Detection and identification of platelet antibodies and antigens in the clinical laboratory

AbstractGlanzmann's thrombasthenia (GT) and Bernard–Soulier's syndrome (BSS) are well-understood congenital bleeding disorders, showing defect/deficiency of platelet glycoprotein (GP) IIb/IIIa (integrin αIIbβ3) and GPIb-IX-V complexes respectively, with relevant clinical, laboratory, biochemical, and genetic features. Following platelet transfusion, affected patients may develop antiplatelet antibodies (to human leukocyte antigen [HLA], and/or αIIbβ3 in GT or GPIb-IX in BSS), which may render future platelet transfusion ineffective. Anti-αIIbβ3 and anti-GPIb-IX may also cross the placenta during pregnancy to cause thrombocytopenia and bleeding in the fetus/neonate. This review will focus particularly on the better studied GT to illustrate the natural history and complications of platelet alloimmunization. BSS will be more briefly discussed. Platelet transfusion, if unavoidable, should be given judiciously with good indications. Patients following platelet transfusion, and women during and after pregnancy, should be monitored for the development of platelet antibodies. There is now a collection of data suggesting the safety and effectiveness of recombinant activated factor VII in the management of affected patients with platelet antibodies.

Download Full-text

Acid-fast bacterium detection and identification from paraffin-embedded tissues using a PCR-pyrosequencing method

Journal of Clinical Pathology ◽

10.1136/jclinpath-2016-204128 ◽

2017 ◽

Vol 71 (2) ◽

pp. 148-153 ◽

Cited By ~ 2

Author(s):

Jian R Bao ◽

Richard B Clark ◽

Ronald N Master ◽

Kileen L Shier ◽

Lynn L Eklund

Keyword(s):

Clinical Laboratory ◽

Cross Reactivity ◽

Rrna Gene ◽

Tissue Sections ◽

Detection And Identification ◽

Formalin Fixed Paraffin ◽

Mycobacterium Genavense ◽

Formalin Fixed Paraffin Embedded ◽

Ffpe Tissues ◽

Group 3

AimsAcid-fast bacterium (AFB) identification from formalin-fixed paraffin-embedded (FFPE) tissues is challenging and may not be readily available to the clinical laboratory. A method to detect and identify AFB from FFPE tissues using PCR and pyrosequencing (PCR-Seq) was developed and evaluated.MethodsThe method was validated using spiked cell-clotted paraffin blocks before use with patients’ specimens. DNA was extracted from tissue sections, and a 16S rRNA gene fragment was amplified and a signature sequence was produced on a PyroMark ID system. Sequences were aligned to established databases for AFB identification. Additional tissue sections were stained and examined for AFB.ResultsBoth sensitivity and specificity were 100% on spiked cell-clotted blocks without cross-reactivity with non-AFB. Of 302 FFPE tissues from patients, 116 (38%) were AFB-stain positive; 83 (72%) of these had AFB identified. The 21 AFB identified included Mycobacterium tuberculosis complex (14 cases), Mycobacterium leprae (3), Mycobacterium genavense (2), Mycobacterium marinum-ulcerans group (3) and 17 other AFB (61). Thirteen cases were AFB-stain indeterminate and 4 were positive by the PCR-Seq method. Of the AFB stain-negative cases, 167 were negative and 6 were positive by PCR-Seq.ConclusionsThe PCR-Seq method provided specific identification of various AFB species or complexes from FFPE tissues.

Download Full-text

DNA amplification and reverse dot blot hybridization for detection and identification of mycobacteria to the species level in the clinical laboratory.

Journal of Clinical Microbiology ◽

10.1128/jcm.30.5.1220-1224.1992 ◽

1992 ◽

Vol 30 (5) ◽

pp. 1220-1224 ◽

Cited By ~ 18

Author(s):

E H Fiss ◽

F F Chehab ◽

G F Brooks

Keyword(s):

Clinical Laboratory ◽

Blot Hybridization ◽

Dna Amplification ◽

Species Level ◽

Dot Blot ◽

Dot Blot Hybridization ◽

Detection And Identification ◽

Reverse Dot Blot Hybridization ◽

Reverse Dot Blot

Download Full-text

Reliable Detection of Group B Streptococcus in the Clinical Laboratory

Journal of Clinical Microbiology ◽

10.1128/jcm.00582-17 ◽

2017 ◽

Vol 55 (9) ◽

pp. 2590-2598 ◽

Cited By ~ 18

Author(s):

Manuel Rosa-Fraile ◽

Barbara Spellerberg

Keyword(s):

Pregnant Women ◽

Clinical Laboratory ◽

Culture Media ◽

Group B Streptococcus ◽

Molecular Techniques ◽

Diagnostic Tools ◽

Species Determination ◽

Flight Mass Spectrometry ◽

Detection And Identification ◽

Group B

ABSTRACTGroup B streptococcus (GBS) is a leading cause of invasive neonatal infections and a significant pathogen in immunocompromised adults. Screening to detect GBS colonization in pregnant women determines the need for antibiotic prophylaxis in that pregnancy. Efficient determination of the GBS colonization status of pregnant women is crucial. Methods that maximize the probability of GBS recovery are needed. The availability of technologies such as matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), molecular techniques, and chromogenic culture media, including Granada-type media, have changed the scenario for GBS detection and identification. This review presents and evaluates novel diagnostic tools, as well as classic identification techniques, for GBS species determination.

Download Full-text

A Nested-PCR-Based Schizodeme Method for Identifying Leishmania Kinetoplast Minicircle Classes Directly from Clinical Samples and Its Application to the Study of the Epidemiology of Leishmania tropica in Pakistan

Journal of Clinical Microbiology ◽

10.1128/jcm.36.10.2877-2881.1998 ◽

1998 ◽

Vol 36 (10) ◽

pp. 2877-2881 ◽

Cited By ~ 140

Author(s):

Harry A. Noyes ◽

Hugh Reyburn ◽

J. Wendy Bailey ◽

David Smith

Keyword(s):

Nested Pcr ◽

Clinical Laboratory ◽

Variable Region ◽

Pcr Primers ◽

Clinical Samples ◽

Leishmania Tropica ◽

Guanidine Thiocyanate ◽

Kinetoplast Dna ◽

Detection And Identification ◽

Pcr Products

A nested PCR was developed to amplify the variable region of the kinetoplast minicircles of all Leishmania species which infect mammals. Each Leishmania parasite contains approximately 10,000 kinetoplast DNA minicircles, which are unequally distributed among approximately 10 minicircle classes. The PCR primers were designed to bind within the 120-bp conserved region which is common to all minicircle classes; the remaining approximately 600 bp of each minicircle is highly conserved within each minicircle class but highly divergent between classes. The nested PCR generated a strong signal from a minimum of 0.1 fg of Leishmania DNA. Restriction digests of the amplicons from the highest dilutions suggested that minicircles from only a limited number of minicircle classes had acted as template in the reaction. One PCR product was directly sequenced and found to be derived from only one minicircle class. Since the primers amplify all minicircle classes, this indicated that as little as 1/10 of one Leishmania parasite was present in the PCR template. This demonstrated that the nested PCR achieved very nearly the maximum theoretically possible sensitivity and is therefore a potentially useful method for diagnosis. The nested PCR was tested for sensitivity on 20 samples from patients from the Timargara refugee camp, Pakistan. Samples were collected by scraping out a small amount of tissue with a scalpel from an incision at the edge of the lesion; the tissue was smeared on one microscope slide and placed in a tube of 4 M guanidine thiocyanate, in which the sample was stable for at least 1 month. DNA for PCR was prepared by being bound to silica in the presence of 6 M guanidine thiocyanate; washed in guanidine thiocyanate, ethanol, and acetone; and eluted with 10 mM Tris-HCl. PCR products of the size expected for Leishmania tropica were obtained from 15 of the 20 samples in at least one of three replicate reactions. The negative samples were from lesions that had been treated with glucantime or were over 6 months old, in which parasites are frequently scanty. This test is now in routine use for the detection and identification of Leishmaniaparasites in our clinical laboratory. Fingerprints produced by restriction digests of the PCR products were defined as complex or simple. There were no reproducible differences between the complex restriction patterns of the kinetoplast DNA of any of the parasites from Timargara camp with HaeIII and HpaII. The simple fingerprints were very variable and were interpreted as being the product of PCR on a limited subset of minicircle classes, and consequently, it was thought that the variation was determined by the particular minicircle classes that had been represented in the template. The homogeneity of the complex fingerprints suggests that the present epidemic of cutaneous leishmaniasis in Timargara camp may be due to the spread of a single clone of L. tropica.

Download Full-text

The Role of the Clinical Laboratory in Managing Chemical or Biological Terrorism

Clinical Chemistry ◽

10.1093/clinchem/46.12.1883 ◽

2000 ◽

Vol 46 (12) ◽

pp. 1883-1893 ◽

Cited By ~ 19

Author(s):

Saeed A Jortani ◽

James W Snyder ◽

Roland Valdes Jr

Keyword(s):

Diagnostic Tests ◽

Clinical Laboratory ◽

Biological Agents ◽

Medical Community ◽

Laboratory Personnel ◽

Specimen Handling ◽

Clinical Laboratories ◽

Detection And Identification ◽

Biological Terrorism

Abstract Background: Domestic and international acts of terrorism using chemicals and pathogens as weapons have recently attracted much attention because of several hoaxes and real incidents. Clinical laboratories, especially those affiliated with major trauma centers, should be prepared to respond rapidly by providing diagnostic tests for the detection and identification of specific agents, so that specific therapy and victim management can be initiated in a timely manner. As first-line responders, clinical laboratory personnel should become familiar with the various chemical or biological agents and be active participants in their local defense programs. Approach: We review the selected agents previously considered or used in chemical and biological warfare, outline their poisonous and pathogenic effects, describe techniques used in their identification, address some of the logistical and technical difficulties in maintaining such tests in clinical laboratories, and comment on some of the analytical issues, such as specimen handling and personal protective equipment. Content: The chemical agents discussed include nerve, blistering, and pulmonary agents and cyanides. Biological agents, including anthrax and smallpox, are also discussed as examples for organisms with potential use in bioterrorism. Available therapies for each agent are outlined to assist clinical laboratory personnel in making intelligent decisions regarding implementation of diagnostic tests as a part of a comprehensive defense program. Summary: As the civilian medical community prepares for biological and chemical terrorist attacks, improvement in the capabilities of clinical laboratories is essential in supporting counterterrorism programs designed to respond to such attacks. Accurate assessment of resources in clinical laboratories is important because it will provide local authorities with an alternative resource for immediate diagnostic analysis. It is, therefore, recommended that clinical laboratories identify their current resources and the extent of support they can provide, and inform the authorities of their state of readiness.

Download Full-text

Rapid method for the detection and identification of mycolic acids in aerobic actinomycetes and related bacteria

Journal of Clinical Microbiology ◽

10.1128/jcm.4.3.284-287.1976 ◽

1976 ◽

Vol 4 (3) ◽

pp. 284-287

Author(s):

S T Hecht ◽

W A Causey

Keyword(s):

Thin Layer ◽

Thin Layer Chromatography ◽

Rapid Method ◽

Clinical Laboratory ◽

Diaminopimelic Acid ◽

Whole Cell ◽

Mycolic Acids ◽

Detection And Identification ◽

Layer Chromatography ◽

Aerobic Actinomycetes

A rapid method for the identification of lipids characteristic of the genera Corynebacterium, Mycobacterium, Nocardia, and the "rhodochrous group" has been developed. Modifications of previously described methods make this procedure suitable for use in the clinical laboratory. Thin-layer chromatography is used to demonstrate the presence of the lipid characteristic of Nocardia spp. (type A) in some corynebacteria, nocardias, and members of the "rhodochrous group." Precipitation in ether and ethanol is used to demonstrate the presence of mycobacterial mycolic acids. Since this procedure can be carried out in less than 2 days and the lipids are extracted from the same batch of cells grown for diaminopimelic acid and whole-cell sugar analyses, it can readily be added to the battery of tests performed in reference laboratories that deal with aerobic actinomycetes and related bacteria.

Download Full-text

Comparison of a Nonradiometric Liquid-Medium Method (MB REDOX) with the BACTEC System for Growth and Identification of Mycobacteria in Clinical Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.37.12.4048-4050.1999 ◽

1999 ◽

Vol 37 (12) ◽

pp. 4048-4050 ◽

Cited By ~ 2

Author(s):

Yung-Ching Liu ◽

Tsi-Shu Huang ◽

Wen-Kuei Huang

Keyword(s):

Clinical Laboratory ◽

Polymorphism Analysis ◽

Clinical Specimens ◽

Detection And Identification ◽

Positive Growth ◽

Redox Indicator ◽

Antibiotic Compounds ◽

Bactec System ◽

Growth Promoting ◽

Medium Method

Early identification of tuberculosis in the clinical setting is of great importance in order for specific therapy to be swiftly initiated. MB REDOX (Heipha Diagnostika), a growth-based medium without radioactive materials, was evaluated and was compared to the BACTEC system for detection of mycobacteria, including the Mycobacterium tuberculosis complex and atypical mycobacteria. MB REDOX consists of a Kirchner medium enriched with growth-promoting additives, antibiotic compounds, and a redox indicator which can be monitored to detect growth of mycobacteria with the naked eye. MB REDOX only detects growth and cannot differentiate the M. tuberculosis complex (M. tuberculosis, M. bovis, and M. africanum) from other species of Mycobacterium. Therefore, PCR-restriction fragment length polymorphism analysis (PRA) was used in this investigation to identify to the species level organisms showing positive growth with MB REDOX. Our data demonstrate the usefulness of MB REDOX for the detection of mycobacteria in clinical specimens. The rate of detection of M. tuberculosis complex with MB REDOX (84.3%) was higher than that with the BACTEC system (68.6%). When combined with PRA for species identification, MB REDOX is easy to perform and is suited to most clinical laboratory settings for the detection and identification of mycobacteria.

Download Full-text