Update on programs for achieving Specialist in Blood Banking certification in the United States: 2023.

A person who has achieved the Specialist in Blood Banking (SBB) certification is a medical laboratory scientist who receives advanced training in blood banking and transfusion medicine and has passed an examination given by the American Society for Clinical Pathology. There are several pathways or "eligibility routes" to qualify for the examination to obtain SBB certification, with the most common route involving enrollment in a Commission on Accreditation of Allied Health Education Programs-accredited SBB program. The goal of this study was to compile information about the current accredited SBB programs in the United States and SBB exam statistics for purposes of assessing changes in the programs and detecting trends in SBB exam takers and pass rates. SBB program coordinators were surveyed about qualitative and quantitative aspects of their programs. Current data, changes over time, and nationally available data were tabulated for comparison. This information may be helpful for all medical laboratory scientists interested in considering further studies and certification in blood banking and transfusion medicine.

SpermCheck Fertility, an immunodiagnostic home test that detects normozoospermia and severe oligozoospermia.

BACKGROUND: A simple and inexpensive home sperm test could be of considerable value to couples attempting to conceive and to men curious about their fertility potential. A two-strip lateral flow immunochromatographic diagnostic device that allows men to evaluate their sperm count at low cost in the privacy of their own homes is described. METHODS: The ability of SpermCheck Fertility to predict sperm counts obtained using a hemacytometer procedure based on standard World Health Organization methodology was assessed. Test results obtained by lay users were also compared with those obtained by trained laboratory professionals, and the ease of use of the device was evaluated in consumer studies. RESULTS: A total of 225 semen samples were analyzed in the method comparison, and the performance of SpermCheck Fertility was excellent with over 96% of all samples correctly classified as normozoospermic (> or =2 x 10(7) sperm/ml), oligozoospermic (5 x 10(6)-2 x 10(7) sperm/ml) or severely oligozoospermic (<5 x 10(6) sperm/ml). Consumer studies with 164 lay users demonstrated that SpermCheck Fertility was easy to use. Lay users and laboratory professionals agreed 95% of the time when reading the same test independently. Overall, the correct response rate on a 20-question survey about the test was over 97%. CONCLUSIONS: SpermCheck Fertility is a simple and reliable immunodiagnostic test that can quickly inform men as to whether their sperm count is normal, low or very low. This home test can assist couples in deciding whether to seek comprehensive clinical evaluation of the fertility status of the male partner.

Equatorial segment protein (ESP) is a human alloantigen involved in sperm-egg binding and fusion.

The equatorial segment of the sperm head is known to play a role in fertilization; however, the specific sperm molecules contributing to the integrity of the equatorial segment and in binding and fusion at the oolemma remain incomplete. Moreover, identification of molecular mediators of fertilization that are also immunogenic in humans is predicted to advance both the diagnosis and treatment of immune infertility. We previously reported the cloning of Equatorial Segment Protein (ESP), a protein localized to the equatorial segment of ejaculated human sperm. ESP is a biomarker for a subcompartment of the acrosomal matrix that can be traced through all stages of acrosome biogenesis (Wolkowicz et al, 2003). In the present study, ESP immunoreacted on Western blots with 4 (27%) of 15 antisperm antibody (ASA)-positive serum samples from infertile male patients and 2 (40%) of 5 ASA-positive female sera. Immunofluorescent studies revealed ESP in the equatorial segment of 89% of acrosome-reacted sperm. ESP persisted as a defined equatorial segment band on 100% of sperm tightly bound to the oolemma of hamster eggs. Antisera to recombinant human ESP inhibited both oolemmal binding and fusion of human sperm in the hamster egg penetration assay. The results indicate that ESP is a human alloantigen involved in sperm-egg binding and fusion. Defined recombinant sperm immunogens, such as ESP, may offer opportunities for differential diagnosis of immune infertility.

Validation of a testis specific serine/threonine kinase [TSSK] family and the substrate of TSSK1 & 2, TSKS, as contraceptive targets.

A family of testis specific serine/threonine kinases, TSSK1-4 and SSTK, in addition to the substrate of TSSK1 & 2, TSKS, have been studied during the past several years in our laboratory. This paper will provide a general background on these kinases through review of pertinent literature and then will summarize data from our laboratory germane to evaluating these kinases as candidate targets for future development of small molecule kinase inhibitors that may serve to regulate male fertility. Bio-informatic and structural analyses of human TSSK1-4 and SSTK indicate that these kinases constitute a unique subfamily belonging to the AMPK branch on the human kinome tree. Expression studies showed that all five kinases and the TSKS substrate are testis abundant, if not strictly testis specific, indicating that tissue specific contraceptive targeting is possible. In situ hybridization further confirmed that mouse TSSK2, SSTK and TSKS are post-meiotic in their expression patterns, a finding that makes them possible targets of reversible contraceptive intervention by preserving spermatogonia and spermatocytes. Our laboratory detected TSSK2, TSKS and SSTK proteins in mature spermatozoa for the first time. TSKS was localized to the centrioles of human spermatozoa, while TSSK2 was observed in the sperm neck, equatorial segment and mid-piece of the sperm tail, and SSTK was localized in the equatorial segment. The interaction and binding between human TSSK2 and TSKS was confirmed by several methods: this substrate and enzyme interaction offers a particularly interesting opportunity for drug design. In vitro kinase assay showed phosphorylation of TSKS by TSSK2. The TSKS phosphopeptide, HGLSPATPIQGCSGPPGS*PEEPPR, was identified by IMAC-LC-FTMS, with serine 285 being phosphorylated (representend by asterisk). These results provide a rationale for high-throughput screening of inhibitors for TSKS phosphorylation and further studies of members of this kinase family as targets for both male contraception and intra-vaginal spermicides.

In search of red blood cells for alloimmunized patients with sickle cell disease.

Patients with sickle cell disease (SCD) typically require transfusions with RBC components, which exposes them to numerous, possible foreign antigens and potentially causes them to produce an antibody or antibodies to the antigens they lack. As transfusion of these patients increases, the likelihood that they will produce an initial antibody or additional antibodies increases. Once a clinically significant antibody is produced, units of RBCs that lack the associated antigen should be transfused. Often patients with SCD present to transfusion service with numerous antibodies in their serum, making the search for compatible RBCs a challenge. The American Rare Donor Program (ARDP) has been used to search for RBCs to meet transfusion needs of this patient population. Between January 2005 and June 2006, approximately 33 percent of the requests to the ARDP for RBC components were alloimmunized patients with SCD. Of these requests, 94.9 percent were completely or partially filled; requests for r"r", Hy-, and E-, hrS- units of RBCs were among the most difficult to fill. This article will discuss the use and effectiveness of the ARDP and testing laboratories associated with the National Reference Laboratory for Blood Group Serology at the American Red Cross in obtaining compatible RBCs for alloimmunized patients with SCD.

Hominoid-specific SPANXA/D genes demonstrate differential expression in individuals and protein localization to a distinct nuclear envelope domain during spermatid morphogenesis.

Human sperm protein associated with the nucleus on the X chromosome consists of a five-member gene family (SPANXA1, SPANXA2, SPANXB, SPANXC and SPANXD) clustered at Xq27.1. Evolved from an ancestral SPANX-N gene family (at Xq27 and Xp11) present in all primates as well as in rats and mice, the SPANXA/D family is present only in humans, bonobos, chimpanzees and gorillas. Among hominoid-specific genes, the SPANXA/D gene family is considered to be undergoing rapid positive selection in its coding region. In this study, RT-PCR of human testis mRNA from individuals showed that, although all SPANXA/D genes are expressed in humans, differences are evident. In particular, SPANXC is expressed only in a subset of men. The SPANXa/d protein localized to the nuclear envelope of round, condensing and elongating spermatids, specifically to regions that do not underlie the developing acrosome. During spermiogenesis, the SPANXa/d-positive domain migrated into the base of the head as the redundant nuclear envelope that protrudes into the residual cytoplasm. Post-testicular modification of the SPANXa/d proteins was noted, as were PEST (proline, glutamic acid, serine, and threonine rich regions) domains. It is concluded that the duplication of the SPANX-N gene family that occurred 6-11 MYA resulted in a new gene family, SPANXA/D, that plays a role during spermiogenesis. The SPANXa/d gene products are among the few examples of X-linked nuclear proteins expressed following meiosis. Their localization to non-acrosomal domains of the nuclear envelope adjacent to regions of euchromatin and their redistribution to the redundant nuclear envelope during spermiogenesis provide a biomarker for the redundant nuclear envelope of spermatids and spermatozoa.

A male genital tract-specific carbohydrate epitope on human CD52: implications for immunocontraception.

The identification of unique sperm surface epitopes that are not expressed or exposed in the female reproductive tract is a key element in the development of antibody-based contraceptives. Western blotting and immunohistochemistry were performed to define the tissue distribution of the S19 epitope, which has been proposed as a target for immunocontraception. S19 is an IgG1 murine monoclonal antibody (mAb) directed to an N-linked carbohydrate epitope on a 15-25 kDa glycoprotein, sperm agglutination antigen-1 (SAGA-1), containing a peptide core identical to that of the lymphocytic surface protein CD52. In this study, the S19 epitope was shown to be absent from human lymphocytes, demonstrating a distinction between this epitope and the CAMPATH epitope that is recognized by an antibody against the terminal tripeptide and GPI-anchor of CD52. Further tissue specificity analysis identified the S19 epitope in the epithelium of the human epididymis and vas deferens, as well as on both epididymal and ejaculated spermatozoa. In contrast, the S19 epitope was absent in the five human female reproductive tract and 18 other somatic tissues tested. These results support the use of the S19 epitope as a contraceptive immunogen and the suitability of the S19 mAb as an intravaginal contraceptive. To test the agglutinating activity of the S19 mAb in a formulation designed for vaginal use, S19 mAb were bound to the surface of Novasomes, a multilamellar liposome delivery vehicle. S19-Novasome formulations agglutinated human spermatozoa and were as effective as unbound S19 mAb, demonstrating the feasibility of spermistatic contraceptives targeted to the male reproductive tract specific carbohydrate epitope.

Co-localization of the inositol 1,4,5-trisphosphate receptor and calreticulin in the equatorial segment and in membrane bounded vesicles in the cytoplasmic droplet of human spermatozoa.

Modulation of the intracellular calcium concentration within mammalian spermatozoa is important in several pre-fertilization events including hyperactivated motility and the acrosome reaction. To identify calcium binding proteins (CBP) potentially regulating these processes, a (45)Ca overlay technique was employed on 2-D blots of human sperm extracts. Microsequencing by Edman degradation and CAD mass spectrometry identified a relatively abundant 60.5 kDa CBP with a pI of 4.2 as calreticulin (CRT). Immunofluorescent labelling with anti-CRT antibodies localized CRT to the acrosome, with highest fluorescence in the equatorial segment, and in the cytoplasmic droplets of 94 and 48% of human spermatozoa respectively. Double immunolabelling experiments demonstrated co-localization of CRT and the inositol 1,4,5-trisphosphate receptor (IP(3)R) in the acrosome, in the equatorial segment, and vesicular structures in the cytoplasmic droplets of the neck region. Electron microscopic immunogold labelling localized CRT to the equatorial segment of acrosome-reacted spermatozoa and to membrane-enclosed vesicles within the cytoplasmic droplet of both acrosome-intact and acrosome-reacted spermatozoa. Localization of the IP(3) receptor to the CRT-containing vesicles, in the sperm neck and to the acrosome, suggests that capacitative calcium entry in human spermatozoa may be regulated from these putative calcium storage sites.

Differential extraction and enrichment of human sperm surface proteins in a proteome: identification of immunocontraceptive candidates.

The objective of this study was to discover previously unknown human sperm surface proteins that may be candidate contraceptive vaccinogens. To this end, methods of concentrating human sperm proteins for microsequencing by mass spectrometry were used, which increased the likelihood of identifying surface proteins. Vectorial labeling, differential extraction and two-dimensional (2-D) gel electrophoresis were employed to identify and isolate proteins accessible at the cell surface. Percoll harvested or swim-up sperm were either solubilized directly or solubilized after surface labeling with sulfo-succinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC-biotin). Comparisons were made of proteins extracted with four lysis buffers: (i) Celis buffer containing 9.8 M urea and 2% Igepal CA-630; (ii) 1% Triton X (TX)-100; (iii) 1.7% TX-114 followed by phase partitioning; or (iv) 1 M NaCl. Blots of proteins separated by high-resolution 2-D electrophoresis were probed with avidin and antibodies to known proteins specific for three domains: the sperm surface (SAGA-1), the acrosome (SP-10), and the cytoskeleton (alpha-tubulin). Celis buffer (45 min) extracted proteins from all three major compartments. However, a 20-s extraction in Celis buffer enriched for several proteins and enabled the identification of several novel peptides by mass spectrometry. Mild extraction with TX-100 or 1 M NaCl solubilized mainly membrane and acrosomal proteins, but not cytoskeletal proteins. Comparison of biotinylated proteins extracted by each method showed that the major vectorially labeled proteins solubilized by Celis buffer were also solubilized by TX-100, TX-114, and 1 M NaCl. Extraction with TX-114 followed by phase-partitioning significantly enriched hydrophobic surface proteins and aided resolution and isolation. Eight protein spots microsequenced following all these extraction methods proved to be novel sperm molecules.

RASA, a recombinant single-chain variable fragment (scFv) antibody directed against the human sperm surface: implications for novel contraceptives.

BACKGROUND: A recombinant single-chain variable fragment (scFv) antibody was engineered to a tissue-specific carbohydrate epitope located on human sperm agglutination antigen-1 (SAGA-1), a sperm glycoform of CD52. METHODS AND RESULTS: cDNAs encoding the variable regions of the S19 [IgG(1)kappa] monoclonal antibody (mAb) were identified, linked, and cloned into the pCANTAB 5E vector. The recombinant anti-sperm antibody (RASA) was expressed in E. coli HB2151 cells as a 29 kDa monomer and, remarkably, also formed multimers of approximately 60 and 90 kDa. RASA reacted with the endogenous SAGA-1 antigen by Western blot analysis, labelled the entire human sperm surface by indirect immunofluorescence, and aggregated human spermatozoa in a tangled (head-to-head, head-to-tail, tail-to-tail) pattern of agglutination, as was also observed with the native S19 mAb. CONCLUSIONS: These results demonstrate that active recombinant antibodies can be produced to a tissue-specific carbohydrate epitope on the human sperm surface, thereby opening opportunities for novel contraceptive agents.

Outer dense fiber proteins are dominant postobstruction autoantigens in adult Lewis rats.

Obstruction of the male reproductive tract commonly results in generation of antisperm autoantibodies. However, only a few of the sperm autoantigens recognized by these antibodies have been characterized. To identify postobstruction rat sperm autoantigens, sperm proteins were separated by two-dimensional(2-D) gel electrophoresis. Spots corresponding to proteins that were stained by at least 50% of postvasectomy rat sera on 2-D Western blots were removed from polyacrylamide gels and microsequenced by tandem mass spectrometry. From a total of 21 spots, 12 contained peptides that matched solely to either of two outer dense fiber proteins, odf1 or odf2. Six additional spots contained peptides comprising odf1 or odf2 and were accompanied by peptides representing other proteins. Only three spots lacked outer dense fiber peptides but did contain sequences of other known proteins. The results indicate that the outer dense fiber proteins odf1 and odf2 are dominant postobstruction autoantigens because they were detected in the majority of the immunoreactive protein spots examined. Possible explanations for this observation include the abundance of outer dense fiber proteins in spermatozoa, slow solubility, which may provide a sustained supply of antigen, and testis-specific expression during spermiogenesis.

Differential nuclear localization of the cancer/testis-associated protein, SPAN-X/CTp11, in transfected cells and in 50% of human spermatozoa.

Cancer-testis antigens (CTAs) represent potential targets for cancer immunotherapy because these proteins are widely distributed in tumors but not in normal tissues, except testes. In this paper, we identify homology of the CTA CTp11 with SPAN-X (sperm protein associated with the nucleus mapped to the X chromosome). On two-dimensional Western blots of human sperm extracts, SPAN-X antibodies recognized 19 spots ranging from 20 to 23 kDa with isoelectric points from 5.0 to 5.5. Differential extraction of spermatozoa demonstrated that the SPAN-X protein is highly insoluble. Only 50% of ejaculated spermatozoa exhibited SPAN-X immunofluorescent staining. Dual localization of the sex chromosomes and the SPAN-X protein demonstrated that an equal number of X- and Y-bearing spermatozoa exhibited SPAN-X staining. In transfected mammalian CV1 cells, the SPAN-Xa and SPAN-Xb proteins were localized to the nucleus and cytoplasm, respectively, by indirect immunofluorescence. On immunoblots of CV1 cells, the SPAN-Xa protein migrated at 15-20 kDa, whereas the SPAN-Xb protein migrated at a higher molecular weight of 21-22 kDa. The SPAN-X protein was ultrastructurally associated with nuclear vacuoles and the redundant nuclear envelope. SPAN-X is the first protein specifically localized to these poorly characterized structures of the mammalian sperm nucleus and provides a unique biochemical marker for investigation of their function in spermatozoa as well as the role of SPAN-X/CTp11 in human tumors.

Spermatid-specific expression of the novel X-linked gene product SPAN-X localized to the nucleus of human spermatozoa.

Formation of mature spermatozoa involves a series of dramatic molecular and morphological changes in the male germ cell lineage. These changes result from the temporally regulated transcription and translation of several testis-specific gene products. Here, we describe a novel, testis-specific protein designated SPAN-X for sperm protein associated with the nucleus on the X chromosome. SPAN-X sequences showed no significant similarity with known cDNA or peptide sequences. The SPAN-X peptide sequences contained three overlapping consensus nuclear localization signals, a high percentage (33%-37%) of charged amino acid residues, and a relatively acidic isoelectric point (pI; 4.88-6.05). Northern analysis of mRNA from multiple human tissues identified a SPAN-X transcript exclusively in the testis. In situ hybridization of human testes sections showed SPAN-X mRNA expression in haploid, round, and elongating spermatids. The SPANX gene was mapped to chromosome Xq27. 1 by fluorescence in situ hybridization and by Southern blot analysis of human/mouse somatic cell hybrids. On Western blots of human sperm proteins, antirecombinant SPAN-X antibodies reacted with broad bands migrating between 15-20 kDa. Immunofluorescent labeling of human spermatozoa demonstrated SPAN-X localization to nuclear craters and cytoplasmic droplets. Expression of SPAN-X, an X-linked gene product, exclusively in haploid spermatids leads to interesting questions regarding the transcription of sex-linked genes during spermiogenesis.

Postvasectomy alterations in protein synthesis and secretion in the rat caput epididymidis are not repaired after vasovasostomy.

Many men who have undergone vasectomy later request vasovasostomy. Unfortunately, significant numbers of these men remain infertile despite the reestablishment of patent ducts. This report examines the possibility that epididymal function remains compromised after vasovasostomy in the rat by examination of quantifiable, in vivo protein synthesis and secretion in the caput epididymidis. Rats were studied 30 days after vasectomy, 30 days after a vasovasostomy (which was performed 30 days after vasectomy), or after sham operations. Epididymal lumen fluids (LF) were collected by micropuncture after 3 hours' in vivo microperifusion of tubules with 35S-amino acids. Proteins were separated by 2-dimensional electrophoresis and were detected by Coomassie blue staining. Synthesized proteins in tubule extract and synthesized and secreted proteins in LF were detected by autoradiography and image analysis. Specific proteins that appeared to be affected by vasectomy-vasovasostomy were identified by internal sequence analysis. LF contained an average of 87 detectable proteins synthesized and secreted in the control caput. Nineteen of the most prominent LF proteins were selected for more focused study. The most prominent proteins were clusterin, cysteine-rich secretory protein (CRISP)-1, and epididymal retinoic acid-binding protein. Among these, CRISP-1 remained reduced in LF after vasovasostomy. Two more minor proteins that remained reduced after vasovasostomy were identified as prostaglandin D2 synthase and phosphatidylethanolamine-binding protein. All 3 of these proteins occur in the epididymides of multiple species and have been associated with sperm fertilizing capacity.

Antisperm autoantibody response is reduced by early repair of a severed vas deferens in the juvenile rat.

OBJECTIVE: To determine whether antisperm autoantibody production after prepubertal vas injury is influenced by immediate repair of the vas compared to delay of the reanastomosis until sexual maturity. DESIGN: Animal study comparing early repair, late repair, and sham-operated groups. SETTING: Research laboratory in a medical school. PATIENT(S): Lewis rats. INTERVENTION(S): After division of the vas deferens in juvenile rats, animals in an early repair group had the vasa repaired immediately by using an absorbable intraluminal stent. Animals in a late repair group had vasa obstructed by ligation until after puberty, when they underwent microsurgical vasovasostomy (age 60 days). MAIN OUTCOME MEASURE(S): Antisperm antibodies were assayed by ELISA. The weights of reproductive organs were determined, and samples of testis were studied by light microscopy. RESULT(S): The antisperm antibody response was less when the vas was repaired immediately than if the repair was delayed until after puberty. There was a low incidence of testicular alteration in the repair groups and none in sham-operated animals. CONCLUSION(S): If the vas deferens is injured or obstructed prepubertally, there may be a benefit to considering immediate repair to reduce the likelihood of developing antisperm autoantibodies, which have been associated with reduced fertility.

FSP95, a testis-specific 95-kilodalton fibrous sheath antigen that undergoes tyrosine phosphorylation in capacitated human spermatozoa.

Protein tyrosine phosphorylation has been associated with both capacitation and motility of mammalian sperm. During capacitation, human spermatozoa undergo tyrosine phosphorylation of a characteristic set of proteins, only one of which has thus far been cloned and localized. We report here the sequence of a fibrous sheath protein of 95 kDa (FSP95) that undergoes tyrosine phosphorylation during capacitation of human spermatozoa and has similarity to sperm A-kinase anchor proteins (AKAPs). FSP95 is both auto- and iso-antigenic in humans as it is recognized by sera containing antisperm antibodies from infertile men and women. The 853-residue protein has a calculated molecular weight of 94.6 kDa and an isoelectric point (pI) of 6.0, and it contains multiple potential phosphorylation sites for protein kinase C and casein kinase II as well as one potential tyrosine kinase phosphorylation site at amino acid 435. The sequence has amino acid homology to mouse sperm fibrous sheath AKAP82 (pro-mAKAP82, 34% identity) and to human sperm fibrous sheath AKAP82 (pro-hAKAP82, 32% identity). The gene encoding FSP95 has 5 exons separated by 4 introns and is located on chromosome 12 at locus p13.3. Northern analysis detected a single transcript of approximately 3.0 kilobases, and Northern dot blot analysis of 50 human tissues revealed FSP95 mRNA expression only in testis. By employing sperm immobilization, indirect immunofluorescence, and immunoelectron microscopy with antisera to purified recombinant FSP95, the protein was localized to the ribs of the fibrous sheath in the principal piece of the sperm tail. FSP95 is the second fibrous sheath protein to be cloned, sequenced and localized in human spermatozoa.

Round spermatid-specific transcription of the mouse SP-10 gene is mediated by a 294-base pair proximal promoter.

Spermiogenesis is the terminal phase of male germ cell differentiation during which haploid spermatids engage in coordinate expression of a number of testis-specific genes, including those specifying acrosomal proteins. To begin to understand the transcriptional regulation during acrosomal biogenesis, we initiated promoter analysis of the gene encoding the acrosomal protein SP-10. SP-10 was previously shown to be transcribed within Golgi-phase round spermatids in the human. The present study characterizes SP-10 gene expression during spermiogenesis in the mouse and identifies regions of the mouse SP-10 (mSP-10) promoter that are capable of driving round spermatid-specific transcription in vivo. Expression of mSP-10 mRNA was initiated in early round spermatids coincident with acrosomal biogenesis and was terminated prior to nuclear elongation. The core promoter of mSP-10 lacked a TATA box but contained a canonical initiator (Inr) element surrounding the transcription start site. Using transgenic mice, we showed that the -408 to +28-base pair (bp) or the -266 to +28-bp mSP-10 5' flanking region is sufficient to direct round spermatid-specific expression of a green fluorescent protein reporter gene. On the other hand, the -91 to +28-bp mSP-10 gene fragment lacked promoter activity in vivo. This is the first functional characterization of a testis-specific gene promoter active in early round spermatids.