE phenotype circulates in the blood. The origin and significance of the low-SCE cells in BS have never been understood, although they are assumed to arise by somatic mutation. In the present investigation, the enigmatic high-SCE/low-SCE mosaicism was investigated by comparing the incidence in several subpopulations of persons in the Bloom's Syndrome Registry who exhibit the two types of cells, and a striking negative correlation emerged: in persons with BS whose parents share a common ancestor, the case in approximately half of registered persons, low-SCE cells are found only rarely; conversely, the mosaicism occurs almost exclusively in persons with BS whose parents are not known to share a common ancestor. Because those who share a common ancestor are predominantly homozygous-by-descent at the mutated BS locus, the negative correlation is interpreted to mean that the emergence of low-SCE cells in BS in some way depends on the pre-existence of compound heterozygosity. A corollary to this is that BS is genetically heterogeneous.

The Bloom's syndrome gene product is homologous to RecQ helicases.

The Bloom's syndrome (BS) gene, BLM, plays an important role in the maintenance of genomic stability in somatic cells. A candidate for BLM was identified by direct selection of a cDNA derived from a 250 kb segment of the genome to which BLM had been assigned by somatic crossover point mapping. In this novel mapping method, cells were used from persons with BS that had undergone intragenic recombination within BLM. cDNA analysis of the candidate gene identified a 4437 bp cDNA that encodes a 1417 amino acid peptide with homology to the RecQ helicases, a subfamily of DExH box-containing DNA and RNA helicases. The presence of chain-terminating mutations in the candidate gene in persons with BS proved that it was BLM.

Somatic intragenic recombination within the mutated locus BLM can correct the high sister-chromatid exchange phenotype of Bloom syndrome cells.

Cells from persons with Bloom syndrome feature an elevated rate of sister-chromatid exchange (SCE). However, in some affected persons a minority of blood lymphocytes have a normal SCE rate. Persons who inherit the Bloom syndrome gene BLM identical by descent from a common ancestor very rarely exhibit this high-SCE/low-SCE mosaicism; conversely, mosaicism arises predominantly in persons who do not share a common ancestor. These population data suggested that most persons with Bloom syndrome in whom the exceptional low-SCE cells arise are not homozygous for a mutation at BLM but instead are compound heterozygotes. Following this clue, we carried out a genotype analysis of loci syntenic with BLM in 11 persons who exhibited mosaicism. In five of them, polymorphic loci distal to BLM that were heterozygous in their high-SCE cells had become homozygous in their low-SCE cells, whereas heterozygous loci proximal to BLM remained heterozygous. These observations are interpreted to mean that intragenic recombination between paternally derived and maternally derived mutated sites within BLM can generate a functionally wild-type gene and that low-SCE lymphocytes are progeny of a somatic cell in which such intragenic recombination had occurred.

Linkage disequilibrium between the FES, D15S127, and BLM loci in Ashkenazi Jews with Bloom syndrome.

Bloom syndrome (BS) is more common in the Ashkenazi Jewish than in any other population. Approximately 1 in 110 Ashkenazi Jews carries blm, the BS mutation. The locus mutated in BS, BLM, maps to chromosome subband 15q26.1, tightly linked to the proto-oncogene FES. We have investigated the basis for the increased frequency of blm in the Ashkenazim by genotyping polymorphic microsatellite loci tightly linked to BLM in affected and unaffected individuals from Ashkenazi Jewish and non-Ashkenazi populations. A striking association of the C3 allele at FES with blm (delta = .422; p = 5.52 x 10(-7)) and of the 145-bp and 147-bp alleles at D15S127 with blm (delta = .392 and delta = .483, respectively; p = 2.8 x 10(-5) and p = 5.4 x 10(-7), respectively) was detected in Ashkenazi Jews with BS. This linkage disequilibrium constitutes strong support for a founder-effect hypothesis: the chromosome in the hypothetical founder who carried blm also carried the C3 allele at FES and either the 145-bp or the 147-bp allele at D15S127.